Topical cidofovir and imiquimod can effectively treat approximately 55% of patients with vulval intraepithelial neoplasia (VIN), thus avoiding the need for surgery. Human papillomavirus (HPV)  This work aimed to determine if the applicability and predictive power of the  HPV E2 methylation and S5 classifier score were measured in fresh tissue samples collected pre-treatment from 132 patients with biopsy-proven VIN grade 3 who participated in a multicentre clinical trial and were randomised to treatment with cidofovir or imiquimod.and HPV 16 or HPV 16  Combining HPV16  Combined HPV  VIN is commonly caused by human papillomavirus (HPV), which is present in around 76% of cases . VIN canSurgical excision has historically been the gold standard of care for patients with VIN but is associated with significant morbidity , 3. WithPrevious research demonstrated that HPV 16 t of VIN . PatientThe potential of HPV genesis) , 10, andEPB41L3. This assay has several potential advantages: it is not reliant on HPV positivity to generate a result (due to inclusion of the EPB41L3 host gene), it detects methylation in three additional HPV genotypes and targets a region  within the past 3 months. Baseline translational research samples were available in addition to the clinical outcome data .2.2A type-specific PCR targeting the HPV 16 6 region  was used2.3HPV DNA methylation testing was restricted to HPV16 positive . PCR products were pyrosequenced using a PyroMark Q96 ID instrument (Qiagen). The pyrosequencing software (Pyromark CpG Qiagen) returns a methylation value (%) and a quality value for each CpG in the sequence; “failed samples” were treated as “missing”, “check samples” were compared with the amplitude of the control results and included if satisfactory.All 132 baseline samples were tested for the components of the S5 classifier as well as HPV16 eviously , 15. In 2.5Statistical analyses were conducted using STATA/SE v16 according to a pre-specified analysis plan. The dataset used in this analysis included all patients from the RT3VIN trial who had both a pre-treatment tissue sample and a post-treatment histological assessment response available.EPB41L3, HPV 16 HPV 16 escribed . In thisous work , the meaS5 The HPV 16 E2 methylation results were compared between laboratories using a scatter and Bland-Altman plot but all other analyses used the HPV 16 E2 methylation results from the QMUL Lab as it was thought to better represent a routine NHS clinical laboratory.HPV positivity was defined as any PapilloCheck HPV genotype or HPV16 For each treatment arm, Wilcoxon rank sum tests were used to compare the distribution of each methylation marker, and chi-square tests to compare the proportion with HPV 16/any HPV positivity, between the group who responded to treatment and the group who did not respond to treatment. Multiple comparisons were accounted using the Benjamini-Hochberg procedure [In order to examine the validity of the pragmatic imputation of missing methylation values as zero, we conducted post-hoc sensitivity analyses to examine the consistency of findings across subgroups of patients: those with missing methylation and no relevant HPV genotype detected (thought to be most likely to be true zeros) and those with missing methylation but relevant genotype detected . 3.3.1EPB41L3, HPV 16 L1 and L2.Table 3.2HPV 16 3.3Using the data from the QMUL laboratory, Table Although HPV 16 lsewhere  i.e. sig3.4A ROC curve of HPV 16 3.5E2 methylation. We found that combining HPV 16 and HPV 16 or HPV 16 E2An HPV 16 ly shown  to defin3.6As described in the methods, in all the above analyses we imputed missing methylation values as 0 and included them in the HPV 16 4.and HPV 16 Cidofovir and imiquimod are topical treatments for vulval intraepithelial neoplasia; when used individually both treatments give complete response in approximately 50% of patients. HPV E2 methylation has been identified as a potential biomarker predictive of response to treatment, which could enable a more personalised approach with increased likelihood of efficacy . A curreThe assessment of HPV 16 on rates . By applIt is noted that the overall HPV prevalence in our cohort (86.4%) is somewhat higher than many studies quoted in the literature, including a systematic review of 2,764 cases of VIN (76.3%) published in 2017 . The higand HPV 16 and HPV 16 To make this biomarker clinically useful, an approach to those patients generating no methylation (missing methylation) result is required. In this analysis we treated missing methylation values as “zero” i.e. low. This may not be biologically accurate since they may represent cases with high levels of viral integration, a feature typically associated with higher levels of methylation. However, we found the same pattern of better response with imiquimod in patients who were HPV 16 positive with missing methylation and in those patients who were HPV 16 negative with missing methylation. Consequently, the data support giving imiquimod to all patients with low and missing methylation, and cidofovir to those with both HPV 16 EPB41L3 were low (5–10%) in VIN patients as opposed to cervical precancer (10–20%) or cancer patients (above 20%) [EPB41L3 which does not seem to differentiate responses in this population. This may reflect differences in the natural history and pathobiology of VIN and CIN.S5 was not a significant marker of response in either imiquimod or cidofovir patients. The S5 classifier was initially designed as a triage tool for detection of CIN and cervical cancer and although it uses HPV and host methylation to do this, it was not designed as a biomarker to predict response to treatment. We noticed that the methylation levels of ove 20%) . It is pand HPV 16 HPV L1 methylation is a component of the S5 classifier and this study demonstrated that low HPV 16 Hypotheses to explain why high methylation is associated with response to cidofovir, and low methylation is associated with response to imiquimod, have previously been described . These iContrary to the case with cidofovir,  targets , 25, 26. targets , so it iIn conclusion, this study refines the use of assessment of HPV methylation as a potential predictive marker in treatment in VIN for all patients from whom a sample is taken. It demonstrates that reproducibility of this method of assessment of methylation, that addition of human genes adds little to the predictive power of the marker panel, and that high levels of HPV gene methylation are consistently associated with good response to treatment with cidofovir and that low or missing levels are associated with good response to treatment with imiquimod. These findings justify validation in a prospective trial.


Cocirculation of multiple human papillomavirus (HPV) infections with low, probably high, and high‐risk genotypes are to be associated with various grades of infections and cancer progression. The oncogenic high‐risk HPVs are distributed and cocirculated throughout the world. This study was investigated to identify HPV genotypes related to genital disorders in unvaccinated women. The subjects were referred from clinics to a molecular lab for HPV testing in Iran as a low‐coverage vaccinated country. HPVs DNAs of cervical scrapping and genital tissue specimens of 1,133 un‐vaccinated women were genotyped using an in vitro diagnostic line probe (reverse hybridization) assay. In addition, phylogenetic trees were constructed on 100 MY09/MY11 polymerase chain reaction (PCR) amplicons of common genotypes of HPV L1 gene by Sanger sequencing. The mean age of the population study was 32.7 ± 8.0 and the mean age of HPV‐positive cases was 31.6 ± 7.8. HPV DNA was detected in 57.8% (655/1133) of women subjects and 42.2% (478/1133) of cases were undetected. Among 655 HPV‐positive cases, 639 subjects (56.4%) were related to defined genotypes and 16 subjects (1.4%) were untypeable. The highest prevalence rate of HPV genotypes was identified in the 25–34 years. The top 6 dominant HPVs in single and multiple genotypes were HPV6 (284/655 [43.4%]), HPV16 (111/655 [16.9%]), HPV31 (72/655 [11%]), HPV53 (67/655 [10.2%]), HPV11 (62/655 [9.5%]), and HPV52 (62/655 [9.5%]). Moreover, single, multiple and untypeable HPV genotypes were diagnosed as follows: 1 type (318/655 [48.5%]), 2 types (162/655 [24.8%]), 3 types (83/655 [12.7%]), 4 types (42/655 [6.5%]), more than 5 types (34/655 [5.3%]), and 1.4% un‐typeable subjects. The sequenced partial L1 gene of HPV genotypes (GenBank databases under the accession numbers: MH253467‐MH253566) confirmed and determined the cocirculated HPV genotypes' origins and addressed helpful insights into the future viral epidemiology investigations. Multiple HPV infections and cocirculation of various oncogenic HPV genotypes among the normal population (women and men) with asymptomatic forms are still challenging in unvaccinated communities. The preventive and organized surveillance programs for HPV screening are needed to be considered and compiled by health policy makers of low or unvaccinated countries. E6 and E7 proteins are the most important genome regions to induce cancer progression. HPVs classification by the International Committee of Taxonomy Viruses (ICTV) is based on pairwise nucleotide sequence identity across the L1 (major capsid protein) open reading frame. The long control genome (LCR) does not encode any genes, and therefore is able to accumulate and tolerate more mutations.Human papillomavirus (HPVs) infections are responsible for several genital, anogenital, oropharyngeal, head–neck cancers, and sexually transmitted infections. The 22.1The 1133 archival cervical scrapping and genital tissue specimens collected from unvaccinated women were referred to a private pathobiology laboratory, Tehran, Iran, from April 2012 to October 2015. The specimens were included vaginal brush 789/1133 (69.6%), vaginal brush with genital wart 248/1133 (21.8%), genital wart 83/1133 (7.3%), formalin‐fixed embedded tissue 4/1133 (0.4%), genital wart with anal wart 3/1133 (0.3%), anal wart 2/1133 (0.2%), vaginal brush with anal wart 2/1133 (0.2%), vaginal brush with anal brush 1/1133 (0.1%), and vaginal brush with genital/anal wart 1/1133 (0.1%). The mean age of the population study was 32.7 ± 8.0 and the mean age of HPV‐positive cases was 31.6 ± 7.8. The clinical data were recorded from medical documents. The cross‐sectional descriptive study was conducted according to the ethical guidelines of the Declaration of Helsinki, and approved by the deputy research of East Tehran Branch, Islamic Azad University, Tehran (No. 28330503961002). The liquid‐based cytology (LBCs) and tissue samples were stored at −20°C until experiments.2.2©), according to the instruction manual. HPV genotyping was performed by an approved CE marked and In Vitro Diagnostic (IVD) Kit including INNO‐LiPA® HPV Genotyping Extra I and II (Fujirebio Diagnostics Inc.). The kit was able to detect 18 HR types , 9 LR types , 1 probably HR type (HPV53), and 5 non HR–LR genotypes .DNA of HPV genotypes was extracted from LBCs and genital tissues using the High Pure PCR Template Preparation Kit (Roche2.3https://blast.ncbi.nlm.nih.gov/Blast.cgi) by the neighbor‐joining method with 1000 bootstrap repetitions to find any similarities and to determine the HPV genotypes origins compared to HPV reference isolates.The HPV‐positive PCR products were amplified using MY09/MY11 primers (3). The positive amplicon around 450 bp was considered HPV‐positive underwent electrophoresis on a 1.5% agarose gel. The gel was stained by ethidium bromide and was visualized under ultraviolet light. An approximately 15 µl of amplicon reactions were sent to Pooya Gostar Gene© for Sanger sequencing by a BigDye Terminator Cycle Sequencing Kit on an ABI Prism 3730/3100 DNA analyzer (Applied Biosystems Inc.). The FASTA files of sequences were trimmed and aligned using BioEdit (version 7.2) and ClustalW software. The phylogenetic analysis was constructed using the MEGA software package (version 6) on multiple alignment outputs that were also performed basic local alignment search tool (BLAST) using NCBI (3HPV genotypes were detected in 655 (57.8%) samples of 1,133 subjects and 478 42.2%) cases were HPV negative. Among 655 HPV‐positive cases, 639 subjects (56.4%) were related to defined genotypes and 16 subjects (1.4%) were un‐typeable. The un‐typeable cases had positive signals on INNO‐LiPA® and PCR MY09/MY11 amplification methods although there were not associated with specific genotypes. The highest prevalence rate of HPV genotypes was observed in the 25–34 years of age group  of HPV isolates are mentioned in Figure 4HPV persistent infection is highlighted to be one of the most common causes of genital disorders throughout the world.The HPV genotypes lineages of phylogenetic outcomes revealed that detected types belonged to different genetic lineages from African, Asian, European, and American populations. Some types are supposed to have existed in specific regions although it seems traveling, tourism, and social communications increase the exposure risk of HPVs' different genotypes. Therefore, the various HPV genotypes were cocirculated between the communalities.5To summarize, the distribution and burden of genital and anogenital malignancies are increasing through the multiple HPV genotypes in the normal population accordingly. Effective screening and diagnostic procedures besides enhanced vaccination strategy are needed to be implemented and extended in low or unvaccinated countries. The main outcome of the current survey addresses helpful insights into molecular epidemiologists and health bodies reports of HPV low‐vaccinated communities for designing and organizing the cancer care screening surveillance programs.Manijheh Vazifehdoost contributed to data analysis and prepared the draft of manuscript. Fatemeh Eskandari performed the experiments, collection, and sorting the specimens. Amir Sohrabi contributed to the conception, design, data interpretation, prepared, and revised the manuscript.The authors declare no conflicts of interest.


