High coverage of human papillomavirus (HPV) vaccination is key to the eventual elimination of infection and cervical cancer. Monitoring coverage, in a digital vaccine registry, is therefore essential to detect problems. For example, misinformation about dangers from HPV vaccine generated in Denmark a decline in coverage among girls age 12–17, from 80% in 2013 down to 37% in 2015, when remedial actions made it rise again. The availability of human papillomavirus (HPV) vaccines and screening tests has raised the possibility of globally eliminating cervical cancer, which is caused by HPV. Cervical cancer is a very common malignancy worldwide, especially among deprived women. High vaccination coverage is key to the containment and eventual elimination of the infection. Public HPV vaccination programmes in Italy and Denmark were swiftly established and are among the most successful worldwide. Still, in both countries, it has been challenging to achieve and maintain the recommended coverage of > 80% in girls. In a well‐studied Italian region, vaccination coverage in girls at age 15 years  reached 76% in 2015 but decreased to 69% in 2018, likely due to work overload in public immunization centres. In Denmark, doubts about safety and efficacy of the HPV vaccine generated a decline in coverage among girls age 12–17, from 80% in 2013 down to 37% in 2015, when remedial actions made it rise again. Insights from these two countries are shared to illustrate the importance of monitoring coverage in a digital vaccine registry and promptly reacting to misinformation about vaccination. CCcervical cancerFVGFriuli Venezia GiuliaHICshigh‐income countriesHPVhuman papillomavirusLMICsmiddle‐income countriesWHOWorld Health Organization1Chlamydia trachomatis, vast human and molecular evidence led to the recognition of human papillomavirus (HPV) as a necessary although not sufficient cause of CC [Many studies throughout the twentieth century suggested that cervical cancer (CC) was related to a sexually transmitted infectious agent. After considering different ‘candidates’ including herpes simplex virus and se of CC . HPV wasIn less than two decades, powerful tools to screen for and prevent HPV infection became available. Three highly efficacious and safe prophylactic vaccines against HPV have been released since 2006, all targeting at least the two most carcinogenic types of the HPV family, that is HPV16 and HPV18 . GreatesThe concurrent availability of vaccines and viral screening tests has raised a real possibility of globally eliminating CC, a very common malignancy among underprivileged women in high‐income countries (HICs) and in the general female population in many low and middle‐income countries (LMICs) , 3. In rHere, we focus on two important issues that can threaten the success of HPV vaccine programmes even in HICs with well‐established and free‐of‐charge immunization programmes run by public national health systems. We will specifically address (a) failure to reach and sustain an adequate coverage, and (b) damage from misinformation about the safety or the efficacy of HPV vaccines. While the Italian case will mainly illustrate the problem of achieving and sustaining high and equal coverage in the target population, the Danish case will be discussed in respect to impact of misinformation about vaccine safety and the subsequent remedial action taken by the Danish Health Authority, the Danish Medical Association and the Danish Cancer Society. We will also emphasize the global importance of these challenges.22.1The implementation of HPV vaccination in Italy , 7, 8 an2.2In Denmark, routine HPV vaccination began in 2009 to girls aged 12 (first birth cohort: 1996). At the same time, girls aged 13–15 (born 1993–1995) received a catch‐up offer free‐of‐charge from October 2008 to the end of 2010. Young women born 1985–1992 were offered the vaccine from September 2012 to December 2013 . InitialIn both Italy and Denmark, the number of doses of HPV vaccine recommended in individuals below age 15 were decreased from three to two in 2014, on account of the demonstration of noninferiority .33.1High coverage is key to the containment and eventual elimination of an infection for which an effective vaccine exists. Coverage does not need to be 100%, but it requires that the vast majority of a population is protected either directly (by being vaccinated) or indirectly (due to the decline of the circulation of an infection following mass immunization). In order to stop the rapid spread of air‐borne infections among entirely susceptible children (e.g. measles), 95% coverage is recommended. For a sexually transmitted virus‐like HPV, adequate coverage may be a little lower at around 80% .Vaccination for HPV had been introduced in more than 100 countries by the end of 2019. World Health Organization (WHO) surveillance data for 75 countries  . HoweverNational HPV programmes that use call/recall system, for example, those in Italy, Denmark, the UK and Australia, typically invite vaccine‐eligible adolescents by birth cohort and, accordingly, monitoring coverage is most commonly done by birth cohort , 8. Whenth birthday (hereafter referred to as ‘at 15’), assuming that each targeted birth cohort has had the opportunity to complete vaccination by that age [For this reason, WHO recommends monitoring coverage in adolescents up to the day preceding their 15that age . Only Authat age  and a fe3.3The following data offer an example of the usefulness of monitoring coverage at age 15 based on data from Friuli Venezia Giulia (FVG), a region in North‐eastern Italy with about 1.2 million inhabitants, excellent health‐quality indicators, and a good digital vaccination registry. By the Full‐course (≥ 2 doses) coverage in girls at age 15, however, reveals a different picture. Coverage rose from 42% in 2009 to 72% in 2012 and 76% in 2015 but declined to 69% in 2018 [3.4All HPV vaccines consist of highly purified virus‐like particles that consist of protein shells of the HPV virus. They contain no viral DNA and, therefore, cannot cause infections or disease. In addition, the safety of HPV vaccine has been thoroughly evaluated in clinical trials and in population surveillance programmes  and has 44.14.1.1More than 80% of Danish girls were fully vaccinated when the HPV vaccine was first introduced in 2009 . In 2015The WHO describes vaccine mistrust as a growing gap emerging between experts and citizens . In Denm4.1.2A joint information campaign launched in 2017 aimed at preventing CC by increasing the uptake of HPV vaccination within the Danish Childhood Vaccination Programme. Extensive preparations preceded a collaborative communications strategy, which combined targeting the press, the social media, doctors and other health professionals, and parents in order to foster an informed decision about HPV vaccination .An important realization arising from these pre‐analyses was that mothers most often decided about vaccination in Denmark . Many moThe communications strategy evolved as described by the WHO: through the definition of the target group and its segments; the understanding of the barriers and motivations of the target group; and the preparation of distinct messages and activities for the various segments of the target group . An earlHowever, this Facebook account was inactivated at a later stage, as it had become dominated by users spreading misinformation . Neverth4.1.3Within the first year of the campaign, and as a result of a more balanced communication through the press, voices criticizing HPV vaccination became less dominant , 22. FigAt the same time, the media debate had become more impartial and supportive of the campaign against misinformation. Indicatively, an independent editor who oversees the interest of the TV viewers rated the aforementioned TV documentary as partial by focusing on girls with unspecified symptoms and not the evidence . Likewis4.2Italy was less affected by mistrust in the HPV vaccine than Denmark. There were initially fears about the issue of vaccinating young girls s against a sexually transmitted disease, but the topic was never really in the spotlight. Some catholic organizations even supported the programme, and there is some evidence that migrants of different ethnic groups participated as often as Italian adolescents . In geneConversely, and contrary to what had happened in Denmark, in the 2010s misinformation and probably intentional fake news in Italy hit hard paediatric vaccinations. False beliefs and conspiracy theories spread on the Internet, and an antivaccination sentiment infected not only parents of small children but also celebrities and politicians . After r5Human papillomavirus vaccine coverage among girls at age 15 in Italy and Denmark is presently among the highest in Europe and worldwide  is of coet al. [Conversely, HPV in Denmark vaccination was mainly threatened by a wave of misinformation that hit an otherwise well‐working HPV vaccination programme. Professionals may not fully appreciate the severe impact of modern sources of misinformation, including the social media, were fake news spread faster than the truth . Unfortuet al.  showed tThe learning from Denmark has been to never leave unanswered questions and to keep in mind the silent audience. Without brave women sharing their personal stories about their lives with CC or intraepithelial dysplasia, the information campaign would be less effective .6https://www.who.int/news‐room/detail/15‐07‐2020‐who‐and‐unicef‐warn‐of‐a‐decline‐in‐vaccinations‐during‐covid‐19). If vaccine mistrust arises, the existence of valuable monitoring tools will help document drops in a programme performance as well as re‐establishing confidence following a crisis. Misinformation can make considerable damages and, therefore, scientists, health workers and media should gather to provide appropriate communication directed to both the ‘brain’ using facts and the ‘heart’ using personal stories.This review emphasizes the need for standardized methods to monitor HPV vaccination nationally and internationally. While coverage by birth cohort is commonly used, we echo WHO’s plea to also measure coverage in girls at age 15 as an indicator of not only uptake but also of timeliness of HPV vaccination. Prioritizing HPV vaccination of girls may become imperative in the COVID‐19 era, during which preventative activities are under added pressure in many countries (The authors declare no conflict of interest.JB and SF equally contributed to the manuscript. JB reviewed the Danish data and SF the Italian data. JB and SF both approved the final paper.


