To examine the impact of self‐reported human papillomavirus (HPV) test result  on a range of psychosocial outcomes.Women and other people with a cervix in Australia aged 25–74 years who reported having participated in cervical screening since December 2017 were recruited through Facebook and Instagram to complete an online survey. The primary outcome measures were anxiety, emotional distress, and general distress.p < 0.001) and emotional distress scores . Concern about test result , perceived risk compared to average women , and cancer worry  were also greater among HPV+ participants than participants testing HPV−. Participants testing HPV+ felt less reassured about their screening result than participants testing HPV− . Participants testing HPV+ had greater knowledge of HPV  and HPV testing  than participants who reported testing HPV−.Nine hundred fifteen participants completed the online survey; 73.2% reported testing HPV negative (‘HPV−’), 15% reported testing HPV positive (‘HPV+’) and 11.8% reported that they did not know/remember their test result (‘HPV unknown’). Compared to participants testing HPV−, participants testing HPV+ had higher mean anxiety Knowledge of human papillomavirus (HPV) is high in individuals who report testing HPV+, but there are still some significant knowledge gapsFuture research is needed to examine how individuals should best receive HPV test results, and what resources should be provided Participants were excluded if they had no capacity to consent, had a previous diagnosis of cervical cancer or had undergone a hysterectomy.2.2Participants were recruited using targeted paid advertising on Facebook and Instagram between 13 and 23 March 2020, to ‘complete a short survey about cervical screening’. Participants could enter a prize draw to win one of twenty $AUD20 vouchers. A web‐link directed participants to read the participant information statement, provide consent to participate (via a tick‐box), and complete a cross‐sectional survey eliciting sociodemographic, clinical, and other background information.2.3Participants completed sociodemographic and clinical information adapted from previous studies of cervical screening attitudes in women/patients.2.3.1Perceived lifetime risk was assessed using ‘What are your chances of developing cervical cancer in your lifetime?’ .2.3.2Worry about developing cervical cancer was assessed using ‘How worried are you of getting cervical cancer in your lifetime?’ .2.3.3Concern about test results was assessed using ‘How concerned do you feel about your recent cervical screening result?’ .2.3.4Reassurance about test results was assessed using ‘How reassured do you feel about your recent cervical screening result?’ .2.3.5Knowledge of HPV was assessed using ‘Before today had you ever heard of human papillomavirus (HPV)?’ (yes/no). Those responding ‘yes’ answered statements assessing knowledge of HPV (16 statements) and HPV testing (5 statements) as ‘true’, ‘false’ or ‘don't know’).2.4With a minimum sample of 1000 participants, and assuming approximately 7.5% of the sample is HPV+ and equal standard deviations of 14.6 on the STAI in both HPV+ and HPV− groups (based on previous work),2.5Analyses were carried out using SPSS v22. Differences in the demographic characteristics between the three result groups  were described descriptively. Variation in anxiety, general distress, emotional distress, knowledge of HPV and HPV testing across the three result groups were explored using ANCOVA. In the anxiety, general distress and emotional distress analyses, we adjusted for age, education, country of birth, relationship status, and cervical screening pre‐December 2017. In the knowledge of HPV and HPV testing analyses, we additionally adjusted for HPV vaccine receipt. After establishing main effects, post‐hoc tests using a Bonferroni adjustment were used to explore differences between groups.p values, were calculated.Multivariate logistic regression models explored whether perceived risk of cervical cancer, cancer worry, and concern and reassurance about test results were associated with HPV test result outcome . Odds ratios, indicating increased or decreased odds for the outcome for each group compared to the HPV− group, with 95% confidence intervals and Data collection was stopped prior to the minimum sample being achieved due to the start of the COVID pandemic, to minimize general anxiety and distress measures being confounded by the pandemic.3n = 7) with an ‘unsatisfactory sample’ were excluded from the analysis. Characteristics of the 915 participants are shown in Table Of the 100,712 impressions , 2302 clicked through to the survey. From this, 1312 began the survey and 922 unique responses were completed and eligible for analysis. Participants  and was highest among the HPV+ group  and lowest among the HPV−  and HPV unknown groups . Post‐hoc tests revealed that participants in the HPV+ group had significantly higher anxiety scores than participants in the HPV− group (p = 0.002). There were no differences between the HPV+ and HPV unknown group (p = 0.098) or the HPV− and HPV unknown groups (p = 1.00).Mean scores for general anxiety, general distress, and emotional distress by HPV status are presented in Table F = 0.75, p = 0.475). Post‐hoc tests also revealed no significant differences between the HPV+ and HPV− groups (p = 0.691), HPV+ and HPV unknown groups (p = 1.00) or the HPV− and HPV unknown groups (p = 1.00).General distress, measured by the GHQ‐12, did not differ across the three HPV result groups  and was highest among the HPV+ group  and lowest among the HPV−  and HPV unknown groups . Post‐hoc tests revealed that participants who reported testing HPV+ had significantly higher emotional distress scores than participants who reported testing HPV− and HPV unknown (both p < 0.001). There was no difference between the HPV− and HPV unknown groups (p = 1.00).Emotional distress, measured by the CSQ, was significantly different across the three HPV result groups . Participants who did not know their HPV status were also more likely to perceive themselves to be at above average risk of developing cervical cancer compared with those testing HPV− .Compared to participants who reported testing HPV−, participants who reported testing HPV+ were significantly more likely to perceive themselves to be at above average risk of developing cervical cancer in their lifetime . There was no difference between the HPV− and HPV unknown group (p = 0.688).Compared to participants who reported testing HPV−, participants who reported testing HPV+ had increased odds of reporting high worry about developing cervical cancer  3.5p < 0.001). There was no difference between the HPV− and HPV unknown group (p = 0.117). Participants who reported testing HPV+ had lower odds of reporting reassurance about their test results , as did participants who did not know their HPV status .Compared to participants who reported testing HPV−, participants who reported testing HPV+ had increased odds of reporting high concern about their test results  and lowest in the HPV unknown group . Post‐hoc tests revealed that participants who reported testing HPV+ had significantly greater knowledge of HPV and HPV testing than participants who reported testing HPV− and HPV unknown . There were no significant differences in knowledge of HPV between the HPV− and HPV unknown groups (p = 0.091), or knowledge of HPV testing between these groups (p = 0.195).Knowledge of HPV and HPV testing differed significantly across the three HPV result groups  and the screening specific measure of emotional distress (CSQ). Elevated anxiety for participants who reported testing HPV+ relative to those reporting testing HPV− is consistent with earlier research immediately following the screening changes in 2017,Although the levels of anxiety are lower among participants who reported testing HPV+ in this sample compared with a previous study,The lower levels of anxiety demonstrated in this sample among participants who reported testing HPV+ compared to a previous Australian sampleIn contrast to anxiety measures, distress specific to cervical screening was higher relative to previous studies.Finally, 12% of participants did not know their HPV result from their latest cervical screening test. This proportion is unchanged from a previous Australian sample in 2017 and remains concerning.4.1Our sample is self‐selected and differs significantly from the Australian population, with an increased proportion of participants reporting testing HPV+ in comparison to the general public ,4.2This study highlights elevated anxiety and emotional distress in individuals testing HPV+, two years following the renewal of the NCSP. Understanding what information individuals receive both prior to and following their cervical screening test is pertinent to ensure information needs are addressed in ways which may also minimise psychosocial impact.4.3Individuals who reported testing HPV+ 2 years following implementation of the renewed NCSP in Australia still reported significantly higher anxiety, concern, and specific distress about test results than those individuals who reported testing HPV−. Future research evaluating the provision of information about HPV, test results and next steps on anxiety and distress, would be valuable.Julia M. L. Brotherton's employer VCS Foundation has received donated human papillomavirus (HPV) tests kits and equipment for unrestricted research purposes from HPV test manufacturers including Roche, Cepheid, Seegene and Becton Dickinson.This study was approved by The University of Sydney Human Ethics Committee (2018/836).Rachael H. Dodd and Kirsten J. McCaffery conceived, designed and developed the methods for the study. Rachael H. Dodd coordinated the running of the study. Kirsty F. Bennett and Rachael H. Dodd contributed to the analysis. Verity Chadwick and Rachael H. Dodd drafted the manuscript. Julia M. L. Brotherton contributed to the content and critical analysis. All authors contributed to the interpretation of the analysis and critically revised the manuscript.Supporting Information S1Click here for additional data file.


