This cohort study investigates the association of human papillomavirus infection status with development of end-stage kidney disease in Taiwan. What is the association of human papillomavirus (HPV) infection with subsequent development of end-stage kidney disease (ESKD) in the general population of Taiwan?In this nationwide, population-based matched cohort study of 152 176 individuals, HPV infection was negatively associated with development of ESKD.These findings suggest that future detailed studies of the mechanisms involved in the association between HPV infection status and decreased risk of ESKD could yield targets for interventions to delay the development of ESKD. The association between human papillomavirus (HPV) infection status and the natural process of kidney diseases has been neglected as an area of research. Further studies are needed to clarify factors that may alter the progression of end-stage kidney disease (ESKD).To describe the rates of ESKD among patients with and without HPV infection.In this nationwide, population-based retrospective cohort study, data were collected from the National Health Insurance Research Database of Taiwan. A total of 76 088 individuals with HPV infection were enrolled from January 1, 2000, to December 31, 2012, and compared with a control group of 76 088 individuals who had never been diagnosed with HPV infection  in the context of the risk of developing ESKD. Statistical analysis was performed between November 2019 and July 2020.International Classification of Diseases, Ninth Revision, Clinical Modification codes.HPV infection was defined according to the The main outcome was ESKD, as recorded in the Catastrophic Illness Patients database. Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% CIs, with the control group as a reference.P < .001), while there was no significant reduction in risk for the other age groups .Of 152 176 individuals , 76 088 individuals (50.0%) had HPV and 463 individuals (0.3%) developed ESKD. Incidence of ESKD was lower in individuals with HPV history than in those without HPV history (3.64 per 10 000 person-years vs 4.80 per 10 000 person-years). In the fully adjusted multivariate Cox proportional hazards regression model, individuals with a history of HPV infection had a significant decrease in risk of ESKD  after adjusting for demographic characteristics, comorbidities, and comedications. In the subgroup analysis, individuals ages 50 to 64 years with HPV infection had a statistically significantly lower risk of ESKD compared with individuals ages 50 to 64 years with no HPV infection (adjusted HR, 0.48; 95% CI, 0.34-0.68; In this study, a history of HPV infection was associated with a lower risk of subsequent ESKD. The mechanism behind this protective association remains uncertain. Future studies are required to clarify the possible biological mechanisms. As health care expenditures continue to increase, public health strategies that maintain costs and quality of care have focused on patients who are at high risk and who account for most health care costs.10 Taiwan has a high prevalence of kidney diseases. In a 2018 study,11 the estimated prevalence of chronic kidney disease (CKD) in Taiwan was 9.1% to 15.5% for CKD stages 3 to 5. Additionally, a previous study in a Taiwanese cohort revealed that the 8-year cumulative incidence in the ESKD rate among individuals with diabetic nephropathy was 14.5%  for people using traditional Chinese medicine and 16.6%  for people who did not use traditional Chinese medicine .12 A 2017 study13 found that in a Taiwanese population, a family history of ESKD was a risk factor associated with developing ESKD, suggesting a moderate level of heritability in the Taiwanese population. In addition to well-known risk factors of ESKD, such as diabetes, hypertension, dyslipidemia, and advanced age, infection-related kidney disease has become a subject of recent research.14 Studies have suggested that viral infections, such as hepatitis B virus (HBV), hepatitis C virus (HCV), HIV, dengue virus, and Hantaan virus, are associated with nephropathies, including vasculitis and immune complex proliferative glomerulonephritis.14 Several large cohorts have documented increased rates of CKD in the setting of HIV infection.16 Studies have also associated HCV infection with histopathological lesions in both native and transplanted kidneys, as well as in CKDs.18End-stage kidney disease (ESKD) remains an important health issue globally.19 Although different HPV subtypes may infect different target cells and the persistence of viral genomes may be different, it is known that 2 HPV viral proteins  play crucial roles in the viral replication strategy for all subtypes.20 These viral proteins mediate oncogenesis progression by preventing apoptosis and causing cell-cycle arrest of the host cells.21Human papillomavirus (HPV), a double-stranded DNA virus, is known to be a distinct risk factor for multiple cancers. The oncogenesis mechanism related to this virus was elucidated in previous studies. This virus exclusively infects epithelium cells, interrupts the normal cell cycle, and triggers uncontrolled cell division.22 Reactivation of oncogenic viruses, such as human herpes virus type 8, Epstein-Barr virus, HBV, HCV, and HPV, is also common among individuals with kidney transplants.23 Kidney transplants are also associated with ESKD, as the reason for kidney transplant is often to improve the quality of life of a person with ESKD.24 Owing to the long preinvasive state of precancer lesions, many HPV-related cancers are preventable with the help of screening and targeted treatment of HPV-related diseases. Thus, pretransplant vaccination and HPV screening in kidney transplant recipients are crucial.25 Because ESKD is frequently a cause of future kidney transplantation and HPV reactivation is an important issue in kidney transplantation, it is reasonable to examine the associations between history of HPV infection and subsequent development of ESKD. To further explore the association between HPV and ESKD, we analyzed a large longitudinal, nationwide cohort.Owing to the occurrence of prolonged immunosuppression, HPV is a common infection in people who have received kidney transplants. Numerous studies have shown that HPV prevalence is significantly increased among individuals with established kidney transplants.26 the identities of individuals with insurance were recoded before analysis. The study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.This cohort study was approved by the institutional review board of the China Medical University Hospital in Taiwan. The need for informed consent was waived because the data were used anonymously and, in accordance with Taiwan’s Personal Information Protection Act,International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM).27This population-based retrospective cohort study used the Longitudinal Health Insurance Database (LHID) 2000, a medical claims database that includes 1 million randomly selected individuals insured from 1996 to 2000 by the National Health Insurance (NHI) system of Taiwan. The LHID was sampled from Taiwan’s National Health Insurance Research Database (NHIRD). In 1996, of 21.52 million residents of Taiwan, 20.04 million individuals (93%) were covered by NHI health care. By 2011, of 23.22 million residents of Taiwan, 23.20 million individuals (99%) were covered by NHI health care. The LHID gives the age and sex distributions of the general population and includes multiple claims files, such as those related to expenditure and order of ambulatory and inpatient care, the prescription of drugs, and the registry for beneficiaries. Together, these data provide information about the diagnoses and details of the use of health care. The diagnostic codes of the claims are recorded according to the ICD-9-CM codes , and the inclusion criteria required at least 3 outpatient visits or any inpatient diagnosis. The HPV group consisted of 76 088 individuals ages 0 to 100 years and newly diagnosed with HPV infection from January 1, 2000, through December 31, 2012; individuals who were diagnosed with HPV from 1997 to 1999 were excluded. The date of the first HPV diagnosis was the index date, and this date was assigned to the matched unexposed individual with the same enrollment criteria. Thus, the study included 76 088 individuals who were newly diagnosed with HPV without an ESKD or kidney transplantation before the index date. These individuals, who were considered exposed, were compared with 76 088 individuals who were not exposed (non-HPV group) and who were matched, via propensity scores matching, by sex, age, index date, comorbidity status, and comedication.HPV infection was defined according to The groups were matched by propensity scores through nearest-neighbor matching, initially to the eighth digit and then as required to the first digit. Therefore, matches were first made within a caliper width of 0.0000001, and then the caliper width was increased for unmatched cases to 0.1. We reconsidered the matching criteria and performed a rematch . For each individual in the HPV group, the corresponding comparisons were selected based on the nearest propensity score. The propensity scores were calculated using the probability of HPV assignment by using a logistic regression model and included the following baseline variables: year of index date, age, sex, comorbidities , and medications . Individuals in the study group and control group were tracked from index visit until an ESKD event, withdrawal from the NHI program, or December 2013.ICD-9-CM codes 401-405), diabetes (ICD-9-CM code 250), hyperlipidemia (ICD-9-CM code 272), coronary artery disease (ICD-9-CM codes 410-414), cerebrovascular disease (ICD-9-CM codes 430-438), CKD (ICD-9-CM codes 585.1-585.5), COPD (ICD-9-CM codes 490-492 and 493-496), chronic liver disease , systemic lupus erythematosus (ICD-9-CM code 710.0), ankylosing spondylitis (ICD-9-CM code 720.0), rheumatoid arthritis (ICD-9-CM code 714.0), Sjögren syndrome (ICD-9-CM code 710.2), HCV infection , HIV infection (ICD-9-CM code 042), and HBV infection .The comorbidities analyzed in this study were hypertension (The outcome was patients with ESKD recorded in the Catastrophic Illness Patients (CIP) database who underwent maintenance dialysis therapy for at least 90 days. In the Taiwan health insurance system, individuals fitting the relevant category of catastrophic illness are classified as individuals who benefit from the catastrophic illness–associated treatment and do not need to pay the fee for that treatment. Verification of the eligibility for CIP certification requires strict investigation. For instance, a patient who has relevant diagnosis codes for ESKD and has received kidney replacement therapy for at least 3 months can apply for the certification.The first ESKD diagnosis of each participant within the study period was used to calculate the risk of ESKD. Follow-up was conducted from the index date until withdrawal from the NHI program or until December 31, 2013. The incidence density of ESKD per 10 000 person-years was calculated in both groups. We used propensity score matching to control for sampling bias. Propensity scores represented patients’ probability of ESKD incidence, and the scores were determined by a multivariate logistic regression model. For balanced covariate distribution, the standardized difference was used to compare the mean of the binary variable between 2 groups in propensity score–matched and nonmatched samples. A standardized mean difference of less than 0.1 indicated a negligible difference in the distribution of a covariate between the HPV and non-HPV groups.P values of .05 or less were considered statistically significant. All data and statistics were processed and analyzed by SAS statistical software version 9.3 (SAS Institute). The statistical analysis was performed between November 2019 and July 2020.To investigate the independent association of HPV with ESKD, a Cox proportional hazard regression analysis was conducted to estimate the hazard ratios (HRs) and 95% CIs after adjusting for full covariates in the study. Variables found to be statistically significant in the univariable model were further examined in the multivariable model. To validate the robustness of the main study findings, a sensitivity analysis was performed by adjusting for demographic characteristics and comorbidities. The Kaplan-Meier curves were plotted to describe the cumulative incidence of ESKD in the 2 groups, and the long-rank test was performed to compare differences between the 2 groups. All statistical tests were 2-sided, and Of 152 176 participants (79 652 [52.3%] women) aged 0 to 100 years , 76 088 individuals (50%) had newly diagnosed HPV infection  and 463 individuals (0.3%) developed ESKD; 76 088 individuals who had no HPV infection  were matched by age, sex, and comorbidities . Among 7In univariate Cox regression analysis and multivariate Cox regression analysis, individuals with HPV infection had a lower risk of developing ESKD compared with those without HPV infection, with an incidence rate of 3.64 per 10 000 person-years vs 4.80 per 10 000 person-years. . The indP < .001), while there was no significant association between HPV infection and ESKD risk for the other age subgroups. The interaction for the age subgroups was not significant. In the sex subgroup analysis, individuals of both sexes with HPV had a lower risk of developing ESKD . However, the