We studied the cases of single and multiple HPV infection and analyzed the correlation with negative cases, and preneoplastic and neoplastic lesions of the uterine cervix with the aim of making a contribution to the prognostic factor under discussion.Nine hundred nine women undergoing second level screening because they had been positive at cervical cytology were enrolled.All the patients underwent colposcopy and cervical biopsy with viral genotyping. We divided mHPV infection based on the number of genotypes present: infections with 2 strains, 3 strains, 4 strains and 5 or more strains.p < 0.05 were considered statistically significant.The analysis of the data was made using the χ2 test. Contingency tables were created to evaluate the correlation between single, multiple and CIN2+ infections. Values with The presence of genotype HPV16 in our study was associated with a 12 times greater risk of developing a high-grade lesion, OR = 12.70. The patients with single infections had the highest incidence of CIN2+ (34.1%) with respect to those with multiple infections (10.6%).When we studied in the mHPV infection the prevalence of the combinations between the genotypes, we found that in mHPV16 infections, the combinations HPV16, 18 and HPV16, 31 were the most frequent (55.5%) in CIN3 lesion.Our results suggest that single HPV infections have a greater risk of developing SCC with respect to multiple infections. Multiple HPV infections are relevant only in the first phase of the lesion (CIN1-CIN2), while they are absent in carcinomas, where infections are of a single genotype. In particular, among multiple infections, HPV16 infection with 2 HR genotypes is associated significantly with CIN2 / CIN3 (21/30) and has 4 times greater risk of developing a high-grade lesion. Thus, it is probable that only specific combinations of HPV  can be associated with a clinically significant impact, while other combinations can simply be correlated because of a common infection or diagnostic method used. Therefore, multiple HPV16 infections with two high-risk genotypes is a major risk of CIN2/CIN3. Human papillomavirus (HPV) Infection of the cervix is a sexually transmitted disease and a significant risk factor for the development of cervical intraepithelial neoplasia (CIN) . HoweverThe prevalence of the various viral genotypes shows considerable differences worldwide  and the The clinical, virological and epidemiological significance of mHPV is still not clear: some reports in the literature suggest a possible role in the development and progression of cervical neoplasia, while other studies  show howFrom January 2014 to November 2017, at the outpatients’ clinic for colposcopy of the University of Catania, Italy, 909 women undergoing second level screening because they had been positive at cervical cytology were enrolled. The data were kept in a database at the University Hospital of Catania, Italy for a retrospective study.The study was carried out in conformity with the Declaration of Helsinki, 1975.women positive at cytology for lesions correlated to HPVwomen with preneoplastic or neoplastic lesions of the uterine cervix diagnosed by a histological examThe inclusion criteria were the following:Women who were pregnant, immuno-depressed or with infections caused by human immunodeficiency virus (seropositive) were not enrolled. The age of the patients was between 16 and 59 years, mean 32 ± 8,3 years. All the patients underwent colposcopy and cervical biopsy with viral genotyping. The histological evaluation was carried out on biopsy samples from colposcopy and/or cone samples from LEEP. Histology was diagnosed according to the OMS classification as CIN2 + for all the cases of lesions CIN2, CIN3 and SCC.In agreement with the LAST Project, we treated CIN1 as a simple viral lesion and we considered only CIN2 + lesions as true preneoplastic lesions.Detection of viral DNA (HPV test) was carried out using Polymerase Chain Reaction (PCR) after the extraction of the cervical sample using Thin-prep. Automatic DNA extraction was carried out using the Nucli Senseasy MAG system  following the HPV 1.1 protocol of the manufacturer. HPV DNA amplification was made by HPV-HS Bio  nested-PCR for the detection of the HPV-DNA sequence inside ORF L1, according to the manufacturer’s instructions. HPV typing was carried out with a probe specific for the most frequent types of HPV . HPV typing identified 11 LR genotypes  and 18 HR genotypes . The positive samples at nested-PCR but negative in reverse line blot for any of these types were considered as undetermined HP. The cervical swab for the HPV test was taken from the endocervical canal and the transformation zone. We divided the multiple infections based on the number of genotypes present: infections with 2 strains, 3 strains, 4 strains and 5 or more strains.p value) of the difference between groups. Contingency tables were created to evaluate the correlation between single, multiple and CIN2+ infections. Values with p < 0.05 were considered statistically significant.The statistical analysis of the data was carried out with the software package SPSS 15.0 . The analysis of the data was made using the χ2 test and, if necessary, Fisher’s exact test was used to calculate the statistical significance  presented a low-grade preneoplastic lesion (CIN 1); 64 women (8.6%) had a moderate-grade lesion (CIN 2), 105 women (14%) had a sever-grade lesion (CIN 3); and 5 women (0.7%) had squamosa carcinomas (SCC). Finally, 115 (15.4%) patients had a negative histological diagnosis (Table .3% preseThere were 360 48.2%) single infections in our group with 123 lesions (34.1%) CIN2+. The typology of lesions with the highest percentage of a single infection was SCC with 100%, followed by CIN3 with 78%, CIN2 with 56.2% and finally negative and CIN1 with 48.6 and 39.5%, respectively. The genotype most frequently found was genotype 16, present in 142/360 cases (39.4%), and was responsible for 76.4% (94/123) of the cases of CIN2+, 21.6% of the cases of CIN1 and 16% of negatives (Table % single In the present study the patients with a single infection from HPV 16 had the highest incidence (76.4%) of CIN2+ with respect to the other HR genotypes (23.5%).In particular, the presence of genotype HPV16 in our study was associated with a 12 times greater risk of developing a high-grade lesion (CIN2+), OR = 12.70; (IC 95% = 7.52–21.42). After genotype 16, the genotype most represented was 31 with 36 cases, of which 11 CIN2+, while the genotypes 18 and 45 were poorly represented with 17 cases of which 8 CIN2+ and 6 cases of 1 CIN3. Other HR HPV found were 33 with 8 cases, of which 2 CIN3, 35 with 4 cases, of which 2 CIN3 and 52 and 58 both with 1 case of CIN2.  multiple infections in our study with an incidence of high-grade lesions of 10.6% (41/387). The typology of the lesion with the highest percentage of multiple infections was CIN1 with a frequency of 60.4% and the negative cases were 50.4%. The percentage decreases more in CIN2 (43.7%) and progressively in CIN3 (22.1%), arriving at zero in carcinomas, characterized exclusively by single infections  with a prevalence of 74.5% (38/51), this was significatively higher in patients with CIN2/CIN3, OR = 5.77 (IC 95% = 2.95–11.26). We studied in particular genotype 16 (Table OR of the single infections from HPV16 associated with CIN2+ was greater than the OR of multiple HPV16 infections Table .Table 5We divided multiple infection by the number of genotypes present (strains) into 2 strains, 3 strains, 4 strains, 5 strains or more. We found that increasing the histological grade of the lesion, decreases the number of HR genotypes present; in fact, CIN3 lesion has two high-risk genotypes in 82.6% of cases (19/23). the remaining 4 cases (4/23) had three genotypes. We did not have any cases of CIN3 lesion with 4 or 5 genotypes.We correlated genotype 16 with the number of strains and the histological diagnosis  in CIN3 Fig. .Fig. 2MFrom the OR analysis, multiple HPV16 infections with 2 high-risk genotypes, with respect to infections with 3 or more genotypes, were significant with an OR = 3.92 (IC% 1.70–9.03) for CIN2+.To date, only a few studies have reported a relationship between mHPV infections and SCC, and the results have not been conclusive. Some studies have suggested a possible role of mHPV in the development of SCC –10, whilFife KH  found, aRolòn  did not The relevance of multiple infections in the last few years has shown an increase in cases thanks to improved PCR sensitivity. In studies published in the 1990s, multiple infections were only 4%, in more recent studies multiple infections have reached 15.7%. The only exception are the women affected by HIV who often present with multiple infections (80%) , in thesThis changes in immuno-competent women in whom the prevalence of mHPV varies from 24.8% to 52,6% between all HPV positive women .In our study, the prevalence of multiple infections was 51,8%, in particular 60.4% in patients with CIN1, to 22% in those with CIN3; the prevalence of multiple infections in the group of patients with a negative histologic exam was 51,3%; we had no SCC cases.The prevalence of multiple infections is high, thus, in negatives and cases of slight dysplasia, CIN2 and CIN3 decreased until it was absent in neoplasia, while the prevalence of infections from a single genotype was lower among negatives and CIN1, rising again in severe dysplasia and carcinoma , while they are absent in carcinomas in which there are only single lesions. Moreover, we found that increasing grades of dysplasia decreases the number of the genotypes present, in fact, transversal analysis of the our data showed that the detection of CIN2-CIN3 in the presence of multiple HPV infections seems to be significantly correlated to the presence of infections due to two strains of high-risk genotypes . In about 95% of invasive cervical tumors positive for HPV, and in all our cases of SCC, only 1 type of HPV was found . This leOur results suggest that single HPV infections have a greater risk of developing SCC with respect to multiple infections. Multiple HPV infections are relevant only in the first phase of the lesion (CIN1-CIN2), while they are absent in carcinomas, where infections are of a single genotype. Also in our study the genotype 16 is the one with the highest oncogenic risk, genotype 16 is significatively correlated with high-grade lesions so much so that some authors consider reserving dedicated guide-lines for HPV16 positive patients .In particular, among multiple infections, HPV16 infection with 2 HR genotypes is associated significantly with CIN2 / CIN3 (21/30) and has 4 times greater risk of developing a high-grade lesion. Thus, it is probable that only specific combinations of HPV  can be associated with a clinically significant impact, while other combinations can simply be correlated because of a common infection or diagnostic method used. Therefore, multiple HPV16 infections with two high-risk genotypes is a major risk of CIN2/CIN3.The limits of our study are connected to the samples studied in as much as the women in our study came from a second level center.Further clinical studies are needed to determine the mechanism of these mHPV infections and if the clinical results are correlated with a particular combination of types of HPV.


