Cervical cancer is an international public health issue. Nearly all cases of cervical cancer are caused by the human papillomavirus. The HPV vaccine prevents more than 75% of cervical cancer. The extent to which adolescent girls' knowledge and uptake of the HPV vaccine have to be investigated in order to build effective promotion strategies and increase the uptake of the vaccine. The evidence that is currently available in this area is controversial and inconclusive. Hence, this study has estimated the pooled proportion of good knowledge, positive attitude, and uptake of the HPV vaccine and its associated factors among adolescent schoolgirls in Ethiopia.2 statistics and Egger’s test, respectively. The PROSPERO registration number for the review is CRD42023414030.PubMed, Google Scholar, AJOL, ScienceDirect, and DOAJ were used to search relevant studies. A total of 10 studies were included. The data were extracted by two reviewers using Microsoft Excel and exported to STATA Version 17 for analysis. A random effects model was applied during the analysis. Heterogeneity and publication bias across the studies were evaluated using IA total of eight studies comprising 3936 study participants for knowledge and attitude and five studies with 2,481 study participants for uptake of HPV were used to estimate the pooled proportions of good knowledge, a positive attitude, and uptake of the HPV vaccine, respectively. The pooled proportions of good knowledge, positive attitude, and uptake of the HPV vaccine were 55.12%, 45.34%, and 42.05%, respectively. Being an urban resident , having good knowledge , and a positive attitude , were significantly associated with the uptake of the vaccine.The pooled proportions of good knowledge, a positive attitude, and uptake of the HPV vaccine were low in Ethiopia. Being an urban resident and having good knowledge and a positive attitude towards the HPV vaccine were significantly associated with the uptake of the HPV vaccine. We recommend increasing adolescent knowledge, positive attitudes, and uptake of HPV vaccination through school-based seminars, health education, and community mobilization.The online version contains supplementary material available at 10.1186/s12905-023-02412-1. Cervical cancer is an international public health issue . GloballNearly all cases of cervical cancer and over 70% of malignancies affecting the vagina, vulva, and oropharynx are caused by the human papillomavirus (HPV) . There aThe HPV vaccine is the most important preventive measure for HPV-associated cancers  and prevThe bivalent HPV vaccine was introduced in Ethiopia on December 3rd, 2018 for girls who are 14 years of age . The vacEven though the HPV vaccine is proven to reduce cervical cancer incidence, many factors influence HPV vaccine uptake. The rate of HPV vaccine uptake has been shown to vary by knowledge and attitude towards HPV vaccine, in addition to other factors such as adolescents' age, social influence, lack of health education on HPV vaccine, and parents' educational status –21. UndeThough many efforts have been made to study knowledge, attitude, and uptake of the HPV vaccine, primary studies reporting the proportion of good knowledge, a positive attitude, and uptake of the vaccine are fragmented. The evidence that is currently available in this area is controversial and inconclusive. Therefore, this meta-analysis aimed to estimate the pooled proportion of good knowledge, a positive attitude, and uptake of the HPV vaccine and its associated factors in Ethiopia. The result of this study will provide important input for policymakers to improve the uptake of the HPV vaccine by creating a positive attitude and good knowledge about the vaccine.This systematic review and meta-analysis aimed to estimate the pooled proportion of good knowledge, positive attitude, and uptake of the human papillomavirus vaccine and associated factors among adolescent schoolgirls in Ethiopia. The protocol of this meta-analysis was registered at  PROSPERO with a registration number of CRD42023414030. A standard PRISMA checklist was used to present the findings , ScienceDirect, and DOAJ). Initially, studies were exhaustively searched by using the full title  and keywords . The Boolean operators "OR" or "AND" were used in conjunction with or independently of one another to connect these keywords to form search terms. In addition, reference lists for all included research were reviewed to identify any missed studies. Furthermore, institutional repositories of Ethiopian universities, primarily those in Gondar, Jimma, Addis Abeba, and Harameya University, were examined for unpublished papers. Searching details for PubMed were as follows: ("knowledge"[MeSH Terms] OR "knowledge"[All Fields]) AND ("attitude"[MeSH Terms] OR "attitude"[All Fields]) AND ("uptake" [All Fields] OR "Practice"[All Fields] OR "utilization" [All Fields]) AND ("papillomavirus vaccines"[MeSH Terms] OR ("papillomavirus"[All Fields] AND "vaccines"[All Fields]) OR "papillomavirus vaccines"[All Fields] OR ("human"[All Fields] AND "papilloma"[All Fields] AND "virus"[All Fields] AND "vaccine"[All Fields]) OR "human papilloma virus vaccine"[All Fields]) AND associated[All Fields] AND factors[All Fields] AND ("adolescent"[MeSH Terms] OR "adolescent"[All Fields]) AND  AND ("Ethiopia"[MeSH Terms] OR "Ethiopia"[All Fields]). The search period was restricted between January 1, 2015 and December 23, 2022  framework.Condition (Co): We included studies that examined at least one or more of the following key outcomes: 1) the proportion or level of good knowledge; 2) the proportion or level of a positive attitude; 3) studies that report HPV vaccination coverage; 4) studies that examined factors associated with HPV vaccine uptake among adolescent schoolgirls.Context (Co): We included studies that were conducted in Ethiopia.Population (Pop): Studies that were done among female adolescents (9–14 years)Study Design: We included cross-sectional and/or case–control studies.Publication status: Both published and unpublished (pre-print) studiesLanguage: We included all studies written in the English language.This systematic review and meta-analysis included studies that fulfilled the following criteria:Qualitative studies and studies with a different study population as well as different outcomes in terms of interests were excluded.The primary outcome was HPV vaccine knowledge, attitude, and uptake rate among adolescent schoolgirls. The secondary outcomes were factors associated with HPV vaccine uptakes such as background characteristics, HPV vaccine attitudes, HPV knowledge, and HPV vaccine availability.Uptake of the HPV vaccine was defined as the proportion of those adolescent schoolgirls who had received at least one dose of the HPV vaccination.Knowledge of adolescent schoolgirls towards HPV vaccine was measured by asking 7 knowledge-related questions and a score greater than the mean was classified as good knowledge , 22–26.Attitude towards HPV vaccination was measured by using seven attitude-related questions and participants with scores above the mean were classified as having a ‘positive attitude’ towards HPV vaccination , 22–27.All studies retrieved through the search were exported to Endnote version 7  software. After duplicates were removed, two authors (DA and NG) independently assessed titles and abstracts for eligibility criteria using Endnote software and manually. During the screening, each study that did not fulfill the inclusion criteria was excluded, and the reasons for the exclusion were documented. A consensus approach with input from the third reviewer (YB) was applied to resolve disagreements. Finally, a modified Newcastle–Ottawa quality assessment tool adapted for cross-sectional studies was used to assess the quality of the studies that fulfilled the inclusion criteria . The quaA predesigned data extraction form was used to extract all necessary data. The data extraction form was prepared in Microsoft Excel and contains the first author's name, publication year, study region, study setting, study period, sample size, the proportion of good knowledge, positive attitude, and uptake of HPV vaccine, as well as an adjusted odds ratio (AOR) with a 95% confidence interval for significant risk factors for the uptake of HPV vaccine S Table. TA random effects model was used to determine the pooled proportion of good knowledge, positive attitude, and uptake of the HPV vaccine and associated factors among adolescent schoolgirls in Ethiopia –30. SubgA total of 109 studies were identified from different international databases and institutional repositories at Ethiopian universities. The Endnote 7 reference manager was used to screen retrieved studies. Then, a total of 99 articles were removed because of unrelated papers to the HPV vaccine, duplicate findings, different study populations, and different outcomes of interest. Ten studies that met the inclusion criteria were finally included in this analysis  and primary schools, with pooled proportions of 70.34% and 78.55%, respectively. Regarding attitude, the pooled proportion of positive attitude was highest in studies conducted at SNNPR and primary schools, with proportions of 53.37% and 52.36%, respectively. Concerning the uptake of the HPV vaccine, the pooled uptake of the vaccine was found to be 42.28% in studies conducted in the Amhara region and 55.50% in studies conducted at primary schools  report in Brazilian  and TanzThe uptake of the HPV vaccine among adolescent schoolgirls in Ethiopia was also higher than in the Demographic and Health Survey report in Uganda. A multilevel analysis from the 2016 Demographic and Health Survey data in Uganda indicated that only 22% of girls aged 10–14 years uptake the HPV vaccine . The varOn the other hand, the uptake of HPV vaccine was lower than a study finding in the USA, which has 62.8% HPV vaccine coverage . This coThis study found that having good knowledge about the HPV vaccine was significantly associated with the uptake of the HPV vaccine. Evidence from a meta-analysis study supports the current finding . This fiFurthermore, this study also found that having a positive attitude about the HPV vaccine was significantly associated with the uptake of the HPV vaccine. A systematic review and meta-analysis study supports the current finding . Other eLastly, being an urban resident was significantly associated with the uptake of the HPV vaccine. This could be justified because adolescent schoolgirls from urban areas may be more aware of the vaccine's benefits, which may have influenced them to take the vaccine. This finding suggests the need for improving health promotion in rural settings and providing access to the HPV vaccine for all adolescents in rural areas.This study should be interpreted in light of the following limitations: The lack of studies from some regions might affect the generalization. In addition, there was heterogeneity across studies, which might affect the pooled estimate of good knowledge, a positive attitude, and the uptake of the human papillomavirus vaccine. Despite doing sub-group analysis and sensitivity analysis to handle the source of heterogeneity, the possible source of heterogeneity was not identified.. Future research should involve investigations of general populations' and parents' knowledge and perception of the HPV vaccine and its effect on the uptake of the HPV vaccine using a mixed-method design.The pooled proportions of good knowledge, positive attitude, and uptake of the HPV vaccine among adolescent schoolgirls were low in Ethiopia. The uptake of the HPV vaccine was significantly associated with good knowledge, a positive attitude, and living in an urban area. We recommend increasing adolescent schoolgirls’ knowledge, positive attitudes, and uptake of HPV vaccination through school-based seminars, health education, community mobilization, mass media, and school mini-media with an emphasis on rural areas. Furthermore, the federal Ministry of Health should design different vaccine delivery strategies for each targeted population and expand the HPV vaccination programs to include all young femalesAdditional file 1.Additional file 2.Additional file 3.Additional file 4.


