According to the recently implemented organized cervical cancer screening program in Germany, women older than 35 years with negative cytology but persistent high-risk human papilloma virus (hrHPV) infection > 12 months should be referred to colposcopy for further evaluation. This study aimed to present and dissect colposcopic and histopathological findings with particular focus on associated hrHPV genotypes.This study is a retrospective analysis of clinical data from 89 hrHPV positive patients with normal cytology who underwent colposcopic examination at a certified dysplasia outpatient clinic in Germany in 2021.n = 68), eight (12%) had cervical intraepithelial neoplasia (CIN) 3 and six (9%) had CIN 2. The proportion of women diagnosed with CIN 3 varied among different hrHPV genotypes .While 38 (43%) women had a normal colposcopic finding, 45 (51%) had minor and 6 (7%) major changes. Thirty-one (35%) of the women were HPV 16 and/or HPV 18 positive and 58 (65%) women were positive for other hrHPV only. Among patients who underwent colposcopy with biopsies (in case of an abnormal finding or type 3 transformation zone, Persistently hrHPV positive women with negative cytology are at increased risk of being diagnosed with CIN 3. As CIN 3 prevalence seems to differ with regard to hrHPV strain, immediate HPV genotyping for risk stratification and subsequent early referral for colposcopy might constitute a feasible strategy. The recognition that a persistent HPV infection is causative for the vast majority of cervical cancers has led to a transition from cytology-based to HPV-based screening programs in an increasing number of countries, either as a stand-alone screening method or in combination with cytology (co-testing) , 2. SeveIn January 2020, almost 50 years after the introduction of an annual opportunistic cytology screening for cervical cancer in Germany, a nationwide organized screening program was implemented. According to the age-dependent screening algorithm, women between 20 and 34 years of age are entitled to an annual cytological examination, whereas women ≥ 35 years should be screened by co-testing with HPV testing and cytology every 3 years. Women with negative cytology (Pap I) and a repeatedly positive HPV test in re-screening after 12 months should be referred to colposcopy within 3 months   . The preColposcopic findings of the cervix and vagina were classified according to the 2011 colposcopic terminology of the International Federation for Cervical Pathology and Colposcopy (IFCPC) . PatientThis study was approved by the Ethics Committee II of the University of Heidelberg, Medical Faculty Mannheim (2022-808-AF 11).p-value < 0.05 was considered statistically significant. Positive predictive values (PPVs) for the most frequently observed HPV strains were calculated to analyze diagnostic performance of HPV testing for detection of high-grade dysplasia.All data were recorded in a Microsoft Excel spreadsheet. Statistical analysis was performed using SAS® software . Categorial data are presented as absolute and relative frequencies. For quantitative normally distributed data arithmetic mean and standard deviation are given. Fisher’s exact test was used to compare the prevalences of cervical dysplasia between HPV 16/18 positive and other hrHPV positive patients. A The data of 89 women were included in this analysis. The mean age of the study population was 49 ± 11 years. Regarding HPV vaccination status, only one patient (1%) was fully vaccinated and one patient (1%) was partially vaccinated. Since the vast majority of women (98%) was not vaccinated, the vaccination status was not taken into account for further analyses.n = 89) and the respective infection pattern (single infection or co-infection) is presented in Table n = 23), followed by HPV 31 in 15% (n = 13) of the cases. Each of the genotypes HPV 18 and HPV 52 was observed in 9% (n = 8) of the patients. 26 patients (29%) were tested positive for pooled hrHPV (other). Since women with multiple infections are counted separately for each HPV type, the sum of the individual HPV types surmounts the total number of included women. On a superordinate level, 31 (35%) of the women were HPV 16/18 positive (HPV 16 + and/or HPV 18 +) and 58 (65%) women were tested positive for other hrHPV only (HPV 16-/HPV 18-/other hrHPV +). Women positive for HPV 16/18 tended to be older than women positive for other hrHPV only, however, this was not statistically significant . Considering the exactly genotyped hrHPV cases, a single infection was present in 84% (54/64) of the women. In 16% (10/64) of the women, a co-infection with different hrHPV strains was detected .The distribution of different hrHPV genotypes among the study population  or it was unsuccessful due to atrophic changes resulting in cervical stenosis . In two women (2%), the obtained biopsy was categorized as non-representative by the pathologist.Table n = 68), three women (4%) had CIN 1, six women (9%) had CIN 2, eight women (12%) CIN 3 and two women (3%) vaginal intraepithelial neoplasia (VaIN) 3. No case of invasive cancer was detected. Higher prevalences for CIN 2 + and CIN 3/VaIN 3 were found in the HPV 16/18 group compared to the other hrHPV group, albeit this difference was not statistically significant . : 46 ± 11 years, p = 0.5756).Out of all women who underwent representative biopsy (n = 68) stratified by hrHPV genotype. Out of the 19 patients who were positive for HPV 16, three (16%) had CIN 2, two (11%) had CIN 3 and two (11%) had VaIN 3. Two out of the six patients (33%) positive for HPV 18 had CIN 3. Six cases of CIN 3 (9%) occurred in patients with a single HPV infection, while two cases of CIN 3 (3%) were recorded in patients with multiple infections . In one patient with CIN 3 no exact identification of HPV genotype was available. In our study population, women positive for HPV 35, HPV 51, HPV 59 or HPV 66 did not show dysplastic changes.Table Positive predictive values of HPV testing for the presence of high-grade vaginal and cervical dysplasia dependent on the HPV strain ranged from 16% 95% CI 7–31) to 46% (95% CI 17–77) for CIN 2 + and 9% (95% CI 3–22) to 33% (95% CI 4–78) for CIN 3/VaIN 3. Highest PPVs for CIN 2 + were observed for HPV 31  and HPV 16  and for CIN 3/VaIN 3 for HPV 18 and HPV 52 . Lowest PPVs were seen for other hrHPV both for CIN 2 +  and CIN 3/VaIN 3  , followed by HPV 31 (15%) and both HPV 52 and HPV 18 (9%). HPV 39, HPV 45 and HPV 51 all had a prevalence of 6%, the other hrHPV types were below 5%. To a larger extent, this distribution is similar to data from a Danish cohort study reporting HPV 16 as the most common type with a comparable frequency to ours (24.2%), also followed by HPV 31 (20.5%) and HPV 52 (20.1%) and with lesser frequency by HPV 51 (14.4%), HPV 33 (13.4%), HPV 39 (11.3%), HPV 45 (11.0%), HPV 56 (10.0%) and HPV 18 (9.5%) . A similIn our study, 43% of the patients had a normal colposcopic result, 51% had minor and 7% had major changes. Data on colposcopic findings among cytology negative, hrHPV positive women are scarce, as the vast majority of studies focus on reporting of colposcopy-guided biopsy results, but not on the colposcopic result itself. In a study by del Pino et al. evaluating the accuracy of initial colposcopy to predict progression to higher-grade CIN, among the cytology negative, hrHPV positive subgroup, 60% of the women had a normal colposcopy, 37% had minor and 3% major changes. However, comparability of those results to ours is limited, as in the aforementioned study all patients who had a diagnosis of CIN 2 or CIN 3 in the biopsy at enrolment were excluded. Interestingly, del Pino et al. found that hrHPV positive women with normal, borderline, or low-grade abnormalities in cytology had a similar risk of progression to high-grade lesions and that the initial colposcopy findings did not provide relevant information on the risk of progression . GeneralWhile we observed only very few CIN 1 cases , prevalence of CIN 1 is reported as high as 21.6% in HPV 16/18 positive women  and 23.9In our study population, 12% of the women with discordant co-testing from whom a biopsy was obtained had CIN 3 . This is well in line with the recently published retrospective findings by Peace et al. reporting of 12.0% CIN 3 in a cytology negative, HPV positive collective with abnormal colposcopic results. However, compared to our study population Peace et al. followed the 2012 ASCCP screening algorithm recommending an immediate referral to colposcopy for HPV 16 or HPV 18 positive cases and repetition of co-testing only for other HPV positive cases . ProspecIn a large Danish prospective cohort study with a follow-up time of up to 11.5 years, 9.7% of the hrHPV positive women with negative cytology at baseline developed CIN 3 + during the observational period. Stratified by hrHPV type and type of infection (single or multiple infection), the highest proportion of women who developed CIN 3 + was observed among those with a single HPV 16 infection (23.3%), followed by those with a single HPV 33 infection (17.9%) or single HPV 31 infection (11.3%). Interestingly, when HPV 16 occurred as a co-infection with another hrHPV type, the proportion of women developing CIN 3 was only 17.2% and for HPV 31 positives in conjunction with another hrHPV type except HPV 16 only 9.7%. Regarding HPV 18, the proportion of women developing CIN 3 did not differ between solely or co-infected women  . In the In our study, prevalence of CIN 3 was 14% for HPV 16/18 positive women who underwent biopsy and 9% for other hrHPV positive. Reviewing literature, CIN 3 prevalence for the former subgroup ranges from 9.1 to 11.8% , 46, 51 In our study population, among the HPV 16 positive women, 11% had CIN 3 and among the HPV 18 positives, 33% had CIN 3. Peace et al. reported a CIN 3 + prevalence of 14.7% among HPV 16 positive cases, yet only of 1.4% among HPV 18 positives . IncludiWe decided to include the cases of VaIN 3 into our study population, as it also is a HPV-associated condition in most cases and the direct precursor of vaginal carcinoma. VaIN is difficult to diagnose as it is a very rare and mostly asymptomatic condition. It can occur alone or simultaneously with cervical or vulvar intraepithelial neoplasia. It has been reported that there is a positive correlation between the grade of VaIN and grade of concomitant CIN, although there is a non-negligible number of women in whom vaginal lesions were found to be more severe than cervical lesions . As espep = 0.082) and concluded that Pap testing could be unnecessary in HPV 16/18 positive women who will undergo colposcopy-guided biopsy to diagnose invasive cervical cancer [Even though we did not detect any cases of invasive cervical carcinoma, other groups found invasive cervical carcinoma in up to 0.5% of the women , 44, 46.For countries undergoing substantial changes in cervical cancer screening, such as Germany, ongoing evaluation of screening results and program effectiveness is essential to highlight any areas for improvement. As hrHPV positive women with negative cytology who have a considerable risk of being diagnosed with CIN 3 are now identified at constantly increasing numbers, refinement of management strategies for this subgroup is crucial. In line with the 2019 ASCCP consensus guidelines, future approaches might comprise a personalized risk-based management, since the individual risk for having or developing CIN 3 is dependent on a combination of past history and current results. For example, a new abnormal test results within 5 years after a negative HPV test or co-test reduces the estimated risk for CIN 3 + by approximately 50%. Thus, incorporating the patient’s history of previous HPV test results into clinical decision-making might help to distinguish high-risk from low-risk patients and subsequently adapt management recommendations . BesidesThere are several limitations to our study that should be noted. Due to the retrospective design it is possible that cases might have been missed, so there is no claim for completeness of the cohort. Furthermore, data regarding HPV genotyping was incomplete. Another shortcoming is the limited number of patients included, which might reduce the statistical power of the results. On the one hand, this is due to the study period of 12 months, which was chosen to enable the provision of current data regarding the recently introduced new cervical cancer screening program in Germany. On the other hand, the limited number of included patients is owed to the single-center design. Therefore, a multi-center prospective study with adequate follow-up is required to further explore the risk of developing high-grade CIN among cytology negative, hrHPV positive patients.Our results suggest that women with discordant co-testing have a significant risk of being diagnosed with CIN 3. As the risk for developing a high-grade CIN might be associated with the underlying hrHPV type, employing instant HPV genotyping and, if necessary, immediate referral for colposcopy may result in substantial improvement in screening management.