With the application of virtual reality (VR), tailored interventions can be created that mirror the traumatic experiences of veterans with post-traumatic stress disorder (PTSD). Visual elements can be mimicked, and auditory and other senses stimulated. In doing so, the degree of immersion can be adjusted to optimize the therapeutic process. Objectively measuring the sensory immersion is key to keep subjects within their personal window of tolerance. Based on this information the therapist can decide manipulate the sensory stimulation embedded in the treatment.The objectives of this study are to explore the different immersive design aspects of VRET that can be modified to influence the experienced presence in veterans with PTSD, and to discuss possible methods of measuring the emotional response facilitated by immersive design aspects and experienced presence.Four design aspects are discussed: system, sensory cues, narrative and challenge. We also report on a user experiment in three veterans that informed on quality and depth of immersion.Believability of the neutral virtual environment was important for maintaining the veterans’ presence within the VR experience. The immersive design aspects that were personalized and supportive in the narrative of the veteran such as music and self-selected images appeared to have a strong influence on recall and reliving of the traumatic events.Finally, in order to increase the therapeutic effect in veterans with PTSD, the highlighted design aspects should be recognized and tailored to maximize immersion in virtual reality exposure therapy.No significant relationships.


Blue sky has been considered to boost outdoor physical activity and social interaction, ameliorate work pressure and life stress, and enhance people's sense of happiness. However, the direct association between blue sky exposure and cardiovascular disease (CVD) still lacks epidemiological evidence. In this study, we aimed to quantify their relationship via a nationwide prospective cohort in China.We extracted the baseline data from the China Hypertension Survey (CHS), by enrolling 22,702 participants aged ≥ 35 years without self-reported medical history of CVD from 14 provinces of China between 2012 and 2015 and followed up from 2018 to 2019. A blue day was marked out with no rain, low cloud cover ≤ climatological mean at each station, and visibility at 2 pm ≥ 21.52 km. We calculated the number of blue days at baseline survey year to evaluate the chronic individual blue day exposure. Cox proportional hazards models were employed to calculate the multivariable-adjusted hazard ratio (HR). We implemented subgroup analyses as well to identify potential effect modifications.A total of 1,096, 993, and 597 incident cases of all-cause mortality, fatal or nonfatal CVD, and stroke occurred during a median follow-up around 5 years, respectively. A 10-day increase in annual blue day exposure was associated with a 3%  and 7% (95% CI: 5–10%) decreased risk of fatal or nonfatal CVD and stroke, respectively. Compared with those exposed to the worst tertile of blue days at baseline, subjects who exposed to the best tertile had a 32% (95% CI: 19–43%) and 43% (95% CI: 29–55%) lower likelihood of developing fatal or nonfatal CVD and stroke, respectively. Negative consistent exposure–response relationships were generally observed between them in the restricted cubic spline model. In the stratified analyses, the cardioprotective effects of blue sky were stronger for females, rural residents, and individuals residing in heavily contaminated areas.This study indicates that blue sky may serve as an independent environmental protective factor against CVD, and informs future policies on fighting air pollution and protecting the blue sky in China. Cardiovascular disease (CVD), including coronary heart disease (CHD) and stroke, is the leading cause of premature mortality and long-term disability worldwide according to the Global Burden of Disease (GBD) study . In receEmerging evidences in recent years suggested that residential blue spaces  could be beneficial for human health and wellbeing –6. An eqDuring 2013–2017 and 2018–2020, Chinese government launched the first 5-year Air Pollution Prevention and Control Action Plan . Wenrollees . Among tnrollees . This stvia cloud cover in the view of meteorology. Thus, it is determined by both environmental and large-scale meteorological conditions  at 378 ground meteorological stations across China during 1980–2018 after strict quality control . Here, wvia death certificates or medical records by the endpoint assessment committee of Fuwai Hospital . In this study, CVD consisted of stroke (ICD−10 code: I60–I61 and I63–I64), coronary heart disease (I20–I25), chronic heart failure (I50), and deaths owing to CVD . We combined the fatal and nonfatal CVD events into the same category, which provided us relatively more cases to analyze, thereby borrowing us enhanced statistical power. Similarly, the fatal  and nonfatal stroke were combined as well.Based on International Classification of Diseases−10th Revision (ICD−10), causes of death and fatal and non-fatal CVD events were coded by well-trained medical staffs at each survey site. These results were further validated 2, normal; 18–24 kg/m2, overweight; and > 24 kg/m2, obesity), hypertension, hypercholesterolemia, diabetes mellitus, family history of CVD, and CVD medication history.At baseline, individuals were surveyed about their demographic characteristics, including age, gender, urban/rural residence, ethnicity (Han or ethnic minorities), geographic region , and education level. Besides, the clinical characteristics of participants were collected as well, including smoking status , alcohol consumption (Yes or No), body mass index (BMI) (< 18 kg/m2.5), nitrogen dioxide (NO2), and ozone (O3) at 15 km × 15 km resolution were derived from assimilations of surface observations from China National Environmental Monitoring Center (R2 = 0.74–0.86). The baseline ambient air pollutant exposures were assigned based on each participant's residential address. Besides, we obtained annual mean ambient temperature, maximum temperature, relative humidity, and altitude from ~2419 meteorological stations across China to denote large-scale meteorological conditions and assigned these variables likewise (http://data.cma.cn/).The annual mean concentrations of ambient fine particulate matter of diameter ≤ 2.5 μm (PMg Center . Aided blikewise . These mhttps://www.resdc.cn/) as surrogates to demonstrate socioeconomic statuses. We also derived normalized difference vegetation index (NDVI) and enhanced vegetation index (EVI) with high spatial (1 km × 1 km) and temporal (monthly) resolutions from the moderate resolution imaging spectroradiometer (MODIS) products , which has been widely used to denote the long-term residential greenness exposure. In addition, we calculated the frequency of extreme heat waves to denote the extent of sun exposure, which is defined as annual number of days when maximum temperature ≥ 90th percentile of 1961–1990 climatology , and population density across survey counties were extracted from the Data Center for Resources and Environmental Sciences for China (matology . LikewisviaANOVA (χ2) test. We calculated the incidence rates  of three health outcomes. Cox proportional hazards models were employed to quantify longitudinal association between blue day exposure and all-cause mortality and cardiovascular events. We interpreted the derived results via two methods. First, we used a 10-day increment in the annual blue day exposure. Besides, we categorized the blue day exposure into three categories . Hazard ratios (HRs) were shown with the worst tertile as the reference group. We first applied the change in estimate approaches combined with directed acyclic graphs (DAG) to select the confounders. Finally, we adjusted for the following lists of covariates: (a) In model 1, we adjusted for age, sex, urbanity, ethnicity, geographic region , educational level, smoking status, alcohol consumption, and BMI; (b) In model 2, we additionally adjusted for hypertension, hypercholesterolemia, diabetes mellitus, family history of CVD, CVD medication history; (c) In model 3, we additionally adjusted for outdoor PM2.5 concentrations and ambient temperature; (d) In model 4, we further adjusted for county-level average years of education, per capita GDP, population density and altitude.We presented the continuous variables with means ± standard deviation (SD), while the categorical variables with absolute numbers (relative percentages). The between-group comparisons of continuous  variables were examined 2), outdoor PM2.5 concentrations , and individual medical history .Given the possible nonlinear association between chronic blue day exposure and all-cause mortality and cardiovascular events, we included the restricted cubic spline (RCS) model in our Cox model. The knot number was determined by Akaike information criterion (AIC), while the nonlinear association was examined via the likelihood ratio test. Point estimates and 95% confidence intervals (CIs) reporting as the 2.5th and 97.5th percentiles of the posterior distributions were calculated. Finally, we implemented a series of subgroup analyses, stratified by age , sex, urbanity, BMI  removing participants who died or got disease within the first year after the baseline survey; (e) excluding subjects whose residential address changed during follow-up to evaluate the possible impact of the movement of study participants; (f) excluding subjects who possessed baseline life-limiting chronic diseases  which could likely be fatal within the next few years; (g) including relative humidity at baseline year additionally as the covariate; (h) including frequency of extreme heat waves additionally as the covariate; (i) including NDVI or EVI additionally as the covariate; (j) including NO2 or O3 additionally as the covariate.To test the robustness of the results in the main analyses, several sensitivity analyses were performed: (a) using another definition of blue day index, which was based on fixed threshold values (22); (b) using another window of blue day exposure ; (c) changing outdoor PMp < 0.05. SAS version 9.4  and R software version 4.1.1  were used to conduct statistical analyses.The statistical significance was delineated at two-tailed 2.5 in general.2.5 or NO2 did not necessarily denote more blue day exposure, which could be due to the modification by large-scale meteorological conditions. We calculated the Pearson correlation coefficients between PM2.5 or NO2 and blue day index for all counties/districts, and the correlations are nonsignificant.ptrend < 0.001) and 43%  lower likelihood of developing fatal or nonfatal CVDs and strokes, respectively. The association in all-cause mortality was not significant . Negative consistent dose-response relationships were observed in blue day exposure and fatal or nonfatal CVD and stroke as well (pnonlinearity < 0.05) and linear-shaped for stroke (pnonlinearity = 0.083).At ~5-year follow-up, the incidence rates of all-cause mortality, fatal or nonfatal CVD, and stroke were 10.49, 9.45, and 5.64 per 1,000 person-years, respectively. In the multivariable-adjusted Cox regression models, a 10-day increase in annual blue day exposure was associated with 3% (95% CI: 1–6%) and 7% (95% CI: 5–10%) lower risks of fatal or nonfatal CVD and stroke, respectively . It sugg as well . The asspinteraction < 0.001) lower odds of developing CVDs, and 63%  fewer strokes. Moreover, a more pronounced protection effect was observed in people living in heavily contaminated areas (PM2.5 ≥ 60 μg/m3), with 37%  fewer CVDs and 52%  fewer strokes in the best tertile compared with the worst one.The results in the main analysis were consistently observed in almost all sensitivity analyses , implyinThis population-based nationwide