A logistic regression model was applied to forecast the association between the predictor and outcome variables. Statistical significance was considered at a 0.05 p-value. Results: The study showed that the overall acceptance of parents to vaccinate their daughters with HPV vaccination was 450 (84.9%). Parents of daughters of male sex , who had only one daughter , whose daughter(s) attended a government school , who had poor knowledge  and who had a negative attitude  were discovered to have a strong correlation. Conclusion: This study found that there was a high level of parental acceptance; attitudes and knowledge about the HPV vaccine are significant in determining their intentions to vaccinate their daughter. Authorities in high-risk areas for cervical cancer incidence should plan and implement strategies by providing health information regarding human papillomavirus vaccination with an emphasis on raising community awareness.Introduction: Human papillomavirus infections are the most prevalent sexually transmitted disease among women worldwide. Cervical cancer is the second-most frequent disease worldwide in terms of incidence and mortality, and it is primarily responsible for fatalities in low- to middle-income nations, including Ethiopia. Objective: To assess awareness, acceptance, and associated factors of the human papillomavirus vaccine among parents of daughters in the Hadiya zone, southern Ethiopia. Methods: From November to December 2021, a community-based cross-sectional study was conducted in the Hadiya zone among parents with daughters in the zone. The study respondents were chosen using a two-stage sampling technique from parents with a 9–14-year-old daughter. An interviewer-administered questionnaire was used to collect data. For analysis, the data were entered into Epidata version 3.1 and exported to SPSS version 25. Variables with a  Human Papilloma Virus (HPV) is the name given to a group of viruses that includes more than 200 related viruses, more than 40 of which are spread through vaginal and anal intercourse and can cause premalignant changes and malignant cancers of the cervix, vagina, vulva, and anus . In bothCervical cancer is the world’s most common cancer in women; every year, approximately 500,000 new cases emerge , accountIn developing countries, the burden of 230,000 cases and deaths  is borne by those who have only the bare minimum of resources to deal with the situation ; despiteCervical cancer is the second leading cause of cancer death among women in Ethiopia. Approximately 7600 new cervical cancer cases are diagnosed each year, with over 6000 deaths from the disease . HPV is Cervical cancer screening is the most effective way for all women to reduce cancer mortality . AlthougAs a result, the World Health Organization (WHO) has developed comprehensive strategies for cervical cancer prevention and control, including HPV vaccination as the primary method of prevention ,11. CervEthiopia introduced the HPV vaccine in 2018 with the help of the Global Alliance for Vaccine and Immunization (GAVI) , which tThere is now an HPV prophylactic vaccine available that protects against most genital warts and cervical cancer. It is also known as the cervical cancer vaccine . It has The WHO recommends two doses of the HPV vaccine for girls aged 9–14 years, and three doses for girls aged 15 years and older, as well as those who are immunocompromised or HIV positive (regardless of age) . HPV vacEthiopia had planned to introduce the HPV vaccine through a routine immunization program for approximately 6 million girls aged 9 to 14 years old ,17. HoweBasic knowledge of women’s pelvic anatomy and the natural history of cervical cancer provides primary- and secondary-level healthcare providers with the knowledge they need to effectively communicate and raise awareness of cervical cancer prevention in women, families, and communities .According to the 2005 CSA report, the Hadiya zone, Gurage, and Kembata Tembaro produced 8364.00 tons of coffee in 2005, accounting for 8.33% of Southern Nation Nationality People Region (SNNPR) output and 3.36% of Ethiopia’s overall output. According to the World Bank report, only 6% of Hadiya people live with electricity, there is a road density of 104.1 km per 1000 square kilometers compared to the national average of 30 km, 0.6 hectares of land for rural households compared to the national average of 1.01 hectares of land, and an average of 0.89 for the SNNPR. There is the equivalent of 0.6 head of cattle; 22.8% of the population is working in non-farm related jobs, compared to 25% nationally and 32% regionally. The average age of first childbirth in the Hadiya zone is 18 years, which is similar to national figures, and the majority of the residents are Christian .The awareness of the parents of daughters on acceptance of the human papillomavirus vaccine was measured by the question “today, had you heard of HPV?” Parents of daughters who answered ‘yes’ to this question were regarded to be aware of HPV ,3,22.The data were collected using interviewer-administered quantitative data. The questionnaire contained questions developed and adapted from various kinds of literature. The necessary permissions were obtained from the owner of the original questionnaire. The questionnaire developed by the investigators contained the following 4 sections: (1) basic demographic characteristic , (2) knowledge, (3) attitude, and (4) information-related factors on vaccination awareness and acceptance of parents of daughters on acceptance of human papillomavirus vaccine. Three days of training were provided for data collection using an interview-administered method by two trained data collectors and one supervisor. The principal investigator reviewed the obtained data to ensure its accuracy, completeness, clarity, and consistency. The questionnaire was written in English, translated into common Amharic to ensure that the items were consistent, and then translated back into English to ensure that the common Amharic was accurate. The questionnaire’s face validity was established.A pre-test was performed before data collection by taking 28 samples out of the study area. The data collection tool’s reliability was evaluated in terms of knowledge, attitude, and acceptance, as well as awareness. To ensure the quality of the data, emphasis was placed on the design of the data collection instrument for its simplicity and standardized community rating scales, validity, and reliability were considered.p-values under 0.25 were added to the multivariable analysis.Data that had been coded was input in EpiData version 3.1 and exported to SPSS version 25 for analysis. Data entry fell under the purview of the principal investigator. Tables, graphs, and charts were used to perform descriptive analysis and report the results in frequency. Variables from the bivariate analysis that had To choose variables for multivariable analysis, bivariate analysis was used. However, multivariable statistical significance was tested at the level of 5%. Using logistic regression, adjusted odds ratios (AOR) and a 95% confidence interval were utilized to confirm the presence and strength of the link between the independent and dependent variables. The Hosmer and Lemeshow tests were used to gauge the model’s fitness. The Variance Inflation Factor (VIF), standard error, and correlation coefficient were used to test for multicollinearity among independently associated variables.Out of the 550 parents of daughters who were eligible to participate in the study, 20 were excluded , resulting in a 96.54% response rate. Out of the 530 parents of daughters included in the study, the majority, 323 (60.9%) of the respondents were female, and almost more than half (323 (60.9%)) of them were in the age category 30–39 years. The mean age of parents of daughters was 38 with (SD) ± (9.45) years and nearly less than three-fourths, 365 (68.9%) of the respondents were Protestant, 354 (66.8%) were urban by residence, 437 (82.5%) were married, 322 (60.8%) had an educational status of degree and above, 354 (66.8%) were civil servants by occupation, and 389 (73.4%) were of Hadiya ethnicity. The majority of the respondents (418 (79.8%)) had more than one child, 355 (67%) respondents were educated in a private school, more than half of respondents, 318 (60%) had information on HPV, and 144 (45.3%) listed the major source of information as television or radio. Almost half, 291 (54.9%) of the respondents reported a monthly income >ETB 4999 (>USD 95.24) .Of the 530 study respondents, the majority 419 (79.1%) with 95% CI  had good knowledge with a mean of 1.7906 and standard deviation of ±0.40729 .Almost more than three-fourths 418 (78.9%) and nearly all most all 499 (94.2%) of the parents of daughters had ever heard about cervical cancer and thought cervical cancer infected only females, respectively. Nearly less than one-fourth 111 (20.9%) and 144 (27.2%) of the parents of daughters knew that cervical cancer produces no signs and symptoms at an early stage and that it is a fast-growing cancer, respectively. Concerning treatment curability, about 418 (78.9%) respondents answered the correct answers about early detection for cervical cancer protection and 371 (70%) respondents were getting a Pap test for early detection of cervical cancer. Almost three-fourths 387 (73%) respondents knew that HPV is the main cause of cervical cancer and the majority 435 (82.1%) knew it was common in women younger than 30 years old. More than three-fourths of respondents knew that HPV vaccination is available for girls aged 9 to 14 years and that cervical cancer risk can be reduced by HPV vaccination 419 (79.1%) and 434 (81.9%), respectively). Almost more than half of respondents knew that HPV infection is a sexually transmitted infection, and that vaccination should be received before sexual debut 307 (57.9%) and 338 (63.8%), respectively. The majority 434 (81.9%) and nearly less than one-fourth 112 (21.1%) of the respondents knew that the persistent infection of high-risk HPV could lead to cervical cancer and 70% of cervical cancer is caused by HPV types 16 and 18, respectively. More than one-fourth 159 (30%) of the parents of daughters were aware of the most common and effective treatment to cure cervical cancer .Out of 530 total respondents, 371 (70%) had positive attitudes with a 95% CI  and a mean of 1.7000, and a standard deviation of ± 0.45869 .This study identified that more than half 291 (54.9%) and nearly three-fourths 387 (73%) of the daughters of parents were willing to regularly consult a medical doctor for cervical cancer screening and those daughters with multiple sex partners would be at higher risk for cervical cancer, respectively. The majority of parents of daughters disagreed that long-term use of contraceptive pills could cause cervical cancer and agreed that the use of condoms could reduce the risk of cervical cancer 413 (77.9%) and 386 (72.8%), respectively. More than half 370 (69.8%) of the respondents would not consult a medical doctor in case of abnormal bleeding between menstrual periods, while 386 (72.8%) parents of daughters agreed that HPV vaccination can prevent cervical cancers. The majority were willing to get a Pap smear test and agreed that a regular Pap smear test helps in the early detection of cervical cancer 434 (81.9%) and 483 (91%), respectively. The majority of respondents 467 (88.1) disagreed to pay for a Pap smear test and 463 (87.4%) respondents agreed that the government should provide free screening programs to reduce cervical cancer prevalence. The majority 434 (81.9%) of parents of daughters disagreed that the HPV vaccine might cause short-term problems, like fever or discomfort .Out of 530 total respondents, 450 (84.9%) parents accepted to vaccinate their daughter for HPV vaccination with a 95% CI  and a mean of 1.1509 and a standard deviation of ± 0.35833  and obstOut of 530 total respondents, 450 (84.9%) parents accepted to vaccinate their daughter for HPV vaccination with a 95% CI  and a mean of 1.1509, and a standard deviation of ± 0.35833 .The majority of the parents of daughters had heard of HPV and had heard of the HPV vaccine 467 (88.1%) and 450 (84.9%), respectively. The majority 450 (84.9%) of the study respondents were willing to accept vaccinating their daughter with HPV vaccination and 32 (6%) had reasons for being unwilling