BackgroundRoutine immunization of both girls and boys starting from nine years of age with the human papillomavirus (HPV) vaccine is the current recommendation. The objective of this retrospective study using National Health and Nutrition Examination Survey data was to evaluate the influence of sociodemographic factors on the series initiation and completion of the HPV vaccine from 2011 to 2020.MethodologyThe chi-square test was used to examine the statistical significance of the association between categorical variables and receipt of the HPV vaccine. The Cochran-Armitage test for trend was employed to assess the statistical significance of temporal trends in risk factors associated with rates of HPV vaccination. These trends were further quantified by a significant rate ratio by comparing them against the most recent survey years.ResultsHPV vaccine uptake was higher in the 9-14-year age group across survey years and had increased for both males and females over that time. The first dose of the HPV vaccine was most likely to be received by the 11-18-year age group. In the most recent survey of 2017-2020, the highest number of vaccination series completion was achieved for Gardasil®.ConclusionsImproved physician efforts and strategies to vaccinate males, low socioeconomic strata patients, and ethnic minorities in more numbers are needed. PreventionThere are three human papillomavirus (HPV) vaccines. The two-valent HPV vaccine, Cervarix®, was licensed for use in females but is no longer available in the United States. The four-valent HPV vaccine, Gardasil®, was licensed for use in females and males and is also no longer available in the United States. The nine-valent HPV vaccine, Gardasil 9®, was approved in February 2015 for routine immunization of males and females and since the end of 2016 is the one currently used in the United States for vaccinating both sexes for the approved age groups [As Gardasil 9® has been in use in the United States since late 2016, there has been reassuring safety monitoring data so far on the vaccine. The vaccine was studied in clinical trials with more than 15,000 males and females. It provides protection against 6, 11, 16, 18, 31, 33, 45, 52, and 58 types of HPV. HPV types 16, 18, 31, 33, 45, 52, and 58 cause vulvar, vaginal, cervical, penile, anal, and oropharyngeal cancers. Condyloma acuminata or genital warts are caused by HPV types 6 and 11. The vaccine also offers protection against the precancerous lesions of the vulvar, vaginal, cervical, and anal regions [ScreeningThe 2018 United States Preventive Services Task Force guidelines recommend screening with cervical cytology alone every three years for women aged 21 to 29 years. Cervical cancer screening in those younger than 21 years is not recommended. With the undertaking of HPV vaccination, the HPV infections and precancer lesions in the 21-24-year age group were lowered considerably, which has led to the screening recommendation by the American Cancer Society to start at a later age of 25 years [Human papillomavirus disease burden globally and in the United StatesIn 2020, cervical cancer was the seventh most common cancer contributing to new cancer cases and the ninth most common cancer contributing to cancer deaths globally. In the United States, the incidence and mortality rate of cervical cancer in 2020 were 28.3 and 2.1 per 100,000 people, respectively ,6. In 20Despite having effective vaccines, the cancer burden can be reduced more efficiently by improving vaccine uptake. We undertook this study to investigate the sociodemographic factors affecting vaccine uptake, which could enable the formulation of targeted measures that can be taken to further improve vaccination coverage among the population.Study design and populationThe data of this study were obtained from the National Health and Nutrition Examination Survey (NHANES) 2011-2020, a nationally representative survey of the resident, civilian, non-institutionalized US population that collects health examination data . The NHAStudy measuresThe objective of evaluating the influence of sociodemographic factors on the series initiation and completion of the HPV vaccine over time was investigated based on the responses to four main survey questions provided in Table This study mainly focuses on the impact of sociodemographic variables, namely, sex, age, ethnicity, education level, and poverty-income (PI) ratio, as provided in Table The PI ratio was categorized based on population distribution of PI ratio by quartiles as provided by Palar et al. , where PThe survey responses to question 1  to 2017-2020 (17.8%) for the age group of 15 years, while for the age group of 9-14 years, the observed increase was approximately 1.66 times  received the highest percentage of the HPV vaccine compared to other ethnic groups in survey years 2017-2020 and 2015-2016 Tables , 4, whilComparing vaccination rates against education, having a “college degree and above” consistently showed higher vaccination rates for all survey years over the “high school graduate” and “less than high school” categories. The improvement in vaccination rate over the survey years was also observed in all categories, with the highest improvement 3.15 times) being found in the high school graduate categories. It should also be noted that the category with “unknown” education information showed the highest vaccination rate in the survey data. The percentage of the HPV vaccine received by the unknown education category had increased from 21.5% in 2011-2012 to 34.5% in 2017-2020  at the age of 11-12 years and vaccination through the age of 26 years if not adequately vaccinated when younger.Further, in the particular age group of 11-18 years, the percentage of females who had taken the first dose of the HPV vaccine was noted to be highest in the survey year 2017-2020, and for males, the percentage was noted to be highest in 2015-2016 Figure . Under iFindings based on the HPV vaccine series completionBased on survey questions 3 and 4, Table It was noted that in the most recent survey of 2017-2020, the highest number of vaccination series completion was achieved for Gardasil®. In the same survey, it was also found that the series completion of Gardasil® and Cervarix® were considerably higher in the age group of ≥15 years compared with 9-14 years but vice versa for the Gardasil 9® vaccine. Under the ethnicity, the highest number of vaccine series completion of Cervarix® was seen in NHB, whereas NHW showed the highest number of vaccine series completion of Gardasil®, Gardasil 9®. Under the income-based categories, the moderate-income group showed the highest number of series completion of Gardasil®.Additionally, Table Based on ethnicity, NHW showed higher number series completion of Gardasil® for all three survey years between 2011-2016, whereas the higher number series completion of Cervarix® based on ethnic groups varied over the survey years. In regard to the PI ratio, the moderate income group showed a higher number of series completion of Gardasil® in 2015-2016 with a gradual increase over the survey years. According to the series completion of Cervarix®, the highest number was noted in the low-income group and moderate-income group for the respective survey years 2013-2014 and 2015-2016.There are certain myths and misinformation around the HPV vaccine that lots of families harbor which seem to hinder vaccination efforts for HPV. One concern is that the child may start having sex early on in life but studies have shown this is not the case . Some faAccording to the NIS-Teen in 2017, 51% of adolescents had not completed their HPV vaccine schedule, and adolescents in rural areas were lagging behind their urban area counterparts in vaccination rates. In 2020, between ages 13 and 17 years, only 61.4% of females and 56% of males were fully vaccinated against HPV. For this age group, Rhode Island was the leading state for the highest coverage rate for fully vaccinated males and females and Mississippi had the lowest coverage rate nationwide . In our According to one study in 2019, it was found that globally, 15% of girls and 4% of boys were fully vaccinated and 20% and 5% received at least one dose of the vaccine respectively . In a stOur study also reveals that when considering the initiation of HPV, the percentage of receiving the first dose of the HPV vaccine was noted to be the highest in the age group 11-18 years. In the particular age group of 11-18 years, the percentage of females who had taken the first dose of the HPV vaccine was noted to be the highest in the survey year 2017-2020, and for males, the percentage was noted to be the highest in 2015-2016.According to a systematic review in 2007, it was found that African American, Hispanic, and white ethnic groups had equal acceptance of the HPV vaccine . AnotherOur study results are in consensus with previous literature in regard to findings on age, gender, and ethnicity. In addition, the results of our study revealed hidden insights about the association and temporal trends of the risk factors education level and PI ratio with the receival of HPV vaccine. Missing data from survey subjects can contribute to non-respondent bias. Our study does show we have made an improvement in HPV vaccination coverage over the past 10 years but we are yet to reach our goal. As per Healthy People 2030, the current target is to immunize 80% of the adolescent population (13-15 years) with the HPV vaccine and 54.4% has been achieved as of 2020 .The authors do recognize that the results obtained by a retrospective study are limited because they cannot prove a causal relationship and a true causal-effect link can only be established by properly conducted prospective studies where there is an option of taking care of biases of different kinds. However, a retrospective study can be helpful for gathering preliminary information and directing the design of upcoming prospective investigations.Various sociodemographic factors influence the series initiation and completion of the HPV vaccine. Physicians focus a lot on the diagnosis and treatment of cervical cancer but better education and more open conversations about the HPV vaccine as a preventative measure by the medical community is a much-needed step. Parental attitudes, beliefs, and barriers play a crucial role in vaccinating children and adolescents. Education at every well visit about the benefits starting even before the recommended age groups will help prime the families’ receptiveness to the vaccine at the appropriate time. Based on the findings of our study, males, low socioeconomic strata patients, populations with less than high school education, 9-10-year age group, and ethnic minorities are the groups that should be focused upon.


