The aim of this study was to investigate the effects of forkhead box protein P3 (FOXP3) intron single nucleotide variants (SNVs) in high‐risk human papilloma virus (HR‐HPV) infection and cervical cancer (CC) malignant lesions. We performed FOXP3 genotyping in 350 patients with CC and 350 healthy controls using the ImLDR multiple single nucleotide polymorphism genotyping technology. The heterozygous mutation TC in rs2294021 decreased the risk of HR‐HPV infection and CC malignant lesions ; the dominant model TC+CC and allele C in rs2294021 decreased the risk of CC malignant lesions . The heterozygous mutation GA, dominant model GA+AA and allele A in rs3761549 also decreased the risk of HR‐HPV infection and CC malignant lesions . Patients with CC and HR‐HPV infection carrying rs2294021 TC and rs3761549 GA had lower expression of FOXP3 protein. Haplotype analysis revealed that T‐C‐A decreased the risk of HR‐HPV infection. Furthermore, we found a significant association between immune cells infiltration and prognosis in patients with CC. Our findings demonstrated that rs2294021 and rs3761549 variants may protect against HR‐HPV and CC malignant lesions by downregulating FOXP3 and that FOXP3 was associated with immune cells infiltration, which affected the prognosis of CC. FOXP3 is characteristically expressed in regulatory T cells (Tregs) and is involved in the activation, proliferation, differentiation and regulation of Tregs.FOXP3 is an essential factor that induces and maintains the unique immunosuppressive properties of Tregs.The forkhead box protein 3  are the most common type of genetic mutation, and variants in the promoter region of a gene can lead to abnormal recognition by RNA polymerase Ⅱ, leading to abnormal gene expression. Furthermore, variants in the intron region of a gene may affect mRNA splicing.FOXP3 variants on the occurrence of CC among Chinese individuals. In addition, we explored the role of immune‐infiltrating cells in the prognosis of patients with CC.Accordingly, in this study, we evaluated the effects of 22.1In total, 350 patients with CC diagnosed at the Affiliated Cancer Hospital of Guangxi Medical University and the Affiliated Hospital of YouJiang Medical University from 2018 to 2019 were randomly enrolled as the case group, and 350 age‐matched healthy women were randomly enrolled as the control group. Before sample collection, all participants were informed of the purpose of the study and provided written informed consent for participation in the study. The inclusion criteria were as follows: all patients were diagnosed with CC, and the diagnosis was confirmed pathologically. The exclusion criteria were as follows: patients who had a history of radiotherapy and chemotherapy for treatment or a history of immune diseases or other cancers. The control participants had no history of immune disease, cervical lesions or HPV infection. The study was approved by the Ethics Committee of the Affiliated Hospital of YouJiang Medical University for Nationalities.2.2FOXP3 gene variants genotyping. In addition, serum was separated from peripheral blood at 2500 g for 10 min and stored at −80℃ until analysis of serum FOXP3 protein levels.Cervical epithelial cytology samples were collected using cytobrushes and stored in 2 ml TE buffer at −4℃ until HPV detection and genotyping once a week. Peripheral blood was collected with EDTA2+ anticoagulant and stored at −80℃ until 2.3Genomic DNA for HPV DNA detection and genotyping was extracted from cytobrushes using DNAzol (YaNeng Bioscience) according to the manufacturer's instructions, and HPV subtypes were then genotyped by polymerase chain reaction (PCR)‐reverse dot blot. The reaction conditions were as follows: 25 µl reaction volume containing primers, dNTPs, Taq DNA polymerase, buffer, UNG enzyme and 5 µl HPV DNA; annealing temperature, 50°C. For the HPV DNA amplification reaction, we used HPV16 DNA as the positive control and a sample without HPV DNA as the negative control to exclude the possibility of contamination. The PCR conditions were as follows: 50°C for 15 min; 95°C for 10 min; 40 cycles of 94°C for 30 s, 42°C for 90 s and 72°C for 30 s; and 72°C for 5 min. The HPV genotype was identified with a blue dot and then was classified into six low‐risk types  and 17 high‐risk types .2.4FOXP3 variants genotyping was extracted from peripheral blood using a TIANGEN kit according to the manufacturer's guidelines. ImLDR multiple single nucleotide polymorphism genotyping technology was performed to detect rs2280883, rs2294021 and rs3761549 in the FOXP3 gene. For rs2280883 and rs2294021, thymine (T) was replaced with cytosine (C), whereas in rs3761549, guanine (G) was replaced with adenine (A). The amplified primer sequences for FOXP3 gene variants were designed using Primer software 3.0, as follows: rs2280883, 5′‐GAAGGAGTTGGGATGGGGTGAT‐3′ (forward) and 5′‐CCATCTCTGCACCTTGCCCTAA‐3′ (reverse); rs2294021, 5′‐ACACATGAGG ACCCTCCACTGC‐3′ (forward) and 5′‐CCCAGCCAGCCAATTAGCAGAT‐3′ (reverse); and rs3761549, 5′‐CGACACCACGGAGGAA GAGAAG‐3′ (forward) and 5′‐CAAACCTGGGTCCTCTCCACAA‐3′ (reverse). The reaction contained 1 µl DNA sample, 0.3 mM dNTPs, 1 µl primers for multiple PCR, 1 µl HotStarTaq polymerase (Qiagen Inc.), 3.0 mM Mg2+ and 1× HotStarTaq buffer. The volume was brought to 20 µl using ultrapure water. The PCR conditions were as follows: denaturation at 95°C for 2 min; 11 cycles of 94°C for 20 s, 65°C for 40 s, and 72°C for 90 s; 24 cycles of 94°C for 20 s, 59°C for 30 s and 72°C for 90 s; elongation at 72°C for 2 min; and final cooling at 4°C. After amplification, the PCR products were purified by incubation with shrimp alkaline phosphatase (Promega) and exonuclease I (Epicentre Company) at 37°C for 60 min and 75°C for 15 min. Then, the reaction of allele‐specific multiplexed ligase was performed with 2 µl PCR product, 0.4 µl of 1 µM 5′ primer, 0.4 µl of 2 µM primer, 0.25 µl thermostable ligase, 1 µl of 10× buffer and 6 µl ddH2O for 35 cycles of 94℃ for 1 min and 56℃ for 4 min. Finally, variants in the FOXP3 gene were identified by capillary electrophoresis.Genomic DNA for 2.5FOXP3 protein levels were detected using an enzyme‐linked immunosorbent assay (ELISA) kit (eBioscience) according to the manufacturer’s instructions. The absorbance value was measured using an enzyme‐labelled metre at 450 nm , and the concentration of serum FOXP3 protein was analysed using a standard curve.Serum 2.6FOXP3 expression groups of patients with CC were analysed and drawn using the ‘limma’ and ‘ggplot2’ packages in R respectively. Survival curves were drawn for each type of immune cells using the ‘survminer’ and ‘survival’ packages in R. In addition, the CIBERSORT, CIBERSORT‐ABS, MCPcounter, XCELL, EPIC, QUANTISEQ and TIMER algorithms were compared with assess cellular components or cell immune responses between high‐FOXP3 expression and low‐FOXP3 expression groups. The differences in immune responses under different algorithms were determined using a heatmap.To reliably evaluate immune cells infiltration in CC, we used CIBERSORT to detect the relative proportions of immune cells in each CC sample. An algorithm with 1000 permutations was adopted to obtain meaningful results, and the expression of immune checkpoint‐related genes and infiltrating levels of immune cells in high and low 2.7χ2) test was used to analyse differences in sociodemographic and clinical characteristics and FOXP3 gene variants between the case and control groups. The Hardy–Weinberg equilibrium was tested using Pearson’s chi‐square test. Binary logistic regression was used to analyse the frequency distribution differences of genotypes and alleles among the HR‐HPV‐positive, HR‐HPV‐negative and control groups. Haplotype analysis was performed using online SHEsis software . The odds ratios (ORs) and 95% confidence intervals (95% CIs) were adjusted for age, smoking status, age at first sexual intercourse, history of abortion and contraceptive use. The expression of FOXP3 protein was analysed using GraphPad Prism 5 . Spearman's correlation was used to analyse the association between FOXP3 expression and immune cells infiltration. The survival time according to immune infiltration status was analysed using the Kaplan–Meier method. Statistical analysis was performed using SPSS Statistics 22.0 and R 3.6.0.Pearson’s chi‐square (33.1p = 0.690). However, women with an age at first intercourse of <18 years (p = 0.002) and who had a history of abortion (p = 0.029) had a higher risk of CC. Persistent HR‐HPV infection was the main cause of CC. Thus, the CC group was divided into HR‐HPV‐positive and ‐negative groups according to HPV infection status. We found that women with an age at first intercourse of less than 18 years (p = 0.016) and who had a history of abortion (p = 0.036) were more susceptible to persistent HR‐HPV infection  were recruited. The flow chart of all subject selection through each stage of the analysis was presented in Figure  p = 0.00 and who  p = 0.00 and who 3.2FOXP3 gene were selected from the 1000 Genomes Project database with minor allele frequencies >5%. The codominant model TC of rs2294021 was associated with a decreased risk of HR‐HPV infection . In addition, the codominant model GA, dominant model GA + AA and allele A of rs3761549 were associated with a decreased risk of HR‐HPV infection  and those patients harbouring the codominant model GA, dominant model GA + AA and allele A of rs3761549 had a lower risk of CC . As shown in Figure FOXP3 expression was significantly negatively correlated with infiltration of dendritic cells . Xiao‐xia Pang: Writing – original draft . Guang‐jing Li: Data curation . Zhi‐hong Chen: Resources . Ming‐you Dong: Methodology (lead); writing – review and editing . Jun‐li Wang: Project administration (lead); writing – review and editing (lead).Fig S1‐S2Click here for additional data file.