Cochrane Reviewers’ Handbook and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2009 guidelines. Moreover, we assessed the quality of each study using QUADAS-2. Results: Twenty-five studies were included in the final analysis. Patients after the treatment of HPV-related gynecological diseases have a significantly higher risk of AC (mean standardized incidence ratio (SIR) = 5.387, mean incidence risk (IR) = 0.096%, mean IR per 100,000 person–years = 10.37) and AIN (mean IR = 23.683%) compared to the population risk. Conclusions: patients with HPV-related gynecological diseases should constitute a group for which an appropriate primary and secondary screening for AC should be introduced.Background: The most important causative agent of neoplasms in the anogenital area is the human papillomavirus (HPV). Due to the anatomical proximity of the genital and anus area and the ease with which HPV infection is transmitted, it seems that patients after the treatment of HPV-related gynecological diseases may have an increased risk of developing a second HPV-related neoplasm anal cancer. The aim of this study was to determine the risk of anal intraepithelial neoplasia (AIN) and anal cancer (AC) among patients after the treatment of HPV-related gynecological diseases. Methods: We conducted a comprehensive review of the available literature from multiple databases. The study was performed following  In 2020, there were 50,865 cases of anal cancer diagnosed worldwide. The cumulative incidence risk of this neoplasm is 0.07 for women and 0.06 for men . HIV-posHPV-related gynecological diseases (HPV-RGDs) are those in which HPV plays a significant role in the pathophysiology. This group includes cervical cancer (CC), which is nearly entirely dependent on HPV infection, vaginal cancer (VaC) and vulvar cancer (VC), which are related to HPV infection in about 78% and 24.9% of cases, respectively , along wIt has been investigated that HPV has the greatest affinity for zones where the epithelium of one type merges into another of a different histological feature, i.e., as in the cervix, where the squamous epithelium joins the one-layer glandular epithelium in the transformation zone . HPV intAnother histologically similar region in the human body is the anal canal, where there is a transformation zone between the glandular and squamous epithelium at the level of the dentate line. Due to its anatomical proximity, this place seems to be particularly vulnerable to the transmission of HPV from infected gynecological organs and, as a result, susceptible to anal intraepithelial neoplasia (AIN) or anal cancer (AC) development. This has been supported by the data of Hernandez et al., which showed that in the case of Hawaiian patients with cervical HPV infection, 13% had concurrent anal HPV infection . MoreoveIn the available research, HPV type 16 was responsible for infections in the perianal area and the anal canal in a subgroup of HIV-negative women in the overwhelming number of cases (about 85%). The second-most common was HPV type 18 followed by HPV types 31/33/45/52/58 . The freConsidering all of the above facts, we formed a research hypothesis that patients diagnosed with HPV disease related to gynecological organs have an increased risk of developing AIN or AC due to the facility of the transmission of HPV infection into the anal canal.The main aim of this study was to determine the risk of AIN/AC in HPV-RGD survivors. The secondary aim was to evaluate which HPV-RGD increases the risk of AIN/AC the most.In order to determine the risk of AIN/AC in the group of HPV-RGD survivors, a systematic review of the literature was performed in accordance with the criteria outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 updated guidelines . A revieThe search strategy consisted of three stages, each conducted by both authors separately. In the first stage, we screened the titles using keywords enclosed in Inclusion criteria: peer-reviewed prospective and retrospective cohort studies, randomized and non-randomized controlled trials; studies about the risk of AIN or AC presented as an indicator of standardized incidence ratio (SIR), incidence risk (IR), or IR per 100,000 person–years (PYs); studies where the analyzed group of participants were survivors of HPV-RGD; studies where the full text of the manuscript was available in English. Exclusion criteria: non-peer-reviewed publications, letters to the editors, systematic reviews, meta-analyses, case reports or case series; non-human subject studies; studies lacking adequate statistics ; studies where there were doubts whether the diagnosis of AIN/AC was confirmed histopathologically.Data were extracted by both authors independently. Extracted data included SIR  term was separately used in publications, this was considered as cervical intraepithelial neoplasia (CIN) 3; thus, CIN3 and CIS were analyzed as one entity. Similarly, the term “low-grade squamous intraepithelial lesion” (LSIL) was considered equal to CIN 1, and “high-grade squamous intraepithelial lesion” (HSIL) was considered equal to CIN 2 or 3. This is in accordance with the current terminology .When two or more publications used data from the same cancer registry and the period of research was similar, for final calculation, we selected data from only one of them (the most numerous), so as not to duplicate the number of patients with a given primary cancer/precancer.To determine the bias risk, each article was assigned to a group of low, high, or unclear bias risk in accordance with the QUADAS-2 recommendations . When deThe search resulted in 7119 records. After the first stage of screening, six thousand nine hundred and fifty-seven records were excluded because of being marked as not human subjects, referring to basic sciences, different types of research than the original research, referring to other malignancies than AIN/AC, or being duplicated. Additionally, one hundred and twenty-five records were excluded after the second stage of screening because the subject of research did not concern the risk of AIN/AC, the results for different diseases were not divided, or the reasons mentioned in the previous stage of screening. Finally, after reviewing the full text of the selected articles, we included 25 articles in our research ,39,40,41The study characteristics of the selected articles are presented in The total number of people diagnosed with CC was 177,984; VC, 9572; and VaC, 2733. In the case of precancerous lesions, there were 110,243 patients after the treatment of CIN 1; 52,151 after the treatment of CIN 2; 447,739 after the treatment of CIN 3; 234 after the treatment of VIN 1; 16 after the treatment of VIN 2; 108 after the treatment of VIN 3; 104 after the treatment of VaIN 1; 13 after the treatment of VaIN 2; and 30 after the treatment of VaIN 3. In several articles, the authors did not divide precancerous lesions into particular subcategories, and therefore, the total number of cases of CIN, VIN, and VaIN was counted. The total number of patients with a medical history of CIN was 682,991 . The total number of patients with a medical history of VIN was 358 and with VaIN was 147. Several of the US studies analyzed data from a similar period from the National Cancer Institute Surveillance, Epidemiology, and End Results (SEER) Program. Moreover, two publications acquired data from the Finish Cancer Registry and three from the Swedish Cancer Register. In these cases, for the final calculation, we used only data from one publication (the one with the most cases). Additionally, data from Chaturvedi et al.’s publication were not included in the final calculation because they are based on 13 popu-lation-based cancer registries in Denmark, Finland, Norway, Sweden, and the United States—in this case, the risk of the duplication of patients was too high.Standardized incidence ratio (SIR). The mean SIR for AC in CC survivors was 3.814 (95% Cl: 1.21–6.41). The mean SIR for AC in VC survivors was 14.55 (95% Cl: 0.15–24.4). The SIR for AC in VaC survivors was 1.8 (95% CI: 0.2–5.3) . The mean SIR for AC in CIN 3 survivors was 5.701 (95% Cl: 2.23–19.2). The mean SIR for AC in CIN 2 survivors was 1.895 (95% Cl: 0.09–4.1). There was no information about SIR for CIN 1, VIN, or VaIN survivors in any of the articles we reviewed. The mean SIR for AC in CIN survivors, without dividing into specific degree of intraepithelial neoplasia, was 4.563 (95% Cl: 0.12–19.2). The mean SIR for all HPV-RGD survivors was 5.387 (95% Cl: 2.99–7.78). Only one study provided data about SIR for AIN and it was 6.68 (95% Cl: 3.64–12.25) in CIN 3 survivors. Incidence risk (IR). In four studies, the authors calculated the IR [d the IR ,25,29,39Incidence risk per 100,000 person–years . The mean IR per 100,000 PY of AC in CC survivors was 9.73 (95% Cl: 8.03–11.43); in VC survivors, it was 37.98 (95% Cl: 22.64–53.32); in VaC survivors, it was 11.78 (95% Cl: 0–29.15); in CIN 3 survivors, it was 5.78 (95% Cl: 5.18–6.38); and in CIN (1–3) survivors, it was 5.37 (95% Cl: 4.85–5.89). The mean IR per 100,000 PY of AC for all HPV-RGD survivors was 10.37 (95% Cl: 9.66–11.08). Only one study had sufficient data to calculate IR per 100,000 PY of AIN, a paper by Ebisch et al., where the risk of AIN 3 in CIN 3 survivors was 6.34 (95% Cl: 5.10–7.90). The summarized results of SIR, IR, and IR per 100,000 PY of AC and AIN after primary diagnosis of specific HPV-RGD are presented in In our study, we confirmed that survivors of HPV-RGD have a higher risk of both AIN and AC in comparison to the general population. Survivors of VC have the highest risk of developing AC, followed by CC survivors and VaC survivors. The risk of developing AC in patients with a history of CIN increased with the severity of CIN—the highest was for CIN 3 and the lowest was for CIN 1.We hypothesize that the risk for VC survivors might be higher than for CC survivors because current CC treatments, mostly including surgical approaches, usually result in total HPV elimination. Furthermore, HPV subtype differences may also be significant. A study by Saraiya et al. showed that the spectrum of the most common HPV subtypes and their proportions detected in the tissues of these cancers differed .Data on the risk of developing AIN after HPV-RGD are much scarcer compared to AC data. Only four studies analyzed the risk of developing AIN after HPV-RGD; in addition, all of them concentrated on patients with precancerous lesions: CIN, VIN, and VaIN ,20,25,38Although we have far less data on the risk of AIN in people with HPV-RGD than on AC, it is evident that the risk of AIN in this subset of patients is substantially higher than AC because HSIL (AIN) is a direct precursor of AC. The large difference between the risk of AIN and AC may be due to the fact that only about one in ten people with AIN develop AC. In a study by Watson et al., on a high-risk group of 72 people with AIN, it was found that 11% of them went to AC in a median time of 42 months. What is more, approximately one-third of this population experienced a decrease in the stage or regression of the disease [The greatest risk of AIN is in the case of a previous diagnosis of VIN, followed by VaIN and CIN. This is most likely owing to the existence of a well-functioning screening for CC, which detects CIN lesions, usually treated using excision of lesions, resulting in HPV elimination . BecauseIn our study review, we showed that patients with a history of HPV-related disease have a significantly increased risk of AIN and AC. These data lead to the concept that these patients, after being treated for the previous HPV-related disease, should be strictly controlled to prevent the development of another HPV-related disease. Unfortunately, currently, there are no recommendations or guidelines that clearly define how such a control should look, how long it should last, and who should be responsible for it. Recently, the International Anal Neoplasia Society (IANS) assembled a Task Force in order to systematize and establish recommendations for AC screening .We think that gynecologists should be responsible for preventing the development of HPV-related disease in the whole anogenital region. A gynecologist is a specialist to whom women regularly schedule for tests of the secondary prevention of CC—cytology and/or a test for high-risk human papillomavirus (hrHPV). The gynecologist takes a swab from the cervix. It is a quick and painless examination, which provides many benefits to patients. More importantly, in the context of this divagation, it is also a perfect moment when a gynecologist can perform a similar procedure and take an additional swab from the anal canal in a group of patients with a particularly high risk of development of AC. Thus, the gynecologist will become the person responsible for detecting such changes and then referring the patient to an anorectal disease specialist in order to implement tertiary prevention.Another important issue that should be raised by gynecologists, in terms of the prevention of other HPV-related diseases, is prophylactic vaccination against HPV. This is applicable even in people of older ages and with HPV-related diseases. It has been shown to be effective in older ages ,48 and iCurrently, the gold standard in AC screening is HRA  Figure . This alDue to the rarity of AC and economic issues, the introduction of routine AC screening, in the case of every patient who has ever been diagnosed with HPV-related cancer or a corresponding precancerous lesion, is impossible even for the richest countries. Therefore, it is important to select the subgroup of patients with the