Most individuals are infected with human papillomavirus (HPV) at least once in their lifetime. Infections with low-risk types can cause genital warts, whereas high-risk types can cause malignant tumors. The aim of this study was to determine the burden of anogenital diseases potentially related to HPV in young women based on German statutory health insurance claims data.We conducted a retrospective claims data analysis using the “Institute for Applied Health Research Berlin” (InGef) Research Database, containing claims data from approximately 4 million individuals. In the period from 2012 to 2017 all women born in1989–1992, who were continuously insured between the age of 23–25 years were identified. Using ICD-10-GM codes (verified diagnosis in the outpatient sector or primary or secondary diagnosis in the inpatient sector) the administrative prevalence  of genital warts (A63.0), anogenital diseases grade I , grade II  and grade III  was calculated .From 2012 to 2017, a total of 15,358 (birth cohort 1989), 16,027 (birth cohort 1990), 14,748 (birth cohort 1991) and 14,862 (birth cohort 1992) women at the age of 23–25 were identified. A decrease of the administrative prevalence was observed in genital warts (1.30% (1.12–1.49) birth cohort 1989 vs. 0.94% (0.79–1.10) birth cohort 1992) and anogenital diseases grade III (1.09% (0.93–1.26) birth cohort 1989 vs. 0.71% (0.58–0.86) birth cohort 1992). In anogenital diseases grade III, this trend was especially observed for severe cervical dysplasia (N87.2) (0.91% (0.76–1.07) birth cohort 1989 vs. 0.60% (0.48–0.74) birth cohort 1992). In contrast, anogenital diseases grade I (1.41% (1.23–1.61) birth cohort 1989 vs. 1.31% (1.14–1.51) birth cohort 1992) and grade II (0.61% (0.49–0.75) birth cohort 1989 vs. 0.52% (0.42–0.65) birth cohort 1992) remained stable.A decrease of the burden of anogenital disease potentially related to HPV was observed in the younger birth cohorts. This was observed especially for genital warts and anogenital diseases grade III. Further research to investigate this trend for the upcoming years in light of varying HPV vaccination coverage for newer birth cohorts is necessary. Human papillomavirus (HPV) infection belongs to the most frequent sexually transmitted infections in men and women worldwide . Nearly HPV types capable of infecting mucosal epithelia are subdivided into low-risk and high-risk types. The low-risk types can lead to anogenital warts (condylomata acuminata). Low-risk types HPV 6 and 11 are responsible for approximately 90% of all anogenital wart cases . WorldwiHigh-risk HPV types can cause malignant conditions such as cervical intraepithelial neoplasia and cervical cancer . AdditioHPV vaccination can prevent certain HPV infections and HPV-related anogenital diseases. The European Medicines Agency (EMA) authorized the first HPV vaccines in 2006  and 2007  . Since 2In Germany, the Standing Committee on Vaccination (STIKO) at the Robert Koch Institute (RKI) is responsible for recommendations on vaccinations. These are then covered by the statutory health insurance (SHI) for all insured persons according to the recommended conditions e.g. in terms of age and gender . School-The aim of this study was to assess the burden of anogenital diseases potentially related to HPV in women at the age of 23–25 based on diagnoses documented in German sickness fund data. The burden of HPV-related anogenital diseases is poorly explored in the birth cohorts who had a chance to receive HPV vaccination directly after its introduction in Germany in 2007. By assessing the first three birth cohorts (1990–1992) who were fully eligible for HPV vaccination according to the first STIKO recommendation and birth cohort 1989 which was partially eligible (this cohort turned 18 in 2007) we aimed to generate insights into the burden of potentially HPV-related anogenital diseases in these specific birth cohorts after the introduction of the HPV vaccination in Germany. HPV vaccination status of the study population could not be evaluated.A retrospective claims database analysis was conducted using the “Institute for Applied Health Research Berlin” (InGef) Research Database. The database comprises anonymized healthcare claims data from approximately 8 million covered lives from about 60 different sickness funds. The data include patient demographics and characteristics, inpatient and outpatient diagnoses, surgeries and diagnostic codes, the healthcare resource utilization and costs of services for the inpatient care, outpatient care, pharmacological therapy, remedies, devices and aids, and sick leave in an anonymized case-by-case individual format. For scientific research projects, an adjusted analysis sample of the InGef database has been created which includes approximately 4 million covered lives structured to represent the German population in terms of age and gender according to the Federal Office of Statistics. The InGef sample represents about 5.5% of the German SHI population, whereas about 85% of the German population is insured by the SHI. It has been proven to have good external validity to the German population in terms of morbidity, mortality and drug use .The STIKO recommendation for HPV vaccination from 2007 included girls between 12 and 17 years. Birth cohort 1989 turned 18 in 2007 and therefore, parts of the birth cohort were too old for the HPV vaccination according to the STIKO recommendation from 2007. The female birth cohorts 1990 to 1992 were the first female birth cohorts that fulfilled the recommended age criteria for HPV vaccination according to the STIKO. Thus, this study included the first three female birth cohorts  which were fully eligible to receive HPV vaccination according to the age recommended by STIKO in 2007 and furthermore, the birth cohort 1989 which was partially eligible to receive HPV vaccination according to the STIKO recommendation. In total, the study population comprised all women in the InGef Research Database from 2012 to 2017 who were born in 1989, 1990, 1991, or 1992. All women had to be continuously observable from the age of 23 to 25, except for women who deceased. Age was determined at December 31st of each year.The identification of potentially HPV-related anogenital diseases was based on International Statistical Classification of Diseases, German Modification ICD-10-GM) codes. All potentially HPV-related anogenital diseases  in the outpatient sector (verified diagnoses) and in the inpatient sector (main and secondary diagnoses) were identified , 16,027 (birth cohort 1990), 14,748 (birth cohort 1991) and 14,862 (birth cohort 1992) women were continuously insured at the age of 23 to 25, also including women who deceased in this age period. Within the 3 years of observation a minimum of five (birth cohort 1990) and a maximum of 10 (birth cohort 1992) women deceased in the age period from 23 to 25 years , followed by genital warts  and anogenital diseases grade III . Anogenital diseases grade II  and especially invasive carcinoma  were less frequently documented . A lower 3-year APR was observed for the younger cohorts (0.94% (0.79–1.10) birth cohort 1992)  in birth cohort 1989 to 1.31% (1.14–1.51) in birth cohort 1992). A peak was observed in birth cohort 1990 (1.60% (1.41–1.80). Among the anogenital diseases grade I, mild cervical dysplasia (CIN I) was most frequently recorded, followed by other specified diseases of anus and rectum (AIN I & II). With a 3-year APR of a maximum of 0.12% for mild vaginal dysplasia (VAIN I) and 0.05% for mild vulvar dysplasia (VIN I) these two anogenital diseases were less frequently recorded compared with CIN I and AIN I & II  in birth cohort 1989 to 0.52% (0.42–0.65) in birth cohort 1992. Among the anogenital diseases grade II moderate cervical dysplasia (CIN II) was most frequently recorded. Moderate vaginal dysplasia (VAIN II) and moderate vulvar dysplasia (VIN II) were hardly recorded ) to birth cohort 1992 ). Among the anogenital diseases grade III, severe cervical dysplasia was most frequently recorded and followed the trend of the pooled anogenital diseases grade III results. . The 3-year APR for invasive carcinomas decreased from 0.12 to 0.06% (0.07–0.19 in birth cohort 1989 and 0.03–0.11 in birth cohort 1992) . We found the highest administrative prevalence for anogenital diseases grade I, followed by genital warts and anogenital diseases grade III among all analyzed birth cohorts. A lower burden of anogenital disease potentially related to HPV was observed in the younger birth cohorts as compared to the older cohort. This was observed especially for genital warts and anogenital disease grade III.The following section discusses the results in the context of HPV vaccination coverage in Germany. HPV vaccination status of the study population could not be evaluated, as the included birth cohort should have received their HPV vaccination in the years 2007 to 2010, which were not available for this analysis. For the analyzed birth cohorts two publications provide estimates on the HPV vaccination coverage. Hense et al. evaluated initiation of HPV vaccination in 2008 based on data from a statutory health insurance and reported 37% of 16 years old girls with at least one dose, which would overlap with birth cohort 1992. In the same publication rates were 33, 19 and 18% for 17-, 18-, and 19-year-old women, respectively (corresponding to birth cohorts 1992–1989) . While tThis next section discusses the results in the context of previous studies on burden of HPV diseases in Germany. One previous German study examined HPV type prevalence  but has The following section discusses the results more specifically in the context of previous studies on HPV vaccination impact in Germany. Single previous reports focused on HPV vaccine impact in Germany and are in line with our results. Deleré et al. found a significant lower HPV 16/18 prevalence in vaccinated women suggesting first effects of the vaccination. Data from the German Pharmacoepidemiological Research Database (GePaRD) also demonstrated a decline of anogenital warts among males and females at the age of 14–24 comparing the timeframe prior to vaccination with the timeframe after vaccination. While the largest decrease (by 60%) was observed for women in the age group 16–20, the incidence ratio in women aged between 21 and 26 years was reduced by 10–20% [This last section discusses further potential limitations of the study in detail, most inherent with the use of health insurance claims data. Claims data are primarily collected for reimbursement purposes. Therefore, only patients who seek physician treatment and cause reimbursement for the health insurance could be identified in the database. Patients without symptoms might not seek medical advice, individuals who do not participate in screening examinations might not be identified, and patients who treat their disease  by themselves or ignore the condition are not recorded in the database. Therefore, this study only presents the administrative prevalence based on reimbursement data and not the clinical prevalence of potentially HPV-related anogenital diseases on a population level. Furthermore, Germany had an opportunistic cervical cancer screening beginning at the age of 20 and thus, most documented diagnoses of intraepithelial neoplasia most likely have been identified during screening and further work-up . The annual and biannual cervical cancer screening participation rate in women 25–29 years old was reported with approximately 60 and 70% in 2011, respectively . As not For the identification of potentially HPV related anogenital diseases we used ICD-10-GM codes, which is the official classification for the encoding of diagnoses in inpatient and outpatient medical care in Germany since 2000 . CliniciCervical cancer screening in Germany is Pap-based for women in their twenties. Documented diagnoses in our analysis are most likely cytologically or histologically defined. No laboratory data was available for this study to demonstrate an HPV association of anogenital diseases. Therefore, it is not possible to distinguish between anogenital diseases, which were caused by an HPV infection and those not caused by an HPV infection. However, it is expected that high-risk HPV infections cause almost all cervical cancers and precancers, approximately 90% of high-grade anal, vulvar and vaginal intraepithelial neoplasia and 30, 70 and 90% of vulvar, vaginal and anal cancers, respectively [Finally, the instrument of ARP is subject to limitations that might influence the results of our study. Data on outcomes might be collected in different ways over time for the different study cohorts. Migration of populations affecting the study cohort might influence the differences between the groups. Seasonal or cyclical variations might result in fluctuations that affect the outcome trend. These limitations are assumed to be negligible in our study as we expect no major changes in the recording behavior of physicians during the study period and the impact of migration will not have affected the SHI, as medical services are paid by other payers . SeasonaIn summary, our results demonstrate the highest administrative prevalence for anogenital diseases grade I, followed by genital warts and anogenital diseases grade III among all analyzed birth cohorts. Even though the HPV vaccination status of the study population was unknown, a decrease of the disease burden of genital warts and anogenital grade III disease was observed in favor of the younger birth cohorts who were fully eligible for HPV vaccination according to STIKO recommendation. Further research is necessary to confirm the observed trend including analyses linked to vaccination status.Additional file 1.


