Sexually transmitted infections (STIs) are one of the world's most severe health challenges. The existence of STIs such as human papillomavirus (HPV) might cause cervical cell changes leading to cervical cancer.This study aims to assess the association of STIs with cervical cytological abnormalities and genital warts among women in northeastern Iran.Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Herpes simplex viruses (1 and 2) were assessed using the real-time polymerase chain reaction method. HPV genital infection was detected based on the principles of reverse hybridization, and cellular changes in the cervix were examined by the liquid-based cytology technique.This cross-sectional study was carried out on 190 women referred to the central laboratory of Academic Center for Education, Culture, and Research, Mashhad, Iran from March to July 2022. The presence of genital infections caused by The mean age of participants was 35.33 High percentages of the participants with non-HPV STIs and HPV infection had normal cervical cytology. It is advised to use STIs and HPV diagnostic tests along with cytology examinations for cervical cancer screening. Chlamydia trachomatis [C. trachomatis], Neisseria gonorrhoeae [N. gonorrhoeae], and Mycoplasma spp.), and protozoa  (2). More than 1 million people are infected with STIs per day (3).Sexually transmitted infections (STIs) are considered a global health problem (1). There are Most STIs infection are asymptomatic and can lead to various outcomes, such as pelvic inflammatory disease, sterility, ectopic pregnancy, congenital infections, and cervical cancer (4). The presence of some STIs facilitates the transmission of other infections and can cause cellular changes that precede some types of cancer (5). Cervical cancer has been one of the most common cancers among females and the 4HPV is one of the most frequent STIs and is normally transmitted through direct contact with infected skin or mucosa (7). Depending on their oncogenic potential, HPV is divided into high-risk (HR) and low-risk (LR) groups. This should be mentioned that the HPV infection is effective risk factor for cervical cancer but it is not enough cause of cancer (8). Most HPV infections are temporary; however, a few cases of infections continue and progress to HR lesions and cancer.N. gonorrhoeae and Mycoplasma genitalium  could change the genital tract flora through repeated infections and provoke cervical cancer. The increase in microbial species in genital tract causes an increase in cervical cancer . Determining the presence of STIs is essential for developing appropriate healthcare strategies that focus on preventive and protective measures to reduce the risk of transmission and their outcomes. The current status of co-infections among Iranian communities is not well known.Some STIs agents such as C. trachomatis, N. gonorrhoeae, M. genitalium, HSV-1/2, and HPV) with cervical cytological abnormalities and genital warts among Iranian women referred to the central laboratory of Academic Center for Education, Culture, and Research, Mashhad Branch, Iran.Therefore, the purpose of the present study was to assess the association between STIs  were obtained from the available medical records. Any of the following factors were considered as exclusion criteria: pregnancy, being in the postpartum period, and having no history of sexual activity.Cervical cytology samples were collected for the liquid-based cytology technique to assay cellular changes. The cervical cytology of each participants was evaluated. The stages of the cervical cytology are classified as negative for squamous intraepithelial lesion or malignancy (NILM), atypical squamous cells (ASC) of undetermined significance (US), ASC-high-grade, low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions, and invasive cervical cancer.The cervical samples were used in molecular experiments for HPV and STIs detection. DNA was extracted from cervical cytology samples using SSNP-2000B nucleic acid extraction system produced by Bioperfectus, China. The DNA solution was stored at -20 C for HPV and STI tests.The HPV DNA test was performed using a high + low papillomastrip kit  according to the manufacturer's protocol. This kit allows the identification of 37 HPV subtypes, including HR: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 69, 73, and 82, and LR: 6, 11, 40, 42, 43, 44, 54, 61, 62, 67, 70, 72, 74, 81, 83, 84, and 91. For STIs detection, real-time PCR with REALQUALITY RQ-STI CT/NG/MG Kit  was used according to the manufacturer's instructions.globin gene. It allows verification of the extraction procedure and detection of PCR inhibition. Real-time PCR was applied for HSV determination using the HSV1/2 RQ kit .The kit includes an internal control consisting of a recombinant DNA fragment of the Written informed consent was obtained from all participants according to the protocol approved by the Ethics Committee of Academic Center for Education, Culture, and Research, Razavi Khorasan Branch, Mashhad, Iran (Code: IR. ACECR.JDM.REC.1401.065).Data analysis was performed using Statistical Package for the Social Sciences, version 22, SPSS Inc., Chicago, Illinois, USA (SPSS). Descriptive analyses including frequencies, percentages, and averages were used to describe the population under study. A Chi-square test or Fisher's exact test and regression analysis were performed for the qualitative variables when deemed necessary. Binary logistic regression was used to evaluate the independent effects of smoking on HPV infection. The statistical significance was set at p A total of 190 women participated in this study, including 128 HPV-positive and 62 HPV-negative women as the control group. The demographic characteristics of the participants are shown in table I. The mean age of the participants was 35.33 The mean age of the HPV-positive women was 34.60 LR-HPV6 was the most common genotype in women with mono-infection with n = 12/68 (17.6%) frequency followed by HR-HPV16 with n = 9/68 (13.2%) and LR-HPV42 with n = 4/68 (5.9%). Furthermore, in women with multiple infections, LR-HPV6 with a frequency of n = 22/60 (36.7%) was more prevalent than other genotypes followed by HR-HPV16 n = 16/60 (26.7%) and HR-HPV52 n = 15/60 (25%). Many of the HPV-positive women were infected with multiple HPV genotypes. In other words, we detected n = 60/128 (46.87%) multiple HPV genotypes in the total participants.C. trachomatis, M. genitalium, and HSV1/2were the STIs detected in this study.No significant association was observed between HPV infection and age in classified age groups, age of marriage in classified marriage age groups, history of abortion, and level of education, as shown in table I. Furthermore, no significant correlation was observed between HPV infection and abnormal cervical cytology, (Table I). Our results showed that smoking is involved in increasing the prevalence of HPV infection. The prevalence of STIs pathogens was significantly different betweenHPV-positive and HPV-negative participants. The prevalence rate of STIs pathogens in the HPV-positive participants was 10.9% (14/128) and it was 1.6% (1/62) in the HPV-negative participants (Table I). M. genitalium  followed by C. trachomatis  and HSV1/2  in the HPV-positive individuals. The most observed Pap smear results were NILM 176/190 (92/6%). Abnormal cervical cytology was detected in 14/190 (7.3%) of the participants, including 10 women with ASC-US (5.2%) and 4 with LSIL (2.1%). All 4 LSIL participants were HPV positive and 7 of the ASC-US individuals were HPV positive. HPV was detected in n = 117/176 (66.5%) women with NILM cytology. Among women with abnormal cytology, n = 11/14 (78.6%) were HPV positive (Table II). However, no significant association was observed between HPV infection and abnormality of cervical cytology. Moreover, no association was found between HPV genotypes (HR and LR) and abnormality of cervical cytology. In addition, STIs or co-infection of HPV and STIs had no significant relationship with an abnormality of cervical cytology (Table II).The most common pathogen was Based on table III, type of HPV  infection is associated with the incidence of warts. The frequency of warts is higher in people who have multiple infections of HR and LR genotypes, and it is more common in people who have an LR-HPV genotype compared to cases with HR-HPV. In addition, abnormal cervical cytology in people with multiple infections of HR and LR genotypes was higher than in the mono-infection group, but this difference was not significant (p = 0.2).LR-HPV6 was the most common genotype in wart sufferers, and it was detected in 25/68 (36.76%) of these women, 11 of whom were identified as having mono-infection and 12 had multi-infection with one or more HR-HPV genotypes while 2 participants showed multi-infection with another LR-HPV genotype. The second prevalent genotype in wart sufferers was HR-HPV16 with a frequency of 10/68 (14.70%), 6 of whom had multi-infection with one or more LR-HPV genotypes.In this study, it was found that the risk of HPV infection increased 5.43 times in the current-smoker cases compared to non-smokers , p C. trachomatis, M. genitalium, and HSV1/2 in 10.9% of HPV-positive participants; however, no significant association was found between the cervical cytological abnormalities and STIs positive in HPV positive group. Because of the low frequency of co-infections between HPV and other STIs, we were unable to make a proper evaluation of the influence of this association in abnormal cytology results. In our study, it was found that the most positive women for HPV and STIs were women In the present study, 34 different HPV genotypes were detected in all HPV-positive women. The most common genotype was LR-HPV6 (26.6%). We detected 3 important STIs including In contrast to the present results, a study found that HR-HPV16 was the most prevalent genotype (49.2%) followed by HR-HPV18 (25.3%), while in the current work, the prevalence of HR-HPV18 was very low (2.3%) (2). In a previous study conducted by the authors, the LR-HPV6 was the most prevalent genotype (43.9%) and HR-HPV18 was not prevalent in comparison with other genotypes with a frequency of 17.7%, which is considerably higher than its prevalence in the current study. This may be due to changes in the trend of prevalent genotypes in the region under study (14). The frequency of multiple HPV-genotype infections was relatively high in the current study (46.87%). One study showed that multiple HPV infections were associated with the occurrence of cytological abnormality (11), which is consistent with this study. Moreover, a significantly higher prevalence of warts was found in participants with multiple HPV infections, and more cytological abnormalities were identified in participants with multiple HPV infections in this study, but they were not significant.C. trachomatis may be a reason for female infertility and clinical manifestations (15). C. trachomatis is a prevalent sexually transmitted bacterial infection (16). Nevertheless, the most common pathogen in our study was M. genitalium followed by C. trachomatis and HSV. In the meta-analysis comprised of 34 studies in Iran in a period from 1998-2015, it was demonstrated that the prevalence of C. trachomatis in women and men was 0-32.7 and 0-23.3%, respectively. The differences were related to different geographical regions, methods of detecting microorganisms, and sample size (15). An investigation that had results similar to our study, detected only one infection with C. trachomatis (0.84%) in the HPV-positive group (17). The rate of HPV infections in cases with abnormal cervical cytology was high (78/6%) in the current study. Another study which had similar findings, detected 78.8% of HPV infections in cervical cancer samples (18).In contrast to the results of another study, no significant association was found between HPV infection or STIs and cervical cytological abnormalities in the present study (2). In a study with similar results to the current study, no significant association was observed between HPV/STIs co-infection and the cervical abnormality (19). In agreement with other studies, our results demonstrated an association between HPV infection and the incidence of warts.We found LR-HPV6 to be the most common genotype among wart sufferers. In an investigation that had similar results to this work, they showed that LR-HPV6 was the most prevalent genotype in wart samples (36.3%) (20). In this research, it was revealed that multiple infections of HR and LR genotypes are more likely to cause warts, and warts are more common in people who have an LR-HPV genotype than in cases with HR-HPV. Consistent with the current results, another study revealed that co-infection of both low- and HR-HPV types increases the risk of developing genital warts . Additionally, similar to the results of the present study, they found that LR-HPV genotypes have a higher risk of genital warts in comparison with HR-HPV types (21). The results of a study showed that the LR-HPV genotype was the major cause of genital warts and HR-HPV infections, and multiple HPV-type co-infections were also common in genital warts. Also, they found HPV6, 11, 52, and 16 as the 4 most common HPV types in genital warts (22). In the current study, HR-HPV16 was the 2Similar to the present results, a study demonstrated that the most prevalent genotype was LR-HPV6 (47%), but unlike the current study, in which HR-HPV16 was detected as the 2As revealed in another study as well as the present study, the risk of HPV infection increases in current smoker individuals in comparison with non-smokers. Similar to the findings of this research, they showed that the risk of HPV infection increases 1.9 times from smokers to non-smokers , p In conclusion, the current study has shown a high prevalence of LR-HPV6 in HPV-positive individuals as well as in cases with warts. It was found that smoking increases the risk of HPV infection. A high percentage of HPV-positive and STI-positive participants had normal Pap smear, so it seems that a Pap smear test alone is not enough for cervical cancer screening, and it is suggested to prepare HPV and non-HPV STI tests as well. According to the present results, higher prevalence rates of HPV and STI were found in women The potential limitations of the current study were sampling method and small sample size. Since the number of participants with abnormal cytology was very small in the current study, more investigations are needed with larger sample sizes in a specific population of women with abnormal cytology.The authors declare that they have no conflict of interest.