With HPV screening the specificity of screening positives has decreased, even with a cytological triage test. Increases in colposcopies and detection of benign or low-grade dysplasia are reported, not least in older women. These results highlight the necessity to find other triage tests in HPV screening strategies, so that women can be more accurately selected for colposcopy, thus minimizing the clinically irrelevant findings.The study included 55- to 59-year-old women who exited the screening with normal cytology, but later in a follow-up test were positive for the HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 and had a cervical cone biopsy done. To model a screening situation with hrHPV-positive women, three different triage strategies, namely, cytology, genotyping and methylation, were performed. The study considered the effect of direct referral to colposcopy for HPV genotypes 16, 18, 31, 33, 45, 52 and 58, and methylation for FAM19A4 and hsa-mir124-2 and/or any form of abnormal cytology.Seven out of 49 women aged 55–59 years with hrHPV had a cone biopsy with high-grade squamous intraepithelial lesion. No triage method found all cases, and when comparing positive and negative predictive value and false negative rate, cytology showed better results than genotyping and methylation.This study does not support a switch in triage strategies from cytology to hrHPV genotyping and methylation for women above 55 years of age yet, but demonstrates the need for more evidence on molecular triage strategies. Where cervical screening programmes are implemented cervical cancer is reduced, since cervical cancer is largely preventable through local treatment of screen-detected cervical precursor lesions, cervical intraepithelial neoplasia . CervicaThe HPV test has high sensitivity but lower specificity to detect high-grade squamous intraepithelial lesion (HSIL), compared to cytology. This is due to the fact that HPV infections most often are transient, and only a minor proportion become persistent and cause dysplasia and cancer , 9. StudCytology as a triage method has shown limited value due to its poor sensitivity, not least among the older age cohorts in the screening , 15. TheGenotyping of HPV could be an alternative triage method in the screening programme, since there is a difference in cancer risk between genotypes, where HPV 16 and 18 genotypes are the most common in cervical cancer . A long-Hypermethylation of certain sites in the human genome have been associated with high-grade cervical dysplasia and cervical cancer  and coulStudies on screening programmes with HPV as primary test and different molecular strategies are few. Older women in these studies need special attention, since in this age cohort colposcopies are more often inconclusive, cytology is not as reliable and whether reactivation of persistent infections – so-called latent infections – develop in the same pattern as newly acquired infections is not clearly established. Methylation and genotyping are objective methods compared to cytology. They show promising results in increasing the specificity in HPV screening in the same way as cytology, and are appealing, as they can be used on self-samples , 26. TheThe study cohort used in this study has previously been published . BrieflyThe current study includes women who participated in the former study and who were positive for the HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in the follow-up test, and had a cervical cone biopsy done at the clinical follow-up. To model a real-life screening situation with hrHPV-positive women, simulation of three different triage strategies – cytology, genotyping and methylation – was performed. The study model considered the effect of direct referral to colposcopy for HPV genotypes 16, 18, 31, 33, 45, 52 and 58 , methylaThis study was approved by the regional ethical committee board in Uppsala, Sweden (D-nr. 2014/121).ThinPrep cytology slides were assessed by one experienced and certified cytotechnician and classified according to the international Bethesda classification system  with atyDNA was extracted from liquid-based cytology samples using QiaAmp DNA mini kit  . HPV detFor methylation analysis the QIAsure Methylation Test Kit  detecting promoter hypermethylation of the genes FAM19A4 and hsa-mir124-2 was used. Extracted DNA was subjected to bisulphite treatment with EZ DNA Methylation-Goldkit . Detection of hypermethylation in any of the two genes resulted in a positive test result.Excel 2010  was used for data collection and evaluation. The performance was evaluated for three screening strategies: cytology, HPV 16/18/31/33/45/51/58 genotyping and FAM 19A4/has-mir124-2 methylation. Positive predictive value (PPV) and negative predictive value (NPV) were calculated for comparison of the accuracy of the test results between the triage methods. Further analysis of the false negative rate (FNR) was done for comparing the triage methods’ likelihood of improperly indicating no presence of dysplasia. Absolute HSIL risk was calculated for the different screening strategies.The study population consisted of 49 hrHPV-positive women from a study of women exiting the screening programme .Of the women included in this study protocol, 49 were hrHPV-positive for HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and/or 68 and had a cone biopsy done. The screening samples were genotyped and analysed for cytology and methylation (Fig. Abnormal cytology was found in 12/49 samples, genotypes resulting in direct colposcopy were detected in 30/49 samples and methylation positivity was found in 35/48 samples; one sample had too little material to analyse after storage in the biobank (Fig. A total of seven HSILs were found in this material where no method alone detected all of the cases. Of these, only three were found by all triage methods. Among the 49 women with a positive HPV-test, nine LSILs were detected. Cytology detected none, methylation six and with genotyping including HPV 16, 18, 31, 33, 45, 52 and 58 four cases were found. When including all 14 genotypes, all nine cases were detected and of them two were methylation negative and all had normal cytology.To further develop the HPV-based cervical screening, future steps could include vaginal self-sampling followed by molecular triage using broad genotyping and methylation for risk stratification. In an attempt to address this question in a pilot strategy, we used a cohort of samples including women 55–59 years of age in 2012–2014 who exited the screening programme with a normal cytology and had a positive follow-up sample including HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, as well as a clinical follow up with histological cone biopsy [Previous studies show that methylation is non-inferior to cytology triage , 31, butThe NPV of hrHPV-positive, methylation-negative samples was 93% for ≥ CIN2 in the article by Bonde et al. , whereasConcerning genotyping in screening, a meta-analysis was published in 2020  with resThis study has not explored a risk stratification with a combination of different triage methods, due to the small study population, but that would be the natural step in continuous work on the subject. With that in mind, a limitation in this study is the few histological HSILs in this study population. This illustrates the need of studies on triage strategies that discriminate different age groups and individualize risk stratification, as well as the need to further investigate the use of combinations of different molecular tests.This study does not support a transition to extended genotyping and DNA methylation with these methods at this time. Rather, it supports the need for further studies with larger cohorts, other methylation methods as well as prospective studies that demonstrate real-life outcomes.


Smoking has been proven to increase the risk of cervical cancer, but it is still controversial whether smoking reduces women’s ability to clear human papillomavirus (HPV) infection. This study investigated the association between smoking behaviors during follow-up and clearance of HPV infection in women with HPV-positive and pathologically normal uterine cervix in China, using a propensity score matching (PSM) analysis.The present prospective study included data from women examined in the Gynecology Department of Shanghai General Hospital from January 2018 to June 2020. Twenty patients who smoked throughout follow-up were selected and matched with 60 patients using the 1:3 PSM method on age, marital status, and whether infected with high-risk HPV (HR-HPV). At each visit, smoking and sexual behaviors were collected. The Kaplan–Meier method and a Cox proportional hazard regression model were used to evaluate the probability of clearing HPV infection within a 2-year follow-up.A total of 80 patients were included in the study, all of whom were infected with at least one HR-HPV type at baseline. Current smokers had a lower likelihood of clearing the HPV infection than current non-smokers, after adjusting for a history of sexually transmitted diseases (STD), HPV infection status, and sexual behaviors during follow-up . Additionally, longer duration, higher frequency and larger doses of smoking correlated with the lower clearance possibility of HPV infection .This study showed that the use of tobacco throughout follow-up could increase the risk of a persistent HPV infection, this risk being higher for smokers with heavier tobacco consumption. Our results should alert HPV-positive women to reiterate the advice to cut-back on or stop smoking. Unfortunately, over 85% of the global cervical cancer burden occurs in low-income and middle-income countries2, with China and India having the heaviest share3. Even though cervical cancer screening programs and HPV vaccination have reduced the incidence and mortality globally, China’s situation is fraught with difficulties, and mortality has even increased in recent years4.Cervical cancer remains the fourth most common cancer among women worldwide, with approximately 604000 new cases and 341000 deaths in 20205. Persistent HPV infection, especially of high-risk types , is considered the necessary cause of almost all cervical lesions and cancer progressions, but this is not the sufficient condition of cervical cancer6. Besides, studies have linked cigarette smoking, even passive smoking, to an increased risk of cervical cancer or its precursor lesions7. Smoking seems to be the most important risk factor, except for HPV infection, for cervical disease.Human papillomavirus (HPV) infection is a common sexually transmitted disease. Most HPV infections are transient and disappear within 12–24 months after infection8. Another study in Britain found no significant connection between smoking and the probability of HR-HPV persistence after six months 9. Nevertheless, similar research was rarely conducted in women with a normal pathological cervical biopsy.The exact results demonstrating the precise impact of smoking on HPV persistence and the corresponding mechanism, however, have not been fully established. Smoking was linked to persistent HPV infection in a study of Romanian women 10. Although the most active smokers are men, women also contributed. Smoking prevalence has reached 3.54% (2.91%–4.18%) among Chinese women10. And in China, the smoking rate among young women (aged 15–24 years) increased by 74.0% from 1990 to 201911.It is reported that 341 million (30%) tobacco smokers lived in China in 201912. Focusing on the natural history of HPV infection in these individuals can allow us to eliminate the distraction of treatments. Our purpose in this analysis was to determine whether there was a correlation between smoking behaviors during follow-up and clearance of HPV infection in women with pathologically normal uterine cervix.Women with HPV-positive and biopsy-negative results are recommended to have at least a one-year follow-up without the requirement for treatment, according to the American Society for Colposcopy and Cervical Pathology (ASCCP)This study was a prospective non-intervention study designed to investigate the natural history of HPV infections in women and its relationship with smoking status and behaviors during follow-up. The clinical data of 4869 patients examined for cervical pathological biopsy in the Gynecology Department of Shanghai General Hospital were assessed between January 2018 and June 2020. All cases provided results of HPV genotyping, cytological examination and cervical biopsy at baseline, among which 3309 cases were HPV-positive and pathologically normal. Follow-up visits were scheduled at intervals of six to twelve months after the cervical biopsy. Infections were followed until the clearance, or until the last visit if the participant remained positive throughout the whole period, or until a diagnosis of higher lesions, which means the follow-up would be interrupted by treatment. At each follow-up visit, patients not only were retested for HPV DNA and genotype, but also completed a brief questionnaire on relevant gynecological events and epidemiological features since the last visit, mainly about smoking and sexual behaviors. According to the inclusion or exclusion criteria, 355 patients were chosen for analysis, among which 20 patients smoked during follow-up.In our study, clearance of HPV infection was defined as when none of the types from the first HPV detection was detected at the subsequent visit. The estimated time to clearance was based on the date of the first negative visit. Persistent HPV infection meant the presence of the same HPV genotype detected at enrollment on one or more consecutive examinations at subsequent visits.Inclusion criteria were: 1) positive for one or more HPV types diagnosed in Shanghai General Hospital at baseline; and 2) colposcopy and cervical biopsy were performed in the same hospital and confirmed normal at baseline. Exclusion criteria were: 1) no follow-up records within two years; 2) underwent any treatment to clear HPV infection during follow-up; 3) unclear final results owing to lack of follow-up; 4) previous hysterectomy or conization; and 5) with other malignant tumors.Information on current smoking behaviors was collected using a standardized questionnaire at each follow-up visit. All patients were classified into current smokers and current non-smokers