to take the HPV vaccine, such as being worried about the price, and the majority 482 (90.9%) of respondents did not accept that they should pay for the HPV vaccination by themselves .p-value < 0.05.Multivariable analysis was used to control potential confounders. Having parents of daughters of the male sex, having only one daughter, having daughter(s) who attended a government school, having poor knowledge, and having a negative attitude were discovered to have a substantial relationship with acceptance of the human papillomavirus vaccine with a In this study, parents of daughters of the male sex were 59.3% less likely to accept HPV vaccination for their daughters as compared to the female sex , parents who had only one daughter were 2.122 times more likely to accept HPV vaccination for their daughters as compared to those who had more than one daughter , parents of daughters who attended a government school were 52.4% less likely to accept HPV vaccination for their daughters as compared to those whose daughter(s) attended a private school , parents of daughters who had poor knowledge were 46.8% less likely to accept HPV vaccination for their daughters as compared to good knowledge , and those parents of daughters who had a negative attitude were 46% times less likely to accept HPV vaccination for their daughters as compared to positive attitudes  .This study was conducted on parents of daughters attending school to ascertain parental awareness, knowledge, attitudes, and views regarding HPV infection, cervical cancer, and HPV vaccination as well as the acceptance of the HPV vaccine. The major purpose of this study was to determine parental awareness, acceptance, and associated factors of the human papillomavirus vaccine in the Hadiya zone, southern Ethiopia.The main finding of this study revealed that, despite the views of some men and parents sending their daughters to government schools, parents indicated they were willing to vaccinate their children. This study finding is also almost nearly two times more than the study conducted in Hong Kong , and 1.1According to the findings, the overall acceptance of HPV vaccinations for their daughters was 84.9%, 3.6 times higher than the study conducted among male baccalaureate students in Hong Kong , 1.1 timThese findings are almost 1.13 times lower than the study conducted in Indonesia , 1.12 tiThe current national drive in Ethiopia for HPV vaccination may have contributed to the high vaccine acceptance rate in the study. During the data collection for this study, the parents were informed of the campaign-based school-based immunization program, and they decided on the vaccine. The high acceptability of the HPV vaccine in our study could be viewed as a chance to expand the country’s school-based HPV vaccination program. The study’s different acceptance rates in Addis Ababa, Gondar, and the Bench-Sheko zone could be partially attributed to the respondents’ varied sociodemographic makeup.Given that the nation has started a school-based vaccination program for these targeted girls, this shows that Ethiopia’s high parental desire to vaccinate their daughters is good news and presents an excellent opportunity as well. Because parents have a significant role in their daughters’ decision to receive the vaccine or not, a greater parental acceptance to have their daughters receive the HPV vaccine will facilitate the vaccination campaign by making more girls available for vaccination.In this study, parents of daughters of the male sex were 59.3% less likely to accept HPV vaccination for their daughters as compared to the female sex , which was almost similar to the study conducted at a premier medical school in India , and theThose parents of daughters who had only one daughter were 2.12 times more likely to accept HPV vaccination for their daughter as compared to the number of parents with more than one daughter . This study found this result to be 1.57 times more than that of the study conducted in Shenzhen, China . This coParents of daughters who attended a government school were 52.4% less likely to accept HPV vaccination for their daughters as compared to those whose daughters attended a private school ; this could be because private schools are funded wholly or partly for the vaccination of daughters for human papillomavirus by a private body.Parents of daughters who had poor knowledge were 46.8% less likely to accept HPV vaccination for their daughters as compared to good knowledge , This study finding was consistent with the study conducted in Gondar town, northwest Ethiopia , almost This study finding is similar to the study conducted in Addis Ababa, Ethiopia , at a teThese disparities may have been brought about by the gap in respondents’ sociodemographic characteristics. This, in our opinion, demonstrates how parents’ acceptance to vaccinate their daughters is influenced by their knowledge of HPV vaccination. Therefore, raising parental knowledge is crucial, and programs should be developed specifically for parents without a formal education.Parents of daughters who had negative attitudes were 46% times less likely to accept HPV vaccination for their daughters as compared to positive attitudes . Parental positive attitude towards HPV vaccine was significantly associated with parental acceptance to vaccinate their daughters. This is supported by other study findings . This shThis study found that there was a high level of parental acceptance to immunize their daughters against the human papillomavirus in the study area. Parents of daughters of the male sex, who had multiple daughters, whose daughters attended government schools, who had poor knowledge, and who had a negative attitude were discovered to have a substantial relationship with acceptance of the human papillomavirus vaccine. Additionally, the parents’ attitudes and knowledge about the HPV vaccine were significant in determining their intentions to vaccinate their daughters.It is advised that there is a need to provide national educational campaigns about the HPV vaccine to the public given that the HPV vaccine is not included in the national immunization schedule. Authorities in high-risk areas for cervical cancer incidence should plan and implement strategies by providing health information regarding the human papillomavirus vaccination, with an emphasis on raising community awareness.The regional and zonal health offices in Ethiopia’s Ministry of Health should develop health information regarding HPV vaccination with a focus on raising community awareness, especially among parents who lack formal education. It is essential to raise community awareness sustainably to secure the long-term acceptability of HPV vaccination. Therefore, it is essential to improve awareness and acceptance of cervical cancer as well as its prevention in the community.To demonstrate the current parental acceptance to vaccinate their daughters in response to the vaccine campaign being run by the Ethiopian MOH and Zonal Health Department, a community-based study with a representative sample of parents from Ethiopia’s rural areas may be feasible. Additionally, it is the first study from a zonal level in Ethiopia to fully characterize parental understanding and acceptance of the HPV vaccination. Since the study is cross-sectional, a causal relationship cannot be shown; nonetheless, recollection and social desirability bias may potentially have an impact.


This sub-analysis aimed to evaluate its efficacy in repairing these cervical lesions and clearing HPV in women aged older than 40 years. This was a multicenter, randomized, open-label, parallel-group, controlled clinical trial. Patients with low-degree HPV-dependent cervical lesions receiving 6-month treatment with the vaginal gel were compared to those with a watchful waiting approach. Among the 41 women analyzed (aged 47.7 years), 31 presented high-risk (HR) oncogenic HPV subtypes, and 14 had 16-18-31 HPV genotypes. After 6 months, normalized cytology and concordant colposcopy were achieved by a greater percentage of treated women. The difference was significant in the total population , and HR-HPV subpopulation . In the HR HPVs-16-18-31 subpopulation, the values were 75.0% and 40.0% (p = 0.293). In the total population, 61.5% of treated patients obtained HPV clearance, compared to 50.0% in the control group. Regarding the HR-HPV subpopulation, these values were 66.7% and 44.4%, respectively. Papilocare® demonstrated significant efficacy in repairing low-degree HPV-related cervical lesions and a positive trend to clear HPV in women older than 40 years old in comparison to the watchful waiting approach.In the PALOMA trial, Papilocare The human papillomavirus (HPV) is one of the most common sexually transmitted infections of the reproductive system . All typIt is estimated that more than 70% of sexually active females will be infected during their lives, and a few will be so more than once. HPV infection is more common among young women ,3. GenerNevertheless, the HPV infection sometimes persists, especially in those subtypes classified as high risk (HR), which increases the risk of cervical intraepithelial lesions and cervical cancer . Besides® , a vaginal gel including Coriolus versicolor and other ingredients, has proven to be effective and safe in repairing low-grade cervical lesions and enhancing HR-HPV clearance [In this context, Papilocarelearance ,13,14,15® versus the control approach was the primary objective of the trial, data from treatment schemes A and B were pooled into a single-treatment group for the main analysis.This analysis represents a sub-study of the PALOMA trial, involving specifically women aged over 40 years. Full details regarding the study design, patients, treatment groups, procedures, and analyzed variables have been reported elsewhere . BrieflyThe primary objective was to evaluate the repair of the cervical-vaginal lesions after 6 months of treatment, defined as normalized cytology and concordant colposcopy observations. Cytology results were classified according to the Bethesda System 2001 . The colThe sample was analyzed by modified intention to treat, defined as the women included in the study that received at least one dose of the vaginal gel and had the baseline value and one posttreatment value available for the variable. Endpoints were analyzed in 3 groups: total population, HR-HPV subpopulation , and HR-HPV 16-18-31 subpopulation. Since the aim of the sub-analysis was to evaluate the efficacy of the vaginal gel, schemes A and B were pooled into a single-treatment arm.p < 0.05. The statistical procedures were performed using the SAS 9.4 software.Continuous variables were expressed as a mean and standard deviation (SD). Categorical variables were shown as absolute and relative frequencies. Comparisons between groups were analyzed using the χ2 or Fisher’s exact test. Statistical significance was established with In accordance with the journal’s guidelines, we will provide our data for the reproducibility of this study in other centers if such is requested.Of 91 total women included in the PALOMA trial, 41 patients were older than 40 years. Among them, 31 presented HR oncogenic HPV subtypes, and 14 had HR-HPV 16-18-31 strains. The mean age was 47.7 (SD = 5.6), 46.9 (SD = 5.3), and 46.2 (SD = 5.5), respectively . After 6 months, normal cytology and concordant colposcopy were achieved by a greater number of women treated with the vaginal gel vs. watchful waiting approach . p = 0.007), and HR-HPV subpopulation . For the HR-HPV 16-18-31 subpopulation, 75.0% achieved repair of the lesions in the treatment group vs. 40.0% in the control group (p = 0.293). The difference was statistically significant in the total population  guidelines recommend observation for low-grade squamous intraepithelial lesion (LSIL) or a lesser lesion . This obThe efficacy and safety of the vaginal gel to treat HR-HPV infection in women over 35 years have been observed previously in one independent retrospective observational study conducted by Gajino et al. . They reCortés et al. have recently presented the results of an observational, prospective, non-controlled study called PAPILOBS  in Spain ,20. The Our study has certain limitations that have been published elsewhere . In thisDespite the limitations and the need for more data from currently ongoing studies, our findings are in line with previous results in women who are at an additional risk of HPV persistence and with difficulty spontaneously clearing the virus because of their age. Due to these facts, the treatment with the vaginal gel should be considered in clinical practice.Furthermore, the investigation of the effects of this vaginal gel on low-grade cervical lesions and HPV clearance is being continued in other clinical trials and observational studies  to consolidate the evidence already shown in the PALOMA study.Coriolus versicolor-based vaginal gel has demonstrated consistent efficacy in repairing low-degree HPV-related cervical lesions in women older than 40 years. Comparisons with the control group were significant in both the total population and HR-HPV subpopulation. A trend for greater HPV clearance was also observed in these groups.After a 6-month treatment period, the