highest risk of AIN/AC and establish cost-effective algorithms concerning AC screening. To do this, the relevant risk factors must be identified.The major issue is the identification of people with disorders in the functioning of the immune system. Patients suffering from HIV, in immunosuppression, or with congenital immunodeficiency disorders constitute a group wherein HPV infection occurs more often and spreads more easily. In this particular group of patients, the time to the development of AIN/AC will be much shorter than in people with a properly functioning immune system ,67. In pSurprisingly, only four of the studies that we have reviewed reported the HIV status of the research participants ,25,27,38Another factor that should be mentioned is the time between the clinical disease and the onset of HPV infection. It has been proven that the risk of CC increases with increasing time since infection in the cervix. The median progression time from CIN 1 to CIN 2/3 is 2–3 years , and theMoreover, the specific type of HPV is another important issue in the case of analyzing the risk of the carcinogenesis process. The time needed for the development of CC in the case of high-oncogenic HPV types  is significantly shorter in comparison with low-oncogenic HPV types. In the case of perianal infection and the development of AC, the pathophysiology is probably similar. However, further research is required to isolate the HPV types with the highest risk of carcinogenesis in the anal canal.The age of the patient is also a valid risk factor. The incidence of AC increases by 2.7% per year, with pronounced increases in age groups 50 years and older . The aveAnother major reason why additional screening in people with HPV-related diseases is important are the problems with the selection of an appropriate method of treatment in people with HPV-related extensive changes in the anogenital area. In such cases, treatment with topical cidofovir or imiquimod may be offered. Unfortunately, their effectiveness is quite low, and relatively, many patients do not tolerate treatment in maximum doses ,78. SurgThe ANCHOR study has proven that the treatment of AIN reduces the risk of future AC in people living with HIV . The nexThe strength of our study is that we performed a systematic review of all relevant publications referring to the subject of research—not only the risk of AC but also what is unique and the risk of AIN in patients who were diagnosed and treated for gynecological HPV-related diseases. Additionally, a critical evaluation of the included articles was performed to provide a level of risk of bias. A summary of all available up-to-date data was also provided. We acknowledge the limitations of this study. The first is that the majority of evaluated publications were based on national registry databases. This kind of research is vulnerable to bias because the potential to under-report or misclassify diseases and procedures is possible. The second is that AC is a rare disease. Moreover, AIN might be significantly under-reported because there are no established screening programs, and HRA, as the best tool to diagnose AIN, is not easily available and is a difficult procedure. Low numbers of both AC and AIN might have a significant impact on the calculation of SIR. The smaller the number of cases of the target disease is, the less precise the SIR calculation is . The thiThe risk of developing secondary AC/AIN is significantly higher in groups of people with primary HPV-RGD than in the general population. Patients who have been diagnosed with VC have the highest risk of secondary AC/AIN. The risk of AIN is much higher in people with HPV-RGD than the risk of AC. Further studies are required to determine the exact risk of AIN in the HPV-RGD subpopulation, particularly in those with CC, VC, and VaIN. Targeted screening programs, including both surveillance  and HPV vaccinations, for AC/AIN, should be developed for patients with HPV-RGD. The true burden of AC/AIN can be determined in prospective studies addressing these specific programs.


According to the SEM, nursing students’ knowledge regarding HPV accounted for 48% of the variance in students’ attitudes. Conclusion: Nursing students’ knowledge regarding HPV vaccination is an important predictor of students’ attitudes regarding HPV.Introduction: One of the most prevalent conditions affecting the vaginal organs is the human papilloma virus (HPV). Human papillomavirus (HPV) knowledge and attitudes have been the subject of numerous studies in Saudi Arabia. However, there are only a few studies that have examined university students’ attitudes and knowledge of the human papillomavirus and the vaccine that is associated with it. Aim: To predict the level of knowledge and attitudes regarding HPV and its related vaccine among undergraduate nursing students. Methodology: This was descriptive cross-sectional research. After being selected from Princess Nourah bint Abdulrahman University’s College of Nursing, 307 nursing students agreed to take part and completed an online survey that was self-administered. Results: The majority of the participants (73.5%) had a low level of knowledge of HPV with a mean score of 2.77 ± 1.78. In addition, more than half of the participating nursing students (57%) had a moderate attitude toward HPV vaccination with a mean score of 51.18 ± 11.16. The study results also verified that there was a highly significant correlation between the nursing students’ demographics and their knowledge and attitudes toward HPV ( Human papillomavirus (HPV) is one of the most common genital viruses. There are one hundred different types of HPV, which spread through skin-to-skin contact, including sexual contact and other types of contact with the genital region, such as touching the genitalia of an infected individual. HPV cannot be spread by coming into contact with objects such as a toilet seat . In most® vaccine from GlaxoSmithKline, Wales; the 4-valent Gardasil® vaccine from Merck Inc., Whitehouse Station; and the 9-valent Gardasil®9 vaccine from Merck Inc., Whitehouse Station, effectively prevent HPV infection and the emergence of cervical cancer [One of the most prevalent malignancies in women is cervical cancer, with developing nations accounting for the vast majority of new occurrences (more than 85%). Invasive cervical cancer and precancerous cervical intraepithelial neoplasia (CIN) are both caused by persistent human papillomavirus (HPV) infection. Between 75 and 80 percent of those who engage in sexual activity will contract HPV at some point in their lifetime. Thankfully, preventive HPV vaccinations have received widespread approval for the primary prevention of cervical cancer. Current research shows that these vaccines, such as the 2-valent Cervarixl cancer ,4,5.Although the majority of HPV infections and precancerous lesions disappear on their own, some persist and develop into aggressive cervical cancer. Cervical cancer is the fourth most frequently diagnosed cancer in women globally and the fourth most common cause of cancer-related death. It is the 10th most prevalent malignancy in Saudi Arabian women between the ages of 15 and 44 ,7. AlthoMoreover, preventative HPV vaccinations for girls ages 11 to 26 have received approval from the Saudi Food and Drug Administration (SFDA) in 2010. Since then, HPV vaccinations have been added to the Saudi National Immunization Schedule’s list of required immunizations for females . In addiA lack of knowledge about HPV and its related vaccine is very common worldwide, especially in Saudi Arabia. A study showed that only 34.5% of Saudi Arabian participants were aware of HPV, compared to 65.5% who had never heard about it. This is a considerably low level of awareness. The prevalence percentage is very high in Saudi Arabia. As studies have shown, 80% of women with cytological abnormalities, >92% of women with cervical cancer, and 31% of the general population are infected with HPV . More thMore than half of the individuals disagreed that HPV is a widespread sexually transmitted disease; furthermore, 63% of participants agreed that the HPV vaccine may prevent warts and cervical cancer, and 53% expressed interest in it. The vaccine was opposed by about 30% of the participants for religious reasons . In termThe human papillomavirus (HPV) is one of the most prevalent STIs, which causes genital infections in about 5.5 million people each year . DependiThe HPV vaccine has been shown to be effective in reducing genital warts and malignancies associated with HPV, particularly cervical cancer . AccordiDarraj’s cross-country analysis (2022)  showed tAnother study showed a positive attitude toward HPV was linked to screening intentions. Informed nurses and midwives might have favorable opinions of HPV vaccination. The recommendation of the vaccine by medical experts will increase its acceptance, according to a comprehensive review of the acceptability of HPV vaccines in sub-Saharan Africa. As a result, effective advice from medical professionals may impact HPV vaccination behavior. The barriers to HPV vaccination according to a group of people consisted of 12 items, such as they thought getting the HPV vaccine was a waste of time, there were no vaccination sites available, and there was not much information available about the HPV vaccine. In addition, they did not know what the HPV vaccine was for, there were no health education programs to promote the HPV vaccination, the vaccine was not necessary. They also said they did not have the money to get the vaccine, they were afraid of the vaccine’s side effects, and there was a risk of contracting the disease . By askiThe HPV vaccine and vaccination in general were viewed favorably by the students. The HPV vaccine was considered to be effective and safe, according to more than 70% of the participants. About 21% of the participants tended to agree that vaccination is not necessary for healthy people. Of the 226 responders, 143 (63.3%) mentioned that there was a possibility of HPV infection even at a young age of 9 to 14 years old. Of the 226 students, 204 (90.3%) emphasized the need for the HPV vaccine to be included in the NIP in the future . The majLow HPV vaccination rates were observed in several studies. It was discovered that just 13.3% of undergraduate students in Hong Kong research had received an HPV vaccination. Only a small number of nurses in Turkey reportedly received the vaccination. The results of this research strongly imply that the issue of low vaccination rates against HPV infection is a global problem. However, health education among medical professionals was found to positively affect the intention to get the vaccine and perhaps suggest it to others in the future in studies conducted in different populations such as Istanbul, Turkey, and South India ,25,26. This study has more recently focused on factors affecting HPV and HPV vaccines, with genital warts seen as a common side effect of some sexually transmitted HPVs, while other side effects can lead to a variety of malignancies, including those of the cervix, vagina, vulva, penis, anus, and oropharynx . A speciOne study by Namakula (2018) reported that cervical cancer is the main consequence of human papillomavirus (HPV) infections. Cervical cancer is the fourth most prevalent cancer in women, affecting 500,000 of them annually and killing an estimated 266,000 people. With an age-standardized incidence rate of 47.5 people per 100,000, Uganda has one of the highest incidences of cervical cancer worldwide. This study evaluated the extent to which female teenagers in the Lira area of Uganda had received the HPV vaccine as well as the contributing factors. A survey of 460 female teenagers was conducted using a mixed-methods technique. The information was gathered through a questionnaire that an interviewer administered. Ten in-depth interviews were conducted and five important sources were spoken with. Uptake was outlined as receiving all three required doses of the vaccination. The respondents’ average age was 13.97 (SD: 1.24) years. There was a 17.61% uptake (81/460). The Lira district had a poor rate of HPV vaccination uptake. Ensuring a steady supply of vaccinations at the vaccination sites, health education aimed at fostering a good attitude about the vaccine, sensitizing teenagers about the vaccine, and undertaking community outreach should be the main priorities of efforts to increase the HPV vaccine uptake .The aim of the study is to predict the level of knowledge and attitudes regarding HPV and its related vaccine among undergraduate nursing students.What is the socio-demographic profile of undergraduate nursing students at Princess Norah bint Abdulrahman University in terms of: age, marital status, GPA, and academic year?What is the level of knowledge of HPV among nursing students?What is the attitude toward the HPV vaccine among nursing students?What are the factors affecting nursing students’ knowledge and attitude regarding HPV?What is the relationship between the nursing students’ demographics and their knowledge and attitude?The researchers aim to assess the level of knowledge and attitudes toward human papillomavirus and its related vaccine among undergraduate nursing students at Princess Nourah bint Abdulrhman University. Specifically, it will answer the following:Alternative hypothesis: There is a correlation between demographics and knowledge and attitudes toward HPV among undergraduate students at Princess Nourah bint Abdulrahman University.Null hypothesis: There is no correlation between demographics and knowledge and attitudes toward HPV among students