based on these questionnaires. Current smokers were defined as those women who smoked during follow-up, including regular smoking and occasional tobacco use, while the others were considered current non-smokers. To further analyze the relationship between smoking behaviors and the capacity to clear HPV infection, the duration (years of smoking), frequency (days of smoking per month), and dose (numbers of cigarettes smoked per day) were also collected.To facilitate analysis and discussion, patients were grouped based on the duration, frequency and dose of tobacco use. Participants were divided into three groups based on their smoking duration: current non-smokers, those who had smoked for 1–10 years, and those for >10 years. Similar limits were used for grouping based on frequency and dose, such as 15 days per month and 10 cigarettes per day.13; 23 HPV types can be detected in total, covering 17 HR-HPV types  and 6 low-risk HPV types . DNA extraction, PCR amplification and result analysis were all conducted according to the manufacturer’s instructions.Testing for HPV was conducted by using a PCR-reverse dothybridization technology, with the help of a PCRRDB HPV genotyping assay in situ.Colposcopies were performed when HPV detection or cytological results were abnormal. The cervical tissue was obtained from a colposcopy biopsy. Suspicious lesions were stained with acetic acid and iodine reagent, and then biopsied on the basis of colposcopy images. The samples were processed with standard histopathological methods and evaluated by at least two pathologists. All colposcopy operations were performed by qualified colposcopy specialists in our center. The pathology results were classified as normal, cervical intraepithelial neoplasia CIN1, CIN2, CIN3, or carcinoma In order to control for the differences in baseline characteristics between smoking and non-smoking groups, we performed propensity score matching (PSM) using the nearest neighbor matching algorithm. Propensity scores of individuals were calculated based on logistic regression analysis with a caliper width of 0.1. Variables used for matching were age, marital status, and whether infected with HR-HPV. Ratio was paired at 1:3 for patients who smoked versus those who never smoked during follow-up, and another 60 eligible participants were identified after matching. Standardized mean difference (SMD) was used to evaluate the balance of the observed covariates across groups before and after matching. Optimal balance on a parameter is generally achieved when the SMD is ≤0.1.The chi-squared test and t-test were used to assess the differences in the distributions of categorical and continuous variables, respectively, between current smokers and current non-smokers. The survival curves of clearing during the follow-up were calculated using the Kaplan-Meier method and compared with the log-rank test. Univariate analyses were conducted to assess factors associated with the clearance of HPV infection. The final multivariable model, a Cox proportional-hazards model, was fitted with a subset of risk variables to control for extra potential confounding effects caused by the women’s characteristics. We estimated the hazard ratios (HR) and corresponding 95% confidence intervals (CIs). The value for p-trend was used to further assess whether heavier smoking was linked to more persistent HPV infection, based on smoking data of duration, frequency and dose. Two-sided p<0.05 values were considered to be statistically significant. All statistical analyses were performed using software package of social science statistical software version 26 .We analyzed the basic demographic information from enrollment and follow-up data before and after matching . Most vaFor all the participants, the mean duration of follow-up was 19.2 months , with an average of 2.5 visits (median=2.0). None of the participants received treatment associated with HPV clearance throughout follow-up. Within two years of follow-up, 56 patients cleared their HPV infection spontaneously, including 10 of 20 current smokers and 46 of 60 current non-smokers. In contrast, 24 individuals were found to have persistent HPV infection by the end of 2 years.To select covariates statistically associated with the risk of persistent HPV infection, we used univariate Cox regression method to analyze 10 factors . None waBased on the HR and corresponding p-value, the duration, frequency, and dose , as well as the current status of smoking , were all significantly linked with HPV persistence. The probability of HPV clearance was lower among current smokers than among current non-smokers, even after adjusting for those aforementioned covariates . Furthermore, longer smoking duration, more frequent smoking, and larger doses of tobacco all had a negative correlation with the likelihood of clearing an HPV infection . It was convincingly demonstrated that the risk of a persistent HPV infection was higher for smokers with heavier tobacco consumption.17, while some paid attention to those with low-grade or equivocal cytological abnormalities19. In terms of this association, until now, there has not been any published research focusing on the pathological findings of cervical biopsies. A study carried out in women with normal cytology in Daqing, a city in China, has found that cigarette smoking was not correlated with type-specific HPV persistence17. However, cytology is known to have a poor sensitivity of approximately 53.0%20. Part of the reasons are that cytological results could be affected by the quality of the sample, the professional ability and the mental state of doctors. And the diagnosis of cervical diseases in all patients depended on cervical histopathology. Compared to the cytology examination, we could demonstrate more accurately that our participants were free of cervical lesions. In our cases, we excluded women with biopsy-positive lesions at baseline from the analysis, aiming to eliminate the interference of any related treatment.To our knowledge, the present study is the first to examine the association between status and specific behaviors of smoking during follow-up and clearance of HPV infection among women with pathologically normal uterine cervix. Previous studies mainly included HPV-positive and cytologically normal participants at recruitmentIn the present study, the 1:3 PSM method was used to match current non-smokers with current smokers to control for possible confounding effects at baseline. After matching, our univariate analysis demonstrated an increased risk for HPV infection persistence in women who smoked compared with those who never smoked during follow-up. Multivariable analysis showed the same results after adjusting for HPV infection status, history of STD and sexual behaviors during follow-up. In addition, we detected a more complex relationship between smoking for a longer time, more often, and at larger doses with a lower likelihood of clearing HPV infection within a 2-year follow-up. These findings indicate that smoking influenced the natural history of HPV infections in women, implying the beneficial effects of smoking reduction and cessation.21 or null22 associations between current smoking and HPV persistent infection. The inconsistency of these studies may be due to different definitions of HPV persistence or clearance, sample sizes, follow-up intervals and durations and so on. A US-based study among young women reported that smoking was protective against persistent HPV infection, but researchers were unsure whether it was because of a biological or a confounding effect21. In another UK-based study, no association between smoking and persistent HR-HPV infection was found, partly on account of the short 6-month follow-up9.As a well-known risk factor for the progression of cervical lesions with HPV infection, smoking was supposed to promote a persistent infection. However, the association has never been clearly evaluated, and some previous studies have found negative25 and the well-accepted advice to quit smoking during follow-up26, indicating that smoking plays an important role in the process of persistent HPV infection in women. Among these studies, only a study based on women aged 18–35 years detected a significant relationship with increasing duration of years of smoking, but not with smoking intensity23.Our results are in accordance with the majority of published studies in this field19. Another study, based on women in the US, indicated that only among HIV-seronegative women the clearance rate for HR-HPV infections was notably lower in ever smokers than in never smokers27.It is worth noting that few studies further explored the correlation between smoking behaviors and HPV infection persistence in the different subgroups. A study based on Korean women found that among alcohol drinkers, women exposed to secondhand smoking at home or in the workplace were at high risk of both 1-year HR-HPV persistence and 2-year HR-HPV persistence. However, among non-alcohol drinkers, there was no association between smoking or secondhand smoking status and these risks28. The non-significant effect of smoking on the detection of E7 protein, an indicator of later stages in the natural history of the infection, also implied that smoking has a more prominent role in earlier stages29. Therefore, the ability of women to clear HPV infections seems to be related to the host immune response.Although the precise mechanism of how smoking influences HPV infection persistence is not clearly understood, there are some speculations. This correlation is more likely to be explained by the effect of smoking behaviors on the immune system rather than its DNA damage, partially because persistence is greatly influenced by host immunity. This hypothesis is also supported by the fact that in about 80% of HPV-infected women, HPV clearance is a rapidly evolving event and occurs before viral integration into the host genome23. In smokers compared with non-smokers, a significant reduction of Langerhans cells in the normal cervical epithelium was observed30. This change could lead to local immunosuppression and facilitate the establishment and persistence of local HPV infection because Langerhans cells are essential in detecting and presenting viral antigens to T cells. It was further found that smoking showed direct inhibiting effects on T cells, increasing the percentage of CD8+ T cells while lowering CD4+ T cells31. As for humoral immune responses, smokers are deemed to have an increased risk of persistent HPV infection by reducing antibodies or preventing the development of antibodies32. In addition, smoking can decrease the activity of the natural killer cells, affecting the innate immune system33. Combined, it has been generally established that current smoking is associated with a decreased capability of the immune system, thus hindering HPV clearance in the cervix.Smoking has been proven to cause a wide range of suppression of immunological functions34, though this phenomenon was not seen in our study. Higher HPV 16 and HPV 18 DNA load was also associated with current smokers35, while the viral load was inversely associated with clearance36. Unfortunately, we did not have information on viral load.Besides suppressing the immune system, smoking is likely to contribute to persistent HPV infection in other ways. Some confounding effects caused by different lifestyles or characteristics that are different in smokers compared with non-smokers may contribute to additional risk to the persistent infection of HPV. For example, more liberal sexual habits and risky sexual behaviors were more familiar among smokers6, the present study might offer a new thought about how smoking contributes to the progression of cervical cancer. As reported previously, tobacco smoke contains carcinogens that could have a direct transformation effect on the cervix, allowing progress to cancer7. Data from the present study indicate that part of the increased risk of progression to disease carried by smoking might be due to its impact on HPV infection persistence and the natural history of HPV infection.Furthermore, considering persistent HR-HPV infection was the most critical factor of cervical lesions and related cancerThis study is the first to examine the correlation between smoking behaviors and HPV infection clearance in women with pathologically normal uterine cervix. This strategy of selecting the participants based on cervical biopsy results was more precise and sensitive than cytology.Nevertheless, our study has several limitations that may have affected the conclusion. First, all patients were diagnosed and followed in a single center, and the restricted sample size did not allow us to conduct subgroup analyses further. Owing to the small number of cases, we gave up assessing the correlation between smoking and persistent infection of different HPV genotypes. Second, some factors were not included in our study. We did not take the specific information on secondhand smoke intake, sleep quality or use of condoms into account since more than half of these women had not completed related content in the questionnaire. We also did not measure viral load at baseline or follow-up. Third, data were based on self-reports from participants who may underreport or overreport their behaviors or attitudes. Our study also lacked a validated biomarker for measuring smoking exposure, such as urine cotinine levels. We were considering introducing this examination at subsequent follow-up visits. As a result, further research is still required to improve these weaknesses.This is the first study to report the association between smoking behaviors during follow-up and HPV clearance in women with normal pathological results of cervical biopsy. The significant finding of this study was that smoking during follow-up is associated with a decreased capacity to clear HPV. Additionally, a reduced likelihood of HPV infection clearance was linked to smoking for longer, more frequently, and at larger doses. Although physicians tend to provide no treatment for women who are HPV-positive and pathologically normal, tobacco use cessation may lead to a faster rate of HPV clearance. Further large-scale and long-term studies are needed to assess these associations in order to assess the future burden of cervical cancer, notably in developing countries.