Human papillomavirus (HPV) infection is an important carcinogenic infection highly prevalent among many populations. However, independent influencing factors and predictive models for HPV infection in both U.S. and Beijing females are rarely confirmed. In this study, our first objective was to explore the overlapping HPV infection-related factors in U.S. and Beijing females. Secondly, we aimed to develop an R package for identifying the top-performing prediction models and build the predictive models for HPV infection using this R package.This cross-sectional study used data from the 2009–2016 NHANES  and the 2019 data on Beijing female union workers from various industries. Prevalence, potential influencing factors, and predictive models for HPV infection in both cohorts were explored.There were 2,259  and 1,593  participants included in analyses. The HPV infection rate of U.S. NHANES and Beijing females were, respectively 45.73 and 8.22%. The number of male sex partners, marital status, and history of HPV infection were the predominant factors that influenced HPV infection in both NHANES and Beijing female cohorts. However, condom application was not an independent influencing factor for HPV infection in both cohorts. R package Modelbest was established. The nomogram developed based on Modelbest package showed better performance than the nomogram which only included significant factors in multivariate regression analysis.Collectively, despite the widespread availability of HPV vaccines, HPV infection is still prevalent. Compared with condom promotion, avoidance of multiple sexual partners seems to be more effective for preventing HPV infection. Nomograms developed based on Modelbest can provide improved personalized risk assessment for HPV infection. Our R package Modelbest has potential to be a powerful tool for future predictive model studies. It is transmitted  contact –3. There contact .HPVs can be broadly divided into low- and high-risk types. Low-risk HPVs  cause non-malignant lesions . While the high-risk HPVs  may lead to various cancers . The onvia this R package.Studies have shown that HPV infection might be associated with sexual behaviors, parity, marital status and age –13. Howehttp://www.cdc.gov/nchs/nhanes/. Since the HPV vaccine was only extensively pursued after 2006 in U.S., we limited our study to female participants in NHANES between 2009 and 2016. The exclusion criteria included: (1) participants aged < 18 or ≥60 years; (2) or with a history of malignancy; (3) or a history of hysterectomy.This was a retrospective cross-sectional analysis in two cohorts. One cohort is from the National Health and Nutrition Examination Survey (NHANES), which is a national cross-sectional study designed to provide nationally representative data on the nutritional and health status of the non-institutionalized, civilian U.S. population. Data in NHANES were collected through participant interviews and physical examinations. All NHANES study protocols were approved by the National Center for Health Statistics (NCHS) Institutional Review Board and all subjects provided written informed consent before data collection. Detailed descriptions of the methodology and data can be accessed at 3 copies/mL of HPV, and the specificity is more than 98%. Detailed content of the questionnaire and the HPV test are shown in Another cohort was from a Beijing Municipal Federation of Trade Unions (BMFTU)-funded cervical screening program for female workers in Beijing. The information needed for this study was sent out to female union members by BMFTU. Females who fulfilled the inclusion/exclusion criteria were invited to take part in this study at Beijing Rehabilitation Hospital. Eligible females were only included as participants in this study after informed consent had been obtained. All participants received questionnaires and HPV genotype testing between April 8, 2020 and May 8, 2020. This study was reviewed and approved by the Ethical Committee of Beijing Rehabilitation Hospital. For this study, the inclusion criteria included: (1) females with a history of having had sex; (2) aged between 20 and 70 years. The exclusion criteria were: (1) history of malignancy; (2) or history of cervical conization or hysterectomy; (3) or history of pelvic radiation therapy; (4) or history of immune disorders; (5) or pregnancy or childbirth within 6 months. For the data analysis of this study, cases with incomplete information were excluded. After completing the questionnaire, the participants received gynecological examinations including HPV genotype test. The detailed procedure of the HPV examination has been described in our previous study . BrieflyFor the NHANES cohort, the participants were randomly divided in a 7:3 ratio into the training and test-evaluation sets  using the “sample” function in R software (version 4.1.3). The training and test sets were used to select features and develop the prediction model. The evaluation set was used to evaluate the performance of prediction model. We also evaluated the prediction model in the combined test-evaluation set. Dividing the data into three data sets  could avoid model overfitting and provide an approach to validate the model .via R packages glmnet (v.4.1-3) and rms (v.6.2-0) on the training set. Features with P-values < 0.05 in both LASSO and multivariate logistic regression analysis were regarded as independent factors associated with HPV infection. R package forestplot (v.2.0.1) was used to graphically represent results of multivariate logistic regression analysis. Same methods were also performed on the Beijing female cohort.For the feature evaluation, LASSO and multivariate logistic regression analysis were performed successively https://github.com/yhx68/best/blob/main/Modelbest_0.1.0.tar.gz) iterates through all possible variables' combinations in a multivariate logistic regression model and outputs all models ordered following high-to-low sorted order based on the Harrells concordance index (C-index); meanwhile, this package can automatically identify the statistically significant variables in multivariate logistic regression among the input variables, and calculate the net reclassification index (NRI) and integrated discrimination index (IDI) of each model compared with the baseline prediction model   . The outThe C-index has a range from 0.5 to 1.0, 0.5 represents random prediction, while 1.0 indicates perfect prediction. IDI and NRI are newer techniques to quantify the improvement of a new model's predictive performance over a baseline prediction model . NRI andThe consensus guidelines  –23 were via R packages DynNom (v. 5.0.1), shiny (v. 1.7.1) and rsconnect (v. 0.8.25). All analysis in this study was conducted using R software (version 4.1.3). To evaluate the nomogram's discrimination ability, the receiver operating characteristic (ROC) plot and area under ROC curve (AUC), C-index, NRI, IDI, calibration curves and decision curve analysis (DCA) were performed in R using rms (v.6.2-0), ROCR (v. 1.0-11), Hmisc (v. 4.6-0), PredictABEL (v. 1.2-4) and rmda (v. 1.6) packages. Finally, online calculators for HPV infection were constructed The study flowchart is shown in For the Beijing female cohort, 1,719 female union workers from various industries participated in this program, following the exclusion of 126 participants with missing data, 1,593 female workers in total were included in the current study's analysis. These participants came from the security industry, governmental agencies, cleaning departments, construction industry, transportation industry, education industry, express delivery industry, property sector and medical industry. Among these 1,593 participants, 8.22%  were HPV positive. In the NHANES cohort, 12 factors were selected for multivariate logistic analysis after LASSO regression, and eight factors  were found to be independent factors significantly associated with HPV infection . NotablyIn the Beijing female cohort, 13 factors were selected for multivariate logistic analysis after LASSO regression, and five factors  were found to be independently associated with HPV infection . HoweverBased on the independently associated factors, the predictive models for HPV infection were constructed (namely log-model). Besides, predictive models based on R package Modelbest were also constructed (namely Modelbest-model). The C-index of the log-models were, respectively 0.792 and 0.691 for the NHANES and Beijing female evaluation sets. While the C-index of the Modelbest-models were, respectively 0.812 and 0.747 for the NHANES and Beijing female evaluation sets. https://cervixuteri.shinyapps.io/HPV1/ is for NHANES cohort; https://cervixuteri.shinyapps.io/HPV2/ is for Beijing female cohort).Taken together, we found number of male sex partners, marital status, and history of HPV infection were factors significantly associated with HPV infection in both the NHANES and Beijing females. Besides, the NHANES cohort revealed that smoking and drinking status were also independently associated with HPV infection. Interestingly, HPV vaccination status in the NHANES cohort showed no significant association with HPV infection. Based on the R package Modelbest developed in this study and the data of NHANES and Beijing females, we built two prediction models, respectively targeted for NHANES and Beijing females, these two models exhibited good prediction performance for HPV infection.It is well known that HPV is transmitted predominately through sexual contact, and our findings  strongly supported this theory. Earlier studies indicated that two-thirds of women were infected with HPV within 2 years of onset of sexual activity and young women with multiple sex partners are at highest risk of HPV infection , 25. ConThe history of HPV infection was also an independent risk factor for HPV infection in both the NHANES and Beijing female cohorts. It is undeniable that, for most people, HPV infection is transient and can be cleared or become undetectable after 1–2 years of infection , 30. NevFor the NHANES female cohort, we also found that smoking status and drinking status were the independent risk factors for HPV infection. These findings are in accordance with the previous studies. Smoking has been confirmed in relation to HPV infection  in mounting studies –33. ReceIn the Beijing female cohort, the smoking status showed no significant association with HPV infection in the multivariate logistic regression analysis, this may be partially due to the small sample size, since only 6.10% of the participants  in the training set were current-smokers. In comparison, in the NHANES training set, 24.10% participants  were current-smokers. The statistical results of the Beijing female cohort would probably be more convincing if the sample size was increased. In the Beijing female cohort, we also found that knowledge about HPV prevention was a risk factor for HPV infection. To some extent, this may reflect the lack of public education about HPV in China, as plenty of women learned about HPV and the HPV prevention strategies after the diagnosis of HPV infections.Based on the NHANES females, we observed no significant correlation between HPV infection and vaccination. This may be due to the HPV vaccine being a newer vaccine with limited application time. The Food and Drug Administration (FDA) only licensed the first HPV vaccine on June, 2006 for females 9–26 years, and the most recent nine-valent HPV vaccine was not approved by the FDA until 2014. Some women may have been