at Princess Nourah bint Abdulrahman University.Research Design: A descriptive cross-sectional quantitative research design was used.Research Setting: The study setting was the College of Nursing—Princess Nourah bint Abdulrahman University.Population: A total of 1014 students from Princess Nourah bint Abulrahman University made up the study population. Participants were undergraduate nursing students.Inclusion criteria were as follows: undergraduate nursing students (enrolled in the nursing program from the 1st year to the 4th year) at Princess Nourah bint Abdulrahman University.Exclusion criteria included students enrolled in internships, midwifery students, diploma and masters’ students, anyone with a life-threatening allergic reaction to the HPV vaccine or any of its components, anyone with a history of severe allergies or a yeast allergy, and patients with moderate or serious disease, those who are not willing to participate in the study.http://www.raosoft.com/samplesize.html (accessed on 16 March 2023).Sampling: The sample included only undergraduates nursing students at the College of Nursing—Princess Nourah bint Abdulrahman University (n = 307) which represented as follow; 1st year represented 51% (n = 156), 2nd year represented 25% (n = 77), 3rd year represented 13% (n = 39), and 4th year represented 11% (n = 35) from participants with 100% response rate. Based on the Epi Info program calculation, with a margin of error of 5%, the confidence level is 95%, sampling attrition 10%, sample size was calculated. So, from the total population of 1014 nursing students, a sample size of 307 undergraduate nursing students was included in the study . The resSampling Technique: The type of sampling was non-probability convenience.The validated Knowledge and Attitude toward HPV questionnaire (KAHPVQ) which was developed by White et al., 2016 and translated into Arabic by Darraj et al., (2022)  was utilThe design of the questionnaire was based on an analysis of pertinent studies ,12,13,14Data were gathered from April to May 2023 with an IRB approval from Princess Nourah bint Abdulrahman University and administrative clearance from the dean of student affairs. The survey was developed online on Google forms and distributed among the study sample through emails via the student affairs offices of the College of Nursing and Deanship of Scientific Research, and WhatsApp groups. The questionnaire took about 15 min to be completed by the participants.t-test and analysis of variance (ANOVA) were used to compare study variables between groups based on socio-demographic characteristics. The correlation coefficient was utilized to examine the relationship between the variables in the study. A multiple regression analysis was performed to predict the knowledge and attitude score in response to demographics. The variables included in the multiple regression models as independent variables were those that were statistically significant  in the correlational analysis, with a correlation coefficient of 100. The structural equation modeling method was also used to analyze the relationship between the research variables. Structural equation modeling is a powerful data analysis technique for investigating complex interactions between multiple analytical variables. It may simultaneously test several mediating and moderating interactions, estimate latent variables using related measures, and deal with practical issues such as non-normality and missing data. Demographics were deemed an independent variable by the authors, while students’ knowledge and attitudes concerning HPV were considered dependent factors. The model’s assumptions were shown to be true. All statistical analyses were performed using IBM SPSS Statistics version 23 and IBM SPSS AMOS version 23. All p-values shown are two-tailed, and the 0.05 threshold was used for statistical significance.IBM SPSS Statistics version 26 and IBM SPSS AMOS version 23 were used for all statistical analyses. Descriptive statistics  were used to quantify demographic characteristics, whereas inferential statistics . Data confidentiality and privacy were sustained and guaranteed. The participants’ written agreement to participate in the research was obtained before the collection of safeguarded data. The study subjects were ensured the right to withdraw from the study.According to According to In addition, p < 0.001, r = 0.579, p < 0.001, and r = 0.483, p < 0.001, respectively). There was also a highly significant positive correlation between overall knowledge and the nursing students’ marital status, GPA, family history of cervical cancer, and receiving the HPV vaccine . However, a highly significant negative correlation was found between overall knowledge and nursing students’ age and academic year . Furthermore, there was a highly significant positive correlation between the nursing students’ overall attitudes and knowledge of HPV vaccination in females, and knowledge of the HPV vaccine in general . There was also a highly significant positive correlation between overall attitude and the nursing students’ marital status, GPA, family history of cervical cancer, and receiving the HPV vaccine . However, a highly significant negative correlation was found between overall attitude and nursing students’ age and academic year .Regarding the correlation matrix in p ≤ 0.001, LL = 2.965, UL = 9.671). The second factor affecting knowledge is a family history of cervical cancer with a direct relationship , . In addition, for attitude toward HPV, the most important demographic factor affecting attitude was marital status . Additionally, there was a direct significant relationship between attitude towards HPV and GPA . However, for the academic year it was a significant negative relationship with attitude regarding HPV . The last factor of the demographic data affecting attitude is family history of cervical cancer . Another important factor is knowledge which had a significant relationship with attitude regarding HPV , .To validate the relationship between the demographic data, their knowledge, and attitudes toward HPV vaccination, a regression analysis was performed, when the demographic data are the independent variables and knowledge and attitudes are the dependent variables p < 0.001; Model fit parameters . Nursing students’ knowledge accounted for the prediction of 48% of the variance in nursing students’ attitudes. All observed variables in the examined model were highly significant at p-value < 0.001 with strong estimates for the study variables .CFI = Comparative fit index; IFI = incremental fit index; RMSEA = Root Mean Square Error of Approximation.Model χ; significance 31.932 *(<0.001 *)Human papillomavirus (HPV) is one of the most common STIs, infecting around 5.5 million people each year . The curThe current study results revealed that nursing students had a low level of knowledge regarding the HPV vaccine. This result may be due to the fact that nursing students have no experience regarding the human papilloma virus, as they did not study it in the academic curricula, there was a lack of educational intervention and awareness campaigns to augment the HPV immunization program, the family was not aware of the importance of vaccination against the human papilloma virus, and there was no awareness at the community level regarding the human papilloma virus and the importance of vaccinations. This study results were supported by White et al., 2016 and Gang Chen et al., (2022)  who reveMore than half of the nursing students had a moderate attitude level toward female immunization, according to the findings of the current study. The outcome may be related to the fact that females may feel that they need more protection and they may perceive that the HPV can be transmitted to the baby if the woman becomes pregnant. These results may be supported by those of He and He (2018) , who demThe current study results revealed that there was a significant correlation between the nursing students’ age and their knowledge and attitude regarding HPV. This result may be due to the growth and development stage of the female nursing students in the age group of adolescence and adulthood as they became interested in women’s health awareness. The current study findings are contradicted by Shakurnia et al.’s (2022)  study reThe current study results revealed that was a significant correlation between the nursing students’ academic year and their knowledge and attitude regarding HPV. This could be due to some students in the lowest academic year having more knowledge than those in the highest years; they may attend workshops and receive more health education at younger ages regarding sexual diseases. According to Baryamoglu Tepe and Ozcorekci (2020) , the uniThe current study results revealed that there was a significant correlation between the nursing students receiving the HPV vaccine and their knowledge and attitudes regarding HPV. When students have knowledge about HPV and how to protect themselves from it, this may influence the intent to receive the vaccine and the rate at which it is obtained. The findings of the research demonstrated by Oh et al. (2021)  was suppThe findings of the current study showed a substantial relationship between nursing students’ knowledge about HPV and their GPA. This result can be attributed to the students’ desire to learn more about the topics covered in their educational courses. This study is supported by the findings of Shakurnia et al., (2022) , which iFurthermore, the current study results were in contradiction with those of Shakurnia et al., (2022) , who fouStrength and limitation: The present study’s findings have greatly added to previous HPV studies. However, the study’s weaknesses should be addressed. Because the sample size was limited to a specific group of people and the participants were recruited from a specific group in a specific context for convenience sampling, the results’ generalizability is limited. One of the study constraints was that the College of Nursing at PNU had a female-dominated educational environment. Furthermore, the current findings were prone to response bias and subjectivity because they were based on self-reported data. Furthermore, this study merely found correlations between study variables; no causal relationship was found. Longitudinal, experimental, and multi-site research may be useful in the future.According to the current study’s findings, there is a considerable lack of information and attitude concerning HPV and its vaccine. Furthermore, nursing students have limited understanding about HPV and its vaccine. Today’s nursing students are the future nurses who will play a vital role in spreading HPV awareness and preventing cervical cancer. In order for students to appropriately inform the public as future healthcare practitioners, they must have a basic understanding of the HPV virus and its vaccine. The study’s findings highlight the importance of educational activities to increase students’ understanding and awareness of HPV in relation to future professions as health care workers.Recommendations: Because of their critical role in public health education, nurses’ awareness and understanding of HPV will be among the primary determinants of health-care utilization. To raise awareness of HPV and its vaccine, a variety of techniques, including educational events and curriculum adjustments, should be implemented. Furthermore, clinician recommendation is an important factor influencing the parents’ decision to vaccinate their children, and school-based vaccination programs increase HPV vaccination rates by increasing vaccine access and the ability to reach a large population, engaging both the family and the clinician, and including the HPV vaccine in the Saudi national vaccination schedule.Practice: the current study findings will help in providing health awareness for the age group ranged from 18 to 26 to enhance their knowledge and understanding regarding the importance of receiving the HPV vaccine and its prevention.Education: the results will help to build and implement different educational programs and training courses about the human papillomavirus and its related vaccine for the specified age group of nursing students.Research: the study results will help the researchers to conduct more scientific research and studies with focus on increasing the number of the sample among female students at the university from various colleges, not just health colleges and among different sectors within the society.Significance of the study in relation to different domains:Nursing Education: This study will highlight the importance of implementing national educational programs about HPV and vaccine campaigns directed toward the public. This action would increase public knowledge of HPV and consequently enhance public attitudes toward the HPV vaccine.Nursing Practice: This study will help enhance the knowledge and understanding of nurses to educate their patients in different community settings including hospitals, clinics, schools, and associations about the importance of taking the HPV vaccine and its preventive effects.Community: The study will promote the HPV vaccine acceptance and use; awareness can be raised in our community to assure better knowledge and achieve higher protection from this virus and its complications by using advertising materials such as performing awareness campaigns in hospitals, malls, and schools.Research: This study may contribute as the base line for further researchers regarding increasing awareness of the virus by applying nursing education sessions for students.