Objective: To investigate the HPV DNA prevalence and genotype distribution among penile cancer in China. To identify association between HPV prevalence, different histological subtypes, tumor stage, tumor grade, demographics, comorbidity, and phimosis incidence trend. Standardized HPV DNA detection and p16INK4a expression were used in a multi-center series of 340 penile squamous cell carcinomas diagnosed from 2006 to 2017.Materials and Methods: HPV DNA detection and genotyping were examined by a validated kit for 23 different HPV subtypes (PCR-RDB HPV test). The cases with positive HPV DNA were additional tested for p16INK4a expression to confirm the HPV infection.Results: Using the PCR-RDB HPV test, overall HPV prevalence was 48.8% (166/340) and that of p16INK4a expression was 45.6%. In this studied population, HPV16 was the most frequent HPV type detected in HPV-positive cancers (76.5%). HPV18 was the second most common type in penile cancers (15.1%). After pathology review, 307 cases were confirmed as invasive penile cancer, and the other 33 were non-invasive caners. The histologic subtypes of warty, basaloid, clear cell papillary, adenosquamaous and pseudohyperplastic were showed high HPV DNA prevalence. Among invasive cancers, no statistically significant differences in prevalence were observed by tumor grade, tumor stage or lymphnode stage at diagnosis. HPV positive penile cancer incidence significantly increase and the phimosis incidence significantly decrease from 2006 to 2017.Conclusions: About a half of penile cancers were related to HPV infection. Our findings highlight the phimosis related penile cancers have been declining, the HPV related in the development of penile cancer and a fully aware of regional differences in HPV genotype distribution are tasks for penile cancer control and prevention. Penile cancer is relatively rare in the developed nations of Western Europe and the United States, with an age-standardized incidence of 0.3–1.0 per 100,000 men . The etiRecently, Alemany et al. reported the largest epidemiologic study to evaluate the role of HPV infection in penile cancer, examining 1,010 penile cancer specimens from 25 countries  penile cancer specimens diagnosed from 2006 to 2017 that were obtained from 5 level three tertiary hospitals in China . Patients with suspiciously invasive penile cancer underwent complete glansectomy, partial or total penectomy. Patients initially diagnosed with pT1b-pT4 or cN1 stage underwent inguinal lymphadenectomy. Patients with more than 2 inguinal lymphnode metastasis, Cloquet's inguinal metastasis, extranodal extension or radiologically suspicious pelvic lymphnode metastasis underwent pelvic dissection. Information about age and year of diagnosis and lymph node metastasis were also obtained from the participating centers. Comorbidities such as smoking, drinking, hypertension, and diabetes were also considered in this study.The first sections of the FFPE samples were used for histopathologic evaluation after hematoxylin and eosin (HE) staining. All cases were reviewed by a specialized pathologist (Peipei Zhang) from Fudan University Shanghai Cancer Center. The pathological evaluation was performed following the WHO criteria proposed in 2016  of tumor cells were selected for further HPV DNA analysis.Our pathologist finally confirmed 307 cases were invasive penile cancer. The other 33 were non-invasive caners, most of them were verrucous type (26/33), and 8 of them underwent inguinal lymphadenectomy.The paraffin samples were processed under strict conditions to avoid potential contamination as described in a previous study. DNA from paraffin-embedded tissue blocks was extracted from 4 to 5 sequential unstained sections, each 4-mm thick . GenomicHPV DNA detection was performed using the Human Papillomavirus Genotyping assay for 23 genotypes from Yaneng BIOscience , which was approved by the China Food and Drug Administration for diagnosis. The system provides a reliable and sensitive clinical reference for HPV detection and has been widely used in cervical cancer screening in China . The sysINK4a expression was evaluated in all specimens. Overexpression of p16INK4a can serve as a surrogate marker for transcriptionally active HPV infection due to its strong correlation with high-risk HPV genotype infection  following the manufacturer's protocol. A pattern of diffuse nuclear and cytoplasmic staining of >70% of the tumor cells was considered positive.Immunohistochemical p16nfection . p16INK4The HPV DNA prevalence was calculated among HPV DNA-analyzed cases. We analyzed the association between the HPV prevalence, age at diagnosis, histopathological subtypes, tumor grade, tumor stage, nutrition [albumin and body mass index (BMI)], comorbidity (hypertension and diabetes mellitus), cigarette smoking, and alcohol abuse. The HPV type-specific information included single and multiple infections. Differences in categorical variables were assessed using Pearson's Chi square test or Fisher's exact test. Mann-Kendall test was performed to examine the significance of the HPV and phimosis prevalence trends. Data analyses were performed with SPSS software v.20.0 . Statistical significance was set at two-sided 0.05.A total of 340 penile cancers were HPV DNA analyzed and included in the final analyses. Among penile cancers, the most frequent diagnosis was usual-type squamous cell carcinoma (74.1%). Less frequently, we identified verrucous (7.6%), warty, sarcomatoid, basaloid, and other types (18.3%). HPV DNA was detected in 128 (50.7%) out of 252 cases of usual type, 29 (72.5%) out of 40 cases of warty and basaloid types, 4 (40%) out of 10 cases of sarcomatoid type, and 5 (41.7%) out of 12 cases of histologically rare cases of penile cancer . No HPV infection was detected in cancers with verrucous features .No statistically significant differences in HPV DNA prevalence for invasive penile cancers detected among different tumor stages, lymph node stages, or tumor grade  The overp = 0.010). No association was observed between HPV type and average age at diagnosis for penile cancers. No statistically significant differences were observed for nutrition status , comorbidity (diabetes and hypertension) or lifestyle  (The HPV infection rates for different age groups were 55.0% (<40 years old), 54.3% (40–50 years old), 59.6% (50–60 years old), 38.1% (60–70 years old), and 34.0% (>70 years old). Thus, penile cancer with HPV infection was more frequently diagnosed in younger patients (rinking) .p = 0.039) and a significant decrease in phimosis  from 2006 to 2017  had a both HPV and p16INK4a positivity. HPV-positive invasive penile cancers were found to have a p16INK4a positivity of 82.9% (136/164), while only 17  HPV-negative cases were p16INK4a positive. Overall, there was 83.1% (138/166) concordance between the two methods.All the specimens were stained to compare p16Limited epidemiologic data are available for the national incidence of penile cancer in China. We provide reference data for the prevalence of HPV infection and the distribution of HPV genotypes among men with penile cancer. To our knowledge, our data provide the first evidence of a high prevalence of HPV infection in patients with penile cancer in China. Our data indicate that the HPV DNA prevalence (48.8%) in China is as high as that in the United States, European Countries and South America , 10. TheHPV prevalence and types have been investigated in fewer studies in Asia compared to Europe, North America and South America. These studies primarily included lesions that were invasive penile cancer and a few cases of intraepithelial neoplasms –17 Supp. HoweverHPV 16 and HPV 18 were the two most common types, with relative contributions of 76.5 and 15.1%, respectively. The combination of HPV types 16 and 18 greatly increased the relative contribution of HPV to penile cancer to 88.6%. Together, the HPV types 16, 18 and 33 further increased the relative contribution to 91.0%, and the results of this study strongly support the restricted genotype contribution in the pathogenesis of penile cancer in China. Our data also confirm the dominant roles of HPV16 and 18 in all types of penile cancers. According to our data, the 2-valent HPV vaccine, approved by the Chinese Food and Drug Administration, indicated a combined prevalence among HPV DNA-positive cases of 88.6% for penile cancers. Recently, the new 9-valent HPV vaccine has been approved, and 95.2% of patients might receive protection from penile cancer.It has been reported that HPV-related tumors are diagnosed in younger patients than non-HPV-related cancers, as has been reported for female genitalia cancers and head and neck cancers . Our finThe etiology of penile squamous cell carcinoma has shifted over the past decade. Our results indicated a significant increase in HPV prevalence and a significant decrease in phimosis. One explanation may be related to increase awareness of early detection of phimosis and prevention of phimosis. The other explanation was the sharp increased population floating in the past decade in China, and HPV are more likely to spread.Our study indicated that HPV DNA may be associated with aggressive tumors, which was similar with a previous report . HPV DNAINK4a expression, which further confirms the PCR-RDB HPV test as a reliable and easy-to-use detection method. This concordance is in agreement with a previous global study (The success of HPV testing in clinical practice is largely dependent on its high sensitivity and negative predictive value. A previous study showed that the PCR-RDB HPV test is a reliable, sensitive, and accurate method for cervical cancer screening , 20. In al study .We adopted the new 2016 WHO classification and applied it to our large cohort. The updated subtypes and grades were applied to 340 cases. We confirmed the HPV differences between the two general groups and found grade discrepancies between pathological subtypes. The connection between subtype and grade may indicate a corresponding HPV positivity rate and warrants further research to determine an individualized treatment approach.INK4a ed information on HPV positivity according to the recent WHO update of pathological subtypes and grades. This study, however, has some potential limitations. The included cases were obtained from five level three tertiary hospitals, mainly on the eastern coast of China, which may represent the developed region in China. Thus, bias could arise due to unbalanced regional development.To our knowledge, our study is the first large multicenter examination of HPV prevalence and the distribution of HPV genotypes in China. The strengths of the study include the use of the same protocol for specimen collection, central pathological review, and classification by an experienced pathologist, and well-standardized virus detection in a single central laboratory using two different markers of viral presence at the levels of viral DNA and p16In conclusion, our study provided information regarding HPV infection in penile cancers in China using robust methods. Approximately half of the penile cancer cases were related to HPV infection, as detected with a PCR-RDB HPV test. As demonstrated in our study population, genotypes 16, 18, and 33 are the predominant HPV types in invasive penile cancer. Our findings highlight the phimosis related penile cancers have been declining. Regional differences in HPV genotype distribution and the carcinogenic impact of HPV indicate the need to be mindful of HPV control and prevention.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the Institutional Review Board of Fudan University Shanghai Cancer Center, The Affiliated Hospital of Qingdao University, Hainan General Hospital, Northern Jiangsu People's Hospital, and Affiliated Hospital of Jiangnan University. The patients/participants provided their written informed consent to participate in this study.DY, YZ, and WG designed research and wrote the manuscript. PZ reviewed the pathology slides. BW, SJ, and YW performed the HPV DNA genotype analysis and immunohistochemical analysis. GZ, JZ, XD, and QW performed data collection and processing of data. All authors approved the manuscript.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.