NCF1) polymorphisms leading to low production of reactive oxygen species (ROS) are strongly associated with autoimmune diseases. We investigated the association of the missense single nucleotide polymorphism (SNP) rs201802880 (NCF1-339) in NCF1 with URSA and explored its function. We performed NCF1-339 SNP genotyping of samples from 152 Chinese patients with URSA and 72 healthy controls using nested PCR and TaqMan assays. ROS production and RELA (NF-κB subunit) expression in the blood of participants with different NCF1-339 genotypes were determined. The frequencies of the wild-type (GG) and mutant (GA) genotypes remarkably differed between the URSA and control groups. The mutant genotype was associated with an increased risk of recurrent abortion. Furthermore, ROS levels in the URSA group with the GG genotype were significantly higher than those in the group with the GA genotype (p < 0.05). RELA expression in URSA patients with the GA genotype was considerably higher than that in control individuals with the GG genotype. These findings indicate that mutations in NCF1 may increase the risk of URSA via the NADP/ROS/NF-κB signaling pathway, which has implications for the diagnosis and treatment of URSA.Unexplained recurrent spontaneous abortion (URSA) is a major concern in reproductive medicine. Neutrophil cytosolic factor 1 ( It occuPrevious studies have shown that abortion results from the immunological rejection of embryos by pregnant women, leading to the failure of embryonic allotransplantation . Some stIn the human genome, neutrophil cytosolic factor 1 (NCF1) is located in the complex 7q11.23 region and encodes the p47phox/Ncf1 protein of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2) complex, which is critical for ROS induction . ROS canIndependently, many groups worldwide have associated the non-synonymous single nucleotide polymorphism (SNP) NCF1-339 rs201802880) with multiple autoimmune diseases, such as systemic lupus erythematosus (SLE), chronic granulomatous disease (CGD), and rheumatoid arthritis –18. Howe02880 wit22.1Case group (URSA group): A total of 152 patients with URSA, admitted to the Guangdong Reproductive Hospital between August 2019 and November 2021, were selected as the case group. Inclusion criteria were as follows: (1) two or more consecutive spontaneous abortions with a gestational age of <12 weeks; (2) normal maternal and paternal karyotypes; (3) absence of reproductive tract malformation, reproductive tract infection, TORTCH(+); (4) regular menstrual cycle, normal basic sex hormones, thyroid function, and fasting blood glucose levels; (5) absence of positive autoantibodies ; and (6) male semen routine examination.Control group: A total of 72 normal women who underwent a medical examination during the same period were selected as the control group. The inclusion criteria were as follows: (1) previous normal birth history (one or more births); (2) no adverse pregnancy history, such as spontaneous abortion, stillbirth, and premature delivery; (3) no history of gestational diabetes, preeclampsia, or other complications of pregnancy, and a regular menstrual cycle; (4) no history or family history of autoimmune and metabolic diseases.g for 10–15 min, and the separated serum was stored in tubes at −80°C.Peripheral blood samples (10 mL) were collected in the morning during non-pregnancy and non-menstrual periods after at least 8 h of fasting. A total of 5 mL of blood was added to anticoagulant tubes, containing 2% ethylenediaminetetraacetic acid, and stored at −80°C. The rest (5 mL blood) was centrifuged at 1,760×Informed consent: Informed consent has been obtained from all individuals included in this study.Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration, and the protocol was approved by the Ethics Committee of Guangdong Institute of Reproductive Sciences .2.2A high-efficiency blood total RNA extraction kit was used to extract RNA , and a DNA extraction kit was used to extract DNA (TIANGEN Cat. No. Dp304-02). DNA and RNA concentrations and the optical density values were determined using an ultra-micro-nucleic acid detector .2.3Whole exome capture: The Illumina TruSeq Exome Enrichment kit was used to capture the whole exome in the peripheral blood DNA of patients. WES: The IllunimaHiseq2500 sequencing platform was used for double-terminal WES of the captured sequences. Recognition of full exon group variation: The best practice recommended by GATK was used to identify SNPs and indels in the entire exon region. Compared with the 1000 Genomes Project data, mutation loci with a distribution frequency of less than 0.01 in the 1000 Genomes Project database were reserved. Four biological function prediction software packages  were used –22.2.4NCF1 sequence using nested PCR and TaqMan assays. For more details, refer to Supplementary Material [To obtain the correct genotypes for the NCF1 variants, we specifically amplified the Material .2.5p-values were less than 0.05.GO enrichment analysis of variant genes was implemented using the clusterProfiler R package, in which gene length bias was corrected. Enrichment was considered significant when the corrected 2.6ELISA kits  were used to detect the concentrations of cytometric factors (ROS) using the double antibody sandwich method. Samples, standard substances, and HRP-labeled detection antibodies were added to the ROS-coated micropores once. Following the incubation, samples were thoroughly washed. The substrate tetramethylbenzidine (TMB) was used for color rendering. TMB was converted to blue under the catalysis of peroxidase and yellow under the action of acid. Color intensity was positively correlated with ROS levels in the sample. We measured the absorbance  using an enzyme-labeled microplate reader  at a wavelength of 450 nm to calculate the concentration using a standard curve.2.7−ΔΔCt method, with β-actin as the internal control. The primers used for the qRT-PCR are listed in The experiment was performed following the instructions in the reverse transcription kit (Cat. No. RR036A). mRNA expression of RELA was measured using the TB Green Premix Ex Taq II Kit  in a StepOnePlus™ Real-Time PCR System with a Tower . Three assays were performed per sample. Data were analyzed using the 22.8t-test was used for data comparison between the two groups, and the rank test was used for data that did not conform to a normal distribution. Statistical significance was set at a p-value of <0.05.The SPSS.26 software was used for data analysis, and GraphPad Prism6 was used for graph construction. The chi-square test was used for comparative analysis of genotypes, and logistic regression analysis was used to calculate the odds ratio (OR) values and 95% confidence intervals (CI) to evaluate the relationship between NCF1 variation and URSA susceptibility. Gene expression, ELISA data, and data grouping were compared, and normality tests were conducted. The homogeneity of variance test was performed for normally distributed data. The 33.1NCF1 had a high mutation frequency in this patient sample (22/53). When 53 samples were verified, it was found that only 15 of the 22 variants had Ncf1 variants, one of which was pure and mutant . Subsequently, the different genotypes of NCF1-339 were analyzed and compared; the results are shown in p < 0.05). The GA mutant was significantly associated with the risk of recurrent abortion . There were only four homozygous mutations (AA) in the URSA group, but the differences were not statistically significant because of the small number of controls included.In addition to the 53 patients with URSA initially included in the WES analysis, we recruited another 99 patients with URSA. The final sample sizes of the patient and healthy control groups were 152 and 72, respectively. The basic demographic data of the URSA and control groups are shown in 3.3p < 0.05). Thus, the GA genotype of NCF1-339 reduced the ROS levels  and control  groups. After group comparisons, we found that ROS levels in the URSA group individuals with the GG genotype were significantly higher than those in the URSA group individuals with the GA genotype, and the difference was statistically significant . In addition, the GA genotype had higher ROS levels than the GG genotype in the URSA group, which may be due to the smaller number of samples included; therefore, the difference was not significant  is located in the active site of NAPDH oxidase in the hot spot mutation region. Of note, there are two pseudogenes close to NCF1: NCF1B and NCF1C (NCF1B/C), which encode truncated, non-functional proteins sharing 98% sequence similarity with NCF1 [80 is locIndependent studies confirm a strong association of the non-synonymous SNP NCF1-339 (rs201802880) with multiple autoimmune diseases, such as SLE, CGD, and rheumatoid arthritis –31. ThisThe NCF1-339 A allele leads to a shift from Arg to His at position 90, located in the phox domain of NCF1, which mediates its binding to the cell membrane. The substitution of evolutionarily conserved Arg90 with an encoded histidine residue was predicted to be deleterious. We, and others, have previously shown that mutation at position 90 reduces the ROS response  and the ROS have numerous downstream signaling factors, among which NF-κB is a dominant transcription factor in the cellular inflammatory response directly involved in the inflammatory response of tissues, while inducing multiple proinflammatory cytokines, including TNF, and the transcription of IL-1 and IL-6, playing a key role in the autoimmune disease process ,37. In tIn summary, we identified a p.Arg90His substitution in NCF1  as a novel risk variant for URSA. NCF1 gene variation may lead to URSA through the NADPH/ROS/NF-kB signaling pathway, which is of great clinical significance. However, this is only a preliminary association study, and further research is required into the specific mechanisms by which the NCF1-339 variant leads to maternal autoimmune disorders and immune imbalances at the maternal–fetal interface, to confirm the association with miscarriage.