prospective study investigated the relationship between chronic blue day exposure and CVD among individuals aged 35 years and older in China. This cohort investigation found that higher levels of blue day exposure were longitudinally associated with lower risks of cardiovascular incidences, especially in females, rural residents, and people living in heavily contaminated areas. These results robustly hold in the sensitivity analyses. Our work may carry potential societal advantages and public health importance. We highlighted that policies to increase blue sky in China may provide opportunities for better cardiovascular fitness.In the present study, we observed a slightly stronger cardioprotective effect of blue sky for females. This gender disparity may be partly explained by the hypothesis that women might be more susceptible to the change of external environments . Besides2.5, are suggested important modifiable risk factors of cardiovascular outcomes   –32, and markers) –35. Secomarkers) . Recent markers) . Thus, emarkers) . Third, markers) . Fourth,markers) .via the RCS model and it could be informative for policymakers to evaluate current progress toward clean air and blue sky, and guide future environmental policy-making and public health advancement in China.During the past decade, growing environmental epidemiological studies focused on the blue spaces , indicating that living near the coast or inland water bodies may serve as a protective factor for human health including CVD (4–6). In the present study, we attempted to extend the concept of traditional blue spaces and probed the potential relationship between blue sky exposure and CVD. The observations in this study provided a novel line of evidence from existing literatures that how the environment would mediate the burden of CVD. Moreover, we obtained a nationally representative dose–response curve for the blue sky–CVD relationship However, owing to the partial nature of our work, it is still too early to provide definitive answer about the beneficial effects of blue sky on human health at this stage. Further studies are required to reveal the relationships between blue sky and other health outcomes and the underlying mechanisms. Besides, it ought to be acknowledged that the definition of blue sky in this study is subjective to some extent given the selection of the three criteria. We would leave such efforts to future studies that could define a more objective blue sky index through technologies such as the satellite remote sensing. Moreover, due to the limited access to observational meteorological, environmental, and human health records, the study region of the present study was confined in China. Future investigations on the potential health benefits of blue skies on world's population living in South Asia, Africa and South America where the air pollution risks are still heavy may be preferable. Furthermore, we have not yet analyzed the respective contribution of air pollution and meteorology/climate to the number of blue days. Additional studies can employ climate model simulations to differentiate the effects of climate from those of air pollution in space and in time (16), which would favor future cost/health-benefit analysis of air pollution reduction programs in China.The major strength of this study is that we identified a potential independent environmental protective factor for CVD, i.e., blue sky. Although blue sky has long been deemed beneficial for cardiovascular health intuitively, here we strived to provide epidemiological evidence and build up their statistical relationship. These results might be instrumental in the attribution of GBD from environmental causes. Besides, we adopted a nationally representative cohort and incorporated a relatively large sample size, which would elevate the statistical power of the findings in this study. Moreover, our study baseline recruitment and follow-up periods were nearly simultaneous with air pollution reduction programs in China that were implemented in 2013–2020, offering the latest high-quality cohort evidence pertaining to the CVD burden associated with the blue sky. Finally, we accounted for a variety of covariates  to adjust for the latent confounding as far as possible.Despite these advantages, the limitations of the present study also merit consideration. First, the present study was essentially an ecological study, and the discrepancy between individual perception and environmental concentrations  may to some extent bias our results. Second, the limited follow-up period of our CHS cohort (median ~5 years) may somewhat weaken the statistical power of our results. Thus, the insignificant association between blue day exposure and all-cause mortality shown in this study does not necessarily mean there is no connection between them and we should treat this result with caution. Future researches of a longer follow-up period would be carried out as our CHS cohort marches forward. Additionally, although we adjusted for the corresponding covariates, a likelihood of residual confounding still exists. For instance, the simple “Yes or No” binary classification may be insufficient to adequately control for stressors related with alcohol consumption. A quantitative classification as (a) no alcohol consumption (never), (b) no alcohol consumption (former), (c) low consumption, and (d) high consumption based on national/international guidelines would be preferable. Therefore, our findings need to be confirmed by future studies in other settings. Last, we cannot rule out the potential confounding biases brought by other covariates such as dietary habits and exercise. These potential mediating, confounding or modifying effects may be important and deserve future studies.In summary, findings from this study offered nationally representative epidemiological evidence for a significant protective association of long-term blue sky exposure with fatal or nonfatal CVD, adjusting for demographic, clinical, environmental, and socioeconomic covariates. This study provided novel insights on probing the environmental determinants of CVD risk. In tandem with previous studies, our work appeals for the continuous implementation of air quality management in China in the future to further protect the blue sky and promote cardiovascular health.The meteorological and environmental datasets used in this study are available from the corresponding author upon request. Requests should be directed to ZW to access the CHS cohort datasets.The studies involving human participants were reviewed and approved by Ethics Committee of Fuwai Hospital. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.HT: conceptualization, methodology, formal analysis, software, visualization, and writing—original draft preparation. XC: methodology, data curation, formal analysis, software, and investigation. CZ: software, investigation, and validation. GH and ZW: conceptualization and funding acquisition. All authors: writing—reviewing and editing.The study is jointly supported by the National Natural Science Foundation of China , and the Youth Innovation Promotion Association of CAS (2021072).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


INK4a (p16). We wanted to determine if HPV-HR or p16 expression is associated with better outcomes in PSCC patients; therefore, we analyzed 143 patients with a diagnosis of PSCC and available tissue was analyzed for p16INK4a expression and HR-HPV status. Patients with p16+ tumors had a significantly longer median cancer specific survival in comparison to the p16-group (p = 0.004), with respective 5-year cancer specific survival probability of 88%  versus 58% . HPV status did not predict survival outcomes. Penile Squamous Cell Carcinoma (PSCC) is a rare but aggressive cancer and approximately 30–50% of cases are associated with high risk human papillomavirus (HR-HPV). HR-HPV infection has been shown to correlate with expression of protein p16INK4a (p16) is highly correlated with HR-HPV expression in other SCCs. To investigate whether the expression of p16 IHC or HR-HPV is associated with survival in PSCC, we conducted a single institution analysis of 143 patients with a diagnosis of PSCC and, available tissue were tested for p16 IHC staining patterns, histological subtype, tumor grade, and lymphovascular invasion (LVI) by an experienced pathologist. HR-HPV status using the Cobas PCR Assay or the RNAScope high-risk HPV in situ hybridization kit were also assessed. Patient characteristics were summarized using descriptive statistics of clinico-pathologic variables. Kaplan–Meier was used to estimate median overall survival (OS), cancer specific survival (CSS) and correlated with HPV, p16, and other study variables. Patients with p16+ tumors had a significantly longer median CSS in comparison to the p16– group (p = 0.004), with respective 5-year CSS probability of 88%  versus 58% . HPV status did not predict survival outcomes. Multivariable analysis with respect to OS and CSS, showed that p16+ status was associated with a lower risk of death , and improved CSS  after adjusting for covariates. In conclusion, tumor p16 status via IHC was an easy to perform independent prognostic factor for OS and CSS that correlates with HR-HPV expression.Penile Squamous Cell Carcinoma (PSCC) is associated with high-risk human papillomavirus (HR-HPV). The immunohistochemical (IHC) test for p16 INK4a (CDKN2A) protein . Overexpression of p16 can be detected by immunohistochemical (IHC) staining and has been found to be a reliable marker for high-risk HPV infection in oropharyngeal SCC and to a lesser extent in PSCC [A major etiologic pathway of penile squamous carcinoma (PSCC) involves high-risk human papillomavirus (HPV). HPV infection occurs in about 30–50% of penile cancers but varies significantly via histologic subtype ,2,3. The in PSCC ,8,9. ThiWe conducted a single institution analysis of patients with PSCC who received treatment at The University of Texas, MD Anderson Cancer Center (MDACC) between 1991 and 2017. Eligible patients were defined as those with PSCC, determined by pathology review (PR), and had tissue available for analysis. We conducted p16 immunohistochemical staining (IHC) and HPV polymerase chain reaction (PCR) testing. The demographic, clinical, and treatment data for each patient were obtained from retrospective chart review from the electronic medical records . Patient® 2.5 LS Probe-HPV HR8 kit  with an automated Leica BOND-III RNA-scope system allowing it to be used in routine clinical care. The pooled probe can recognize high risk HPV genotypes 16, 18, 31, 33, 35, 45, 52, and 58 as a “pooled result” as either negative or positive but does not provide the specific genotype. In each run, appropriate controls for the RNAscope 2.5 LS probe  were used. HPV detection was performed using Cobas PCR HPV assay on all samples. However, in a subset of samples (44) Cobas provided an invalid reading and the high-risk HPV in situ hybridization assay (HPV-ISH) was used as an alternative strategy on these samples to clarify HPV status. Cobas HPV testing  was performed in a similar manner to published methods  using fo® p16 Histology, clone E6H4, Ventana Medical Systems, Tucson, AZ, USA). p16 staining patterns of 0, 1, 2, and 3 were classified using previously described categories [Histological subtype, tumor grade, LVI (lympovascular invasion), and p16 staining patterns were confirmed by a genitourinary pathologist (PR). For p16 IHC, all samples were tested, and stained on a BenchMark Autostainer  as described by the manufacturer’s protocol using a prediluted mouse monoclonal antibody  was used for statistical analysis. Abbreviations for 95% confidence interval (95% CI) or interquartile range (IQR) used throughout the text. The distribution of each continuous variable was summarized by its mean, standard deviation, and range. Categorical variables