interaction for the sex subgroups was not significant. In the comorbidities subgroup analysis, compared with matched individuals without HPV infection, those with HPV infection and any comorbidity had a decreased risk of subsequent ESKD development . However, the interaction for the comorbidities subgroup was not significant. Among people with HBV infection, there was no significant association of HPV infection with ESKD risk , but there was a significant reduction in ESKD risk for individuals with HPV infection and no HBV infection . However, the interaction was not significant by subgroup for HBV. Among people without HCV, there was a significant negative association of HPV infection with ESKD , but there was no reduction in ESKD risk for individuals with HPV infection and HCV infection. There was also a significant interaction by HCV subgroup (P for interaction = .04) (P = .003)  . The Kap = .003) .In this retrospective cohort study using nationwide population-based data over 12 years, a prior diagnosis of HPV was associated with a 28% reduction of risk of developing ESKD. The age cluster phenomenon, with 80.0% of the patients in the HPV study group age 49 years and younger, may be due to the increased prevalence of HPV among younger age groups. Furthermore, stratified analysis revealed that HPV infection was associated with a decreased risk of ESKD in both sexes and for people aged 50 to 65 years. HPV infection may be an independent and distinct factor with a protective association with the development of ESKD even after adjusting for baseline characteristics, comorbidities, and comedications. However, an interaction between HCV status and HPV infection status associated with the development of ESKD was also observed in this study. In the presence of HCV, the decreased association between HPV and ESKD was not observed. The underlying mechanism to explain how HPV infection may decrease the risk of developing ESKD remains unclear. Further studies verifying the potential protective association between HPV infection and ESKD are warranted.28 HCV infection is associated with a higher incidence and faster progression of CKD, which may support our findings. Adequate anti-HCV therapy in patients with CKD improves long-term kidney and patient survival, so clinical suggestions of anti-HCV treatment are warranted.29 The interaction between HPV and HCV in oncogenesis was not investigated thoroughly in previous studies to our knowledge. We know that HPV and HCV involve p53 tumor suppressor pathway deregulation.30 In a 2016 case-control study of head and neck cancers,31 HCV was found to be associated with nonoropharyngeal and HPV-positive oropharyngeal head and neck cancers. That study by Mahale et al31 also suggested that the HPV oncoproteins E6 and E7 directly inhibit type 1 interferon signaling, which is crucial for HCV intracellular replication. Thus, HPV may facilitate the proliferation of HCV in oropharyngeal cells. Moreover, an oncogenic synergistic role in head and neck cancer development was suggested based on the induction of p53 degradation by HPV E6 protein and Rb degradation by the HCV NS5B protein.31 Although HPV and HCV seem to share similar oncogenic pathways, their interaction in kidney function remains unclear. Further investigations are still needed to elucidate the associations among HPV, HCV, and ESKD.Additionally, the interaction of HCV with HPV and the subsequent risk of ESKD warrants further study. Our stratified analysis suggested that HCV may be a mediator between HPV infection and the risk of ESKD. HCV infection is a documented risk factor for CKD and progression to ESKD. Based on a 2018 review,We identified a possible association between HPV infection and ESKD, although the underlying mechanism remains unclear. The unselected large sample size, long-term follow-up period (up to 12 years), and use of population-based data were advantages of using the national database. Several rigorous statistical models were applied in our study. Additionally, strict inspection of applications for certification helped ensure the accuracy of ESKD diagnosis acquired from the catastrophic diseases database. As is mandatory in Taiwan, ESKD diagnosis was confirmed by 2 nephrologists who reviewed the original medical records, laboratory data, and imaging findings.ICD-9-CM codes for diabetes, hypertension, hyperlipidemia, COPD, coronary artery disease, and cardiovascular disease. Third, no reports on laboratory factors, such as serum creatinine level or estimated glomerular filtration rate, were available in NHIRD. However, a 2015 study32 mentioned the acceptable accuracy of CKD diagnostic codes using the standard CKD definition (estimated glomerular filtration rate <60 mL/min/1.73 m2) and a regional hospital data set for validation .This study had several limitations. First, our cohort study design was subject to biases regarding confounding adjustment. Despite using 2 statistical models to adjust for confounding factors, bias may have occurred. Second, risk factors, such as lifestyle and family history of ESKD, are not recorded in the NHIRD. To minimize bias, we applied proxy measures to adjust other associated, unrecorded information, such as Fourth, the study may have been had a selection bias, and one of the major concerns was avoiding such bias. Presumably, many of the patients with HPV had been infected for a long period before detection. This limitation makes matching on the index date difficult. The look-back period before the index date was made sufficiently long, from 1995 to 2000, and HPV events of all age groups (from 0-100 years) were included. Inevitably, some patients with asymptomatic HPV infection do not submit claims for medical services, and thus, these patients may have been classified into the comparison group in this study. However, if HPV is associated causally with subsequent ESKD, patient misclassification would bias the estimated HRs toward the null.In this population-based cohort study, the presence of HPV infection was associated with a reduction of the risk of developing ESKD. An interaction between HCV status and HPV infection status associated with the development of ESKD was also observed in this study. Future detailed studies of the mechanisms involved may yield targets for interventions that could prevent or delay the development of ESKD.


The detection and location of pipeline leakage can be deduced from the time difference between the arrival leak signals measured by sensors placed at the pipe access points on either side of a suspected leak. Progress has been made in this area to offer a potential improvement over the conventional cross-correlation method for time delay estimation. This paper is concerned with identifying suitable sensors that can be easily deployed to monitor the pipe vibration due to the propagation of leak noise along the pipeline. In response to this, based on the low-frequency propagation characteristics of leak noise in our previous study, polyvinylidene fluoride (PVDF) wire sensors are proposed as a potential solution to detect the pipeline leak signals. Experimental investigations were carried out at a leak detection pipe rig built in the Chinese Academy of Sciences. Their performances for leak detection were shown in comparison with hydrophones. It is suggested that with special considerations given to aspects pertaining to non-intrusive deployment and low cost, the PVDF wire sensors are of particular interest and may lead to a promising replacement for commercial leak noise transducers. High quality municipal utilities are fundamental to sustaining modern life and economic growth. Recently, a great challenge involving water loss in the stock of water distribution network, has confronts China’s buried infrastructure . AccordiLeak localization is the identification of the position of a suspected leak prior to excavation and repair, which is normally coordinated in parallel to leakage management, i.e., monitoring and control. Since the second half of the nineteenth century, many leak detection techniques have been developed, primarily including acoustic, chemical tracers, infrared thermography, ground-penetrating radars, and in-line leak detectors ,6,7,8,9.It is now common practice that the acoustic methods based on cross-correlation are relatively effective for pinpointing leaks in metallic pipes; however, difficulties are often encountered when applied in plastic pipes, even in an acoustically quiet environment, since leak noise in plastic pipes attenuates more heavily at lower frequencies. Substantial research has been carried out by the present authors for leakage localization in buried plastic pipes, with attention paid to improvement over the conventional cross-correlation method for time delay estimation (TDE) . MoreoveContinuous  pipe network monitoring systems have commenced more recently to take the leak detection and location methods into a whole new sphere, which requires the deployment of a wireless sensor network to work steadily in extreme environments of high humidity and high corrosion ,21,22. CIn this paper, PVDF wire sensors are developed and their performance for pipeline leakage detection and location are assessed. It is organized as follows. Acoustic leak detection equipment operates at low frequencies, where the axisymmetric (n = 0) fluid-borne (s = 1) wave is responsible for propagation of leak noise . Gao et As suggested in , the traα and For an elastic surrounding medium, the coupled equation of motion dominated by the s = 1 wave can be obtained by .(3)[Ω2−fined in . Settingve speed . FurtherBy substituting Equation (4) into Equation (2), the relationship between the internal acoustic pressure and the pipe radial displacements is obtained byg31 is much higher than other piezoelectric materials. The piezoelectric property of the PVDF has been studied in previous research ,31,32,33Q generated by the PVDF wire sensor is given by [S is the conductive electrode area. The performance of the PVDF wire sensor can be analyzed with a one-dimensional, lumped parameter approach, since the PVDF piezo film is very thin and the deformation in the direction of length is much larger than those in the directions of width and thickness. Thus, it is termed a wire sensor. The piezoelectric equation can be obtained bygiven by (9)Q=d31Since the PVDF wire sensor was wrapped around the pipe, the circumferential strain in the pipe wall is given bySubstituting Equations (7) and (10) into Equation (9), the pressure sensitivity of the wire sensor  is given byAs can be seen from Equation (11), the pressure sensitivity was proportional to the winding number of the pipe. It was also governed by the pipe material. For typical in-air metal and plastic pipes, as suggested in , the proWhen a pipe leak occurs, the leak noise will travel in both directions along the pipeline through the pipe wall and the water, which can be captured by leak noise transducers installed on the pipe wall and inside the pipe bracketing the leak, as shown in D is the distance between the two sensors; c is the propagation speed of leak noise given by Equation (6); and D and the propagation speed c, the problem of leakage localization was now transformed into the estimation of time delay k is the wavenumber given by Equation (4), which can be re-expressed as c and Referring to ained by (14)S12, and no pipe bulging occurs, the pressure sensitivity can be considerable for plastic pipes. This opens up new possibilities for the detection of water leaks in plastic pipes. However, in practical applications, a limitation for such sensors is that both aforementioned scenarios involving slippage and pipe bulging are likely to occur, particularly when the adhesives degrade over time, thus potentially reducing the pressure sensitivity dramatically. To the authors’ knowledge, their effects on the pressure sensitivity, to date, cannot be quantifiable. Further research is required to evaluate the performance for sensing the leak noise signal in real pipeline network. In this paper, the use of the PVDF wire sensors has been investigated for pipeline leakage localization. The performance of the wire sensor has been studied with respect to pressure sensitivity for monitoring the pipe vibration due to leakage. Leak noise measurements have been performed in a PE water pipe in the laboratory under controlled conditions. Theoretical analysis with experimental validations has confirmed the effectiveness of the wire sensors for the detection and location of pipeline leakage by comparing the performance of the PVDF wire sensors and commercial hydrophones. Two sets of measurements have been made, associated with pressure sensitivity and leak localization on the in-air and in-water pipe rig. From the measurements of pressure sensitivity on the in-air pipe, it has been demonstrated that good pressure sensitivity can be achieved by the selected PVDF wire sensors with possible improvement over the sensitivity by adopting higher winding numbers. To overcome the undesirable effects of the supports on the wave behavior, leak localization measurements have been done on the in-water pipe. Frequency analysis has clearly shown the effectiveness of the wire sensor for accurate location of the pipe leakage. Construction of a buried PE water pipe is under way, and more measurements in practical situations will be reported at a future date. This work may offer an alternative to leak noise transducers due to it easy employments and low cost.