Prostate cancer is a major cause of cancer-related deaths globally, yet its causes remain unclear. While human papillomavirus (HPV) is known to be associated with other cancers, such as cervical cancer and anal cancer, investigation of its connection to prostate cancer has yielded mixed results. This systematic review aimed to assess the relationship between HPV and prostate cancer using Bradford Hill’s criteria. Out of 482 studies screened from PubMed, 60 were included and evaluated. Although the strength of association was not strong and certain criteria were not met, the review identified plausible biological mechanisms and reported the presence of HPV in prostate cancer tissues. However, the overall quality of evidence remains low, and further high-quality studies are needed to establish a definitive link. These findings have implications for understanding prostate cancer and guiding future research efforts.Globally, prostate cancer is the fifth most common cause of cancer-related death among men, and metastatic castration-resistant prostate cancer has a high cancer-related mortality rate. However, the aetiology of this disease is not yet fully understood. While human papillomavirus (HPV) has been associated with several types of cancer, including cervical, anal, and oropharyngeal cancers, studies investigating the relationship between HPV and prostate cancer have shown mixed results. This systematic review aimed to evaluate the causative association between HPV and prostate cancer using Bradford Hill’s criteria. A comprehensive search of PubMed was conducted, and 60 out of 482 studies were included in the review. The included studies were evaluated based on nine Bradford Hill criteria, and information on the identification and transmission of the virus and potential oncogenic mechanisms was also extracted. The strength of association criterion was not met, and other criteria, such as consistency and coherence, were not fulfilled. However, biological plausibility was supported, and potential oncogenic mechanisms were identified. While some studies have reported the presence of HPV in prostate cancer tissues, the overall quality of evidence remains low, and the association between HPV and prostate cancer is weak. Nevertheless, the prostate is a potential reservoir for the transmission of HPV, and the HPV E6 and E7 oncoproteins and inflammation are likely to be involved in any oncogenic mechanisms. Further studies with a higher level of evidence are needed to establish a definitive link between HPV and prostate cancer. Propionibacterium acnes [Prostate cancer (PCa) is the second most common form of cancer among men, with an estimated 1.4 million new cases and 375,000 deaths worldwide in 2020 ,7. Benigum acnes ,11,12.HPV is one of the most common sexually transmitted infections globally, and at least 12 so-called high-risk HPV types have been identified as human carcinogens . HPV 16HThe relationship between HPV and PCa has been the subject of much debate and research in recent years, with results from various studies being contradictory. To clarify the connection between these two factors, statistical meta-analysis has been employed ,19,20. HOur investigation is premised on the idea that high-risk HPV types could play a causative role in PCa. As HPV infections can be prevented through vaccination, investigating their potential role in PCa is of critical importance.A comprehensive examination of available research studies was carried out following the PRISMA guidelines for Preferred Reporting Items in Systematic Reviews and Meta-Analyses . A searcThe initial search resulted in 482 papers. We screened the abstracts of the 482 papers in terms of the inclusion and exclusion criteria and relevance to the study . All absThe relevant 60 papers were screened for quality using the Newcastle–Ottawa Quality Assessment Scale  Wells e, while tTo extract data to assess the causal relationship between HPV and PCa incidence, each paper that scored above 3 on the Newcastle–Ottawa scale was selected and scrutinised using the nine (9) central postulates of the Bradford Hill criteria. These nine (9) criteria are Strength, Consistency, Temporality, Biological Gradient, Specificity (now defunct), Biological Plausibility, Coherence, Experiment and Analogy. To aid this review, we added specific criteria for means of transmission, identification of the virus, and oncogenic mechanisms to the Bradford Hill criteria.A total of 60 original papers were reviewed to investigate the causative role of HPV in PCa. Among the 60 studies, 11 (18%) showed a positive association between HPV presence and the development of PCa. Another 10 studies (17%) were uncertain and suggested further research was necessary, while the remaining 39 studies found no association.The analogy criterion is one of the nine criteria proposed by Bradford Hill to assess the causal relationship between an exposure and an outcome. It involves comparing the relationship between two phenomena to a previously established causal relationship. This criterion assumes that if two phenomena are similar in relevant ways, they may have similar causal relationships . In thisIn the case of the relationship between HPV and PCa, the analogy criterion may be useful in exploring potential causal links. HPV is a well-established causal factor in several types of cancer, including cervical cancer, penile cancer, anal cancer, vaginal cancer, vulvar cancer, and cancer of the head and neck; for recent reviews, see ,86. TherIn addition, while cervical dysplasia and cervical carcinoma are often regarded as different stages of the same progressive disease, PCa and BPH are regarded as two separate diseases, with high-grade Prostatic Intraepithelial Neoplasia (PIN) acknowledged as the main precursor of invasive prostatic adenocarcinoma . In the The analogy criterion can also be useful In exploring potential differences in the nature of the HPV DNA in malignant and pre-malignant lesions. The viral genome often persists in genital neoplasms in an integrated state, and this is a key event in the development of cervical cancer. Integration is not detected in benign lesions and clinically healthy tissues that test positive for HPV . SimilarThere are limitations to the use of the analogy criterion in exploring potential causal relationships. For example, it is important to ensure that the similarities between different diseases are not superficial and that the mechanisms by which exposure may cause an outcome are comparable. It is also important to consider the possibility of confounding factors and other potential explanations for the observed association. The similarities between PCa and cervical cancer in the way that different types of HPVs act suggest that further investigation into the potential role of HPV in PCa is warranted.Plausibility refers to the idea that there must be a theoretical basis to support a potential association between a virus and a disease . This reCoherence is similar in that the cause-and-effect story should make sense with all knowledge available to the researcher . HoweverEstablishing the identity of an exposure is crucial in determining a causal relationship between an exposure and outcome. Different methods are used to identify HPV in different studies, each with its strengths and limitations. Polymerase chain reaction (PCR) is a widely used method for detecting the HPV genome, wherein specific primers are used to amplify and detect a DNA fragment of interest. In all, 51 studies used PCR as a detection method; a summary and the primer sets are presented in Alternative methods of identifying HPV in PCa have been explored because of the limitations of PCR and Southern blotting. In situ PCR has been used to detect high-risk HPVs in the nuclei of PCa cells and is less susceptible to contamination compared to standard PCR . HybridiSerology is another method used to identify HPV infection, by detecting antibodies to HPV capsid proteins in blood samples. One advantage of serology is that it can detect any prior exposure to HPV since antibodies can persist for a lifetime, even after the clearance of HPV DNA . HoweverTo supplement the information obtained from HPV detection, immunohistochemistry (IHC) can be used to detect specific proteins in the samples, such as the E6/E7 oncoproteins and tumour markers. This additional method can provide a wider picture of the oncogenic processes and of how HPV may interact with other cellular components. For example, oncogenic HPV E7 proteins were identified through IHC in 112 (75%) of 150 PCa cases . MoreoveUnderstanding the possible reasons for discrepancies in results between studies is crucial for accurately assessing the role of HPV in PCa development. This review highlights the importance of considering differences in laboratory techniques when interpreting the results of HPV detection in PCa studies.Strength of association typically refers to the degree to which a larger association indicates a more likely causal relationship. In this review, the strength of association is evaluated based on whether a statistically significant result was found. However, not all studies performed statistical tests, so this information is not available for all of them.p = 0.001).All accessible published studies based on serology were included, indicating no selection bias in this review. In this systematic review 13 studies used serology as a detection method . Only thSero-epidemiological studies do not provide a clear case for HPV as a cause of PCa. In contrast, HPV-related cervical cancer shows a significant increase in the prevalence of HPV serum antibodies among patients compared to healthy individuals .p values < 0.05 [p values, with some just stating “no significant findings” or “p > 0.05” , is often linked to sexual behaviour and to an increased risk of anogenital tumours. Interestingly, sexual behaviours including early age at first intercourse and multiple sexual partners may also increase the risk of PCa ,103. A sp-value > 0.05 [Interestingly, Saudi Arabia’s population has low incidence rates of PCa, which may be associated with genetic and environmental factors, including circumcision, religionreligious practices, and dietary habits . This mae > 0.05 ,31,34,58e > 0.05 . The deme > 0.05  was selfOverall, while a possible link between HPV and PCa through sexual activity is suggested, the evidence is not conclusive, and there may be other factors at play.Temporality is an essential causal criterion and states that exposure must precede the onset of disease for a relationship to be causal. Many of the studies in this review are