Human papilloma virus (HPV)-associated cancers and their precursors are increasing worldwide. The purpose of our study was to investigate HPV subtypes in anogenital and oral samples to analyze the distribution and frequency of high- and low-risk HPV in the cervix, vagina, vulva, anus, and oral cavity. Identification of special HPV subtypes for these areas may help to increase the prognostic value of HPV screening for early detection of precursors or cancers being highly HPV-attributable. HPV genotyping with the EUROArray HPV test was performed in 509 patient samples from our dysplasia consultation. In addition to the well described HPV, e.g., HPV 16 and HPV 31, we detected further HPV subtypes with higher incidences in the investigated areas  which may be evident for precursors and cancers of all areas.Background: Cancers and intraepithelial lesions of different anogenital areas as well as oral cancer are associated with human papilloma virus (HPV) infections. Methods: In this study cervical, vaginal, vulvar, anal, and oral samples were taken from 509 patients visiting our dysplasia consultation clinic. HPV genotyping was performed using the EUROArray HPV test. Results: Positivity of HR HPV was found in 60.4–64.3% of anogenital and 14.6% of oral samples. HPV 16 showed the highest incidence in all investigated areas. In cervical and vaginal samples HPV 31 was detected second most, while in vulvar, anal, and oral samples HPV 53 was the second most common subtype. HPV 18 was found lower in all areas, while HPV 51, HPV 52, and HPV 73 were detected higher than expected from published data. A good concordance between cervical, vaginal and vulvar samples was examined for most of the HPV. HR HPV infection was higher in cervical cancer  and high-grade intraepithelial squamous lesions  compared to low-grade SIL  and normal samples (44.8%). Conclusion: In addition to the well described HPV subtypes, we found others with high incidences in the investigated areas which may be evident for HSIL and CC of those areas. Human papilloma virus (HPV)-associated cancers have increased worldwide in the last years. In addition to cervical cancer (CC), where more than 95% of all cases are attributable to HPV infections , other aHPV-associated cancer develops after a persistent infection with oncogenic HPV genotypes of the HR subtypes including carcinogenic genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, and possibly carcinogenic genotypes HPV 68, 73, and 82, according to the definition of the International Agency for Research on Cancer (IARC)  p = 3.4 × 10−28), for cervical/anal 4.58 ([95% CI: 2.91–7.27] p = 6.3 × 10−11), and for cervical/oral 1.88 ([95% CI: 1.09–3.42] p = 0.03).Being HPV positive at the cervix extensively increased the chances of being positive at other sites: Age-adjusted odds ratio for cervical/vaginal association was 28.39 .Considering the new nomenclature  we also p = 1.850 × 10−5; OR = 1.034, 95% CI = 1.018–1.050). For every year of age, the odds of HPV positivity increased by 3%. In the extended set of 310 patients, a statistically significant association between age and HPV positivity could be shown, too .HPV prevalence in the genital and anal areas decreased with age with the highest prevalence among women ≤39 years, while the oral HPV prevalence remained constant with minor fluctuations . In 477 p = 0.033; OR = 3.211, 95% CI = 1.097–9.402), but not with CIN or CC.In 310 patients, earlier use of hormonal IUD was associated with positivity of HPV , Eastern Europe (21.4%), and Latin America (16.1%) showed the highest prevalence and Western Europe one of the lowest (9.0%). The HPV prevalence, as well as the burden of the HPV related diseases differ considerably by and within areas of countries according to several factors, including both extrinsic and intrinsic factors .HPV is ranked as the second most common pathogen leading to gynecological disorders and the first in causing female cancers. Cervical cancer, which is the third most common female malignancy worldwide, is the most common disease resulting from infection with the 18 known HR HPVs , followed by anal, vulvar, penile, vaginal, and oropharyngeal cancers . AdditioWorldwide, the five most common HPV subtypes among women with normal cervical cytology were HPV 16 (3.2%), HPV 18 (1.4%), HPV 52 (0.9%), HPV 31 (0.8%), and HPV 58 (0.7%) while the corresponding prevalence for the European continent were HPV 16 (4.8%), followed by HPV 31 (2.3%), HPV 18 (0.9%), HPV 39 (0.8%), and 33 and 66 (0.6% each) . In our The most common HPV subtype in vulvar swabs was HPV 16 at 53.9% followed by HPV 53 (17.7%) and HPV 31 (15.8%). In a study by De Sanjose et al. including 587 VIN and 1709 vulvar cancer patients, the percentage for HPV 16 at 72.5% was the most common subtype. The next most frequent HPV of their study was HPV 33 (6.5%) and HPV 18 4.6%) with percentages comparable to those found in our study (HPV 33 6.2% and HPV 18 4.6%) .6% with . The metDepending on the examined cohort, varying frequencies of HPV subtypes will be detected. In a meta-analysis, the overall HPV prevalence was 67.8%, 85.3% and 40.4% among 90 VIN 1, 1,061 VIN 2/3 and 1873 vulvar carcinomas, respectively; 100%, 90.1% and 69.9% among 107 VaIN 1, 191 VaIN 2/3 and 136 vaginal carcinomas; and 91.5%, 93.9% and 84.3% among 671 AIN 1, 609 AIN 2/3 and 955 anal carcinomas, respectively . HPV 16 In a study by Insinga and coworkers—referring to a defined population (review of 22 U.S. studies) with cervical cancer and its pre-stages—the top two HPV types contributing to disease were HPV 16/66 (15.3%) in CIN 1, HPV 16/31 (61.9%) in CIN 2/3, and HPV 16/18 (79.2%) in CC . In our In line with other studies, we showed that the most predominant HPV in either disease is HPV 16, and we identified HPV 31 as the second most common HR HPV, especially in HSIL and CC ,25. CompIn contrast to many other studies , we deteIn our study, HPV 53 was frequently detected in all investigated areas with percentages ranging from 11.3% (cervix) to 18.9% (anus). A study by Gong et al detected 8.8% of HPV 53 in a total of 1259 patients with cervicitis (10.0%), LSIL (9.0%), HSIL (7.3%) and CC (0.0%) . In our Another aim of our study was to compare the HPV infection and subtypes of one patient in different anogenital areas and investigate a possible concordance of the different subtypes. As expected, we found the highest concordance in cervical, vaginal and vulvar samples, especially for the most common HPV subtypes HPV 16, HPV 31, and HPV 52. Another study group reported a similar prevalence for any carcinogenic HPV type in vaginal and cervical specimens compared to our study [In addition to 16 and 31, less frequent HPV subtypes such as 51 and 73 that were detected more often in anal samples, displayed a substantial or moderate concordance with cervical, vaginal as well as vulvar samples. Similar results were reported by Nasioutziki and coworkers who showed that the cervical HPV infections of women with prevalent cervical dysplasia, correlated with the HPV types detected in their anal canal . No or jAge-specific HPV distribution worldwide as well as in Western Europe presents with a first peak at younger ages ,33, whicWe showed that in a set of 310 patients earlier use of hormonal IUD was associated with positivity of HPV, but not with CIN or CC. Lekovich and coworker reported that hormone-containing IUDs could be associated with decreased HR HPV infection clearance and possibly increased acquisition compared to copper-containing IUDs . In a poA limitation of the study is the mixed cohort of patients visiting our dysplasia consultation resulting in the small number of samples from cancer patients and especially from patients with INs of entities other than cervical .The purpose of our study was to analyze a broad set of HR HPV and LR HPV in five areas for a better elucidation of HPV infections. This should add information on the spreading of infections between areas by analyzing co-infections with identical HPV subtypes. Furthermore, the correlation of infections with clinical data should provide further knowledge about incidences of HPV-attributable cancers and their precursors. We found, according to other studies, HPV 16 as the most common HPV and HPV 31 as second most in cervical and vaginal samples. HPV 53, which is not included in most other tests, was second most common in vulvar, anal and oral samples. HPV 53 is marked as possibly carcinogenic and is not well investigated. Our study found HPV 53 in CC and preferentially in HSIL samples. HPV 73 is an HR HPV and is also not covered by most other tests. We found HPV 73 with highest percentages in anal samples and with good concordance between the different areas. Further studies of these specific subtypes could be useful to clarify the importance of HPV 53 and HPV 73 in neoplasia areas next to the cervix.In conclusion, the testing of many specific subtypes is valuable for the association of specific HPV subtypes with lesions and their precursors in various areas.