In this study, all patients with recurrence were hr-HPV-positive with the same genotype as that before treatment. In patients who were hr-HPV negative after treatment, no recurrence was observed. Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is a distinct subtype of head and neck cancer. Here, we investigated how frequently brushing remained high-risk (hr)-HPV positive after treatment and whether patients with positive post-treatment brushings have a higher recurrence rate. Following the end of treatment of patients with initially hr-HPV positive OPSCC, surface brushings from the previous tumor site were performed and tested for hr-HPV DNA. Of 62 patients with initially hr-HPV DNA-positive OPSCC, seven patients remained hr-HPV-DNA positive at post-treatment follow-up. Of the seven hr-HPV-positive patients at follow-up, five had a tumor relapse or tumor progression, of whom three died. The majority of patients (55/62) was HPV-negative following treatment. All HPV-negative patients remained free of disease ( Approximately 85% of adults acquire a human papillomavirus (HPV) infection in their life. Most HPV infections are transient, asymptomatic, and eliminated by the immune system ,2. HowevIt is unclear whether hr-HPV persists in oropharyngeal tissues in patients with hr-HPV-positive OPSCC following cancer treatment and which consequences this might have. Zhang and coworkers collected blood at diagnosis and post-treatment in 64 patients with p16-positive OPSCC to test for serum antibodies to E6 and E7 proteins of HPV 16. At diagnosis, most patients were seropositive to HPV 16 E6 (85%). In the post therapeutic samples, HPV 16 antibody levels decreased slowly over time, but only three patients became seronegative . In anotFakhry and coauthors used oral rinse samples for HPV detection in 396 patients with oral and oropharyngeal cancer, of which 51% were HPV-positive before therapy. After treatment the HPV prevalence decreased. In patients who received surgical resection, the HPV prevalence decreased from 69.2% to 13.7%. In a subset of patients who required postoperative radiotherapy, the HPV prevalence decreased from 70% to 38% after surgery and then to 1% after radiotherapy. HPV detection in oral rinses was performed several times for patients who received radiotherapy. The median time to clearance was 42 days . The only factor significantly associated with reduced clearance was current smoking. HPV-positivity with the same genotype was detected after treatment in 14.3% of initially HPV-positive patients and among these patients, the cumulative incidence of recurrence was 45.3%. HPV DNA detection after completion of therapy was significantly associated with increased risk of recurrence and death . In cervical dysplasia, hr-HPV infection can persist following treatment and promote disease recurrence. Söderlund-Strand et al. performed a long-term follow-up study obtaining cervical samples for HPV DNA testing and cytological analysis from 178 women with abnormal smears referred for conization. Three years after treatment 3.1% of women were persistently HPV-positive with the same HPV genotype as before treatment. Recurrent or residual cervical intraepithelial neoplasia (CIN) in histopathology was found among 9 (5.1%) women during follow-up. All these women had a type-specific HPV persistence. The authors concluded that only type-specific HPV persistence predicted recurrent or residual disease .In a previous publication, we reported that surface brushings from oropharyngeal cancer reliably detect HPV-DNA. In 53 patients with OPSCC, sensitivity and specificity of the brush test was 86% (95%CI: 70–95%) and 89% (95%CI: 65–99%) . Also, BIn this study, we examined whether hr-HPV is still detectable in surface brushings after treatment in patients with initially hr-HPV-positive OPSCC. Moreover, we compared the course of disease in patients with and without post-treatment hr-HPV in oropharyngeal brushings.During the study period, 74 patients with hr-HPV DNA-positive OPSCC before treatment were included. From 12 patients, no post-treatment brushings were available because they missed the follow-up or received follow-up in another hospital. The mean age of the remaining 62 patients with post-treatment brushings was 61 years. Forty-eight patients were male, and 14 were female. Most patients had UICC stage IVa and were treated with radiochemotherapy . After tBefore treatment, the most common HPV subtype was HPV 16 . In 60/6The post-treatment brushing was taken during complete response in one of the five patients positive with the same genotype. This patient with initially cT4cN2cM0 oropharyngeal cancer A was disIn two patients, post-treatment brushing was positive for another hr-HPV genotype than before therapy. The genotypes were HPV 16 and 33 before therapy and, in the same order of patients, HPV 68b and 16 thereafter. These two patients were in full remission after treatment, no recurrence or tumor persistence was stated in these patients. After treatment, one hr-negative patient was positive with the low-risk type HPV 6, and 54 patients were negative for all investigated HPV strains. p = 0.0007; OR 244.2; 95% CI: 10.4 to 5757.7). All post-treatment hr-HPV-positive patients were also p16-positive at initial diagnosis. Interestingly, all patients with recurrent or progressive tumor (5/62) were hr-HPV positive with the same genotype than before therapy. Three of these five patients died because of the recurrence or tumor progression 10, 12, and 35 months after diagnosis. In contrast, in the two hr-HPV positive patients after treatment without recurrence, another hr-HPV genotype was detected than before treatment. In 4/7 patients with positive post-treatment hr-HPV DNA detection, a recurrence was observed, and in 1/7, tumor progression was observed . In contp = 0.025). Another associated factor was advanced primary tumor T-stage (p = 0.031). No association was observed between post-treatment hr-HPV-positivity and UICC stage, treatment modality and radiotherapy dose. Post-treatment hr-HPV positivity was also not associated with PD-L1 expression, patient-reported smoking status, and consumption of alcoholic beverages at initial diagnosis. Post-treatment hr-HPV positivity was more frequent in patients whose primary tumor expressed epidermal growth factor receptor (EGFR) . EGFR exBefore therapy HPV 16 was the most common genotype  and HPV 18 the second most common . Other genotypes were HPV 33  and HPV 35 . HPV 70, 66, 58, 82, 31, and 40 were detected each in a single patient or additionally to HPV 16 as a multiple infection. As mentioned before, after therapy five patients were hr-HPV-positive with the same genotype than before therapy. Only in these patients a recurrence or tumor progression was stated. The genotypes HPV16 were detected in three patients, and HPV 33 and HPV 18 were each detected in a single patient after therapy. As HPV 18 and HPV 33 are rare in the population of HPV-positive OPSCC patients, we suspect a virus persistence instead of a new infection. In this study, we questioned whether patients with hr-HPV DNA-positive OPSCC remain hr-HPV DNA-positive after treatment and if post-treatment hr-HPV DNA at the initial tumor site is associated with the rate of disease persistence or recurrence. Before and after treatment, brushings were taken from the oropharynx, including the surface of the previous tumor site and tested for hr-HPV-DNA. Post-treatment brushings were available in 62 patients. Overall, 88.7% of hr-HPV-positive patients were hr-HPV negative at follow-up. In seven patients, hr-HPV after treatment was detected, and all patients hr-HPV-positive for the same genotype developed a recurrence or tumor persistence. Detection of hr-HPV at follow-up was associated with a substantially increased risk for persistent or recurrent disease .Post-treatment hr-HPV positivity and persistent or recurrent disease are rare events in hr-HPV-related oropharyngeal carcinoma. Accordingly, the results on potential influencing factors are based on a low number of patients and should be considered with caution. However, our results are in line with previous data. p  =  0.003), as was recurrence-free survival  [Also, Hanna and coworkers described a significant decrease in post-treatment E7 antibody levels in the salivary glands of patients with OPSCC . Rettig   0.001) . ®9  during full remission, hoping to reduce virus activity. However, he developed a locoregional recurrence. Interestingly, 2/7 patients were HPV-positive after therapy with a different hr-HPV genotype, these two patients were in full remission and experienced no recurrence. It seems that a new hr-HPV infection occurred and the risk in these patients for recurrence after