Treatment of cervical intraepithelial neoplasia (CIN) is associated with an increased risk of preterm delivery (PTD) although the exact pathomechanism is not yet understood. Women with untreated CIN also seem to have an increased risk of PTD. It is unclear whether this is attributable to human papillomavirus (HPV) infection or other factors. We aimed to investigate whether HPV infection shortly before or during pregnancy, as well as previous treatment for CIN, is associated with an increased risk of PTD and other adverse obstetric and neonatal outcomes.n = 1,044,023). The study population had a mean age of 30.2 years (SD 5.2) and a mean body mass index of 25.4 kg/m2 (SD 3.0), and 44% of the women were nulliparous before delivery. Study groups were defined based on cervical HPV tests, cytology, and histology, as registered in the Swedish National Cervical Screening Registry. Women with a history of exclusively normal cytology  were compared to women with positive HPV tests  or abnormal cytology  within 6 months prior to conception or during the pregnancy, women treated for CIN3 before delivery , and women with CIN2+ diagnosed after delivery . Study groups were compared concerning obstetric and neonatal outcomes by logistic regression, and comparisons were adjusted for socioeconomic and health-related confounders. HPV infection was associated with PTD , preterm prelabor rupture of membranes (pPROM) , prelabor rupture of membranes (PROM) , and neonatal mortality . Treatment for CIN was associated with PTD , spontaneous PTD , pPROM , PROM , intrauterine fetal death , chorioamnionitis , intrapartum fever , neonatal sepsis , and neonatal mortality . Women with CIN2+ diagnosed within 3 years after delivery had increased PTD risk . Limitations of the study include the retrospective design and the fact that because HPV test results only became available in 2007, abnormal cytology was used as a proxy for HPV infection.This was a retrospective population-based register study of women with singleton deliveries registered in the Swedish Medical Birth Register 1999–2016  is associated with an increased risk of preterm delivery (PTD), preterm prelabor rupture of the membranes (pPROM), and neonatal mortality. However, the exact biological mechanism is still unknown.Women with untreated CIN also seem to have an increased risk of PTD. It is unknown whether HPV infection can cause PTD or other adverse obstetric outcomes.Although ascending bacterial infection is an established leading cause of PTD, there is limited knowledge concerning whether PTD after excisional treatment for CIN is associated with infectious complications.This retrospective population-based register study (1999–2016) found that HPV infection shortly before or during pregnancy is associated with an increased risk of PTD, pPROM, prelabor rupture of the membranes, and neonatal mortality.Treatment for CIN was also associated with these adverse outcomes and with an even higher risk for PTD and pPROM.This study presents new evidence that previous treatment for CIN is associated with an increased risk of maternal and neonatal infectious complications.Although we cannot claim causality due to the register-based study design, our results suggest that pregnancies after treatment for CIN should be regarded as high-risk pregnancies and counseled accordingly.These results support the idea that strategies to mitigate HPV infection, such as vaccination programs, may be beneficial for maternal and neonatal pregnancy outcomes. Human papillomavirus (HPV) infection is the most common genital infection in women of reproductive age. Persistent genital infection with high-risk HPV is causally associated with cervical cancer and its precursor, cervical intraepithelial neoplasia (CIN) . The widThe mechanism behind excisional treatment increasing the risk of PTD remains unclear. The hypotheses include increased risk of ascending bacterial infection, HPV-induced immunomodulation, and acquired “mechanical weakness” secondary to loss of cervical tissue . AlthougThe magnitude of the increase in risk for PTD attributed to treatment differs between studies ,10–15 anIn summary, the risk of PTD increases after treatment for CIN, but the underlying mechanism is still not known. Women with untreated CIN also seem to have an increased risk of PTD, but it has not been established whether this risk is attributable to HPV infection during pregnancy. Neither the associations between untreated HPV infection in pregnancy and PTD and other obstetric and neonatal outcomes, nor the association between previous treatment for CIN and infectious pregnancy complications, have as yet been studied in a comprehensive population-based study. In this large Swedish national population-based study, we aimed to investigate whether HPV infection shortly before or during pregnancy is associated with an increased risk of PTD and other adverse obstetric and neonatal outcomes. We also aimed to study the association between previous treatment for CIN and PTD and other adverse obstetric and neonatal outcomes, with special focus on infectious complications at birth.This study is a retrospective population-based study using data from different Swedish health and population registers. Data linkage between the registers was performed with the unique personal identification number held by each resident of Sweden. The Swedish Medical Birth Register (MBR), established in 1973 and managed by the Swedish National Board of Health and Welfare, is the object of compulsory registration and thus comprises all live births and stillbirths in Sweden at 22 completed weeks of gestation and up . The infThis study is reported as per the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline (2 (SD 3.0); 44% of the women were nulliparous before delivery. Of these, 400,583 women had at least 1 delivery that fulfilled the criteria for inclusion in a study group .A reference group was defined, consisting of women who had a history of exclusively normal cervical cytology results until the end of the study period and at least 1 sample taken in the 3 years preceding the included delivery .HPV infection (cytology): abnormal cytology .HPV infection (HPV test): positive cervical HPV test .Subsequent CIN2+ group: at presumed risk of persistent HPV infection based on a histological diagnosis of CIN2 or CIN3, adenocarcinoma in situ (AIS), or cervical cancer any time after delivery .Treated group: histologically diagnosed CIN3 before conception. Since there is no national Swedish register covering treatment for CIN, CIN3 was used as a proxy for treatment as these women are always treated in Sweden, a policy that has been consistent over the study period , 1986–1995 , and 1996–2016  .In the treated group, the interval from treatment to delivery was determined, as was the interval from delivery to CIN2+ diagnosis in the subsequent CIN2+ group.The primary outcome was PTD at 22–36 weeks of gestation (154–258 days). Gestational age was retrieved from the MBR using the best estimate, i.e., ultrasound determination when available and last menstrual period or estimation of gestational age at delivery ward in the remaining cases. Subanalyses for early PTD  and for very early PTD  were performed.Secondary outcomes were pPROM (determined according to ICD-10 codes in the MBR) and spontaneous PTD .Further studied outcomes comprised prelabor rupture of membranes (PROM) in term pregnancies (≥37 weeks of gestation), chorioamnionitis, intrapartum fever, neonatal sepsis, Apgar score < 7 at 5 minutes, intrauterine fetal death, neonatal mortality (1–28 days), and small for gestational age (SGA), defined as birthweight less than −2 SD according to the Swedish reference curves  S4 Tabl.The following background variables were retrieved from the MBR and from Statistics Sweden registers: age at delivery, BMI, smoking before and during pregnancy, marital status, education level at delivery, employment at delivery, disposable household income in the 3 years preceding delivery, country of birth, parity, infant’s sex, and assisted reproduction, as well as chronic renal disease, diabetes, epilepsy, and chronic hypertension as reported in antenatal care records.https://www.r-project.org/) and SPSS software . A significance level of 0.05 was applied throughout.Descriptive data are presented as number and percentage for categorical variables and as mean (SD) and median (interquartile range) for continuous variables. Analyses were performed using R  and the last delivery before diagnosis of CIN2+ in the subsequent CIN2+ group. The first eligible delivery after treatment in the treated group was compared to the last delivery in a woman included in the other exposure groups. This was done to better match for age and parity, since women in the treated group naturally were older than women in the other exposure groups. Women who had both a delivery after treatment and a previous delivery included in any of the other exposure groups were excluded from the treated group when the treated group was compared to the other exposure groups.HPV infection can exist for a long time before diagnosis of CIN2+. Therefore, the subsequent CIN2+ group was compared to the reference group after stratification for interval (0–3 years or >3 years) to diagnosis of CIN2+, based on the median interval from delivery to CIN2+ diagnosis of 3.2 years. The 2 groups were also compared with each other.Women with a delivery included in the subsequent CIN2+ group followed by a delivery included in the treated group were included in paired analyses with conditional logistic regression analysis.When comparing the HPV infection (HPV test) group with other groups, only deliveries after 2006 were included, since no HPV tests were registered before that year. In order to test whether cytology was a valid substitute for a positive HPV test, the 2 HPV infection groups were compared to each other. In these analyses, women with both a positive HPV test and abnormal cytology were excluded from the HPV infection (cytology) group.2], normal weight [18.5–24.9 kg/m2], overweight [25.0–29.9 kg/m2], obese ≥30.0 kg/m2], missing), marital status , country of birth , infant’s sex , smoking , highest disposable household income in the 3 years preceding delivery (population divided into tertiles for every year), education level , and assisted reproduction . The paired analyses were adjusted for BMI, marital status, infant’s sex, smoking, and assisted reproduction, with the group definitions defined above.The multivariable analyses were adjusted for potential confounding factors, including background variables with an uneven distribution between the groups, i.e., year of delivery , maternal age , parity , BMI  and also adjusted for gestational age.In all stratified analyses, differences in odds ratios between strata were analyzed by an interaction term between the stratum and the group in logistic regression.The study was approved by the Regional Ethical Review Board at the University of Gothenburg (registration number 614–15). In this retrospective register-based study, no consent was obtained from the participants since the data were analyzed anonymously.n = 32,457 women) were older than 35 years, and similarly 10% in the subsequent CIN2+ group  and 12% in the HPV infection (cytology) group  were older than 35 years, while age over 35 years was more frequent in the HPV infection (HPV test) group  and the treated group . Nulliparity was most frequent in the treated group  and HPV infection groups  and less frequent in the reference group  and subsequent CIN2+ group . Smoking during pregnancy was less common in the reference group  than the exposure groups .Background factors for the study population are presented in The comparisons presented below for different obstetric outcomes are adjusted logistic regression analyses; unadjusted analyses are presented in In the total dataset of 1,787,842 deliveries in 1,044,023 women, 87,727 deliveries (4.9%) were PTD and 62,951 deliveries were spontaneous PTD (3.5%).p < 0.001), but an increased risk was also found in the HPV infection (cytology) group  and the HPV infection (HPV test) group  (p < 0.001) and the HPV infection (cytology) group had aOR 1.18  (Compared to the reference group (4.6%), the treated group had the highest risk of PTD  ; Fig 2A.