Magnetic resonance imaging (MRI)-associated prostate-specific antigen density (mPSAD) and MRI predictive models have been proposed for improving the selection of candidates for prostate biopsy among men with suspected prostate cancer (PCa). While the calculation of mPSAD only requires a simple division, the individual risk assessment of PCa using the available risk calculators is also a swift process. We aim to compare the clinical usefulness of mPSAD and an MRI predictive model that utilises the same predictors as the recently developed and externally validated Barcelona MRI predictive model (MRI-PMbdex).p < 0.001. MRI-PMbdex showed net benefit over biopsy in all men when the probability of csPCa was greater than 2%, while mPSAD did the same when the probability of csPCa was greater than 18%. Thresholds of 13.5% for MRI-PMbdex and 0.628 ng/mL2 for mPSAD had 95% sensitivity for csPCa and presented 51.1% specificity for MRI-PMbdex and 19.6% specificity for mPSAD, with p < 0.001. MRI-PMbdex exhibited net benefit over mPSAD in men with prostate imaging report and data system (PI-RADS) <4, while neither exhibited any benefit in men with PI-RADS 5. Hence, we can conclude that MRI-PMbdex is more accurate than mPSAD for the proper selection of candidates for prostate biopsy among men with suspected PCa, with the exception of men with a PI-RAD S 5 score, for whom neither tool exhibited clinical guidance to determine the need for biopsy.This study is a head-to-head comparison between mPSAD and MRI-PMbdex. The MRI-PMbdex was created from 2432 men with suspected PCa; this cohort comprised the development and external validation cohorts of the Barcelona MRI predictive model. Pre-biopsy 3-Tesla multiparametric MRI (mpMRI) and 2 to 4-core transrectal ultrasound (TRUS)-guided biopsies for suspicious lesions and/or 12-core TRUS systematic biopsies were scheduled. Clinically significant PCa (csPCa), defined as Gleason-based Grade Group 2 or higher, was detected in 934 men (38.4%). The area under the curve was 0.893  for MRI-PMbdex and 0.764 (95% CI: 0.774–0.783) for mPSAD, with  Early detection of clinically significant prostate cancer (csPCa) decreases the specific mortality of PCa . The clahttps://mripcaprediction.shinyapps.io/MRIPCaPrediction/ (accessed on 5 March 2022). For the first time, the performance of an MRI-PM has been analysed with regard to PI-RADS categories, showing a net benefit over biopsy for all men in each PI-RADS < 4. The designed RC also incorporates the novel option of selecting the csPCa probability threshold [PSAD is a classic tool that improves the specificity of serum PSA . Howeverhreshold , which mhreshold  and imprhreshold . Furtherhreshold . Four-Kahreshold ,43,44,45hreshold ,47,48, Shreshold ,50,51,52hreshold , and thehreshold .This study aims to compare the clinical usefulness of mPSAD and MRI-PMbdex for the proper selection of candidates for prostate biopsy in a sizable, multicentre population of men with suspected PCa, according to PI-RADS categories.A head-to-head comparison of mPSAD and MRI-PMbdex was designed from a multicentre trial conducted in the metropolitan area of Barcelona. The trial involved 2432 men with suspected PCa due to a serum PSA > 3.0 ng/mL or an abnormal DRE. MRI-PMbdex was created from the development cohort of 1486 men studied at Vall d’Hebron Hospital (VHH) and the external validation cohort of 946 men from Parc de Salut Mar (PSM) as well as Hospital Germans Trias i Pujol (GTiPH). This study was conducted between January 1, 2006, and 31 December 2019 . Pre-bio2) was calculated from the pre-biopsy serum PSA and the prostate volume reported in the pre-biopsy MRI, and individual generation of csPCa likelihood was expressed as percentages ranging from 0 to 100%.The development of MRI-PMbdex and the individual generation of csPCa likelihood was expressed as percentages ranging from 0 to 100%. mPSAD  equipment was used in VHH, Diamond Select Achieva  in PSM, and Nova Dual  in GTiPH. The acquisition protocol included T2-weighted imaging (T2W), diffusion-weighted imaging (DWI), and dynamic contrast-enhanced (DCE) imaging, in accordance with the European Society of Urogenital Radiology guidelines . In eachAll men from the participant institutions underwent 2 to 4-core mpMRI-TRUS cognitive fusion-guided biopsies of suspicious lesions and 12-core TRUS systematic biopsies when the PI-RADS reported in the pre-biopsy mpMRI was 3 or higher, while 12-core TRUS systematic biopsies were performed when the PI-RADS was lower than 3 . In eachThe biopsy samples were sent separately to each pathology department, where an expert uro-pathologist analysed the biopsy specimens and, after identifying the PCa, reported the International Society of Uro-Pathology (ISUP) Gleason-based Grade Group (GG) . ComplexThe endpoint measurements were the rates of detected csPCa and missed csPCa, in addition to the frequency of avoided prostate biopsies and iPCa.p < 0.05 were considered statistically significant. The statistical analyses were computed using the R programming language v.4.0.3 , and SPSS v.25  was used.The reporting recommendations for tumour marker prognostic studies (REMARK)  and the The characteristics of the entire population are summarised in p < 0.001), with the exception of PCa family history, which showed a non-significant trend that ranged from 4.7% in men with PI-RADS 1 to 8.3% in men with PI-RADS 5 (p = 0.287). On comparing the characteristics of men with PI-RADS 1 and those with PI-RADS 2, it was found that only the frequency of repeat biopsies was significantly higher among the latter ; all other characteristics were similar between the two categories. Between PI-RADS 2 and PI-RADS 3, the frequency of PCa detection increased from 20.3% to 30.5% (p = 0.028), while the frequency of csPCa and iPCa exhibited no significant increase. Men with PI-RADS 4 were significantly older than those with PI-RADS 3, had a higher PSA and mPSAD, had a lower prostate volume, and exhibited an abnormal DRE more often. The frequency of repeat biopsies, PCa family history, and iPCa detection was similar in both PI-RADS 3 and PI-RADS 4 categories; however, a significant increase in csPCa frequency was observed (p < 0.001). Moreover, in PI-RADS 5 compared with PI-RADS 4, the frequency of repeat biopsies and iPCa was low, while the frequency of csPCa and overall PCa increased significantly (p < 0.001). The frequency of repeat biopsies as well as iPCa also decreased, while the frequency of all PCa and csPCa increased significantly (p < 0.001).The characteristics of the men according to PI-RADS categories as well as comparisons between each pair of consecutive PI-RADS are presented in p < 0.001 (The efficacy of MRI-PMbdex in detecting csPCa was higher than that of mPSAD. The AUC was 0.893 (95% CI: 0.880–0.906) for MRI-PMbdex and 0.763 (95% CI: 0.774–0.783) for mPSAD, with  < 0.001 A. The MR < 0.001 B.2, respectively, and their corresponding specificities were 51.1% and 19.6%, respectively, with p < 0.001 (p < 0.001). In relation to the aggressiveness of missed csPCa, both mPSAD and MRI-PMbdex missed 2.4% of PCa with ISUP-GG ≥ 3 (22/934). Furthermore, mPSAD missed 1% of csPCa with ISUP-GG ≥ 4 (9/934), while MRI-PMbdex did not miss any of them.The thresholds of MRI-PMbdex and mPSAD with 95% sensitivity for csPCa were 13.5% and 0.628 ng/mL < 0.001 . MRI-PMbp < 0.001 (Among the 550 men with negative mpMRI (PI-RADS < 3), the MRI-PMbdex exhibited an AUC of 0.824 (95% CI: 0.756–0.892), while mPSAD exhibited an AUC of 0.685 (95% CI: 0.610–0.761), with  < 0.001 A. MRI-PM < 0.001 B.The selected thresholds of MRI-PMbdex and mPSAD provided sensitivities of 67.3% and 90.9% as well as specificities of 87.7% and 21.4%, respectively, in men with PI-RADS < 3. The MRI-PMbdex avoided 82.2% of prostate biopsies, whereas mPSAD avoided 20.2%. However, the probabilities of missed csPCa were 32.7% and 9.1%, for MRI-PMbdex and mPSAD, respectively. MRI-PMbdex did not detect 0.9% of PCa with ISUP-GG = 3 (5/55), while mPSAD did not detect 0.2% (1/55) .p < 0.001 , while mPSAD exhibited an AUC of 0.696 (95% CI: 0.641–0.750), with  < 0.001 A. Thus,  < 0.001 B.The selected thresholds of MRI-PMbdex and mPSAD provided sensitivities of 78.9% and 92.7% as well as specificities of 52.1% and 20.5%, respectively, in men with PI-RADS 3. MRI-PMbdex avoided 46.8% of prostate biopsies, while mPSAD avoided 18.3%. However, the probabilities of missed csPCa were 21.7% and 7.3%, respectively. mPSAD did not detect 2.8% of PCa with ISUP-GG ≥ 3 (3/109), whereas MRI-PMbdex did not detect 10% (11/109) .p < 0.001 , while mPSAD showed an AUC of 0.742 (95% CI: 0.709–0.775), with  < 0.001 A. MRI-PM < 0.001 B.The selected thresholds for MRI-PMbdex and mPSAD provided sensitivities of 98.6% and 94.3% as well as specificities of 12.2% and 16.2%, respectively, in men with PI-RADS 4. MRI-PMbdex avoided 6.5% of prostate biopsies, while mPSAD avoided 10.7%. Moreover, the rate of missed csPCa was 1.4% for MRI-PMbdex and 5.7% for mPSAD. The rate of missed PCa with ISUP-GG ≥ 4 was 0.9% (5/439) for mPSAD and 0% for MRI-PMbdex .p < 0.001 , while mPSAD showed an AUC of 0.761 (95% CI: 0.698–0.825), with  < 0.001 A. Neithe < 0.001 B.The selected thresholds for MRI-PMbdex and mPSAD provided sensitivities of 100% and 97.3% as well as specificities of 4.6% and 20%, respectively, in men with PI-RADS 5. MRI-PMbdex avoided 0.8% of prostate biopsies, while mPSAD avoided 5.6%. The rate of missed csPCa was 0% for MRI-PMbdex and 2.7% for mPSAD. Moreover, mPSAD did not detect 4 PCa with ISUP-GG ≥ 4 .First, the characteristics of suspected PCa regarding PI-RADS categories are considered. The similar characteristics of men with PI-RADS 1 and PI-RADS 2, in addition to similar csPCa detection rates 9.8% vs. 10.6%) and high iPCa over detection rates above 50%) in both subsets, justify considering these two PI-RADS categories as a negative mpMRI. The observed 90% negative predictive value of mpMRI was in the recently reported ranges  .6% and h,12. The 0% in bot2 into some MRI-PM [PSAD was introduced by Benson et al. in 1992 to improve the specificity of serum PSA in distinguishing men with localised PCa from those with benign prostatic enlargement ,63. Shore MRI-PM ,32,33,75e MRI-PM ,77,78,79e MRI-PM ,80. Nevee MRI-PM ,81. The Our study is the first to compare mPSAD and MRI-PM in a head-to-head manner for improving the selection of candidates for prostate biopsy in suspected PCa men after mpMRI. The compared MRI-PMbdex shared the same predictors of csPCa as the recent Barcelona MRI-PM. MRI-PMbdex and mPSAD avoid the inconveniences of modern markers as they are free of cost, do not require sample procurement, and require very little time for the assessment. This study was carried out on a sizable, multicentre population representing a metropolitan area. In all participant institutions, the criteria for PCa suspicion and diagnostic approach adhered to the EAU PCa guidelines . MRI-PMbThe limitations of this study include the design, which is prone to sampling and selection biases. Furthermore, no concordance analysis was performed by the pathologists, radiologists, and urologists involved in the study. In addition, the inter-variability that is inevitable among researchers involved in multicentric studies also exists. Moreover, prostate biopsies and mPSAD suffer from intrinsic limitations related to the heterogeneity of prostate cancer and prostate volume. The lack of histopathological analysis of surgical specimens is another limitation, although not every PCa patient undergoes radical prostatectomy. While the definition we adopted for csPCa is the most common one, it may not represent the true csPCa. Moreover, as the MRI-PMbdex is not the developed Barcelona MRI-PM, the results cannot be attributed to this model. Hence, further studies analysing the clinical usefulness of mPSAD and MRI-PMs according to PI-RADS categories are needed.It will be important in the future to include the results of radiomics and radio-genomics to improve the next generation of MRI-PMs. It appears likely that we will have more powerful predictors than PI-RADS and a more refined definition of csPCa based on genomic expression in addition to the current the pathology of prostate biopsies or surgical specimens ,97.MRI-PMbdex, which used the same predictors as the recent Barcelona MRI-PM, outperformed mPSAD in the proper selection of candidates for prostate biopsy. MRI-PMbdex exhibited net benefit over mPSAD in men with PI-RADS ≤ 4. However, regarding men with PI-RADS 5, neither tool showed clinical benefit over biopsy. The clinical usefulness of tools that improve the selection of candidates for prostate biopsy in the entire population does not represent those observed in each PI-RADS category.