HNSCC) is less well understood. The objective of this study was to identify prognostic classification markers in CUPHNSCC. Therefore, we investigated a consecutive cohort by multivariate modeling and testing for HPV DNA, mRNA, and p16INK4a (p16) expression. In 31% of CUPHNSCC, p16 was overexpressed, and high-risk HPV DNA was detected in 18/32 (56.3%) of them, which was mostly consistent with mRNA detection. In contrast to oropharyngeal cancer, detection of p16 without additional detailed HPV testing appears to be more appropriate for the classification of CUPHNSCC. Three risk groups can be stratified based on performance status and p16, but additional factors may become important in future data or for cases with particular risk profiles.Human papillomavirus (HPV) association is the most important predictor of survival in squamous cell carcinomas in the head and neck region (HNSCC). The role of HPV in cancer of unknown origin at this anatomic site (CUPHNSCC), much less is known. Methods: We assessed a consecutive cohort of CUPHNSCC diagnosed from 2000–2018 for HPV DNA, mRNA, p16INK4a (p16) expression, and risk factors to identify prognostic classification markers. Results: In 32/103 (31%) CUPHNSCC, p16 was overexpressed, and high-risk HPV DNA was detected in 18/32 (56.3%). This was mostly consistent with mRNA detection. In recursive partitioning analysis, CUPHNSCC patients were classified into three risk groups according to performance status (ECOG) and p16. Principal component analysis suggests a negative correlation of p16, HPV DNA, and gender in relation to ECOG, as well as a correlation between N stage, extranodal extension, and tobacco/alcohol consumption. Conclusions: Despite obvious differences, CUPHNSCC shares similarities in risk profile with OPSCC. However, the detection of p16 alone appears to be more suitable for the classification of CUPHNSCC than for OPSCC and, in combination with ECOG, allows stratification into three risk groups. In the future, additional factors besides p16 and ECOG may become important in larger studies or cases with special risk profiles.Background: Human papillomavirus (HPV) status is the most important predictor of survival in oropharyngeal squamous cell carcinoma (OPSCC). In patients with cervical lymph node metastases of squamous cell carcinoma of unknown origin (CUP HNSCC, provided that other etiologies are excluded.Metastases from which the primary site is unknown are called carcinoma of unknown primary (CUP). In head and neck cancers, the most common histological type is squamous cell carcinoma (HNSCC), which is also the case in 53–90% of CUP in this anatomical region ,2,3. HisHNSCC, a substantial proportion of cases also appear to be associated with HPV [INK4a (p16). Overexpression of p16 has been introduced in the AJCC-8/UICC-8 staging system as a surrogate marker for classifying HPV-associated OPSCC and CUPHNSCC [Infection with high-risk (HR) human papillomavirus (HPV) is causative for a subgroup of HNSCC, particularly oropharyngeal squamous cell carcinoma (OPSCC). An increasing incidence of HPV-related HNSCC is reported in several countries worldwide ,7,8. In with HPV ,10,11. DCUPHNSCC .HNSCC is considerably better [HNSCC and OPSCC are thought to be related diseases [The prognosis is significantly better in HPV-related than in HPV-negative OPSCC, and risk models have shown that HPV is the most important prognosticator in these patients ,14,15,16y better ,17,18,19diseases  because diseases ,21. Nevediseases . In 5–20diseases ,25,26,27diseases , the ovediseases .HNSCC by analyzing p16 expression and detecting HPV DNA and mRNA. In addition, we sought to identify prognostic classification markers in CUPHNSCC multivariate using recursive partitioning (RPA) and principal component analysis (PCA) [HNSCC.In this context, we aimed to determine the HPV prevalence in CUPis (PCA) ,29 to idHNSCC (according to ICD10) and treated at our hospital  between 2000 and 2018. The study was performed retrospectively in accordance with the local ethics committee . Exclusion criteria were an absence of written informed consent, histologically confirmed primary tumor outside the cervical lymph nodes, or unavailable or insufficient amounts of formalin-fixed, paraffin-embedded samples from the pre-therapeutic tumor biopsy or tissue from tumor resection. The absence of primary tumors in the head and neck region was confirmed by panendoscopy with bilateral diagnostic tonsillectomy and biopsies from the nasopharynx and base of the tongue. In addition, all patients since 2011 received pretherapeutic 18F-fluorodeoxyglucose positron emission tomography/computed tomography as part of the diagnostic workup. Follow-up examinations included systematic inspection of the entire oral cavity, oropharynx and neck, ultrasonography of the cervical lymph nodes, and computed tomography or magnetic resonance imaging scans at regular intervals. All examinations were performed according to the guidelines of the German Guideline Program in Oncology (GGPO) valid at the time of the examinations.Patients: We retrieved clinical data of all patients diagnosed with CUPHNSCC was treated by upfront surgery (neck dissection) and/or by radio- (RT) or chemoradiotherapy (RCT) (n = 4) received cisplatin or mitomycin C (MMC) and 5-fluorouracil (5-FU) in two cases each. Patients treated by surgery and adjuvant RCT (n = 37) received cisplatin or carboplatin , MMC + 5FU (n = 4) or 5-FU + Cisplatin (n = 5). In three cases, the chemotherapeutic agent was not specified.Therapy and risk factors: CUPpy (RCT) . TreatmeWhen patients presented, tumors were classified by pathological or clinical stages  according to the International Union Against Cancer (UICC) TNM classification valid at the time of diagnosis ,31. The HNSCC was determined by immunohistochemical detection of p16 expression using the CINtec Histology Kit (Roche mtm Laboratories) and by analyzing DNA extracted from archived formalin-fixed, paraffin-embedded tumor tissue for mucosal high-risk (HR)-HPV DNA, as we described previously [n = 20) that tested negative for both. Briefly, amplification of HPV E6*I (approximately 65 base pairs) was used to determine HPV transcriptional activity, and ubiquitin C (85 base pairs) was used as a cellular marker to control mRNA quality and confirm assay validity.HPV status: HPV status of CUPeviously ,33. The eviously ,34. All p-values (test type = ‘Bonferroni’). Principal component analysis (PCA) [p ≤ 0.05 for all tests unless otherwise indicated. With the exception of RPA, p-value adjustment for multiple testing was not performed due to the exploratory nature of the study.Statistics: Recursive partitioning analysis (RPA) was performed by using the function ctree  of the R (Version 4.0.4) package partykit, Version 1.2–12 . The stois (PCA) ,29 was uHNSCC stratified by p16 status are shown in HNSCC were less likely to have increased tobacco and alcohol consumption, advanced N stage (>2b), and extranodal extension (ENE). In contrast, our data show an increased male-to-female ratio and worse performance status (ECOG) in patients with p16-negative CUPHNSCC, although without reaching statistical significance (HNSCC (p = 0.057). When treated with curative intent, most cases underwent upfront surgery with adjuvant radio-  or chemoradiotherapy , with no significant differences according to p16 status . HR-HPV DNA was detected in 21/103 (20.4%) of all samples but not in 14/32 (43.8%) of p16-positive samples. HR-HPV mRNA assays confirmed the DNA-based results in most cases. The mRNA assay was valid in all samples analyzed but did not detect HPV mRNA in seven samples positive for HR-HPV DNA  samples A. The in-HPV DNA A. ConsidHNSCC  to capture the most important aspects of the data by reducing complexity and to assess possible relationships between factors independent of survival. All factors previously used for the RPA were included, and we used the first two components (1 and 2) for a two-dimensional visualization A. The loIn PCA, a value for components 1 and 2 was calculated for each individual case, describing its position in a two-dimensional space B. In simHNSCC in our consecutive cohort overexpressed p16, but HPV-related carcinogenesis was confirmed by DNA detection in only 17.5% of all cases. This discrepancy is remarkable but also known from the literature. For example, in a systematic review, the estimated rate of HPV association in CUP patients decreased from 40~60% to 17 and 39%, respectively, when HPV mRNA or -DNA was tested in addition to p16 [We found that 31% of CUPn to p16 . Comparen to p16 , and ourn to p16 ,37,38,39HNSCC—possibly due to a lower probability of contamination during resection of “sterile” lymph nodes compared with samples from the oral cavity, which could still contain productive HPV infections. A lack of detection of HR-HPV DNA in p16-positive samples, on the other hand, could be due to the reduced integrity of DNA in archived samples. However, we excluded samples in which the β-globin gene could not be amplified, so the effect of aging should be small. This is confirmed by our results of the HR-HPV mRNA assay. Although mRNA is more unstable than DNA, the assay was valid in detecting the control mRNA in all samples analyzed. Only in one case with p16 overexpression was HR HPV-mRNA detectable, but no DNA. This does not particularly support the assumption that these cases are indeed HPV-associated. Despite the relatively small size of our cohort, the overall performance of the two assays (HR-HPV DNA and mRNA) was reasonably comparable, and their results largely agreed. This argues against fundamental technical/methodological problems in detecting HPV in archival samples. On the other hand, we might assume that p16 expression in the absence of HR-HPV DNA may be due to the nature of carcinogenesis in CUPHNSCC. HPV-independent overexpression of p16 is known from other tumors [HNSCC samples is quite low (but sufficient for detecting the control PCR product) and, therefore, may not be sufficient for the detection of HPV. This would imply that at least some of the p16-positive/HR-HPV DNA-negative samples are positive for both, i.e., HR-HPV DNA false-negative. This is also supported by our survival data  are the most important factors and sufficient to stratify patients into three risk groups with significant differences in OS. This is consistent with our previous results for OPSCC, although HPV DNA/RNA status is more important there. In subjects with good to moderate performance and p16 negative CUPHNSCC, we could not verify N status as another important factor, as was the case in OPSCC [HNSCC subgroups. Nevertheless, PCA shows that ENE, smoking, and alcohol consumption correlate with N status, which is likely the next important factor in RPA. We acknowledge that the RPA allowed us to identify only the most important factors here, but this does not preclude the identification of other relevant factors when larger cohorts become available in future analyses.In most cases of cervical lymph node metastases where another primary origin can be ruled out, and especially in HPV-associated CUPor OPSCC ,27. In tHNSCC  with similar published cohorts [HNSCC is rare  [HNSCC show some differences: we found a negative correlation between ECOG and p16 status for CUPHNSCC, whereas, in OPSCC, a correlation seems overall unlikely. On the other hand, ECOG correlated together with T- (and N stage) in OPSCC, which cannot be investigated for CUPHNSCC. In CUPHNSCC, we found a likely correlation between the N stage, along with tobacco and alcohol use, which was unlikely in OPSCC [The main limitation of this study is the overall small sample size of our CUP cohorts ,48, and  tumors) ,49,50. I tumors)  and CUPHin OPSCC .HNSCC is not completely understood. In this consecutive cohort analyzed for HPV DNA, mRNA, and p16 expression, and by multivariate modeling, we found that CUPHNSCC patients could be classified by performance status (ECOG) and p16. Despite obvious differences, CUPHNSCC shares similarities in risk profile with OPSCC. In contrast to OPSCC, the detection of p16 alone  appears more appropriate for the classification of CUPHNSCC. Three risk groups of patients with CUPHNSCC and significant differences in OS can be stratified based on ECOG and p16, though additional factors may become important in future studies or cases with particular risk profiles.The role of HPV and risk factors in CUP