= 0.001) ; Fig 2B.= 0.001) .p < 0.001) and spontaneous PTD , the HPV infection (cytology) group , and the HPV infection (HPV test) group  . Compared to the reference group (1.5%), the risk of pPROM was increased in the treated group  ; Fig 3. = 0.001) , althoug= 0.001) . Compare= 0.001) ; Fig 3.p < 0.001) (p < 0.001) (p = 0.002). The associations for the HPV infection (cytology) group were oriented in the same direction but were not statistically significant . Women in the treated group had an increased risk of PROM at term compared to the reference group   and comp< 0.001) . Moreovenificant .In the study population, 41,323 infants were born SGA (2.3%). Data were missing for 4,028 deliveries (0.2%). There was no difference between the reference group and any of the exposure groups in regard to SGA risk .In the study population, 22,968 babies were born with a 5-minute Apgar score < 7 (1.3%). The treated group had higher risk of low Apgar score compared to the reference group  and to tp = 0.006) and in the treated group , while it was decreased in the subsequent CIN2+ group   reported cases of intrauterine fetal death in the study population. Compared to the reference group (0.2%), the risk was increased in the HPV infection (cytology) group  . This de= 0.026) .p < 0.001), intrapartum fever , and neonatal sepsis  compared to the reference group  deliveries were complicated by chorioamnionitis, 10,832 (0.6%) by intrapartum fever, and 12,923 (0.7%) by neonatal sepsis. The treated group had an increased risk of chorioamnionitis , but not in the term delivery group . There was no clear increase in infectious complications in the HPV infection groups  and women without pPROM , but was significantly higher in women with pPROM (p = 0.032). Similarly, the increase in risk of neonatal sepsis in the treated group compared to the reference group was significantly higher in women with PTD with pPROM  than in women with PTD without pPROM  (p = 0.015 for the interaction).Stratifying for pPROM in the PTD cases revealed that the aOR for chorioamnionitis was higher in the treated group than in the reference group, in both women with pPROM , as well as in the HPV infection (cytology)  and HPV infection (HPV test)  groups, compared to the reference group . Neonatal mortality was increased in the treated group  and HPV test .After additional adjustment for PTD, the increased risk of neonatal mortality disappeared in the treated group, suggesting that the risk increase had been mediated by gestational age. However, the risk was still increased in the HPV infection groups, i.e., cytology  than in women without infectious complications  (p = 0.015 for the interaction).Of those with neonatal mortality, 4.1% in the reference group and 14.9% in the treated group also contracted chorioamnionitis, and 6.4% in the reference group and 25.5% in the treated group had infants with neonatal sepsis. For an illustration of the relationship between neonatal mortality, infectious complications, and PTD, see p = 0.007), spontaneous PTD , and pPROM  were increased after treatment  and PROM  in the HPV infection (HPV test) group compared to the HPV infection (cytology) subgroup with no HPV test, no significant differences in outcomes were found between the HPV infection groups  , perhapsIt is still unclear whether the HPV infection itself, common confounders, or a genetic predisposition is the causal link behind the association between CIN and PTD. However, our results suggest that HPV infection might be an important factor. Women developing CIN2+ after delivery also had a small increased risk of PTD and spontaneous PTD, although it was not significant in the subgroup of women who were diagnosed more than 3 years after delivery. This may possibly reflect an ongoing HPV infection during pregnancy and delivery in women with earlier diagnosis of CIN2+ after birth. These findings contradict the hypothesis of a common genetic susceptibility to both persistent HPV infection and PTD or an association merely due to confounding.To the best of our knowledge, this is the largest study to date examining the associations of HPV infection shortly before or during pregnancy and treatment for CIN with obstetric and neonatal outcomes. It comprises an entire population, and we believe it compares the largest number of deliveries before and after treatment for CIN in the same women published so far.As this is an observational study, causality cannot be established and residual confounding cannot be ruled out despite thorough adjustments. However, the risk increases for PTD, spontaneous PTD, pPROM, and chorioamnionitis found in the treated group were supported by similar findings in the paired analyses, in which 2 deliveries in the same woman were compared.The definition of HPV infection in this study has some potential limitations. To begin with, relying on positive screening tests may lead to an underestimation of the true incidence and prevalence as many infections can occur and resolve between testing. A small minority of HPV tests might have been positive due to exclusive detection of low-risk strains, although the tests used at Swedish laboratories predominantly specifically detect high-risk HPV. Since HPV test results only became available in 2007, abnormal cytology was used as a proxy for HPV infection, as in earlier studies ,38. A SwThe use of HPV tests in cervical cancer screening has increased during the last decade, which will facilitate large population-based analysis of the relationship of HPV infection and pregnancy outcomes in the near future. Our results support general HPV vaccination programs, and future studies in vaccinated cohorts will ascertain whether PTD and neonatal mortality decrease. Additional experimental studies are also needed to establish the causal pathways linking HPV infection and treatment for CIN to PTD and other adverse obstetric outcomes. This may be a key to understanding the biological mechanism leading to PTD. We suggest that pregnancies after treatment for CIN should be regarded as high-risk pregnancies.This large population-based study demonstrates that women with HPV infection shortly before or during pregnancy have an increased risk of PTD, pPROM, PROM, and neonatal mortality. Treatment for CIN was associated with further increased risk of PTD and pPROM and was also associated with PROM, neonatal mortality, and an increased risk of maternal and neonatal infectious complications. These results suggest that pregnancies after treatment for CIN should be regarded as high-risk pregnancies and counseled accordingly. Furthermore, our results support general HPV vaccination programs.S1 STROBE Checklist(DOC)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S7 Table(DOC)Click here for additional data file.S8 Table(DOC)Click here for additional data file. 7 Jan 2021Dear Dr Wiik, Thank you for submitting your manuscript entitled "Treated and untreated human papillomavirus infection is associated with preterm delivery and neonatal mortality - a Swedish population-based study" for consideration by PLOS Medicine.Your manuscript has now been evaluated by the PLOS Medicine editorial staff and I am writing to let you know that we would like to send your submission out for external peer review.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. 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For instructions see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it. Please ensure that the paper adheres to the PLOS Data Availability Policy  in the subtitle . We suggest: “Associations of reated and untreated human papillomavirus infection with preterm delivery and neonatal mortality: A Swedish population-based study” or similar.2. Abstract: Methods and Findings: Please provide some of the key summary demographics of the population.3. Abstract: Methods and Findings: Please quantify the main results .4. Abstract: Methods and Findings: In the last sentence of the Abstract Methods and Findings section, please describe the main limitation(s) of the study's methodology.5. Abstract: Conclusions: Please address the study implications without overreaching what can be concluded from the data; the phrase "In this study, we observed ..." may be useful.6 .Abstract: Conclusions: Please avoid language implying causality (“Treatment for CIN increases risks ruther and seems to confer a risk…”)https://journals.plos.org/plosmedicine/s/revising-your-manuscript#loc-author-summary7. Author summary: At this stage, we ask that you include a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract. Please see our author guidelines for more information: 8. Main text: Throughout: Please do not include spaces within brackets of in-text citation  and please place the brackets before the punctuation .9. Introduction: Please do conclude the Introduction by mentioning your study objective/hypothesis, but this does not need to be in a separate “Objectives” section within the introduction.10. Methods/ figure 1: Is it possible to give additional clarification/explanation of the 600,000+ excluded women (did not fulfil criteria of any group) from Fig 1 flow chart (reasons for exclusion)?11. Methods: Please add the following statement, or similar, to the Methods: "This study is reported as per the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline (S1 Checklist)."12. Methods: Did your study have a prospective protocol or analysis plan? Please state this (either way) early in the Methods section.a) If a prospective analysis plan  was used in designing the study, please include the relevant prospectively written document with your revised manuscript as a Supporting Information file to be published alongside your study, and cite it in the Methods section. A legend for this file should be included at the end of your manuscript. b) If no such document exists, please make sure that the Methods section transparently describes when analyses were planned, and when/why any data-driven changes to analyses took place. c) In either case, changes in the analysis-- including those made in response to peer review comments-- should be identified as such in the Methods section of the paper, with rationale.13. Results: Please provide more quantitative description of the background differences in study groups .14. Results: For all results presented in the text, please provide both the 95% CIs and p values.15. Results: “Small for gestational age and Apgar score” section: please clarify which groups are meant by “The groups did not differ…” in the second sentence of this paragraph.16. Results: “HPV infection groups” section: please report the result for this association “Except for a higher risk of pPROM and PROM in the HPV infection (HPV test) group” and specify the comparison group.17. Discussion: Please present and organize the Discussion as follows: a short, clear summary of the article's findings; what the study adds to existing research and where and why the results may differ from previous research; strengths and limitations of the study; implications and next steps for research, clinical practice, and/or public policy; one-paragraph conclusion. 