CCNA1, NDN, CD1A, DCC, p16, GADD45A) are associated with tumor recurrence and survival, in a well-characterized, prospective, cohort of 346 HNSCC patients.HPV-associated HNSCCs have a distinct etiologic mechanism and better prognosis than those with non-HPV associated HNSCCs. However, even within the each group, there is heterogeneity in survival time. Here, we test the hypothesis that specific candidate gene methylation markers  in all HNSCC patients (NDN hazard ratio (HR): 2.35, 95 % CI: 1.40-3.94; CD1A HR: 1.31, 95 % CI: 1.01-1.71). Stratification by HPV status revealed hypermethylation of CD1A was associated with better OST and recurrence/persistence-free time (RPFT) , while hypomethylation of CCNA1 was associated with increased RPFT in HPV (+) patients only .Methylation markers were differentially associated with patient characteristics. DNA hypermethylation of NDN and CD1A was found to be significantly associated with increased overall survival time in all HNSCC patients. However, stratification by the important prognostic factor of HPV status revealed the immune marker, CD1A, and the cell cycle regulator, CCNA1 to be associated with prognosis in HPV (+) patients, specifically. Here, we identified novel methylation markers and specific, epigenetic molecular differences associated with HPV status, which warrant further investigation.This study is the first to describe novel epigenetic alterations associated with survival in an unselected, prospectively collected, consecutive cohort of patients with HNSCC. DNA hypermethylation of The online version of this article (doi:10.1186/s12885-015-1806-8) contains supplementary material, which is available to authorized users. Heavrcinomas . The overcinomas , 5. Low rcinomas  and freqThe incidence of HPV-associated HNSCC has steadily increased, especially in younger patients, while incidence of non-HPV associated HNSCC has declined in recent years –10. HPV-Gene-specific DNA methylation has been increasingly recognized as a contributor to the molecular heterogeneity of HNSCC , 20. SevCCNA1, NDN, CD1A, DCC, GADD45a, and p16) are associated with tumor recurrence and survival, in a well-characterized, prospective cohort of HNSCC patients with extensive epidemiologic, clinical and outcome information, who were treated by a single group of clinicians with a homogenous treatment approach. This approach allows careful consideration of the epigenetic biomarkers in the context of epidemiologic and clinicopathologic characteristics that influence overall and recurrence-free survival.Here we test the hypothesis that specific candidate gene methylation markers  approaches every incident, previously untreated HNSCC patient to participate in longitudinal epidemiology studies. This unselected study population represents 28 % of incident HNSCC cases in the state of Michigan. From November 2008 through June 2012, subjects were screened for eligibility and 92 % (n = 513) of subjects approached signed a written, informed consent. Consented subjects completed a baseline questionnaire of demographics, epidemiologic characteristics, and behavior modules. Comorbidity data were abstracted from the medical record and graded by severity  using the Adult Comorbidity Evaluation of 27 conditions organized by 12 systems (ACE-27). Research assistants collected formalin-fixed, paraffin-embedded (FFPE) HNSCC tissue blocks and detailed pathophysiological and clinical data annually until death or the patient was lost to follow-up. This study was approved by the Institutional Review Board of the University of Michigan Medical School.The FFPE tissue blocks were collected pretreatment from three possible sources: (1) a biopsy obtained from an outside hospital, (2) a biopsy performed at the University of Michigan hospital, and/or (3) from surgery performed at the University of Michigan. Tissue acquired from the three sources yielded at least one sample for 88 % (n = 450) of the subjects.An expert head and neck pathologist (JM) confirmed tumor histology and screened representative blocks for areas of >70 % cellularity and minimal necrosis. Seventy-two percent (n = 369) of all subjects had sufficient tissue and DNA to yield methylation results. Of these, 15 subjects were excluded for tumors arising from rare sites or non-squamous histology  and 7 subjects were excluded for indeterminate HPV status. One subject was lost to follow up. This resulted in a total of 346 subjects used in the methylation analyses, representing 67 % of the 513 eligible participants screened.All patients were followed prospectively at designated intervals by clinicians at the University of Michigan or through contact with referring physicians. The median follow-up period was 27 months for survival and 24 months for recurrence (range: 1-54 months). Number of patients alive and followed for OS at 1, 2 and 3 years were 307, 90 and 85 patients, respectively. Number of patients alive and followed for RPFT at 1, 2 and 3 years were 242, 129 and 53 patients, respectively. Deaths were captured through the Social Security Death Index, yearly survey updates, notification from family, and medical record reviews. Survival time and events were censored as of 4/30/13. Recurrence and persistent disease events were confirmed updated annually during a chart review at every subject’s yearly anniversary of their date of initial diagnosis.CCNA1 (cyclin A1) was chosen for further testing based on its potential for clinical relevance and the discovery analysis that identified regions of their promoters to be significantly differentially methylated in head and neck cancer patients by HPV status [NDN (necdin) and CD1a (cluster of differentiation 1a) were also differentially methylated in this discovery analysis, however they were not significant, potentially due to small sample size. NDN is an imprinted gene previously implicated in epithelial ovarian, bladder, breast, colorectal, and urothelial cancers, as well as premalignant lesions such as vulval intraepithelial neoplasia and Barrett’s oesophagus, although has not been studied in the context of HNSCC [CD1A was the first immune gene found to be differentially methylated in the discovery analysis. CD1A methylation has not been previously studied in HNSCC, however significant hypermethylation