infected with HPV prior to HPV vaccination, nevertheless, the vaccine is not therapeutic and may only prevent future infection. In addition, the Roche HPV linear array applied in the NHANES females can detect 37 HPV types, but currently available HPV vaccines can prevent at most of nine types of high-risk HPV infection. It is also possible that a few women did not develop effective immunity response after vaccination. This suggests that, for sexually active women, HPV vaccination is only part of a successful strategy for preventing HPV infection and cervical cancer, and that regular cervical screening and safer sex approaches still play an integral role.Nomograms, as a reliable and convenient tool for individual risk prediction, have been widely used in the medical field and for guiding clinical decision , 38. TheHowever, there are several limitations to this study: First, its main limitation is that it is retrospective. Second, the predictive models built in this study lack external validation. For example, the research on the Beijing female cohort was conducted among Beijing female union workers who live in areas with a great availability of medical services and are provided ThinPrep cytology test and HPV testing yearly. This characteristic of the cohort limits the generalizability of the Beijing-female-based predictive model to other regions/populations. In comparison, the NHANES are a series of nationally representative cross-sectional surveys for community-dwelling U.S. population using a complex multistage probability sampling design, which allows for generalizability of the NHANES-based predictive model to the U.S. population. Third, the study covers a small sample size, which may lead to some factors being insignificant in the present study. The heterogeneity in study design and populations between NHANES and Beijing female cohorts is another limitation, which was partly reflected in the differences in HPV detection methodologies . The enormous population heterogeneity prevents any standardization of data across these two populations. However, this study was not designed to compare the HPV infection rates between different cohorts, but to find the overlapping risk factors for HPV infection among different regions and construct targeted prediction models for different regions.Collectively, in this study, we found number of male sex partners, marital status and history of HPV infection were significantly correlated with HPV infection in both NHANES and Beijing female cohorts, and the multi-cohort findings increase the credibility and generalizability of our conclusion. Beyond that, we developed an R package (Modelbest) which could serve as a powerful tool for identifying the top-performing prediction models in future studies. The Modelbest-developed nomograms in this study showed good predictive performance and can provide risk assessments for HPV infection that are both personalized and evidence-based.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study involving Beijing female participants was reviewed and approved by the Ethical Committee of Beijing Rehabilitation Hospital. The participants provided their written informed consent to participate in this study. Ethics approval and informed consent were not required for the analysis of the publicly available NHANES data.HYu, BZ, YZho, and HYa designed the research. YZha performed HPV testing. WL, YL, FZ, XG, ZT, and HLi conducted questionnaire survey. HYa, YX, RG, and HLiu analyzed data and developed R package Modelbest. HYa wrote the manuscript. HYu, BZ, and YZho proofread and revised the manuscript. All authors contributed to the article and approved submitted version.


Background: About 87% of head and neck cancer (HNC) patients  are infected with human papillomavirus (HPV). Recent studies have demonstrated a significant correlation between HPV infection and nutritional disorders in HNC patients. Therefore, we formed a hypothesis that nutritional disorders or their severity in HNC patients may be associated with the occurrence of HPV infection due to known molecular differences in involved tissue. This literature review aimed to evaluate the influence of HPV infection on the occurrence and severity of nutritional disorders in HNC patients. Materials and Methods: The PubMed database was used to search papers with the keywords “HPV”, “HNC”, and “nutritional disorders” in different variants and combinations. Conclusions: The data available in the discussed papers indicate, among other things, that HPV−positive patients may be at higher risk of malnutrition, critical weight loss, and necessity for gastrostomy after radiotherapy or chemoradiotherapy (C-RT). It should be highlighted that despite some studies demonstrating positive results, currently available data regarding the influence of HPV infection on the occurrence and severity of nutritional disorders in HNC remain limited and inconclusive, and thus further research on this issue is warranted. Among all disturbances of nutritional state, cachexia and sarcopenia seem to have the most serious health consequences. In head and neck cancer (HNC), nutritional disorders develop in approximately 90% of patients . In turnCachexia (wasting syndrome) is considered an irreversible, multifactorial process characterized by a progressive loss of skeletal muscle mass accompanied or not by a decrease in adipose tissue. Another commonly noted symptom is a general deterioration in mental state, which, in the case of oncological patients, often determines worse quality of life and prognosis . CachexiIt is estimated that in patients with an advanced stage of cancer, cachexia occurs at a frequency of 60–80%, while in 20–40%, it is considered a direct cause of death . In abouSarcopenia is characterized by an accelerated loss of muscle mass and function. It is linked to a higher risk of falls, functional limitations, frailty, and morbidity ,7. SarcoIn the case of HNC, the most obvious explanation of cachexia- and/or sarcopenia-related weight loss is the location of the tumor in the head and neck region, the main site of food intake. Anatomical localization of the tumor and its surgical implications are undisputed grounds for the initiation of cachexia development; however, several other factors should not be underestimated, such as the influence of host–tumor interactions on inflammation, the endocrine system, metabolism, and many other processes ,10,11,12Generally, cachexia is a complex process that involves several known mechanisms. It was established that the development of cachexia is associated with an imbalance between anabolism (decreased) and catabolism (increased) in muscle and adipose tissue. In a situation where mediators participating in anabolic processes , testosterone, and glucocorticoids) are lacking or the receptors that bind them are damaged, a predominance of catabolism over anabolism occurs . In cachIt needs to be highlighted that in cancer patients, cachexia is commonly accompanied by sarcopenia. Therefore, its pathomechanism is in many aspects very similar to cachexia. The systemic inflammatory response has been associated with muscle wasting in cancer patients. In sarcopenia, an increased level of numerous proinflammatory cytokines, i.e., CRP, IL-6, IL-1β, and IL-8, are observed. Other mechanisms of muscle wasting include inhibition of myoblast differentiation, enhanced autophagy, and derangements in the renin–angiotensin–aldosterone system (RAAS) .Papillomaviridae family and represents a group of viruses that infect the epithelial surface of the skin and mucous membranes. The WHO has classified HPV types as belonging to a high-risk group or low-risk group of cancer development [Human papillomavirus (HPV) belongs to the elopment ,18,19,20Persistent HPV infection (with high-risk types) is considered the main factor leading to precancerous or cancerous transformation in several types of tissue, including the head and neck region, lower genital tract, and above all in the uterine cervix. It should be noted that only 10–15% of patients with high-grade cervical dysplasia are HPV−negative. HPV infection is responsible for about 70% of cervical cancer. Cancerous transformation is caused by precursor dysplastic lesions of the cervical epithelium. Cervical cancer is diagnosed in a significant percentage of women in developing countries. Importantly, the prognosis for patients with cervical dysplasia who receive appropriate treatment remains excellent ,22. MoreThe primary cause of cancer in about 87% of HPV−positive HNC patients is HPV16 . HPV−posHPV replication processes involve eight open reading frames (ORF). The proteins encoded by these genes are divided into two groups: early proteins (E1–E7) and late proteins  ,30. The This literature review aimed to evaluate the potential influence of HPV infection on the frequency or severity of nutritional disorders in HNC patients.p < 0.05.We researched all papers published in the literature (in the PubMed database), with the following keywords: “head and neck cancer,” “HNC and HPV”, “cancer cachexia”, “sarcopenia”, “HPV infection in cancer cachexia”, “HPV immune response”, “survival in patients with HPV−related HNC”, “HNC and malnutrition”, “HPV and nutritional disorders”, “HPV and sarcopenia” and “HPV and “weight loss”. For the purposes of this review, the incidence and risk of nutritional status disorders depending on the HPV status in some cases were calculated based on the available data extracted from cited publications. Statistical analysis was performed using the odds ratio (OR) test with a 95% confidence interval (95% CI) and results were considered statistically significant at Studies on experimental models and those focused on the molecular changes related to HPV infection seem to support a hypothesis that HPV infection is not only associated with a higher risk of malignancy but can also potentially affect the development of nutritional disorders in the course of neoplastic disease. As described by da Costa et al., mice infected with HPV16 had muscle mass loss associated with the activation of the NF-κB pathway. In the above experiment, six-week-old female K14-HPV16 mice were used. The study used hemizygous (HPV16 +/−) and wild-type (HPV16 −/−) mice. In the group that was not exposed to the rutin or curcumin, a significant decrease in muscle mass was observed in the hemizygous group compared to the wild-type variant group. The HPV16 +/− animals experienced systemic inflammation and cachexia, but whether this was due to the dysplastic lesions or HPV itself was not shown. This study lacked an HPV−negative but dysplasia-positive control, and thus the associated cachexia may not be solely attributable to HPV alone. One of the study’s main limitations was that it does not allow us to conclude that the inflammation was related to HPV infection. Indeed, in the discussion, the authors state that epidermal dysplasia (and not HPV per se) is correlated with a robust systemic inflammatory response that is known to be associated with cachexia .The presence of HPV infection leading to the neoplastic transformation can be crucial for the expression and activation of the NF-κB pathway in many cancers . The actp < 0.0001), IL-1β (p < 0.0001) and TNF-α (p < 0.0001) were found [The secretion of IL-1 and TNF-α can have a major impact on the regulation of the food intake process. These cytokines affect the increase of corticotrophin-releasing hormone (CRH) production, which leads to impairment in nervous regulation of food intake and modification of sensitivity to glucose. What is more, it can modulate the functioning of the digestive tract and determine nutritional satiation through the nervous system . An evalre found .Data, results, and conclusions of all publications discussed below are summarized in p = 0.6524; OR = 1.41, 95% CI: 0.26–7.54, p = 0.6885) as well as one month  after treatment. Three months after treatment, HPV−positive patients had a significantly higher rate and insignificantly higher risk  of malnutrition (B or C category). Weight loss, depression, poor quality of life, and adverse events were all secondary outcomes. Additionally, in the last week of C-RT, the HPV−positive group had insignificantly lower risk of 5% weight loss . On the other hand, after 1 month  and 3 months  after treatment, HPV−positive patients had insignificantly higher risk of losing more