18. References: Please double check the formatting . Please provide an English translation for ref 26, 28, 29, 30.19. Table 2, Table 3, Table, 4, and Table 5: Please present actual p values rather than representing with asterisks. Please provide the unadjusted results in addition to the adjusted analyses .20. Figures 2 and 3: Please include the p values with the arrows, if possible. As mentioned for the Tables, the unadjusted analyses should be included, whether in the main text or supporting information.21. S5 Table: Please provide p values for each comparison, rather than indicating p values with asterisks. Please provide the unadjusted analyses.22. STROBE checklist: Thank you for including the checklist. It would be appropriate to refer to locations in the text for each item with an additional column on the right-hand side of the table- When completing the checklist, please use section and paragraph numbers, rather than page numbers.Comments from the reviewers:Reviewer #1: This retrospective population-based register study of women with singleton deliveries registered in the Swedish Medical Birth Register 1999-2016, aims to investigate whether HPV infection in conjunction with pregnancy, as well as previous treatment for CIN, is associated with an increased risk of PTD and other adverse obstetric and neonatal outcomes.Comments:There are some typos in the text that need rectifying.The STROBE checklist is suitably included in the supplementary material.Descriptive data are appropriately presented, and the comparison of exposure groups by logistic regression  is a technically valid and robust approach.Are there other factors that may be associated with PTD that can be included as potential confounders within the models? Alcohol or drug abuse, for instance? Table 1 is very informative. Did the authors complete any statistical testing to explore if differences between groups are significant or not?Table 2 is an efficient and effective way to communicate the main results.Perhaps more caution should be applied when drawing inferences from the paired analysis. These results might not be generalisable due to the specific nature of the cohort analysed. For instance: Might multiple births be affecting the outcomes? Or, the effect of having treatment after at least one prior delivery? Are there other potential intra-woman confounders in this analysis ?The main limitations have been acknowledged, including the fact that this in an observational study , the potential for confounding, and the possibility of misclassification.Overall, this is a thorough study looking at different subgroups and their outcomes over a large cohort and incorporating a good use of visualisation in the figures which helps to clearly communicate the research findings.Reviewer #2: This is a large population-based study that investigates in detail the association between HPV infection, CIN or CIN treatment and PTB. The authors have put a lot of effort and have conducted a comprehensive analysis by including all possible comparison groups  and performing all possible comparison combinations including self-matching. The authors very nicely associate their findings with previous studies, and suggest potential mechanism of PTB based on their findings. Although the main findings of this study have been suggested by other studies, findings were conflicting or not conclusive before as the authors describe in the manuscript. This is the largest and most comprehensive study so far trying to cast light on the long-standing debate whether HPV infection per se increases the risk of PTB, and therefore I highly recommend it for publication in PLoS Medicine.I have only some minor comments to the authors:-Line 34: Replace "outcome" with "outcomes"-In the HPV infection group, I do not understand what the authors mean by "between six months prior to conception and delivery". I think that they mean "within six months prior to conception or during pregnancy".I suggest rephrasing throughout the text.-Additionally, in abstract the various comparison groups are not very clear. I understand that there is a word limitation, but if possible, authors could describe the different groups in more details, especially the timing of the abnormal test/intervention. By reading the abstract only, it is not very clear for example whether the HPV positivity group includes women with HPV prior to pregnancy, during pregnancy, or both.-Page 7, lines 16-17: I suggest adding citations for the statistical software that the authors have used.-Page 7, line 32: Replace "Subsequent" with "subsequent"Reviewer #3: This is a well-written substantive retrospective paper based on over 1 million births in Sweden. 1. p.5 Line 29. Women with exclusively normal cytology. Does that mean that they had documented negative HPV tests? I ask because if they did not, how do you know that they were not also HPV positive which would put them in the comparison group. When the term Lifetime is used -what does that mean? Is it the time prior to delivery? 2. p.6, line 13: It is possible that women who have CIN2+ at a young age can clear spontaneously. Did you exclude because they got categorized into the other exposure groups?3. How did you account for parity? Were women who had multiple births counted as one person or was each birth counted? This could mean a women with multiple preterm births could be counted multiple times with the same characteristics over and over again.4. P2, line 31: With the high prevalence of HPV infection in anyone who has ever been sexually active, it may be more appropriate to say, "Women with detectable HPV infection..."Any attachments provided with reviews can be seen via the following link:[LINK] 5 Mar 2021AttachmentResponse to reviewers.docSubmitted filename: Click here for additional data file. 19 Apr 2021Dear Dr. Wiik,Thank you very much for re-submitting your manuscript "Associations of treated and untreated human papillomavirus infection with preterm delivery and neonatal mortality: A Swedish population-based study" (PMEDICINE-D-20-06245R2) for review by PLOS Medicine.I have discussed the paper with my colleagues and the academic editor. I am pleased to say that provided the remaining editorial and production issues are dealt with we are planning to accept the paper for publication in the journal.The remaining issues that need to be addressed are listed at the end of this email. Please take these into account before resubmitting your manuscript:[LINK]***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***In revising the manuscript for further consideration here, please ensure you address the specific points made by the editors. In your rebuttal letter you should indicate your response to the editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. If you haven't already, we ask that you provide a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract.Please also check the guidelines for revised papers at plosmedicine@plos.org) if you have any questions or concerns.We expect to receive your revised manuscript within 1 week. Please email us , which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.Please ensure that the paper adheres to the PLOS Data Availability Policy  such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.plosmedicine@plos.org.Please note, when your manuscript is accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you've already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosmedicine@plos.org.  If you have any questions in the meantime, please contact me or the journal staff on We look forward to receiving the revised manuscript by Apr 26 2021 11:59PM.   Sincerely,Caitlin Moyer, Ph.D.Associate Editor PLOS Medicineplosmedicine.org------------------------------------------------------------Requests from Editors:1. Abstract: Methods and Findings: Please clarify if the OR reported in the text here are the adjusted ORs.2. Abstract: Methods and Findings: Page 3, Line 1-2: We suggest revising the limitations sentence as “Limitations of the study include the retrospective design and the fact that because HPV test results only became available in 2007, abnormal cytology was used as a proxy for HPV infection.”3. Author summary: What did the researchers do and find? In the first bullet point of this section, we suggest removing “...is, so far, the largest published and the first to demonstrate…”4. Author summary: What do these findings mean? We suggest providing some clarification on the final bullet point in this section, for example “These results support the idea that strategies to mitigate HPV infection, such as vaccination programs, may be beneficial for maternal and neonatal pregnancy outcomes.” or similar.5. Methods: Page 6: Lines 6-8: Please clarify this sentence to: “We did not publish the analysis plan but the overall exposure, outcomes, confounders and analyses were planned in 2015 by the research team based on hypotheses drawn from previous studies.”6. Methods: Page 6, line 19: Please change to “30.2 years”7. Results: Page 17, line 4: We suggest replacing “non-significant” with “not statistically significant” or similar.8. Discussion: Page 21: Lines 25-26: We suggest revising to: “However, our results suggest that HPV infection might be an important factor.”9. Discussion: Page 22: Line 1-2: Please revise to: “To the best of our knowledge, this is the largest study to date examining the associations of HPV infection in conjunction with pregnancy and treatment for CIN with obstetric and neonatal outcomes.” Please also temper the second sentence (compares the largest number…) at lines 3-4 with “to the best of our knowledge” or similar.10. Discussion: Page 22, Line 11: We suggest changing “...implies an obvious