Dementia is an oxidative stress-related disease. Coenzyme Q10 is a nutrient that occurs naturally in the human body and acts as an antioxidant. The purpose of this study was to investigate the relationships of coenzyme Q10 status, biomarkers for dementia (amyloid β and tau protein), and antioxidant capacity in patients with dementia.Eighty dementia patients aged ≥60 years and with a mini mental state examination (MMSE) score ≤ 26 were enrolled. The levels of coenzyme Q10, total antioxidant capacity (TAC), amyloid β, and tau protein were measured.p < 0.05). Coenzyme Q10 status was significantly correlated with the values of TAC, MMSE score, amyloid β-42, and amyloid β-42/40 ratio (p < 0.05) but not with tau protein. Additionally, a high proportion of moderate dementia patients were found to have low coenzyme Q10 status (p = 0.07).A total of 73% of patients had a low coenzyme Q10 status. Patients with low coenzyme Q10 status had a significantly higher level of serum amyloid β-42 and amyloid β-42/40 ratio (Patients with dementia suffered from coenzyme Q10 deficiency, and the degree of deficiency was related to the level of amyloid-β and antioxidant capacity. Since adequate level of coenzyme Q10 may delay the progression of dementia, monitoring coenzyme Q10 status in patients with dementia is necessary. Dementia has become a critical public health issue. The latest report of the World Health Organization estimated that more than 55 million people are living with dementia worldwide, and the number is growing every year . The patCoenzyme Q10 is an essential electron carrier in the mitochondrial electron transport chain for the generation of adenosine triphosphate . CoenzymEighty patients with dementia were recruited from the Chung Shan Medical University Hospital, which is a medical center in Taiwan. Patients with dementia were diagnosed by a neurologist and psychiatrist based on brain magnetic resonance imaging or computed tomography scan to confirm the absence of structural lesions. The inclusion criteria were age ≥60 years and mini mental state examination (MMSE) score ≤ 26. The MMSE questionnaire included six domains, such as orientation, registration, attention and calculation, recall, language, and visual construction. The sum of all item scores on the MMSE was 30, with higher scores indicating better cognitive function. As an MMSE score > 20 was defined as mild dementia and an MMSE score ≤ 20 was defined as moderate dementia . We excl® duo control, Heidenheim, Germany) was used to measure blood pressure. Height and weight were measured to calculate body mass index (BMI). Waist circumference was measured with a tape measure. In addition, we used vacutainers with K2-EDTA anticoagulant  or without anticoagulant to collect the venous blood sample after 12 h of fasting in patients with dementia. Plasma, serum, and red blood cell (RBC) samples were separated after centrifugation at 3,000 rpm for 15 min at 4°C. The white blood cell (WBC) sample was collected by using RBC lysis buffer and stored at −80°C until analysis. An automated chemistry analyzer  was used to analyze the levels of triglycerides (TG), total cholesterol (TC), low density lipoprotein-cholesterol, high density lipoprotein-cholesterol (HDL-C), fasting glucose, glycated hemoglobin, and high-sensitivity C-reactive protein.The demographic data of patients with dementia, including age and gender, were collected by a questionnaire. A digital electronic sphygmomanometer , and the wavelength of the detector was set at 275 nm. The standard of coenzyme Q10 was purchased from Sigma-Aldrich  as the external standard to apply the calibration curve to measure the level of coenzyme Q10. In the method of coenzyme Q10 in this study, the linearity with correlation coefficients of the standard curve was 0.9999. The mean of intra- and inter-assay coefficients of variability for coenzyme Q10 were 3.4 and 2.8%, respectively. The mean analytical recovery of coenzyme Q10 was 102.0%. The reference range of plasma coenzyme Q10 was 0.5–1.7 μM in adults, and the individuals with plasma coenzyme Q10 level lower than 0.50 μM were defined as having plasma coenzyme Q10 deficiency (High performance liquid chromatography (HPLC) with an ultraviolet detector was used to measure the level of coenzyme Q10 in plasma and WBC . The WBCficiency .A Trolox equivalent antioxidant capacity assay was used to analyze the total antioxidant capacity (TAC) in serum and RBC . The 2,2We used an enzyme-linked immunosorbent assay kit to quantify the levels of amyloid β-42 , amyloid β-40 , and tau protein  in serum, and followed the manufacturer’s instructions.t-test or the Mann−Whitney rank sum test. Spearman’s rank order correlation coefficient was calculated to examine the correlations between coenzyme Q10 status and antioxidant capacity and biomarkers for dementia in patients with dementia. The differences in coenzyme Q10 status between mild and moderate dementia patients were examined by the Mann−Whitney rank sum test; the differences in the proportion of moderate dementia between low and high coenzyme Q10 status were examined by the Chi-square test and the Fisher’s exact test. Statistical significance was set at p ≤ 0.05.All data were analyzed by using SigmaPlot software  in the present study. The mean ± standard deviation (median) or percentages are shown for continuous variables or categorical variables, respectively. The Shapiro−Wilk test was used to examine the normality of distribution for the data. The differences in demographic data, antioxidant capacity, and biomarkers for dementia between the two groups stratified by coenzyme Q10 status were examined by Student’s 2. The median values of waist circumference were 91.8 and 89.3 cm in male and female, respectively, which were higher than the reference values for waist circumference . In hematology, the fasting glucose level was higher than the normal reference value (fasting glucose < 5.6 mmol/L). Regarding coenzyme Q10 status, the median level of plasma coenzyme Q10 was 0.36 μM. Approximately 73% of patients with dementia had plasma coenzyme Q10 deficiency, with coenzyme Q10 level lower than 0.50 μM. We therefore stratified by coenzyme Q10 status based on plasma coenzyme Q10 (0.5 μM) and found that patients with low plasma coenzyme Q10 status had significantly lower lipid profiles, such as TC (p = 0.02) and HDL-C (p < 0.01).p = 0.06), similarly shown in patients with low plasma coenzyme Q10/TC (p = 0.08) and low WBC coenzyme Q10 levels (p = 0.07). In addition, patients with low WBC coenzyme Q10 level exhibited a significantly lower level of RBC TAC than those with a higher level of WBC coenzyme Q10 (p = 0.04). Regarding biomarkers for dementia, patients with low plasma coenzyme Q10/TC (p = 0.05) or low WBC coenzyme Q10 levels (p = 0.02) exhibited a significantly higher level of serum amyloid β-42. A similar trend was found in the serum amyloid β-42/40 ratio in patients with low plasma coenzyme Q10/TC (p = 0.08) or low WBC coenzyme Q10 (p = 0.01), while patients with low level of WBC coenzyme Q10 had a slightly lower level of serum amyloid β-40 (p = 0.08). However, there was no significant difference in the level of tau protein after stratified by coenzyme Q10 status. In the MMSE score, patients with low coenzyme Q10 status had a significantly lower MMSE score than those with high coenzyme Q10 status .r = −0.25, p = 0.02), serum amyloid β-40 , and amyloid β-42/40 ratio  were significantly correlated with the level of serum TAC, as well as in the patients with moderate dementia  and amyloid β-42/40 ratio  and the amyloid β-42/40 ratio . However, there was no significant correlation between coenzyme Q10 status and tau protein  or low level of plasma coenzyme Q10/TC  showed a slightly higher proportion of moderate dementia than those with high coenzyme Q10 status . However, the middle-aged and elderly without osteoarthritis in our previous study without assessing MMSE scores, so we did not make comparisons in the present study. Therefore, it is necessary to monitor coenzyme Q10 status in patients with dementia, and dietary supplementation with coenzyme Q10 could be considered to increase coenzyme Q10 to normal levels.This study was the first to investigate the relationship between coenzyme Q10 status and biomarkers in patients with dementia. The amyloid hypothesis of the pathogenesis of dementia was proposed by John Hardy and David Allsop in 1991 . AmyloidThe oxidative stress hypothesis of dementia pathogenesis has been widely discussed . Growingr = 0.32, p < 0.01; glycated hemoglobin: r = 0.28, p = 0.01) and triglycerides  in patients with dementia. Glycemic disorders have been identified as a key risk factor for dementia . The patients/participants provided their written informed consent to participate in this study.P-SC, H-HC, T-JL, and C-HY performed the study and recruited the subjects. P-SC performed the data analyses. J-CP helped perform the study and analyzed the sample. P-TL conceived the study, participated the design, and coordinated the study. P-SC and P-TL drafted the manuscript. All authors read and approved the final manuscript.