therapy is as low as in post-treatment HPV-negative patients. However, higher numbers of patient are needed for further evaluation. Although, only few studies analyzed HPV-positivity after treatment in OPSCC patients and although the number of patients in our study is low, post-treatment HPV-positivity seems to be a strong predictor for overall survival. An inclusion of this observation in the clinical management in patients diagnosed with hr-HPV-positive OPSCC should be discussed. Post-treatment HPV detection in the early follow-up period can be a possible new standard to evaluate and predict the clinical course of those patients. As the brush test does not require biopsies and is easy to perform in awake patients without anesthesia, this is a very suitable and simple test method for HPV-DNA detection. Post-treatment hr-HPV-positive patients may require a very close clinical and radiological follow-up, these patients can be at high risk for a poor overall survival. One of our patients with post-treatment HPV-positivity received an HPV vaccination with GardasilAlso, in patients with cervical cancer, persistent HPV infection is associated with an increased risk for recurrence . In a stIn our study HPV 16 was the most common genotype before therapy, other genotypes like HPV 18, 33, and 35 were rare. Two patients were pre- and post-treatment positive for comparatively rare HPV genotypes 18 and 33. As reinfection with these rare types is unlikely, we assume tumor virus persistence rather than new HPV infection. Our findings about pretreatment HPV genotypes are in line with other studies. Chatfield-Reed and coauthors reported that of 99 hr-HPV positive HNSCC patients, 75.6% were positive for HPV 16 and 3% for HPV 18. In this study, 16.2% were positive for the genotype HPV 33, which we detected in only 1.9% . Fossum In this study, post-treatment hr-HPV positivity was associated with clinical T-stage at diagnosis and tumor EGFR expression. An association of post-treatment hr-HPV positivity and primary tumor EGFR expression has not yet been reported. However, this observation is based on very few patients. EGFR protein over-expression has been reported in 70–100% of HNSCC, but 46–72% of OPSCC ,22,23. AWe did not observe an association of smoking and post-treatment hr-HPV positivity. This might be due to the low number of patients. Active smoking was admitted by 8/45 patients. Kero et al. reported a correlation between persistent oral HPV infection and smoking in 131 men who were sampled by serial oral scrapings. Genotype-specific HPV persistence was detected in 18/129 men. The mean persistence time ranged from 6 months to 30.7 months. The authors concluded that most of the persisting oral infections in males were caused by HPV 16, and smoking increased the oral hr-HPV persistence . AnotherHowever, the most significant observations of this study are that the majority of patients are hr-HPV-negative after therapy and that post-treatment hr-HPV positive patients seem to have an increased risk of tumor persistence or recurrence.Limitations of the study are that not all OPSCC patients were enrolled consecutively in the study. From 12 patients, no post-treatment brushings were available because they missed the follow-up or received follow-up in another hospital. This includes also patients who may have had a relapse. Moreover, several otorhinolaryngologists were involved in sampling, but all were instructed how to collect samples. In this study, the brushings were obtained in some patients under general anesthesia during restaging panendoscopy and in some patients awake during clinical follow-up. The brush test is a simple test method and does not require tumor biopsies for HPV-DNA detection, future clinical usefulness of this test includes an oropharyngeal HPV screening for populations at risk, e.g., immunosuppressed elderly population. Partners of HPV-positive OPSCC patients, who are often concerned about oral HPV transmission, can also be tested by oropharyngeal brushings. The aim of the study was to determine how many patients with hr-HPV-DNA-positive oropharyngeal carcinoma before treatment have positive hr-HPV-DNA detection in the former tumor region after completing therapy. We were also interested in whether post-treatment hr-HPV-DNA detection is related to disease recurrence. This prospective study was conducted in accordance with the GCP guidelines. Written informed consent was obtained from each patient who agreed to participate in this study following detailed explanation of the procedural workflow. Prior to any patient enrolment, the study had been approved by the institutional board in charge, i.e., the ethics committee of the Medical University Innsbruck, Austria. The respective reference number was 1147/2018. The study was conducted in full accordance with the principles expressed in the Declaration of Helsinki.Patients with incident oropharyngeal cancer positive for HPV-DNA before treatment were included. Virus DNA detection was performed by tumor surface brushings and/or by HPV-DNA isolation from formalin-fixed and paraffin-embedded (FFPE) tumor biopsies . All pat® HC2 DNA Collection Device, Qiagen, Hilden, Germany) of the tumor surface was taken. The brush test reliably detects HPV-DNA in patients with OPSCC, sensitivity and specificity of this method is 86% (95%CI: 70–95%) and 89% (95%CI: 65–99%) [Before the start of the treatment, all patients underwent routine endoscopy under anesthesia, including tracheobronchoscopy, esophagoscopy, and laryngopharyngoscopy. During this examination, a cytology brush (Digene 65–99%) . The bruAfter completion of therapy, a second surface brushing was taken from all subsites of the oropharynx, including the initial tumor site. This second surface brush was obtained during follow-up examinations at least 12 weeks after therapy during a restaging endoscopy or during clinical follow-up. ® easyMAG®, Biomerieux, Marcy-l’Étoile, France) [Nucleic acid extraction was performed within two days of arrival of the samples at the laboratory. The DNA extraction was performed in a fully automated manner (NucliSens France) ,28. A to®, Padova, Italy) [®, Padova, Italy). Using the 2′-deoxyuridine 5′-triphosphate/uracil-DNA N-glycosylase dUTP/UNG system, which degrades non-specific residual RNAs, contamination due to carry-over was minimized. The detection limit of the test kit is 1000 viral copies per ml for HPV 16 and HPV 6, which is in accordance with the required detection limit by the World Health Organization (WHO) and the international HPV reference center [For the detection of HPV DNA, real-time polymerase chain reaction (PCR) was used based on the amplification of the L1 open reading frames (ORF). As an internal control for the availability of cell material, a PCR for the household beta-globin gene was performed in parallel. The HPV DNA was considered positive if the fluorescence signal appeared before the 40th cycle . In the , Italy) . The gene center . Based oe center ,33.® Histology V-Kit, Roche Diagnostics, Basel, Switzerland). EGFR was detected by a rabbit monoclonal IVD certified antibody (clone: A20-E) of Diagnostic Biosystems , PD-L1 was detected by a rabbit monoclonal antibody (clone:E1L3N®) of Cell Signaling Technology , both of these primary rabbit monoclonal antibodies were used in a 1:200 final dilution in a Ventana Discovery staining system. Staining was completed using universal secondary antibody solution, hematoxilin counterstaining, and the DAB MAP Kit . The tumor cell areas were evaluated by one experienced observer. Specimens were judged p16-positive if ≥60% of the cells in tumor areas revealed immunohistochemical reaction products. Staining of fibroblastic stroma cells was not counted. EGFR staining was scored based on previous publications as a positive membrane reaction in tumor cell nests: “0”: no staining, “1”: 1–9%, “2”: 10–49%, “3”: over 50% of the cells positive [Five-micrometer-thick paraffin sections were dewaxed, and antigens were retrieved in an automated staining system . A commercial in vitro diagnostic (IVD) certified assay containing ready-to-use prediluted mouse monoclonal antibody was used for p16 detection (CINtecpositive ,35. PD-Lpositive . Frequency data are tabulated and analyzed with Fisher’s exact test or with the Kruskal–Wallis test for ordered alternatives . To calcIn this study, most patients with initially hr-HPV-positive OPSCC were HPV-negative after treatment, none of these patients experienced a recurrence. The five patients with recurrence or tumor progression were all post-treatment hr-HPV-positive with the same genotype. No recurrence was observed in two post-treatment hr-HPV-positive patients with a different genotype than at diagnosis.