than 5% of their body weight, while HPV−positive patients had insignificantly lower risk of weight loss > 10% in the last week of treatment  and 1 month after C-RT . At three months after therapy, HPV−positive patients had significantly higher risk of losing more than 10% of their body weight . Moreover, similarly to most studies, mortality after 2 years was significantly higher in HPV−positive compared to HPV−negative patients . The risk of weight loss and malnutrition depending on HPV status was calculated for the purposes of this study based on the extracted data [In the only currently available prospective study, conducted by Harrowfield et al., data were obtained from 83 OPSCC Caucasian patients (84.3% of patients were HPV−positive) undergoing C-RT. The patient-generated subjective global assessment (PG-SGA) was used to assess nutritional status as the primary outcome and loss of weight as the secondary outcome. At the beginning of the treatment, a lower incidence of malnutrition in HPV−positive patients was noted. On the other hand, HPV−positive patients had an insignificantly higher incidence and risk of malnutrition (B or C category) in the last week of RT . Critical weight loss (CWL) was observed in 86% of patients, and the majority of them were HPV−positive (92.6%). Such factors as HPV status (positive) and C-RT were found to be predictors of CWL. Moreover, in the HPV−positive group, significantly higher risk of CWL (≥5%)  was noted. A significant percentage of HPV−positive patients required PEG (68.6%), mostly in the form of reactive feeding tubes (RFT) (43.6%). Among patients in whom feeding tube dependence (FTD) (prophylactic feeding tubes—PFT) was used, HPV−positive patients showed significantly higher mean weight loss . When only the HPV−positive patients were analyzed, it turned out that the patients using FTD (PFT and RFT) had significantly higher mean percentage weight loss compared to the ones in whom parenteral nutrition (PN) was not used at all . HPV−positive patients had insignificantly higher risk of FT (PFT and RFT) . Moreover, insignificantly lower risk of PFT in HPV−positive group  was noted. While patients with HPV infection had insignificantly higher risk of RFT . HPV−positive patients had insignificantly lower risk of CWL during PFT  and insignificantly higher risk of CWL during RFT . Nevertheless, the main weakness of this study is that there were only 10 HPV−negative patients (10%) as a comparison group and only 6 patients (13%) in the HPV−positive group have definitive radiation. In the case of 22 patients (22%), HPV status was unknown. The risk of weight loss and FTD depending on the HPV status was calculated for the purposes of this study based on the extracted data [Treatment-naïve HPV−positive OPSCC patients are less likely to suffer from dysphagia or tumor-induced odynophagia . On the p = 0.008). In the LIRi group, total weight loss was significantly higher compared to the HRi , and HIRi  groups. Moreover, in the LIRi group, percentage weight loss during FT use was significantly higher compared to the HRi  and HIRi  group. However, the authors suggest that the poor nutritional outcomes in HPV−positive patients were associated with a lack of adherence to the given PFT recommendations observed in this group, resulting in suboptimal feeding tube utilization. The percentage of weight loss and weight loss during FT depending on the HPV status was calculated for the purposes of this study based on the extracted data [The influence of HPV infection on weight loss (according to the risk of prolonged feeding tube use) was analyzed by Anderson et al. in a retrospective study on 101 Caucasian patients with OPSCC. Based on the T-stage and N-stage features, the study group was divided into three subgroups: high-risk , high-intermediate risk , and low intermediate risk  of prolonged feeding tube use. All patients were treated with definitive IMRT (with or without concurrent chemotherapy). HPV infection was confirmed in 58.4% of patients. In the study group, good adherence to PFT recommendations was noted (87%). Interestingly, in the LIRi group compared to the HRi and HIRi groups, a significantly higher proportion of HPV+ patients were noted . The authors also cite other studies in which higher organ toxicity and dysphagia in patients with HNC who were p16 positive were noted. On the other hand, increased toxicity may explain increased rates of gastrostomy tube insertions, which in turn may suggest only an indirect association between HPV infection and malnutrition. However, a few limitations of this study need to be raised. Increased gastrostomy tube rate in p16+ patients was only in the low-risk cohort, which had a very small number of patients: only 14 (23.7%) were HPV−positive. Moreover, the authors analyzed the association of p16 status and PEG only in 19 (26.8%) patients who met the criteria for proactive gastrostomy. However, it should be highlighted that a direct comparison of malnutrition incidence by HPV status was not performed by the authors. The risk of gastrostomy depending on the HPV status was calculated for the purposes of this study based on the extracted data [In patients with HNC, complications related to the location of the tumor and the type of treatment are observed very often. The methods of PN include gastrostomy involving the insertion of a feeding tube into the stomach before starting treatment. The assessment of the presence of p16 in tumor tissue (surrogate of HPV16 infection) and its usefulness in the prediction of the need for gastrostomy was performed by Brown et al. in a retrospective study on 269 Caucasian patients diagnosed with oral cancer (30%), OPC (34%) and other types (36%) of HNC. Patients enrolled in the study were classified as being at high risk (88 people) or low risk (181 people) of malnutrition. In the case of 59 patients (36.2%) the HPV status was confirmed by immunohistochemistry. The risk of the necessity of gastrostomy was significantly higher in p16-positive patients . HPV−positive patients were characterized by significantly higher pretreatment BMI compared to HPV−negative patients . Additionally, in the HPV−positive group, an insignificantly lower risk of sarcopenia  was observed. Similarly to other studies, significantly lower risk of disease-free survival shortening  and overall survival shortening  were noted in HPV−positive patients. The risk of sarcopenia depending on HPV status was calculated for the purposes of this study based on the extracted data [In contrast, Tamaki et al. conducted a retrospective study of 113 patients with OPC. HPV infection was confirmed in 85 (75.9%) patients. Among the study group, 32 (28.3%) patients had sarcopenia. The pre-treatment BMI and skeletal muscle index (SMI) values were used to divide patients as sarcopenic or nonsarcopenic. Patients with sarcopenia were more often older (63.5 vs. 57.6 years), female (76.5% vs. 53.1%), and had lower mean BMI , while the absence of HPV infection was noted in 37 HNC patients (28.68%). HPV status was reported only for oropharynx cancer cases and a few oral cavity cases where p16 staining was performed. Interestingly, the presence of sarcopenia was diagnosed in 47.83% of HPV−positive patients and in 67.57% of patients without p16 protein expression (p = 0.0517). Therefore, there was a significantly lower risk of sarcopenia developing in HPV−positive patients when compared to the control group . The risk of sarcopenia depending on HPV status was calculated for the purposes of this study based on the extracted data [A retrospective study by Stone et al. was performed in a group of 260 Caucasian patients with HNC. Sarcopenia was diagnosed in 144 patients (55.4%). Based on the method used in the study, sarcopenia was identified when the L3 index was lower than 38.5 cmted data .p = 0.4170; OR = 0.66, 95% CI: 0.29–1.50; p = 0.3228). Moreover, there was no difference in overall survival between HPV−positive patients with or without sarcopenia . The limitation of this study concerns the lack of a direct comparison of the incidence of sarcopenia depending on HPV status  [In a retrospective study performed by Olson et al., the data were obtained from 245 OPSCC Caucasian patients before treatment (RT or surgery). HPV infection was confirmed in 197 patients (87.6%). After body composition analysis (SMI), 135 patients (55.1%) were diagnosed as sarcopenic, whereas 100 patients (44.9%) were classified as nonsarcopenic. Risk of sarcopenia was insignificantly lower in HPV−positive patients  .p = 0.02), had reduced incidence of restricted diet  and feeding tube dependence (FTD) . Moreover, only HPV status was found to be a significant predictor of decreased swallowing dysfunction. In comparison to HPV−negative, HPV−positive OPSCC patients had significantly reduced risk of late (after 24 months after treatment) swallowing impairment after C-RT , while at 55 months of follow-up, HPV+ patients were more likely to resume a normal diet . The numbers, percentages, and results of statistical analysis were calculated for the purposes of this study based on the extracted data [A retrospective study performed by Naik et al. included 147 Caucasian patients with OPSCC who were treated with standard three-field C-RT. HPV infection was confirmed in 130 patients (88.4%). Patients with HPV−positive OPSCC at last follow-up were more likely to resume a normal diet  is played by HPV oncoproteins (mainly E6 and E7) contributing significantly to genetic changes in host tissue and the increase in proinflammatory cytokine secretion associated with infection. On the other hand, HPV infection evades the immune system and leads to immunosuppression. However, patients with HPV−positive OPC have a better response to RT or C-RT, so this may presumably produce an increase in therapy-related inflammation with the consequent increase of inflammatory cytokines. Therefore, we are looking for an answer to the question about the potential relationship between HPV infection and the development (or severity) of nutritional disorders  in patients with HNC. It should be highlighted that to the best of our knowledge, to date, this is the first review systematizing knowledge about the influence of HPV infection on nutritional disorders in HNC. The HPV infection observed in patients with HNC may result in increased secretion of the same inflammation mediators, which have a key role in the development of nutritional disorders. Interestingly, before the start of treatment, BMI in HPV−positive HNC patients is usually higher. However, those patients, although rarely diagnosed with dysphagia, are characterized by higher weight loss during treatment and more often require PEG. In patients with HNC, HPV infection by changing the host genome, including the functioning of protein products of genes encoding the metabolism-modifying factors and cancer-associated inflammation can lead to malnutrition/cachexia risk change (probably increase). Unfortunately, the data in the papers quoted in this review do not provide strong evidence that HPV itself is contributing to nutritional deficiencies. In many of the studies discussed, there were no  HPV−negative patients serving as a control group, and thus direct comparisons in cachexia between HPV−positive and HPV−negative patients have not been performed and we cannot make an overarching statement that HPV−positive patients have a higher risk of cachexia (or higher risk of more severe cachexia). Although treatment is a factor contributing to the development of nutritional deficiencies in this group of patients, the presence of HPV infection may reduce the risk of this process. Interestingly, the available data suggest that HNC HPV−positive patients may have a lower risk of sarcopenia. However, it should be taken into account that the available data also in this case are limited and inconclusive. Therefore, it should be further investigated what key mechanisms of development of cancer cachexia and or sarcopenia are affected by the presence of HPV oncoproteins. The knowledge acquired may in the future improve the individualization of the treatment , which will translate into a measurable benefit for the patient. However, this requires taking into account that the risk and severity of cachexia or other nutritional disorders may be associated with the occurrence of the molecularly distinct—HPV−positive—HNC.