underestimation…” to “...may lead to an underestimation of the true incidence…” or similar.11. Discussion: Page 22, Line 27-28: Please revise to “For the paired analyses it was not possible to adjust for maternal age and parity and these results must thus be interpreted with caution.”12. Discussion: conclusions: Page 23: Line 9-11: Please revise to avoid causal implications: “Treatment for CIN was associated with further increased risk of PTD, pPROM, PROM and neonatal mortality and was associated with increased risk of maternal and neonatal infectious complications. These results suggest it may be beneficial to regard pregnancy after treatment for CIN as high-risk pregnancies and counselled accordingly.”https://journals.plos.org/plosmedicine/s/submission-guidelines#loc-references13. References: Please check journal name abbreviations . Please remove the italics from ref 17, and 27. Please use the "Vancouver" style for reference formatting, and see our website for other reference guidelines 14. Figure 2. Please give an overall title for Figure 2, noting the differences between panels a and b. 15. Figure 4 and Figure 5. Please provide the numbers in addition to percentages.16. Table 5: Please note that the line numbers are overlapping with the right-side column for the last 5 rows.17. Supporting information: If possible, please include each item as a separate file.18. S8 Table: It is not clear what part of the table this reference is supporting Any attachments provided with reviews can be seen via the following link:[LINK] 24 Apr 2021AttachmentRebuttal letter .docSubmitted filename: Click here for additional data file. 29 Apr 2021Dear Dr Wiik, On behalf of my colleagues and the Academic Editor, Jenny E Myers, I am pleased to inform you that we have agreed to publish your manuscript "Associations of treated and untreated human papillomavirus infection with preterm delivery and neonatal mortality: A Swedish population-based study" (PMEDICINE-D-20-06245R3) in PLOS Medicine.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. 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If the press office is planning to promote your findings, we would be grateful if they could coordinate with http://www.plos.org/about/media-inquiries/embargo-policy/.We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at Thank you again for submitting to PLOS Medicine. We look forward to publishing your paper. Sincerely, Caitlin Moyer, Ph.D. Associate Editor PLOS Medicine


In Germany, HPV vaccination of adolescent girls was introduced in 2007. Nationally representative data on the distribution of vaccine-relevant HPV types in the pre-vaccination era are, however, only available for the adult population. To obtain data in children and adolescents, we assessed the prevalence and determinants of serological response to 16 different HPV types in a representative sample of 12,257 boys and girls aged 1–17 years living in Germany in 2003–2005.Serum samples were tested for antibodies to nine mucosal and seven cutaneous HPV types. The samples had been collected during the nationally representative German Health Interview and Examination Survey for Children and Adolescents in 2003–2006. We calculated age- and gender-specific HPV seroprevalence. We used multivariable regression models to identify associations between demographic and behavioral characteristics and HPV seropositivity.We found low but non-zero seroprevalence for the majority of tested HPV types among children and adolescents in Germany. The overall seroprevalence of HPV-16 was 2.6%, with slightly higher values in adolescents. Seroprevalence of all mucosal types but HPV-6 ranged from 0.6% for HPV-33, to 6.4% for HPV-31 and did not differ by gender. We found high overall seroprevalence for HPV-6 with 24.8%. Cutaneous HPV type seroprevalence ranged from 4.0% for HPV-38 to 31.7% for HPV-1. In the majority of cutaneous types, seroprevalence did not differ between boys and girls, but increased sharply with age, . Associations between behavioral factors and type-specific HPV prevalence were determined to be heterogeneous.We report the first nationally representative data of naturally acquired HPV antibody reactivity in the pre-HPV-vaccination era among children and adolescents living in Germany. These data can be used as baseline estimates for evaluating the impact of the current HPV vaccination strategy targeting 9–14-years-old boys and girls.The online version contains supplementary material available at 10.1186/s12879-022-07028-8. Infections with human papillomaviruses (HPVs) are among the most common sexually transmitted infections but can also be transmitted perinatally. HPV belongs to the diverse Papillomaviridae virus family and comprises over 200 different types, which can be further categorized based on different parameters –3. HPV tChildren and adolescents before sexual debut can also be affected by HPV infections. Typical disease manifestations of HPV infection in children are skin warts , commonlThere are only few HPV serology data in children available, even though research on HPV infections in children already started more than 50 years ago –19. ReseTo investigate the prevalence of HPV infections, DNA testing is used as the reference standard for the detection of current HPV infections . While DOur study aimed to determine age-specific HPV seroprevalence in a representative sample of 12,257 boys and girls aged 1–17 years living in Germany in the years 2003 to 2005. As HPV vaccination of girls has only been introduced in Germany in 2007, our results show naturally acquired antibody reactivity in the pre-HPV vaccination era.Archived serum samples were obtained from the German Health Survey for Children and Adolescents (KiGGS) carried out by the Robert Koch Institute (RKI) from 2003 to 2006 . This crThe study was conducted according to the Federal and State Commissioners for Data Protection guidelines and was approved by the Charité University Medicine Berlin ethics committee and the Federal Commissioner for Data Protection. Informed written consent and agreement were obtained from the parents of all participants.In the KiGGS study, a total of 17,641 children and adolescents aged 0–17 years were interviewed and medically examined. Of those, 14,386 participants aged 1–17 years provided a blood sample. As we could not test around 15% of the sera samples because they were already used up in the core study , or could not be successfully tested in the laboratory for reasons like insufficient bead count (n = 84), the overall number of serum samples available for this study were 12,257 (85.2%).Standardized self-administered questionnaires were used to obtain information on socio-demographic and lifestyle variables. Questionnaires were filled out by the parents of all children (1–17 years) and by the children themselves (> 10 years). Age was categorized into seven groups . Region of residence was split into two groups, with ‘West Germany’ and ‘East Germany’, considering the former boarders of the German Democratic Republic and the Federal Republic of Germany from 1949 to 1990. Urbanity was categorized as rural (< 5000 residents), small city , medium-sized city  and large city . The socioeconomic status was based on a parental socioeconomic status (SES) index, including information about education, occupational status, and income of both parents separately. The highest index score was used for the overall household SES. Based on the household SES index, children were categorized into ‘low’, ‘medium’ and ‘high’ SES .S-transferase (GST) capture immunoassay in combination with fluorescent bead technology as previously described [In 2016/2017, serum specimens were tested for antibodies to the major capsid (L1) protein of 16 different HPV genotypes at the German Cancer Research Center (DKFZ) in Heidelberg. Serological testing was performed by a glutathione escribed . We usedFor calculating seropositivity, MFI values were dichotomized as positive or negative based on previously established type-specific cutoff-values. Seropositivity was defined as the proportion (%) of positive tested sera. The cut-off values were established with sera of a cohort of 125 young Korean women who were HPV DNA negative and self-reported to never have had sexual intercourse . The folTo assure representativeness of the data at the national level, survey weights were calculated and applied to all estimates, adopting the study sample (providing HPV antibody test results) to the population structure of Germany in 2003 in terms of age, gender, state, size of municipality, education and German/non-German nationality and the regional distribution between East and West Germany. These survey weights, which accounted for the stratified and clustered sample design of the survey, were applied throughout the statistical analyses.Weighted seroprevalence was calculated for all 16 types separately and for the following groups of HPV types: types included in the bivalent vaccine , types included in the quadrivalent vaccine , and types included in the nonavalent vaccine . Group-specific seroprevalence was calculated as the weighted proportion of participants seropositive to at least one of the HPV-types included in one group. Additionally, MFI were plotted against the percentile for each HPV type individually stratified by gender and age for analyzing antibody reactions without relying on a specific cutoff.2 or Fishers Exact test. Chi2-tests were used to test for statistical significance in categorical variables (p < 0.05) and logit transformation was applied to calculate confidence intervals (95% CI).Differences regarding demographic and behavioral characteristic of participants stratified by gender were evaluated by using chiWe calculated prevalence ratios (PRs) using Poisson regression models to identify factors independently associated with HPV seropositivity for HPV-6, HPV-11, HPV-16, HPV-18 and for at least one of the cutaneous HPV types  tested for in our analysis (HPV-cut). We used PR instead of Odds Ratios to obtain more interpretable association estimates . The modData management and statistical analysis were conducted using Stata, Version 14 . Percentile plots were created with R Studio, Version R version 3.6.0 (2019-04-26).Overall, 12,257 serum samples of children and adolescents aged 1–17 years with valid HPV serology from the pre-HPV-vaccination era were included in the analysis . The highest value  was observed in the age group 4–6 years. HPV-6 seroprevalence decreased thereafter to 16.6% (95% CI 14.5–19.1%) in the age group 16–17 years. HPV-6 was the only mucHPV which differed by gender with an overall seroprevalence of 23.1% (95% CI 21.5–24.7%) in girls and 26.4% (95% CI 24.9–28.0%) in boys (p < 0.001). HPV-11 resembled HPV-6 regarding age distribution with highest seroprevalence in the age group 4–6 years.HPV-16 was 2.6% (95% CI 2.2–3.0%). It remained relatively stable in the youngest age groups with 1.5% (95% CI 0.9–2.6%) among the 1–3 years-old and 2.1% (95% CI 1.4–3.1%) among the 12–13 years-old and increased thereafter to 3.1% (95% CI 2.2–4.2%) and 4.4% (95% CI 3.4–5.8%) among the age groups 14–15 and 16–17 years, respectively.The overall seroprevalence of HPV-18 seroprevalence had a low variability across age groups, with highest seroprevalence in the oldest group . Overall, lowest seroprevalence was found for HPV types 33, 45, 52, and 58, with slightly higher values in older age groups. HPV-31 showed the highest seroprevalence  in the youngest age group, strongly decreasing at higher age.Overall, 6.1% (95% CI 5.5–6.8%) of the participants were seropositive for at least one of HPV-16 or HPV-18, both types targeted by the bivalent vaccine  (data not shown). 