Human papillomavirus (HPV) detection and genotyping were performed by PCR and immunohistochemistry. Sixteen samples were inconclusive for HPV detection. In total, 93% of the specimens exhibited HPV positivity, which increased with patient age. The most detected high-risk (HR)-HPV type was HPV16 (41.2%) followed by HPV18 (16%). The immunostaining of cytoplasmatic βA-activin and follistatin was higher than nuclear immunostaining in all cervical epithelium layers of the CIN1, CIN2, CIN3, and SCC groups. A significant decrease (p < 0.05) in the cytoplasmic and nuclear immunostaining of βA-activin was detected in all cervical epithelial layers from the control to the CIN1, CIN2, CIN3, and SCC groups. Only nuclear follistatin immunostaining exhibited a significant reduction (p < 0.05) in specific epithelial layers of cervical tissues from CIN1, CIN2, CIN3, and SCC compared to the control. Decreased immunostaining of cervical βA-activin and follistatin at specific stages of CIN progression suggests that the activin–follistatin system participates in the loss of the differentiation control of pre-neoplastic and neoplastic cervical specimens predominantly positive for HPV.The activin–follistatin system regulates several cellular processes, including differentiation and tumorigenesis. We hypothesized that the immunostaining of βA-activin and follistatin varies in neoplastic cervical lesions. Cervical paraffin-embedded tissues from 162 patients sorted in control ( Cervical cancer is the fourth most common type of cancer among women, resulting in an estimated mortality rate 2018–2020) of 311,000 women worldwide 8–2020 of. HoweverE1, E2, E4, E5, E6, and E7 genes, and the long control region (LCR), encoding the L1 and L2 capsid-binding proteins [E6 spliced isoforms (E6 *) have been hypothesized to play a role in HPV-induced carcinogenesis [HPV is a small non-enveloped circular DNA virus classified into two main categories: HR-HPVs, which include the HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, with HPV16 and HPV18 being the most prevalent and oncogenic ones, and low-risk (LR)-HPVs, which include the HPV types 6, 11, 40, 42, 43, 54, 61, 70, 72, and 81, that are primarily associated with the development of benign genital and skin wart lesions [proteins . In addiWomen are frequently exposed to mucosal HPV infections during their first sexual intercourses; however, most of the of sexually active women infected by HPV during early adulthood (>90%) will resolve the HPV infection via actions of the host immune system- within 2 years. If persistent, cervical HPV will lead to the development of pre-neoplastic or neoplastic lesions of the cervix . The mecActivins are growth factors belonging to the transforming growth factor (TGF)-β superfamily, which is extensively implicated in the onset of several proliferative disorders. Activins, which are also considered to be peptide hormones or cytokines, are polypeptide dimers bound by a disulfide bridge. There are five types of activins formed by two β-dimers each, namely, activin A, formed by two βA subunits (βA-βA), activin B (βB-βB), activin AB (βA-βB), activin C (βC-βC), and activin E (βE-βE). Importantly, activin A is the isoform of greater physiological importance in humans . ActivinFollistatin and inhibins are two major regulatory elements preventing activins from functionally activating their transmembrane receptors . FollistActivins can be secreted by several cell types and have distinct actions in different cells and tissues , includiTo date, knowledge about the role of the activin–follistatin system in the progression of cervical cancer is limited . It has n = 15. LSIL was designated as CIN-1 (n = 38). HSIL lesions were subdivided in two groups, CIN-2 (n = 37) and CIN-3 (n = 39), whereas the neoplastic cervical lesions were SCC (n = 33). Of importance, since the minimum age range of the HSIL cases enrolled in our cohort was 17 years and the CIN2 and CIN3 terminology is preferred in young women (aged < 30 years) due to higher regression rates in the former [This is a retrospective cohort study that includes 162 cervical biopsies of paraffin-embedded tissues (PETs) selected from the archival tissue bank of a large anatomical and histopathological laboratory analysis . Histopathological diagnoses were performed between 2004 and 2006 (therefore the population included in our cohort did not receive HPV vaccination). Cervical biopsies were sorted into five different study groups. The control group consisted of tissues with no cervical lesions; e former , we subdPET-cervical tissues were cut into 5 μm serial cross-sections and stained with H&E  for histopathological analysis or subjected to immunohistochemistry analysis for βA-activin or follistatin using protocols previously described ,38,39 wiHistopathological diagnosis of cervical lesions and SCC was revised by three experienced pathologists, blinded to groups, employing established criteria . Three rFor immunohistochemistry, slides were deparaffinized in xylene, rehydrated in a decreasing alcohol series, and subjected to antigen retrieval by boiling in 0.01 M sodium citrate buffer (pH 6.0) solution for 30′ in a water bath at 100 °C. After cooling at room temperature (RT), slides were blocked using a PBS (1×)/BSA  solution for one hour (RT). This was followed by incubation at 4 °C overnight in a 1:1500 dilution of anti-βA-activin  or anti-follistatin  primary antibodies. The next day, a non-specific reaction to endogenous peroxidase was blocked by incubating the slides with a solution of hydrogen peroxide (3%)/methanol for 30′ (RT). Next, sections were incubated with biotinylated anti-mouse secondary antibody for βA-activin  or anti-rabbit secondary antibody for follistatin , for one hour (RT). Between each step, slides were washed three times (for 10′ each) with PBS 1×. Slides were incubated for 30 min (RT) with streptavidin–HRP (horseradish peroxidase)- and subjected to chromogenic detection of HRP activity by 3,3′-diaminobenzidine (DAB) reagent . Sections were counter-stained with Harris’ haematoxylin. Primary antibody replacement with normal serum provided negative controls.Cytoplasmatic and nuclear immunoreactivity for βA-activin and follistatin was examined in different histological layers of healthy and pathological cervical tissue by visual assessment using optic microscopy . The immunoreactive score (IRS) of βA-activin and follistatin  was calc®, São Paulo, SP, BRA). The PCR amplification of the β-globin gene was used to control the integrity of the extracted DNA by using a previously described hemi-nested PCR protocol [β-globin gene using cervical human lysate as a DNA template. The PCR product obtained in the first reaction was used as a template in the second PCR amplification, performed with PC03 (5′ ACACAACTGTGTTCACTAGC 3′) and PC04 (3′ CCACTTGCACCTACTTCAAC 5′) primers, able to amplify from the β-globin gene a 100 bp DNA fragment internal to the DNA fragment first amplified. The employed PCR conditions  are described in To assess the HPV infective status of the cervical biopsies, 10 µm thick paraffin-embedded sections were obtained per sample as previously described . Importaprotocol . BrieflyHPV DNA detection was undertaken by a nested PCR protocol as described previously . For theHPV-positive samples were genotyped for the detection of the HR-HPVs 16, 18, 31, 33, 35, 52, and 58 using both hemi-nested PCR and conventional PCR protocols. DNA detection from HPVs 16, 18, 52, and 58 was performed by hemi-nested PCR protocols developed in our laboratory, using primers and PCR conditions previously described, with minor modifications . For the® . This step was undertaken in a two-step washing procedure enclosed in a 100 °C steamer for 30′ each, followed by 2 washes in distilled water, RT, for 5′ each. The NovoLink kit®  was used for p16 immunohistochemistry. Endogenous peroxidase activity and a nonspecific protein block were undertaken by slide immersion in specific NovoLink kit® solutions for 10′ each (RT). Sections were washed twice in 1×-PBS for 5′ and incubated for 1 h (RT) with p16 monoclonal primary antibody diluted in 3% 1×-PBS/BSA at 1:100 . After the incubation step, sections were washed 3 times in 1× PBS for 5′ (RT). Tissue sections were subsequently treated with the post primary antibody, Block-solution, for 50′ (RT), washed twice with 1× PBS for 5′ (RT), exposed to the Novolink polymer  or 30′ (RT), and re-washed (3 times) in 1× PBS for 5′ (RT). Immunoreactivity was visualized using diaminobenzidine 3,3 (DAB) chromogen. Specimens were counter-stained with Harris’ Hematoxylin  and evaluated by light microscopy . CIN specimens were deemed positive for p16 if nucleo-cytoplasmic staining in the cervical squamous epithelium was strong and negative if nucleo-cytoplasmic staining was weak/sparse. Positive controls consisted of SCC specimens that were positive for both HPV DNA and HPV DNA genotyping. Negative controls were performed by omitting the primary antibody.CIN1, CIN2, and CIN3 tissue specimens that were negative for both HPV DNA and HPV DNA genotyping analysis were evaluated for p16 immunostaining, a surrogate marker for HR-HPV . Tissue p < 0.05.Data distribution was analyzed by the D’Agostino and Pearson normality test. Differences among groups for βA-activin and follistatin IRS were assessed by the Kruskal–Wallis test for every component of the cervical epithelium and stroma followed by Dunn’s test for non-parametric multiple comparisons. All statistical analyses were performed using R-statistics computer software. Data are presented as the mean ± standard error of the mean (SEM), and differences were considered significant if The mean patient age of all groups was 39.2 ± 15.6 years (range: 17–90). As expected, the group that exhibited the highest mean age was the SCC group (50.3 ± 15.6), whereas the group with the lowest mean age was CIN1 (30.85 ± 10.38) . The preWe conducted a detailed histopathological