The first HPV vaccines licensed targeted two HPV types responsible for most cervical cancers. A 9-valent vaccine (9vHPV), targeting 5 additional types, was introduced in 2016 and is currently the only HPV vaccine available in the United States. Previous studies demonstrated high rates of HPV infection in Alaska Native (AN) women. We sought to measure prevalence of high risk HPV types in AN women undergoing colposcopy and to determine those preventable by vaccination.For this cross-sectional study, we recruited women who were undergoing colposcopy for clinical indications at Alaska Native Medical Center to obtain cervical brush biopsy samples. Specimens were shipped to Atlanta, Georgia for DNA extraction, HPV detection, and typing using L1 PCR with type-specific hybridization to detect 37 HPV types.p < 0.001) HPV 16 or 18 were detected in 77% (17/22) with CIN 3, 53% (24/45) with CIN 2, and 36% (67/184) with CIN 1. (p < 0.001).Four hundred eighty eight specimens from 489 women were tested. At least one HPV type was found in 458 (94%) specimens. Of 458 participants who were HPV positive, 332 (72%) had two or more types. At least one type targeted by 9vHPV was detected in 95% of participants with CIN 3 (21/22), 82% with CIN 2 (37/45), and 65% with CIN 1 (119/184). (A substantial proportion of AN women attending colposcopy clinic had evidence of HPV 16/18 infection, as well as other high risk types targeted by 9vHPV. At least one 9vHPV type was detected in 62% of the participants overall, and 95% of participants with CIN3. AN women are expected to benefit from vaccination against HPV 16/18, and will have greater benefit from 9vHPV. Information from this study could be used to develop public health strategies to increase vaccine uptake, or to track HPV genotype prevalence over time. Human papilloma virus (HPV) infection is common among young Alaska women and these infections cause many cases of cervical cancer among Alaska Native people –5. In a The worldwide HPV prevalence in cervical carcinomas is 99.7% ; more thIn a 2000 study cervical cancer specimens from 53 Alaska Native people, 32 Greenland Native people and 34 Danish Caucasians from 1980 to 1989 were analyzed by polymerase chain (PCR) for HPV types 16, 18, 31, 33, and 45 [Vaccines against HPV types 16 and 18 have been in use since 2006 in the United States . The quaIn February 2015 ACIP recommended 9 valent HPV vaccine (9vHPV)  as one of three HPV vaccines that can be used for routine vaccination. 9vHPV is a noninfectious, virus-like particle (VLP) vaccine . SimilarStatewide HPV Vaccine for Alaska Natives: Projects and Evaluations (SHAPE) developed this project with the goal of establishing HPV genotype surveillance among a risk population. We sought to obtain a baseline of HPV prevalence that could be used in future evaluations to determine changes in HPV vaccine-type prevalence after vaccine introduction.A multi-agency Alaskan working group was established in 2006 to develop projects to improve HPV vaccine uptake and evaluate the impact on cervical cancers and other health outcomes. The group called The SHAPE group undertook a cross-sectional study to determine HPV genotypes in AN women undergoing colposcopy for abnormal cervical screening results at ANMC in 2009. ANMC, located in Anchorage, Alaska, is a tertiary care center for the Alaska Native Health Consortium which also serves as the primary and secondary health facility for approximately 140,000 Alaska Native and American Indians people.All non-pregnant Alaska Native Health System beneficiaries 18 years old or older undergoing indicated colposcopy at the ANMC from October 1, 2009 – September 30, 2011 were eligible to participate. The colposcopy was indicated based on the American Society for Colposcopy and Cervical Pathology cervical cancer screening consensus algorithms. Exclusion criteria included age less than 18 or pregnancy. A routine urine pregnancy test was done prior to colposcopy. Basic demographic data, tobacco use, and vaccination status were obtained though the electronic medical record at the time of the colposcopy. There was no information collected for women who refused enrollment.After informed consent, a clinician-collected cervical sample was obtained at the time of pelvic examination. After visualization of the cervical os, the sample was taken with a Digene Cervical Sampler brush . The brush was placed in specimen transport medium  and stored in the ANMC colposcopy refrigerator, then transported on ice to the CDC Arctic Investigations Program in Anchorage, Alaska USA and was frozen. Specimens were batched and shipped on dry ice to CDC in Atlanta, Georgia, USA for extraction, HPV detection, and typing using the Roche linear array test as previously described . SpecimeP-values less than 0.05 were considered statistically significant and all tests were 2-sided. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 were considered the 13 high risk types [The de-identified data were analyzed with proportions of patients with HPV types compared using a nonparametric test for trend across ordered groups. sk types . Analysin = 4), then the second specimen was used. One additional woman was excluded because she did not have a cervix. Four hundred and eighty eight specimens were included in the final analysis.Five hundred and twenty specimens from 489 women were collected and tested. In the 31 women who were tested twice, the first study specimen was included, unless no biopsy obtained at that time (Self-reported ethnic groups of the sample included: Inuit/Yupik 238 (49%), Aleut 7315%), Athabascan /Tlingit /Haida 149 (31%), and other 28 (5%). The mean age of the participants was 29.7 years. The largest age group was less than 25 years old, 40%, followed by 25–29 years (25%).  had received any HPV vaccine and only twenty one (4.3%) had completed a three dose series at the time of the colposcopy. No significant difference was found in HPV type distribution or diagnostic category between participants who received any vaccine doses and those who received no vaccine. There was no significant difference in HPV type distribution or diagnostic category between tobacco using and non-tobacco using participants.The most common cytology referral diagnosis was atypical squamous cells of undetermined significance 231 (47.3%), followed by low grade squamous intraepithelial lesion , high grade squamous intraepithelial lesion , atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion , and unknown .An ectocervical biopsy was obtained from 438 (90%) women and an endocervical curettage was performed in 402 (82%). There were 42 (8.6%) loop electrosurgical excision procedure (LEEP) specimens. CIN 3 was found in 22 (4.5%) specimens; CIN 2 , CIN 1 , metaplasia , other , no biopsy , and insufficient .One or more of the 37 HPV types were found in 458 (94%) specimens. Of the 458 participants who were HPV positive, 332 (72%) had two or more types. Among those with any HPV detected, the mean number of HPV types per participant was 2.6. Table  Three huOf the 9vHPV high-risk types, type 16 was found in 127 (26%) of all participants; type 31 in 64 (13%); type 58 in 59 (12%); type 52 in 58 (12%); type 45 in 40 (8%); type 18 in 39 (8%); and type 33 in 15 (3%).  AN women. 76 16%) had 2 types, 19 (4%) 3 types, and 4 (0.8%) 4 types. There was a mean of 0.88 9vHPV types per participant. (Table 6% had 2 There was no significant difference in HPV genotype distribution or diagnostic category by vaccination history or by history of tobacco use. Table .p < 0.001). (Table p < 0.001).One or more of the 9vHPV types was detected in 95% with CIN 3 21/22), 82% with CIN 2 (37/45), and 65% with CIN 1 (119/184)  [The distribution of HPV types in this AN group was similar to the pattern reported in American Indian women in the Northern Plains , 16. In Of the 458 AN participants who were HPV positive, 332 (72%) had two or more types, with an mean of 2.6 HPV types per specimen. This is a similar pattern to previous findings in AN women in 1980–1989 in which multiple types were found in 43% of AN women [This study has some important limitations. The participating women were a convenience sample of all patients presenting to colposcopy clinic. However, the clinical staff estimated they enrolled approximately 95% of eligible patients, minimizing selection bias. Thus, these results are likely generalizable to Alaska Native women with abnormal cytology who receive care at Alaska Native Medical Center. The proportion of women who had received any doses of HPV vaccine series was very low and there was no difference in HPV genotype distribution by vaccine status. So, although this group was not entirely vaccine naïve, these data effectively represent the HPV genotypes in a population with abnormal cervical screening prior to use of HPV vaccine.This study provides data which may help to plan future evaluations of HPV genotype prevalence and vaccine impact among this clinical population. For example, 532 participants 18 years or older would be needed to detect a change from a baseline prevalence of HPV 16/18 of 31 to 25%, using an alpha = 0.05, beta = 0.10. One hundred fifty seven participants 18 years or older would be needed to detect a decreased rate to 20% for HPV 16/18 positive. For comparison, assuming that the HPV type changes would be more rapidly detected in participants < 25 years of age, only 272 participants would be necessary to detect a change from a baseline rate of 34% for HPV 16/18 to 25%; whereas 106 participants < 25 years of age would be needed to detect a decrease of HPV 16/18 from the baseline of 34 to 20%. From these calculations, focusing on younger women would improve efficiency and reduce the impact of the study on clinical services. Consideration could also be given to using recruitment strategies other than continuous enrollment, such as the first week of the month.A substantial proportion of AN women attending colposcopy clinic have HPV 16/18, as well as the other high risk types in 9vHPV - 31/33/45/52/59. While any of the 9vHPV types were detected in 62% of the participants overall, this increased to 95% of participants with CIN3. AN women would be expected to benefit from vaccination against HPV 16/18, and would be expected to have greater benefit from 9vHPV. In 2014, the CDC reported that 81.2% AN adolescent females aged 13–17 years received one dose of HPV vaccine, while only 58.3% received all three HPV vaccine doses as recommended by ACIP at that time [