We recently developed a test based on the Agilent SureSelect target enrichment system capturing genomic fragments from 191 human papillomaviruses (HPV) types for Illumina sequencing. This enriched whole genome sequencing (eWGS) assay provides an approach to identify all HPV types in a sample. Here we present a machine learning algorithm that calls HPV types based on the eWGS output. The algorithm based on the support vector machine (SVM) technique was trained on eWGS data from 122 control samples with known HPV types. The new algorithm demonstrated good performance in HPV type detection for designed samples with 25 or greater HPV plasmid copies per sample. We compared the results of HPV typing made by the new algorithm for 261 residual epidemiologic samples with the results of the typing delivered by the standard HPV Linear Array (LA). The agreement between methods (97.4%) was substantial (kappa = 0.783). However, the new algorithm identified additionally 428 instances of HPV types not detectable by the LA assay by design. Overall, we have demonstrated that the bioinformatics pipeline is an accurate tool for calling HPV types by analyzing data generated by eWGS processing of DNA fragments extracted from control and epidemiological samples. Detection and typing of the family of human papillomaviruses (HPV) in samples collected in epidemiologic studies remains an important tool for monitoring the impact of HPV vaccines. These studies provide data assuring that, of the more than 200 types currently recognized, the prevalence of types targeted by the vaccines decreases among those vaccinated appropriately, and that other types do not show an increase (type replacement). Currently, only the most common genital types (less than 40) are identified by commercially available typing assays. The advent of next generation sequencing (NGS) methods raises the possibility that questions relating to type variants, integration status, and the role of additional HPV types could be combined with vaccine monitoring studies. Recently we have developed an enriched whole genome sequencing assay (eWGS), an NGS assay for HPV using Agilent SureSelect and RNA baits covering the entire genomes of 191 HPV types to enrich the fraction of HPV target sequences [phiX174 (accession NC_001422), used as a control in Illumina sequencing runs, and Escherichia coli, an anticipated contaminant in biologic samples.Sequences of 286 HPV genomes, 183 types recognized by the International Committee on Taxonomy of Viruses and 103 candidate types, were obtained from the Papillomavirus Episteme Database (PAVE) in February 2018 , includiWe used short read sequences from eWGS of 383 samples divided into four sets  containiAll samples were sequenced using the earlier published eWGS method . The RNAThe HPV typing pipeline  takes asE. coli), allowing only 1% sequence mismatch per read. These reads were filtered out. Information about reads aligned to the globin gene (2041–3480 segment) that was used as sample control was recorded at this step. The STAR read alignment algorithm  was usedIn the next step, the remaining reads were mapped by the STAR algorithm to reference genomes of HPV types. To account for possible HPV variants, the alignment allowed up to 10% sequence mismatch. To account for sequences that may be split as the circular HPV genome is linearized, we used duplicated sequences of the reference HPV genomes. Reads simultaneously aligned to genomes of several reference HPV types were filtered out.The read pairs from each sample were grouped by their mapped HPV type and each group was analyzed independently using a machine learning pattern recognition algorithm implementing a well-established support vector machine (SVM) approach . For supWe introduced the rate of distinct read pairs to help identify false detection due to index swapping that occurs as a side effect of parallel sequencing of several samples in the same Illumina flow lane . The eWGTo clarify this point, let assume that a total number of read pairs isN1 is the number of unique read pairs, N2- the number of read pairs repeated twice, etc., and kN the number of read pairs repeated the largest number of times—k. Then, the number of distinct read pairs isHere M/N.Now, the rate of distinct read pairs is defined as In what follows when we refer to a number of read pairs mapped to a genome of particular HPV type, we mean the number of distinct read pairs. total number of aligned read pairs mapped to a given HPV genomeaverage depth of alignment of the read pairs to the given HPV genomeaverage coverage of the given HPV genome by aligned read pairsrate of distinct read pairsThe following features were used as input to the SVM classifier:The rate of distinct read pairs for HPV types known to be in the designed and control samples  in Sets 1–4 was largely in the range of 0.1 to 0.6 . TherefoA pattern recognition algorithm must distinguish between reads originating from an HPV type truly present (true positive reads) and those originating from assay noise . The read data from control/defined samples with known HPV type composition are used to determine parameters of the pattern recognition algorithm that best discriminate between true and false reads and retain high sensitivity . The four feature vectors from the whole training data set are divided into positive feature vectors from HPV types known to be present (true positive) and negative feature vectors or those that would identify an HPV type known to be absent . The process of training an SVM method is used to find a separation surface dividing the feature space into positive and negative sub-spaces.Within the 122 samples used in training (from Sets 1–4), 28 were HPV negative while 94 included one or more HPV types resulting in a total of 263 instances of known HPV types. The mapped reads identified 256 true HPV type instances and 1030 false HPV type instances. The seven true HPV instances with no mapped reads occurred in samples with the lowest HPV concentrations. The feature vectors with 256 true and 1030 false labels were used in the classifier training and testing. We used an open source package LibSVM  csie.nt. The SVMFor the radial basis function (RBF) kernel, we had to select two parameters (C and γ). The selection procedure was done by five-fold splitting of the whole feature vector data into training and development sets and running an exhaustive search on a grid in the C and γ space . The C aInterestingly, when the number of the read pairs mapped to the genome of a particular HPV type was above 100, we observed that the value of the depth feature became linearly dependent on the number of read pairs . In addiFor instances of HPV types with 100 or less read pairs the RBF kernel SVM used all four features. The threshold of 100 was to some extent arbitrary as in the vicinity of this threshold both methods worked with about the same level of accuracy (data not shown). For each sample, read pairs mapped to a particular HPV type were processed independently of those mapped to other HPV types and the decision to use 4-feature or 2-feature SVM was made for each type. The optimized bioinformatics pipeline was applied to the 261 epidemiologic samples  previously assayed with LA. For the 37 types detected by LA, type-specific agreement rate and kappa coefficients (k) between LA and the eWGS pipeline results were calculated using GraphPad . AgreemeDetection and typing accuracy of the SVM based bioinformatics pipeline was evaluated in the control/designed samples used for SVM training. In Set 1, prepared with high HPV copy numbers, all HPV types were correctly detected without false positive predictions. In sample 11 from this set containing Caski DNA we observed the highest HPV copy number, resulting in 2,403,579 HPV16 read pairs. Notably, HPV16 was detected in all samples in the same flow cell lane, including negative controls , but were not detected in the second lane of the flow cell. Presence of HPV16 false reads could be explained by index swapping , and theSet 2 included four replicates designed to simulate infection with multiple HPV types per sample and to determine reproducibility and the limit of detection of eWGS assay [The total number of instances of known HPV types in designed samples of Sets 3 and 4 was 52 (38 in Set 3 and 14 in Set 4). The pipeline correctly detected all 52 instances of HPV types . Howeverk = 0.783 . The proportion of positive agreement was 80% and proportion of negative agreement 98.5%. The eWGS results for the 261 epidemiological samples  gave 6848 instances of HPV types by mapped reads. Of these, 1124 instances fell within the 2-feature SVM algorithm and 4217 required the 4-feature SVM . We evalThere were 117 instances in which HPV detected by LA were not identified by eWGS assay pipeline. Among these were 31 instances with no reads and 68 instances with less than 100 reads mapped to the expected HPV type. In the remaining 18 instances in which more than 100 reads were mapped, the SVM algorithm missed particular HPV types because the distinct read pairs rate was slightly above the linear kernel SVM separation line .Among the 133 discordant instances in which the eWGS assay detected an HPV not identified by LA, 42 had more than 500 reads mapped to the HPV type. On the other hand, there were 22 instances in which the eWGS assay detection was based on less than 100 mapped reads. The eWGS assay detected 176 different HPV types, including an additional 428 instances of HPV types not included in the LA assay . The 176Data from Sets 1–3 were previously analyzed manually using threshold values in number of mapped reads, depth of coverage, and percentage coverage . The autThe SVM training on a set of 1000 samples was taking several minutes. This time was insignificant in comparison with total time required for analysis of a set of samples. Calculation of the SVM features for a single sample from raw reads did take ~5 min. Therefore, for example, the time needed for analysis of the whole Set 3 with 64 samples on one CPU is about 5 h.Our results demonstrated that eWGS data generated for HPV typing could be efficiently processed by a new machine learning method that employed an SVM classification algorithm in a fully automated pipeline. The pipeline uses open source tools that will make it easier for others to adopt. Compared with the manual method that relied on simple thresholds , the autThe SVM parameters were developed using eWGS data from 122 defined/control samples used as training sets. The algorithm was initially examined using the defined/control sample data and further validated using eWGS data from 261 epidemiologic samples with prior typing results . The algE. coli, an anticipated contaminant. After mapping the remaining read pairs to the genomes of 286 HPV types those reads that had equal similarity to genomes of two different HPV types were eliminated as well.The Illumina read pairs from the eWGS assay were processed by removing those that could originate from the human genome or i.HPV read pairs from one sample incorrectly assigned to another sample (index swapping);ii.read pairs similar to some HPV genomes but originated from unknown sample contamination.The read pairs that appeared as an input to the SVM algorithm still contained two types of noiseIn our experiments, index swapping was observed to be the main source of noise complicating HPV typing. In a prior published study, the index swapping (bleeding) rate in multiplexed sequencing experiments was estimated to be ~0.3% , similarHPV3 was not used in any design/control samples but was the most frequently detected among HPV types that were not supposed to be present. The same HPV3 sequence, with characteristic single nucleotide polymorphisms, was present in epidemiological samples. The presence of HPV3 as a contaminant is likely. While most false positive results in control samples could be explained by index swapping, others could be due to sample contamination by extraneous reads with similarity to parts of some HPV genomes.Interestingly, the number of read pairs larger than ~300 per HPV genome was sufficient to cover and assemble the full genome . In such