characterization of all sample tissue specimens included in our cohort. As expected, the cervical specimens of the control group exhibited distinct histological layers with germinative cells in the basal layer and the absence of dysplasia and HPV infection signals in the stratified epithelium . CIN1 hiTo determine the prevalence of HPV infection in our cohort, we assessed the presence of HPV DNA in cervical PET specimens diagnosed with CIN1-3 and SCC. In the present study, out of the 162 initial cervical specimens, 16 samples were inconclusive for HPV detection; therefore, all HPV detection analyses were undertaken in 146 specimens of the cohort. Almost sixty percent (59.6%) of the specimens (87/146) were positive for HPV DNA. The HPV DNA positivity rate for the CIN1 group was 46.7% (14/30). In CIN2, we detected 51.5% HPV DNA positive samples (17/33). In CIN3, we found 75% HPV DNA positivity (27/36), whereas, in the SCC group, 90.6% of the samples (29/32) were positive for HPV DNA .Next, we genotyped HPV types more frequently associated with the progression of pre-neoplastic lesions . We weren = 131; n = 3) in CIN1, 30.3% (n = 10) in CIN2, 52.8% (n = 19) in CIN3, and 68.8% (n = 22) in SCC specimens.Furthermore, some specimens were positive for more than one HR-HPV type. As expected, there were no HR-HPV types genotyped in the control group. HPV 16 genotype was the most prevalent in 41.2% (54/131) of the genotyped samples with cervical lesions n = 131; , followen = 9), CIN2 (n = 14), and CIN3 (n = 6)-negative specimens for both HPV DNA and HPV DNA genotyping, using p16 immunoreactivity—a surrogate marker for HR-HPV [We further investigated HPV positivity in CIN1 , CIN1 (n = 38), CIN2 (n = 37), CIN3 (n = 39), and SCC (n = 33) groups. βA-activin was detected in the entire cervical epithelium and stromal components in different stages of cervical cancer progression , CIN2 (p < 0.001), CIN3 (p < 0.001), and SCC (p = 0.028) were lower than that of the control group. Similarly, the cytoplasmic IRS values of βA-activin in the CIN1 (p = 0.002), CIN2 (p = 0.003), and SCC (p = 0.028) groups were lower than that in the control group. In the intermediate layer of the control group, we found lower nuclear (p = 0.0039) and cytoplasmic (p = 0.0004) IRS values of βA-activin compared to those of CIN1. In the superficial layer, the nuclear IRS values of CIN1 (p = 0.0052) and CIN2 (p = 0.004) were lower than those in the control group. Accordingly, compared to the control, cytoplasmic IRS was lower in the CIN1 (p = 0.013) and CIN2 (p = 0.024) groups. In columnar epithelium, we found lower nuclear (p = 0.003) and cytoplasmatic βA-activin IRS values (p < 0.001) in CIN1 compared to the control. Similarly, CIN1 had lower cytoplasmatic IRS than that in the CIN2 (p < 0.001) and CIN3 (p = 0.0029) groups. In blood vessels, nuclear IRS in the CIN1 (p < 0.001), CIN2 (p = 0.0189), CIN3 (p < 0.001), and cancer (p < 0.001) groups was lower than that in the control group. In addition, cytoplasmic IRS was lower in the CIN1 (p < 0.001), CIN2 (p = 0.0233), CIN3 (p = 0.01), and cancer (p = 0.001) groups compared to the control. Concerning the inflammatory cell infiltrates, nuclear IRS in the CIN1 (p < 0.001), CIN2 (p = 0.03), CIN3 (p = 0.017), and cancer (0.0288) groups was lower than that in the control, whereas the cytoplasmic IRS of the CIN1 group had lower IRS compared to the control (p = 0.0047), CIN2 (p = 0.001), and CIN3 (p = 0.0192) groups.Next, we evaluated the IRS of βA-activin in different groups . The oveFollistatin tissue localization was detected in all cervical epithelial layers, as well as in the stromal components of cervical specimens from the control group through all stages of progression into SCC . In the p < 0.001). In the intermediate layer, nuclear IRS was lower in CIN1 compared to controls (p = 0.045). In the superficial layer, follistatin IRS in the cytoplasm and the nucleus were lower in CIN2 compared to the control group (p < 0.05). In contrast, nuclear follistatin IRS in CIN1 was lower compared to controls (p < 0.05). In the columnar epithelium, follistatin IRS was decreased in the nucleus of CIN1 and CIN2 compared to controls (p = 0.002). In addition, blood vessel nuclear follistatin was decreased in CIN1, CIN2, and SCC compared to controls (p < 0.05). Finally, in a comparison of the nuclear follistatin IRS of inflammatory cells, the nuclear IRS of SCC (p = 0.037) was lower than the IRS observed in the control group.Compared to the control group, no IRS differences in cytoplasmic and nuclear follistatin were identified in the cervical basal layer of CIN1 to CIN3 and SCC . HoweverIn the present study, we investigated the localization pattern and expression profile of βA-activin and follistatin during different stages of cervical cancer progression (from CIN1 to SCC) in unvaccinated women predominantly positive for HR-HPV infection.We observed an increase in the mean patient age along with the progression of cervical lesions from CIN1 to SCC. These results are in agreement with previous studies reporting greater risk of cervical cancer lesions in older patients . In the Several molecular and epidemiological studies conducted over the past 40 years elucidated that SCC development is significantly related to persistent HR-HPV infection ,3. In thOur cohort’s HPV positivity rate gradually increased during cervical lesion progression. Our results agree with a systematic study  reportinThe most prevalent HR-HPV types involved in the progression of intra-epithelial lesions to cervical cancer are HPVs 16, 18, and 31 ,63,64. IOur study is the first to analyze βA-activin and follistatin expression profiles in the stratified epithelium layers and components of cervical human specimens. In normal cervical tissue, we detected positive immunostaining of βA-activin in all layers of the cervical epithelium, both in the nucleus and in the cytoplasm, suggesting that activin A and/or AB or inhibin A may control the proliferation and differentiation of the normal cervical stratified epithelium and stroma. There is limited information about the effect of activins, inhibins, and follistatin controlling cervical epithelial cell proliferation and differentiation. However, it has been demonstrated that activin A inhibited cell proliferation and increased the cellular volume of the PHM1 myometrial cell line . FurtherOur study observed an overall decrease in cytoplasmic and nuclear βA-activin IRS in different stages of cervical lesion progression. This important finding suggests that lower expression of activins A and/or AB or inhibin A may be involved in cervical lesion progression as early as the initial stages of CIN1 development. It has been reported that activin A cervical fluid levels decreased after surgical removal of the lesion , suggestOur current results showing decreased βA-activin IRS in SCC specimens agree with previous findings reporting decreased activin βA and βB subunits in the epithelium of cervical adenocarcinoma compared to a healthy cervix . Of impoWe also identified lower cytoplasmic and nuclear immunostaining of βA-activin in the blood vessels of CIN1, CIN2, CIN3, and SCC compared to the control group. The effect of activin A regulating different aspects of angiogenesis is controversial since reports have described activin A may have either pro-  or anti-Immunolocalization of βA-activin was also detected, mainly in the nucleus of mononuclear cells of the inflammatory infiltrates in the control group , while a decrease in βA-activin IRS was verified in subsequent stages of CIN progression. The activity of several inflammatory cells, such as macrophages, can be stimulated by activin A, which can induce a pro- or an anti-inflammatory effect depending on the cellular maturation stage ,70. For It is important to note that activin A is related to immune responses to viral infections. An increase in tissue activin A may be related to the inhibition of the viral replication of the hepatitis C virus, human cytomegalovirus, and Zika virus. In the chronic stages of hepatitis A and B and H1N1 infection, an increase in the local expression of activin A is related to liver lesions’ chronicity and fibrosis . Here, wWith respect to follistatin, it participates in the tumorigenesis, metastasis, and angiogenesis of different tumors through its interaction with members of the TGF-β superfamily, especially activins ,78. In aImportantly, the decrease in the follistatin nuclear immunoscore occurred in the intermediate layers between the control group and CIN1 and in the superficial layer, between the control group and CIN1/CIN2. Although we did not observe any alterations in the follistatin immunoscore in the basal layer, changes identified in the upper epithelium may have originated in the basal layer. In cervical blood vessels, nuclear follistatin immunostaining decreased in all groups compared to the control, whereas nuclear follistatin IRS in inflammatory cells decreased in the SCC group. Follistatin is secreted by endothelial cells when stimulated by pro-inflammatory cytokines . TherefoWe conclude that HPV16 was most prevalent in the cervical lesions of the unvaccinated population herein investigated and that βA-activin and follistatin IRS decrease in pre-neoplastic and neoplastic diseases of the human cervix, predominantly positive for HPV. Since great effort is currently being undertaken to develop therapeutic agents that modulate activin signaling , underst


Human papillomaviruses are small DNA tumor viruses that infect cutaneous and mucosal epithelia. The viral lifecycle is linked to the differentiation status of the epithelium. During initial viral infection, the genomes replicate at a low copy number but the mechanism(s) the virus uses to control the copy number during this stage is not known. In this study, we demonstrate that the tyrosine kinase focal adhesion kinase (FAK) binds to and phosphorylates the high-risk viral E2 protein, the key regulator of HPV replication. The depletion of FAK with a specific PROTAC had no effect on viral DNA content in keratinocytes that already maintain HPV-16 and HPV-31 episomes. In contrast, the depletion of FAK significantly increased HPV-16 DNA content in keratinocytes infected with HPV-16 quasiviruses. These data imply that FAK prevents the over-replication of the HPV genome after infection through the interaction and phosphorylation of the E2 protein. HPV replication is linked to the differentiation status of the infected epithelium ,2. Initi6GGT sequences at the viral origin of replication (ori) to support genome replication [Basal levels of the viral E2 protein are required to initiate viral replication ,14. The lication . E2 is rlication ,20,21,22lication . We idenlication . Pyk2 shlication .FAK is a tyrosine kinase involved in cell migration, adhesion, proliferation, and angiogenesis . FAK is Since FAK is expressed in keratinocytes , and we We previously demonstrated that Pyk2 was nuclear and that it bound the HPV-16 and 31-E2 proteins . FAK hasThe FAK protein is both nuclear and cytoplasmic. We performed immunofluorescence for FAK in the cervical HPV-16 keratinocyte dysplastic cell line W12 clone 20850. Although we