Type-specific prevalence of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, and HPV45 in penile cancer was estimated.The authors searches identified 1836 non-duplicate records, of which 73 relevant papers (71 studies) were found to be eligible. The pooled HPV DNA prevalence in penile cancer  was 50·8%. A high pooled HPV DNA prevalence was seen in basaloid squamous cell carcinomas and in warty-basaloid carcinoma. The predominant oncogenic HPV type in penile cancer was HPV16 followed by HPV6 and HPV18. The pooled HPV DNA prevalence in penile intraepithelial neoplasia  was 79·8%. The pooled p16INK4a percent positivity in penile cancer  was 41·6% (p <0·0001), with a high pooled p16INK4a percent positivity in HPV-related squamous cell carcinoma as compared with non-HPV-related squamous cell carcinoma. Moreover, among HPV-positive cases of penile cancer, the p16INK4a percent positivity was 79·6%, compared with 18·5% in HPV-negative penile cancers. The pooled p16INK4a percent positivity in penile intraepithelial neoplasia  was 49·5%.In this interesting manuscript the authors concluded that a large proportion of penile cancers and penile intraepithelial neoplasias are associated with infection with HPV DNA (predominantly HPV16), emphasising the possible benefits of HPV vaccination in men and boys.


Addressing the burden of HPV-associated diseases among men is increasingly becoming a public health issue. The main objective of this study was to determine HPV prevalence among a healthy community-based Malaysian men.This was a cross-sectional study that recruited 503 healthy males from 3 community-based clinics in Selangor, Malaysia. Genital and anal samples were collected from each participant for 14 high risk and 2 low risk HPV DNA detection and genotyping. All participants responded to a set of detailed sociodemographic and sexual behaviour questionnaire.The median age at enrolment was 40 years old (IQR: 31–50). The anogenital HPV6/11 prevalence was 3.2% whereas high risk HPV prevalence was 27.1%. The genital HPV prevalence for HPV6/11 was 2.9% while high risk HPV was 18.8%. HPV6/11 prevalence in the anal canal was 1.6% and high risk HPV was 12.7%. HPV 18 was the most prevalent genotype detected in the anogenital area. There was a significant independent association between genital and anal HPV infections.Anogenital HPV infection is common among Malaysian men. These findings emphasize the ubiquity of HPV infection and thus the value of population-wide access to HPV prevention. Human papillomavirus (HPV) infection is the most frequent sexually transmitted infection among men and women. HPV is classified into the high risk (HR) and low risk (LR) genotypes. The LR types including HPV 6 and 11 are the main cause of genital warts whereas HPV 16 and 18 are the more carcinogenic genotypes. HPV is also linked to approximately 50% of penile cancer, 88% of anal cancer and up to 56% of oropharyngeal cancer cases . While tThis was a cross-sectional study that had recruited 503 healthy males via convenience sampling from September 2014 to February 2016. These participants were recruited from 3 community-based clinics in Selangor, Malaysia and most of them attended the clinics as a companion to their family members that required medical attention or routine check-ups. Participants aged 18 to 60 years that agreed to self/doctor’s anogenital sampling were recruited for the study. Informed consent form was obtained from all participants. The exclusion criteria were acute illness or never having been sexually active. This study was approved by the Medical Research Ethics Committee (NMRR-13-444-14609), Ministry of Health Malaysia and the University of Malaya Medical Ethics Committee (MREC989.32).Anogenital samples were obtained using a brush . Genital samples were collected by swabbing the shaft skin of penis for at least ten times. For anal sample collection, brush was inserted into the anus and turned for at least ten times. Self-sampling or doctor’s sampling was performed according to participant’s preference using the same sample collection method. All the brushes were then soaked in a tube containing 2ml of ThinPrep PreservCyt Solution  for preservation of cells during transportation. All participants responded to a set of questions on detailed sociodemographic and sexual behaviour data. The interview was carried out by a trained study staff member in private.All collected samples were kept at room temperature before extraction. Cells were pelleted before DNA extraction using DNeasy Blood & Tissue Kit  as per manufacturer instructions within 2 weeks of sample collection. The final volume of extracted sample was 100ul. DNA samples were stored at -20 ℃ before transporting to Beijing, China for HPV genotyping. HPV genotyping was performed using the BGISEQ-100 (Beijing Genome Institute (BGI)-assembled Ion Proton Sequencer from Life Technologies, South San Francisco, California, USA) as previously described . AmplifiDemographic and sexual behaviour information such as age, age at sex debut, and number of lifetime sexual partner was recorded as continuous data. Marital status, ethnicity, highest attained education level, household income, circumcision status, smoking and alcohol drinking history were also recorded. Anogenital HPV positive infection was defined by detection of any HPV DNA genotypes at least one of the sites. HPV genotypes targeted by bivalent vaccine was defined as HPV16/18, those targeted by quadrivalent vaccine were HPV16/18/6/11 whereas nonavalent vaccine targeted genotypes were HPV16/18/6/11/31/33/45/52/58. The prevalence estimates were described using percentage. Association between HPV infection and variables were evaluated using Pearson Chi Square Test or Fisher Exact Test when 20% or more of the cell count were less than 5. Variable with p-value less than 0.2 in the univariable analysis and variables that were deemed important from the literatures were included in the multivariable analysis to determine the independent risk factor for HPV infection. A p-value of <0.05 was considered as statistically significant. All analyses were performed using IBM SPSS Statistics Version 20 and Microsoft Excel 2016.A total of 503 healthy male subjects were enrolled in this study, 29 of them were excluded due to incomplete data/unable to provide samples . The median age at enrolment was 40 years old (IQR: 31–50). The main ethnic groups were Malay , followed by Chinese  and Indian . Most participants were heterosexual , married  while 72.4% (343/474) were smokers and 62.9% (297/472) reported to have been circumcised. The final analytic set with valid HPV results was not statistically different from the total enrolled population in this study.A total of 474 anogenital samples were collected from the study population. However, 17.9% (85/474) of genital samples and 22.2% (105/474) of anal samples could not be tested due to insufficient amount of DNA, resulting in 389 genital samples and 369 anal samples with valid HPV results for analysis. A total of 314 subjects provided both genital and anal samples with valid HPV results for analysis. There was a significant difference in term of sample sufficiency between self-collected samples and clinician-collected samples , with the highest HPV prevalence (32.1%) in the youngest age group (18–24) . The anoThe associations of HPV infection and sociobehavioural characteristics are shown in Tables To date, this is the first community-based HPV prevalence study among healthy men in Malaysia. The overall anogenital HPV prevalence for 14 high-risk (HR) genotypes and 2 low-risk (LR) wart-associated genotypes among healthy heterosexual male population in Malaysia was 29.6% with the highest prevalence demonstrated in the youngest age group (32.1%). Genital HPV prevalence was 21.1% and the HPV prevalence at the anal canal reported to be 13.6%. HPV18 was the most prevalent HPV genotype in both genital site and anal canal.In a meta-analysis that included 40 publications on HPV detection in men, the prevalence of genital HPV infections was reported to be variable from 1.3% to 72.9% . HR-HPV To date, there is limited data on the prevalence of anal HPV infections among healthy, heterosexual male populations. The HIM study that had included more than 1300 men from the United States, Brazil and Mexico had reported a comparable anal HPV prevalence (12.0%) in the heterosexual male population . AnotherThere is a significant independent correlation between genital HPV infection and anal HPV infection (p<0.001) in this study. 20.4% (19/93) of the HPV positive individuals had concurrent infection at genital area and anal canal and 63.2% (12/19) of them had the same HPV genotypes in both anal and genital sites. This finding is similar to a study by Pamnani et al (HIM Study) who reported a higher risk of acquiring anal HPV infection following genital HPV infection in a heterosexual male population, indicating a possibility of self-inoculation or other non-penetrative mechanism as the route of HPV transmission . AnotherSmoking and alcohol intake were reported to be the risk factors for HPV infection among men in the HIM study , 35. TheThere are some limitations in our study. By recruiting from only 3 clinics in Selangor and Kuala Lumpur, our sample may not adequately capture the variability of male HPV prevalence in Malaysia. Additionally, we also note the difference in term of racial composition in this study compared to the Malaysian population. As this is the first Malaysian study investigating HPV prevalence among healthy men, future studies with larger sample size and wider coverage should be conducted to verify the finding. We note that our data were comparable to international studies using population-based sampling. We do note a substantial rate of sample failure from both genital and anal swabs which might be resulting from inefficient sampling method as the sampling device was designed specifically for self-collected cervico-vaginal screening , and if this failure was not random according to HPV infection status may have led to biased prevalence estimates. As with all similar studies, measures of sociodemographic and sexual behaviours were self-reported and could be subjected to response desirability and recall bias.In conclusion, the combined HR HPV and wart associated HPV6/11 prevalence among men in Malaysia was 29.6% . These findings are comparable to other countries. The most prevalent HPV genotypes  found in the male population reflect the genotype distribution in Malaysian women. While continuous high vaccine coverage in Malaysian women is likely to confer indirect positive impact on prevention of HPV infection and associated cancers in men through herd immunity, the data from this study will be important to support policymakers in Malaysia in optimization of their HPV elimination plans which will require balancing the cost-benefit of multiple HPV control strategies, such as gender-neutral vaccination and improved screening for the many cohorts of women infected prior to vaccine introduction.