case-control-based, and in these instances, the temporality criterion is not met. Because of the design of a case-control study, it is not possible to look back in time before the cancer was diagnosed and therefore not possible to prove that HPV infection preceded cancer onset. Only six studies in this review meet the temporality criteria and they report conflicting evidence for a link between HPV and PCa; they are a mix of serology and PCR-based studies.p < 0.05) [The first study to meet this criterion is a nested case-control study using a Finnish serum bank. The study reported the relative risk of PCa when seropositive for HPV18 as 2.59 (CI 95% = 1.17–5.75) and found the association between HPV18 and PCa to be highly significant ( < 0.05) . The ris < 0.05) . In fact < 0.05) . Consist < 0.05) .For these four studies, the conflicting results cannot always be explained. The Finnish and Nordic studies are very similar in terms of methodology and use the same threshold levels for the ELISA assays. The Nordic study includes three different cohorts from three different countries, Norway, Finland, and Sweden, and tests for associations between and within countries. This indicates that geographical region is also not a cause of differing results, as both used Finnish patients. The main difference between the two studies is the sample number, and the Nordic study likely had a higher statistical power that was able to exclude results because of chance. The inverse relationship between HPV18 seropositivity and PCa found in the Nordic study needs further analysis. The negative findings of the US studies could be down to the difference in timings between serum collection and diagnosis. In the 2010 study, blood was collected closer to diagnosis at the second visit. The 2007 study took blood between 1993 and 1995, and the follow-up period was a diagnosis in 2000. In contrast, the two Scandinavian studies collected serum several years before diagnosis and so an early HPV oncogenic mechanism may or may not have been detected.p < 0.001). In contrast, a Swedish study conducted by Bergh et al. [Two studies that also meet the temporality criterion used archival formalin-fixed paraffin-embedded (FFPE) tissue samples rather than serum. The first study was conducted in an Australian population of men with benign prostate biopsies who, 1 to 10 years later, developed PCa . In all,h et al.  used proh et al. .The positive findings from studies that meet the temporality criteria are consistent with a ‘hit and run’ hypothesis for the role of HPV in PCa. This suggests that HPV acts early in the oncogenic process, supported by its presence in benign prostate tissue years before cancer diagnosis  and is rIn conclusion, temporality is a key Bradford Hill criterion; however, it is hard to test for. As results between these studies are conflicting, further studies need to be conducted that meet this criterion to prove that exposure precedes disease.To become cancerous, cells need to acquire specific characteristics, known as the “Hallmarks of Cancer” . These iThe HPV E6 and E7 oncoproteins play a crucial role in cancer development by inactivating host tumour-suppressor proteins, p53 and pRb, respectively, and subsequently inducing genome instability and leading to cell proliferation, immortalisation, and malignant transformation . AnMany of the studies discussed in this review used benign prostatic hyperplasia (BPH) tissue as a control, but BPH is also associated with inflammation. This presents a limitation because using BPH patients as controls may make it difficult to draw meaningful conclusions if both conditions have a shared cause . If inflp = 0.102) [p > 0.05). Therefore, based on this study alone, it cannot be concluded that HPV infection confers oncogenic potential associated with differing miRNA expression in PCa. The expression of miRNA-150-5p has been implicated in promoting cancer by encouraging cell migration and invasion; this miRNA is upregulated in recurrent ovarian cancer [In addition, to expand the scope of the investigation, microRNAs (miRNAs) and cell regulator proteins, such as survivin, Bcl-2, and c-Myc, can be tested. miRNAs are a family of small, endogenous non-coding RNAs that regulate a wide variety of biological processes and have been found to be dysregulated in a range of cancers . They ca= 0.102) . The stun cancer . Anothern cancer ,118,119.n cancer . Thus, tp-value < 0.0001), which supports the role of HPV in the degradation of this crucial tumour suppressor in these tissues [Specific cell regulator proteins were also found to be differentially expressed in the HPV-positive PCa group compared to the HPV-negative PCa group . A stati tissues . Survivi tissues . Overexp tissues , highlig tissues . Cancer According to Anwar et al. (1992), HPV18 infection was observed in 12 out of 15 HPV-positive PCa cases with bone metastasis, which accounts for 80% of the cases . In addip < 0.005) [p values of 0.014, 0.003, and 0.0008, respectively [Several studies included in this review suggested that HPV may be linked to later stages of the disease, disputing the ‘hit and run hypothesis’ by associating virus presence with clinical data such as the Gleason score (GS). The GS is a system used to grade PCa, with higher grades indicating more abnormal cells and a greater likelihood of cancer growth and spread. Positive associations between HPV and high GS suggest that the virus may play a later role in carcinogenesis by increasing inflammation in prostate tissue, which may lead to tumour development and metastasis. However, the results are conflicting. The first study to support this hypothesis reported a significant increase in HPV infection levels with increasing GS (< 0.005) . This stectively ,35,73. Hectively ,43,48, aectively . The stuRecent investigations have focused on the emergence of apolipoprotein B mRNA editing enzyme, catalytic-polypeptide-like (APOBEC) enzymes as a protective mechanism against viral infections ,127,128.Despite these findings, the role of HPV in prostate carcinogenesis remains unclear, as there are conflicting reports among studies. The ‘hit and run’ hypothesis opposes the later-acting mechanism, which is observed by correlation with a higher GS and metastasis. The use of immunohistochemistry (IHC) proposes interesting mechanisms for how HPV may interact with cellular components, but more research is needed to confirm these findings . MoreoveVarious studies examining this topic have yielded inconsistent results, which may be due in part to differences in methodology. While there have been previous discussions about different techniques for detecting HPV, this section will specifically examine discrepancies in the collection and storage of tissue samples .The initial investigation on HPV DNA in prostate tissue relied primarily on TURP for sample collection, with suprapubic prostatectomy (SPP) used for two samples . The stuAnother study used a microdissection approach that excluded urethral mucosa and found no HPV DNA in PCa cases , while aIt is conceivable that high occurrence in the initial detection of HPV and PCa was due to contamination from urethral tissue. Nonetheless, it is more likely that the small sample size was the actual cause, as evidenced by other TURP-based studies that did not produce positive results. Most studies in this review shifted away from the TURP method, particularly for PCa cases, which implies that contamination is not a significant confounding variable to consider in this review.p = 1.00) [Another difference in study methodology is the way in which tissue is stored. Most studies used FFPE blocks, fresh, or frozen tissue. In the earlier studies, fresh or frozen tissue was primarily used, but most studies in this review used FFPE blocks as the storage method . The use = 1.00) .In conclusion, the studies reviewed here are inconsistent with each other in terms of HPV detection rates, HPV detection methodology, and tissue collection and storage methodology. Although some studies found these differences to have non-significant effects on the results, it is not clear what the true reasons are for discrepancies among studies. Perhaps a review that focuses on studies using solely the same techniques would mean the consistency criterion could be met, but this would require many further studies.The original Bradford Hill criteria state that a dose–response relationship supports a causal association. However, for HPV and PCa, demonstrating different exposure dosages is difficult as most studies only report whether the virus is present. A few studies in this review did consider the HPV copy number per cell ,33,50,56Bradford Hill described this criterion as evidence drawn from experimental manipulation . In thisTo conclude, in this review, experimental manipulation of HPV as an exposure was not tested for and cannot be commented on for causal inference in PCa. Notably, expression of HPV16 E6 and E7 was shown to immortalise prostate epithelial cells , which pAlthough the evidence available to date provides interesting insights into the potential role of high-risk HPV in PCa, it is not yet conclusive enough to meet key Bradford Hill criteria such as strength of association, consistency, and coherence. Additionally, the complex nature of HPV transmission and oncogenic mechanisms, coupled with the difficulty of testing criteria such as temporality, biological gradient, and experiment in this context, make it challenging to draw definitive conclusions.While there are some similarities between PCa and cervical cancer, they also have significant differences that limit the extent to which analogies can be drawn between the two. Although the identification and transmission of HPV provide valuable information regarding the virus’s plausibility as a causative agent in PCa, the conflicting nature of the literature prevents us from drawing a definitive conclusion about the causal role of HPV in this disease.However, it is possible that the prostate serves as a reservoir for HPV transmission. If high-risk HPV does play a role in prostate carcinogenesis, it is likely through the actions of the viral oncoproteins, the promotion of inflammation, which could potentially contribute to the oncogenic process at various stages, and/or damage to host DNA. Further studies are required to gain a better understanding of this topic, potentially focusing on homogeneous study designs and result analyses. Therefore, while the evidence to date suggests a possible causal role for HPV in PCa, it is not yet strong enough to use the term “highly likely” without further research to support this claim.