observed that FAK and Pyk2 were predominantly cytoplasmic, we also observed nuclear staining .To determine the effects of FAK on viral genome replication during the maintenance stage, we used W12 cells clone 20850. When cultured with J23T3 feeders, this cell line maintains the viral HPV-16 episomes. We confirmed the episomal status using an exonuclease V assay . W12 celTo determine whether FAK depletion affects viral replication during the amplification stage of viral lifecycle, we treated CIN612 cells which contain episomal HPV-31 with 2 mM calcium to induce keratinocyte differentiation. BI-0319 induced FAK degradation in both proliferating and differentiated cells A. BI-031Our next goal was to determine if FAK influenced early viral replication initially after viral infection. In monolayer cell lines derived from HEK293, L1 and L2 self-assemble into virions and efficiently package co-transfected viral DNA (quasivirus) or any reporter DNA less than 8 Kb  ,37,38. HWe packaged HPV-16neo (L1 and L2 genes were replaced with a neomycin cassette) DNA into HPV-16 L1/L2 capsids (VLPs). NIKS cells were infected with mCherry pseudovirus and HPV-16 quasivirus at the time of BI-0319 treatment. We did not observe differences in mCherry protein levels after infection A with FAIn this study, we demonstrate the importance of FAK activity on initial HPV replication. Similar to previously studied Pyk2, FAK binds to and increases HPV-E2 tyrosine phosphorylation. Pyk2 siRNA and treatment with a FAK/PYK2 dual specificity inhibitor, PF-431396, significantly increased HPV DNA content in cell lines that maintain viral episomes . HoweverViral particles bind to heparan sulfate and activate FAK by phosphorylation at Y397 through the α6 integrin receptor . The treWe observed a modest increase, which was, however, not statistically significant, in viral genome content after FAK PROTAC treatment in differentiated CIN612 cells. Like during initial replication, replication during differentiation is not dependent on the host cell cycle. Our data suggest that FAK is an important regulator during bursts of replication. Calcium treatment-induced differentiation can increase FAK autophosphorylation and kinase activation . These eFAK mRNA and protein expression were significantly higher in cervical cancer cell lines when compared to moderate to well differentiated squamous cell carcinoma . The treAfter infection in the basal epithelium, the virus must undergo replication at a low copy. E2 was one of the first transcripts expressed at 4 h post infection using native HPV-31 virions . Viral rThe following plasmids were used: pCDNA3-FLAG-HPV-31 and -16 E2, 16pSheLL (gift from J. Schiller), GFP-Pyk2 WT and KD , and pGF2. HEK293TT (from J. Schiller and C. Buck), J23T3 fibroblast feeders, HaCaT (from N. Fusenig), SiHa, and HEK293TTF (from R. Roden) were cultured in Dulbecco modified Eagle medium  with 10% fetal bovine serum  and penicillin/streptomycin . W12 cells (from P. Lambert), NIKS (from B. Allen-Hoffman), and CIN612 cells from (L. Laimins) were grown in F or E medium with mitomycin-C-treated J23T3 fibroblast feeders. Quasiviruses were produced as previously described [All cell lines were maintained at 37 °C and 5% COescribed  using thescribed  or pmCheescribed . BI-0319Cells were plated on a coverslip in 6-well dishes and immunofluorescence analysis was performed as previously described . FAK (CeCells were transfected with PEI (2 mg/mL) and lysed, and immunoprecipitation and immunoblotting were performed as previously described  Inputs aCT – Undigested CT)–(Digested  × 100.Cells were resuspended in PBS and isolated by using the DNeasy blood and tissue kit (Qiagen). For the exonuclease V digestion, 500 ng of DNA was either treated with exonuclease V (New England Biolabs) or left untreated for 4 h at 37 °C. The enzyme was inactivated at 70 °C for 20 min. Next, 50 ng of the digested or undigested DNA was quantified using real-time PCR. HPV-16/31 LCR and actin primers were previously described ,53.{The t-test was used to find the significant value.All experiments were repeated at least three independent times, except when stated otherwise, and results were averaged. The Student’s two-way


Although there was a difference in the expression of p53 between specimens that were positive and negative to toluidine blue, the correlation did not reach a significant level. Based on the results from this study, vinegar has a lower sensitivity than toluidine blue but a higher specificity for oral cancer screening. The results of the clinical screening using vinegar correlated with the expression of Ki67 at the cellular level.The aims of this study were to investigate the sensitivity and specificity of toluidine blue and/or vinegar in oral cancer screening and to examine the correlation between clinical screening using toluidine blue and vinegar and the expression of the tumor marker p53 and proliferation marker Ki67, respectively. The study consisted of 87 patients with suspected oral squamous cell carcinoma lesions. Toluidine blue and/or vinegar were applied to the lesions, followed by biopsies. The tissues were diagnosed histopathologically and underwent immunohistochemical process for p53 or Ki67. The results revealed that the sensitivity and specificity of oral cancer screening using toluidine blue were 93% and 46%, respectively; whereas the sensitivity and specificity using vinegar were 85% and 81%, respectively. A statistically significant correlation between the use of vinegar and the expression of Ki67 ( Both toluidine blue and vinegar were applied to a third group of 24 patients.For the application of toluidine blue, a cotton bud soaked in 1% acetic acid was used to clean the lesion prior to the application of toluidine blue with a different cotton tip for 30 s. Subsequently, a third cotton bud soaked with 1% acetic acid was used to remove any excess toluidine blue on the lesion. The patient then rinsed out their mouth. A positive lesion was the one which color changed to blue, while a negative lesion was with no change. The area was then photographed and an incisional biopsy was performed at the blue-stained area.For acetic acid application, a gauze soaked with vinegar was applied to a clean and dry lesion for one minute. The researcher noted the changes and took picture of the lesion. A positive finding was designated when a lesion changed to opaque white. When a lesion did not change or changed to transparent white, a negative finding was indicated. An incisional biopsy was performed at the area that had turned opaque white.For the 24 patients to which both substances were applied, application of vinegar was conducted first, followed by the application of toluidine blue. A positive finding was indicated for a lesion with color changed to both opaque white and blue, while a negative finding was a lesion not demonstrating these combined changes. An incisional biopsy was performed at the area that turned opaque white and had stained blue. If the areas that had stained blue and turned opaque white were not coincident, incisional biopsies were performed at both areas. If the lesion did not stain or change color, a biopsy was performed at the central area of the lesion.The specimen was routinely processed for histology, and sections were cut and then stained with hematoxylin and eosin for histopathological  diagnosis. For tissues obtained from patients receiving toluidine blue application, the next tissue section was used for the immunohistochemical study of tumor marker, p53 . For tisp-value less than 0.05 was considered statistically significant.Sensitivity and specificity of toluidine blue and/or vinegar for oral cancer detection were calculated. The correlation between the results of the vinegar application and the final diagnoses was determined using Fisher's exact test. Differences in the percentage of p53/Ki67 positive cells between toluidine blue/vinegar positive and negative lesions were compared using the Mann–Whitney test. A p = 0.000 and p = 0.004, respectively).As seen in p = 0.198) (in situ (9/81.8%), and oral squamous cell carcinoma (24/88.9%) (p = 0.018) (= 0.198) . The res= 0.198) , Dysplas= 0.198) . In cont= 0.198) . We foun4/88.9%) . The per4/88.9%) . The pos= 0.018) .The patients in our study were 61.8 ± 11.8 years old with a male to female ratio of 6 to 5 is comparable with that of other studies . Betel nWhen we compared the sensitivity and specificity of toluidine blue in the present study to the sensitivity and specificity of toluidine blue used for oral cancer screening from other studies –24, our The association between oral cancer screening using toluidine blue or vinegar and final diagnoses indicated that toluidine blue and vinegar reacted better with malignant or dysplastic tissues than with normal tissues, warranting the application of toluidine blue and 5% acetic acid in oral cancer screening. As vinegar has a comparable sensitivity to toluidine blue but a higher specificity in oral cancer screening, future study evaluating the use of vinegar for the detection of oral premalignant lesions in rural communities will be of value.in situ, and epithelial dysplasia was significantly higher than less affected specimens. This revealed that the immunohistopathological diagnosis of oral premalignant and malignant lesions are correlated with the number of cells positive to Ki67 antibody. Our findings further warrant the application of both p53 and Ki67 antibody reaction for oral cancer treatment planning and prognosis determination.Our results revealed that the observable transformations due to vinegar use in oral cancer screening and the results of final diagnoses were associated with a significant difference in Ki67 positive cells between tissues that were positive to vinegar screening and those that were not. Although the number of p53 stained cells were higher in specimens that were positive to toluidine blue than those negative to toluidine blue, the difference did not reach a significant level. This could stem from the difficulty in separating lesions that are truly positive to toluidine blue  and false positives  as stated by a recent publication that pale blue lesions have no histological significance . The num