The AHCC supplement is a proprietary, standardized extract of cultured lentinula edodes mycelia  in 50 women over 30 years of age with confirmed persistent high-risk HPV infections for greater than 2 years. Patients were randomized to placebo once daily for 12 months (N = 25) or AHCC 3-g supplementation by mouth once daily on empty stomach for 6 months followed by 6 months of placebo (N = 25). Every 3 months, patients were evaluated with HPV DNA and HPV RNA testing as well as a blood sample collected to evaluate a panel of immune markers including interferon-alpha, interferon-beta (IFN-β), interferon-gamma (IFN-γ), IgG1, T lymphocytes, and natural killer (NK) cell levels. At the completion of the 12-month study period, patients on the placebo arm were given the option to continue on the study to receive AHCC supplementation unblinded for 6 months with the same follow-up appointments and testing as the intervention arm.Fifty women with high-risk HPV were enrolled, and 41 completed the study. Fourteen (63.6%) of the 22 patients in the AHCC supplementation arm were HPV RNA/HPV DNA negative after 6 months, with 64.3% (9/14) achieving a durable response defined as being HPV RNA/HPV DNA negative 6 months off supplementation. On the placebo arm, two (10.5%) of 19 patients were HPV negative at 12 months. In the twelve placebo arm patients who elected to continue on the unblinded study, 50% (n = 6) were HPV RNA/HPV DNA negative after 6 months of AHCC supplementation. At the time of completion of the study, there were a total of 34 patients (22 blinded and 12 unblinded) who had received AHCC supplementation with an overall response rate of 58.8% that cleared HPV persistent infections. At the time of enrollment, the mean IFN-β level was 60.5 ± 37.6 pg/ml in women with confirmed persistent HPV infections. Suppression of IFN-β to less than 20 pg/ml correlated with an increase in T lymphocytes and IFN-γ and durable clearance of HPV infections in women who received AHCC supplementation.Results from this phase II study demonstrated that AHCC 3 g once daily was effective to support the host immune system to eliminate persistent HPV infections and was well tolerated with no significant adverse side effects reported. The duration of AHCC supplementation required beyond the first negative result needs more evaluation to optimize success for durable outcomes. The suppression of the IFN-β level to less than 20 pg/ml correlated with clearance of HPV infections and merits further evaluation as a clinical tool for monitoring patients with HPV infections.clinicaltrials.gov/ct2/, identifier NCT02405533 Ther lesions . However lesions , 5.Although HPV vaccination is effective in the prevention of HPV infections, it has little benefit for the treatment of patients already infected with HPV . There a®, Amino Up, Ltd., Sapporo, Japan) that was developed in Japan in 1992; the compound is primarily composed of α-glucan components. Several animal and human studies have reported a variety of therapeutic effects, including antioxidant and anticancer activities and modulation of the immune system to prevent the infectious processes of both viral and bacterial infections  as documented by the primary gynecologist’s records, and deemed otherwise healthy. Patient demographic information was collected including the number of lifetime sexual partners, contraception methods, and periodic pregnancy testing throughout the study. AHCC and matching placebo were generously provided by Amino Up, Ltd. .This was a phase II randomized, double-blind, placebo-controlled study with post-study unblinded AHCC . At 12 months, if on placebo the control arm patients were still HPV positive, then patients were offered to continue on AHCC unblinded to gain more information on its toxicity and efficacy. The purpose of “crossover” after unblinding time point was to also help enhance recruitment and enrollment since AHCC is a readily available nutritional supplement.Patients were monitored once every 3 months during the study with a clinic visit to review the pill diary to assess compliance, evaluate any toxicity experienced over the previous 90 days, and collect HPV samples The primary outcome of this trial was to evaluate clearance of persistent high-risk HPV infection determined by HPV DNA-negative test results achieved while receiving AHCC supplementation and maintained for 3, 6, and 12 months post-completion of the AHCC supplementation compared to receiving placebo. At 12 months, after receiving AHCC supplementation for 6 months, if patients were still HPV DNA positive, it was considered a treatment failure or no response (NR), and they went off study. If negative after completion of 6 months of AHCC supplementation and 6 months of placebo, patients continued on study for another 6 months (two visits) to confirm they remained HPV negative and to evaluate the durability of the response. A complete response (CR) was defined as those patients who were HPV RNA and HPV DNA negative at the time of completion of AHCC supplementation and remained HPV RNA and HPV DNA negative throughout the 12 months of follow-up off AHCC supplementation. A partial response (PR) was defined as those patients who were HPV RNA and HPV DNA negative at the time of completion of AHCC supplementation but then tested either HPV RNA or HPV DNA positive at one or more visits in the 12 months of follow-up off AHCC supplementation. Group 2 served as the control for all time points for the duration of the study. Safety and efficacy data were collected in the patients who elected to continue on unblinded AHCC supplementation for 6 months.® liquid gynecology specimen vial . Twelve milliliters of whole blood was collected in EDTA  tubes from all patients at every visit. All blood samples were centrifuged at 1,500 rpm for 10 min, and plasma samples were separated and stored at −80°C within 6 h of collection until further use. Peripheral blood mononuclear cells (PBMCs) were isolated from remaining blood by density gradient centrifugation over Ficoll Lymphocyte Separation Medium  using the manufacturer’s protocol. The PBMCs were cryopreserved in a freezing medium containing 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) in liquid nitrogen (−196°C) until further use. FBS was purchased from GIBCO Invitrogen Co. . The DMSO was purchased from Sigma-Aldrich Co. .At each visit, a gynecologic  specimen was collected by three methods—cytology brush, cytology broom, and cervical spatula—to ensure an adequate sample and transferred to a ThinPrepHPV samples were evaluated initially with HPV E6/E7 RNA testing  at the UTHealth McGovern Medical School Molecular Pathology laboratory. HPV RNA-negative results were confirmed with COBAS HPV DNA testing  at Methodist Molecular Pathology laboratory to detect the presence of HPV DNA and rule out latent HPV infections.Ready-to-use sandwich ELISA kits for the detection of human Interferon-alpha and beta were purchased from R&D Systems . Human interferon-gamma (IFN-γ) and IgG1 ELISA kits were purchased from Affymetrix eBioscience . Sandwich ELISAs for the detection of total IgG1, interferon-alpha, interferon-beta, and interferon-gamma were performed according to the manufacturer’s protocol. For each series of immune marker determinations, a standard curve was constructed with known concentrations of these markers provided with the kits. Plasma concentrations of these immune markers at each time point were calculated from standard graphs and were compared with baseline concentrations.Five mouse anti-human antibodies were used for the identification and quantification of human natural killer cells (NK cells): CD56 APC clone, B159; CD3 PerCP-Cy5.5 clone, UCHT1; CD2 PE clone, RPA 2.10; CD7 FITC clone, M-T701; and CD45 APC-H7 clone, 2D1. All antibodies and fluorescence-activated cell sorting (FACS) lysing solutions were purchased from BD Biosciences . For flow cytometry analysis of human NK cells, the PBMCs cryopreserved at every time point were gently thawed and centrifuged with 5 ml of a complete medium at 1,500 rpm for 5 min at 4°C. The cell pellets were re-suspended in 100 µl of FACS buffer  with 5% FBS). For staining of the cells, one set of cells from each sample was incubated with 5 µl of CD45 APC, and the second set of cells was incubated with 50 µl of a cocktail of five antibodies for 15 min in the dark at room temperature as per the manufacturer’s protocol. Following the incubation, 1 ml of FACS lysing buffer was added to all tubes and was further incubated for 5 min in the dark at room temperature. To stop the lysis of cells, 1 ml of PBS was added to each tube immediately after incubation. All tubes were then centrifuged at 1,500 rpm for 5 min, and cells were re-suspended in 500 µl of FACS buffer and stored at 4°C.The stained cells were acquired using LSR II (BD Biosciences), and Diva software was used for analysis. With the use of forward and side scatter, the lymphocyte population was gated on low side scatter and CD45 bright population while acquiring the 50,000 gated events. The NK cell population was then identified as  cells. The percent of T cells and NK cells from the lymphocyte gate was obtained for the final analysis.In this population, women with persistent HPV infections, the expected eradication or clearance of HPV infection on its own is zero to 10%. The target success rate based on pilot study data was 50% at 6 months after the end of AHCC supplementation and 12 months after the end of supplementation. Based on these proportions, 10% clearance in the absence of supplementation and 50% clearance with supplementation, a power analysis was conducted using data simulation in Mplus 7.2 through a multi-step process . The proFifty women with HR-HPV were enrolled in this study, and 41 completed the study ; the compound is primarily composed of α-glucan components as compared to most mushroom extracts, which are composed of β-glucan components. AHCC exhibits unique immune modulation to downregulate the hyper-stimulated IFN-β level resulting in negative feedback to kick-start the release of IFN-γ and T lymphocytes needed to clear chronic viral infections (Pleurotus sajor-caju) have potent immunomodulatory activity  for the publication of any potentially identifiable images or data included in this article.JS served as principal investigator of the clinical trial and was responsible for study concept, protocol design, data analysis, clinical interpretation of data, writing, and finalization of the manuscript. AG, LM, and BR served as the primary research team responsible for patient screening, clinic visits, sample collection, and analysis and participated in the writing and review of the manuscript. JF served as a clinical collaborator on this study and participated in protocol design, clinical interpretation of data, and final review of the manuscript. JL served as a clinical collaborator on this study and participated in the clinical interpretation of the data and final review of the manuscript. YB served as a research collaborator participating in the interpretation of the sample of analysis, protocol design, and review of the final manuscript. RO served as a research collaborator participating in the interpretation of the sample of analysis and implications for future monitoring assays. He participated in the review and finalization of the manuscript. TB served as a clinical collaborator on this study and participated in protocol design, clinical interpretation of data, and final review of the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This