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file. 3 Nov 2020PONE-D-20-07025Prevalence and Sociodemographic Correlates of Anogenital Human Papillomavirus (HPV) Carriage in A Cross-Sectional, Multi-Ethnic, Community-Based Asian Male PopulationPLOS ONEDear Dr. Woo,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Dec 18 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. 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For instructions see: We look forward to receiving your revised manuscript.Kind regards,Giovanni Delli CarpiniAcademic EditorPLOS ONEAdditional Editor Comments:Even if the reported statistics is correct, the tables should be improved for better clarity; consider additional assistance by a medical statistician.Journal Requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf2. Thank you for stating the following in the Financial Disclosure section:"This study is funded by Merck Sharp & Dohme to YLW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." ext-link-type="uri" xlink:type="simple">https://www.msd-malaysia.com/home/"URL of funder: We note that you received funding from a commercial source: Merck Sharp Dohme.relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc.Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” . The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This is an interesting and important study, yet I do have following concerns.1. The representativeness of the sample: the authors recruited 503 males based on convenience sampling. Besides the sample size being small, the authors failed to discuss the representativeness of the sample; for example, Chinese ethnicity comprise about 24% of the sample: what’s the racial composition of the Malaysian population? Also, they were sampled from 3 hospitals; how representative of those 3 hospitals?2. I have some issue with Table 4. Statistical significance was not achieved in most of the categories, which might be related with their small sample size (thus insufficient power)., Also, some categories have number of patients around 10 or 20, raising concerns regarding the reliability of the results.Reviewer #2: The manuscript entitled: “Prevalence and sociodemographic correlates of anogenital human papillomavirus (HPV) carriage in a cross-sectional, multi-ethnic, community-based Asian male population”, analyze HPV prevalence among healthy Malaysian men and the association with sociodemographic features.The work is interesting since a large cohort was analyzed. Healthy men were recruited from three clinics in Malaysia and 474 were available for the study. Samples were taken (self or doctor’s sampling) from the anogenital region, anal canal, and genitals; and a questionnaire with sociodemographic and sexual behavior data was applied. Samples were processed for DNA extraction and HPV genotyping with a kit that detects 14 high-risk and 2 low-risk HPV types. Finally, 389 genital samples and 369 anal samples had an HPV status result.Within the relevant results is that:-Clinician collected samples were less effective for DNA sufficiency than self-collected, showing a significant difference.-The overall anogenital HPV prevalence was 29.6%, being 27% for high-risk types and 3% for low-risk, with the highest prevalence in the younger men's group. HPV18 was the most commonly detected type.- From the HPV positive men 24.7 % had anal positivity only; 54.8 % had genital positivity only and 20.4% were positive for both genital and anal regions, having the 63% of them the same viral type. Therefore, a significant independent association between genital and anal HPV infection was found.- HPV prevalence was higher among men having sex with men, although in the multivariable analysis an independent association was not found.- Significant associations with HPV positivity were found for smoking or drinking, but were not found to be independent risk factors for HPV infection.- No significant associations was found between HPV prevalence and the othersociodemographic characteristics or sexual behavior analyzed, although it is observed that there is a lot of missing data, which can give an uncertain result.The statistics presented in tables is well performed.The discussion is appropriate where the results obtained in the literature concerning other populations are adequately compared. Limitations of the study are also stated.Comments:A figure legend is missing in Figure 1.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  27 Nov 2020Additional Editor Comments:Funding and Competing interest statements:I confirm and approve the following:Funding:https://www.msd-malaysia.com/home/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This study is supported by Merck Sharp Dohme funding to YLW . Our study cohort comprised of 52.3% of Malay, 23.4% of Chinese and 20.9% of Indian. We note the differences of racial distribution in our study cohort as compared to the population therefore have added this point at the study limitation paragraph by adding this sentence below at Page 19, Line 257-258.“Additionally, we also note the differences in the racial composition of the study cohort compared to the Malaysian population.”This cohort was part of another larger study aiming to determine the HPV prevalence among Malaysian therefore the study cohort were sampled from these clinics. These clinics are located at urban and suburban areas of Selangor and Kuala Lumpur, which are among the most densely populated states in Malaysia. However, we agreed that these clinics could not adequately represent the whole Malaysian population. We emphasized this study limitation by including the following at Page 19, Line 255-260 as shown below:“By recruiting from only 3 clinics in Selangor and Kuala Lumpur, our sample does not represent the male HPV prevalence in Malaysia. Additionally, we also note the differences in the racial composition of the study cohort compared to the Malaysian population. As this is the first Malaysian study investigating HPV prevalence among healthy men, future studies with larger sample size and wider coverage should be conducted to verify our findings.”2. I have some issue with Table 4. Statistical significance was not achieved in most of the categories, which might be related with their small sample size (thus insufficient power)., Also, some categories have number of patients around 10 or 20, raising concerns regarding the reliability of the results.Thank you for your comment. For your information, the sample size for this study was calculated using a formula by Daniel WW 1999 with an expected prevalence of 65% based on the HIM study’s finding  and margin error of 5%. The required sample size was 350.We do note that statistical significance was not achieved in most of the categories and agree that this might be due to the small number in some of the subcategories. This was explained as a study limitation at Page 19 Line 254-259 as shown below:“By recruiting from only 3 clinics in Selangor and Kuala Lumpur, our sample does not represent the male HPV prevalence in Malaysia. Additionally, we also note the differences in the racial composition of the study cohort compared to the Malaysian population. As this is the first Malaysian study investigating HPV prevalence among healthy men, future studies with larger sample size and wider coverage should be conducted to verify our findings.”In addition, we have added a paragraph to discuss further on the findings in Table 4 at Page 19 Line 242-252, please kindly see below:Smoking and alcohol intake were reported to be the risk factors for HPV infection among men in the HIM study . The proposed biological pathway for this is the detrimental effect of tobacco and alcohol on immunity which may lead to inefficient elimination of virus but more epidemiologic data are needed to confirm this hypothesis . In our study, we did not find an association between smoking and alcohol consumption to HPV acquisition. This finding was in consistent with other published evidence [38]. A plausible explanation is that increased risk of HPV infections among smokers and drinkers is often confounded by promiscuous sexual behaviours . Numerous studies reported early age of sexual debut and increased number of lifetime partner as significant risk factor for HPV infection among men . In our study, none of these sexual behavioural factors were found to be independently associated with HPV infection. This is most likely due to the small sample size of our study cohort. Reviewer #2: 1. The manuscript entitled: “Prevalence and sociodemographic correlates of anogenital human papillomavirus (HPV) carriage in a cross-sectional, multi-ethnic, community-based Asian male population”, analyze HPV prevalence among healthy Malaysian men and the association with sociodemographic features.The work is interesting since a large cohort was analyzed. Healthy men were recruited from three clinics in Malaysia and 474 were available for the study. Samples were taken (self or doctor’s sampling) from the anogenital region, anal canal, and genitals; and a questionnaire with sociodemographic and sexual behavior data was applied. Samples were processed for DNA extraction and HPV genotyping with a kit that detects 14 high-risk and 2 low-risk HPV types. Finally, 389 genital samples and 369 anal samples had an HPV status result.Within the relevant results is that:-Clinician collected samples were less effective for DNA sufficiency than self-collected, showing a significant difference.-The overall anogenital HPV prevalence was 29.6%, being 27% for high-risk types and 3% for low-risk, with the highest prevalence in the younger men's group. HPV18 was the most commonly detected type.- From the HPV positive men 24.7 % had anal positivity only; 54.8 % had genital positivity only and 20.4% were positive for both genital and anal regions, having the 63% of them the same viral type. Therefore, a significant independent association between genital and anal HPV infection was found.- HPV prevalence was higher among men having sex with men, although in the multivariable analysis an independent association was not found.- Significant associations with HPV positivity were found for smoking or drinking, but were not found to be independent risk factors for HPV infection.