This study aims to investigate the association of immune markers with high risk human papillomavirus 16 (HPV 16) infection status and to evaluate the prognostic value of programmed death ligand-1 (PD-L1) in patients with oropharyngeal squamous cell carcinoma (OPSCC).This retrospective study collected 50 cases of HPV positive and HPV negative OPSCC from January 2011 to December 2015. The correlation of CD8 + tumor infiltrating lymphocytes (TILs), programmed death-1 (PD-1), and PD-L1 expression with HPV 16 infection status was analyzed via immunofluorescent staining and quantitative real-time PCR.P = 0.003; 5-year disease specific survival [DSS], 73% vs. 44%, P = 0.001). The expressions of immunity related makers were significantly higher in the HPV + group than the HPV − group . Positive CD8 + TIL and PD-L1 were independent factors for better prognosis of OPSCC . Kaplan–Meier survival analysis indicated that patients with TILs of high HPV + /CD8 + expression were more likely to have better prognosis than those with TILs of low HPV + /CD8 + expression , TILs of high expression of HPV − /CD8 + , and TILs of low expression of HPV − /CD8 + . Furthermore, HPV + /PD-L1 + OPSCC patients had significant better prognosis compared to patients with HPV + /PD-L1 − , HPV − /PD-L1 +  and HPV − /PD-L1 − .There was no significant difference in the baseline data between the two groups. Patients with HPV + OPSCC had better prognosis compared to HPV − patients (5-year overall survival [OS], 66% vs. 40%, HPV + OPSCC had a significantly better prognosis, and PD-L1 expression was elevated in HPV + OPSCC. PD-L1 positivity might be related to the better prognosis of HPV + OPSCC.This study provides a theoretical basis and baseline data for the application of immune checkpoint inhibitors in head and neck tumors. Besides, the response to immunotherapy are different between patients with HPV + and HPV − OPSCC [Oropharyngeal carcinoma is one of the most common malignant tumors in head and neck, and more than 90% of them are squamous cell carcinoma . Human p − OPSCC , 9.Tumor infiltrating lymphocytes (TILs) contribute to immune response in tumor immune microenvironment, and the infiltration degree of CD8 + TILs is correlated with the clinical prognosis in OPSCC . In addiThe objective of this study was to investigate the expression of CD8 + TIL, PD-1, and PD-L1 in OPSCC, and analyze their correlations with clinical prognosis of different HPV status.This study retrospectively involved 100 cases of patients with OPSCC, which included 50 cases of HPV + OPSCC and 50 cases of HPV- OPSCC. The patients were recruited from Peking Union Medical College Hospital from 2014 to 2018, and they had been followed up for more than 5 years until death. The correlation of PD-L1 expression with HPV 16 infection status was analyzed using propensity score matching (PSM). The clinical parameter of patients was collected, including gender, age, living habit, and clinical stage. The pathological characteristics were also collected. All patients were graded according to the TNM grading criteria specified by the American Joint Committee on Cancer (AJCC) in 2010 . All patInclusion criteria: (1) all patients met the diagnostic criteria in the guidelines of The 2017 WHO Classification of Head and Neck Tumours ; (2) patExclusion criteria: Patients with the following diseases or conditions were excluded: (1) severe diseases such as immune system disease; (2) inability to cooperate; (3) other kinds of malignant disease; (4) infection in oropharyngeal or other inflammatory diseases that may affect the results of the study.The DNA in the tumor tissue samples were extracted using QIAamp DNA FFPE Tissue Kit . Rotor Gene 6000 and SYBR Premix Ex Taq Kit  were applied for quantitative real-time PCR (qRT-PCR) for detection of HPV 16 DNA. Primers and FAM-MGB labeled probes (TaqMan) were designed and optimized to amplify the HPV16E6 region. The HPV 16-positive cervical cancer cell lines CaSki and SiHa (ATCC-LGC-HTB-35) were used as positive controls and calibrators for the assay. The detection threshold for HPV-positive status was set, and samples were deemed positive if the threshold was met in duplicate runs.Paraffin sections about 5 μm thickness were prepared by conventional methods, and then, immunofluorescence staining was performed after dewaxing. Antigen repair was performed in EDTA buffer in microwave. The slices were added primary antibody CD8 (dilution 1:1000), PD-1 (dilution 1:1000), PD-L1 (dilution 1:100), and incubated overnight at 4 °C. After sections were washed, fluorescent-labeled secondary antibody (dilution 1:300) was incubated, and then, FITC reagent (CD8 sections) and CY3 (PD-1 and PD-L1 sections) were dropped, and autofluorescence quencher was added after incubation in the dark. Finally, DAPI was re-stained. All the sections were sealed by fluorescence decay–resistant medium and recorded in fluorescent microscope, and each was viewed by two experienced pathologists who was blinded to the patients’ clinicopathologic information. A pathologist (M. H.) assessed the expression of PD-L1. PD-L1 positivity was defined as membrane staining in 20% of tumor cells.PD-L1 positivity was defined as membrane-expressing PD-L1 tumor cells accounting for more than 5% of tumor cells . PD-1 poP < 0.05 was considered statistically different.All of the analyses were conducted using SPSS 22.0, and all of them were bilateral tests. In this study, 50 patients with HPV + OPSCC were selected and matched with 50 cases of HPV − OPSCC by PSM, including age, gender, smoking, tumor, primary site, treatment method, and clinical stage. All binary variables were analyzed by chi-square test (or Fisher’s test). Ordered categorical variables were analyzed by Mann–Whitney rank sum test. Kaplan–Meier method was used for univariate analysis of overall survival (OS) and disease-specific survival (DSS), and Cox proportional risk model was used for multivariate analysis. P > 0.05; Table In HPV positive group, there were 47 (94.00%) male and 3 (6.00%) female patients, of which 12 (24.00%) patients were younger than 55 years old. Thirty-one patients (62.00%) were smokers, and 28 (56.00%) patients were drinker. Forty-three patients (86.00%) with 0–1 point of Charlson comorbidity score and 7 (14.00%) patients were with higher. There were 16 (32.00%), 21 (42.00%), and 13 (26.00%) patients with high, moderate, and low differentiation of cancer in HPV positive group. Among these, 32 (64.00%) patients were with tonsil cancer and 18 (36.00%) patients were with cancer in root of tongue. There were 20 (40.00%) patients with stage I–II cancer and 30 (60.00%) patients with III–IV cancer. Of these, 28 (56.00%) patients received surgery and 22 (44.00%) did not. In HPV negative group, there were 45 (90.00%) male and 5 (10.00%) female patients, of which 13 (26.00%) patients were younger than 55 years old. Thirty-three patients (66.00%) were smokers, and 24 (48.00%) patients were drinker. Forty patients (80.00%) with 0–1 point of Charlson comorbidity score and 10 (20.00%) patients were with higher. There were 27 (54.00%), 13 (26.00%), and 10 (20.00%) patients with high, moderate, and low differentiation of cancer in HPV positive group. Among these, 33 (66.00%) patients were with tonsil cancer and 17 (34.00%) patients were with cancer in root of tongue. There were 14 (28.00%) patients with stage I–II cancer and 36 (72.00%) patients with III–IV cancer. Of these, 35 (70.00%) patients received surgery and 15 (30.00%) did not. There was no significant difference in these factors between the two groups , and PD-1 was often seen in tonsillar squamous cell carcinomas (P = 0.052). CD8 + TILs were highly infiltrating in high differentiation (P = 0.029) and tonsillar squamous cell carcinomas (P = 0.004), but unrelated to other clinical pathological features including gender, smoking, alcohol, tumor stage, and complications , and the 5-year DSS rate was 73% and 44% in these two groups, respectively (P = 0.001). The Cox multivariate regression analysis showed that HPV positivity , high infiltration of CD8 + TILs , PD-L1 positivity , smoking , complications , the low degree of differentiation , and treatment method  were independent prognostic factors of DSS. The multivariate analysis of OS was similar to the DSS. Positive HPV, high infiltration of CD8 + TILs, PD-L1, complications, and degree of differentiation were all independent prognostic factors, but smoking and treatment regimen were no longer independent factors. Besides, the expression of PD-1 had no effect on prognosis , high expression of HPV − /CD8 + TILs , and low expression of HPV − /CD8 + TILs  , HPV − /PD-L1 +  and HPV − /PD-L1 −   and HPV − /PD-1 −   Fig. .Fig. 3KaHPV infection is one of the important pathogenic factors of oropharyngeal cancer, causing about 31% of oropharyngeal cancer worldwide . HPV + oHowever, the mechanism about that patient with HPV + OPSCC has a better prognosis remains unclear. Some scholars attributed the better prognosis to increased sensitivity to chemoradiotherapy . PatientP = 0.012) [Currently, there are a number of studies on the relationship between HPV-infection-related tumors and their immunophenotypes. One study showed that patients with HPV + cervical cancer had high serum levels of PD-L1 which could be diagnostic indicator for identifying HPV + cervical cancer . Another= 0.012) , and a m= 0.012) . Balermp= 0.012) . After d= 0.012) , 38. Ano= 0.012) . Solomon= 0.012) . Later, = 0.012) . Moreove= 0.012) . In this= 0.012) . This stThese contradictory results may be partly due to the small sample size and partly due to the lack of uniformity of the analytical methods, including variability in the immunohistological tests between observers, the lack of standardized antibodies to determine PD-L1 expression, and the different thresholds for defining positive expression. At the same time, the expression of PD-L1 in tumor cells may change dynamically in different stages of the disease, and the detection results may be affected by the time of biopsy. Moreover, tumors are heterogeneous, and the expression of PD-L1 may be different in different locations of the same lesion and among different lesions. The results of this study suggested that positive PD-L1 was an independent prognostic factor for improving OS and DSS. Meanwhile, patients with HPV + /PD-L1 + were more likely to have a significantly better prognosis than those with HPV + /PD-L1 − , HPV − /PD-L1 + , and HPV − /PD-L1 − .These contradictions and inconsistencies may be related to the following reasons: (1). Virus-associated tumors are caused by virus-associated oncoproteins, and usually, the tumor mutation load is low or moderate. The emergence of corresponding neoantigens to virus infection may cause stronger immune response. Many virus-associated tumors show strong immune response and overexpression of PD-L1 ; (2). AfOur study had several limitations. First, immunofluorescence staining is a semi-quantitative detection, which cannot perfectly display the expression level of each marker. Second, even though a relatively large cohort was analyzed, compared to previous studies, because of the favorable prognosis of OPSCC, only a few events occurred in the entire cohort. This might limit the power of the study to determine the prognostic role of PD-L1 expression. Third, the inherent nature of a retrospective study makes it impossible to explore any dynamic changes in PD-L1 expression.In conclusion, the results of this study confirmed that the prognosis of patients with HPV + OPSCC is significantly better compared with those with HPV − . CD8 + TILs, PD-L1, and PD-1 were more highly expressed in patients with HPV + OPSCC. The infiltration degrees of CD8 + TILs and PD-L1 expression were independent factors for the prognosis. The favorable prognosis of patients with HPV + OPSCC might be related to different immune microenvironment. This study also provided a theoretical basis and baseline data for the application of immune checkpoint inhibitors in head and neck tumors.