28.9% (95% CI 27.5–30.4%) were seropositive for at least one of the types covered by the quadrivalent vaccine  and around a third  were seropositive for one of the nonavalent vaccine types .Seroprevalence data and MFI distributions of cutHPV are presented in Fig. CutHPV seroprevalence increased nearly steadily from youngest to oldest age groups. We found the strongest increase in HPV-1 seroprevalence, ranging from 2.1% (95% CI 1.1–4.0%) in 1–3-years-old to 55.7% (95% CI 51.7–59.6%) in 16–17-years-old among girls and from 0.9% (95% CI 0.4–1.8%) in 1–3-years-old to 47.7% (95% CI 43.4–52.0%) in 16–17-years-old among boys.Taken all seven tested cutaneous HPV types together, 46.0% (95% CI 44.7–47.3%) of the participants were seropositive for at least one HPV-cut type (data not shown). Seroprevalence for HPV-cut increased sharply from 16.0% (95% CI 13.5–18.9%) in the age group 1–3 years to 57.5% (95% CI 54.4–60.6%) among children aged 10–11 years. Thereafter, seroprevalence for HPV-cut remained relatively stable and increased only slightly to 66.9% (95% CI 64.2–69.5%) in age group 16–17. No seroprevalence difference was observed between females and males in overall HPV-cut (p = 0.087).36.6% (95% CI 35.1–38.2%) of the study population was seronegative for all 16 investigated HPV types. Around a third  were seropositive for one HPV type, and 16.8% (95% CI 15.8–17.7%) were seropositive for two HPV types. 7.4% (95% CI 6.7–8.2%) of the study population were seropositive for three HPV types, and 6.5% (95% CI 5.9–7.1%) were seropositive for more than three HPV types. In general, multiple HPV seropositivity was influenced by the high proportion of HPV-6 seropositivity. No gender difference was observed for multiple HPV seropositivity (p = 0.475).The percentage of children being seronegative to any HPV types decreased from 57.2% (95% CI 53.1–61.1%) among 1–3-years-old to 28.8% (95% CI 26.1–31.6%) among 10–11-years-old children and decreased only slightly thereafter to 25.7% (95% CI 23.3–28.3%) among the oldest age group (16–17 years)  and HPV α9 types 52 and 58 (r = 0.75). Moderate correlation was observed among α7 types 18 and 45 (r = 0.46) and among α9 types, with HPV types 31/33 (r = 0.39), 33/52 (r = 0.49), and 33/58 (r = 0.49).Regarding cutHPV, moderate correlation was found only between HPV types 8 and 38 (r = 0.44), 8 and 49 (r = 0.47), and 38 and 49 (r = 0.43).We analyzed possible associations of demographic variables with type-specific seropositivity. The weighted crude and adjusted PR for HPV-16 and HPV-cut can be found in Tables HPV-16 model, only age and region of residence were significantly associated with seropositivity. Children of older age  and children living in West Germany  were more likely to be seropositive compared to younger age groups and to those living in the eastern part of Germany.In the fully adjusted HPV-cut model, a significant association with HPV-cut seropositivity was only observed for age, region of residence, urbanity, and migratory background of parents in the multivariable model. The strongest association with HPV seropositivity for cutHPV was seen for age, with a PR increase from 1.6 (95% CI 1.3–1.9) among the youngest age groups to 4.2 (95% CI 3.5–5.0) among the oldest. Living in East Germany, compared to West Germany, was associated with a slightly lower seropositivity . The same was true for living in medium  or large cities , compared to living in rural areas, as well as for migratory background of parents , compared to non-migratory background.While several associations, like number of household members, or number of sunburns, were significant in the univariable Region of residence was the only variable which was significantly associated with HPV seropositivity in all five regression models. Children living in West Germany were more likely to be seropositive for HPV-6, HPV-11, HPV-16, HPV-18, and HPV-cut, compared to children living in East Germany. HPV-6 and HPV-11 were the only serotypes for which number of siblings  or number of household members  were significantly associated with a slightly higher seropositivity  transmission, reporting varying risk of transmission between 4 and 22%. Maternal transmission was addressed by several research papers , 27, 59,Others have analyzed the age-dependent increase of mucosal HPV seroprevalence in older children and adolescents which was assumed to reflect sexual transmission of HPV due to onset of sexual activity. However, mucosal HPV prevalence was also detected in children of other age groups, irrespective of sexual transmission , 18. SubType-specific HPV seroprevalence in children showed a considerable variability in previous studies. HPV-16 seroprevalence ranged from 0.0% in a study with 128 children aged 0–9 years in Australia  to 10.9%In the same study, HPV-16-seroprevalence was highest (5.2%) among infants aged 0–0.5 years and again high with 6.1% and 3.2% among children aged 7–10 (n = 165) and 10–13 years of age (n = 124) . A bimodAn age distribution similar to that of HPV-16 seroprevalence was observed for HPV-18, HPV-45, HPV-52 and HPV-58. The opposite was true for HPV-6, HPV-11, and HPV-31: the highest seroprevalence was observed in younger age groups, followed by lowest HPV seroprevalence in the oldest age groups. This is not in line with results from another study, even though a comparison is limited as younger age groups are combined broadly and numbers are low within those age groups in the study from Australia . HoweverUsing HPV-specific antibodies as a measure of previous infections poses several challenges. While it has been shown that mucosal HPV antibodies can serve as an indicator of previous HPV infections , varyingIt has been discussed whether the development of HPV antibodies  in early childhood protects from HPV infection or HPV associated diseases in later life , 77, 78.In Germany, HPV vaccination is recommended for girls by the Standing Committee on Vaccination (STIKO) since 2007 . In 2018We observed no difference in HPV seropositivity in children regarding most of the demographic factors, like gender, or socioeconomic status. The significant higher prevalence ratio for HPV-16 seropositivity in the age groups 15–17 years is in line with other publications and an expression of the sexual debut and the increase in sexual contacts. Slightly higher seroprevalence was found in West Germany compared to East Germany both for mucHPV 6, 11, 16 and 18, and for HPV-cut. The regional difference mirrors the old state border between the Federal Republic of Germany and the German Democratic Republic (1949 till 1990). Therefore, it is a potential factor of underlying socio-cultural differences between the two regions and described as a potential factor for prevalence differences of infectious diseases before .There are a few conflicting studies about a potential association between obesity, measured mostly as body mass index, and HPV infections –89. In oWe observed a pronounced association of cutHPV seroprevalence with age. This result is in line with other studies, showing a strong increase of cutHPV in early years of childhood , 93. TheDue to the wide range of different mucosal and cutaneous HPV types included in the assay, the determination of seroprevalence largely depends on the applied cutoff value. There is a variety of serological HPV antibody detection methods, a lack of a universal applicable reference, and no standard cutoff values for type-specific HPV serology. Therefore, serological HPV studies often use a most likely negative cohort as the negative reference to calculate cutoff values. This, however, assumes negativity in this cohort and is challenging if the ‘true’ status is unknown. Our data was based on previous used cutoffs, established in a cohort of South Korean young women who claimed to have never had sexual contacts before and used for other studies with the same serological assay before , 44. HowAnother potential limitation is antibody cross-reactivity for phylogenetically closely related HPV types, which could lead to an overestimation of seroprevalence for some types . However, there is no substantial cross-reactivity across phylogenetic species  or genera, so the majority of our results are expected to reflect type-specific results.To our knowledge, this is the first study on HPV antibody seroprevalence of cutaneous and mucosal HPV types among a large, representative sample of children and adolescents aged 1–17 years living in Germany. As a result of our study, we found varying age distributions in seroprevalence, dependent on type and tropism and we found low but non-zero seroprevalence for most tested mucHPV among children and adolescents. For HPV-16, only age and regional differences were associated factors with seropositivity. Compared to mucHPV, seroprevalence of cutHPV were higher and generally increased with age. Our study results provide population-based HPV seroprevalence data among children and adolescents and are an important data source of prior cumulative exposure to HPV in different age groups. Our data can serve as additional baseline data to understand the nature of HPV infections among children and adolescents and help evaluating the impact of the HPV vaccine introduction.Additional file 1: Figure S1. Selection criteria of HPV types.Additional file 2: Figure S2. Percentile plot of the antibody reaction  of muscosal HPV types HPV-6, HPV-11, HPV-16, HPV-18, HPV31, HPV33, HPV45, HPV52, HPV58, HPV seroprevalence study .Additional file 3: Figure S3. Percentile plot of the antibody reaction  of cutaneous HPV types HPV1, HPV4, HPV8, HPV10, HPV38, HPV41, HPV49, HPV seroprevalence study .Additional file 4: Figure S4. Multiple HPV seropositivity by age, HPV seroprevalence study .Additional file 5: Table S1. Seroprevalence of individual mucosal human papillomavirus types by gender and age, HPV seroprevalence study .Additional file 6: Table S2. Seroprevalence of individual cutaneous human papillomavirus types by gender and age, HPV seroprevalence study .Additional file 7: Table S3. Regression estimates for associated factors with seropositivity for HPV-6, HPV seroprevalence study .Additional file 8: Table S4. Regression estimates for associated factors with seropositivity for HPV-11, HPV seroprevalence study .Additional file 9: Table S5. Regression estimates for associated factors with seropositivity for HPV-18, HPV seroprevalence study .