- No significant associations was found between HPV prevalence and the othersociodemographic characteristics or sexual behavior analyzed, although it is observed that there is a lot of missing data, which can give an uncertain result.The statistics presented in tables is well performed.The discussion is appropriate where the results obtained in the literature concerning other populations are adequately compared. Limitations of the study are also stated.Thank you for your comments.We have added a paragraph in discussion to further discuss on the associations between sociodemographic and sexual behavioural factors (results in Table 4). Please refer to Page 19, Line 242-252 as shown below:Smoking and alcohol intake were reported to be the risk factors for HPV infection among men in the HIM study . The proposed biological pathway for this is the detrimental effect of tobacco and alcohol on immunity which may lead to inefficient elimination of virus but more epidemiologic data are needed to confirm this hypothesis . In our study, we did not find an association between smoking and alcohol consumption to HPV acquisition. This finding was in consistent with other published evidence [38]. A plausible explanation is that increased risk of HPV infections among smokers and drinkers is often confounded by promiscuous sexual behaviours . Numerous studies reported early age of sexual debut and increased number of lifetime partner as significant risk factor for HPV infection among men . In our study, none of these sexual behavioural factors were found to be independently associated with HPV infection. This is most likely due to the small sample size of our study cohort. We understand the concern regarding the reliability of the finding due to some missing data and small number in some of the categories. Therefore, we have edited the study limitations to clarify at Page 19, Line 254-259 as shown below:“By recruiting from only 3 clinics in Selangor and Kuala Lumpur, our sample does not represent the male HPV prevalence in Malaysia. Additionally, we also note the differences in the racial composition of the study cohort compared to the Malaysian population. As this is the first Malaysian study investigating HPV prevalence among healthy men, future studies with larger sample size and wider coverage should be conducted to verify our findings.”2. Comments:A figure legend is missing in Figure 1.Thank you for pointing this out.The figure legend is added in Figure 1. Fig 1. Sites of HPV infection distribution among HPV-positive subjects (n=93). 20.4% of the HPV-positive subjects had concurrent genital and anal HPV infections whereas more than half of them (54.8%) had only genital HPV infection.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 7 Jan 2021Prevalence and Sociodemographic Correlates of Anogenital Human Papillomavirus (HPV) Carriage in A Cross-Sectional, Multi-Ethnic, Community-Based Asian Male PopulationPONE-D-20-07025R1Dear Dr. Woo,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Giovanni Delli CarpiniAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: I Don't Know**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #2: I consider that the authors responded adequately to the comments and the manuscript is now suitable for publication.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  Carriage in A Cross-Sectional, Multi-Ethnic, Community-Based Asian Male Population Dear Dr. Woo:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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Triticum aestivum, 2n = 42) or durum wheat  and the intermediate wheatgrass . These lines were characterized by the vigorous regrowth for at least 3 years. The one- and 2-year-old plants had higher grain yield potential than the 3-year-old perennial plants. The decline of grain yield was associated with plant age-related effects on yield components. The perennial wheat lines were all resistant to both Heterodera avenae and H. filipjevi, the two distinct cereal cyst nematode species that occur in China, except that line SX12-787 exhibited moderate resistance only to H. avenae. The dual-purpose perennial wheat lines were evaluated for quality values of both defoliated grass and harvested grains in the form of amino acid profile, mineral concentration, and contents of protein and fiber. Difference in the quality profile was observed between the perennial lines. These perennial lines had an overall improved quality levels over those of the perennial wheat control Montana-2 (T. turgidum × Th. intermedium) and the annual wheat cultivar Jinchun 9. The amplification profiles of the molecular markers provided molecular evidence for the introgression of alien chromatin. Genomic in situ hybridization detected 16, 14, 14, and 12 Th. intermedium chromosomes in lines SX12-480 (2n = 48), SX12-787 (2n = 56), SX12-1150 (2n = 56), and SX12-1269 (2n = 54), respectively, in addition to either 32 or the complete set of wheat chromosomes. The four perennial wheat-Th. intermedium lines described here provide valuable sources of perennial wheat for the dual-purpose application of both grain and forage.Perennial wheat is considered to be a practical way to increase the flexibility and profitability of sustainable agricultural system, as it can be either a forage grass or a grain crop. Four perennial wheat lines SX12-480, SX12-787, SX12-1150, and SX12-1269 were developed from a series of interspecific crosses between common wheat ( Recent increase in the global attention has been paid to the development of perennial crop alternatives to annual crops . EnrichmThinopyrum spp. (Th. intermedium (Host) Barkworth and D. R. Dewey leading to the development of cultivar Kernza as a dual-purpose forage and grain crop (T. turgidum L. var. durum (Desf.); 2n = 28] with Agrotriticum intermediodurum Khizhnyak (syn. Th. intermedium)  .Thinopyrum derivatives are highly resistant to various fungal and viral diseases, such as the rusts , Fusarium head blight (caused by Fusarium graminearum Schw.), powdery mildew , eyespot , Barley Yellow Dwarf Virus (BYDV), Wheat Streak Mosaic Virus (WSMV) and its vector wheat curl mite Aceria tosichella (Keifer) (Heterodera spp.) is one of such parasites that causes significant yield losses in cereal crops and has become an important constraint on wheat production in many parts of the world  Stelter is an emerging threat to wheat production  . Howeverhe world . This rohe world . Heterododuction . The nemoduction . Monoculoduction . Extensioduction . Many of species . Very rece versa . Taken tilipjevi .Th. intermedium lines were able to achieve approximately 75% of the grain yield of the annual wheat cultivar in the first year; however, these lines were hardly able to regrowth beyond the first harvest . To breed for the dual-purpose perennial wheat, emphasizes on consistent grain yield potential and improved forage biomass can offset inherent physiological constraints on grain production to a profitable and sustainable extent. Simultaneously, constantly high degree of resistance to soil-borne diseases is foremost objective to breed for superior perennial wheat.We developed four wheat-Th. intermedium lines are shown in Th. intermedium//DR116) with a Th. intermedium accession. Lines SX12-480, SX12-1150, and SX12-1269 were developed from the same cross between an octoploid partial amphiploid line  and a hexaploid partial amphiploid Xiaoyan 505 . The Th. intermedium accession and the durum wheat DR46, DR116, and DR1022 were provided by the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China. The Chinese wheat cultivars Wenmai 19 (Lankao 4/Wen 2540) and Jinchun 9 , and perennial wheat cultivar Montana-2  from the United States were used as the controls in the assessments of CCN resistance and quality parameters, respectively. An accession of Th. intermedium  and common wheat cultivar Chinese Spring  were used to extract DNA for preparation of the probe or the blocker in the GISH analysis.The pedigrees of the four perennial wheat-1.The perennial wheat lines were developed and assessed on the sandy loam soil at Dongyang Agricultural Experimental Station, Shanxi Agricultural University, Jinzhong, Shanxi province . The local weather condition belongs to the temperate continental arid climate with an annual precipitation of 418 – 483 mm. The long-term annual mean temperature is 9.8°C, the mean frost-free period is 158 days, and the average temperature in winter ranges from −12°C to 12°C H. avenae (pathotype Ha43) (H. filipjevi (pathotype Hfc-1)   and H. fe Hfc-1)  were evae Hfc-1) . The rooe Hfc-1) .Perennial wheat lines were grown in plots consisting of 3 m-long row and spaced 0.4 m that were arranged in a randomized complete block design with three replicates. Twenty-five seeds were sown in a row in early October 2011. Defoliation was performed twice during June (both grain and forage harvest) and November (forage cut preparing for over-wintering) each year, afterwards regrow ability was observed. The agronomic performances were investigated in three consecutive years. Depending on the agronomic traits, evaluation was performed before the first grain and forage harvest at June, or immediately after harvesting. The “first-year plant” refers to individuals during their first growing season (2011–2012), the “second-year plant” denotes plants that show persistent regrowth during the second growing season (2012–2013), and the “third-year plant” represents plants displaying post-harvest regrowth habit for three consecutive years during the third growing season (2013–2014). From each line, plant height (cm), spike length (cm), and number of spikelets per spike were recorded in ten randomly sampled individuals from each plot during the growing seasons of 2011–2012, 2012–2013, and 2013–2014. Plant height was determined from the ground level to the top of the spike, and spike length was measured from the base of rachis to the top of the spike. In addition, number of spikelets per spike was enumerated and the spikes were threshed in a bench micro-thresher to determine thousand-kernel weight (g). The common practices of irrigation , fertilizers , and pesticides were conducted following the local farm system.The end-use quality of the perennial wheat lines, including 16 amino acids, crude fat, crude protein, crude fiber, ash, phosphorus, iron, selenium and carotene were analyzed by the Inspection and Testing Center for Quality of Cereals and Their Products (Harbin), Ministry of Agriculture and Rural Affairs, China. Forage of the one-year old plants for each line was defoliated and samples were dried at 70°C for 48 h. Forage dry matter (300 g per sample) was taken for quality analysis. Grains  from the first-year plants and common wheat cultivar Jinmai 9 were collected for quality measurements. Method for determination of amino acid profile in forage dry matter was described in the National Standard (GB/T) 18246-2000 . MeasureXedm 28, Xedm16, and Xedm105), one sequence characterized