were summarized in terms of their frequencies and percentages. The associations between the binary outcomes and other factors were assessed using Fisher’s Exact test or Wilcoxon Rank sum test as appropriate. Kappa coefficients were used to evaluate the agreement between different p16 definitions and HPV status. The primary endpoints included CSS and OS. CSS was defined as the time from diagnosis until the time of death related to PSCC or censored at the last follow-up. OS was defined as the time from diagnosis until the time of death or censored at the last follow-up. The last follow-up date was defined as the last time the patient was seen by the clinical team within our health care system or the last time the patient corresponded with the hospital to update their disease status if they were being followed by outside providers. We estimated the survival outcomes for the overall cohort using the Kaplan–Meier method, comparing the survival outcomes among subgroups of patients by p16 and HPV status using logrank test. The Cox regression proportional hazard model was used for multivariate analysis, predicting OS and CSS based on the backward model selection. The variables that showed potential significant effect on OS and CSS in univariable models were considered in the initial multivariable model. n = 32, 97%) received the combination chemotherapy regimen with cisplatin, ifosfamide and paclitaxel (TIP). The median follow-up time for the study was 5.8 years , during this period, 70 (49.7%) patients died, 42 (29.8%) patients died from PSCC, 11 (7.8%) patients were alive with disease, and 60 (42.6%) patients remained alive without evidence of disease. One hundred forty-three patients were identified. Median age at diagnosis for this cohort was 58.9 years  and the median time between onset of symptoms and date of diagnosis was 6.5 months . Patient characteristics are summarized in Forty-seven patients (32.9%) were positive for HR-HPV with 45 (31.5%) positive for p16 using our HS IHC analysis method . Our HS The association between HPV and p16 status and clinicopathological features are shown in p-value of 0.007 while p16 status showed non-significant impact on OS with HR = 0.60  with a p-value of 0.068. Using staining pattern 3 as p16 positive, p16 status was significant in CSS with HR = 0.32  and p-value of 0.03 while p16 status showed non-significant impact on OS with HR = 0.63  and p-value of 0.145. Therefore, p16 status is significant in CSS using either our HS analysis or the staining pattern method. However, we do observe lower p-values with our HS analysis method and a better kappa coefficient therefore, p16 status based on our HS method was used in all other analyses. To determine how our p16 HS analysis compared to the previously published staining pattern 3 IHC cut off  plots for OS and CSS stratified by p16 and HPV status are shown in INK4a protein (p16) is upregulated and has been detected in high risk HPV related malignancies utilizing IHC [PSCC is a rare but lethal cancer whose biology is driven, at least in part, by differences in high risk HPV status ,17,18. Szing IHC ,6,8,9,17zing IHC ,20. The p = 0.004). These findings were clinically relevant and feasible to document utilizing IHC and a binary cut off value  for p16 status. Of note, p16 positivity may exert its prognostic significance among patients with more advanced PSCC  as the correlation with better survival outcomes was significant among those with advanced versus lower stage disease . Among advanced disease PSCC patients the use of multimodality therapy is certainly more prevalent. It is plausible but not proven that p16 as a potential marker of HPV driven malignancy may correlate with response to multimodal therapies such as chemoradiation or chemotherapy. In one recent study patients with advanced PSCC that were HPV+ exhibited significantly greater overall survival when treated with radiotherapy when compared with the HPV− cohort [The expression of p16 was found to be superior to all other clinical, HPV genotyping, or IHC parameters for predicting prognosis for patients with OPSCC ,22. We f− cohort .Our study highlights the importance of p16 staining in PSCC and how this relatively simple methodology using IHC compares to the more costly HPV testing technologies. To our surprise HPV status was not shown to have the same prognostic ability as p16 staining in the overall cohort but was significantly associated with an overall survival benefit among patients with advanced primary tumor stage. A recent meta-analysis by Sand and colleagues of PSCC studies  that assIn our 143 patient samples, HPV and p16 status were discordant in 20 samples. All HPV testing was initially run using the Cobas system and a subset that gave invalid results  were anap-value 0.007) utilizing our HS method over a previously published staining pattern method  [A novel finding of our study is that the use of a binary cut off of 75% staining was a simple method to assess p16 positive or negative status. A stronger overall agreement between HPV and p16 status was found using our HS method versus the staining pattern method as measured by the kappa coeffecient. Considering its prognostic value we noted a stronger correlation with CSS  . HoweverThe primary limitations of this study are its retrospective nature in the setting of a tertiary referral center and heterogeneous treatment regimens. To minimize these effects, we analyzed the importance of a variety of known prognostic covariates on survival outcome. Clinical and pathologic correlates of advanced disease were significant predictors of both OS and CSS. However, after adjusting for all variables, p16 remained an important independent predictor of both OS and CSS. Thus, the present study provides further evidence that p16 IHC is a rapid, easy to perform test that has a high sensitivity and high negative predictive value for assessing HPV status in PSCC. As such p16 status serves as an important prognostic marker and excellent first line test to potentially describe the biology driving a given patient’s PSCC. We validate p16 status as a valuable independent survival biomarker in PSCC that correlates with HPV driven malignancy.


Background: Few data are currently available on the treatment of patients with HPV infection. In particular, there is no agreement on the use of antioxidants in these patients. Ellagic acid and annona muricata appear to improve HPV clearance in infected women. However, it is presently unknown whether they could enhance the clearance of HPV infection in infertile male patients. Aim: To evaluate the effects of a commercially available combined compound containing ellagic acid and annona muricata on semen quality in patients with documented papillomavirus (HPV) infection, and on the frequency of HPV DNA detection in seminal fluid after treatment. In addition, anti-sperm antibodies and the percentage of spermatozoa with fragmented DNA were evaluated. Materials and methods: This was a retrospective case-control study including patients attending our center for infertility. Fifty selected patients who were positive for high risk (HR)-HPV with available semen analysis results were consecutively enrolled. Patients were classified into two groups, according to the clinician’s decision to either administer ellagic acid 100 mg and annona muricata 100 mg (combined tablet formulation) for a period of three months , or to re-evaluate HPV DNA after a period of active surveillance only  . Results: Group A patients had a mean age of 31.0  ±  11.0 years, while Group B was 33.0  ±  8.0 years old (p  > 0.05). After three months of treatment with ellagic acid and annona muricata, all conventional seminal parameters improved more significantly in Group A than in Group B patients: sperm concentration = 45 mil/mL vs. 20 mil/mL (p < 0.05); sperm progressive motility = 45% vs. 18% (p < 0.05); and normal sperm morphology = 18% vs. 6% (p < 0.05). After the treatment, the frequency of persistence of HPV DNA in the seminal fluid was significantly lower in Group A patients compared to those in Group B . Finally, after 3 months, Group A showed a significant reduction in anti-sperm antibodies and in the percentage of spermatozoa with fragmented DNA. Conclusion: The results of this study demonstrate, for the first time, the effects of a commercially available combined compound containing ellagic acid and annona muricata on semen quality in patients with HR-HPV infection, and that this therapy is also associated with a significant reduction in the persistence of HPV DNA in the seminal fluid. Papillomavirus (HPV) infection has been observed with high clinical frequency in infertile patients for many years now . The epiLimited data are currently available on the treatment of patients with HPV infection. Vaccines are the first choice to promote an antibody response and achieve a faster clearance of the infection . HoweverViral and bacterial urogenital infections are known to negatively impact the oxidative balance, thus worsening seminal parameters . CliniciEllagic acid is a phenolic compound with antioxidant, chemopreventive, and antiviral activities. Annona muricata is a type of fruit tree with anticancer and antioxidant activities ,18,19,20Therefore, this study aimed to evaluate the effects of a three-month treatment regime using ellagic acid (100 mg) and Annona muricata (100 mg) on both semen quality and frequency of HPV DNA detection in patients with high-risk (HR) oncogenic HPV infection. Finally, we evaluated the percentage of patients with anti-sperm antibodies, and the percentage of spermatozoa with fragmented DNA, before and after therapy.This was a retrospective case-control study performed using the clinical charts of men who consulted the Division of Endocrinology, Metabolic Diseases and Nutrition, at the University of Catania, for infertility.We consecutively screened the clinical charts of patients attending the outpatient clinic from January 2018 to April 2021. We selected the first 50 patients who were positive for HR-HPV and had undergone semen analysis at the time of the infection.Exclusion criteria were as follows: the presence of non-HPV-related male accessory gland infection (MAGI), severe oligozoospermia (sperm concentration < 5 mil/mL), any endocrine disease including any type of diabetes mellitus, cigarette smoking, being overweight/obese, mean testicular volume < 12 mL (measured using the Prader’s orchidometer), and any drug use. In particular, to exclude the presence of bacterial MAGI, symptomatic patients and those with certain signs at the physical examination  underwent sperm and urethral swab cultures. Patients with positive cultures or with signs/symptoms strongly suggestive of bacterial infection were excluded.Patients were classified into two groups according to the clinician’s decision at the time of their visits on whether to administer ellagic acid (100 mg) and annona muricata (100 mg) or not. The two molecules were given in a single tablet formulation for three months , or the patient was re-evaluated for HPV DNA presence after three months of active surveillance only  .The clinical charts contained information on sperm parameters that were analyzed at the Unit of Endocrinology, Metabolic Diseases and Nutrition, University of Catania. For each patient and control, semen samples were collected by masturbation into a sterile container after 2–7 d of sexual abstinence, and were analyzed immediately after liquefaction. Each sample was evaluated for seminal volume, pH, sperm count, progressive motility, morphology, and round cell concentration according to the 2010 WHO guidelines . The evaSDF was evaluated by flow cytometry using an EPICS XL . DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. The negative control was obtained through the non-addition of terminal deoxynucleotidyl transferase to the reaction mix, while the positive control was obtained by pre-treating spermatozoa with 1 mg/mL of RNase-free deoxyribonuclease I  at 37 °C for 60 min before labeling.