In 2020, the first generic isosulfan blue product for injection  was approved by the National Medical Products Administration of China. This multicenter, single-arm, phase III, open clinical validation trial was organized by the Chinese Society of Breast Surgery to evaluate the efficacy and safety of this domestically produced generic isosulfan blue injection for sentinel lymph node biopsy (SLNB) in patients with early breast cancer (BC) in China.Inclusion criteria. (1) A full understanding of the purpose, nature, and methods of the study, voluntary participation, and signing of the informed consent form; (2) age 18 to 70 years, of either sex; (3) histopathological diagnosis of invasive BC or carcinoma in situ, with the clinical Tumor-Node-Metastasis stage of cT1–2N0M0; (4) no history of radiotherapy on the breast or chest wall; (5) no surgical contraindications on preoperative examination; (6) ability to maintain good communication with the investigator and understand and comply with the requirements of the clinical trial.Exclusion criteria. (1) Allergy to the investigational drug or its excipients, triphenylmethane or related compounds, or other dyes, or a history of allergies to two or more drugs, foods, etc; (2) severe or uncontrollable disease, such as severe cardiovascular or cerebrovascular disease , severe pulmonary insufficiency, psychosis, bronchial asthma, and allergy; (3) surgery or neoadjuvant chemotherapy for BC on the affected side; (4) any form of breast augmentation surgery; (5) liver or kidney dysfunction; (6) current pregnancy or breastfeeding or a pregnancy plan that made the patient unwilling to take effective contraceptive measures, or a plan to donate eggs from the screening period to within 3 months after drug administration; (7) positivity for any of the indicators in the infectious disease screen; (8) participation in any other clinical trial within the past 3 months.Steps of the SLNB procedure [Supplementary Figure 1]. Within 5 min before the operation, the first domestically produced generic isosulfan blue injection  was subcutaneously injected into the mammary areola or the periphery of the mass at 1 to 3 points. After massage, the skin was incised. The surgeon visually located and photographed the blue-stained lymphatic vessels and blue-stainedsentinel lymph nodes (SLNs). The total number of resected SLNs and the number of blue-stained SLNs were recorded in the pathology department, and the surgical procedure for axillary lymph node dissection (ALND) was selected based on the pathological report. The vital signs and hematologic indicators of the subjects were monitored 1 h (±0.5 h) before drug administration, during the operation, 3 days after the operation, and during the safety follow-up period (14 ± 2 days after drug administration). The occurrence and treatment of adverse events were monitored and recorded throughout the trial period.Statistical methods. Statistical analysis of the population: The efficacy and safety (endpoint indicators) were evaluated based on the per-protocol set. Statistical analysis was performed using SAS . Descriptive statistical methods were used for quantitative and count indicators. The one-sided significance level was at 0.025.1–2N0M0 were enrolled in this study, all of whom were female, aged 21 to 70 years, with a median age of 50 years. Pathological types: There were 123 cases (82.0%) of invasive BC, 27 cases of carcinoma in situ (18%). Surgical procedures: There were 53 cases (35.3%) of breast-conserving surgery, 91 cases (60.7%) of simple mastectomy plus SLNB, and six cases (4.0%) of nipple–areola complex–sparing mastectomy and immediately reconstruction. A total of 117 patients (78.0%) received SLNB, and 33 SLNB-positive patients (22.0%) received completion ALND.A total of 150 patients with cTSuccess rate of lymphatic vessel detection and the accuracy of SLN detection in SLNB after generic isosulfan blue injection in these 150 patients with early BC .Discussion. SLNB is a well recognized, standard surgical procedure to assess the axillary status of patients with clinically axillary-negative BC. Commonly used SLNB tracers include blue dyes , radionuclides, nanocarbon, and fluorescent dyes (indocyanine green). Methylene blue is the most commonly used tracer in China due to its low cost and high accessibility. The detection rate and false-negative rate of SLNB have been clinically confirmed. Patent blue is an isomer of isosulfan blue. The detection rate of SLNB using patent blue alone has been 83.2%, but patent blue has not been marketed in China. The clinical application of radionuclides is limited because radionuclide management  must be performed by qualified physicians. Nanocarbon is also a lymphatic tracer for clinical application. The detection rate and false-negative rate of SLNB using nanocarbon was 99.1% and 4.1%, respectively, though the clinical application of nanocarbon is hampered by its high price and permanent pigment deposition due to the difficulty of metabolizing nanocarbon. The detection rate of SLNB using indocyanine green alone was 97%, and the detection rate of SLNB using indocyanine green plus blue dye reached 99.6%. However, indocyanine green imaging requires special fluorescence imaging equipment, which is cumbersome to operate and difficult to apply widely in hospitals.vs. radionuclide alone to trace SLNs in cN0BC patients, and the results showed that there was no difference in the detection rate between the two (P = 0.58). The endpoints of a phase III clinical trial were the effectiveness and safety of lymphatic detection, with adequate and inadequate detections of lymphatic vessels as indicators to evaluate the effectiveness, and the results showed that isosulfan blue is a reliable tracer for lymphatic vessel detection in SLNB.In contrast, isosulfan blue injection has the advantages of simple and convenient use, high detection rate, and high accuracy so it has been widely used as an SLNB tracer in many countries. A prospective randomized controlled trial compared the differences between the use of a radionuclide plus isosulfan blue The results of this study showed that the success rate of lymphatic vessel detection with the first domestically produced injectable generic isosulfan blue in China was 98.0%, and the success rate and the accuracy of SLN detection were 94.0% and 96.0%, respectively, which were superior to the results of existing studies. In this study, we found that using the first domestically produced generic isosulfan blue injection alone as the tracer for SLNB, two or more SLNs were detected in 94% (141/150) of the patients, a total of 382 blue-stained SLNs were finally confirmed in pathological sections. The results suggest that more SLNs could be detected using the first domestically produced generic isosulfan blue injection in China, with high accuracy. There is nothing to do with isosulfan blue because these complications such as fever, difficulty urinating, wound inflammation, and so on are also observed in routine BC surgery and anesthesia. Three serious adverse events occurred in three patients (2.0%), including one case (0.7%) of deep venous thromboembolism (VTE), one case (0.7%) of cerebral infarction, and one case (0.7%) of postoperative wound infection. The incidence of VTE after breast surgery is relatively low. In this study, the incidence of VTE was 0.7%, and the drug administration was already done when VTE occurred, so the VTEs might not have been related to the investigational drug. One patient had underlying diseases that were perioperative risk factors, namely, hypertension and coronary heart disease, before the operation. On the 4th day after discharge, the patient had mobility and language impairment and was diagnosed with cerebral infarction, which improved after symptomatic treatment. This case of cerebral infarction was related to the underlying diseases and long-term atherosclerosis. The investigational drug is physiologically inert, has no pharmacologic effect, and cannot enter the blood through the blood vessel wall. Hence, this adverse event was definitely not related to the investigational drug. Another patient developed an incision infection on the 5th day after the operation. After antibiotics and regular incision cleaning and dressing changes, the patient recovered. This adverse reaction was also not related to the investigational drug because the incision became infected long after drug administration (the drug is excreted through the urine within 24 h of drug administration). In summary, the three serious adverse events observed during this study were definitely not related to the use of the investigational drug.Like other blue dye tracers, isosulfan blue has the risk of allergic reactions, including spontaneously resolved erythema and urticaria, though some patients need intensive care unit treatment.The results of this multicenter, single-arm, open validation test show that the first domestically produced generic isosulfan blue injection in China as an SLNB tracer has a good success rate and accuracy of lymphatic vessel detection and SLN detection, with few side effects and spontaneously resolved mild symptoms. The generic isosulfan blue injection has the advantages of ease of use, high convenience, and good accessibility, and it is safe and effective for SLNB in Chinese patients with early BC.


Results: We included 569 in the study. Most participants were single (65%), females (83%), with a university-level of education (78%). The recorded knowledge score for all participants was 1.99 (out of 10). About half of the participants denied that HPV is a common sexually transmitted infection. Further, 53% were interested in the HPV vaccine, and 63% of participants acknowledged that the HPV vaccine could prevent warts and cervical cancer. About 30% of the participants opposed the vaccine due to religious reasons. Conclusion: The results of our study highlight the inadequate level of knowledge concerning HPV infection, even among highly educated people. Thus, by promoting the HPV vaccine acceptance and use, awareness can be raised in our community to assure better knowledge and achieve higher protection from this virus and its complications.Background: Human Papillomavirus (HPV) is the most common sexually transmitted infection that affects teens and adults in their early 20 s. Screening and HPV vaccination are important preventive measures to reduce cases of HPV and associated complications. Studies about HPV knowledge and vaccine acceptability are scarce in Saudi Arabia. This study evaluated participants’ knowledge, attitude, and acceptability concerning HPV and the HPV vaccine in Jazan Province, Saudi Arabia. Methods: A cross-sectional study was conducted using a pretested survey that assessed knowledge and attitude toward HPV and the HPV vaccine among the population of Jazan Province, Saudi Arabia. Responses were collected from adults between January and March 2022. Data were analyzed using a  Human papillomavirus (HPV) is the most common cause of sexually transmitted infection globally . HPV is Despite the existence of a safe and effective vaccine against HPV, there has been an increase in HPV cases and its associated complications in the last few years . ImplemeThe World Health Organization (WHO) estimates that the HPV vaccine will save more than 4 million women’s lives in low- and middle-income countries over the next decade . In thisEducation plays a crucial role and enhances knowledge about HPV, and studies have revealed that students who belong to health-sciences faculties, including applied medical sciences and medicine, have better knowledge and perception of HPV vaccination, and those with higher education usually have better knowledge of HPV .Studies about HPV prevalence, knowledge, and HPV vaccine acceptance are absent in Jazan province, southwestern Saudi Arabia, which has almost 2 million inhabitants. Therefore, we aimed in this study to assess the level of knowledge of HPV and the acceptability of the HPV vaccine among adult people who reside in Jazan Province.This was a cross-sectional investigation conducted between January and March 2022 that targeted the population of Jazan, southwestern Saudi Arabia.A semi-structured validated questionnaire  was compThis study targeted Jazan residents, mainly females 18 and older, regarding HPV