The variables that showed a barrier effect for HPV vaccination were age , and immigrant origin . The variable that showed a facilitating effect for HPV vaccination was HPV PCR .HPV vaccination coverage rates can vary depending on several factors. The main objective of this study is to identify possible independent prognostic factors that have an impact on HPV vaccination in a rural community, specifically related to sexual and reproductive health. A case–control, retrospective, community-based study was carried out on women aged 15 to 40 in the primary health centers of Southern Catalonia’s Terres de l’Ebre region, Spain, from 1 January 2020 to 31 December 2022. A random sample of 520 women with an average age of 29.3 (SD 7.8) years old was included in the study. Independent prognostic factors: age OR 0.680 (95% CI: 0.635–0.729,  The human papillomavirus (HPV) vaccine is a critical component of the World Health Organization’s (WHO) 2030 Objectives program. This initiative places a strong emphasis on achieving high vaccination rates among women, with a specific target of attaining a 90% vaccination coverage rate among 15-year-old females .Regions undergoing demographic changes, such as the one in which this study was conducted, especially due to immigration phenomena, rurality, and socioeconomic characteristics ,6,7,8,9,On one hand, it is known that vaccination coverage rates vary due to numerous factors : income On other hand, facilitating factors have been described for vaccination such as practicing safe sex and undergoing regular sexual and reproductive health screenings . LikewisEffectively addressing these challenges necessitates a multifaceted approach that acknowledges the wider context of rural communities, takes into account their unique obstacles, and caters to their specific needs. The primary objective of this study is to ascertain potential autonomous prognostic determinants that impact HPV vaccination within a rural community, with a specific focus on aspects related to sexual and reproductive health.A retrospective, case–control, community-based study was carried out on women aged 15 to 40 in the primary health centers of Southern Catalonia’s Terres de l’Ebre region, Spain, with data available up to 31 December 2022.The Terres de l’Ebre region  compriseThe study’s target population encompassed 24,415 women aged 15 to 40 residing in the Terres de l’Ebre region, constituting 94.7% of the total number of women in that age group registered within the territory.https://www.imim.es/ofertadeserveis/software-public/granmo/, accessed on 23 January 2023). The STROBE checklist was used for the review of items included.For this case–control study, a randomized sample size of 520 participants between the ages of 15 and 40 years, who are registered in the territory and have an active medical history in any of the healthcare centers within it, was calculated. The study aimed to include 182 cases of vaccinated patients and 338 controls of unvaccinated patients. The calculations were based on the following parameters: alpha risk of 0.05, beta risk 0.15, and a bilateral contrast to detect a minimum odds ratio of 0.5. A rate of 0.3 exposed in the control group is assumed, and a loss of 10% in follow-up has been estimated. The POISSON approach has been used .−Women who have been residents for at least five years in one of the four regions of the Terres de l’Ebre territory , regardless of their place of birth.The inclusion criteria were as follows:−Patients without active or open clinical history and/or with insufficient data or inaccessibility to said data.−Disease with vital prognosis of less than one year.The exclusion criteria were as follows:The vaccination status was classified as “correctly vaccinated” if the individual received either 2 doses before their 15th birthday or 2 or 3 doses after their 15th birthday, as well as for immunocompromised patients ,37,38,39The participants in this study were categorized into two groups based on their vaccination status, as per the criteria outlined by the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC) ,37. Sinc-Demographic variables: age, ethnic origin, immigrant origin, and educational level.−Variables related to cervical cancer screening: first registered cervical cytology, result of the first registered cervical cytology, first registered cervical PCR HPV test, and result of the first registered HPV PCR test.−Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Ureaplasma spp., and Mycoplasma spp.Variables related to sexually transmitted diseases: first registered PCR sexually transmitted disease (STD) test in the vagina or cervix, result of the first registered PCR STD test in the vagina or cervix, and history of any diagnosis of an STD or HIV in the medical record. The STDs that are included in the test available at our laboratory are: −The variables related to sexual and reproductive health included factors such as childbirth, breastfeeding, voluntary termination of pregnancy or abortion, contraception methods, and the use of condoms.−Other clinical variables: smoking and immune status.Age was divided into intervals in The variables have been categorized based on the individual’s vaccination status, captured at the time of HPV vaccination, and the results have stratified into different age groups or periods.All study participants belong to the Catalan Institute of Health (ICS), which manages the area through 11 Primary Care Centers (EAPs). Data were obtained retrospectively through an anonymized database provided by the Service of Technology and Communication. The data are recorded using the International Classification of Diseases (ICD-10) and sent to the principal investigator in an anonymized format. Data were supervised and analyzed according to the General Data Protection Regulation of Spain/Europe as of 1 February 2017. The study was conducted in accordance with the most relevant standards on data handling, experimental context with patients, ethics, and data protection and privacy, following Directive 95/46/EC . All collected data were compiled and stored in a dedicated repository specifically created for this study. The repository was then handed over to the principal investigator for analysis. Prior to conducting the study, the research protocol underwent thorough ethical evaluation and received approval from the Ethics Committee of the Jordi Gol Primary Care Research Institute, with the registration number 21/064-P.The Health Plan of the Terres de l’Ebre Healthcare Region 2021–2025 : This stThe HC3 Patient Episode Dataset for Catalonia : This dataset contains demographic and clinical information on inpatient and outpatient care in Catalan hospitals. It provides details on patient episodes of care, including hospital admissions, outpatient visits, and diagnostic tests.The shared clinical information database of the 11 primary care teams managed by the Catalan Health Institute: This database includes clinical data, symptoms, tests, diagnoses, comorbidities, prescribed medication, referrals to secondary and tertiary care, and the alive/dead status of 97.7% of the residents in the territory.Pharmacological variables collected from the Integrated System of Electronic Prescription (SIRE): This dataset captures information on prescribed medications.Data provided by the Statistics Institute of Catalonia: This dataset includes information on gross household income per capita relative to the Catalonia average 100%), population density per square kilometer, and aging index compared to Catalonia (100%) ,8,10.0%, populThe datasets utilized for this project include:Data on these factors was automatically collected whenever possible or manually collected otherwise.The vaccination coverage rate was calculated by dividing the number of patients with complete vaccination by the total target population. Data are presented using frequencies and percentages for categorical variables and using means and standard deviations for continuous variables, according to region, vaccination status, and age. To detect differences between the two groups, the chi-square test for categorical variables and the Mann–Whitney U test for continuous variables were used.p < 0.05 established, and 95% CI. The association between variables in the logistic regression model was evaluated using the area under the curve (AUC) of the receiver operating characteristic (ROC) curve. By utilizing the kappa concordance methodology, researchers assessed the agreement between the outcomes derived from the multivariate regression analysis and the real vaccination status against HPV. Specificity was employed to determine the proportion of true negative results  among all individuals who were not vaccinated, while sensitivity measured the proportion of true positive results  among all individuals who were vaccinated. All analyses were performed using the IBM SPSS Statistics 20 statistical package.The study employed binary logistic regression using the backward stepwise method of Wald to identify the independent predictive variables associated with HPV vaccination. The backward stepwise method of Wald was used to eliminate variables that did not significantly contribute to the model while identifying those that were significantly associated with vaccination. The results were presented in the form of odds ratios (ORs) and their 95% confidence intervals. The ORs enabled the determination of the probability of vaccination for each predictive variable while considering the effect of other variables in the model. The variables analyzed included age, smoking, ethnic origin, immigrant origin, offspring, breastfeeding, contraception, cervical cytology, HPV PCR test, and STD PCR test. The association between variables was calculated using the OR, with a statistical significance level of p < 0.001), contraception usage (p = 0.008), parenthood status (p < 0.001), and the practice of breastfeeding (p < 0.001). In the logistic regression analysis, the age , immigrant origin , and HPV PCR  emerged as independent facilitators of HPV vaccination.A randomized sample of 520 cases was included, with an average age of 29.3 (SD 7.8 years old). HPV vaccination displayed a significant association with various factors, including regular Pap smear screenings (p = 0.044) and immigrant status (p < 0.001) are shown in relation to vaccination status.Statistically significant differences between vaccinated vs. unvaccinated groups are shown for average age in p < 0.001).A total of 175 (33.6%) individuals were classified as correctly vaccinated. Among