study was supported by the NIH-NCI Small Grants Program for Cancer Research (1R03CA212935), which had no involvement in the study design, data collection, data analysis, and interpretation or writing of the manuscript or its publication.JS was a recipient of various unrestricted research grants supporting preclinical studies on AHCC prior to 2014 from Amino Up, Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Molecular studies about carcinomas of the oral cavity and oropharynx demonstrate the presence of human papilomavirus genome in these tumors, reinforcing the participation of human papilomavirus in oral carcinogenesis.This study aimed to determine the prevalence of human papilomavirus and genotype distribution of HPV16 and HPV18 in oral cavity and oropharynx carcinomas, as well as their association with clinical characteristics of the tumors.This is a retrospective study, with clinical data collected from 82 patients. Human papilomavirus detection was conducted on specimens of oral cavity and oropharynx carcinomas included in paraffin blocks. Patients were assisted in a cancer reference center, in the central region of Brazil, between 2005 and 2007. Polymerase chain reaction was used for the detection and genotyping of human papilomavirus.Among the patients evaluated, 78% were male. The average age of the group was about 58 years. Risk factors, such as smoking (78%) and alcohol consumption (70.8%) were recorded for the group. HPV DNA was detected in 21 cases  of which 33.3% were HPV16 and 14.3% were HPV18. The presence of lymph node metastases and registered deaths were less frequent in human papilomavirus positive tumors, suggesting a better prognosis for these cases; however, the differences between the groups were not statistically significant.The results obtained in the present study, with respect to the presence of the high-risk HPV16 and HPV18 genotypes, highlight the importance of human papilomavirus vaccination in the control of oral cavity and oropharynx carcinomas. Head and neck cancers (HNC), including oral cavity and oropharynx carcinomas, are the sixth most common cancer types in the world, with an annual estimate of 633,000 new cases and 355,000 deaths.Oral and oropharynx carcinomas account for more than 80% of the total HNC cases,5Age, gender, and tumor-node-metastasis (TNM) tumor staging, which includes the extension of the tumor, the presence of lymph node metastases, and distant metastasis, are the main prognostic factors for oral cavity and oropharynx carcinomas.8The treatment of SCC of the oral cavity and oropharynx is usually accomplished by surgery or radiotherapy, alone or associated, and may also include the use of chemotherapy as an alternative to improve the chances of cure.10Smoking and alcohol consumption are considered the main risk factors for oral cavity and oropharynx cancer.14HPV is a sexually-transmitted infection; therefore, factors such as early initiation of sexual activity, high number of sex partners, and the practice of unprotected oral sex are included as risk factors for HPV infection in the oral cavity and oropharynx mucosa.16The prevalence of HPV in oral cavity and oropharynx SCC is the focus of several studies worldwide; HPV16 is considered the most prevalent and relevant genotype in the epidemiology of these carcinomas.20This study aimed to investigate the prevalence and genotypic distribution of HPV16 and HPV18 in oral cavity and oropharynx carcinomas, as well as their possible association with the clinical characteristics of the tumors.The study was approved by the Research Ethics Committee, under No. 13580613.5.0000.0031/2014. It was a retrospective cross-sectional study that used data collected from medical files and analysis of paraffin blocks containing specimens of oral cavity and oropharynx carcinomas. The selection was first accomplished by analyzing the records of the Pathology Department at the Hospital, and only patients with histological diagnosis of SCC of oral cavity and oropharynx were included. Initially 312 cases of SCC diagnosed in the period from 2005 to 2007 were selected. After eliminating duplicates, 174 cases with available clinical records were selected. Patients who received chemotherapy/radiotherapy before surgery were excluded, resulting in 108 cases. Paraffin blocks from 108 cases were histologically examined, and specimens with exiguous amount of tumors were excluded, resulting in 82 cases that were selected for DNA extraction and HPV DNA detection.Genomic DNA was purified from tumor samples fixed in formalin and included in paraffin. The samples were dewaxed in xylene and washed in ethanol according to standardized protocol. DNA was isolated by using the commercial Wizard Kit (Promega). The presence and the integrity of the DNA were verified by the amplification of a 99 base pair (BP) fragment from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), by using polymerase chain reaction (PCR).2, 2 mM/L of each deoxyribonucleotide (dNTPs), 2.5 μM/L of each oligonucleotide primer, 0.25 U Taq polymerase  and ultra pure water in sufficient quantity for the final volume. Cycling conditions included: preheating for 1 min at 94 °C, followed by 40 cycles of: 94 °C for 1 min, 1 min at 45 °C, and 1 min at 72 °C, with a final extension of 5 min at 72 °C. Positive and negative controls were used in each reaction.HPV DNA was detected by PCR. The set of primers used was SPF 1/2 (short PCR fragment). The SPF 1/2 amplifies a fragment of 65 pb of the L1 region of HPV genome. This set of primers allow the detection of 39 high and low oncogenic risk HPV: 6, 11, 13, 18, 26, 30, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 72, MM4, MM7, 73, 74, and MM8. The genotypes detected by SPF 1/2 are those that infect the mucous membrane.2, 2 mM/L of each dNTPs, 2.5 μM/L of each oligonucleotide primer, 0.25 U Taq polymerase , and ultra pure water in sufficient quantity for the final volume. Cycling conditions included: preheating for 1 min at 94 °C, followed by 40 cycles of: 94 °C for 1 min, 1 min at 45 °C, and 1 min at 72 °C, with a final extension of 5 min at 72 °C. PCR with HPV18 primers was performed in a final reaction volume of 25 μL, with 5 μL of purified DNA, 2.5 mM/L MgCl2, 2 mM/L of each dNTPs, 2.5 μM/L of each oligonucleotide primer, 1.25 U Taq polymerase,  and ultra pure water in sufficient quantity for the final volume. Cycling conditions included: preheat for 3 min at 94 °C, followed by 35 cycles of: 1 min at 94 °C, 1 min at 53 °C, and 1 min at 72 °C, with a final extension of 3 min at 72 °C.HPV16 and HPV18 genotyping was performed by PCR for all HPV-positive tumors. Two sets of primers that amplify part of E7 region of each HPV genome were employed.® spreadsheets. Clinical and histological data of the group, as well as the presence of HPV genome, HPV16, and HPV18 genotypes were analyzed by using Fisher's exact test and the chi-squared test. Values of p ≤ 0.05 were considered statistically significant. Positive and negative controls were used in each reaction.All the patients’ data were transcribed to Microsoft ExcelClinical and histological characteristics: a group of 82 cases of SCC of oral cavity and oropharynx were selected and evaluated with respect to clinical and histological characteristics . Among tp = 0.08). Distant metastases were not detected in HPV-positive cases, while one distant metastasis was described in the HPV-negative cases (p = 0.46). With respect to histological grade, 85.7% of HPV-positive cases showed moderate to high grade of differentiation. A greater number of deaths were registered in the HPV-negative group (39.3%) compared to the HPV-positive group (19.1%), though this difference was not statistically significant (p = 0.11). With respect to HPV genotyping, seven samples (33.3%) were HPV16 positive, while three samples (14.3%) were HPV18 positive. Significant differences between HPV16 and HPV18 tumors were not detected in this study.HPV detection and genotyping: HPV DNA was detected in 21 cases  16.9–36.6), of which 33.3% were HPV16 and 14.3% were HPV18. In this study analyzing 82 cases of SCC of oral cavity and oropharynx diagnosed in a cancer reference center in the central region of Brazil. HPV DNA was detected in 21 cases 25.6%), of which 33.3% were HPV16 and 14.3% were HPV18. These results support the hypothesis that a subgroup of oral cavity and oropharynx carcinomas is HPV related..6%, of wThe association between HPV infection with oral cavity and oropharynx SCC emphasizes the importance of introducing specific molecular tests into oral cancer prevention practices, in order to evaluate the presence of the virus and the possibility of expanding anti-HPV vaccine in the male population.24In the present study, 54.9% of the patients were younger than 59 years. Several studies describe the age range of the patients with carcinoma of oral cavity and oropharynx as similar to the value described in the present study.29Smoking and alcohol consumption are described as the main risk factors for carcinomas of the oral cavity and oropharynx.33The profile of patients with carcinoma of the oral cavity and oropharynx associated with HPV tends to be characterized by a group of younger patients, aged less than 60 years, non-smokers or light smokers, non-drinkers, and with more promiscuous sexual behavior; however, these data are controversial.18The prognosis of oral cavity and oropharynx carcinomas is uncertain and difficult to predict.10vs. 57.4%), suggesting a better prognosis for these tumors; however, such differences were not statistically significant.A better prognosis for HPV-positive oral cavity and oropharynx carcinomas has been described in a few studies, including well-differentiated tumors, with less risk of lymph node metastasis and distant metastasis.HPV16 and HPV18 are described as the most prevalent genotypes in oral cavity and oropharynx carcinomas, and their association with these carcinomas seems to be relevant.Recent reports highlight that vaccination campaigns are an efficient solution for the control of the HPV-associated carcinomas.In the present study, the presence of HPV was detected in 25.6% of the cases, including types 16 and 18, leading to the conclusion that HPV is also associated with the development of oral cavity and oropharyngeal carcinomas. This study presented an important limitation with respect to clinical and behavioral data of the patients. In the medical files, such data were scarce, including sexual behavior, oral hygiene, history of sexually transmitted diseases, and patient follow-up, among others. These difficulties are inherent to retrospective studies, since the researchers rely only on the information present in medical records that can often be lost, incomplete, or unclear.30The authors suggest a prospective study, with more efficient data collection and patient follow-up, allowing a more accurate and complete survey, with a larger number of cases from different oncology centers in the country, in order to improve the knowledge of HPV carcinogenesis in oral cavity and oropharynx carcinomas.This study confirms the higher prevalence of HPV DNA in oral cavity and oropharynx carcinomas, especially in males (78%), with an average age of 58 years, and a high frequency of smokers and alcohol drinkers.The prevalence of HPV DNA genome in the samples analyzed was 25.6%, and among the positive cases, 33.3% were HPV16 and 14.3% were HPV18, highlighting the association of high-risk HPV in oral cavity and oropharynx carcinogenesis.The authors declare no conflicts of interest.