The process of HPV-mediated oncogenesis in HNSCCs is not fully understood. DLG1 and SCRIB protein expression levels and localization changes were evaluated in a number of HPV16-positive and HPV-negative OPSCCs and seem to be associated with malignant transformation. Moreover, loss of SCRIB expression inversely correlates with higher grade tumors, and this is much more evident in the presence of HPV16 E6. This could serve as a potential marker in predicting development of OPSCCs.The major causative agents of head and neck squamous cell carcinomas (HNSCCs) are either environmental factors, such as tobacco and alcohol consumption, or infection with oncogenic human papillomaviruses (HPVs). An important aspect of HPV-induced oncogenesis is the targeting by the E6 oncoprotein of PDZ domain-containing substrates for proteasomal destruction. Tumor suppressors DLG1 and SCRIB are two of the principal PDZ domain-containing E6 targets. Both have been shown to play critical roles in the regulation of cell growth and polarity and in maintaining the structural integrity of the epithelia. We investigated how modifications in the cellular localization and protein expression of DLG1 and SCRIB in HPV16-positive and HPV-negative histologic oropharyngeal squamous cell carcinomas (OPSCC) might reflect disease progression. HPV presence was determined by p16 staining and HPV genotyping. Whilst DLG1 expression levels did not differ markedly between HPV-negative and HPV16-positive OPSCCs, it appeared to be relocated from cell–cell contacts to the cytoplasm in most samples, regardless of HPV16 positivity. This indicates that alterations in DLG1 distribution could contribute to malignant progression in OPSCCs. Interestingly, SCRIB was also relocated from cell–cell contacts to the cytoplasm in the tumor samples in comparison with normal tissue, regardless of HPV16 status, but in addition there was an obvious reduction in SCRIB expression in higher grade tumors. Strikingly, loss of SCRIB was even more pronounced in HPV16-positive OPSCCs. These alterations in SCRIB levels may contribute to transformation and loss of tissue architecture in the process of carcinogenesis and could potentially serve as markers in the development of OPSCCs. Head and neck squamous cell carcinomas (HNSCCs) are the most common form of head and neck cancers and are the sixth most common cancer worldwide, with more than 930,000 new cases and more than 460,000 deaths in 2020 ,2. HNSCCHPVs are small non-enveloped double-stranded DNA viruses associated with almost 100% of cervical cancers, 70% of anogenital cancers, 40–60% of OPSCCs and 5% of non-OPSCCs ,5. HPV cThe process from the initial infection to cancer development in the head and neck (HN) region is still poorly understood. It appears to be much shorter than in the cervix/uterus where it usually takes 12–15 years before a persistent HPV infection may result in development of cervical carcinoma . Unfortun = 65), HPV16-positive oropharyngeal cancer (n = 21), HPV-negative oropharyngeal cancer (n = 36), and healthy tonsillar tissue (n = 8). The tissue samples were obtained from the archives of the Department of Pathology and Cytology, University Hospital Dubrava  and the Department of Pathology and Cytology University Hospital Centre, Zagreb, Croatia . All the tissue samples were previously fixed in 10% buffered formalin and embedded in paraffin. The study was conducted on FFPE HNSCC samples , according to the manufacturer’s instructions. The concentration of the isolated DNA was measured using NanoPhotometer® N60 (Implen GmbH), and the quality was validated by PCR using primers generating 99bp long beta-actin fragments [From each FFPE sample, five to seven slices (10 µm) were used for DNA isolation. DNA was isolated using a commercial kit  and SPF 10 (~65bp) ,27. PCR ® Histology kit system (Roche Holding AG) and EnVision® + Dual Link System-HRP (Agilent).Endogenously expressed DLG1 and SCRIB were detected in FFPE tissue sections using mouse monoclonal antibodies SAP 97 (2D11) and Scrib (C-6)  at 1:20 dilution. Biotinylated secondary antibodies were provided in commercial kits CINtec® Histology kit system  and compared with the HPV status of the corresponding tissue. Endogenous expression of DLG1 and SCRIB was detected using EnVision® + Dual Link System-HRP  according to the manufacturer’s instructions. Negative controls for each sample were processed in the same way, except that the primary antibody was replaced with the negative control solution provided (CINtec® Histology kit system), in p16 staining or antibody diluent . The intensity of DLG1 and SCRIB immunostaining was graded and scored by three independent pathologists as follows: 0 (no staining), 1+ (low intensity), 2+ (medium intensity), and 3+ (strong intensity).Slices (7 µm) of the FFPE tissue sections were mounted on pretreated glass slides, deparaffinized in xylene substitute BioClear , and rehydrated using a graded ethanol series . The expression of p16 was analyzed using the CINtecDescriptive statistics were used to evaluate differences in DLG1 and SCRIB expression and localization in HPV-positive and HPV-negative OPSCCs.A Kruskal–Wallis test was used to evaluate possible significant differences between the designated groups, HPV- OPSCCs grades 1–3 and HPV16+ OPSCCs with Dunn’s multiple comparison posttest. The ratio of DLG1- or SCRIB-positive staining in tumor cells was evaluated by three independent pathologists.Pearson’s χ2 test was used for evaluation of any significant differences in DLG1 or SCRIB localization between designated groups: HPV- OPSCCs grades 1–3 and HPV16+ OPSCCs. All statistical analyses were performed using GraphPad Prism 5 software.A total of 66 OPSCC and 8 normal, non-cancerous and HPV-negative tonsillar tissue samples were collected. DNA was isolated, and PCR detection of HPV16 E6 was performed, as described in Materials and Methods. Of 66 OPSCC samples, 21 were classified as HPV16-positive and 36 as HPV-negative OPSCC. The remaining nine samples were excluded, as they were either inconclusive due to insufficient DNA or were HPV-positive but not HPV16-positive.The expression of p16 protein was investigated using IHC in all samples, including normal tonsillar tissue . As expeBefore investigating any potential differences in the expression levels and localization of SCRIB and DLG1 during malignant progression in the oropharynx, we investigated their patterns of expression in eight non-cancerous tonsillar tissue samples, using IHC . The speWe started by analyzing 36 HPV-negative OPSCC samples whose HPV negativity was confirmed by genotyping. These samples were divided into three groups as classified by a pathologist; G1: well differentiated (7/36), G2: moderately differentiated (10/36), and G3: Poorly differentiated or undifferentiated (19/36). Representative examples of each group are shown in In short, only marginal changes in levels of DLG1 expression were seen in all three groups of HPV-negative OPSCCs when compared to cancer-free healthy tonsillar tissues. However, the presence of relocated, and thus functionally altered DLG1, could potentially be caused by modifications of DLG1 at the post-transcriptional and/or post-translational levels, rather than being caused by genomic mutations in this set of tumors. Although some mutations in the PDZ-2 domain of DLG1 have been found in breast cancer, there is still no clear explanation of their effects, if any, on tumor development and/or progression. However, a possible explanation is that these PDZ-2 domain mutations in DLG1 might prevent it from forming complexes with other proteins such as APC and PTEN ,32, thusSince cell polarity regulators SCRIB and DLG1 have been previously characterized as cellular targets of E6 oncoproteins , we wantp = 0.0003, p = 0.0214, More interestingly, in those samples where SCRIB was still detectable, it was localized solely in the cytoplasm of cancer cells, suggesting that relocation of SCRIB from cell–cell contacts may contribute to cell transformation in these tumors. These results are in line with previous studies showing that epithelial cell differentiation , variousIn the past 30 years, HPV infection has been recognized as a causative agent of HPV-positive OPSCCs, with HPV16 being responsible for almost 90% of them ,5. Numer® histology kit. Similarly, 3/36 (8.3%) of HPV-negative samples were p16-positive. As expected, our results reaffirmed p16-positivity in both HPV-negative and HPV-positive OPSCCs, additionally supporting the need for new biomarkers, more closely correlating with HPV positivity, and more predictive of disease development in oropharyngeal tumors. DLG1 was shown to be implicated in the process of malignant transformation, since loss of polarity is recognized as a fundamental step in carcinogenesis [In our combined analysis of PCR genotyping and p16 IHC staining, we found that 3/21 (14.3%) of the total HPV16-positive samples analyzed were p16-negative when stained using the CINtecogenesis . Furtherogenesis . The facogenesis ,38, whilogenesis ,37,38. Togenesis ,37. Likeogenesis .p = 0.0214) only from that in grade 3 HPV-negative tumors. However, the slightness of changes in relocation suggests that HPV16 E6 does not markedly affect localization of DLG1, and therefore the phenotype could be a result of other, unknown, processes during cancer progression, such as the acquisition of certain mutations, like G338R, I348V, G338R/I348V, and L329R/G330R in the DLG1 PDZ-2 domain in both HPV-negative and HPV-positive tumors during the process of oncogenesis, as has been seen in breast cancer. Furthermore, these PDZ-2 domain mutations in DLG1 seem to disable its interaction with the tumor suppressors APC and PTEN, emphasizing the importance of DLG1 functions and how alterations in its protein expression and localization might contribute to the process of oncogenesis [Therefore, in this study we first wanted to determine whether similar changes occur in OPSCCs and, if so, whether changes