The biological importance of human papillomavirus (HPV) in the field of medicine – related to cervical carcinogenesis – has been extensively reported in the last decades. For the first time, a direct correlation between cause and effect to explain a cancer development was completely achieved in medical research. Consequently, the Nobel Prize was awarded to HZ Hausen in 2008 for his efforts to understand the effects of persistent infection of oncogenic types of HPV and malignancy transformation. The aim of the present review was to summarize the principal elements of HPV characteristics and their importance in oncology. It is established that HPV is the main etiologic agent for the development of cervical cancer. With the evolution of diagnosis and molecular biology, many tools have become essential for an early diagnosis and thereby, considerably reducing mortality. Molecular biology continues to advance and provide new perspectives with the use of reverse-transcription PCR in automation and genotyping through next-generation sequencing. This article aims to provide an overview of what is currently used in HPV diagnostic and research and future perspectives with the help of technologies such as next-generation sequencing for screening and vaccination. Papillomavirus are a large family of virus that infect animals and humans. The most studied and known papillomavirus is the human type. There are more than 150 types of human papillomavirus (HPV) . HPV is HPVs are phylogenetically grouped in five different genera: α-, β-, γ-, mu- and nu-HPVs . The alpThe high-risk HPVs (HR-HPVs) have been classified as an oncogenic risk by the International Agency for Research on Cancer , and comprise the types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. Within these, type 16, followed by type 18 are the most oncogenic . Most HPHPVs classified in the beta group includes 54 HPV types, which have skin cell tropism and are responsible for most types of benign skin lesions and warts . In someThe gamma subgroup includes the majority of the known HPVs, with 99 types and even though presenting the same β-HPV biology and infection mechanism, their biological activities are poorly studied and deserve more investigation. They infect human skin and mucosal tissues of the oral cavity and its seroconversion appears to be slow and to increase with age ,15. The The HPV biological cycle begins as a microtrauma in the mucosa, which allows the virus infection into basal cells of the epithelium. In approximately 3 weeks, it is already possible to detect HPV DNA copies in the basal epithelium as circular DNA, the episomal form. The HPV can develop strategies to avoid immune surveillance, especially the high-risk types .During the basal cell differentiation into epithelial cells, HPV carries many copies of DNA in episomal form in the cell cytoplasm of the cells ,14. At tThe evolution of the virus requires a host to allow DNA replication and viral DNA production . In a seE1, E2, E4, E5, E6 and E7, which carry out regulatory functions [L1 and L2 genes and encode the major and minor capsid proteins, respectively [L1 and E6 genes, which is involved in controlling the gene expression and replication in the host cells [The HPV genome is divided into three different regions based on functional properties and location. The ‘early’ genes (E) comprise the group of genes unctions ,4. The ‘ectively . The L1 ectively ,4. A thist cells ,23. In tst cells .E1 and E2 genes are responsible for promoting the integration of the HPV genome in human DNA. Through these regions, the HPV has the ability to evolve and progress during the life cycle. E1 binds to E2 forming a dimer of hexamers, when connected, they are able to control viral replication by controlling the host's cellular DNA replication mechanism and inducing the transcription of HPV DNA virus. It is important to draw attention to the fact that the integration of HPV is not always constant, it is necessary that E2 methylation takes place, to found increase of E6/E7 expression [In the early region, the pression ,24.In addition, both E4 and E5 are involved in the early stages of HPV viral replication cycle . Recent E6 and E7 are the last genes to be activated by stimulating cell growth, inhibition of differentiation and inducing chromosomal instability. The HPV E6 and E7 DNA integration allow the development of a new tumor, which is going to proliferate and invade new tissues [ tissues ,25,26. BL1 and L2 genes, are resposible to produce HPV nonenveloped capsid [The late region, composed by d capsid . HPV infd capsid ,27. Onced capsid ,24. L2 iE6 and E7 genes during HPV genome replication [E6 and E7 oncogenes modulate important signaling pathways – from the host cells cell cycle, to apoptosis and immortalization – allowing viral replication and persistence and becoming the only viral genes that are always retained in cancer cells [After the infection, HPV can integrate into the human genome. The E2 protein loses part of its region and also its function, by controlling lication . The E6 er cells ,30.E6 and E7 oncogenes, which are able to integrate into human DNA inducing molecular changes promoting the neoplasm development [In the development of cervical cancer (CC), the most important HPV-related human cancer, some genes are downregulated, while others are upregulated by HPV elopment .E6 and E7 oncogenes. Tumor suppressor p53 is a regulatory gene that is involved in diverse cellular processes such as, apoptosis, DNA repair and cell cycle. The E6 and E7 oncogenes are capable of inactivating this gene, allowing tumor development [The protein p53 is one of the main affected by elopment . Specifielopment ,32.Rb [Another gene that is also important to HPV carcinogens is Rb . The Rb Rb .TERT is another important oncogene. It encodes the catalytic subunit of telomerase enzyme and is crucial to maintenance and regulation of the length of the telomeres [The elomeres ,35. In nelomeres . Telomerelomeres ,37.CC is the most important HPV-related human cancer. The association of HPV in CC was first described by HZ Hausen in 1977, for which he was awarded the Nobel Prize in Physiology and Medicine in 2008 . CC is tUsually, HR-HPVs cause transient infections that are eventually eliminated over a period of several months by the immune system. If some genetic changes, discussed above, occur in the viral genome and in the infected host cells, a transient infection may be persistent . If not ®, which is a registered trademark of the GlaxoSmithKline  group of companies and targets HPV types 16 and 18, which are most common HR-HPV causing CC; quadrivalent, produced by Merck Sharp & Dohme Corp.  with a commercial name Gardasil®, which in addition to preventing HPV 16 and 18, also immunizes against HPV 6 and 11; and more recently, the Gardasil expanded, a nanovalent type, which targets the quadrivalent HPV types, plus the types 31, 33, 45, 52 and 58 [Vaccination is considered the main frontline program to CC prevention, as primary care . Three t2 and 58 ,18. Immu2 and 58 .The secondary prevention strategy for CC is based on screening strategies to detect CIN alterations. The WHO recommends that screening for CC should be performed at least in the target age group (30–60 years), through cytology and visual inspection (VIA) with the use of acetic acid. In women, who test negative on cytology or VIA, the screening interval should be every 3–5 years. In women, who test negative on an HPV test, rescreening should be done after a minimum interval of 5 years .After the long application of pap smear and histology, new technologies to assist in the screening for CC have emerged. Before real-time PCR be applied on a large scale for diagnosis, hybrid capture was a very important tool for HPV screening. It is a technique developed with the advent of molecular biology capable of detecting the presence or absence of HPV virus DNA from low- and high-risk groups using chemo luminescent substrate to produce light, an important step toward the diagnosis with more specificity when compared with conventional cytology .® 4800 platform  [HPV typing becomes an important tool in primary screening for further clinical management. Through available technologies capable of genotyping HPV, it is possible to treat patients from another therapeutic perspective. The challenges are still being overcome, mainly in countries with limited financial resources and health policies . DespiteCA, USA) , a systeCA, USA) , becauseThe first platform widely used and applied on a large scale for the detection of the HPV virus using real-time PCR technology was Cobas 4800. It has completely autonomous sample preparation with real-time PCR technology, for amplification and detection of HPV, developed by Morris and Nightingale, later commercially explored by Roche . The tesAdditionally, several studies performed on commercially-available HPV DNA detection assays have determined that HPV assays do not detect the same infections in women undergoing primary screening: approximately 30–50% of positive cases show discordance in the specific HPV infection detected. The Validation of HPV Genotyping Tests is an important framework, its function is to validate the HPV assays available on the market, through Validation of HPV Genotyping Tests clinical protocols and trials and be considered safe and valid for the clinic and diagnosis, minimizing the differences from one protocol to another .E6 gene in 74.4% of the samples, when compared with the old sequencing deposited in GeneBank [Therefore, the variation in percentage between one HPV molecular test and another may not only be related to analytical or technical aspects, but also to the viral target sequence selected to detection. According to Galati and collaborators, that studied variations in HPV 16 sequencing in cervical samples, it was possible to observe a significant difference in single nucleotide polymorphisms (SNPs) in the GeneBank . These fGeneBank .Modern DNA sequencing methods have enabled complete genomic characterization of cancers on an unprecedented scale. The so-called ‘next-generation sequencing’ (NGS) is enabling the simultaneous analysis of millions of fragments of DNA. The full HPV genotyping is one of the key points of NGS: to detect other genotypes that may increase oncogenic potential in women protected against HPV 16 and 18 infections (through vaccination) .It is known that HPV detection assays based on NGS are capable of detecting different variants of oncogenic HPVs. A set of novel sequencing platforms, such as from Illumina  and Thermo Fisher Scientific  completely transformed the scenario, by allowing extensive throughput, while greatly reducing the necessary time and cost of any sequencing endeavor. In the face of increasing migration, these insights will aid identifying high-risk populations for CC screening be more effective. In addition, with NGS assays it is possible to select HPV DNA sequences with maximum sensitivity and specificity for the 14 high-risk genotypes .In this scenario, it is possible to compare conventional cytology and HPV testing. Conventional cytology has less of an automated management, requiring a trained professional to analyze samples microscopy, and therefore, classify the lesion of the squamous epithelium according to the smear on a slide. HPV testing has been gaining more space, because it’s easier handling in diagnosis with automation methods and precision in the identification of oncogenic types of HPVs in possible lesions, leading to a faster and more effective process .New technologies are being used more and more with a greater approach within research. The market follows this evolution and has modernized and developed technologies, such as real-time PCR, making it accessible to anyone.With the advancement of the application of NGS technology, it is possible to increase the reach and access diverse populations in an effective way, mainly in the early detection of HPV, in which the economic factor is closely linked to prevention. The present study has a perspective that the increasingly advanced technology is a potential ally in prevention, designing the sequencing of all types of high-risk HPV and also in its subtypes. Extrapolating in addition to CC lesions, it is known that persistent HPV infection in other organs and tissues can also develop other types of tumors.E6 and E7 genes, it opens a new perspective for the production of new vaccines for HR-HPV and will become essential in monitoring both vaccinated and unvaccinated women. In addition, an increased of quality in CC screening by the design of potential rapid and efficient molecular tests may improve the detection of CINs and decrease the mortality rates from this type of cancer.With the advancement of research on HPV genotyping, mainly in the Different NGS assays applicable to HPV genotyping are under development. This includes the use of different primer design, different NGS platforms and different bioinformatics analysis . In addi