Moreover, the expression of ADCY7 increased significantly in high-risk human papillomavirus (HR-HPV) infection but not in HPV-A5/6 species. The area under the receiver operating characteristic curve (AUC) of ADCY7 was 0.897, and an optimal cut-off was 0.435. Furthermore, ADCY7 had the highest OR ) for detecting CIN 2+, followed by HPV genotyping, TCT, and age , (0.370–8.137), and (0.171–4.694), respectively). Moreover, this study indicated that higher ADCY7 levels could be a suitable predictor for poor prognosis in cervical cancer due to immune cell infiltration. A new auxiliary predictor of CIN2+ in cervical cytology specimens is ADCY7 ≥ 0.435. Furthermore, it may be a promising prognosis predictor and potential immunotherapy target for the combined treatment of cervical cancer and possibly further block HR-HPV persistent infection.The effect of cervical cancer immunotherapy is limited. Combination therapy will be a new direction for cervical cancer. Thus, it is essential to discover a novel and available predictive biomarker to stratify patients who may benefit from immunotherapy for cervical cancer. In this study, 563 participants were enrolled. Adenylate cyclase 7 (ADCY7) mRNA was detected by real-time quantitative PCR (qPCR) with cervical cytology specimens. The relationship between ADCY7 and cervical intraepithelial neoplasia in grade 2 and higher (CIN2+) was analyzed, and the optimal cut-off values of the relative expression of ADCY7 mRNA to predict CIN2+ were calculated. In addition, the clinical significance of ADCY7 in cervical cancer was determined by the Kaplan–Meier Cox regression based on the TCGA database. The mean ADCY7 mRNA expression increased significantly with cervical lesion development, especially compared with CIN2+ ( Cervical cancer is the fourth most common type of cancer in women . AccordiPersistent infection with high-risk human papillomavirus (HR-HPV) is a high-risk factor for cervical precancerous lesions and cervical cancer ,6. HPV vImmunotherapy is an emerging treatment option that might be a novel option to improve the prognosis of these patients. It has recently achieved great preclinical and clinical success through immune checkpoint inhibitors (ICIs) ,13,14. IRecently, some research  pointed This present study aimed to evaluate the predictive value of ADCY7 expression in cervical cancer. We investigated whether the expression of ADCY7 could be a valuable biomarker to predict CIN2+ with cervical cytological specimens collected non-invasively. Additionally, this study demonstrated the prognosis of ADCY7. The results provide novel insights into the active role of ADCY7 in HPV infection and cervical cancer, thereby highlighting a potential mechanistic basis whereby ADCY7 influences immune cell interactions with tumors.We included 563 participants from the Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics and Gynecology and Pediatrics, Fujian Medical University from December 2018 to May 2022. The populations had to reach the following criteria: (1) age above 20 years; (2) sexually active; (3) no history of cervical cancer, CIN, or HIV infection; and 4) did not undergo a hysterectomy or cervical surgery. The Hospital Ethics Committee approved the research of Fujian Provincial Maternity and Children’s Health Hospital, an affiliated hospital of Fujian Medical University (2022KYLLR03050), and all individuals participating in this study signed written informed consents.® PreservCyt® solution  for cytology examination. The storage conditions of the samples are detailed in our previous study [Exfoliated cervical cells were collected from cervical canals using a cytobrush. The samples were collected in vials containing preservation solutions for HPV DNA testing or in bottles of ThinPrepus study ,29.Cytological samples were blindly examined, independent of the results from the other assays, by two experienced cytopathologists. The results were reported according to the Bethesda 2001 system . If the The colposcopy and needle biopsy in women who were HPV-16/18 positive, with or without abnormal cytology ), were performed according to the guidelines. Women with a punch biopsy diagnosis of more than high-grade squamous intraepithelial lesions (HSIL) underwent conization by the cold knife or loop electrosurgical excision procedure cone biopsy (LEEP). Specimens were fixed in 10% formalin and routinely processed for paraffin embedding. Then, according to standard methods, 4 µm thick tissue sections were cut and stained with hematoxylin and eosin.RT-qPCR was carried out according to the protocol. ADCY7 PCR was used with primer pairs 5′-GAT GTA CGT CGA GTG TCT CCT-3′ and 5′-CTT TGT CCA TGC GTC GAA CA-3′. GAPDH was used as an experimental control for sample quality and adequacy during the PCR process. GAPDH PCR was applied with primer pairs 5′-GGT GTG AAC CAT GAG AAG TAT GA-3′ and 5′-GAG TCC TTC CAC GAT ACC AAA G-3′. Relative levels of ADCY7 mRNA were quantified by qPCR and calculated by the 2−ΔΔCT method. We analyzed the OS, DSS, and PFI according to different characteristics to individualize the prognosis prediction in cervical cancer patients. All clinicopathological data were acquired from TCGA-CESC datasets, including 306 cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) sample tumor tissues and 13 normal tissues .https://timer.cistrome.org, accessed on 20 January 2023) is a database used for the analysis of tumor-infiltrating immune cells and various gene expression levels in different types of cancer [The Tumor Immune Estimation Resource (TIMER2.0)  from healthy specimens. Furthermore, we compared the impact of ADCY7 levels on the incidence of CIN2+ using a multivariate Cox analysis. Different risks were estimated using odds ratios (ORs) and 95% confidence intervals (CIs). In all statistical analyses, p < 0.001). The analysis of the 563 cervical tissue samples indicated that 143 (25.400%) were standard, 116 (20.604%) were classified as CIN1, 202 (35.879%) were categorized as CIN2/3, and 102 (18.117%) were diagnosed as cancer. As shown in p < 0.001, ANOVA test)  A. MoreovADCY7 expression is a valuable predictor for the detection of ≥CIN2. To predict CIN2+ lesions, ROC analyses of ADCY7 mRNA levels were performed. ADCY7 mRNA levels were evaluated individually . We obseHigher ADCY7 mRNA levels predict a poor prognosis in cervical cancer. p = 0.001, p = 0.005, p = 0.044, p < 0.05)  D–T. Accop-values were below 0.001. These results demonstrate that ADCY7 mRNA levels play an essential role in the immune infiltration in cervical cancer. In addition, this research revealed that ADCY7 mRNA levels were significantly connected with immune inhibitors, as shown in Immune infiltration is a crucial factor related to tumor progression. In cervical cancer, ADCY7 mRNA levels are associated with immune cell infiltration levels using TIMER platforms. This study demonstrated that cervical specimens with an over-expression of ADCY7 mRNA were significantly associated with CIN2+ lesions. Thus, the present study may provide information on a novel and valuable biomarker to predict the development of CIN2+ with non-invasive methods. Interestingly, this study demonstrated that a high expression of ADCY7 suggested HR-HPV infection. In this study, ADCY7 mRNA levels increased significantly with the development of the cervical lesion. Moreover, ADCY7 could be a valuable biomarker to predict CIN2+. The AUC for ADCY7 mRNA levels was 0.898. Moreover, an optimal cut-off for ADCY7 mRNA levels was 0.435. Additionally it was demonstrated that higher ADCY7 mRNA levels (≥0.435) had the highest OR ). Thus, this suggests that ADCY7 mRNA levels in cervical specimens can be a valuable biomarker to predict CIN2+ with non-invasive procedures. Our results may offer a helpful basis for the accurate cytological diagnosis of cervical pre-malignancy and cancer in future transitional research.Furthermore, we also revealed that the expression of ADCY7 mRNA levels increased significantly in HR-HPV positive, HPV A7 species, HPV A9 species, HPV-16/18 positive, HPV-16 positive, and HPV-18 positive samples, but not in the HPV A5/6 species. We divided the high-risk HPV types into three groups: the HPVA5/6 species contains HPV 51, 56, and 66; the HPVA7 species contains probes for HPV 18, 39, 45, 59, and 68; and the HPVA9 species contains probes for HPV 16, 31, 33, 35, 52, and 58. It has been reported that persistent infection with high-risk human papillomavirus (HPV) is a high-risk factor for cervical precancerous lesions and cervical cancer . This stNearly 95% of cervical cancer patients have a high risk for HPV infection . HoweverADCY7 encodes a membrane protein. It is involved in extracellular signaling into intracellular reactions . It is rp = 0.001), DSS , and PFI. Similarly, Giorgio Bogani. et al. [Our study revealed that higher ADCY7 mRNA levels were significantly related to poor OS  and unresponsiveness to immune checkpoint blockade (ICB) therapy . In addiIf treatments for persistent HPV infection are developed, persistent HPV infection will become less frequent, significantly reducing the occurrence of high-grade intraepithelial neoplasia and cervical cancer in the cervix. Therefore, immunotherapy against HPV may have important clinical implications for preventing persistent HPV infection. This study indicates that targeting ADCY7 can serve as a new direction for immunotherapy for cervical cancer and further block HR-HPV persistent infections. This study fills the gaps in the primary and clinical research of the ADCY7 protein in the field of cervical cancer; provides theoretical support for the prevention, diagnosis, and treatment of cervical cancer; and seeks new targets for the treatment of cervical cancer. It provides valuable clinical data for ADCY7 in human biological function research, with good innovation and progress. However, there are some limitations, as follows: Firstly, we did not verify the relationship between ADCY7 expression levels and the prognosis of cervical cancer based on our clinical data in this study, which is worth further study. Secondly, the specimen size was not big enough. Therefore, increasing the sample size may be necessary in the future. This study revealed that ADCY7 expression levels significantly increased in cervical cytology and is strongly related to CIN2+ lesions. ADCY7 expression levels can be a suitable biomarker to predict CIN2+. Moreover, higher ADCY7 expression levels predict a poor prognosis in cervical cancer due to the promotion of the expression of the immune checkpoint. In addition, ADCY7 mRNA levels may be an auxiliary indicator for HR-HPV infection. These results may provide a valuable research direction for the combined treatment of cervical cancer with immunotherapy and possibly further block HR-HPV persistent infection.