® Genomic DNA Kits . Real-time PCR (RT-PCT) was used for DNA amplification, using specific primer pairs for the amplification of the L1 region of the viral genome, which is particularly preserved in the different HPV genotypes  Genotyping, Experteam s.r.l., Venezia, Italy). As an internal amplification control, the human gene thiosulfate sulfurtransferase mapping to the region 22q13.1 was simultaneously amplified.According to the clinical records, each enrolled patient underwent HPV DNA testing using the procedure described below. The semen sample underwent DNA extraction using PureLinkTo identify high-risk HPV genotypes , fluorescent PCR Multiplex using fluorescent primer pairs for the target amplification region (E6 and E7), specific for each different HPV viral type searched, was employed. Human polymorphic system short tandem repeats (STRs) served as internal controls and were amplified simultaneously with the viral genotypes. The visualization of the amplified products was performed by means of fluorescent capillary electrophoresis . The Standard Gene Scan 500 LIZ  was used for the detection of the amplified products.t-test or the Mann–Whitney U-test was applied according to the normal or non-normal distribution of the data, respectively. The statistical analysis was performed using MedCalc Software Ltd. , version 19.6–64 bit. A p-value lower than 0.05 was accepted as statistically significant.The results are reported as mean  ±  SD throughout the study. The normal distribution of the variables was evaluated using the Shapiro–Wilk test. The Student Since most of the data support the concept that sperm motility is the parameter most likely to be affected by HPV infection, we chose this parameter for power calculations. A 32% sperm progressive motility was assumed for the control, based on the WHO 2010 manual. An absolute reduction of 25% was viewed to be of sufficient clinical significance to change practice. The study required a total of 34 participants (17 per group) to preserve 90% power in order to detect an absolute difference of 5% in a two-sided test, taking into account a dropout rate of 5%.This study was conducted at the Division of Endocrinology, Metabolic Diseases and Nutrition, in the university teaching hospital “G. Rodolico-San Marco”, at the University of Catania . The protocol was approved by the internal Institutional Review Board, and written informed consent was obtained from each participant after a full explanation of the purpose and nature of all procedures used. The study was carried out according to the principles expressed in the Declaration of Helsinki.The results of this study are reported in After three months of treatment, the percentage of patients with persistence of HR-HPV in Group A—those who received ellagic acid (100 mg) and annona muricata (100 mg) - was found to be significantly lower than that in Group B—those who did not receive drug treatment. Patients in Group A showed a significant increase in sperm concentration, sperm progressive motility, and percentage of spermatozoa with normal morphology, compared with those in Group B. The percentage of patients with anti-sperm antibodies was found to be significantly lower in Group A patients compared with those in Group B. Lastly, the percentage of spermatozoa with fragmented DNA was found to be significantly lower in Group A patients compared with those in Group B.The results of this study showed that the use of the combination of ellagic acid (100 mg) and annona muricata (100 mg) was associated with an improvement in the semen quality of infertile patients with high-risk oncogenic papillomavirus infection, as well as with an increased clearance of the infection, a reduction in the percentage of patients with anti-sperm antibodies, and a reduction in the percentage of spermatozoa with fragmented DNA.Ellagic acid is a bioactive plant-based polyphenolic molecule, present in several flowering plants, especially in eudicotyledons . It is cAt physiological pH, ellagic acid interacts with several species of free radicals, and this is an important property, given the prevalence of these molecules in the biological environment. In addition, it can be considered to be an uncommon and versatile antioxidant, because it is continuously reproduced after deactivating two free radicals per cycle, providing a “free radical scavenging cascade”. Thus, ellagic acid ensures continuous and high protective effects against oxidative stress, even at low concentrations .Alongside ellagic acid’s antioxidant effects, several authors have suggested other mechanisms by which ellagic acid interferes with biological processes under conditions of oxidative stress. These include metal chelation , bindingThe evidence that ellagic acid is active against oxidative stress has mostly been demonstrated through in vitro and in vivo studies . Most clEllagic acid has been shown to have antitumor effects via the following mechanisms: apoptosis induction, blocking of tumoral proliferation, prevention of viral infection, and interference with inflammation . ExperimThus, the cytoprotective, anticytotoxic, and antioxidant effects of ellagic acid contribute to its anticancerogenic action, as demonstrated by an increase in therapy tolerance. Ellagic acid is primarily an antioxidant molecule, and it has been extensively studied for different positive health effects, among which is its ability to modulate the body’s defense system against pathogens such as viruses . HPV belThe therapeutic approaches employed in the treatment of HPV-related cancers, such as surgery, chemo- and radiotherapy are considered insufficient . EvidencThe antiviral and chemoprotective effects of ellagic acid in a group of women with HPV cervical infection were evaluated by Morosetti and colleagues. In this study, the administration of ellagic acid (160 mg daily) for 12 months resulted in a protective effect on cervical cells, whereas no effects on viral clearance and virus integration were found in either the control or treatment groups. Among the mechanisms hypothesized, it seems that apoptosis stimulation, promotion of DNA repair, and inhibition of the cell cycle underlying the antioxidant capacity of ellagic acid can counteract the damage caused by oxidative stress, thus guaranteeing effective maintenance of the body’s natural defense mechanisms, as well as it having an organ-cytoprotective action . A randoTo our knowledge, this is the first evidence regarding the use of phytotherapy in the treatment of male infertility associated with HPV infection with high oncogenic risk. Our research will continue with the study of more numerous case series, and we will later attempt to define further molecular mechanisms that can help to better explain the results obtained.


TAP1 and TAP2 polymorphisms, TAP1, and TAP2 genes expressions, and protein levels in cervical cells presenting different degrees of pre-cancerous lesions in 296 immunocompetent women infected or not by HPV. TAP SNPs were genotyped by Sanger sequencing, and gene expression by real-time PCR. Aneuploidy was determined by DNA index using flow cytometry. TAP-1 and TAP-2 tissue expressions were evaluated by immunohistochemistry. The Asp697Gly SNP of TAP1 presented a risk for cellular aneuploidy (P=0.0244). HPV+ women had higher TAP-2 mRNA (P=0.0212) and protein (P<0.0001) levels. The TAP2D and TAP2E haplotypes were associated with the risk for aneuploidy and pre-cancerous lesions. In conclusion, nucleotide variability at the peptide binding region of peptide transporter genes, particularly of the TAP2 gene, may influence the HPV-peptide transportation from the cytosol to the endoplasmic reticulum, increasing the susceptibility to the development of high-grade cervical lesions.Human papillomavirus (HPV) is the major pathogen for cervical lesions. The evasion mechanism of the immune response and persistence of HPV infection can be influenced by polymorphisms (SNPs) in genes associated with transporter associated with antigen processing (TAP), which may change the peptide binding affinity or the TAP expression impacting the efficiency of peptide transport in the secretory pathway, and the presentation of peptides to cytotoxic T lymphocytes. This study aimed to evaluate the role of the  In the ng (TAP) .TAP genes may influence the selection of peptides bound to HLA-I molecules, modulating the diversity of peptide transport from cytosol to ER (TAP genes (TAP1 and TAP2) are close to the peptide-binding site, and some are in the protein transmembrane domain  (The presence of single nucleotide polymorphisms (SNPs) in ol to ER . Most SNe domain . These crthritis , systemirthritis , esophagrthritis , multiplrthritis , and tubrthritis . Additiocer (CC) .TAP gene variability.Human papillomavirus (HPV) is a sexually transmitted infection, accounting for 99.7% of CC cases . In BrazTAP genes and evaluated the TAP genes expression and protein levels in cervical samples obtained from women infected or not by HPV.To understand the contribution of TAP on HPV-induced cervical lesions, we analyzed five SNPs at the coding regions of 22.1We enrolled consecutively 296 women (38.6 ± 8.9 years) at the Cervical Pathology outpatient clinic of the Instituto de Medicina Integral Professor Fernando Figueira (IMIP) and Colposcopy Service at the Centro Integrado de Saúde Amaury de Medeiros (CISAM) in Recife (Northeast Brazil), between April 2016 and October 2018. All patients were from the metropolitan region of the Recife capital and were 18 years or older. They were recruited before undergoing colposcopy and oncotic cytology examination, offered free of charge to every woman in the public health service by the National Cervical Cancer Surveillance Program of the Brazilian Health Ministry. Thus, the research team did not have access to information about a previous diagnosis of cervical lesion or HPV infection at the time of recruiting the volunteers for the study. All participants signed the informed consent form. The Ethics Committee of the Aggeu Magalhães Institute approved this study under the protocol CAAE:51111115.9.0000.5190. Clinical and laboratory data of the patients were obtained from the hospital records Table 1.2.2After answering a clinical questionnaire, the patient’s peripheral blood was collected to study the association of inherited gene variants with precancerous cervical disease. Then, during the colposcopy examination, we collected a sample of exfoliative cervical cells for molecular diagnosis of HPV infection, evaluation of the DNA ploidy, and gene expression studies. Therefore, they were linked to the cytological and histological examination of the day they were collected.In the presence of atypia, a cervical biopsy was performed for further histopathological and immunohistochemical analysis, justifying why a small number of women were evaluated for TAP protein expression in the cervical lesions. The colposcopy results were classified according to the nomenclature of the International Federation for Cervical Pathology and Colposcopy, IFCPC . The absL1 gene confirmed the HPV infection, and the sequence of the L1 fragment defined the HPV type. Of the 296 women evaluated, 189 were negative for HPV infection (HPV-), and 107 were positive for HPV (HPV+). Of these, 11 (10.3%) were infected with low-risk HPV , and 96 (89.7%) with HrHPV .Genomic DNA was extracted from cervical samples according to the methodology reported by Medeiros et al. (2022) . PCR ampThe DNA ploidy of the exfoliative cervical cells was determined as previously reported . A DNA i2.3gDNA extraction using the DNAzol reagent . Conventional PCR used specific primers designed according to the target region at TAP1 (Genbank ID: 6890) (TAP2 (Genbank ID: 6891) genes. Primers and reaction