amplified region (SCAR) marker (SCAR1248), and one marker obtained from repetitive DNA sequence from Th. elongatum (2P1/2P2) were used to characterize alien chromatin in perennial wheat lines  based protocol , and 6 μL sterilized ddH2O. The profile of DNA amplification was initial denaturation at 94°C for 3 min, followed by 35 cycles of 1 min at 94°C, 47–52°C ramp annealing for 45 s, 1 min for extension at 72°C, and a final extension step at 72°C for 10 min (Primer pairs for the three simple sequence repeat (SSR) markers  as a probe, and sheared genomic DNA of Chinese Spring was used as a blocker. Antidigoxin Rhodamine and 4,6-diamidino-2-phenylindole  were loaded on slides prior to dark incubation. Hybridization signals were visualized under a Leica fluorescence microscope . Photos were taken by a Leica digital camera (Model DFC480). The detailed GISH protocol was described by Seeds of the perennial wheat lines were germinated in Petri dishes at 23°C for 24 h, 4°C for 48 h and 23°C for 27.5 h. Root tips 1–1.5 cm in length were pretreated in ice water (0–4°C) for 24 h, and then fixed in ethanol-acetic acid (3:1) for 24 h at room temperature. The fixed root tips were squashed in 45% acetic acid after staining with 1% acetocarmine for 2 h. Chromosome numbers were enumerated under a light microscope . Genomic DNA of Analysis of variance (ANOVA) for the phenotypic responses to two CCN species was performed considering genotype, year, CCN species, genotype by CCN species, genotype by year, year by CCN species, and genotype by year by CCN species interactions as the main factors. The ANOVA for the agronomic traits was performed comprising genotype, year, and genotype by year interaction as the main factors. The ANOVA for forage and grain quality characteristics was performed considering genotype as the main factor. The Fisher’s Least Significance Difference (LSD) was used to perform multiple comparisons for differentiating the significant difference among genotypes and among plant ages  on the means of each parameters examined. Statistical analysis was performed in the IBM SPSS Statistics for Windows, Version 26.0 .1 to F3, the targets of selection focused on the traits associated with perennial growth habit, such as non-shattering heads, high fertility, and ability of regrowth in the fields. The F1 hybrids showed a vigorous regrowth habit and were often sterile. Some F1 plants were able to persist adequately even for more than 7 years in the field and developed numerous tillers, but only a few tillers produced viable seeds. Then, the F2 and F3 plants were obtained by either self-pollination or backcrossing to common wheat. However, the persisting life cycle was converted into annual growth habit in some F2 and F3 plants. In parallel, the F2 plants with perennial growth habit were obtained via backcrossing to hexaploid partial amphiploid lines or Th. intermedium. Importantly, the fertility of the perennial plants was restored. A wide range of variation in the major agronomic traits, such as spike and seed morphology, was observed at the early generations . Among them, lines SX12-480, SX12-787, SX12-1150, and SX12-1269 were able to regrow from the crowns of plants after forage removal  model was carried out for the number of cysts on roots of the perennial lines and the control wheat cultivar Wenmai 19 challenged by 014–2015 . The resH. avenae is the main CCN species. In the growing season 2013–2014, the perennial wheat lines were highly resistant (line SX12-1269) or moderately resistant (lines SX12-480 and SX12-1150) to H. avenae. Although line SX12-787 displayed moderate level of susceptibility, the number of cysts (11.1 ± 3.6) was significantly lower than the susceptible control, an annual wheat cultivar Wenmai 19 (32.3 ± 5.8) (P < 0.05)  relative to that of susceptible control (23 ± 5.2) (At the field of Zhengzhou (ZZ),  < 0.05) . The fie3 ± 5.2) .H. filipjevi in the field of Xuchang (XC) in 2013–2014, lines SX12-480, SX12-1150, and SX12-1269 were able to mediate high levels of resistance and had significantly large impact on reducing the number of female cysts. Line SX12–787 also conferred resistance to H. filipjevi, but to a smaller extent .Upon natural infestation with r extent . In the sistance . Taken tP < 0.05) (P < 0.05)  . There w < 0.05) . Compare < 0.05) . Further < 0.05) .In parallel, to understand the grain yield consistency of perennial lines, a multi-year field study was conducted to compare the agronomic performance of the one-year old plants to the 2- and 3-year old ones . CompariThe first-year perennial lines, intermediate wheatgrass and an annual wheat cultivar Jinchun 9 were evaluated in the form of crude protein, crude fat, crude fiber content, the amino acid profile and the micronutrient contents .Forage harvested from the perennial lines had comparable concentrations of crude protein (96.9–99%), crude fat (75.1–101.6%), and crude fiber (95.4–96.4%) with those obtained from the commercial perennial wheat cultivar MT-2 . The levTh. intermedium. Oppositely, forage obtained from Th. intermedium was a better source of selenium (Se). The concentrations of P and Se in the perennial grains were significantly richer than in the annual grains (P < 0.05). Among the perennial lines, SX12-480 was particularly intriguing due to high P and Se concentrations in grains and was significantly more than those in MT-2 (Micronutrient concentrations for phosphorus (P) and carotene were found to be higher in the defoliated grass of perennial wheat lines SX12-1150 and SX12-787 than those produced from MT-2. Iron (Fe) level was enormously improved in the perennial lines compared to their perennial donor,  in MT-2 . VariatiP < 0.05). Line SX12-787 was slightly lower in histidine and had significantly higher level in lysine. Wheatgrass was a great source of valine, phenylalanine and leucine (P < 0.05), whereas isoleucine content was the lowest. Line SX12-1269 had the highest level of lysine and threonine, but leucine level was found to be the lowest. Line SX12-480 ranked in the middle grades in the essential amino acid profile among the perennial lines . Moreover, grains harvested from SX12-1150 were the best sources of alanine, arginine, aspartic acid, glutamic acid, glycine, proline, and serine, which covers majority, if not all, of the non-essential amino acids   . Besidesectively . Marker romatins .Th. intermedium total genomic DNA as a probe and the Chinese Spring total genomic DNA as a blocker to detect the Th. intermedium chromosomes in the perennial wheat lines. Lines SX12-787 and SX12-1150 had a mitotic chromosome number of 2n = 56. Fourteen chromosomes showed lilac-pink fluorescence signals, and 42 chromosomes were counterstained with blue fluorescence signals, indicating that both SX12-787 and SX12-1150 contained 14 Th. intermedium chromosomes and 42 wheat chromosomes  for the 3-year-old perennial genotypes relative to one- or 2-year-old genotypes were lower in general. However, spike length and the yield components  remained consistent over time in the perennial wheat lines SX12-1150 and SX12-1169. In many cases, yield declines have been observed in perennial cereals , althougF. culmorum (W.G. Smith) Sacc.], crown rot (caused by F. pseudograminearum O’Donnell & T. Aoki), common root rot [caused by Cochliobolus sativus (Ito & Kuribayashi) Drechs. ex Dastur], and damping-off/root rot (caused by Pythium spp.) , resulting in large amount of biomass loss. Little information has been available on the resistance to common root pathogens and pests in perennial donors, as well as their derivatives. So far, only few screening has been publicly available on the phenotypic responses of wheat-Thinopyrum hybrids to CCN  pathogens, such as foot rot [caused by um spp.) . The epium spp.) . Cox et s to CCN . The wheTh. intermedium and Th. elongatum (Host) D. R. Dewey, are well-known for their beneficial characteristics including improved nutritional values (Th. intermedium and Th. elongatum in hybrid breeding was contributed by their superior end-use quality and nutritional profile. Partial amphiploids derived from different Thinopyrum species were found to have higher levels of carotenoids, soluble polyphenols, alkilresorcinols, and better gluten digestibility than common wheat (Thinopyrum intermedium seed was reported to have a higher concentration of protein and almost all essential amino acids than wheat (Th. intermedium over common wheat was highlighted in terms of protein and fiber contents (During the last decades, the mineral and protein concentrations of annual wheat cultivars have shown a continuous decline coinciding with the significantly increased grain yield . Common l values . The widon wheat . Thinopyan wheat . The supcontents . NotablyTh. elongatum (Th. intermedium has already been available (Perennial growth habit is a quantitative trait and is mediated by multiple genes on different alien chromosomes . The genlongatum . It is pvailable , which pTh. intermedium entries (2 and F3 derivatives were selected for advancing to next generations (The perennial wheat generally has unique morphological traits that are quite divergent from annual wheat. Usually, the wild perennial species have performed poorly for some domestication traits, such as small spike and seed size, which need to be rectified in perennial wheat varieties . Common  entries . Thousan entries . The incerations . Howevererations .Th. intermedium lines, which showed the remarkable capability to regenerate and persist at least 3 years in the field. Promising resistance was identified to both species of H. avenae and H. filipjevi that are prevalent in China in these lines. They had superior amino acids and other nutrient compositions for forage and grain over the common wheat control. Analyses of Thinopyrum genome-specific molecular marker and GISH indicated that these lines had 12–16 Thinopyrum chromosomes. Currently, these perennial wheat lines have been evaluating at multiple geographic locations to test their adaptability to different harsh environments. Meanwhile, new perennial wheat lines are being made using these lines as parents. The development of perennial species has been proposed as an approach to improve the sustainability of agriculture, conserving natural resources while producing food and forage.We have developed four perennial wheat-The datasets generated for this study are included in the article or are available on request to the corresponding authors.LC and YS conceived and designed the research. LC, YR, YZ, QG, ZT, YN, WY, and YS conducted the experiments and analyzed the data. LC and HL wrote the manuscript. 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.