The presence of and the causative role of high‐risk human papilloma virus (HPV) is a subject of controversy in oral squamous cell carcinoma (OSCC). The disagreement can be related to the misclassification of OSCC as oropharyngeal squamous cell carcinoma and/or lack of standard detection methods. This study aimed to examine the presence of transcriptionally active high‐risk HPV in a homogenous Norwegian cohort of primary and second primary OSCC of the mobile tongue .Tissue microarrays containing formalin‐fixed and paraffin‐embedded cores of 146 OTSCC from the anterior 2/3 of the tongue (n = 128 primary and n = 18 second primary) from a multicentric Norwegian cohort were examined for the presence of high‐risk HPV by DNA‐ and RNA‐in situ hybridization (ISH) assays and p16 immunohistochemistry.E6/E7 mRNA) was not identified in any of the OTSCC specimens. In parallel, no tumors were positive for HPV by DNA ISH. Although, 61 (42%) OTSCC demonstrated p16 positivity with varying staining intensity and subcellular localization, only two cases demonstrated strong and uniform p16‐staining (both cytoplasmic and nuclear) in >70% of cancer cells. The absence of transcriptionally active high‐risk HPV in this cohort of OTSCC indicates that high‐risk HPV is an unlikely causative factor in the present material.Transcriptionally active HPV ( Here, squamous cell carcinoma (SCC) accounts for the majority of malignant tumors  technique has become increasing popular and it allows direct visualization of viral transcripts in routinely processed tissues, thereby reflecting the active HPV infection  is the primary etiological factor in oropharyngeal squamous cell carcinoma (OPSCC) in the Western world  in OTSCC.The current work represents a sub‐study of a joint initiative  between the four University hospitals in Norway treating OSCC . In this multicenter study, all conventional OSCC cases diagnosed between January 1, 2005 and December 31, 2009 at the four Norwegian university hospitals treating head and neck cancer  were retrospectively identified. Using ICD‐10 codes (C02‐C06) except for codes C05.1 and C05.2 , a total of 608 OSCC patients were identified from the electronic health record. Two hundred and seventy‐three OTSCC with clinical data were identified. Of the 273 OTSCC, 146 (128 primary + 18 second primary) were included in this study . For scanned images with inadequate focus, the original glass slides were examined by a Leitz Aristoplan microscope.Tissue microarrays were constructed from formalin‐fixed, paraffin‐embedded (FFPE) tissue blocks in a fully automated tissue microarray machine . Two to four tissue cores  with a diameter of 2 mm were arrayed on the recipient paraffin blocks. The stained TMA‐sections were scanned  and evaluated using the CaseViewer™ software (2.3Immunohistochemistry (IHC) was performed on TMAs on a Ventana Benchmark Ultra automated immunostainer , using a mouse monoclonal antibody clone E6H4 . Bound antibody was detected by the biotin‐free ultraView Universal DAB Detection Kit (VMS #760‐500). A known p16‐expressing OPSCC was used as a positive control. Sections incubated with phosphate buffer saline (instead of primary antibody) and with isotype‐matched control  were used as negative controls. For details, see 2.4Automated in situ hybridizations were carried out on TMAs on a Discovery XT (VMS) instrument using Research ISH UltraMap XT procedure and Ventana products (INFORM HPV III Family 16 Probe (B), #800‐4295). For details, see Blinded for the clinicopathological data, the HPV DNA ISH results were scored by TMS and DS. The in situ results were interpreted following the manufacturer's guidelines (Interpretation Guide for Ventana INFORM HPV Probes ISH). Any blue nuclear dots in the tumor cells were regarded as positive staining and all of the samples were classified binary as either positive or negative.2.5Automated RNA in situ hybridizations were carried out on a Discovery Ultra  using the fully automated RNAscope VS HRP assay . Standard protocols were used for the deparaffinization followed by heat pretreatement using Discovery CC1 and mRNA sample prep protease treatment. For details, see Blinded for the clinicopathological data, the HPV RNA ISH results were scored by TMS and DS. A positive HPV test result was defined as punctate staining that localized in the cytoplasm and/or nucleus of malignant cells. The RNA ISH staining was scored according to advanced CELL diagnostics guidelines, as described in the 2.6Descriptive statistics , where applicable, were calculated for continuous and categorical variables using the Statistical Package for the Social Sciences (spss) 26.0 for Windows .3This study adheres to the REMARK criteria  OTSCC showed p16 positivity with varying staining intensity and subcellular localization  for routine diagnostics of HPV‐related oropharyngeal squamous cell carcinomas/head and neck carcinomas . However, both of the p16 positive OTSCC were negative for HPV DNA and RNA ISH. This suggests that the p16 expression in those cases might be related to non‐HPV mechanisms and supports the view that p16 is not a reliable surrogate marker for HPV in OTSCC . This is an interesting observation since p16 positive OPSCC usually are non‐keratinizing. However, another non‐keratinizing carcinoma included in this study was p16 negative.A general increase in the incidence of OTSCC has been reported globally with a shifting trend toward female and/or younger patients with OTSCC  diagnosed from 2005 until 2010 were found to be positive for high‐risk HPV. In parallel, only two OTSCC were p16 (Score 3) positive. Our results suggest that high‐risk HPV is an unlikely causative factor in the present material and will not influence future biomarker studies utilizing the current material.None declared.Appendix S1: Supporting InformationClick here for additional data file.


Squamous cell carcinomas of the head and neck are the subject of numerous current studies, especially in view of the increasing incidence of tumors induced by human papillomavirus (HPV) and the latest changes to the TNM classification of oropharyngeal squamous cell carcinoma (OPSCC). In addition to HPV status, the presence of extranodal extension of lymph node metastases represents an important risk and prognostic factor, which has now been integrated into the staging algorithm of the eighth edition of TNM classification for HPV-negative OPSCC. In the past numerous studies had shown a lack of prognostic significance of extranodal extension in HPV-associated tumors. However, extranodal extension–as a possible risk factor even in HPV-positive OPSCC–remains an important subject of current studies, which are now particularly characterized by high numbers of cases. In this paper, diagnostic methods and the prognostic significance of extranodal extension in surgically treated HPV-positive OPSCC are presented and discussed based on relevant literature, and the results of current publications are summarized. Further development of diagnostic criteria and procedures as well as international standardization of clinical diagnostics of extranodal extension should be encouraged. Several studies demonstrate that extranodal extension results in worse survival outcomes even in HPV-positive tumors, in contrast to results of previous studies. Consequently, whether the prognostic significance of extranodal extension is not actually relevant to outcome and the staging algorithm of HPV-positive OPSCC should be questioned and further analyzed. The role of HPV in OPSCC has gained a great deal of attention in recent years. In addition to its causative role, HPV infection also proved to have a clear prognostic value . With thp = 0.048) (p = 0.002)  . By mean= 0.048) . Patient= 0.002) . In addi= 0.002) .Although these subcategories have not yet been applied for the purpose of pN classification, they are recommended by the AJCC for data collection and analyses and find application in recent studies . In addiIn addition to the widely used diagnostic methods involved in postoperative histopathologic examination, various imaging techniques can be applied, such as ultrasound, magnetic resonance imaging (MRI), and computed tomography (CT). Clinical diagnosis of ENE of lymph node metastases presupposes that clear, defined criteria are met . ClinicaFor a long time there have been strong demands for standardization and further development of investigation methods and internationally recognized diagnostic criteria for the imaging modalities of ultrasound, CT and MRI . Only rep < 0.01; 95% CI = 0.64–0.68)  . HararahFurther studies and more prolonged use of the new TNM classification are required to show how far the new clinical N classification, or particularly the clinical diagnosis of ENE defined therein, can succeed in everyday clinical practice and result in reliable identification of ENE status or whether new diagnostic methods will prevail in future. Understaging or upstaging of patients not surgically treated should be avoided where the prognostic influence of ENE is proven. Improving the modalities for clinical diagnosis of ENE and standardizing ENE diagnosis in general will thus continue to be the objective over the next few years.PubMed. Using the PubMed Search Builder the following search term was created: ((((((((((extracapsular spread[Title/Abstract]) OR perinodal spread[Title/Abstract]) OR transcapsular spread[Title/Abstract]) OR extranodal spread[Title/Abstract]) OR extracapsular extension[Title/Abstract]) OR extranodal extension[Title/Abstract]) OR perinodal extension[Title/Abstract]) OR transcapsular extension[Title/Abstract])) AND (((hpv[Title/Abstract]) OR human papilloma virus[Title/Abstract]) OR p16[Title/Abstract])) AND (((((head and neck squamous cell carcinoma)) OR hnscc) OR oropharyngeal squamous cell carcinoma) OR opscc). The process of literature research is illustrated in The aim of this paper is to provide a structured overview of current study results on the topic “Prognostic influence of ENE in surgically treated HPV-positive OPSCC.” In order to investigate a possible prognostic influence of ENE in HPV-positive OPSCC, a literature research was conducted with Before screening the records, the following inclusion criteria were defined: (a) OPSCC, (b) patient collective contains HPV-positive tumors, (c) surgically treated collective/Neck Dissection (with or without adjuvant therapy), (d) ENE-Status available, (e) statement on the prognostic influence of ENE, (f) original research paper. Full-text articles assessed for eligibility were screened for further relevant publications. The publications included in analysis were screened for their study results referring to the prognostic impact of ENE in HPV-positive OPSCC. The relevant results as well as characteristics of the studies and number of cases are shown in Numerous studies in the past have shown that the presence of ENE additionally worsens the prognosis of patients with HNSCC , 46–50. Furthermore, the correlation between ENE and locoregional recurrence and distant metastases has been studied in recent years. For example, Myers et al. showed in their publication of 2001 on 266 patients with squamous cell carcinoma of the tongue that ENE was the most significant prognostic parameter for the risk of regional recurrence and distant metastases in their population (p < 0.001)  .n = 73, adjuvant CRT: n = 69), they published a lack of significant impact of ENE on overall and disease-specific survival  , one of their findings was that the number of lymph node metastases (≥5)—but not ENE—was an independent prognostic factor  . In 2015n = 101 patients–postoperative radiation therapy: n = 100, postoperative chemotherapy: n = 44)  , and disease-specific survival (DSS) in surgically treated HPV-positive OPSCC ( n = 44) . In fact n = 44) . In thei n = 44) . In 2015 n = 78) .n = 111, adjuvant chemotherapy: n = 40).In this connection Maxwell et al. reached the following conclusion in their 2013 publication : In thein = 139, 65.5% HPV-positive) was also the purpose of the study by Klozar et al. published in 2013  (p = 0.0021) and advanced T-classification represent significant predictors of survival in HPV-negative surgically treated OPSCC (most patients treated with adjuvant RT/RCT based on NCCN guidelines). While ENE nearly reached significance in the univariate analysis of HPV-positive OPSCC (p = 0.0553), multivariate analysis revealed that ENE was not significantly associated with survival (p = 0.7644)  with neck dissection as part of primary definitive treatment—also confirmed that ENE is not a negative prognostic factor for HPV-positive OPSCC (in respect of RFS and DSS) . Kaczmar et al. showed that ENE does not correlate with increased risk of local as well as distant progression in HPV-positive OPSCC  in univariate analysis  , DSS, and OS] (n = 138) . In addin = 138)  showed t 0.7644) . Kharytaand DSS)   . Table 1p = 0.008)  .p = 0.012; ENE not included in the multivariate analysis) T: n = 67).The univariate analysis of the study by Meyer et al. , which en = 642, surgery + RT: n = 1,005, surgery + CRT: n = 1,773) (p < 0.001) (p = 0.004); pN2: ENE-negative 88%, ENE-positive 77% (p = 0.061)]  for the years 2010–2014  . As well< 0.001) . Upon st 0.061)] .n = 306, adjuvant CRT: n = 498). Patients who met the defined inclusion criteria were identified