knowledge and attitude. Data were collected using the English and Arabic language questionnaire that requires acceptance by the participants. The questionnaire was distributed through social media to reach into selected age group. Data were manually verified, entered, and analyzed using the Statistical Package for the Social Sciences (SPSS version 25). The analysis was used to assess the relationship between the variables.http://www.raosoft.com/samplesize.html (accessed on 1 November 2021). This calculation was based on the prevalence of knowledge of 50% (because no previous studies have been conducted in Jazan) and a 95% confidence interval, with an error rate of less than 5% in a population of two million. However, we included 569 participants to increase the significant power of this study.The sample size proposed for this study was calculated to be 384 participants using The ethical approval to conduct the project was granted by the Jazan University Ethics Committee, with approval number REC-43/05/076 on 6 December 2021. We conducted this study following the ethical guidelines of the Helsinki Declaration and the local guidelines of the National Committee of Bioethics, Saudi Arabia. Participants in this study signed a consent to participate before data collection. All collected data were kept confidential and used for only the purpose of research. Also, the questionnaires did not include participants’ personal information or any other identification methods. All the participants were given the right to continue or withdraw at any time from the study.Five hundred sixty-nine participants completed the questionnaire. Most participants in the study were female (83%). The mean age of participants was 26 years. Depending on marital status, the majority were single (65%), while 32% were married participants, and the rest were divided into divorced (2%) and widows (1%). Regarding educational level, the majority hold a diploma and bachelor’s (78%), while 19% have a high school level and only 3% hold a master’s and doctorate. More than half participants were students (57%), followed by employed (25%), and the rest were either looking for jobs or retired. These data are summarized in The total knowledge score percent of our participants is 20%. Half of the participants knew that HPV was a common sexually transmitted infection (49%), and only 38% acknowledged the relationship between cervical cancer and HPV . FurtherIn Half of the participants agreed that males should be given the human papillomavirus vaccine to prevent possible partner HPV infection and its consequences (54%). About 34% of participants thought there is no need to vaccinate males against HPV if females are already vaccinated against HPV . About 6HPV is a common sexually transmitted disease and a common cause of genital warts and cervical cancer . To overDespite being incorporated into females’ routine immunization schedules in the national immunization schedule in Saudi Arabia ,19. HPV Religion and religious practices are important factors that could affect the knowledge level concerning HPV and acceptance of the HPV vaccine. In our study, about 30% of the participants were opposed to the HPV vaccine for religious reasons . It has Despite being one of the few studies in Saudi Arabia that discussed knowledge of HPV and the acceptability of its vaccines, our study bears many limitations. The data have been collected by a self-administered online survey, a method with a known reporting bias. However, due to the conservative nature of our community and the relativity of this topic to sexual aspects, we preferred to limit the interaction with participants as most of them were females (83%). Therefore, data collection was actively distributed via an online self-reported questionnaire, and its distribution depended on the authors’ networks. As such, most of the participants were young, students, and female. It would be insightful to ask participants if they have been diagnosed with HPV and received the HPV vaccine or included questions about smoking as an important cervical cancer risk factor. However, we know that national awareness about HPV increased recently, i.e., a few months before conducting this study , and theHPV vaccines, in addition to other preventive methods, play a vital role in reducing the number of anogenital cancers and HPV-related conditions. Overall, vaccines’ efficacy and safety profile indicate that they will provide significant health benefits to the population. As more data on the efficacy of these vaccines against HPV-related head and neck cancer becomes available, this study highlights the low level of knowledge among both male and female populations from the southwestern region of Saudi Arabia about HPV infection and its vaccine. The lack of knowledge was reported even among highly educated people since HPV infection is a preventable disease. Recently, health officials have started promising national HPV awareness campaigns to increase public awareness of HPV and the importance of its vaccine. Consequently, we emphasize the importance of educating and raising awareness among women about the risk factors of cervical cancer, as well as the importance of screening programs. A well-designed health education program about cervical cancer and the advantages of screening and vaccination would raise awareness among the Saudi population.


Human papillomaviruses (HPVs) cause a substantial amount of human disease from benign disease such as warts to malignant cancers including cervical carcinoma, head and neck cancer, and non-melanoma skin cancer. Our ability to model HPV-induced malignant disease has been impeded by species specific barriers and pre-clinical animal models have been challenging to develop. The recent discovery of a murine papillomavirus, MmuPV1, that infects laboratory mice and causes the same range of malignancies caused by HPVs provides the papillomavirus field the opportunity to test mechanistic hypotheses in a genetically manipulatable laboratory animal species in the context of natural infections. The E6 and E7 proteins encoded by high-risk HPVs, which are the HPV genotypes associated with human cancers, are multifunctional proteins that contribute to HPV-induced cancers in multiple ways. In this review, we describe the known activities of the MmuPV1-encoded E6 and E7 proteins and how those activities relate to the activities of HPV E6 and E7 oncoproteins encoded by mucosal and cutaneous high-risk HPV genotypes. Human tumor viruses contribute to at least 15% of the world’s total cancer burden . In partHPV can be classified into various genera based on their genome sequence . PracticThe high-risk, mucosal HPV E5, E6, and E7 genes encode proteins with oncogenic activities and have been extensively studied. They target cellular processes that have been linked to the hallmarks of cancer ,29. The High-risk mucosal HPV-induced disease is driven by the activities of three viral proteins, E5, E6, and E7. They play critical roles in the replication of HPV and their oncogenic activities reflect their viral replication functions. The high-risk” HPV E5, E6, and E7 oncogenes are well characterized and extensively studied. The high-risk HPV E5 protein activate the EGF signaling pathway . IncreasIn addition to inhibiting p53 and pRB, the high-risk HPV E6 and E7 proteins also contribute to viral pathogenesis through other mechanisms. Both the high-risk HPV E6 and E7 proteins have been implicated in the inhibition of the differentiation process . ExpressCutaneous HPVs do not encode an E5 protein; however, cutaneous HPV E6 and E7 proteins have a number of pro-oncogenic activities including the ability of a subset of cutaneous β-HPV E6 and E7 oncoproteins to immortalize keratinocytes ,91,92,93A key tool for studying disease is the utilization of a pre-clinical small animal model to translate findings from tissue culture-based studies to in vivo studies. Small animal models provide the opportunity to investigate key activities of oncogenic virus activities in a physiologically relevant setting and to test therapeutics. However, as pointed out earlier, the species specificity of HPVs has been a strong barrier in the ability to study viral pathogenesis. Some animal models, however, have been available to circumvent this barrier.A major accomplishment in the papillomavirus field was the development of HPV transgenic mouse models wherein HPV genes are expressed in epithelial cells from cellular promoters such as the human keratin K14 promoter ,114. SucTransgenic animal models for the cutaneous HPV8 and HPV38 associated with cancer in EV patients have documented the carcinogenic activities of these HPVs ,32,33,34As pointed out in the previous section, transgenic mouse models have been powerful tools in our understanding of the biological activities of the HPV oncoproteins proteins. In most of the models, however, the viral genes are ectopically expressed from a heterologous promoter and in every cell of a given tissue . This stIn 2011, a naturally occurring papillomavirus was discovered in a colony of nude mice in India . This muExciting studies examining the biological activities of MmuPV1 E6 and E7 are shedding light into how specific biochemical activities of the E6 and E7 proteins contribute to neoplastic disease induced by MmuPV1. MmuPV1 falls within the Pi genus, which contains other rodent PVs and is more closely related to the cutaneous β- and γ-HPVs than the mucosal α-HPVs . These sINK4B [R130A, did not induce skin disease [R130A mutant does not impair NOTCH signaling nor the ability of MmuPV1 E6 to inhibit keratinocyte differentiation [R130A mutant may also affect interactions with other LXXLL motif containing cellular proteins including the IRF3 transcription factor, paxillin (PXN) and the E6AP (UBE3A) ubiquitin ligase. Therefore, it cannot be ruled out that loss of interaction with such proteins may also contribute to the observed phenotype indicating that additional studies are required [Affinity purification coupled to proteomic analyses of MmuPV1 E6 in human cells demonstrated that MmuPV1 E6 protein binds MAML1, a key co-activator of NOTCH signaling, as well as the SMAD2/SMAD3 transcription factors that mediate the transcriptional output of TFG-β signaling . Of noteINK4B . SimilarINK4B . The maiINK4B . ConsistINK4B . These aINK4B . Experim disease . Additiontiation ,183. Therequired . MoreoveD90A, markedly inhibited viral pathogenesis in that papillomas arose later and were significantly smaller [D90A mutant MmuPV1 [Despite lacking an LXCXE motif which is known to mediate the ability of many HPV E7 proteins to bind the tumor suppressor, pRB, affinity purification/proteomic analyses of MmuPV1 E7 in human cells revealed that MmuPV1 E7 can interact with pRB ,112,182 Proteomic analyses of MmuPV1 E7-associated cellular proteins in human cells also identified several other candidate cellular targets of MmuPV1 E7. One that is of particular interest is the non-receptor tyrosine phosphatase PTPN14 that is a known binding partner of many HPV E7s, including high-risk mucosal HPV E7s . DetermiCollectively the research on MmuPV1 E6 and E7 have already provided important insights into how specific interactions of the viral E6 and E7 proteins may contribute to viral pathogenesis.Unlike the “high-risk” mucosal HPV E6, MmuPV1 E6 does not target p53 for degradation . InsteadS, that is defective for E2F1-EZH2 complex binding has been shown to render mice susceptible to the development of cancer, specifically lymphomas and hepatocellular carcinoma [As with MmuPV1 E6, MmuPV1 E7 has been shown to interact with at least two known targets of HPV E7 oncoproteins, the pRB and PTPN14 tumor suppressors ,77,182 . Given tarcinoma . Considearcinoma ,192. A narcinoma . It willMany of the non-canonical pRB activities remain poorly studied and whether and how they may affect keratinocyte biology remains largely unknown. Additionally, pRB has also been implicated in the maintaining genome stability, chromosome condensation, and DNA damage repair (non-homologous end joining and homologous repair) ,196,197.As discussed previously, the high-risk HPV E7 proteins have pRB independent activities that play a role in promoting tumorigenesis as mutant forms of E7 that cannot interact with pocket proteins can still transform tissue culture cells