conditions are shown in TAP1 and TAP2 genes used the SeqMan v.7.0 program  for the genotype identification. We evaluated the SNP rs1135216 in exon 10 at TAP1 and the SNPs rs2228396 in exon 10, and rs4148876 and rs241447 in exon 12 at TAP2 that are in the peptide binding site; and the SNP rs1800454 in exon 6 at TAP2 related to the peptide transport.Peripheral blood mononuclear cells (PBMC) were isolated by gradient density using Ficoll-Paque™ Plus reagent  and submitted to D: 6890)  and TAP22.4® reagent and cDNA synthesis using the M-MLV RT reverse transcriptase enzyme (Invitrogen) following the manufacturer’s protocol. Specific primers designed to amplify the mRNA of the TAP1 (ID: NM_000593.5) and the mRNA of the TAP2 (ID: NM_000544) genes used the Oligo IDT software. The TAP1 and TAP2 expression was quantified in duplicates by qPCR in the QuantStudio™ 5 instrument (Applied Biosystems) following the reaction parameters described in GAPDH) gene expression . The formula for each sample was: ΔCq = target average Cq, reference average Cq. The relative changes in gene expression used the Fold-change = 2-ΔCq.The total RNA extraction of exfoliative cervical cells was performed using the TRIzolpression . Primers2.5® , and visualized with 400x magnification in an inverted microscope  equipped with a camera and a 4.7.4 Image Analysis Program . Immunohistochemistry (IHC) reaction used the Dako EnVision™ FLEX+ Kit  following the manufacturer’s instructions. After deparaffinization and hydration, the slides were subjected to heat-induced antigen retrieval with Tris/EDTA Buffer pH 9 for 30 minutes. After the inactivation of endogenous peroxidase by the peroxidase-blocking reagent, the slides were incubated in a humid chamber, at room temperature, with the following primary antibodies: rabbit polyclonal anti-TAP-1  and rabbit polyclonal anti-TAP-2 . Phosphate-buffered saline (PBS) replaced the primary antibody as a negative control. The sections were incubated for 20 minutes with the EnVision™ FLEX/HRP, and the reaction was visualized with the chromogen 3,3’-diaminobenzidine tetrahydrochloride (DAB) solution. The cells were counterstained with Harris’ hematoxylin. Two experienced pathologists blind-evaluated the labeling for TAP-1 and TAP-2. Brown staining detected in the cytoplasm of cells was considered positive for TAP-1 or TAP-2. Three photos of each slide were captured per patient. The protein levels were estimated by the Gimp 2.10 software , considering the intensity of pixels in the stained area of the image.Biopsies of the cervical lesions of 40 women were fixed in 10% buffered formalin and embedded in paraffin. Slides of 4μm tissue sections were stained with hematoxylin and eosin (H&E), assembled with Entellan2.6https://wpcalc.com/en/equilibrium-hardy-weinberg/). The linkage disequilibrium (LD) was evaluated by the r2 statistic between different polymorphisms using the Haploview software (https://www.broadinstitute.org/haploview/haploview).The description of the categorical variables used absolute number and frequency (%), and the comparisons were performed using Fisher’s exact or the Chi-square tests. Comparison of continuous variables between two groups used the student-T or the Mann-Whitney-U tests. In contrast, comparisons of three or more groups were analyzed using the ANOVA or Kruskal-Wallis tests. The decision to use parametric or non-parametric tests was based on the distribution of the variables using the Kolmogorov-Smirnov normality test. The statistical analyses used the GraphPad Prism program (version 5.0) for Windows . The association between genetic frequencies with susceptibility to the disease was determined by calculating the Odds Ratio (OR) using Fisher’s exact test, considering P<0.05 as significant. The Hardy-Weinberg equilibrium (HWE) expectations were assessed using specific calculator software (33.12 = 0.0399). Noteworthy, carriers of the rare GG genotype were at increased risk for cervical cell aneuploidy  (The p.Asp697Gly (rs1135216) allele and genotype frequencies were not associated with the cytological and histological lesions or with the presence of HPV infection in 290 women , and the genotypes distribution fitted the HWE equilibrium expectations (χ=0.0244) Table 2.TAP1 mRNA expression in exfoliative cervical cells of 261 women showed no significant difference in the median levels of TAP1 transcripts for the following groups of comparison: aneuploidy vs. diploidy ; HPV+ vs. HPV- ; cytological  or histological  alterations; and TAP1 p.Asp697Gly genotypes  (The =0.6790) E–H.Overall, the TAP-1 protein was higher in CIN 1 lesions compared to BL (P=0.0280) A. Carri3.2TAP2 alleles and genotype frequencies are shown in 2 = 1.7), but the genotypes of the SNPs Ala565Thr (rs2228396) (χ2 = 57.2), the Arg651Cys (rs4148876) (χ2 = 83.5), and the Thr665Ala (rs241447) (χ2 = 268.9) did not fit the expected proportions of HWE. In addition, the SNPs rs241447 (Thr665Ala) and rs2228396 (Ala565Thr) are in linkage disequilibrium (D’=100).The TAP2 gene expression, stratified according to the alleles of each of the four studied TAP2 SNP , showed no significant results , dominant , and recessive  models Table 3.TAP2 mRNA levels in exfoliative cervical cells. However, overall TAP2 mRNA levels did not differ accordingly to the severity of histological lesions    , and increased the risk of CIN  (data not shown). Interestingly in women with HSIL, the rare homozygous p.Thr665Ala GG genotype was associated with a significant decrease in The TAP-2 levels increased according to the severity of the cervical lesion and were significantly higher in tissues with CIN 3 compared to CIN 1 (P=0.0483) or BL (P=0.0001) Figure 3TAP2 p.Val379Ile, p.Ala565Thr, p.Arg651Cys, and p.Thr665Ala SNPs in HPV+ compared to HPV- women. The HPV infection increased the TAP2 mRNA expression in exfoliative cervical cells and the levels of TAP-2 protein in the cervical lesion of carriers of the common homozygous p.Val379Ile GG, p.Ala565Thr GG, p.Arg651Cys CC, and p.Thr665Ala AA genotypes. However, women carrying rare genotypes responded differently to the HPV infection. Carriers of the rare p.Val379Ile A allele showed a significant increase in tissue TAP-2 protein, while carriers of the p.Ala565Thr A allele showed no changes in TAP-2 levels. Additionally, this genotype was associated with higher TAP-2 protein levels than that observed in carriers of the common genotype (p.Ala565Thr GG) without HPV infection , formed by the combination of wild-type alleles, followed by the TAP2C haplotype (8.5%) Table 4.The TAP2E and TAP2D haplotypes diverge only in the position of the p.Val379Ile SNP, with no difference in mRNA expression. However, they are great protein producers, and the TAP2D showed the highest protein level (P=0.0004) Figure 5TAP2 mRNA and protein levels. The rare p.Thr665Ala allele G is the unique difference observed about the TAP2A haplotype sequence, suggesting its role in lowering the TAP2 expression.The TAP2B haplotype was associated with low TAP2 gene; ten were considered rare because of their low frequencies. The TAP2A/TAP2A diplotype (54.4%) was associated with protection against aneuploidy , while TAP2B/TAP2B diplotype (7%) was associated with a 5.6-fold increase in the risk of HSIL . The TAP2A/TAP2D diplotype (6%) was associated with protection against CIN , suggesting the effect of the TAP2A haplotype was dominant  showed the lowest protein level, and those who encompassed the TAP2A haplotype combined to TAP2D (P=0.0018) or TAP2C (P=0.0101) haplotypes showed increased protein levels (TAP2 mRNA transcripts than that HPV- (P=0.0155), and a higher median of TAP-2 protein levels was found in HPV+ women with TAP2A/TAP2C diplotype when compared to HPV- women (P<0.0001)  was similar among Europeans (14.4%), Africans (23.3%), Asians (16.5%), Latin Americans with Afro-Caribbean ancestry (16.3%), and Latin Americans with European and Native American ancestry (17.6%)  .We demonstrated that women who carry the G allele in homozygosis (3%) have a higher risk of having aneuploidy in cervical cells Table 2.TAP1 and HLA-I expression by HPV-16 E7 and HPV-18 E7 oncoproteins  of the TAP-2 protein, and the p.Ala565Thr, p.Arg651Cys, and p.Thr665Ala SNPs code for amino acids in the cytosolic nucleotide-binding domain (NBD2) of TAP-2  or protection alleles (*0501 and *0603), showing less difference in TAP2 alleles distribution between cases and controls among carriers of the DQB1 risk alleles, suggesting a hitchhiking effect of TAP2 with DQB1 alleles  analyzed TAP1 and TAP2 expression using TCGA (The Cancer Genome Atlas) data and found higher expression in cervical carcinoma tumors in HPV+ samples than in normal control tissue (TAP1 and TAP2 expressions were also higher in EBV-associated gastric carcinoma samples than in normal control tissues or other gastric carcinoma subtypes (TAP2 expression and a concomitant increase in protein levels. The rare allele A of the p.Val379Ile SNP in the TAP2C haplotype elevates the TAP-2 level in HPV-infected tissue and CIN 3 samples.The TAP2A/TAP2A carriers responded to HPV infection, increasing the l tissue . TAP1 ansubtypes . InteresTAP2 expression, possibly mediated by p.Val379Ile SNP allele A, may contribute to cervical cancer progression from precursor lesions (The increase in TAP2 expression and protein level may be related to greater recognition and processing of the viral peptide, increasing the adaptive immune response . Neverth lesions .Some viruses have an immune response evasion mechanism based on the release of inhibitory molecules, which decrease TAP levels . In the The main limitation of our study was the low number of biopsies, which compromised the TAP-1 and TAP-2 protein analyses, reducing the possibility of multiple comparisons. Nevertheless, the evaluation of mRNA expression in exfoliative cervical cell samples allowed the analysis of a more significant number of samples, increasing the strength of the observed association. In addition, we chose to use the DNA index in the final association analysis because aneuploidy is related to the integration of the HPV genome into cervical cells, and the increase in cervical dysplasia has a prognostic value for cervical cancer .5TAP2 gene allowed the identification of low, intermediate, and high TAP-2 producers in women. We found the association of some haplotypes with aneuploidy and the presence of HSIL. We showed the increase of TAP-2 protein in HPV+ and CIN 3 cervical lesions, a physiologic mechanism for eliminating infected and damaged cells. In addition, we addressed the possible mechanism of immune evasion exerted by HPV in modulating the expression of TAP variants, which may favor viral persistence, contributing to the evolution of the disease to malignancy. Further functional studies will confirm the relationship between TAP gene variants and TAP2 expression, HPV infection, and disease outcome.The results obtained with the diplotypes formed by the four SNPs in the https://doi.org/10.6084/m9.figshare.21263343, https://doi.org/10.6084/m9.figshare.21265785, https://doi.org/10.6084/m9.figshare.21265848.v1.The data presented in the study are deposited in the Figshare repository, and accession links and DOIs can be found below: The studies involving human participants were reviewed and approved by The Ethics Committee of the Instituto Aggeu Magalhães approved this study under the protocol CAAE:51111115.9.0000.5190. The patients/participants provided their written informed consent to participate in this study.FM, NL-S conceived, designed the study, did the formal analysis, and wrote the paper. FM, MS, NS, TG, RG, FG, LP conducted the experimental work. FM, LP, MR, SW, MM followed-up patients and performed cytopathological and coloscopy evaluations. NL-S and ED applied for financial support and managed the project. All authors contributed to the article and approved the submitted version.