In the landscape of healthcare disparities and the marginalized status of men who have sex with men (MSM) in Malaysia, understanding the dynamics surrounding HPV vaccination is of paramount importance. The purpose of this study is to examine the knowledge and attitudes of MSM regarding HPV vaccination and to identify factors that may hinder or facilitate its uptake. The findings will contribute to the development of targeted interventions to promote HPV vaccination and reduce the burden of HPV-related health issues among Malaysian MSM. Between May 2019 and September 2022, an online cross-sectional survey was conducted to collect data through popular social media platforms targeting MSM in Malaysia. A multivariable logistic regression model was employed to investigate the associations between HPV vaccination intention and various influencing factors. Out of the total 411 respondents in the study, 266 (60.3%) indicated an intent to receive the HPV vaccination, falling under the categories of “certain to happen”, “very likely”, and “likely”. The average knowledge score for participants was 6.82  out of a total possible score of 13. In the multivariate logistic model, participants who identified themselves as bisexual  and gay/homosexual  showed a greater inclination to receive the HPV vaccine compared to heterosexual participants. High intent to be vaccinated for HPV infection was positively and significantly associated with a high level of knowledge . In the multivariable model, there was no significant association between all variables of attitudes towards HPV infection and HPV vaccinations and the intention to receive HPV vaccination. Study participants reported a low level of susceptibility to HPV infection despite their perception that HPV infection is severe. Two-thirds of participants expected to encounter stigma in healthcare settings during future implementation of HPV vaccination programs. This study underscores the importance of improving HPV vaccine acceptance among Malaysian MSM due to the moderate acceptance level observed. In Malaysia, promoting HPV awareness, enhancing risk perception, and addressing stigma and sensitivity surrounding HPV vaccination may be beneficial in increasing the vaccination willingness among MSM. The Human Papillomavirus (HPV) is a sexually transmitted infection (STI) affecting both men and women. Infection with HPV is widely recognized as a significant risk factor for the development of anal cancer . AccordiIn addition to its association with anal and penile cancers, HPV has also been identified as a causative factor in various types of malignancies affecting the head and neck region. HPV infection is responsible for a minimum of 3% of oral cancer cases, 12% of pharyngeal cancer cases, and a significant proportion ranging, from 30% to 60%, of oropharyngeal carcinoma cases ,8. FurthIn Malaysia, cervical cancer ranks as the fourth most prevalent cancer  and stanThe implementation of HPV vaccination programs has demonstrated effectiveness in reducing the prevalence of HPV-associated diseases, thereby reducing the disease burden. Various factors may influence the acceptance of the HPV vaccine among MSM. These factors include knowledge regarding HPV and the vaccine, awareness of the associated health risks, concerns regarding vaccine safety and efficacy, social and cultural influences, and access to healthcare services ,15,16,17In 2010, Malaysia launched a national vaccination program with the objective of providing three doses of HPV vaccines to all 13-year-old girls. In 2015, the program transitioned to a two-dose regimen. Importantly, this program has achieved a population coverage rate of over 80% . The maiThe primary objective of this study is to evaluate HPV vaccination acceptance among MSM in Malaysia. In addition, the study will investigate knowledge and attitudes towards HPV and HPV vaccination acceptance among MSM. Our study hypothesizes that demographics, sexual behavior, sexual identity, and stigma in healthcare settings concerning MSM receiving the HPV vaccine may influence acceptance. The study seeks to inform the development of effective strategies to promote vaccine uptake among the MSM population.A cross-sectional online survey was conducted from May 2019 to September 2022. Eligibility criteria for participants included: (i) being 18 years of age or older, (ii) being Malaysian citizens, and (iii) identifying as biologically male and reporting engaging in sexual activity with other men within the past 12 months. Several recruitment methods were utilized to engage participants, focusing on leveraging popular social media platforms within the MSM community in Malaysia, including platforms such as Grindr and Hornet. The survey link was strategically posted on these platforms, ensuring maximum visibility among the target audience. Furthermore, social media profiles of LGBT-friendly non-governmental organizations were utilized, and peer referrals were also employed to reach and engage potential participants. In addition, a network of peer referrals was established, encouraging participants to refer their peers to participate in the survey. These collective approaches were employed to ensure a diverse sample of MSM in Malaysia was reached and engaged in the study.Interested individuals were given a web link to access the online survey hosted by Qualtrics  . Before n = Z2 × P × (1 − P)/d2 [2 × 0.5 × (1 − 0.5)]/0.052).The sample size was calculated using the formula:  − P)/d2 . The samA self-administered questionnaire in English was developed with reference to the relevant previous studies in the literature ,28,29. TParticipant demographic characteristics collected in the study included age, ethnicity, highest educational attainment, occupation, and income. Participants self-reported their sexual identity, sexual behaviors, symptoms of STIs, and any history of being diagnosed with an STI.To evaluate the participants’ knowledge regarding HPV and HPV infections, a 13-item questionnaire was utilized in this study. The questionnaire required participants to indicate whether a given statement was “true”, “false”, or “don’t know”. For scoring purposes, a correct response was assigned a value of one, while an incorrect or “don’t know” response received a value of zero. The scores for each item were summed up, and those who scored equal to and higher than the median value were classified as having good knowledge, while those who scored lower were classified as having poor knowledge.To assess the attitude of the participants towards HPV infection, a six-item questionnaire was employed in this study. The questionnaire in this study utilized a 4-point Likert scale to measure participant responses. The scale included options ranging from “very high” to “very low” and from “strongly disagree” to “strongly agree”. Attitudes towards HPV vaccination were measured using 11-item questions. Similarly, a 4-point Likert scale was used to measure participant responses. The scale included options ranging from “strongly disagree” to “strongly agree”.Participants were queried regarding the perception of stigma and discrimination in healthcare settings surrounding the vaccination against HPV for gay and bisexual men, with the options for response being either “yes” or “no”. The acceptance of the HPV vaccine was assessed using a rating scale ranging from 1 to 7, with 1 indicating “no chance” of receiving the vaccine and 7 indicating a “certainty to happen”. Participants were asked to indicate their intention to receive the HPV vaccine based on this scale. The study questionnaire is provided in This study was approved by the University of Malaya Research Ethics Committee (UMREC). Approval code: (UMREC)-UM.TNC2/UMREC—493.p < 0.05), were conducted to determine factors associated with the intention to take the HPV vaccine. Odds ratios (OR), 95% confidence intervals (95% CI), and p-values were calculated for each independent variable. The “certain to happen”, “very likely”, and “likely” categories were merged to denote the intent category. The “no chance”, “very unlikely”, “unlikely”, and “moderate chance” categories were merged to denote the non-intent reference category. A Hosmer–Lemeshow test was performed to check the model’s goodness of fit [p-value of less than 0.05 was considered statistically significant.Frequency tables, charts, and proportions were used for data summarization. The proportion and its respective 95% confidence interval (CI) were calculated. Cronbach’s alpha was calculated for the knowledge items to assess reliability in terms of internal consistency. The knowledge scale had good internal consistency, with a Cronbach’s alpha of 0.919. Univariate analyses followed by a multivariable logistic regression analysis, including all factors showing significance  belonged to the professional and managerial group .Most participants (73.7%) self identified as gay, homosexual, or belonging to people like us (PLU) as their sexual orientation. Under half of the participants (48.3%) reported engaging in condomless anal intercourse within the past six months. Additionally, 25.2% of the participants reported experiencing symptoms of STIs at some point, while 26.8% had a history of STIs within the past year.As indicated in With regard to attitudes concerning HPV vaccination, 69.2% reported that they lacked sufficient information about the HPV vaccine to make a decision regarding vaccination. A majority of participants also concurred that the long-term potential side effects of HPV vaccination remain unclear. Approximately 32.7% perceived taking HPV vaccination as a sign of promiscuity, while 47.6% acknowledged that HPV vaccination is a sensitive topic. A considerable number of participants expressed agreement that recommendations for HPV vaccines from friends (70.8%) and healthcare providers (69.2%) can positively influence their intention to get vaccinated against HPV. The majority of participants (75.4%) anticipate the presence of stigma in healthcare settings when it comes to gay and bisexual men receiving the HPV vaccine.The MSM population faces a high risk of HPV infection, and targeted HPV vaccination is the most cost-effective public health measure for prevention. Studying HPV vaccine acceptance among the MSM population in Malaysia, a country with a Muslim majority and conservative values, is crucial because it sheds light on the potential challenges and opportunities associated with implementing effective HPV prevention strategies in such cultural and social contexts.The knowledge score from this study reveals gaps in participants’ understanding of the transmission of HPV. For example, a significant number of participants were unaware that genital HPV can be transmitted through skin contact or non-sexual contact . Many weIn this study, participants perceived low susceptibility to HPV infection, even though they recognized its severity. The findings regarding attitudes toward HPV infection underscore the importance of emphasizing the risks associated with HPV infections. Low-risk perception and susceptibility have been identified as barriers to HPV vaccine acceptance among men and MSM ,34,35. WIn general, the participants showed varied attitudes to HPV vaccination. This study underscores the heightened risk of HPV infection among MSM. It is evident that a substantial proportion of MSM expressed a moderate willingness to receive the HPV vaccine, underscoring the need for intensified efforts to promote and encourage HPV vaccination within this demographic. The research underscores the pivotal role of knowledge in influencing vaccination willingness among MSM. While attitudes toward HPV infection and vaccination may not be significant predictors of vaccination intention, certain misconceptions have been identified. Therefore, to enhance vaccination rates, it is crucial to prioritize the dissemination of accurate information about HPV and the HPV vaccine while fostering positive attitudes.Many participants reported that inadequate information about the HPV vaccine hindered their decision-making in vaccination. Additionally, the long-term potential side effects of HPV