in the expression levels and/or localization of DLG1 are dependent upon HPV status. In healthy and non-infected tonsillar tissue, DLG1 was expressed both in the cytoplasm and at cell–cell contacts, mostly in the parabasal, intermediate layer of stratified squamous epithelium, but was absent from the basal and superficial layers. Although these results differ somewhat from studies of normal cervical epithelium, which found DLG1 in the basal and parabasal, but not in the superficial, layers ,38, thisogenesis ,32.We then examined the same set of samples for any changes in the expression and localization of SCRIB, which is the preferred PDZ-domain containing target of HPV16 E6 . Unlike In healthy and non-infected FFPE tonsillar tissues, we observed SCRIB expression both in the cytoplasm and at cell–cell contacts; like DLG1, it was mostly in the parabasal layer of the stratified squamous epithelium and was absent from the basal and superficial layers. This differs from The Human Protein Atlas, where SCRIB is reported to be expressed in the basal and intermediate layers of squamous epithelia but is absent from the superficial layer. The lack of basal layer SCRIB expression in these samples could be the result of inflammatory processes prior to tonsillar removal or to potential age differences between the patients. In the HPV-negative OPSCCs, SCRIB was expressed at low to medium levels in almost all the well- and moderately-differentiated samples; only one sample showed SCRIB levels that were similar to those seen in normal tonsillar tissues. SCRIB was predominantly localized in the cytoplasm of cancerous cells and absent from cell–cell contact areas. However, in moderately-differentiated HPV-negative OPSCC, we observed a complete loss of SCRIB protein in 2/10 samples. There was a greater loss of SCRIB protein in poorly-differentiated tissue samples. This is consistent with studies on breast cancer cells and on mutant transgenic mice, showing that cytoplasmic SCRIB promotes cancer development by affecting subcellular localization of PTEN and activation of the Akt/mTOR/S6 kinase signaling pathway ,48. SimiThis is the first study to analyze the cause-and-effect relationship of DLG1 and SCRIB protein expression levels in a panel of HPV-negative and HPV-positive histologic oropharyngeal cancer samples. Importantly, we show a marked, differentiation-dependent reduction in SCRIB levels in the HPV-negative tumor samples, especially in grade 3 tumors, which show similarities with HPV16-positive OPSCC . No suchThe findings of this study provide novel insights into alterations in DLG1 and SCRIB protein expression levels in a subset of HPV-positive and HPV-negative OPSCCs. We observed an increase in cellular relocation of DLG1 to the cytoplasm in tumor samples, regardless of HPV-presence, implying that deregulation of DLG1 normal function is likely to be linked with disease development. However, there was an obvious significant down-regulation in SCRIB expression levels, and this was more pronounced in poorly differentiated higher grade tumors. Interestingly, this effect was even more evident in HPV 16 E6-positive tumors. Therefore, these results imply that loss of SCRIB could serve as a potential late-stage marker in oropharyngeal carcinogenesis.


This study is focused on the epidemiological characteristics and biomolecular mechanisms that lead to the development of precancerous and cancerous conditions of oral lesions related to Human Papilloma Virus (HPV) infections. Current evidence from the literature demonstrates the role of HPV in potentially malignant oral disorders. Therefore, the underlying biomolecular processes can give arise, or contribute to, benign lesions as well as to oral carcinogenesis. Oral diseases, their symptoms and therapy have already been observed and described by the ancient doctors with a scientific approach by Hippocrates of Kos (Ἱπποκράτης ὁ Κῷος 460–370 BC), Galen of Pergamum , Oribasius of Pergamum , and others [Helicobacter pylori, viruses (such as those belonging to the Papillomaviridae and Polyomaviridae family) and fungi  are associated with some types of cancer  ,4,5. Mosf cancer  [1,6,7,86 Dalton [Helicobacterpylori) can cause malignant tissue degeneration over time [HPVs are members of the Papillomaviridae (PV) family. To date, over 450 distinct types of human (HPV) belong to this virus family. The HPV viral body consists of a circular, double-stranded, and supercoiled DNA whose length is 7900 bp and a weight limit of approximately 5.2 × 106 Dalton ,12,13,14ver time ,12,13. Hver time ,14,15.Most of these viruses are low risk and are associated with benign lesions such as papillomas, warts, wedges, and Heck disease . Malignant lesions by carcinogenic HPV are anal, genital , and some types of oral. HPV tumor risk strains are mostly associated with epithelial dysplasia and squamous cell carcinomas and metastasis. The genotypes 1, 2, 3, 4, 6, 7, 10, 11, 13 are found in various type lesions of the oral cavity ,14. An aThe receptor for the virus to enter cells is integrin A6. The receptor involved in virus entry to the host cells is integrin A6 and HPV DNA is found in apoptotic epithelial cells of the oral mucosa of clinically healthy individuals. The HPV DNA was found in apoptotic epithelial cells of the oral mucosa of clinically healthy individuals. They play an important role in the disorders of intercellular connections. They also transmit information related to cell survival. In a SCC immunohistochemical study, immunohistochemically integrin α6β4 has been shown to be involved in early recurrence and tumor remodeling. The expression of α6β4 is lower in squamous cell carcinoma SCC than in the normal epithelium ,34,35,36Viral DNA is associated with cell histone proteins to form a chromatin-like complex. Regions of attack on histone proteins are the scaffold attachment regions (SARs) or matrix attachment regions (MARs). The HPV genome organized into three unequal regions: E (early), L (late) region, and the non-coding LCR. The E region represents more than 50% of the genome and is responsible for regulating transcription, virus replication, and activating the lytic cycle ,40. ThesThe E1 is required for virus replication. The E2 binds to E1 and together they activate the replication of viruses. The E4 contributes to facilitate and improve viral replication (controlling the maturation and release of the virus) and is the first expressed in the late stages of infection and is subsequently followed by L1 and L2 proteins . In fact, E4 protein can be as an important biomarker in diagnosis of the infection ,42. The Thus, the oncogenic activity of the virus is due to the ability of the viral proteins E6 and E7 to integrate into host DNA. These two proteins promote tumor growth by inactivating two tumor suppressor proteins retinoblastoma pRb and tumor protein 53 (gene TP53), that regulate the transition from the G1 phase of the cell cycle to S ,51. CellUbiquitin ligase [Overexpressed E6 binds and cleaves the post-synaptic density proteins (PDZs) strands , protein tyrosine phosphatase non-receptor type 13 (PTPN13), PATJ, and MAG1  that lead to the host cell metaplasia. The E6 inhibits apoptosis, impairs the transcription process, and increases the life cycle of cells. The E6 in turn participates in the cleavage of “wild” type of p53 through a three-dimensional complex consisting of E6, E6AP (E6 associated protein), and TP53 ,53. Furtn ligase . Insteadn ligase . E7 is an ligase . The P53n ligase ,59,60.Finally, the proteins E6 and E7 responsible for carcinogenesis modulate the epigenetic mechanisms. Epigenetic dysregulation is the change in gene expression that is not caused by variations in the DNA sequence. Epigenetic is represented by three main processes: histone modifications, non-coding RNA, and DNA methylation. Together, these mechanisms can exert their effects by directly regulating DNA transcription, damage/repair and replication, controlling RNA levels and post-transcriptional stability, normalizing protein translation, or causing post-translational changes in proteins ,61. ThusThere is increasing evidence that HPV can act in combination with cigarette smoking and alcohol to cause oral cancer ,63. The Finally, the oral microbiota eubiosis plays an essential role in maintaining health in the oral cavity and in the general physiology of the human body. Several viruses are present in the oral cavity, such as HPV, Paramyxoviridae (mumps virus), Rhabdoviridae, and others that are mainly related to mouth and upper airways diseases. Most of the virus sequences detected were like those of bacteriophages, which may not be surprising given the density and variety of the bacterial community found in the mouth. To date, the role of a probable interaction between them has not been thoroughly studied with the beneficial role of probiotics in microbiota dysbiosis ,70,71,72From literature data, it appears that (HPV) infection plays an important role in the risk of precancerous lesions of the oral mucosa, and therefore in the dysplastic and malignant transformation of the lesions. Furthermore, the ability of external carcinogens such as smoking, and alcohol may be risk cofactors in the development of carcinogenesis. Indeed, the potential role of HPV in oncogenic processes in the oral cavity remains an element of update regarding prevention and treatment. HPV is then associated with malignant evolutions and, therefore, with different types of the oropharynx. Thus, carcinogenic HPVs such as 18, 56, 58, and others have been implicated in the pathogenesis from studies conducted in the onset of the cancerous lesion. Therefore, also predisposing factors for the development of cancer can be hygiene and dysbiosis of the oral microbiota. The most widely accepted hypothesis by scientists points to occasional HPV infection in combination e.g., with chronic tobacco or alcohol use as the most likely cause of oral cancer pathogenicity along with genetic predisposition.