via the NCDB for the years 2010–2012 (p = 0.01) (p = 0.046) , An et al. do not question the cut-off of ≥5 lymph node metastases as a prognostic parameter for HPV-positive OPSCC. However, they do advocate evaluation of both parameters as potential prognostic factors—especially since a higher number of lymph node metastases is associated with the presence of ENE with a greater probability  . No sign = 0.01) . Further= 0.046) . Hence t= 0.046) . For HPV= 0.046) . Given cbability .p = 0.033) was observed  (p < 0.001) with 5-year overall survival of 92.6% (95% CI = 90.5–94.7%) for negative ENE status compared to 84.0% (95% CI = 80.7–87.4%) for ENE-positive tumors (p < 0.001). The pathologic N stage—or hence the number of positive lymph nodes—was significantly associated with patients' outcome (pN2 vs. pN1: HR = 1.53)  in comparison with N1/ENE-negative OPSCC (HR = 1.00) (p = 0.002) as ENE-negative pN1 tumors. Bauer et al. thus concluded in the course of their evaluation that ENE is prognostic irrespective of the number of positive lymph nodes and that the combination of ENE status and number of positive lymph nodes (pN category) particularly leads to an improved picture of mortality risk  . The unin = 1,262, adjuvant CRT: n = 1,501, unknown: n = 78) (p < 0.001) and the multivariate analysis , the presence of ENE is associated statistically highly significantly with a deterioration of overall survival in HPV-positive tumors and it should be included in future staging algorithms  . In theigorithms .p < 0.0001)  (p = 0.033), tumor-specific survival (p = 0.165), progression-free survival (p = 0.42), and DFS (p = 0.04)  . Among o 0.0001) . In addi: n = 1) . Their m = 0.04) . The res = 0.04) . Further = 0.04) .As a whole the results described above contrast with the conclusions of the 2016 review by Mermod et al. which included an analysis of the prognostic significance of histopathologically proven ENE in HPV-positive OPSCC , An et aThe possible influence of tobacco consumption of patients was not analyzed because of the lack of recording in the NCDB. The influence of nicotine consumption on the risk and prognostic profile of HPV-positive OPSCC, however, is a relevant parameter according to the results of Ang et al.  and shouAll in all, the current study results from various publications presented here emphasize that ENE is a risk and prognostic factor, including HPV-positive OPSCC, which to date has not been integrated into the staging algorithm of the TNM classification. Possible reasons for the different results of the studies mentioned are discussed by Bauer et al. , An et aexplicitly focus on this issue, as well as partly limited comparability, we did not choose a systematic review, but a structured review according to the PRISMA guidelines. We performed our literature research as structured and traceable as possible. Furthermore, no quality assessment of the included studies was carried out. Despite the focus on surgically treated collectives, it can be assumed that the therapy algorithms  vary to a certain extent from institute to institute, which influences the comparability of the studies. As histopathologically proven ENE is an accepted risk factor in HNSCC in general and de-escalation strategies in HPV-positive tumors have not been integrated into clinical practice, the majority of patients of all discussed studies should have been treated by adjuvant radiotherapy. As a topic that is currently gaining more and more interest, a future meta-analysis—including possible additional publications of the next months/years—should be considered.Limitations of this work are that only studies that explicitly examined ENE in HPV-positive OPSCC  were included—therefore other possibly relevant research results, which were not identified through literature research or references, could have been missed. The aim of this publication was to provide the reader with an overview of the current status of research in this field in a structured form. Due to a limited number of studies that positive tumors in the TNM classification system should be reevaluated in detail in the context of prospective multicenter studies. According to the study results presented here, it also seems necessary to record ENE in the tumor documentation for HPV-positive tumors. Furthermore, it is also the extent of ENE  that should be examined and documented as it represents an additional prognostic factor. The methods applied  in the clinical diagnosing of ENE have limited sensitivity and specificity. In this regard initial attempts at computer-aided analysis of image data should be pursued. Furthermore, clinical diagnostic criteria should be standardized overall at the international level.Whether the prognostic influence of ENE of lymph node metastases can actually be ignored for HPV-JK came up with the idea and planned the manuscript. AB and JK collected the data. AB drafted the manuscript and performed the systematic review and its analysis. AB provided the original data presented. SZ and KE provided expert opinion on histopathology and high resolution histopathologic pictures. IM helped with editing the manuscript. GP, CB, and JK reviewed the manuscript. All authors finally checked the manuscript and provided critical review of its content.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.


Male infertility is responsible for about 50% of the cases, and the various causes of male infertility may be classified in pre-testicular , testicular, and post-testicular  causes. Sexually transmitted infections (STI) are increasingly widely accepted by researchers and clinicians as etiological factors of male infertility. In particular, several recent reports have documented the presence of HPV in seminal fluid and observed that sperm infection can also be present in sexually active asymptomatic male and infertile patients. In this review, we aimed to perform a systematic review of the whole body of literature exploring the impact of HPV infection in natural and assisted fertility outcomes, from both an experimental and a clinical point of view. Starting from  Through immunofluorescence techniques, some authors clarified both the mechanism of sperm-HPV binding and its exact localization (Human Papillomavirus (HPV) is the etiological agent of the most common sexually transmitted infection worldwide, with an estimated 6.2 million new cases annually . HPV comlization , 13. Thelization , 14–16.in-vitro studies reporting the possible role of HPV infected spermatozoa on blastocyst development and trophoblastic invasiveness. Moreover, we summarized the diagnostic and therapeutic strategies available in infertile couples aimed to improve the reproductive outcome.The first aim of this review was to summarize the implications of HPV semen infection on the following topics: i) effect on sperm parameters, ii) development of anti-sperm antibodies, iii) impact on both natural and assisted reproductive outcomes, such us pregnancy rate and miscarriage rate. We also discussed in vitro studies focused on the effects of HPV infection on oocyte fertilization, blastocyst development, and trophoblastic cell invasiveness. Also, studies describing the adjuvant administration of the HPV quadrivalent vaccine Gardasil  as a possible strategy to promote HPV clearance from semen in infected males, were included. An accurate analysis of the references of the main works was successively performed. We considered data from eligible studies separately, according to different topics: “HPV and impairment of sperm parameters,” “HPV and development of anti-sperm antibodies,” “HPV and impairment of natural and assisted fertility outcome.” Manuscripts were selected according to the use of the following methods: semen parameters and the related alterations were defined according to WHO laboratory manual for examination and processing of human semen (in situ hybridization (FISH) techniques. The evaluation of apoptotic events, related to HPV, in spermatozoa and embryo was detected through a terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay (TUNEL) test, DNA-Comet assay, DNA Disk chip assay, and Cell Death Detection ELISA assays or SCSA (flow cytometric technique). Sperm washing procedures relied on two-layer isolate colloid wash, test-yolk buffer procedures, swim-up procedure, modified swim-up with enzymatic treatment  and discontinuous Ficoll gradients. Foresta et al. , sperm apoptosis, sperm DNA alteration and infertility. In the included studies, all known causes of male infertility had been ruled out. We also included experimental an semen  and Hamian semen ; HPV-DNAa et al.  used the(PRISMA) flow diagram which shows the process of study selection. The reported evidences ranged between 1997 and 2020. Some papers evaluated more than one topic. Among these manuscripts, 14 papers focused on the clinical impact of HPV detection in semen related to the alteration of sperm parameters    suggesteIn addition to the possible impairment of sperm parameters, different kind of semen infections have been associated with the development of anti-sperm antibodies (ASAs). Also HPV semen infection, has been recognized as a risk factor for the ASAs , 41. In Due to its ability to affect seminal parameters and to induce ASAs development, HPV semen infection seems to be a significant risk factor for male infertility. In this regard, many original studies, reviews and meta-analyses reported that HPV semen infection is related to reduced fertility both in natural and assisted conceptions. However, the exact mechanisms responsible for this condition have been never definitely assessed , 44, 45.vs. 15%) (vs. 15%) . On the vs. 15%) , 36. Howin-vitro studies have shown a negative influence of HPV infection upon several aspects of human fertility. For example, Gomez et al., in 2008, reported that HPV transfected blastocyst and trophoblastic cells were affected by a reduction in decidua invasion capacity, potentially responsible for a failure of maternal uterine wall invasion by trophoblastic cell, subsequent placental dysfunction and adverse pregnancy outcome (in-vitro fertilization). To better understand this process, Foresta et al. performed an in-vitro study evaluating the ability of the virus- infected sperm to transfer HPV DNA and capsid proteins to oocyte during fertilization. After transfecting a human sperm with a plasmidic episome containing HPV E6 and E7 proteins, they used the hamster egg-human sperm penetration test to show the ability of infected sperm to transfer the capsid protein L1 to oocyte and the expression of E6 and E7 viral protein in the fertilized oocyte , asthenozoospermia, presence of ASA, positive medical history for HPV infection or evidence of ongoing HPV-related diseases. From the above reported literature, we can conclude that HPV semen infection can impair sperm quality and induce ASAs development, thus reducing couple fertility, particularly when viral DNA is present on the sperm surface. The impact of HPV genital tract infection in females is less clear, however the presence of the virus in embryos has been related to reduced pregnancy rate and increased abortion rate.via assisted reproduction.In the present systematic review, we summarize the state of the art on the link between HPV infection and couple infertility and suggest a flow-chart for the management and counselling of infertile couples in whom the male partner has HPV semen infection. The aim of this clinical management is to ensure the best reproductive outcome, both natural and through ARTs, particularly in those men without any other factor explaining infertility other than the presence of HPV. In the light of the large body of literature showing that HPV semen infection has a negative impact on reproductive outcome, we are strongly convinced that the time has come to follow this path. Further studies are required in order to demonstrate that the described strategy and, in particular, the clearance of HPV from semen is able to improve the delivery rate of infertile couples unable to conceive either naturally or The present review comprehensively summarizes, in a systematic way, the results of studies analyzing the correlation between HPV semen infection and male idiopathic infertility and provides the state of the art on the diagnostic and therapeutic strategies available in infertile couples aimed to improve the reproductive outcome. The main limitation of this analysis is that many of the studies, included in this review, did not consider important factors such as infertility duration and age of the female partner. Moreover, not all the studies clearly ruled out all known causes of infertility.AG, FM, and CF designed the study. LT, FM, GG, and AC collected the data. LT and FM interpreted the results and drafted the report. 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.