the vaccinated patients, the average number of doses administered was 2.54 (SD 0.59). Moreover, the probability of successfully finishing the entire vaccination schedule once initiated exhibited a 25-fold increase , except for the mean age in the different results of the HPV PCR test (p = 0.125).Significant statistical differences  were obsp = 0.793). No statistically significant differences were found in the mean age of individuals who had undergone an STD PCR test or not or in the various outcomes of the test , while the average age of those with at least one test recorded was 28.9 years old (SD 7.5) (the test .p < 0.001); use of contraception (p = 0.008); parenthood (p < 0.001); breastfeeding practice (p < 0.001); and regular Pap smear screenings (p < 0.001).Significant disparities  were obsp = 0.170) as shown in p < 0.001) as shown in p = 0.002).Related to other variables, no significant differences were found in terms of immune status alterations , specificity of 0.93 (95% CI 0.91–0.96), positive predictive value of 0.87 (95% CI 0.82–0.92), and negative predictive value of 0.90 (95% CI 0.87–0.93). p < 0.001). These findings underscore the significance of considering age, immigrant status, and accessibility to cervical cancer screening when addressing the issue of HPV vaccination access.Eventually, three target risk groups were identified related to challenges in accessing HPV vaccination: women over 25 years old, women of immigrant origin, and women without regular cervical cancer screening, and the probability of successfully finishing the entire vaccination schedule, once initiated, exhibited a 25-fold increase , contraception usage (p = 0.008), parenthood status (p < 0.001), and the practice of breastfeeding (p < 0.001). In the logistic regression analysis, the age , immigrant origin , and HPV PCR  emerged as independent facilitators of HPV vaccination.A randomized sample of 520 cases was included, with an average age of 29.3 (SD 7.8) years. HPV vaccination displayed a significant association with various factors, including regular Pap smear screenings  and immigrant origin , which were found to decrease the likelihood of HPV vaccination, and HPV PCR  as a facilitator for HPV vaccination. Also, the accessibility to the initial vaccine greatly enhances the likelihood of completing the full vaccination schedule, increasing it by a remarkable 25 times. These findings highlight opportunities to improve HPV vaccination rates.The study identified as barriers age (OR 0.68, 95% CI 0.63–0.72,


Background: The persistence of the HPV infection is a risk factor in the integration of viral DNA in the host genome, leading to transforming events. The lack of therapies for HPV-persistent infections determine an unmet medical need. Methods: We enrolled forty patients with persistent HPV infections and cervical lesions and divided them into two groups. The women in the treated group received 200 mg epigallocatechin gallate (EGCG), 400 µg folic acid (FA), 1 mg vitamin B12, and 50 mg hyaluronic acid (HA) for 12 weeks. The control group received no treatment. Results: 40 patients completed the study. Fifteen out of 20 women in the control group still had an LSIL at the end of the study. One woman had a decrease in the DNA load, while six had no change and eight had an increase in DNA content. In the treatment group, 17 out of 20 women achieved a full viral clearance. These women showed no cytological or histological evidence of lesions following the treatment. Conclusions: Our data highlight the possible effect of such combination on LSIL. Therefore, the evidence reported here supports the potential to carry out further randomized placebo-controlled studies with an adequate number of patients to verify our results. Human papilloma virus (HPV) is a family of double-strand DNA virus that can be transmitted through various types of contact, including skin-to-skin, skin-to-mucosa, and mucosa-to-mucosa. HPVs are non-enveloped viruses that generally penetrate the cells via a clathrin-dependent mechanism. A feature of papilloma viruses is the lack of coding regions for any DNA polymerase in their genomes. In order to replicate by taking advantage of the host’s polymerase, the target tissues of these viruses are those characterized by active and continuous replication. Therefore, HPV is a family of viruses that affect skin and mucosa, particularly genital, oral, and anal. HPV infections are diagnosed especially in women, and the estimates suggest that 80% of sexually active women contract this infection during their lifetime .In the greater part of the cases, HPV infections resolve spontaneously, without any relevant clinical manifestation. In the case of clinical manifestations, HPV is responsible, in most cases, for benign lesions such as skin warts or condylomas, the latter occurring in the affected mucosa. These benign lesions usually heal spontaneously although in some cases they may require treatment, usually consisting of ointments or minor procedures. More rarely, HPV infections affecting the mucosa can give rise to malignant lesions and evolve into cancers. Indeed, HPVs are classified based on their risk of transforming healthy cervical tissue into a cancerous mass. The most accurate classifications identify two levels of risk, determining to a classification divided in high-risk strains and low-risk strains . Among the possible HPV-induced cancers, the most burdensome for women’s health globally is cervical cancer. Cervical cancer is the 4th most common cancer worldwide, with 604,127 new cases and 341,831 deaths in 2020. Considering the female population, cervical cancer is the 2nd most common cancer in the world in women aged between 15 and 44 years [The persistence of the infection has different definitions. Generally, persistence is defined as the detection of the same virus strain affecting a patient in two temporally distant moments. The time interval between the two tests shifts from nine months to two years . The perAccording to the WHO histopathological classification system, cervical intraepithelial neoplasms (CIN) are characterized by alterations such as mild dysplasia (CIN1), moderate dysplasia (CIN2), severe dysplasia (CIN3), and carcinoma in situ (CIS) . A revisDespite numerous advances in HPV research and screening campaigns with Pap smear and HPV-DNA test, HPV-persistent infections have no specific treatments, an issue that remains to be solved. In this perspective, developing new therapeutic strategies to act against the persistence of HPV infection is crucial. Several non-pharmacological molecules are increasingly gaining importance in the treatment of a plethora of tumors, including those induced by papilloma virus ,17,18,19Tumors are multifactorial diseases, relatable to environmental, food, genetic and epigenetic factors. Indeed, the lack of several nutrients may contribute to cancer development. Among them, literature reports the crucial role of folic acid (FA) and vitamin B12 in HPV-positive patients ,27. FA and vitamin B12 can prevent HPV proliferation by controlling DNA methylation. In particular, such methylation prevents the transcription of several genes, including those involved in virus persistence . In the context of HPV infection, hyaluronic acid (HA) is crucial to repair lesions. HA is essential to activate several pro-inflammatory factors that allow wound healing . HA acceIn this study, we aimed to test the efficacy of the association of EGCG, FA, vitamin B12, and HA in patients with persistent HPV infections and cervical lesions.ClinicalTrials.gov (Ref. No. NCT05625308). Following Institutional Review Board approval, patients were asked to sign an informed consent explaining the purpose of the study and the study protocol. The inclusion criteria were (1) females aged 30 to 45 years, (2) HPV-persistent infection for more than two years before entering the study, defined as positivity to the same HPV genotype in two different HPV-DNA tests performed at least two years apart, (3) PAP test indicating LSIL or ASC-US cytology, and (4) colposcopy and biopsy confirming LSIL. The exclusion criteria included (1) cervical cancer patients, (2) diagnosis of HSIL, (3) previous surgical procedures for HPV-induced transformation , (4) concurrent uterine pathologies, and (5) pregnancy or intended to seek pregnancy in the next three months. This study was a pilot, open label, controlled clinical trial. Participants were recruited at Clinica ALMA RES from June 2022 to August 2022. The study was approved by the Institutional Review Board of Clinica ALMA RES (Ref. No. 014/2022) and conducted according to the ethical principles of the Declaration of Helsinki. The trial was registered on ®, Farmares, Italy). After three months from the study start, all the patients included in the study underwent a new complete medical examination, including PAP test, HPV-DNA test, and colposcopy. Moreover, in the case of positivity to colposcopy, we performed a biopsy to examine the exact extent of the lesion. All women received cervical smears, HPV-DNA test, and colposcopy with biopsy to confirm the HPV infection. The primary outcome of the study was the clearance from the viral infection, which is inversely related to virus persistence rate. Therefore, the main outcome was the rate of negative responses to the HPV-DNA test in patients with a history of positivity to HPV-DNA test and LSIL longer than two years. The secondary outcomes were negative response to PAP test and to biopsy analyses. As the combination of EGCG, HA, FA, and vitamin B12 had never been tested before in a clinical trial, we could not perform a power analyses to determine a sample size. Therefore, we could not select a sample size to be enrolled for our pilot study. Patients who wished to enter in the treatment group received a box containing all the tablets for a 12-week treatment with one tablet per day via oral route. Conversely, patients who decided to enter in the study as control underwent no treatment but followed a regimen of clinical surveillance as suggested by the Italian guidelines. Tablets given to the patients in the treatment group consisted of a combination of EGCG 200 mg, HA 50 mg, FA 400 μg, and vitamin B12 1 mg . Patients were also encouraged to report any other side effects.p value < 0.05.During PAP test, the swab was transferred to a glass microscope slide, fixed in 95% ethanol, and treated using the Papanicolaou′s solution 2a orange G . The same anatomical pathologist examined all the glasses and classified the results according to the Bethesda