and cause neoplastic disease in transgenic animals ,123,199.R130A mutant (the LXXLL motif binding mutant) [R130A mutant would suggest that other LXXLL binding partners of MmuPV1 E6 contribute to viral pathogenesis and a complete complementation would suggest binding of MAML1 through an LXXLL motif, which inhibits NOTCH signaling, is the only activity that contributes to viral pathogenesis. Similar studies can also be performed by infecting mice that have been genetically alter TGF-β signaling to clearly determine the importance of inhibiting TGF-β signaling for viral pathogenesis. Similar complementation studies can be performed to understand the importance of specific biological activities of MmuPV1 E7 including its interaction with pRB. Utilizing animals that encode an pRB mutant that lacks non-canonical activities of pRB, i.e., pRBS mutant mice, infection with the RB-binding defective E7D90A mutant MmuPV1 could reveal whether loss of non-canonical activities of pRB complement for the loss of pRB binding and provide evidence on which non-canonical activities of pRB play a role in MmuPV1 pathogenesis [The MmuPV1 mouse model has already provided important insights into the biological activities of E6 and E7 which are relevant for HPV pathogenesis and potentially carcinogenesis. Future work is still needed to better understand the interplay between interactions with cellular targets and effectors, in particular understanding how these interactions promote proliferation and other aspects of neoplastic disease outside of inhibiting differentiation. The development of transgenic animal models that express the MmuPV1 E6 and E7 oncoproteins have the potential to determine if these viral oncogenes are carcinogenic on their own or require co-factors, i.e., estrogen, UV, or others, to drive cancer development in mice. Additional studies can also be performed to determine how other early viral proteins contribute, including the viral transcriptional activator E2, to these phenotypes. An outstanding question is which activities of the MmuPV1 E6 and E7 oncogenes promote proliferation and increases E2F activity since MmuPV1 E7 clearly does not perform this activity through its interaction with pRB . Continu mutant) . Utiliziogenesis . Studies


In England, bivalent vaccination (Cervarix) against high-risk human papillomavirus (HR-HPV) genotypes 16/18 was offered in a population-based catch-up campaign in 2008–2010 to girls aged 14–17 years. These women are now entering the national cervical screening programme. We determined the impact of catch-up bivalent vaccination on their screening outcomes.We studied the overall and genotype-specific screening outcomes in 108,138 women aged 24–25 (offered vaccination) and 26–29 years (not offered vaccination) included in the English HPV screening pilot between 2013 and 2018.p < 0.001), with estimated vaccine effectiveness of 87% (95% CI: 82–91%). The detection of any CIN2+ halved from 6 to 3% (p < 0.001), with an estimated vaccine effectiveness of 72% (95% CI: 66–77%). The positive predictive value of a colposcopy for CIN2+ decreased for both low-grade (p < 0.001) and high-grade (p = 0.02) abnormalities on triage cytology. The decreases in screen-detected abnormalities at age 26-29 were of a substantially smaller magnitude.At 24–25 years, the detection of high-grade cervical intraepithelial neoplasia (CIN2+) associated with HPV16/18 decreased from 3 to 1% (These data confirm high effectiveness of bivalent HPV vaccination delivered through a population-based catch-up campaign in England. These findings add to the rationale for extending screening intervals for vaccinated cohorts. Approximately 70%  of cerviThe National Health Service Cervical Screening Programme (NHS CSP) invites women for their first cervical screening 6 months before their 25th birthday. This means that the first cohorts in the catch-up campaign eligible for vaccination were invited for screening in March 2015. In 2013, a national pilot of primary screening with HR-HPV testing, followed by cytology if HR-HPV positive, was launched in six CSP laboratories. The pilot included ~1.3 million women whose primary screening test was either a HR-HPV test or liquid-based cytology (LBC) . In routThe aim of this study was to determine the impact of the catch-up vaccination programme with the bivalent vaccine administered at the age of 14 years and older on screening outcomes in the CSP pilot of HR-HPV testing for primary cervical screening. We report (a) the prevalence of all HR-HPV and, for a subset where this is known, of genotype-specific 16/18 infections, (b) the proportion of women referred to colposcopy, (c) the proportion of women diagnosed with CIN1, CIN2+, CIN3+, and cervical cancer detected by screening, and (d) the positive predictive value (PPV) of colposcopy for CIN2+.The pilot has been described in detail –10. In sWomen screened with HR-HPV tests were returned to routine recall if they tested negative. Samples found to be HR-HPV positive were sent for cytology triage. Women with non-negative cytology were referred to colposcopy at baseline. Non-negative cytology was defined as borderline changes in squamous or endocervical cells or worse, which is approximately consistent with atypical squamous or glandular cells of undetermined significance or worse in the Bethesda 2014 classification. Women with negative cytology had early recalls at 12 and 24 months, although those results could not yet be included in the analysis owing to few women who had undergone early recall after having been first screened in 2016 or later.Data from the first  round of primary screening by HR-HPV testing were retrieved from the laboratory information systems. These data were available until 31 December 2016 for women screened at 26 years of age or older, and until 31 December 2018 for women screened at 24–25 years of age. For all women with positive HR-HPV tests regardless of their age, follow-up histology diagnoses were available until December 2018. All diagnoses were reported as assigned by the routine services. It was assumed that tests were performed for primary screening if the laboratory had no record of an LBC or HR-HPV test within the previous 2 years and/or the test itself was not marked as a response to a recent abnormality. All other tests were excluded from the analysis. Women were also categorised according to the decile of the Index of Multiple Deprivation (IMD). The IMD is an area-based standard measure of deprivation in England . In order to determine a woman’s IMD, her postcode at the time of screening was linked to the Lower Layer Super Output Area code in conjunction with the government’s English indices of deprivation report from 2015 . A smallNeither screened women nor the public was involved in the conduct of this study. A study to evaluate women’s psychological responses to HR-HPV testing was embedded in the pilot and its findings have been separately reported –14.The last of the six laboratories began reporting data to the pilot in August 2013, and women were included in the analysis if they were screened in September 2013 or later. We reported the annual proportions of women  with HR-HPV infections detected at baseline screening, HR-HPV infections in combination with non-negative cytology , diagnosis of CIN1, CIN2+, CIN3+ or cervical cancer, either squamous or glandular, after referral to colposcopy at baseline , and the PPV of colposcopy for a CIN2+ diagnosis on histology. For the subset of the data from the four genotyping laboratories, we stratified these outcomes according to whether the infecting HR-HPV genotypes were those included in the vaccine (HPV 16/18) or not (12 “other” HR-HPV genotypes). Infections with “other” genotypes were counted independently of any co-infections with HPV 16/18; however, for the analyses of CIN2+ and CIN3+ associated with these “other” HR-HPV genotypes, cases of 16/18 co-infections were excluded, as the latter were the more likely drivers for the development of a CIN lesion. The PPV of colposcopy for CIN2+ was calculated as the proportion of women who underwent a colposcopy because of an HR-HPV-positive baseline test with non-negative triage cytology, with a diagnosis of CIN2+.adj). Analyses were reported separately for women aged 24–25, 26–27, and 28–29 years at screening.The annual trends in the proportions of screened women who had abnormalities detected by screening were tested using log-binomial regression using the glm command with binomial family and log link in Stata 15.0. These models included year as a continuous predictor. All models were adjusted for (a) IMD decile, because women from more deprived backgrounds are known to be more likely to develop CIN and cervical cancer , and (b)The observed time trends in screening abnormalities at the age of 24–25 years were compared with the increasing vaccination coverage in the population. Data on the individual vaccination status of women included in the pilot were not available for analysis. As before , the ageadj provided an estimate of the relative differences in the prevalence of screening abnormalities between a fully vaccinated population (100% vaccination coverage) and an unvaccinated population (0% vaccination coverage) for all years combined (2013–2018). The vaccine effect was then estimated by subtracting the PRadj for the vaccination coverage from 1 (i.e. 1 − PRadj) to estimate the reduction in the risk due to vaccination for women who were screened at age 24–25 years. An example of this calculation is presented in the Supplementary Information. To test the robustness of this model, we separately estimated vaccine effectiveness by assuming that the proportion of vaccinated women among those screened was 20% higher than in the analysis described above , and a population with an increasing proportion of women who received the vaccination as part of the catch-up campaign and a decreasing proportion of women who were eligible for this vaccination but did not obtain it (later years). As the information on the individual women’s vaccination status was not available, we could not estimate the relative differences in screening outcomes between vaccinated and unvaccinated women (i.e. the vaccine effectiveness) directly. Instead, we used log-binomial regression models that included a continuous variable for the assigned vaccination coverage  to estimate prevalence ratios  adjustedStata version 15 and RStudio version 1.1.463 were used for the analyses.As part of the pilot, 64,274 women aged 24–25 years were screened using HR-HPV testing between September 2013 and December 2018 Table .Table 1Op for trend, adjusted for IMD and laboratory: <0.001). The decrease in the proportion was more pronounced for vaccine genotypes 16/18, where the prevalence in 2018 decreased by about three-quarters compared with the prevalence observed in 2013–2014 . The estimated effectiveness of the vaccine (i.e. the estimated relative difference between vaccinated and unvaccinated women) was around 40% against HR-HPV infections overall, and around 90% against HPV 16/18 infections . The observed proportion of women with CIN1 among those screened oscillated around 2% (p = 0.03). The proportion of those with CIN2+ approximately halved from 6 to 3% (p < 0.001), as did the proportion with CIN3+, which halved from 4 to 2% (p < 0.001). The degree of protection exerted by the vaccine (i.e. the effectiveness) was estimated to be 70–80% for all high-grade CIN lesions  decreased from 13% in 2013–2014 to 9% in 2018 : −91 to 93%).Although the unadjusted observed detection of cervical cancer at screening decreased by about 75% between 2013 and 2018 (from >0.1% to <0.05%), the trend did not reach statistical significance. The Figure p < 0.001) and high-grade (p = 0.02) cytological abnormalities.The PPV, for CIN2+, of a colposcopy after a positive HR-HPV primary screening test with non-negative triage cytology decreased from 46% in 2013–2014 to 32% in 2018 . For prevention of HPV 16/18 infections, for example, the estimate decreased from 90% (95% CI: 89–92) to 86% (95% CI: 84–88); for prevention of CIN2+ associated with HPV 16/18 infections, it decreased from 87% (95% CI: 82–91) to 82% (95% CI: 76–86). Similarly, time trends adjusted for IMD and site without specifying any assumption on vaccine effectiveness exhibited, in general, highly