VIA] was done as an initial screening method for cervical cancer. The cervical swab was then collected using L-Shaped FLOQSwabs in eNAT nucleic acid preservation and transportation medium. A Pap test was done to determine the cytological profile. Nucleic acid was extracted using STARMag 96 ProPrep Kit on SEEPREP32. A Real-time multiplex assay was performed to amplify and detect the HPV L1 gene used for genotyping. The data were entered into Epi data version 3.1 software and exported to STATA version 14 for analysis. A total of 901  women were screened for cervical cancer using VIA and 832 women had a valid co-testing (Pap test and HPV DNA testing) results for further process. The overall prevalence of hr HPV infection was 13.1%. Out of 832 women, 88% of them had normal and 12% had abnormal Pap test results. The proportion of high risk HPV was significantly higher among women with abnormal cytology  and younger age . Among 110 women with hr HPV, 14 genotypes  were identified while HPV-16, -31, -52, -58, and -35 genotypes were highly prevalent. The high risk HPV infection continues to be a significant public health problem among women 30–35 years old. The presence of high-risk HPV irrespective of genotypes is highly correlated with cervical cell abnormalities. Genotype heterogeneity is observed suggesting the importance of periodic geospatial genotyping surveillance for vaccine effectiveness.Cervical cancer is a vaccine-preventable sexually transmitted disease. In the year 2020, there were an estimated 604,000 new cases and 342,000 deaths worldwide. Although its incidence is global, it is much higher in sub-Saharan African countries. In Ethiopia, there is a scarcity of data about the prevalence of high-risk HPV infection and its association with cytological profiles. Therefore, this study was conducted to fill this information gap. A hospital-based cross-sectional study was conducted from April 26 to August 28, 2021, and enrolled 901 sexually active women. Socio-demographic and other relevant bio-behavioral and clinical data were collected using a standardized questionnaire. Visual inspection with acetic acid [ In 2020The prevalence of hr HPV infection in sub-Saharan African countries is unevenly distributed, ranging from 10.7%  to 97.2%Infection with human papillomavirus (HPV) is the primary cause of cervical cancer . Around Genital/mucosal types are of the alpha-PV genus and are classified into oncogenic (high-risk) or non-oncogenic (low-risk) types based on their involvement in malignant lesions. Genotyping of the virus defines by the genetic sequence of the protective outer shell or capsid made of a protein called the Late gene 1 L1; . AccordiCurrently, three licensed HPV vaccines constructed using L1 capsid antigens: 9-valent HPV vaccine , quadrivalent HPV vaccine , and bivalent HPV vaccine  are available . Hence, 2.2.1.A health facility-based cross-sectional study was conducted from April 26 to August 28, 2021, in three cities  in Ethiopia . These h2.2.The source populations of the study were all women who live in eastern Ethiopia and who have started heterosexual intercourse. Women between the ages of 30 and 65 years , who hav2.3.2.3.1.Socio-demographic and other relevant bio-behavioral data  were collected through a face-to-face interview using pre-designed and pre-tested structured questionnaire. A hospital checklist was used to collect a clinical data .2.3.2.VIA method for cervical cancer. During VIA examination, women with an invisible transition zone were excluded from the study. A sterile plastic spatula was inserted into the vagina to visualize the cervix. Then, 5% acetic acid was applied to the cervix and monitored the changes for 1 min. A sharp, distinct, well-defined, dense  aceto-white area with or without raised margins define as a positive test  and make a smear on the slide. The smear was fixed on the slide using ethanol and stained according to standard protocols . Then, t2.3.4.Endocervical and ectocervical cells were collected from the cervical canal using an L-shaped Endo/Ectocervical FLOQSwab®  cytobrush. The brush was then placed into a 2 ml eNAT nucleic acid collection and preservation vial  for HPV DNA detection and genotyping. The collected cervical cells were transported to Child Health and Mortality Surveillance (CHAMPS) Ethiopia project, Haramaya University, and Armauer Hansen Research Institute (AHRI), Addis Ababa laboratories and stored at -80°C until further processed.2.4.in vitro diagnostics) automated Liquid Handling Workstation . The extracted DNA was eluted with 70 μl of elution buffer. Parallel detection and genotyping of HPV was carried out using Anyplex™II HPV HR kit  which can detect and genotype 14 h HPV . A multiplex Real-time assay was performed to amplify the HPV L1 gene for genotyping and Human housekeeping gene as an endogenous internal control [IC] which can ensure the purification of DNA, verification of PCR reaction, and clarification of cell adequacy from each specimen. CFX96TM Real-time PCR System (Bio-Rad) experiment setup was used for the detection of 14 types of HPV using 5 μl of template DNA in a total volume of 20 μl. The 14 HPV types detection and genotyping were done in five fluorescent channels , each with individual parameters for target detection and validity; channel 1 HPV-66/-45/-58, channel 2 HPV-51/-59/-16, channel 3 HPV-33/-39/-52, channel 4 HPV-35/-18 and, channel 5 HPV-56/-68/-31.An aliquot of cervical swab [200 μl] was used to extract nucleic acid using STARMag 96 ProPrep Kit  on SEEPREP32™  of the College of Health and Medical Sciences, Haramaya University, Ethiopia (Reference Number: IHRERC/212/2020) and the Armauer Hansen Research Institute Ethics Committee (Reference Number: PO/20/20) approved the protocol.3.3.1.VIA screening method. The majorities were urban residents (86.9%) and married (87.1%), while more than half of the study participants were unemployed (65.3%), unable to read and write (54.2%), and over 18 years of age at the time of their first marriage (68.4%) and first sexual intercourse  were initially screened using e 66.8%; .3.2.VIA screening and Pap smear test, 654 (72.6%) women had negative and the remaining 247 (27.4%) positive VIA results. But, during the Pap smear test, 60 (6.7%) women were excluded from the study due to “unsatisfactory/unreliability test result for the evaluation of cervical epithelial cell abnormalities.” Therefore, we included only 841 women who had normal  and abnormal  Pap test results for further analysis. Out of 101 women with abnormal cytology results, 98 (97%) had Low-grade squamous intraepithelial lesions (LSIL), and 3 (3%) had High-grade squamous intraepithelial lesions . There was also a significant difference in the proportion of hr HPV detection between the VIA-positive and negative women , 54 women had unsatisfactory Pap test results and 9 women had invalid HPV DNA testing results. Further, the results of 6 women for both the Pap test and HPV DNA testing were invalid. Therefore, the co-testing results of 832 women were used for further analysis. The overall proportion of hr HPV infection was 13.1%, and the rate of hr HPV detection was significantly higher in women with an abnormal Pap test result compared to women with a normal Pap test   was observed in women aged 30 to 35 years. In addition to that, the proportion of hr HPV infection decreased as the age of the women increased, and statistically, it has a significant association (= 0.018) .3.4.In the study, 14 genotypes were identified among 110 women whose hr HPV DNA was detected. Among these, 80 (72.7%) women had a single genotype and the remaining 30 (27.3%) women had more than one (multiple) genotype. In addition, Hr HPV-16 (31.8%), -31(19.1%), -52(11.8%), 58(10.9%), and -35(10%) were the most frequently detected genotypes .3.5.p value <0.25 and entered them into the multivariate logistic regression model.We first explored the main factors associated with hr HPV infection in this study using a binary logistic regression model. Then, we selected only associated factors with a p = 0.004). Similarly, the odds of getting the hr HPV infection among women who had more than one sexual partner is higher than women who had a single sexual partner .Accordingly, depending on their potential risk with different sex partners the odds of getting the hr HPV infection among women with single marital status is higher than married women  was observed in women between 30 and 35 years of age, while the prevalence of infection was comparatively lower in women over 40 years of age. The age-specific prevalence of the disease in the current study is consistent with the results of previous studies . This isAlong with women’s age, the other strongest factor influencing the natural history of the disease is the presence of hr HPV (particularly HPV-16 and -18 genotypes) infection . These t5.The hr HPV infection continues to be a major public health problem among women of 30–35 years old. Although the prevalence was high in younger women, the age-specific HPV infection prevalence declines as the age increase. The presence of hr HPV irrespective of genotypes is highly correlated with cervical cell abnormalities. Genotype heterogeneity is observed suggesting the importance of periodic geospatial genotyping surveillance for vaccine effectiveness.