System. For HPV-DNA testing, we used Hybrid Capture 2 (HC2) test kits  according to the manufacturer’s instructions. We considered samples with a relative light unit (RLU) higher than 1 as positive to HPV-DNA test. We compared age, height, weight, Body-Mass Index (BMI), parity, and time of virus persistence between the two groups at baseline to obtain statistically matching samples of patients. We analyzed data through student’s T-test for parametric distribution of continuous data. We analyzed outcomes with a dichotomous variable through chi square test. All data were analyzed using GraphPad Prism Software. We considered statistically significant a We assessed a total of 57 women for eligibility. Twelve were excluded as they did not match our inclusion criteria or matched the exclusion criteria. Four women declined to participate, and the remaining 41 women with HPV infection were enrolled and asked about their preferences to enter in the intervention (20 women) or in the control (21 women) groups. No patient was further excluded; however, we had a dropout from the study as a patient in the control group was lost to follow-up. No patient in the treatment group reported an interruption of the treatment. p = 0.000137). With regards to viral DNA presence, HPV-DNA test revealed a clearance rate following the twelve week period equal to 85.0% and 25.0% in the intervention and in the control group, respectively . Chi squResults of the PAP test, HPV-DNA test, and biopsy before and after the treatment or the 12-week period are presented in By analyzing the results in relation to the genotype, we could calculate the clearance rates among the different genotypes. Among the 17 women in the treatment group that proved to have a viral clearance, eight women had HPV-18, four had HPV-16, two had HPV-33, two had HPV-45, and one had HPV-52. Therefore, the clearance rate was 80% in the case of HPV-18 (eight patients out of 10) and 80% in the case of HPV-16 (four patients out of 5).Concerning the side effects, patients under treatment with 200 mg of EGCG, 400 mcg of folic acid, 1 mg of vitamin B12 and 50 mg of hyaluronic acid for 12 weeks reported minor or null side effects. Only one patient reported the occurrence of gastrointestinal disturbs during the first 4 weeks of treatment. Patients reported no other adverse events. To the best of our knowledge, this is the first clinical study investigating the role of EGCG, FA, HA, and vitamin B12 in papillomavirus-persistent infection. Moreover, this study provides the first insight into the therapeutical activities of natural compounds in such clinical picture. Indeed, we found out that an oral treatment with tablets containing 200 mg of EGCG, 400 mcg of FA, 1 mg of vitamin B12, and 50 mg of HA for 12 weeks reduces the rate of viral persistence. Despite our clinical trial providing the first evidence on such combination, the effects of EGCG alone, folic acid alone, hyaluronic acid alone, and vitamin B12 alone are already described in the literature ,35,36,37Zou and colleagues  carried Another study proved that EGCG treatment reduces the expression of both E6 and E7. This finding is pivotal to understand the efficacy of EGCG in reducing the malignant transformation rate of cervical lesions in HPV positive patients, as E6 and E7 increasingly abolish the control of the cells on their own apoptotic processes. Therefore, by reducing E6 and E7 expression, EGCG alone is able to induce a slight reduction in the chance of viral persistence. Moreover, there is weak evidence on the ability of EGCG to prevent viral DNA inclusion in the human genome, but this evidence still needs to be further validated . In another in vitro study, the authors treated two HPV18-positive cell lines derived from foreskin keratinocytes and vulvar intraepithelial neoplasia with EGCG for 24, 48, and 72 h. Their results indicate that EGCG treatment significantly downregulates E6 and E7 viral proteins, promoting the degradation of both the proteins through the proteasome. Moreover, such reduction in the expression of viral oncogenes increases the expression of pro-apoptotic factor p53 and pRB. Consequently, the proliferation of the keratinocytes and the vulvar neoplasia cells decreased, restoring a near-healthy phenotype. Moreover, EGCG displayed an important inhibitory activity on E4 expression. E4 is the viral protein responsible for viral DNA replication; therefore, EGCG treatment also blocks the replication of the virus, contributing to reduce the extent of the infection . From a clinical point of view, a study involving 90 women with different-graded cervical lesions revealed that EGCG oral treatment has chemo-preventive effects in vivo. The authors prescribed a treatment consisting of an oral tablet containing 200 mg of EGCG daily, examining the response to the treatment, considered as the histological absence of lesions. The results report an improvement in 60% of the cases following oral treatment; in addition, the authors report a positive response in 71% of the patients with severe dysplasia, namely HSIL . A following study on the efficiency of EGCG involved 226 men and 227 women with anogenital condyloma. The authors prescribed a treatment with topical applications of an ointment consisting of green tea extracts and containing a variable amount of EGCG (50–72%). Following the application of the ointment three times per day for 16 weeks, the authors examined the complete clearance rate following the treatment. They report that 53% of the cases had a total disappearance of the lesions after EGCG treatment. Moreover, they state that the treatment is far more efficient in women than in men, with a complete clearance rate equal to 60% of the cases in women and 45% of the cases in men. In addition, they report that 78% of patients treated with EGCG ointment had a clearance equal to or higher than 50%. The authors also state that the effects of EGCG on the regression of condylomas derives from its block on cell proliferation, inducing apoptosis and leading to a regression or total disappearance . FA and vitamin B12 also displayed important properties in the case of HPV infection. Piyathilake et al.  carried Sedjo et al.  examinedHyaluronic acid is particularly important in the case of HPV infections for tissue repair and homeostasis. HA plays an important role in restoring the integrity of epitheliums and mucous membranes. In the case of HPV-positive patients, due to its effects in maintaining the integrity of mucosa, HA is effective in preventing the entry of viral particles into the cells, blocking the infection process. Riemma et al.  also proHyaluronic acid is a well-known moisturizer, and literature highlights that its effects may differ based on the molecular weight of the molecules. Indeed, HA is a chain composed of hundreds of condensed disaccharides of glucuronic acid and N-acetyl-glucosamine. Therefore, the activities of such polymers may differ depending on the number of repetitions. Indeed, literature highlighted that high-molecular-weight hyaluronic acid (weighting more than 500 kDa) has anti-inflammatory properties, while low-molecular weight hyaluronic acid (weighting less than 500 kDa) has lubricant properties. Indeed, the HA that was comprised in the formula that we tested is called very low-molecular weight hyaluronic acid, which has a molecular weight lower than 10 kDa. Literature evidence supports the use of very-low molecular weight HA in the case of wounds or abrasions. This is based on the evidence on the molecular activities of a particular type of HA, which is known to deeply penetrate into the wounds and abrasions, filling the injured tissues that HPV would penetrate; thus, promoting the repair . Our study aimed to test the efficacy of the combination of EGCG, FA, vitamin B12, and HA. Our results confirmed the evidence on the single molecules, also providing information about the potential efficacy of such combination against HPV-persistent infections. Our data suggest that the oral supplementation with such combination for 12 weeks leads to a positive response in HPV-positive women with viral persistence. From a cytological point of view, seven out of eight patients with ASC-US and 10 out of 12 patients with LSIL displayed positive responses after treatment. From a histological point of view, 17 out of 20 patients with LSIL displayed a negative response to the biopsy following the treatment. Therefore, our data also suggest that the combination of EGCG, FA, vitamin B12, and HA may represent a promising treatment for suppressing HPV persistence. Interestingly, we obtained matching results with PAP tests, HPV-DNA tests and biopsies, suggesting an eradication of the viral infection and a matching positive effect on the lesions. We also stratified the results based on the HPV genotypes; however, we could not include a significance analysis due to the small sample size representing the different genotypes. Nonetheless, when considering the overall response rate, our data indicate that such treatment with the combination of EGCG, FA, vitamin B12, and HA can be considered as an effective therapy, as patients reported no side effects.Our results provide evidence on a new possible treatment for HPV-persistent infection. However, this study involves only a small number of patients; therefore, our result cannot confirm the efficacy of the treatment. Based on the evidence we produced, we hope to prompt research on these molecules in treating women affected by LSIL. As this is a pilot study involving a small sample size, we strongly encourage further double-blind, randomized, and placebo-controlled studies to verify our preliminary results.Although the population size of this study is small, our clinical observation provides the first evidence on the combination of EGCG, FA, vitamin B12, and HA in treating LSIL. Moreover, our observations indicate that such combination of natural molecules may represent a promising treatment for women with persistent HPV infection. Despite our promising results, our findings should be verified in larger cohort studies with an appropriate randomization plan and a placebo treatment as control. Based on our findings, further studies may involve an adequate number of patients to confirm with certainty our results. In summary, our study suggests that a combination of EGCG, FA, vitamin B12, and HA may exert preventive effects against the progression from mild dysplasia to a more severe stage of cervical neoplasia. However, more structured studies are needed to verify our preliminary results.