vaccination remain unclear for many participants. HPV vaccination has been available for a significant period, but study participants highlighted that the long-term potential side effects of HPV vaccination remain unclear. Therefore, awareness interventions that provide accurate information about the safety of HPV vaccination with supporting clinical data are crucial in promoting public understanding and acceptance of the vaccine. Most importantly, intervention should enlighten participants that HPV vaccines have undergone extensive clinical testing and have been proven safe and effective in preventing HPV infections. Numerous large-scale clinical trials involving thousands of participants have been conducted to evaluate the safety of HPV vaccines before they were approved for public use. Additionally, the HPV vaccine’s safety has been confirmed through post-marketing surveillance studies and by data from national immunization programs . Of impoAddressing sensitivity towards HPV vaccination is crucial in Muslim countries, where sexual-related issues  remain a cultural taboo . In thisThe study participants displayed a moderate level of acceptability towards the HPV vaccine, with approximately 60% of them falling into the category of intending to vaccinate. Our study adds to the evidence indicating that MSM have an average acceptance of HPV vaccination . No signIn this study, a higher level of knowledge significantly predicted HPV vaccine acceptance. Similarly, numerous other studies have consistently reported that an increased awareness of HPV is positively associated with a greater willingness to undergo vaccination among men . The finSeveral study limitations should be acknowledged. Firstly, the use of an online survey may introduce bias, as participation relies on self-selection and access to the internet. Consequently, individuals who lack internet access or do not engage in social media may be underrepresented in the sample. Secondly, the study employed convenience sampling, further potentially biasing the sample toward individuals who are more active online or open about their sexual orientation. Moreover, the data collected may be influenced by reporting bias, as participants may be hesitant to divulge sensitive information related to their sexual behaviors and attitudes. These limitations, coupled with the relatively small sample size of 441 participants, pose challenges in generalizing the findings to the broader MSM population in Malaysia, especially given the unknown size of this population. Despite these limitations, this study provides valuable insights that can inform future research and interventions aimed at enhancing HPV vaccination rates among the MSM population in Malaysia.MSM are at an elevated risk of HPV infection. The study indicates that most MSM showed a moderate willingness to be vaccinated against HPV, indicating the need to enhance efforts to promote and encourage HPV vaccination among this population. In this study, knowledge played a crucial role in influencing vaccination willingness among MSM. While attitudes towards HPV infection and vaccination may not be significant predictors of vaccination intention, some misconceptions were identified in the study. Therefore, to increase vaccination rates, it is important to prioritize the promotion of HPV and HPV vaccine knowledge, alongside enhancing positive attitudes. The findings emphasize the need for comprehensive strategies to address sensitivity issues and enhance the acceptance of HPV vaccination. A culturally sensitive approach is essential in dispelling misconceptions about the vaccine’s association with promiscuity. HPV vaccination can be combined with MSM-related services, such as STI counseling and testing, to enhance accessibility and acceptability among this population. The study’s findings carry significant implications for public health interventions targeting HPV prevention and control strategies for MSM in Malaysia.


Despite the global vaccination campaign to prevent HPV-related morbidity, HPV vaccination uptake remains unacceptably low in the developing world, like Ethiopia. For strong interventional measures, compiled data in the field is required which is otherwise missed in the Ethiopian context. Therefore, this systematic review aimed to provide an estimate of the HPV vaccination uptake, mothers‘ willingness to vaccinate their adolescent girls, and associated factors in Ethiopia.Articles were systematically searched using comprehensive search strings from PubMed/Medline, SCOPUS, and grey literature from Google Scholar. Two reviewers assessed study eligibility, extracted data, and assessed the risk of bias independently. Meta-analysis was performed using STATA v 14 to pool the vaccination uptake and mothers‘ willingness toward HPV vaccination in Ethiopia.We included 10 articles published between 2019 and 2022 covering reports of 3,388 adolescent girls and 2,741 parents. All the included articles had good methodological quality. The pooled estimate of the proportion of good knowledge about HPV vaccination and the agreement of girls to get the vaccine was 60% (95%CI: 59–62) and 65% (95%CI: 64–67), respectively. The pooled estimate of vaccination uptake of at least one dose of HPV vaccine among girls was 55% (95%CI: 53–57). Positive attitudes to the vaccine, higher maternal education, and having knowledge about HPV and its vaccine were reported as statistically significant predictors. On the contrary, not having adequate information about the vaccine and concerns about possible side effects were reported as reasons to reject the vaccine. Likewise, the pooled estimate of mothers who were knowledgeable about HPV vaccination, who had a positive attitude, and willing to vaccinate their children were 38% (95%CI: 36–40) 58% (95%CI: 56–60), and 74% (95%CI: 72–75), respectively.Knowledge about the HPV vaccine among girls and their vaccination uptake is suboptimal that falls short of the 2030 WHO targets. Therefore, stakeholders need major efforts in rolling out vaccination programs and monitoring their uptake. Social mobilization towards primary prevention of HPV infection should focus on adolescents. The existing strategies need to address the predictors of uptake by educating girls and parents. The prevalence of cervical cancer (CC) is steadily increasing in low-income countries and causes significant morbidity and mortality. Cervical cancer is one of the emerging public health challenges in Ethiopia. The incidence and prevalence are increasing from time to time . AccordiThe global scale-up HPV vaccination, HPV-based screening, and treatment of precancerous lesions are the recommended interventions to curb the burden of CC. The World Health Organization (WHO) has set a vision of a world where CC is eliminated as a public health problem using the life-course approach. By 2030, each country should meet the 90-70-90 targets; i.e. (1) 90% of girls shall be fully vaccinated with HPV by age 15 years, (2) 70% of women shall be screened with a high-performance test by 35 years of age, and again by 45 years, and (3) 90% of women identified with cervical disease shall receive treatment .Though Ethiopia has issued a guideline to meet the global targets, the life-course approach for CC prevention is at an early stage . Most woBecause HPV infection is transmitted sexually, the quadrivalent recombinant HPV vaccination strategy targets female adolescents aged 9 to 14 years, for whom the first vaccination dose should be administered before a sexual encounter. Despite the global vaccination campaign to prevent HPV-related morbidity, HPV vaccination uptake remains unacceptably low. The uptake of vaccination of young girls is low in low- and middle-income settings (13%) [Since the vaccination program targets girls aged 9–14, the success of vaccination depends on the parental decision and their willingness to vaccinate their daughters. The involvement of parents in the decision to take the HPV vaccine for their children is very crucial for the acceptability and utilization of the vaccine . GeneralA systematically compiled nationwide data on HPV vaccination uptake in Ethiopia is missing which is otherwise important for strong interventional measures. Therefore, the current study was conducted to assess HPV vaccination uptake among female adolescents and to describe the knowledge and willingness of the HPV vaccine and associated factors among parents of female adolescents in Ethiopia.This systematic review addresses the following questions;What percentage of Ethiopian adolescent girls received the HPV vaccine?How much do parents know about vaccines and how willing were they to vaccinate their kids?The aim of this review was to describe the HPV vaccine uptake among female adolescents and to assess the knowledge and willingness of parents to vaccinate their adolescent girls.Study design: descriptive studies that reported the HPV vaccine uptake among female adolescents and the knowledge of women on this vaccination. Participants: female adolescent girls and parents of children. Setting: we included studies with the outcome of interest reported in Ethiopia. Language and publication: We included peer-reviewed published articles and unpublished preprints written in the English language.Studies were selected based on the following criterion: This review was done following the PRISMA guideline (Supplementary file 1). A computerized systematic strategy was adopted to search for articles in PubMed/Medline and SCOPUS. The last search was conducted on 20 April 2023. A manual search from Google Scholar and Google databases was also carried out for grey literature. The search terms were developed in line with the Medical Subject Headings (MeSH) thesaurus using a combination of key terms which are derived from the research question. The reference lists of retrieved articles were probed (forward and backward searching) to identify articles that were not retrieved from the databases’ manual search. The first two authors; AD and DM searched the articles independently.The domains of the search terms were: ‘HPV vaccination’, ‘vaccination for cervical cancer’, ‘female adolescents’, ‘vaccination uptake’ ‘parental knowledge’, ‘parental willingness‘, and ‘Ethiopia’. We combined these terms using the Boolean operator “OR”, and “AND” accordingly. The full search strategy for the two databases is annexed in Supplement 1.Studies that reported the HPV vaccine uptake among female adolescents and the knowledge and willingness of parents to vaccinate their children were included regardless of their year of publication. Searched articles were directly imported and handled using EndNote X9 citation manager . Based on the PRISMA procedure, duplicated articles were excluded and the titles and abstracts of the remaining papers were screened sequentially for inclusion in full-text evaluation by the first two authors. Differences between the reviewers were resolved through discussion.The extracted data items include the name of the first author, publication year, age range/mean of the study participants, sample size, variables related to the HPV vaccine uptake, and the knowledge and willingness of parents about