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Institutional Health Research Ethics Review Committee (IHRERC) of the College of Health and Medical Sciences, Haramaya University, Ethiopia and the Armauer Hansen Research Institute Ethics Committee. The patients/participants provided their written informed consent to participate in this study.AS, AdM, AnM, BS, RH, and AbA participated in proposal development, data collection, laboratory works, data analysis, and manuscript writing. TG conducted clinical examination, sample collection, and supervision of midwives during sample collection. AdA and AB participated in the cytological examination. DA participated in nucleic acid extraction, laboratory protocol review, HPV detection, and HPV genotyping. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Men should not be overlooked in research on human papillomavirus (HPV) and its associated genital diseases. This is because men infected with HPV are not only at higher risk of genital cancers, but also increase their partners’ risk of HPV infection and reinfection through sexual contact. Herein, we summarized the state of knowledge regarding the prevention and treatment of HPV infection in men as well as the possible effects of the prevention and treatment of HPV in men on their female partners. Condom use, smoking cessation, male circumcision, and HPV vaccination for men each play an important role in preventing HPV infection within heterosexual couples. Additionally, men could choose to test for certain types of HPV, such as the oncogenic HPV16 or HPV18 strains, as part of a routine screening program when their partner is positive for HPV. Although there is no recognized treatment for HPV infection as of yet, immunotherapy drugs, such as toll-like receptor agonists, therapeutic HPV vaccines, and immune checkpoint inhibitors, have shown promising results in clinical trials and in actual clinical practice. HPV infection in men also increases the risk of cervical cancer in their female partners. Because of the high partner concordance for HPV demonstrated in prior research, the prevention and treatment of HPV in men should be explored more comprehensively in future research. Human papillomavirus (HPV) is the most common reproductive tract virus. HPV belongs to the Papillomaviridae family. Every year, HPV causes 630,000 cancer cases in men and women , thereby posing a serious threat to public health on a global scale. In 2012, HPV-related malignancies accounted for 8.6% of all cancer cases in women and 0.8% of all cancer cases in men . The MarHPV is divided into broad high-risk (hr-HPV) and low-risk types (lr-HPV) according to carcinogenicity. Infection with hr-HPV can cause head and neck cancer and oropharyngeal cancer in addition to anogenital cancer. In contrast, benign skin lesions, such as genital warts, are generally attributed to lr-HPV . AlthougRecent studies suggest that men can take a series of measures to reduce the incidence of certain HPV types, for example, men should be vaccinated against HPV   and circAt present, the joint prevention and treatment of HPV in men and women  is not at the core of the screening and treatment protocols that are currently in place. However, cross-infection between couples results in persistent HPV infections more easily than in other situations, thereby elevating the risk of developing high-grade cervical lesions and eventually cervical cancer . Hence, Sexual contact is the primary transmission route for HPV. Males act as both virus carriers and vectors and this is an important component of the epidemiological chain for HPV . FurtherThe fact that cross-infection occurs between members of a couple should not be ignored, as this is one of the reasons for the poor control of HPV evident in the literature and in clinical practice. Condoms are effective in physically isolating HPV infection, and men who do not use condoms have higher rates of HPV infection . AccordiSmoking is a known independent risk factor for HPV infection. Schabath and colleagues successively demonstrated that current smoking overall as well as current smoking with a history of greater than five pack-years of smoking was associated with a higher incidence of HPV infection  and a lower probability of infection clearance in men . ResearcNumerous studies have demonstrated that male circumcision (MC) is effective in reducing the incidence of multiple HPV infection strains in men , therebyTo date, 107 countries have introduced HPV vaccination programs, among which developed countries  have high vaccine implementation coverage; in contrast, in middle and low-income developing countries, the scale of HPV vaccine introduction has not yet been satisfactory . It is tA growing number of studies on gender-neutral HPV vaccination have emerged, and investigators have suggested that HPV16 eradication in the general population is predicted when 75% coverage of early adolescents (both boys and girls) is achieved . We addiNevertheless, barriers still exist to achieving prevalent HPV vaccination in men, including but not limited to a lack of knowledge regarding HPV, prejudices against the vaccine, various sociodemographic and religious factors, fear of side effects, and concerns about cost . ConsequThere are three types of HPV vaccines: a bivalent HPV vaccine, four-valent HPV vaccine, and nine-valent HPV vaccine. The bivalent vaccine protects against HPV16 and HPV18; the four-valent vaccine protects against HPV6, HPV11, HPV16, and HPV18; and the nine-valent vaccine protects against HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58. These three types of vaccines are all effective against HPV16 and HPV18, including HPV-related cancers, as it can be prevented effectively through the use of vaccination campaigns, as the majority of these cancers are caused by HPV16.According to the recommendation of the Advisory Committee on Immunization Practices , femalesAs most HPV infections clear spontaneously without intervention, a positive result does not indicate the need for immediate treatment of the patient or his or her sexual partners. Nevertheless, asymptomatic HPV infection in men is thought to be an important cause of ongoing transmission to female partners, and HPV infection in men increases the risk of cervical cancer in women . HPV infHPV testing for men should remain a focus within future research and clinical endeavors. We recommend several models herein: 1) men could choose to test for HPV when their female partner is positive for HPV ; 2) HPV screening programs for men should be developed as part of an easy and routine program; and 3) certain types of HPV infection, such as HPV16 and HPV18, should be highlighted because these infection strains are linked to the majority of malignancies of the penis, anus, and head and neck as compared with other HPV strains.Current treatment is focused on addressing individual HPV-related lesions, such as cervical cancer, vulvovaginal cancer, and penile cancer. Surgery, radiotherapy, chemotherapy, and targeted therapy are widely used in clinical practice. There is presently no standardized treatment for HPV infection only.However, numerous studies regarding immunotherapy for HPV infection, a treatment modality that aims to achieve therapeutic goals by restoring local immune cell function, have been emerging as of now. The specific mainstream immunotherapy approaches currently under evaluation are described later in this report.in situ of the penis as an alternative treatment option to surgery, with fewer adverse events and tumor recurrence  are considered ideal targets for cervical cancer immunotherapy . TherapeImmune checkpoints and their ligands have been found to be constantly upregulated in the tumor microenvironment of diverse malignancies, representing a major obstacle in the initiation of the body’s effective innate anti-tumor immune response . Immune Reciprocal transmission of HPV is prevalent between couples. In a previous study conducted by Burchell and colleagues that evaluated couples in which both partners were positive for any type of HPV, 87% were found to be concordant for at least one type of HPV strain . CompellAmong heterosexual partners, female patients diagnosed with cervical intraepithelial neoplasia have been shown to increase the risk of HPV infection in their sexual partners . AlthougMen are an important component of the cycle of the transmission of sexually transmitted diseases, and that HPV-positive men are also responsible for their female partners’ reinfection status . As carrHence, in addition to various screening and preventive efforts, the co-treatment of HPV for male and female partners is extremely crucial to slow down HPV transmission. Moreover, partner co-therapy for HPV infection should gradually be included within clinical treatment practices and formal medical guidelines informing clinical decision-making.HPV infection in men and the effects of HPV infection on their partners are increasingly being emphasized in the medical literature, in ongoing epidemiologic and clinical research efforts, and in clinical practice. Since sexual transmission is the main route of HPV infection, condoms can help prevent HPV transmission by physically isolating contact with the mucous membranes of the skin. Smoking has also been recognized as an independent factor contributing to HPV infection, and quitting smoking is one of the known measures for preventing HPV. Moreover, studies have successively proven that MC and male vaccination exert a crucial effect on preventing HPV in female sexual partners.We also note that, on the one hand, HPV infection in men may be a potential risk factor for cervical cancer in women. On the other hand, the high concordance of HPV types in partners suggests that cross-infection is a barrier to HPV control. Therefore, in summary, the treatment of HPV infection in men should undoubtedly be addressed equally as that for women.Early