Head and neck cancer (HNC) is a highly prevalent and heterogeneous malignancy. Although extensive efforts have been made to advance its treatment, the prognosis remained poor with increased mortality. Human papillomaviruses (HPV) have been associated with high risk in HNC. TP53, a tumor suppressor, is the most frequently altered gene in HNC, therefore, investigating its target genes for the identification of novel biomarkers or therapeutic targets in HPV-related HNC progression is highly recommended.Transcriptomic profiles from three independent gene expression omnibus (GEO) datasets, including 44 HPV+ and 70 HPV- HNC patients, were subjected to integrative statistical and Bioinformatics analyses. For the top-selected marker, further in-silico validation in TCGA and GTEx databases and experimental validation in 65 (51 HPV- and 14 HPV+) subjects with histologically confirmed head and neck squamous cell carcinoma (HNSCC) have been performed.A total of 498 differentially expressed genes (DEGs) were identified including 291 up-regulated genes and 207 down-regulated genes in HPV+ compared to HPV- HNSCC patients. Functional annotations and gene set enrichment analysis (GSEA) showed that the up-regulated genes were significantly involved in p53-related pathways. The integrative analysis between the Hub-genes identified in the complex protein-protein network and the top frequent genes resulting from GSEA showed an intriguing correlation with five biomarkers which are EZH2, MDM2, PCNA, STAT5A and TYMS. Importantly, the MDM2 gene showed the highest gene expression difference between HPV+ and HPV- HNSCC (Average log2FC = 1.89). Further in-silico validation in a large HNSCC cohort from TCGA and GTEx databases confirmed the over-expression of MDM2 in HPV+ compared to HPV- HNSCC patients (p = 2.39E-05). IHC scoring showed that MDM2 protein expression was significantly higher in HPV+ compared to HPV- HNSCC patients (p = 0.031).Our findings showed evidence that over-expression of MDM2, proto-oncogene, may affect the occurrence and proliferation of HPV-associated HNSCC by disturbing the p53-target genes and consequently the p53-related pathways. This makes HNC the sixth most frequent disease overall . Squamou-) ones , 6. HPV-gulators .+ HNC of worldwide cases whereas the remaining are caused by mainly HPV33, HPV35, HPV52, HPV45, HPV39, HPV58 , assembly (L1 and L2) and other accessory proteins . HPV16 and HPV18 contributed to around 85% of HPV9, HPV58  by escap9, HPV58 . Thus, t9, HPV58 . HPV-assrognosis , 14, 15.rognosis , therefoTP53 gene, is a transcription factor that regulates several cellular processes, including inflammation, cell senescence, cell metabolism, autophagy, and regulation of aberrant cell survival and death  database. Only gene expression datasets that had been performed in HNC patients’ tumor samples and involved the HPV status were selected. In total 114 samples were analyzed, including 44 HPV+ and HPV- HNC patients, the LIMMA package of R was applied (p) < 0.05 were considered as significant DEGs between HPV+ and HPV- HNC patients. Overall patient’s transcriptome data, for a given gene, and the maximum expression value of the multiple probes were considered. The processed expression datasets were merged into a common expression matrix based on the gene names.Quality control analysis, technical heterogeneity and batch effect exclusion, data background correction and data normalization are performed for all HNC samples. To detect the differentially expressed genes (DEGs) between HPV applied . Genes t+ and HPV- HNC patients. Genes were annotated based on their associated Biological Processes (BP), Molecular Function (MF), and Cellular Component (CC) gene ontologies.We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using hypergeometric tests to uncover the putative biological roles of the DEGs between HPV+ and HPV- HNC patients. Normal and absolute gene set enrichment analyses were conducted on the DEGs as stated before (https://www.gsea-msigdb.org). In addition to the subset of p53 target genes which was collected from online pathway databases, publications and through bioinformatics search  was performed to identify gene sets that were differentially regulated between HPVd before . The ana+ and HPV- HNC patients, the STRING database (v11.5) was used (https://string-db.org/). The DEGs were mapped, and the corresponding PPI pairs were identified with the highest confidence interaction score of 0.9. Next, to identify the target genes, the PPI network was visualized using Cytoscape software (https://cytoscape.org/). In the network, nodes showing a higher number of links are apt to preserve the stability of the whole network based on the edge-connectivity concept. The cytoHubba application (v0.1) was used to find the top hub nodes ordered by degree of interactions using the Degree method.To assess the potential Protein-Protein Interaction (PPI) network of the DEGs between HPVin-silico transcriptomic analysis, a total of 65 patients with oral squamous cell carcinomas (OSCC) were collected, including 14 HPV+ and 51 HPV- OSCC samples. The case classification of HPV status was based on the immunostaining of the p16 protein which is considered as a surrogate marker to diagnose high-risk HPV infection in OSCC according to the College of American Pathologists  classification (Sixth edition). All clinical information regarding the pathological tumor node metastasis (pTNM) staging of the original tumor, the level of cancer invasion and metastasis, the resection margin of cancer, the perineural invasion (PNI) and the lymphovascular invasion (LVI), were obtained from the patient’s histopathology reports.Each tumor’s invasive front was identified, and a donor paraffin block’s core (0.5 cm) was considered for the experiment. From the 65 OSCC samples, 16-17 cores per block of tissue microarrays (TMA) were placed on four paraffin blocks. Initial H&E slides that subsequently revealed a limited tumor area were deleted from the following procedure analysis.Paraffin sections were cut at 4 um thickness, placed on positively charged slides, and dried in an oven for 30 minutes at 70°C. Deparaffinization, rehydration, and target retrieval were performed in the PT Link (Dako) using a 3-in-1 procedure. Details of the immunohistochemistry (IHC) staining protocol for the used antibodies for p16, MDM2 and p53 were described in http://gepia.cancer-pku.cn/), which represents the gene expression alterations in large cohort cancer samples, and the Tumor IMmune Estimation Resource (TIMER) (https://cistrome.shinyapps.io/timer/), which presents a comprehensive analysis of tumor-infiltrating immune cells and UALCAN resource , which gives access to different publicly available cancer OMICS data. The survival analysis of the MDM2 gene was evaluated in HNSCC patients with high mutation burden, using the Kaplan-Meier plots.The potential role of MDM2 in HPV-associated HNSC was further evaluated based on the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) data resources using the Gene Expression Profiling and Interactive Analysis database (GEPIA) . Statistical significance between the protein expression and clinical parameters was analyzed using the t-test. Pearson’s correlation was applied to evaluate the possible correlation between protein expression and different clinical parameters across HPVp < 0.05) between the HPV+ compared to HPV- HNSCC patients across the different datasets  , Transcriptional Regulation by E2F6 (p= 1.09E-09) and Regulation of cell cycle process (p= 9.99E-09), DNA replication and repair including DNA repair pathways (p= 2.06E-08), Chromatin organization (p= 3.6E-11) and Histone modification (p= 8.36E-08) and in the p53 tumor suppressor protein regulation (p= 5.02 E-05) including the Transcriptional Regulation by TP53 (p= 2.15E-09), PID P53 Downstream pathway and Regulation of cellular response to stress (p= 2.15E-09). While the down-regulated genes were mainly annotated in the VEGFA-VEGFR2 signaling pathway (p= 9.18E-09), Hemostasis (p= 6.71E-08) and Focal adhesion (p= 1.38E-07).To assess the biological functions related to the identified DEGs, functional annotation and pathway analysis have been performed for GO and KEGG analyses. The up and down-regulated genes were separately annotated. The top 20 enriched terms including the related up/down-regulated genes in HPV+ vs HPV- HNSCC patients, the PPI-network interactions were constructed at the highest confidence of 0.9, defined as the minimum required interaction score , with PPI enrichment p-value < 1.0E-16, consisting of 58 nodes was mainly associated with the DNA metabolic process and E2F regulation pathway. Cluster 3 (colored in blue), with PPI enrichment p-value = 2.97E-12, consisting of 38 nodes was related to cellular senescence and transcription regulation by TP53. The proteins and their node degree related to each cluster are listed in PCNA, MCM5, MCM3, MCM2, POLA1, RPA2, MCM6, PRIM1, RFC5, RBBP4, DUT, TYMS, RRM1, EZH2, LIG1, CHAF1A, RBBP7, MDM2, SUZ12, and SMC2  in the patients with HPV-related HNSCC . The details of these significant gene sets are listed in NOTCH1 (24 occurrences) and CAV1 (23 occurrences), which reinforces the previous analysis showing the important subset of p53-target genes in HPV-associated HNSCC.To gain a better understanding of the potential distinctness in molecular signatures and biological mechanisms between HPVEZH2, MDM2, PCNA, STAT5A and TYMS   compared to normal tissues (44 samples) using data from TCGA and GTEx in GEPIA. Importantly, the mRNA levels of MDM2 expression were significantly higher in HNSCC tissues than in normal tissues (p < 0.05)  and in normal samples (p = 2.81E-07), however, only a slight difference was observed between HPV- HNSCC patients and normal samples (p = 0.03) , p = 0.046) is correlated with poor overall survival in HNSCC patients , p = 0.002) in HNSCC HPV+ patients, while no significant association (p = 0.263) was reported between the MDM2 expression and the overall survival of the HNSCC HPV- patients . + and HPV- OSCC patients , (+ and HPV- OSCC patients (p = 0.44) has resulted between HPV+ and HPV- OSCC patients . As shown in Lack of knowledge about key genes and molecular pathways that drive the onset and progression of HPV-associated HNSCC has prevented early identification and therapy despite the growing body of research on the disease. Therefore, using bioinformatics analysis helps to get an in-depth understanding of the molecular keystones of HPV-associated HNSCC progression which are extremely needed for prediction, early detection, and therapy. To date, computational and high-throughput approaches have become more powerful and relevant to identify biomarkers in cancer study that helps in the understanding of carcinogenesis by integrating omics data . Among s+ and HPV- -HNSCC. Importantly, we showed that p53-target genes, among others MDM2 was a central biomarker, potentially deriving the differential signature of significant molecular pathways between HPV+ and HPV- HNSCC patients which is consistent with previous findings showing that MDM2 genetic aberrations and related pathways play a central role in the carcinogenesis of head and neck cancers in addition to a particular correlation with HPV status  difference between HPV+ and HPV- HNSCC. MDM2, a proto-oncogene, is amplified in 25–40% of human malignancies. The reported frequency of MDM2 overexpression is high in HNSCC, ranging from 40 to 80%. MDM2 can be overexpressed via either gene amplification, improved transcription, or post-transcriptional mechanisms . These findings provide considerable insight into the prognostic role of MDM2 in HPV+ HNSCC patients.The immunohistochemistry validation in HPV+/- related cancers. Although mutant p53 follows the same routes as wild-type p53, in HPV- HNSCC the p53 mutation prevents transcription activation from occurring downstream and affects the p53 target genes. However, in HPV+ HNSCC the process of HPV oncoprotein-mediated disruption of the cell cycle is different. In vivo investigations have demonstrated that MDM2 and HPV E6 utilize distinct methods to degrade p53  and HPV+ cells in The connection between p53 and MDM2 is essential for controlling cell growth and apoptosis but with different processes in HPVrade p53 . HPV E6 rade p53 , 40. MDMrade p53 . In anotrade p53 , and thurade p53 . Consequpression , 47, 2, pression , 49. It pression . Moreovepression . In ligh- HNSCC, HPV+ HNSCC has a good prognosis, with 5-year survival rates averaging between 75% and 80%, since these cancers react better to chemotherapy and radiation (p = 0.002) is correlated with improved overall survival in HPV+ HNSCC patients compared to HPV- HNSCC patients (p = 0.263). The inverse relationship between HPV status and high MDM2 expression is consistent with several studies showing that HPV+ HNSCC patients appear to have a significantly improved response to radiotherapy and chemotherapy, in addition to a lower risk of second primary cancers than the HPV- . Additionally, we revealed a negative correlation between MDM2 expression and smoking (Pearson correlation = -0.12) in HPV-associated OSCC patients. Notably, the effects of MDM2 genetic alterations were more prominent in never-smokers and never-drinkers , to show a positive association between MDM2 expression and HPV status  can be found below: GEO: GSE3292, GSE6791 and GSE55542, TCGA and GTex: GTEx Analysis V8 (dbGaP Accession phs000424.v8.p2).The studies involving human participants were reviewed and approved by the Research Ethics Committee at Tawam Hospital (REC: AA/AJ/556). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Conceptualization: AB, RH and NA-R. Data curation: AB and NA-R. Bioinformatics analysis: AB. Funding acquisition: RH and NA-R. Methodology: AB, DF, MA, AQ, and ZA. Supervision: RH and NA-R. Writing - original draft: AB and NA-R. Writing, review and editing: AB, ZA, RH and NA-R. All authors contributed to the article and approved the submitted version.