statistically significant decreases in the proportions of women with screen-detected abnormalities.Between September 2013 and December 2016, 16,864 women aged 26–27 years had a primary screening test, as did 27,000 women aged 27–28 years  in the age group 26–27 years and by about one-seventh  in the age group 28–29 years, while it decreased by three-quarters in the age group 24–25 years (as shown above). The prevalence of “other” 12 HR-HPV genotypes (in combination) remained stable throughout the observation period .In both age groups, decreases in overall HR-HPV prevalence were small, if observed at all (p = 0.01 for both CIN2+ (from just <4% to just >2%) and CIN3+ (from 3 to 2%)). The decreases were less pronounced at 28–29 years of age . At 26–27 years of age, there was a suggestion of an increase in CIN2+  and CIN3+  associated with “other” HR-HPV genotypes .The proportion with high-grade CIN associated with HPV 16/18 decreased by about one-third in the age group 26–27 years  showed that, regardless of the level of deprivation, fully vaccinated women were more likely to be screened . If the Our study relied on routinely reported diagnoses and high-grade CIN histology was not tested to confirm aetiological associations with specific HR-HPV genotypes. By default, for women with genotyped results, we assumed that CIN was associated with “other” HR-HPV infections if no HPV 16/18 infections were detected in the same screening sample. The apparently increasing trend of high-grade CIN in cohorts not offered vaccination at ages 26–27 years) may be a consequence of this definition in the context of changing epidemiology of multiple infections involving HPV 16/18, as co-infections of HPV 16/18 and “other” HR-HPV genotypes were more frequent before vaccination . On the –27 yearsAs far as the study designs can be compared, the patterns observed in the English pilot in cohorts that were offered vaccination between the ages of 14–17 years were consistent with those observed in previous studies. , 28–32 IBefore our analysis, English data were only available from settings such as chlamydia screening or from sexual health clinics. There, the effect of the bivalent vaccine administered at 15–17 years of age against HPV 16/18 infections was estimated to be a 49% reduction [These data indicate an impact of the HPV vaccine both beyond the vaccinated cohorts and beyond the vaccine genotypes. The decrease in high-grade CIN associated with non-vaccine genotypes indicated the presence of partial cross-protection against “other” HR-HPV genotypes, while a decrease in the prevalence of HPV 16/18 infections in older women not included in the catch-up campaign probably indicates some degree of herd protection to those older women. Both effects have already been observed in other routine settings , 26, 28.Several years ago, concern was expressed about the use of cytology for screening vaccinated women, fearing that a decreasing prevalence of cervical lesions would lead to a deterioration in the PPV for CIN2+ . Such deOther programme services that can be expected to be affected by vaccination are those related to cytological evaluation in the triage of HR-HPV infections. While the need for cytology capacity decreased by about 85% when the programme substituted LBC with HR-HPV testing [Neither our data nor the data from Scotland  showed aIn conclusion, our data provide further evidence that the impact of vaccination against HPV 16/18 delivered through a catch-up campaign is impressive. Within the English CSP, these findings add to the rationale for a review of the case for extended screening intervals for cohorts offered vaccination.AppendixReproducibility Checklist


HPV vaccine hesitancy is a key barrier to HPV vaccination. Using a population-based survey of HCPs practicing in Texas we determined the association between formal training of HCPs and perceived self-efficacy in counseling HPV vaccine-hesitant parents and adult patients. A total of 1283 HCPs completed the survey, with 879 providing vaccination services to pediatric patients and 1018 providing vaccination services to adult patients. Among HCPs included in this study, 405 of 577 (70%) and 315 of 505 (62%) perceived they were very/completely confident in counseling HPV vaccine-hesitant parents and adult patients, respectively. Compared to HCPs who received no training, those who received formal training in HPV vaccination promotion or counseling had 2.56  and 2.84 times higher odds  of perceiving that they were very/completely confident in counseling HPV vaccine-hesitant parents and adult patients, respectively. Additionally, increasing years of practice and volume of patients seen were positively associated with being very/completely confident in counseling HPV vaccine-hesitant parents and adult patients. On the other hand, nurses were less likely than physicians to be very/completely confident in counseling HPV vaccine-hesitant parents. To increase HPV vaccination uptake, HCPs should receive tailored training to improve their self-efficacy in addressing HPV vaccine-hesitancy. Human papillomavirus (HPV) is the most common sexually transmitted infection in the United States (U.S.) . Infecti7 However, HPV vaccine hesitancy by parents and adult patients continues to undermine efforts aimed at increasing HPV vaccination uptake rates [There is overwhelming evidence to support the safety and efficacy of HPV vaccines for preventing HPV infection and associated diseases. To date, over 135 million doses of HPV vaccines have been administered in the U.S., with robust data from the Vaccine Adverse Event Reporting System (VAERS) confirming the safety of these vaccines . Betweenke rates ,9. Even ke rates . For exake rates . This rake rates .HPV vaccine hesitancy is a significant barrier to HPV vaccination initiation and completion . StudiesThis was a cross-sectional study based on the data from a state-wide HPV survey conducted by The University of Texas MD Anderson Cancer Center. The survey was administered online between January and April 2021. Email addresses of HCPs practicing in Texas were acquired from the LexisNexis Master Provider Referential Database . HCPs cuThe dependent variable was HCP’s perceived self-efficacy or confidence in knowledge and ability to counsel hesitant parents of pediatric (9–18 years) patients as well as adult (>18 years) patients. We had two dependent variables assessed separately using the following survey questions: How confident are you in your knowledge and ability to counsel: (1) Parents who are hesitant to vaccinate their child, (2) HPV vaccine-hesitant adult patients (>18 years)? Possible responses to both questions were “Not at all”, “Somewhat”, “Moderate”, “Very”, or “Completely.” Each dependent variable was operationalized as a binary variable, 0 = “Not at all/Somewhat/Moderate” and 1 = “Very/Completely”.The primary independent variable was the formal training of HCPs. This was assessed based on the survey question, “Have you received formal training in HPV vaccination promotion or counseling ?” This was a binary variable: yes versus no.The following HCP-related factors were also assessed: age , race/ethnicity , HCP’s sex, and practice location . The zip code of primary work addresses reported by HCPs was used in determining their practice locations by linking the Federal Information Processing Standard (FIPS) Codes with the 2013 Rural-Urban Continuum Codes (RUCC) . Other pp-value < 0.05 was considered statistically significant.The distribution of formal training, socio-demographic and practice-related factors of HCPs was described across the strata of HCPs’ perceived self-efficacy or confidence in counseling HPV vaccine-hesitant patients using proportion and Pearson’s Chi-square test. This was presented separately for HCPs who reported perceived self-efficacy in counseling hesitant parents of pediatric patients and adult patients. We had two separate regression models for each dependent variable: HCPs who counsel parents and HCPs who counsel adult patients. We predetermined to include variables in our models based on the literature and relevance to our study question. Univariable and multivariable logistic regression analyses were used to estimate the odds of HCPs’ self-efficacy in counseling HPV vaccine-hesitant parents and adult patients while adjusting for all covariates included in the final model. We adjusted for HCPs’ age, sex, race/ethnicity, region of practice, years in practice, number of patients seen per week, role in practice, and facility type in each multivariable logistic regression analysis. Furthermore, we assessed the association between covariates and HCPs’ perceived self-efficacy in HPV vaccination hesitancy counseling. All analyses were performed using Stata/IC version 15.1. A two-sided A total of 1283 HCPs completed the online survey. The overall response rate was 7%, and there was no significant difference between respondents and non-respondents concerning provider type and sex of HCPs. Of the total HCPs who completed the survey, 614 (48%) practiced in facilities that served both adult and pediatric patients, 404 (31%) served only adult patients, and 265 (21%) served only pediatric patients.Of the 879 HCPs who provided vaccination services to pediatric patients, 577 (66%) responded to the question on perceived self-efficacy in counseling HPV vaccine-hesitant parents of pediatric patients (9–18 years) . Of thesResults from multivariable logistic regression analysis  showed tAmong the 1018 HCPs providing vaccination services to adult patients, 505 (50%) responded to the question on perceived self-efficacy in counseling HPV vaccine-hesitant adult patients (>18 years) . Of thesResults from the multivariable logistic regression analysis  showed tIn this population-based study, we evaluated the association between the formal training of HCPs in HPV vaccination promotion or counseling and their perceived self-efficacy in counseling vaccine-hesitant parents and adult patients. Our study revealed that about a third of the HCPs did not perceive themselves as having high self-efficacy in counseling HPV vaccine-hesitant parents or adult patients. The low self-efficacy seen in this study is consistent with previous studies, which also found a low confidence level among HCPs regarding HPV and HPV vaccines ,14,26. HImportantly, our study found a positive association between the formal training of HCPs in HPV vaccination promotion or counseling and their perceived self-efficacy in counseling HPV vaccine-hesitant parents and adult patients. This finding aligns with studies that found an increase in provider self-efficacy following the implementation of HPV vaccination training interventions ,30,31. OWhile our study found a similar level of perceived self-efficacy among nurses and physicians in counseling HPV vaccine-hesitant adult patients, nurses had a low perceived self-efficacy in counseling vaccine-hesitant parents compared to physicians. Undoubtedly, nurses remain key stakeholders in addressing vaccine-hesitancy. In the U.S., nurses often provide vaccination services and play an essential role in vaccine advocacy, counseling, and recommendation . With inFindings from this study are subject to some limitations. This is a cross-sectional study, and causality cannot be inferred. Additionally, this study may be prone to information bias from the subjective ascertainment of HCPs’ self-efficacy in counseling hesitant parents or adult patients. Although the response rate reported in our study was low, it is not uncommon to see low responses for online surveys involving HCPs, particularly in surveys of frontline HCPs, mostly in non-academic health settings . HoweverIn conclusion, about a third of HCPs had low perceived self-efficacy in counseling HPV vaccine-hesitant parents and adult patients. Nurses were less confident in counseling HPV vaccine-hesitant parents compared to physicians. Formal training of HCPs enhances their self-efficacy in addressing HPV vaccine hesitancy among parents and adult patients. Additionally, HCPs’ self-efficacy in counseling hesitant parents and adult patients increased with years of practice and counseling several hesitant patients. To increase HPV vaccination uptake, HCPs should receive tailored training in HPV vaccine hesitancy counseling, especially nurses and HCPs with few years in practice.