Human papillomavirus (HPV) contributes to sexually transmitted infection, which is primarily associated with pre-cancerous and cancerous lesions in both men and women and is among the neglected cancerous infections in the world. At global level, two-, four-, and nine-valent pure L1 protein encompassed vaccines in targeting high-risk HPV strains using recombinant DNA technology are available. Therapeutic vaccines are produced by early and late oncoproteins that impart superior cell immunity to preventive vaccines that are under investigation. In the current review, we have not only discussed the clinical significance and importance of both preventive and therapeutic vaccines but also highlighted their dosage and mode of administration. This review is novel in its way and will pave the way for researchers to address the challenges posed by HPV-based vaccines at the present time. Papillomaviridae  is reported as one of the the most prevalent sexually transmitted infection (STI), having a significant detrimental impact on the social life of an individual . Most ofaviridae . The ciraviridae Figure 1HPVs infects basal cells of epithelial tissue and causes either malignant or benign lesions of the mucosal layer of the gastrointestinal tract, upper aerodigestive tract and the anogenital tract, and skin . CervicaCervical cancer caused primarily by HPV infection is one of the largest causes of death in women in southeast Asia . HPV16 iAs observed in numerous global areas, the three types of currently licensed HPV vaccinations include nine-valent, trivalent, and bivalent vaccines, which are effective in lowering HPV infection and HPV-related illness incidence . GardasiIn the current review, we have drawn a comparison between the currently available vaccines, Cervarix and Gardasil, emphasizing the clinical importance of vaccines that are being produced in recent times and shed light on their ongoing clinical trials. Moreover, we have discussed the various therapeutic vaccines and mentioned the challenges faced concerning vaccine coverage, the safety issues of the vaccines, dosage, and the administration routes. Finally, we presented the alternative therapies that are available to tackle HPV infection. This review provides a detailed idea about molecular therapies and cellular landscaping of the vaccines.The vaccines against HPV are constituted with the aid of recombinant technology using empty protein shells, called virus-like particles (VLPs) of the major capsid protein of papillomavirus, L1  Figure. The recGardasil, manufactured by the American company Merck & Co., is a quadrivalent HPV vaccine and was among the first to be approved by the FDA . GardasiSimilarly, Cervarix, manufactured in the UK by GSK, is a bivalent HPV vaccination that was licensed by the European Medicines Agency (EMA) in 2007 and the FDA in 2009. Cervarix offers protection against the most frequent HPV oncogenic genotypes (types 18 and 16), which are responsible for roughly 70% of cervical malignancies . As an ain situ (AIS), cervical intraepithelial neoplasia 3 (CIN 3), and CIN 2 effectively  vector that expresses HPV16 E6/E7, CIN 2/3, IL-2, in clinical response . At the Lactobacillus plantarum, Listeria monocytogenes, and Lactococcus lactis). A clinical phase I study of Listeria-based vaccine (Lm-LLO-E7) in 15 cervical cancer patients showed an unadorned effect in 40% of patients (L. monocytogenes (Lm) is an auspicious live vector that acts as an adjuvant to design and enhance the effect of the vaccine by inducing the macrophages through antigen processing via MHC I and MHC II pathways  displayed a 70% decrease in abrasion to CIN 2. All the patients showed positive outcomes without any adverse side effects of HPV E7 cell-mediated immunity in disease eradication  that targeted HPV antigens through DCs was used, resulting in E6/E7-specific induced IFN-g-producing T-cell response in cervical intraepithelial neoplasia 3 (CIN 3) patients. After 36 weeks of therapy, the proliferation of polyfunctional HPV-specific CD8 T cells, enhancement of cytolytic activity, and the synthesis of effector molecules without any severe side effect at any dose was observed . ® has been clinically tested for non-small cell lung and prostate cancer for stimulated long-term humoral and cellular immune responses  from infected donor tissue or pathological sample . The bionfection , 129.HPV16/18-positive advanced cervical cancer patients treated with HPV16 E6 (arm A) or HPV E7 peptide (arm B) showed evoked immune response, with 63% for HPV16 E6 and 58% respectively Table 2.DC-based whole-cell HPV vaccination is a common and emerging therapeutic vaccination to treat virus-induced cancers . DC-basein vivo conditions, amplified expression of immune modulator proteins was reported in tumor cells succeeding in HPV vaccination. Activation of specific genes for the cytokine IL-2, IL-12, and granulocyte-macrophage colony-stimulating factor (GMCSF) was noticed. However, in clinical trials, these vaccines have been examined to suppress cancer progression, especially for the E6 and E7 malignant tumors. In a clinical study of patients having metastatic melanoma, the instant response of whole cell vaccination along with tumor regression in 50% of patients was reported  showed a 98% (CI 86–100) efficacy against an HPV16- or HPV18-susceptible population having cervical intraepithelial neoplasia grade 2 or 3 and adenocarcinoma under in situ conditions  or subcutaneously (SC) to evoke the immune system and generation of antigen-specific antibodies . Skin isGlobally, the market for the HPV vaccine has been growing and numerous commercially available products are clinically used to cause inhibition of carcinoma related to HPV infections . In 2006In 2013, an HPV vaccination statistical analysis of the United States showed that 57% of the population aged 13 to 17 years take a single vaccination dose and 38% take triple doses. However, in Australia, 9-year HPV vaccination data from 2007 to 2015 displayed that in the female population of age 15 years, the vaccination rate for single dose was 85.6% and that for the triple dose was 77.4%, which was significantly much higher than that in the United States. Therefore, many non-government agencies are conducting awareness programs, sponsoring medical campaigns, and connecting with society to campaign about HPV vaccinations. Based on this program information, data collection, and outcome, it was reported that HPV illness is mostly found in developing countries, and only 15% of these countries have executed a health program for HPV vaccination .The vaccination trials for 10 years at pre-licensure and post-licensure help monitor and assess confirmation of the commercialized HPV vaccination . It was via HPV infection [RB1] [LK2]. In the current review, we have covered the currently available vaccines against HPV infection and the vaccines that are under clinical studies against HPV  funded by the Ministry of Education (NRF-2020R1I1A2066868), the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1A5A2019413), a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HF20C0116), and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HF20C0038).The authors acknowledge the respective departments and institutions for providing facilities and support. The authors would also like to appreciate Biorender.com for their software.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cervical Cancer (CC) is a vaccine-preventable disease, and it is treatable if diagnosed early and managed properly. However, it is the fourth most common cancer in women worldwide with about 604,127 cases and 341,831 deaths in 2020. In Italy, it represents the fifth most common cancer in women under 50 years of age with about 2,400 new cases in 2020. The CC elimination is today a global public health goal published by the World Health Organization (WHO) in 2020 and a commitment of the European Union that has included it in Europe's Beating Cancer Plan. Therefore, urgent action is needed, at international and national level, to implement value-based interventions regarding vaccination, screening and timely management of the disease. Our study aims to describe the state of the art of Human Papilloma Virus (HPV) prevention in Italy and to get a consensus on indicators for monitoring the progress toward CC elimination at national level.The study envisaged the following activities: research and synthesis of the evidence on strategies and actions for CC elimination at regional Italian level; identification of indicators to monitor such strategies/actions; organization of a multi-stakeholder consensus to reach the agreement on main indicators to be used in Italy.As for HPV vaccination coverage, the last Italian available data  showed that it was way below the target (95%) with full cycle vaccination coverage ranging from 6 to 61.7% in female adolescents and from 5.4 to 55.4% in male adolescents (2008 birth cohorts). The coverage rate of CC screening is variable with a range of 61.7–89.6%. Furthermore, coverage rates due to organized screening programs (excluding out-of-pocket screening) shows a range from 20.7 to 71.8%. The mapping of the Italian Regions highlighted an important regional heterogeneity in respect to organizational/operational issue of HPV vaccination and CC screening. Indicators for monitoring CC elimination strategies have been drawn from the Australian experience and distinguished by disease outcomes, vaccination coverage, screening participation and treatment uptake. The highest consensus was reached for the following indicators: CC incidence; detection of high-grade cervical disease; CC mortality; full cycle vaccination coverage; screening participation; high-grade cervical disease treatment rates; CC treatment rates.The assessment of the current status of CC elimination as overarching goal beyond the achievement of vaccine, screening and treatment targets represents the first step for the identification of interventions to be implemented to accelerate the path toward CC elimination. Based on this and following the WHO call, a value-based approach is proposed to untangle the full benefit of HPV-related cancers elimination strategies and identify priority and best practices. Human Papilloma Virus (HPV) has been recognized as a carcinogenic agent since 1995  and HPV More than 95% of CC is due to the HPV .About 604,127 new CC cases are diagnosed annually worldwide with about 342,000 deaths each year (estimations for 2020); this cancer represents the 4th leading cause of woman cancer and the 2nd most common cancer in women aged 15–44 years in the world .CC is a preventable disease, through HPV vaccination and screening, and it is treatable if diagnosed early and managed properly . HoweverFollowing the WHO call, in February 2021, the European Commission (EC) published the Europe's Beating Cancer Plan with the aim of promoting a common fight against the cancer in all European Union (EU) Member States. One of the proposed initiatives concerns precisely the elimination of CC and other HPV-related cancers through the achievement of the 90-90-90 targets by 2030 .In Italy, the current National Immunization Plan (NIP) 2017–2019 provides free HPV vaccination in girls and boys aged 12 years of age and sets a 95% target vaccination coverage . AdditioIn a current context characterized by increasing economic pressure, health systems worldwide face challenges related to the need to ensure access to high quality healthcare for all citizens. Therefore, evidence-based tools to support a value-based decision-making process are needed also in the prevention field , 15. Undwww.salute.gov.it), of the National Institute of Health (www.epicentro.iss.it) and of the National screening observatory  -, regional websites, and the website of the Italian Group for Cervical Cancer Screening (GISCi) (www.gisci.it), were queried using the following