intervention in men can protect against the transmission of HPV infection, while also reducing the incidence of cervical cancer in female partners and facilitating the treatment of female patients with HPV. However, there is still much to learn about the prevention and treatment of HPV and its related malignant diseases. We strongly recommend that this disparity be investigated in male patients as well in female patients, and that this should become a key focus and hotspot for future research.KZ contributed to the manuscript drafting and final approval. YH contributed to manuscript revising and critical discussion. ZL provided practical suggestions and critically revised the manuscript. All authors have read and approved the final manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the Medical Science and Technology Project of Sichuan Provincial Health Commission (No. 21PJ050).We acknowledge the staff of the Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Chengdu, People’s Republic of China, for the methods instruction of the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This review covers the significant new developments in the pathological classification of gynecological tumors. Many of these were included in the updated World Health Organization Classification of Female Genital Tract Tumours, published in 2020. Topics include the compelling evidence that a large majority of extrauterine high‐grade serous carcinomas arise from the fallopian tube; the Cancer Genome Atlas (TCGA) Classification of endometrial carcinomas; the discovery that most so‐called synchronous endometrial and ovarian endometrioid carcinomas represent metastasis from the endometrium to the ovary; and the division of cervical, vaginal, and vulval carcinomas into clinically meaningful HPV‐associated and HPV‐independent types. Newly described tumor types are covered, including endometrial and ovarian mesonephric‐like adenocarcinoma, uterine sarcoma types associated with specific molecular abnormalities, and gastric ‐type adenocarcinomas of the endometrium and vagina. Important molecular events in ovarian sex cord–stromal tumors are also discussed. Review of significant new developments in the classification of gynecological tumors; many are included in the 2020 World Health Organization Classification of Female Genital Tract Tumours. The evidence is compelling, both in sporadic cases and cases associated with germline 3TP53 mutation as an early founder event, and mutational analysis of ovarian and peritoneal HGSC with concurrent STIC has shown these to harbor identical TP53 mutations in the vast majority of cases.TP53 mutation in tumors at different sites is strong evidence for clonality, as the probability of an identical mutation occurring simultaneously at multiple sites is extremely low. It can be summarized that there is irrefutable evidence that almost all HGSC arises from a single tumor clone and that multiple foci of disease do not result from a multifocal origin or a field effect.It was previously thought that HGSC could arise at multiple independent sites through some form of “field effect.” However, it has been convincingly shown that HGSC at different sites are clonal, with one site representing the primary and the others being metastatic. Almost all extrauterine HGSC exhibit 4In 1983, in a seminal and widely cited paper, Bokhman proposed that there were two broad types of endometrial carcinoma, type I and type II.The current 2020 WHO Classification of endometrial carcinomas,–serous carcinomas with no inclusion of other high‐grade carcinomas.POLE (ultramutated), microsatellite instability , copy‐number low , and copy‐number high (serous‐like). It was demonstrated that the four molecular types are of prognostic significance, with POLE tumors having the best prognosis  and copy‐number high the worst.POLE ultramutated (7%).In 2013, TCGA published a seminal comprehensive molecular study of 373 endometrial carcinomas; the study was restricted to endometrioid, serous, and mixed endometrioidPOLE‐mutated (good actors) and the copy‐number high (poor actor) neoplasms.POLE mutation analysis may not be necessary in typical low‐grade endometrioid carcinomas since POLE mutations are not common in this group and these neoplasms would not qualify for adjuvant therapy unless they are advanced stage at diagnosis. Typical serous carcinomas may also not require POLE mutation analysis. CTNNB1 mutation analysis (or perhaps beta‐catenin immunohistochemistry as a surrogate), L1 cell adhesion molecule (L1CAM), and p53 may be of value in low‐grade endometrioid carcinomas in identifying those cases likely to have a worse outcome.POLE mutation analysis is not available in most pathology laboratories and, as such, development of an appropriate infrastructure will be required; this will likely entail centralization of testing in a limited number of reference laboratories. It is also clear that the molecular classification will be complementary to morphology since parameters such as depth of myometrial invasion, lymphovascular space invasion and cervical and nodal involvement, which are prognostically significant, can only be identified on morphological examination.Since the delineation of the four molecular types and the demonstration of prognostic significance, there has been an explosion of studies investigating how to incorporate molecular typing into routine reporting of endometrial carcinomas, and a simplified combined morphological–molecular classification  rearrangements.Up until relatively recently, almost all uterine sarcomas were considered to represent leiomyosarcoma, low‐grade endometrial stromal sarcoma, undifferentiated sarcoma, and rare “heterologous” sarcomas, such as rhabdomyosarcoma. However, the last few years have witnessed the description of several “new” uterine sarcoma types, such as high‐grade endometrial stromal sarcomas associated with 8The 2014 WHO Classification of cervical squamous cell carcinomas (SCC) divided these neoplasms into essentially meaningless morphological types, such as keratinizing, nonkeratinizing, basaloid, warty, papillary, squamotransitional, verrucous, and lymphoepithelioma‐like. These represent morphological variations rather than tumor types and, in practice, most pathologists did not use these categories, which suffered from lack of reproducibility and were of no prognostic significance. The 2020 WHO Classification categorizes cervical SCC into HPV‐associated and HPV‐independent types.9Traditionally (including in WHO 2014), cervical adenocarcinomas were classified based on morphology and were divided into clinically meaningless and poorly reproducible categories with no biological basis, including villoglandular, endometrioid, and serous. Analogous to cervical SCC, cervical adenocarcinomas are now categorized in WHO 2020 into HPV‐associated and HPV‐independent types Table . Most ceHPV‐associated adenocarcinomas are typified by easily identifiable mitotic figures and apoptotic bodies and almost always exhibit diffuse block‐type immunoreactivity with p16. Subtypes of HPV‐associated adenocarcinoma include usual type and mucinous type; the former encompasses villoglandular and micropapillary variants and mucinous type encompasses stratified mucin producing carcinoma, intestinal, signet ring, and NOS variants.HPV‐independent types of cervical adenocarcinoma are gastric type (the most common), mesonephric, and clear cell.Serous carcinoma of the cervix is not included in the 2020 WHO Classification. Most tumors previously diagnosed as such represent usual‐type HPV‐associated cervical adenocarcinomas with papillary architecture and high‐grade nuclear features or direct involvement by or a drop metastasis of a tubo‐ovarian or endometrial serous carcinoma.10Similar to the cervix, vaginal and vulval SCC are now divided into HPV‐associated and HPV‐independent types in WHO 2020.TP53 mutations.TP53 wild type and there is growing evidence that these may have an intermediate prognosis between HPV‐associated SCC (best prognosis) and HPV‐independent TP53 mutated neoplasms (worst prognosis).Similarly in the vulva, traditional histologic typing of vulval SCC has been superseded by HPV status as the major determinant of classification. HPV‐independent SCC have a worse prognosis with significantly worse recurrence‐free and overall survival compared with HPV‐associated SCC.There have also been changes to the classification of primary vaginal adenocarcinomas with the description of new entities such as HPV‐associated and gastric‐type adenocarcinomas,11FOXL2 mutations in well over 90% of cases,DICER1 mutations; these may be somatic or germline, the latter signifying DICER1 syndrome.CTNNB1 or less frequently APC mutations and is occasionally an extracolonic manifestation of familial adenomatous polyposis.In WHO 2020, the classification of ovarian sex cord–stromal tumors is largely unchanged from the prior classification.SMARCA4SMARCA4 is part of the SWI/SNF complex, which is implicated in the pathogenesis of a growing number of other malignancies. Demonstration of this mutation and/or loss of immunohistochemical staining with SMARCA4 (BRG1) may, in the correct morphological context, be crucial in the diagnosis of this highly aggressive neoplasm.SMARCA4 mutation testing.Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT), which is included in the category of miscellaneous ovarian neoplasms in WHO 2020, has been shown to be characterized by deleterious germline or somatic mutations in a single gene The author has no conflicts of interest to declare.The author is responsible for the design and writing of the paper.