the vaccination. The data were extracted from the included articles using piloted Excel data extraction sheet developed by the first author.The validity and methodological quality of all included studies were assessed using the Joanna Briggs Institute Critical Appraisal Checklist for prevalence data (Supplementary file 3). The tool consists of nine criteria that were checked as ‘yes’, ‘no’, ‘unclear’, or ‘not applicable’. After carefully evaluating the included articles against each criterion, studies were finally classified into three groups; a study that fulfilled > 80% of the criteria were considered as ‘good quality’. Similarly, a study that scored 50–80% and < 50% were rated as ‘fair’ and ‘poor’ quality, respectively. Fortunately, all the included studies scored > 80 and were judged as methodologically good.I2 test and I2 = > 50% was considered as high heterogeneity among the results of the included studies. Additionally, to assess the presence of publication bias, a funnel plot was performed. To assess the influence of individual studies on the pooled data, a sensitivity analysis was performed.Descriptive statistics, such as simple counts, ranges, and percentages were used to present the synthesized data. A systematic narrative synthesis was provided in which summary results were presented using text and tables. To pool the overall vaccination uptake and mother’s knowledge, meta-analysis was performed using STATA v 14  using a random effect model. Predictors were presented in the description. A predictor is considered for inclusion when two or more articles reported it as a statistically significant factor. The heterogeneity of the included studies was assessed using the the proportion of adolescent girls who have received the HPV vaccine.the included studies used a series of different items to measure adolescent girls and mothers‘ knowledge of HPV vaccination. The level of knowledge was measured using items related to risk factors for cervical cancer, the benefit of the vaccine, their attitude, and the prevention methods of cervical cancer. Studies used the cumulative mean score of the participants about cervical cancer to measure their knowledge. Based on this, they labeled poor knowledge for those who had scored less than the mean and good knowledge for those who had scored greater than or equal to the mean value.From the computerized systematically searched databases and other sources, a total of 44 articles were retrieved and sequentially screened for inclusion in the analysis using the PRISMA flow chart (Supplementary file 2). Ten articles met our inclusion criteria and were included in the systematic review and meta-analysis.The characteristic of the included studies is summarized in Table The included studies –19 were The pooled estimate of the proportion of good knowledge about HPV vaccination and the agreement of adolescent girls to get the vaccine were at 60% (95%CI: 59–62) and 65% (95%CI: 64–67), respectively. The included studies reported the proportion of vaccination uptake between 44.1%  and 66.5The statistically significant predictors for the uptake of the HPV vaccine by adolescent girls are indicated in Table Four studies reported mothers‘ willingness to vaccinate their children. The pooled estimate of mothers who were knowledgeable about HPV vaccination and those who had a positive attitude to vaccinating their children were 38% (95%CI: 36–40) and 58%(95%CI: 56–60), respectively Table .Parents having media exposure, having good knowledge about HPV infection, and positive attitude towards HPV vaccination were reported to be important predictors to vaccinate their adolescent girls Table .The global scale-up of HPV vaccination, HPV-based screening, and treatment of precancerous lesions are the recommended interventions to curb the burden of cervical cancer worldwide. This particular review summarized the findings of articles containing information about the level of adolesent girls HPV vaccination uptake and mothers willingness to vaccinate thier children in Ethiopia.In our review, the included studies reported the proportion of vaccination uptake between 44.1%  and 66.5If HPV vaccination and cervical screening are scaled up, the annual cervical cancer incidence can be brought down by significant levels. HPV-based vaccination in Ethiopia was started in October 2018 for school girls. The available vaccine in the country is Gardasil-4, which targets HPV6, 11, 16, and 18 . AccordiThe level of HPV vaccination uptake in different African countries was also reported to be low. For example, a study in Nigeria showed that the level of knowledge of adolescent girls on HPV vaccines was low and the vaccination uptake was 2.1-4%. The most common reason given for not having taken the vaccine was unawareness of the vaccine , 25. SimAnother systematic review on the uptake of HPV vaccination among adolescent girls showed that the vaccination uptake rate for at least one dose varied significantly among countries, ranging from 2.4% in Hong Kong to 94.4% in Scotland . SimilarThe difference in the reports of the HPV vaccination uptake across these studies might be due to disparities in socioeconomic status, health information access, and tools used to measure the acceptance of HPV vaccination. Specifically, the higher vaccination uptake in the developed world might be because girls might have good accessibility to the HPV vaccine with all the necessary information.The acceptance of the HPV vaccination in our review is low which might be due to poor health information access, less involvement of girls participating in health-related school clubs, and sociocultural influence on the female gender. In our review, the pooled estimate of the proportion of good knowledge about HPV vaccination and the agreement of adolescent girls to get the vaccine were 60% and 65%, respectively. These figures might be directly related to the low uptake of the vaccine. A review article in Sub-Saharan Africa also showed that there was a relatively higher level of willingness to get the HPV vaccine, but low levels of knowledge and awareness about the HPV vaccine was reported .The influence of knowledge and perceptions of HPV vaccination suggests the importance of tailored health education on HPV immunization. Therefore, actions aimed at creating a positive attitude toward the HPV vaccine, sensitization of adolescents about the vaccine, and conducting community outreaches are timely areas of interventions that the stakeholders should consider. As social media outlets are becoming an increasing source of information in Ethiopia, using these platforms HPV related information, attitudes, and behavioral-related information can be easily made available to the people at large.The United States, Australia, and Canada were the first countries to implement HPV vaccination as part of their national immunization programs since 2006 . In contOur review also identified factors associated with HPV vaccination practice among adolescent girls. The included studies in our review reported different statistically significant predictors for the uptake of the HPV vaccine by girls. Positive attitudes to the vaccine, higher maternal education, and having knowledge about HPV and its vaccine were reported by two or more articles as important predictors to take the vaccine. On the contrary, not having adequate information about the vaccine and concerns about possible side effects were reported as reasons for not taking the vaccine. A similar study in Nigeria reported that The most common reason given for not having taken the vaccine was unawareness about the HPV vaccine (98%) , 25.Proper strategies to overcome these barriers are needed to ensure successful vaccination uptake. There is no single solution to increase vaccination uptake and different approaches may be better suited to certain populations. Hence, it is suggested that barriers to the uptake of the vaccine should be addressed, and that school-based sexual health education of HPV infection and vaccine promotion should be practiced in Ethiopia . A studyA similar study in Latin America indicated that there were several reasons attributed to low vaccination uptake among adolescent girls: limited knowledge of HPV and HPV vaccine, misguided safety concerns, high cost, cultural barriers, and the COVID-19 pandemic . There iWith regard to mothers‘ willingness to vaccinate their children, the pooled estimate of mothers who were willing to vaccinate their adolescent girls was 74%. Similar studies in Nigeria reported that 70-79.2% of mothers of adolescent girls demonstrated a willingness to vaccinate their daughters against HPV. Those mothers who were unwilling to vaccinate their adolescent girls reason out that it may encourage sexual promiscuity . LikewisIn our study, the pooled estimate of mothers who were knowledgeable about HPV vaccination and those who had a positive attitude to vaccinate their children were 38% and 58%, respectively. Similarly, in Nigeria, only 19.0% of mothers had good knowledge about cervical cancer prevention strategies . FurtherFor the HPV vaccination uptake barriers include parents‘ knowledge, perceptions, and attitudes toward the HPV infection . In our It is reported that health education intervention was found to be effective at improving the parental willingness to vaccinate their adolescents with the HPV vaccine. A multipronged approach in educating the parents of adolescents about the benefit of the HPV vaccine in reducing and preventing the infection and its effects. Educated mothers might feel confident to vaccinate their daughters against HPV by retrieving medical information on the risks of contracting HPV infections .To the best of our knowledge, this systematic review reported the latest summarized finding of HPV vaccination uptake and parental willingness to vaccinate adolescent girls in the Ethiopian context. However, our findings should be interpreted with caution due to some drawbacks; the studies were reported from only some regions of the country. The absence of data from the rest of the regions in the country might compromise our conclusion. The other snare of this review is the presence of high heterogeneity between the included articles.In this study, the level of HPV vaccination practice among adolescent girls in Ethiopia was 55%. The pooled estimate of mothers who were willing to vaccinate their adolescent girls was 74%. The current levels of vaccination in the population of adolescent girls fall short of the 2030 WHO targets. Therefore, stakeholders need major efforts in rolling out vaccination programs and monitoring their uptake. Social mobilization towards primary prevention of HPV infection should focus on adolescents and mothers. Community education on cervical cancer and its prevention to increase awareness is necessary. Moreover, efforts should be made to enhance awareness about HPV vaccination through mass and social media outlets and other health education means towards HPV vaccination. School-based clubs should be considered to provide adolescent-friendly information to create basic awareness and as a result to improve the observed low level of HPV vaccination uptake in Ethiopia.