Vaccination programs with the current prophylactic HPV vaccines started in most countries around 2008 with introduction of the bivalent Cervarix HPV16/18 vaccine, rapidly followed by Gardasil (HPV6/11/16/18) and, finally, Gardasil 9 (HPV6/11/16/18/31/33/45/52/58), from 2015. Many studies have now confirmed their ability to prevent infection with vaccine-covered HPV types, and the subsequent development of either genital warts and/or cervical neoplasia, although this is clearly more effective in younger women vaccinated prior to sexual debut. Most notably, reductions in the prevalence of vaccine-covered HPV types were also observed in unvaccinated women at the same geographical location, presumably by sexual dissemination of these changes, between vaccinated and unvaccinated women. Furthermore, there are several studies that have demonstrated vaccine-associated HPV type-replacement, where vaccine-covered, high-risk HPV types are replaced by high-risk HPV types not covered by the vaccines, and these changes were also observed in vaccinated and unvaccinated women in the same study population. In light of these observations, it is not entirely clear what effects vaccine-associated HPV type-replacement will have, particularly in older, unvaccinated women. There are now many reports that indicate that prophylactic vaccination with Cervarix or Gardasil, prior to sexual debut and HPV infection, is most effective at preventing both infection and the subsequent development of cervical intraepithelial neoplasia (CIN), specifically caused by the HPV types covered by each vaccine . Indeed,With respect to vaccine efficacy, it is noteworthy that there are >50 HPV types known to infect genital epithelium, where ~14 are high-risk (HR) and the others are either probable high- or low-risk (LR). Moreover, established HPV infections of one type can influence susceptibility to infection with others by stimulating cross-type immunity  and supeThe answers to the preceding questions are not straightforward, since natural cross-type HPV immunity interacts with vaccine-induced, cross-type protection . When thThe findings discussed, thus far, have analysed the effects of vaccination on HPV type prevalence, irrespective of cervical dysplasia, and it is very clear that this should also be evaluated. In this regard, a very recent study carried out in Northern Italy assessed the HPV type and CIN status of 5807 women, aged between 21–65 years, with abnormal pap smears, who attended for colposcopy over a 15-year period, between 2005 and 2019 . AnalysiSince cervical cancer usually takes ~10 years to develop, the aforementioned studies analysed the effects of the vaccines on HPV type prevalence and the incidence of CIN as proxies for the subsequent development of cancer. In spite of this, it would be expected that the incidence of invasive disease would at least remain stable or show a moderate decline post-vaccination. However, using the United States Cancer Statistics (USCS) database, a study on cervical cancer incidence was carried on 15–29 year old women in the US, between 1999–2017 .The current trend of increased incidence of cervical cancer in Sweden  has alreCost-effectiveness analyses (CEA), carried out on the current HPV vaccines, is dependent on the length of protection, degree of cross-type protection, and extent of any HPV type-replacement observed . Thus, iCollectively, the aforementioned studies clearly demonstrate vaccine-related direct and indirect herd effects, where changes in the incidence of vaccine- and non-vaccine-covered HPV infections were transferred between vaccinated and unvaccinated women over time. Most significantly, it will be important to carefully monitor the potential impact of these alterations in older unvaccinated women, where there is no vaccine-related cross-type protection, and there may be increased potential for iatrogenic outcomes.


Lactobacillus in CVM was more to be frequently identified in SIL-positive women as compared to SIL-negative individuals. Our findings suggested that significant CVM differences occurred when SIL developed to HSIL which was caused by persistent HPV infection.Lower female genital tract is colonized by a variety of microbes  which associate with the risk of genital infection. This study characterized CVM for 149 Chinese women with different status of human papillomavirus (HPV) infection and squamous intraepithelial lesion (SIL): no HPV infection (HPV-), HPV infection without significant SIL (HPV+NoSIL), HPV infection with low-grade SIL (HPV+LSIL) and HPV infection with high-grade SIL (HPV+HSIL). Analysis results showed CVM has dramatically changed in HPV+HSIL group when compared to HPV+LSIL group, but it exhibited no significant differences between HPV- and HPV+NoSIL groups as well as between HPV+NoSIL and HPV+LSIL groups. In consistence, random forest analysis found more notable differences in HPV+HSIL vs HPV+LSIL comparison than in other comparisons. In addition, depletion of  Persistent human papillomavirus (HPV) infection is the highest risk of invasive cervical cancer (ICC) . Given tLactobacillus is prevalent in healthy women who had no genital infections , HPV+NoSIL (HPV-positive but no significant SIL), HPV+LSIL (HPV-positive and low-grade SIL) and HPV+HSIL (HPV-positive and high-grade SIL) groups, and to interpret the association of CVM with HPV infection as well as SIL level. Our findings should provide extensive insights into the correlation between CVM and HPV infection for Chinese women.This study was approved by the Ethics Committee of Peking University Shenzhen Hospital (registration number: 2021-006).2O2, leukocyte esterase, neuraminidase, β-glucuronidase or acetylaminoglucosidase . The vaginal swabs were preserved in a 2 ml sterile tube and stored at -80 centigrade within 30 minutes after collection for sequencing in six months.Women older than 18 years and sex-exposed were eligible to be enrolled. Exclusion criteria were as follows: pregnant or lactation; post-menopause; with a history of cervical ablation or resection surgery or hysterectomy or pelvic radiotherapy; suffering from autoimmune diseases or HIV infection; taking immunosuppressants, oral antibiotics, vaginal douching; having sex one week before colposcopy; having positive results for H® Preservative Solution. Then the Cobas 4800 platform , which is approved by FDA for HPV testing, was applied to detect 14 high-risk HPV genotypes via collected cell samples: HPV16, HPV18 or other 12 high-risk HPV genotypes (HPV 31/33/35/39/45/51/52/56/58/59/66/68). For pathological diagnosis, we used directed biopsies for all visible lesions, and random biopsies at the squamocolumnar junction in normal quadrants. The highest grade among the multiple biopsies from each quadrant was recorded as the final diagnosis. Pathological diagnosis were reported as negative, cervical intraepithelial neoplasia (CIN) grade I, II, III, or cancer. According to the criteria of the Bethesda system, CIN I and CIN II/III were classified as LSIL and HSIL respectively.Cervical exfoliated cells were obtained by cervical brushes and were preserved in 20ml SurePathMicrobial DNA was extracted by Dneasy PowerSoil Pro Kit . The DNA concentration and purity were estimated by 1% agarose gels on Agilent5400 . Then we amplified 16S rDNA V4-V5 hypervariable regions  of 16S rRNA gene and determined the quality of PCR products . Qualified DNA libraries were then sequenced by 250bp read length based on Illumina NovaSeq platform .pwr package. In this study, we randomly enrolled the patients with/without HPV infection, and furtherly divided the patients into four groups. The power and significant one-way level w set as 0.8 and 0.05 respectively, and the large ANOVA effect size was 0.4. Then the sample size was estimated to be >= 24 for each group. Given inter-individual variations for vaginal microbiota, we enrolled more than 24 patients in each group. Raw sequencing data were filtered and annotated as previously reported  years old), 40 were HPV-positive and had no significant SIL  years old), 28 were HPV-positive and pathological diagnosed with LSIL  years old), and 51 were HPV-positive and pathological diagnosed with high-grade HSIL  years old). For SIL-negative and -positive women who was HPV-positive, HPV16 infection (including mixed infection with other HPV genotypes) represented 12.9% and 44.3% respectively, HPV18 infection (including mixed infection with other HPV genotypes) represented 5.7% and 11.4% respectively, and the other 12 high-risk HPV infection accounted for 47.1% and 59.5% respectively. All recruited women did not smoke, and had no menopause as well as genital infections other than HPV.Principal coordinate analysis (PCoA) indicated slight differences of microbial samples between HPV-, HPV+NoSIL, HPV+LSIL and HPV+HSIL group (Lactobacillus-dominated (LD) and non-Lactobacillus-dominated (NLD) CVM respectively (Gardnerella (median(IQR), 17.89% vs 0.03% in LD CVM), Fannyhessea % vs 0.02% in LD CVM), Megasphaera % vs 0.006% in LD CVM), Prevotella % vs 0.003% in LD CVM), Sneathia % vs 0% in LD CVM), and Streptococcus % vs 0.05% in LD CVM)  signally accumulated, including OTU877, OTU883 and OTU893 -associated anaerobes, such as  reports , enabled reports . Sneathitoxicity . This watoxicity . The aboLactobacillus-dominated CVM, which represented higher proportion in HPV- group in our study and other reports  can be found below: This study was approved by the Ethics Committee of Peking University Shenzhen Hospital (registration number: 2021-006). The patients/participants provided their written informed consent to participate in this study.Conceptualization, RW, HD, and WD. Methodology, CG, JH, HC, DW, and WD. Software, QZ and WD. Formal analysis, QZ and WD. Resources, RW and HD. Data curation, SL. Writing—original draft preparation, WD and CG. Writing—review and editing, RW and HD. Visualization, WD. Supervision, RW and CL. Funding acquisition, WD and RW. All authors contributed to the article and approved the submitted version.This work was supported by Shenzhen High-level Hospital Construction Fund(grant number: YBH2019-260), Shenzhen Key Medical Discipline Construction Fund(grant number: No.SZXK027), Sanming Project of Medicine in Shenzhen (grant number: No.SZSM202011016) and Scientific Research Foundation of Peking University Shenzhen Hospital (grant number: No.KYQD2021075).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.