Under different thickness parameters, the effects of side profile parameters on the streaming are similar. But the maximum streaming velocity is proportional to the frequency squared and inversely proportional to the viscosity. Besides, the ratio factor g and sidewall profile angle In microchannels, microstructure-induced acoustic streaming can be achieved at low frequencies, providing simple platforms for biomedicine and microfluidic manipulation. Nowadays, microstructures are generally fabricated by photolithography or soft photolithography. Existing studies mainly focused on the projection plane, while ignoring the side profile including microstructure’s sidewall and channel’s upper wall. Based on the perturbation theory, the article focuses on the effect of microstructure’s sidewall errors caused by machining and the viscous dissipation of upper wall on the streaming. We discovered that the side profile parameters, particularly the gap (gap  Acoustic Streaming is a non-zero time-averaged flow due to the nonlinearity created by acoustic waves in fluid propagation . It formBecause of the chip’s size effect, the viscous layer’s dissipation between the fluid and the wall dominates in microfluidic acoustic streaming, and the resulting acoustic streaming can be defined as the boundary layer streaming . BubblesMicrostructure-induced flow braking of laminar flow in microchannels has received extensive attention in recent years. The fluids in microchannels have characteristic sizes ranging from tens to hundreds of microns, and their Reynolds number dominate . Therefodominate . The shadominate , while adominate . In addidominate . Even thdominate . For shadominate . Moreovedominate . Even thdominate . The micdominate . The curdominate , which dMicrostructures are typically fabricated directly by photolithography or by molding technology using a photolithographic positive mold. The light projection surface can be better guaranteed, but considering the light scattering and uneven light intensity along the thickness, the sidewall profile isn’t ideal  in real . In the (200 μm) . Howeverh (describing the microstructure features), and the gap g parameter  are introduced. Since the thickness of the viscous layer f). It should be noted that the study is aimed at the case where there is a space  , we desiw, and height h. For the width w, the diameter of the micropillars has little effect on the streaming, so we ignore the effect of the w parameter and set h, set g , first and second order quantities, marked as subscripts 0, 1 and 2, respectively. For example:of sound . where ξs is the unit mass entropy), and eliminating the zero-order and the first-order higher-order term, the continuity, the momentum, and the energy equation are in the first-order form [Without acoustic waves, the parameters of the fluid are considered constant, while micro perturbations occur when the acoustic waves are present. Assuming that the perturbation is linear, all parameters can to be extended to the first order, as der form :(7)∂tρ1=The zero-order speeds can only have a considerable effect at unreasonably high-speed background flow (up to 1000 mm/s) , hence wFor the linearization of above equations, the isothermal compression coefficient quations  and 11)kt, the iuations  and  and kt, quations –(9) takeZero-order parameters are considered constants and take values in the reference state. Combined with known boundary conditions, such as quations –17)..p1 or V1Although the first-order field has been obtained, considering that the NS equation is nonlinear, the parameters need to be extended to the second-order, as er field . And theThe second-order velocity is much smaller than the first-order velocity and can usually be ignored. When time-averaged is considered, the first-order velocity is zero, while the second-order is not. When the time-averaged function quations  and 19)〈A〉=1/τ∫0The numerical simulation is based on PT, so the boundary condition setting is done in two steps. The initial conditions of the second-order field are achieved by the inheritance of solutions from the first-order field. Our study is based on the assumption that there is no background flow and the first-order acoustic field wall condition is hard wall, refer to Mesh independence test is required in numerical discreteness to obtain the optimal mesh. To reduce computation, fluid domain mesh is divided by region, including high gradient regions near the wall and bulk domain region. For high gradient regions, We set multiple the boundary layer mesh and measure the maximum element size length R to calculate the relative error respectively. The evaluation of the mesh independent test was performed at the basic boundary layer thickness  of the upper wall differ in number, position, and intensity of vortexes. Classified by the number and shape of the vortexes, the mode can be divided into five phases as the gap increases: space limitation, high-speed vortexes development, maximum action area development, high-speed vortexes fusion, and all vortexes fusion. Due to the space constraints of The streaming pattern consists mainly of reverse symmetrical vortexes, with the right-half shown in g increases, showing a tendency to initially decrease rapidly and stabilize slowly. Furthermore, using the curve fitting, we found that the g. The g can achieve the maximum effective area of streaming, called optimal gap. When comparing different When considering the upper wall, streaming enhances especially at small gap, and the effective areas also change significantly, see The reduction in rsion as . At presAs the According to previous analyses, angle reduction increases g on streaming at various heights. We set g on the streaming under various heights. As show in h under the same g. In addition, we set height-normalized equivalent velocity h is approximately equal. But when the gap is small, the As an important factor in boundary streaming, height is worthy of attention. In this section, we investigate the effects of sidewall profile angle h on the effective area. We combine the respective effective area h on h has a large influence, causing a peak fluctuation in the effective area. h not only generates larger effective areas, but also requires larger optimal clearance. Furthermore, with the change of the gap under various h, the model all have five stages, as in h has more viscous dissipation area, which will improve the strength and effective area of streaming. When the gap is small, h. When h, because more viscous dissipation requires more space to release. Thus, the effective area is affected by gap and height.The higher h, which is related to the viscous boundary layer. When discussing the effect of g and The streaming is controlled not only by side profile (geometry) but also by frequency and viscosity related to g and In this section we set uency in . But theg on f, where g is the equal, f, O is the operational coefficient. For the fixed operational parameters, O is a constant. re of Va , V2max=θAs shown in g and This section will consider another boundary layer thickness factor, viscosity g increases, g is small, the g, From Based on perturbation theory, we develop a 2D simulation model to analyze the effects of side profile and boundary layer thickness on acoustic streaming. Using the coupling boundary of fluid-soild, we define three parameters to characterize the streaming, including the mode, maximum streaming velocity, and effective area. Review and summarize the main conclusions as follow:h, the greater the optimal gap is required.For the side profile parameters, our model predicts that the presence of the upper wall makes the streaming more intense and the pattern more complex. For the boundary layer parameters, the effect on the mode is small, but the effect on the quantitative parameters is obvious. The effect of viscosity and frequency on the streaming is opposite. The higher the viscosity the lower the perturbation intensity of the streaming, but the wider the perturbation region, which is the opposite of the frequency. In conclusion, this study focuses on the effect of side profile on streaming. We use no-slip boundary condition and sidewall angle to introduce upper wall and machining error respectively, which will be closer to reality. Moreover, it also provides a theoretical basis for designing the upper wall’s position to adjust the streaming. Subsequently, this study explored the effect of side profile under various boundary layer thicknesses to expand the adaptable range. Our findings may optimize various applications of microstructure-induced streaming, such as particle manipulation, cell rotation, and micromixing. Sharper sidewall profile angles result in greater streaming velocity at the tip, promising fast target manipulation. Compared with no upper wall, the mode of the streaming is more complicated existing multiple pairs of vortices under a certain gap, which will improve the micro-mixing efficiency.