search terms: HPV elimination strategies, HPV vaccination uptake and cervical screening. The mapping process was performed by six researchers independently. Then, the items reported in A two-pronged method was used to conduct the study. First, a search of documents and data on HPV vaccination and CC screening policies and strategies in the 20 Italian regions was conducted from June 2021 to March 2022. For this purpose, institutional websites—such as those of the Ministry of Health , an otolaryngologist and a general practitioner, all referents of their respective scientific societies.The board was involved in two virtual meetings and was requested to answer an online survey launched through Google Platform in between the two meetings. In the first virtual meeting, in December 2021, the research working group shared collected information and data and introduced the indicators to monitor CC elimination in Italy. Regarding indicators, the Australian report on progress toward the elimination of CC was considered . AustralFor these reasons, the 11 Australian indicators grouped into four components  were proposed to the group of Italian experts in order to evaluate their applicability and usefulness in Italy.The online survey was aimed at collecting experts' positions in respect to the utility of the 11 indicators (binary response: yes/no). The survey was live for four weeks to allow all experts to take part and a reminder was sent one week prior to the deadline to ensure maximum participation.In the second virtual meeting, results of the survey were shared and a final agreement was reached though a plenary discussion led by three experts, scientific managers of the project .These activities were completed on March 9, 2022.The main results of our study are reported in the following sections: HPV vaccination in Italian regions, screening for CC in Italian regions, and results from the experts' consultation.The results of the mapping of policies and strategies in Italian regions are summarized in Despite the efforts to reach the goal of 95%, the last Italian available data  showed that vaccination coverage was way below the target with full cycle vaccination coverage ranging from 6 to 61.7% in female adolescents (2008 birth cohort), and from 5.4 to 55.4% in male adolescents (2008 birth cohort). The screening coverage data refer to 2020, except for Lombardy whose available data are updated to 2019. The coverage rate of CC screening is variable  with a rAt the time of our analysis, the transition from the Pap test to HPV-DNA as the primary test for women aged 30-35 proposed by the National Prevention Plan 2020–2025  was stilAs of January 2022, target populations for HPV-DNA test differ across the regions. In 13 (65%) regions, HPV-DNA testing is offered to women older than 30 years of age, whereas in seven (35%) the target population is represented by women older than 35 years of age. Recently, the Puglia region disclosed that it will perform the HPV-DNA test starting from the age of 25 (start of the program from September 2022).At the time of data analysis, the presence of a regional coordination for the screening is evidenced in all regions and A.P. except for Campania region.The presence of a regional DTCP specific for CC management was available in eight regions (38.1%).Eventually, regarding the rescheduling of the screening for women vaccinated with a complete cycle, within the National Prevention Plan 2020–2025  is givenThe response rate to the online survey was 100%. The results regarding the agreement on the utility of the 11 indicators drawn from the Australian experience are reported in This paper reported the status of vaccination and screening policies and strategies in Italy highlighting two main important issues: the first one is that Italy must still work to achieve the targets of the WHO Global Strategy whereas the second one is that Italian regions show an undue variability that might slow down the achievement of the targets.Considering the actions proposed by the WHO strategy and in particular the goal of vaccinating 90% of girls with HPV vaccine by age 15 years, our data shows that if the provision of vaccination to the target group is satisfied in all Italian regions, coverages are still not optimal. Furthermore, referring to the other NIP indications, namely vaccination in MSM and in women aged 25 years old, only slightly more than half of the regions offer the HPV vaccination to the MSM, and still not all regions actively offer vaccination at the first screening.However, it is important to point out that some Italian regions have extended the vaccination to other targets at risk, such as, for example, women treated for HPV-related lesions.On the contrary, with respect to the possibility to access vaccination in co-payment all regions are aligned, with someone even providing for the extension to the male population. In addition, worthy of note is to report that, at the time of our analysis, two Italian regions (Lombardia and Piemonte) reserve free of charge vaccination to life for women who have returned to the primary target, a strategy that can facilitate the improvement of vaccination coverage.Nevertheless, as vaccination coverage in Italy is still very far from the 90% target set by WHO it is necessary to implement targeted actions aimed at implementing health education, as also proposed in the WHO strategy .Indeed, the Italian national health system should work on the integration of different approaches, including personalized reminders, information and educational activities aimed at increasing adolescents', parents' and healthcare professionals' (HCPs) awareness and knowledge about HPV infection and vaccination, training programs for HCPs on communication strategies with parents and adolescents, and facilitated access to vaccination also including vaccination programs in schools  as done Australia, thanks to the primary and secondary prevention strategies implemented , 20, is In respect to screening, in Italy, the coverage is largely variable across regions and A.P. Furthermore, considering that the objective set by the WHO refers to the HPV-DNA test, it must certainly be highlighted that yet not all the Italian regions have completed the process of implementation of the HPV-DNA test within screening programs. Moreover, also in the regions where the HPV-DNA test is offered, an extension of 100% is not always achieved. It should be also noted that adherence to the screening with HPV-DNA testing is still extremely variable among different Italian regions. It follows that even in respect to screening there is still a lot to work. Surely greater regional coordination action, currently present in almost all regional realities, would allow for a more homogeneous offer.Despite the effectiveness and cost-effectiveness of prevention interventions, investment in disease prevention remains low in many countries . Among tAccording to this value-based perspective, the involvement of all stakeholders—governments, scientists, healthcare professionals, patients and citizens, providers and industries—is the key to implement high-value health care . SimilarThe lack of nationwide data on the whole HPV-related diseases epidemiology and treatment indicators could undermine the assessment of the quality and the performance of the health system. In fact, a fundamental action for a proper governance of healthcare and health systems as a whole is the identification and the routine use of indicators, as done in Australia for monitoring CC elimination. Our survey with the experts revealed the utility and applicability of the Australian indicators also in Italy, even though some critical issues have been pinpointed in respect to the availability and access to data in particular in respect to disease outcomes and treatment. In this respect, the active and informed involvement of all relevant stakeholders  will plaIn September 2022, the document “Roadmap to accelerate the elimination of cervical cancer as a public health problem in the WHO European Region 2022–2030” was also published with the aim to implement the Global strategy to accelerate the elimination of CC as a public health problem in the European Region . This doOur study is in line with the guiding principles of the WHO European Region roadmap, in particular with regard to the definition of indicators for monitoring CC elimination strategies.The importance of indicators was also emphasized in the Europe's Beating Cancer Plan. In fact, the European Cancer Inequalities Registry  was creaAccording to data reported by the European register, Italy is with France and Bulgaria among the European countries with the lowest HPV vaccination coverage. On the other hand, for the screening indicator, Italy in line with the European average .The Australian experience and our study emphasized that attention should be paid also to other indicators to comprehensively monitor the attainment of the targets proposed by the Global strategy for the CC elimination.Our study has some limitations. First, collected data are not updated to the last year. Furthermore, a selection bias could not be completed ruled out even though the mapping process was performed by six researchers independently and based on specific criteria. Eventually, the heterogeneity of data limits the possibility to further elaborate on information and issue more definite findings. Nevertheless, in our opinion, this first Italian regional mapping on prevention interventions for the CC control could help bringing forward the assessment and the appraisal of the health policies in this field from the point of view of both academic research and supranational, national and local decision-makers.The mapping of the Italian Regions highlighted that HPV vaccination coverage and cervical screening coverage are still too low to achieve CC elimination by 2030; furthermore, an important regional heterogeneity was shown in respect to primary and secondary prevention policies, strategies and implementation status. Therefore, our study highlighted room for improvement regarding several issues, as the highlighted heterogeneity imply great differences in terms of access and equity.The assessment of the status of achievement of vaccination, screening and treatment goals and the identification and constant assessment of specific indicators for monitoring the progress toward CC elimination are fundamental actions to be able to respond to the WHO call. To achieve the goals proposed by the global strategy for the CC elimination, all available means must be used, also in Italy, focusing on a comprehensive approach in favor of value-based effective interventions of prevention and best practices to be implemented at regional level.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Conceptualization and methodology, project administration, and funding acquisition: GEC. Definition of the search strategy, supervision, and data review: GEC and CdW. Search of documents on HPV vaccination and CC screening policies and strategies in Italian regions: MTR, FD'A, CC, MS, RM, and AP. Data analysis: MTR and FD'A. Writing—original draft preparation: MTR, FD'A, GEC, and CdW. Writing—review and editing: GEC, RR, RPdV, and CdW. All authors have read and agreed to the published version of the manuscript.This study was financed by an unconditional grant from MSD Italia S.r.l. The sponsor had no role in conducting or designing the study, collecting, analyzing, interpreting the data, and the writing the manuscript. Universitá Cattolica del Sacro Cuore contributed to the funding for this publication with funds from UCSC-Line D.1 2022.All the authors worked as consultants of VIHTALI , Spin-Off of Università Cattolica del Sacro Cuore , and which received funds from MSD Italia S.r.l. The sponsor had no role in conducting or designing the study.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.