Squamous cell carcinoma (SCC) from an unknown primary tumor (SCCUP) accounts for 2.0%–5.0% of all head and neck cancers. SCCUP presents as enlarged cervical lymph nodes without evidence of a primary tumor upon physical examination. Primary site detection is important to target treatment and avoid treatment-related morbidity. In this review, we discuss updates in SCCUP management. Diagnostic workup should focus on localization of the primary tumor in SCCUP. Initial workup centers on neck biopsy to confirm the presence of SCC. Given the increasing incidence of HPV-related SCC in the oropharynx, HPV testing is crucial. An HPV-positive status can localize the tumor to the oropharynx, a common site for occult tumors. Imaging includes neck CT and/or MRI, and PET/CT. After imaging, panendoscopy, palatine tonsillectomy or diagnostic transoral robotic surgery can facilitate high rates of primary tumor localization. Primary tumor localization influences treatments administered. SCCUP has traditionally been treated aggressively with large treatment fields to all potential disease sites, which can induce weight loss and swallowing dysfunction. As a result, primary localization can reduce radiation fields and provide possible de-escalation to primary surgical management. Advances in intensity-modulated radiation therapy and dose management also have the potential to improve functional outcomes in SCCUP patients. Given the improved prognosis associated with HPV-positive SCCs, HPV tumor status may also inform future treatment de-intensification to reduce treatment-related toxicity. The global incidence of head and neck squamous cell carcinoma (HNSCC) is anticipated to increase by 30% to 1.08 million new cases annually by 2030  is a new technology that highlights neo-angiogenesis to provide superior visualization of mucosa compared to standard endoscopy , 12. StuBiopsy of the neck mass ascertains the presence of SCC over other etiologies. Fine needle aspiration (FNA) is the first-line tool as it is minimally invasive and cost-effective . FNA shoCore needle biopsy (CNB) uses a cutting needle piston, which obtains a larger tissue sample to preserve the native histologic architecture . One metIn up to 10% to 15% of cases, FNA may be insufficient in supplying enough diagnostic material . ExcisioINK4a, a marker for HPV E7 oncogene expression (via HPV DNA in situ hybridization (ISH)  detection of p16pression . Among pon (ISH) . Currenton (ISH) . A limiton (ISH) . Howeveron (ISH) . Since uon (ISH) . High tuon (ISH) .HPV negative tumors can be further tested for EBV using ISH, which can localize the tumor to the nasopharynx. One retrospective study showed that among patients with EBV-positive nodes, 51.7% of the primary sites were in the nasopharynx .Imaging is essential to identifying a primary tumor, and suspicious sites on imaging are biopsied. Due to its availability and low cost, contrast-enhanced computed tomography scan (CT) of the neck with contrast is commonly the first-line imaging tool . Magnetivia PET/CT has been reported as ranging from 17% to 55.2% (18F-fluorodeoxyglucose-positron emission tomography (PET) scans are another key imaging modality for identifying primary sites in SCCUP patients. A study comparing the diagnostic accuracy of PET alone with integrated PET and CT (PET/CT) demonstrated that PET/CT had a significantly higher primary detection and positive prediction rate compared to PET alone . Primaryto 55.2% , 30. Othto 55.2% .To pathologically confirm the primary site, panendoscopy and/or tonsillectomy can be performed. Panendoscopy includes direct laryngoscopy, bronchoscopy, and esophagoscopy performed under general anesthesia, allowing for inspection of at-risk mucosa . Only sivia panendoscopy in patients with negative imaging, some argue that this benefit to only 10% of SCCUP patients must be considered against the disadvantages of panendoscopy, including the cost and risks of general anesthesia  or protons are preferred , 50. IMRGrewal et al.  comparedHPV status has important prognostic significance, which influences the appropriate SCCUP treatment. It is well-known that HPV-positivity is a strong, positive prognostic factor for oropharyngeal SCCs . PossiblOther studies have investigated the prognosis of HPV-positive SCCs in areas outside of the oropharynx (non-OPSCC). Ko et al.  suggest Major advances have been made in the past two decades to improve SCCUP treatment, including TORS development to increase primary detection and IMRT adoption to reduce treatment morbidity. Future challenges to improving SCCUP outcomes include increasing specialized care access, improving long-term functional outcomes, and incorporating HPV tumor status into treatment de-escalation when appropriate.Primary tumor detection plays a critical role in a treatment regimen and subsequent outcomes, and a full diagnostic workup is outlined in Improvements in long-term swallowing and functional outcomes for SCCUP patients are still needed. While IMRT is adopted as the primary radiation therapy for SCCUP, improvements to its administration can reduce toxicity . LaVigneGiven evidence supporting the favorable prognosis in HPV-positive HNSCC, an HPV-positive status has the potential to inform treatment deintensification among SCCUP patients. While current guidelines do not yet specifically discuss the use of an HPV status to de-escalate treatment, several de-escalation trials for HPV-related disease have recently been published or are underway , 67. DatStudy conception and design, writing: TG, SK, Literature review, analysis, writing: SK and PS, Pathology images, writing: GL. All authors contributed to the article and approved the submitted version.TG is supported by 1KL2TR001444 through NIH.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Suboptimal adolescent human papillomavirus (HPV) vaccination rates have been attributed to parental perceptions of the HPV vaccine. The internet has been cited as a setting where misinformation and controversy about HPV vaccination have been amplified.We aimed to test message effectiveness in changing parents’ attitudes and behavioral intentions toward HPV vaccination.We conducted a web-based message-testing experiment with 6 control messages and 25 experimental messages and 5 from each of the 5 salient themes about HPV vaccination . Themes were identified from previous web-based focus group research with parents, and specific messages were developed by the study team using content from credible scientific sources. Through an iterative process of message development, the messages were crafted to be appropriate for presentation on a social media platform. Among the 1713 participants recruited via social media and crowdsourcing sites, 1043 eligible parents completed a pretest survey questionnaire. Participants were then randomly assigned to 1 of the 31 messages and asked to complete a posttest survey questionnaire that assessed attitudes toward the vaccine and perceived effectiveness of the viewed message. A subgroup of participants  with unvaccinated children aged 9 to 14 years was also assessed for their behavioral intention to vaccinate their children against HPV.969=3.03, P=.003), which was associated with increased intention to vaccinate among parents of unvaccinated children aged 9 to 14 years . At the thematic level, we identified 4 themes (themes 2-5) that were relatively effective in increasing behavioral intentions by positively influencing attitudes toward the HPV vaccine . On the message level, messages that provided scientific evidence from government-related sources  and corrected misinformation  were effective in forming positive perceptions toward the HPV vaccination messages.Parents in the experimental group had increased positive attitudes toward HPV vaccination compared with those in the control group (tEvidence-based messages directly countering misinformation and promoting HPV vaccination in social media environments can positively influence parents’ attitudes and behavioral intentions to vaccinate their children against HPV. Human papillomavirus (HPV) is a group of >200 viruses that can infect all genders . HPV infSince the HPV vaccine was approved in 2006, the prevalence of misinformation on social media as well as the lack of educational campaigns regarding HPV vaccines have contributed to HPV vaccine hesitancy and refusal -11. WithHealth communication and education programs play a vital role in countering misinformation regarding HPV vaccines. According to the CDC, strategic mass media communication is one of the best approaches to addressing a specific public health issue ,20. Web-In this study, we compared different message themes related to HPV vaccine hesitancy and refusal and examined the themes and messages that were more effective in promoting HPV vaccine uptake. We hypothesized that exposure to HPV vaccine uptake messages will influence parents’ behavioral intention to vaccinate their children against HPV by improving their attitudes toward the vaccine.We recruited participants through paid advertisements on Facebook and Amazon’s Mechanical Turk (MTurk). MTurk is a web-based labor market with more than 500,000 anonymous workers worldwide, where requesters distribute tasks and registered workers complete them. MTurk has been widely used for various research purposes such as surveys, cognitive tasks, and web-based experiments ,22. ProsOnce directed to the message-testing experiment, participants completed a pretest survey  before this study. Through focus group discussions, we identified 5 salient themes to address in our messages on HPV vaccination.Safety, side effects, risk, and ingredient concerns and long-term or major adverse eventsDistrust of the health care systemHPV vaccine effectiveness concernsConnection to sexual activityMisinformation about HPV or HPV vaccineWe developed 5 experimental messages for each theme and 6 control messages on e-cigarettes. The experimental messages were grounded in scientific information from published articles and reputable health organizations . The experimental messages were designed to help the audience understand HPV and HPV vaccination and included a call to action to vaccinate their children against HPV.Message-framing tactics were applied to change parents’ attitudes and behavioral intentions toward HPV vaccination. To avoid confounding effects, we controlled for elements that were not part of the experimental manipulation designs. First, we controlled the length of the messages to no more than 130 words and the message source across all 31 messages to be identical and neutral . Second, we controlled the message structure: each message started with introductory statements, included tailored statements specific to the message theme, provided a photo or link to a video that was congruent with the message, and ended with the same call to action. We countered common myths and misinformation and added the correct information. This message structure is consistent across all stimulus messages.The research team conducted multiple iterative review processes with content experts in the fields of adolescent health, cancer prevention, and health communication before finalizing the messages . Throughbad-good, harmful-beneficial, useless-useful, and unsafe-safe  with an additional option indicating “I don’t know.” To measure attitudes toward HPV vaccination, we used 9 items from a study by Kim and Niederdeppe  and modiTo assess the vaccination status of their children against HPV, we first asked, “Do you have any sons (or any daughters)?” Among parents who indicated having sons or daughters, we asked “How old are your sons (or daughters)? Please select all that apply.” Multiple choice options were available to indicate the ages of sons or daughters .” For those who reported having sons or daughters aged between 9 and 14 years, we asked, “Has your 9-14 year-old son (or daughter) been vaccinated for HPV? If you have more than one son (or daughter) 9-14 years old, please answer about the son (or daughter) who had the most recent birthday.” Four response options were available, including “Yes, s/he has received two or more HPV vaccine shots,” “Yes, s/he has received one HPV vaccine shot,” “No, s/he has not received any HPV vaccine shots,” and “I don’t know.” For parents who indicated not receiving any HPV vaccine shots or “I don’t know,” we displayed the following behavioral intention item on a 5-point Likert scale, “Thinking about the same 9-14 years old son (or daughter), how likely is it that s/he will receive the HPV vaccine in the next 12 months?” The response options were 1=very likely and 5=very unlikely. Greater values indicate a lower intention to not vaccinate their unvaccinated children against HPV.During the posttest survey, in addition to remeasuring the items in the pretest survey, such as attitudes toward HPV vaccination, participants were guided to answer a series of additional posttest items, including manipulation check questions to ensure participants had viewed the messages, as well as message perceptions. To understand message perceptions, we adapted 5 items from the message sensation value scale  and 4 itAn identical behavioral intention question was displayed for a subgroup of parents who indicated at pretest having unvaccinated children aged 9 to 14 years  at Dartmouth College, and the institutional review board reliance has been approved by Virginia Commonwealth University and Dartmouth College . The study team obtained a waiver for documenting participant signatures during the consenting process; an information sheet about the study was provided to prospective participants, and they were asked to click We first conducted descriptive statistics on demographic variables with the sample and by comparing experimental and control groups. These subgroup comparisons were conducted for pretest variables, including prior knowledge and response efficacy beliefs measures. Mean-based composite scores were generated to assess attitudes toward HPV vaccination on the pretest  and posttest survey items . The attitude change scores were calculated by subtracting the pretest composite values from the posttest composite values for each participant . Greater values indicate more positive changes in attitudes toward HPV vaccination. Five posttest items assessing the sensation value of the viewed messages on a 7-point Likert scale were averaged to form a composite score . Greater values indicate a more positive sensation toward the messages. A total of 4 posttest items measuring perceived message effectiveness on a 5-point Likert scale were averaged to form a composite score . Greater values indicate a greater perceived effectiveness of the message.For thematic-level analyses, a path-modeling approach was used to model persuasion pathways from message exposure to attitudinal changes toward HPV vaccination, which indirectly influenced behavioral intentions to vaccinate. For the eligibility of thematic-level analyses, which included behavioral intention measures for HPV vaccination in the next 12 months measured at the posttest survey, we only included a subset of data from 189 parents with children aged between 9 and 14 years who had not received any HPV vaccine shot. We used 5 dummy variables to generate exogenous constructs in the path model. The control condition was used as the reference group. Path coefficients from dummy-coded variables to the attitudinal change construct were the magnitude of the associations compared with those from the reference group (control group).For individual message-level analyses, we used the ANOVA with a Bonferroni correction within each theme. Composite scores of measures assessing attitudes toward HPV vaccination, sensation values of the viewed message, and perceived message effectiveness were evaluated in the ANOVA framework to statistically identify relatively more effective messages within each of the 5 thematic blocks.A total of 1674 respondents provided consent and participated in the eligibility screening survey, and 1043 (62.31%) were eligible to participate in the study. Among the 1043 participants, 45 (4.31%) left the study before being randomized to a message, and an additional 3 participants left during the posttest questionnaire, leading to a final sample size of 998 for pretest data analysis and 995 for the posttest data analysis . The aveNearly 88%  of the participants reported having heard of HPV before the study, and 83.3% (827/993) reported that they had heard of the HPV vaccine. At the pretest survey, 14% (140/998) of participants indicated that they did not know about the efficacy of the HPV vaccine in the prevention of cervical cancer, and 28.8% (287/998) of participants did not know of the efficacy of the HPV vaccine in preventing mouth or throat cancer .969=3.03, P=.003). Parents’ behavioral intention to vaccinate their children against HPV was significantly associated with the increase in positive attitudes toward HPV vaccination .At a group level , experimental messages about the HPV vaccine significantly increased positive attitudes toward HPV vaccination compared with the control messages about e-cigarettes . At the thematic level, according to the results from path modeling, messages countering 4 themes—theme 2: distrust of the health care system, theme 3: HPV vaccine effectiveness concerns, theme 4: connection to sexual activity, and theme 5: misinformation about HPV or HPV vaccine—were more likely to increase behavioral intention to vaccinate, in part because of the increased positive attitudes toward the vaccine  and perceived message effectiveness . Within theme 3 countering HPV vaccine effectiveness concerns, the message ID c  reporting statistical evidence of the HPV vaccine’s effectiveness in preventing cancer  was perceived more effective than the message ID a  that stated the importance of HPV vaccination to effectively treat HPV infections , F4,161=3.32, P=.01. Within theme 4, although the ANOVA test reported that the mean values of the 5 messages differed , the significance did not emerge when a Bonferroni correction was applied. For theme 5, which focused on countering misinformation about HPV or HPV vaccination, the message ID e  correcting misinformation about HPV vaccine recommendations  was more persuasive than the message ID d  addressing the misinformation that the HPV vaccine can cause ovarian failure and fertility issues .Within each theme, the influence of each message on changing vaccine attitudes, sensation values of the message viewed, and perceived message effectiveness were examined. For example, at the individual message level, compared with message IDs b, d, and e in theme 1, the message ID c in theme 1, which directly countered safety concerns on the HPV vaccine with an educational video that addressed HPV vaccine effectiveness, significantly increased the sensation value  toward the message effectiveness , addressing HPV vaccine effectiveness concerns, educating on perceived connection to sexual activity, and correcting common myths about HPV vaccination. Given that parental views are often altered because of misinformation that is easily accessible on social media or web-based communities, our study demonstrates that evidence-based messages that directly correct misinformation may be effective in enhancing public knowledge and attitudes about HPV vaccination.As shown in the ANOVA results, certain messages within each thematic block were more effective than other messages in generating positive perceptions of the message viewed. For example, messages that provided numeric evidence from credible sources  were perceived more persuasive than messages that did not refer to a credible source . It should also be noted that there was no message that generated adverse effects or boomerang effects of persuasion, such as stimulating the opposite stance ,28.We acknowledge that attitudes and behavioral intentions toward HPV vaccination are likely to differ according to the level of issue involvement , the HPVThe prevalence of myths and misinformation about HPV and HPV vaccination on social media influences parents’ decisions about HPV vaccination ,32. Our Parents’ hesitancy toward vaccinating their children against HPV was, in part, because of psychological barriers, such as skepticism about HPV vaccine effectiveness and distrust toward the health care system, which was influenced by misinformation about HPV vaccination. On the basis of the salient themes that emerged as parents’ reasons not to follow HPV vaccine recommendations, most of the themes we used in our message designs focused on addressing the lack of knowledge and misunderstandings about HPV vaccine recommendations. For example, we developed messages that directly countered the misinformation that the HPV vaccine is lucrative for health care systems and pharmaceutical industries, which had led some parents not to follow the HPV vaccine recommendations. We also developed multiple messages that directly countered misinformation regarding the side effects and safety issues associated with the HPV vaccine. This type of misinformation has been prevalent on social media, which has been a contributing factor in vaccine refusal and hesitancy among parents ,35.It should be noted that our parent sample was slightly younger, more educated, and poorer than the nationwide samples of parents reported by the Pew Research Center’s Internet and American Life Project and the US Census Bureau Social and Economic Supplement Surveys ,37. For In fact, multiple replication studies support the utility of web-based samples such as MTurk for experimental studies. Through a series of 15 replication experiments comparing results from MTurk convenience samples to probability samples, Coppock  confirmeOur study methods were designed to enhance our understanding of the theory-based causal process  and ecological validity  measured after message exposure could be driven by immediate recency effect and priming effect that can diminish over time . To measSecond, for our path analyses, we generated dummy-coded exogenous variables to examine persuasion pathways from the conditions  and also from the 5 thematic-level variables, as shown in If you had a child aged between 9 and 14, would you consider HPV vaccination in the next 30 days?”). Instead, we asked about the true behavioral intention for HPV vaccination only among parents with children aged between 9 and 14 years who had not yet vaccinated their children against HPV . Future experimental research may replicate the path analysis and further test potential moderators or mediators discussed in the literature. Identifying moderators and mediators involved in the persuasion pathway for HPV vaccination uptake will help to identify effective communication tailoring strategies.Finally, although most of our analyses were based on a sample of over 900 participants, the final path model was based on a subset of the sample (n=189) with children aged between 9 and 14 years, who did not receive any HPV vaccines at the moment, to reflect the age range for the HPV vaccination most recommended by the CDC; that is, we did not ask a hypothetical question to all participants (eg, “Our study highlights that strategic communication efforts in social media environments can offer the opportunity to change parental attitudes and behavioral intentions toward HPV vaccination. Given how ubiquitous social media platforms are today, promoting evidence-based messages—such as ours—on social media may play an important role in promoting accurate health information and enhancing knowledge and attitudes about HPV and HPV vaccination for cancer prevention. We found that when harnessing social media platforms for public health communications, directly countering dominant misinformation themes and providing accurate science-based information can be particularly effective in promoting HPV vaccination uptake.