of CD1B, CD1C, CD1D and CD1E has been found in HPV (+) HNSCC tumors compared to HPV(-) tumors [DCC , GADD45 (growth arrest and DNA damage 45) and p16 (cyclin-dependent kinase inhibitor) were all previously found to be hypermethylated in HNSCC and were chosen for their role as tumor suppressors and potential involvement with HPV [Our group recently completed a discovery-based study designed to identify novel prognostic epigenetic biomarkers for patients with HNSCC , 23. CCNV status . NDN (neof HNSCC –29. CD1A) tumors . DCC  according to the manufacturer’s protocol. DNA concentration and purity was measured with a NanoDrop spectrophotometer . Sodium bisulfite treatment was performed on 250 ng of DNA using the Epitect Bisulfite Kit  according to the manufacturer’s recommended protocol.HPV status was determined by an ultrasensitive method using real-time competitive polymerase chain reaction (PCR) and matrix-assisted laser desorption/ionization mass spectroscopy, as described in Tang et al. . MultiplDCC, CD1A, GADD45 and NDN, were designed using PyroMark Assay Design 2.0 software across 5, 2, 5 and 3 CpG sites, respectively . The methylation assay for p16 was adapted from Shaw, et al. and covered 4 CpG sites [CCNA1 was sequenced using the Sequenom EpiTyper, a MALDI-TOF mass spectrometry based platform across 4 CpG sites. Primer sets are shown in Additional file CCNA1, and HotStar Taq® Master Mix Kit  for all other genes, with a forward and reverse primer concentration of 0.2 mM and 30 ng of bisulfite-converted DNA. Confirmation of PCR product quality and absence of contamination was established from 2 % agarose gels with ethidium bromide staining. Fifteen microliters of each PCR product was combined with the respective sequencing primer and methylation analysis by pyrosequencing was conducted using the Pyromark™ MD System (Biotage) according to manufacturer’s protocol, including single strand binding protein (PyroGold reagents). Four bisulfite and four pyrosequencing controls were generated by mixing unmethylated and methylated control DNA  to obtain controls with 0 %, 30 %, 60 % and 100 % methylation. Each sample plate was run with all controls. If methylation values of controls were incorrect, all samples on plate were re-run. Measurement of all samples for every methylation marker selected was not possible if there was insufficient quantity of total extracted DNA. Average methylation across all CpG sites measured for each gene were used in all statistical analyses as strong associations between CpG sites of each gene has been previously shown [Methylation assays for promoter regions of pG sites . The proly shown .Overall survival time (OST) and recurrence/persistence-free time (RPFT) were calculated beginning at date of diagnosis. An OST event was defined as death from any cause. For RPFT, an event was defined as any recurrence  of the tumor or persistence of the tumor after definitive treatment. In the case of persistence, an RPFT of 1 day after diagnosis was assigned. In the case of death prior to recurrence, a subject was censored for RPFT at the last known date recurrence-free.Univariate analyses, including Kruskal-Wallis and Wilcoxon-rank tests, were conducted to test for differences in methylation of each gene by clinico-pathological and epidemiological characteristics. The Kaplan-Meier method was employed to estimate survival time distributions and graphically visualize time-to-event outcomes for overall survival time (OST) and recurrence/persistence-free time (RPFT) by methylation. Methylation of each marker was categorized into quartiles for the Kaplan-Meier curves, with quartile 1 containing the lowest values and quartile 4 containing the highest  to arrive at a parsimonious model, with the stipulation that stage and disease site would remain in the model regardless of their significance. Variables introduced for potential inclusion were: age, HPV status, ACE comorbidity score , tobacco use , and alcohol use . The final clinical model for both OST and RPFT included age, tumor stage, disease site, and HPV; the final clinical model for OST included comorbidity score in addition. After data exploration, significant violation to the proportional hazards assumption was observed for HPV. Stratified Cox proportional hazard models were performed that allowed differing baseline hazard functions for HPV+ and HPV- groups accounting for the non-proportional hazards observed in our data. These stratified Cox proportional hazards models included the same adjustment covariates as the unstratified version, and included an additional interaction term between HPV and methylation marker to test for differences in the associations of the biomarker with OST or RPFT across HPV status. Finally, each methylation marker was added to both the stratified and un-stratified version of the clinical Cox models to assess associations between the marker and outcome after covariate adjustment. All methylation measurements were standardized to interquartile ranges (IQR) of each respective marker , a more conservative Bonferroni threshold for significance was calculated as p < 0.004 (0.05/12) and reflected in superscripts in the Cox model results   as well as similar recurrence-free survival times (KM (95 % CI): 85 % ; 80 % , respectively, p-value = 0.59; Additional file p-value = 0.02). Distributions of patient characteristics by HPV status are listed in Table Patients with HPV (+) tumors n = 135) were on average younger than patients with HPV (-) tumors n = 211)  patients and mean age = 61.4 years, SD: 12.3 years for HPV (-) patients, 11  tumors compared to HPV (-) tumors. Increasing number of total pack-years across all tobacco types was significantly associated with decreased methylation of NDN and CD1A. Tobacco use  was also considered separately as these data were complete and more reliable than pack-years for most patients . Interestingly, former smokers had lower methylation of these genes compared to never or current smokers. Trends of each methylation marker differed significantly across disease site. Methylation of NDN was higher in patients with higher stage tumors , while a trend for CCNA1 methylation with stage was U-shaped, with higher methylation at stages I and IV . As comorbidity status increased, methylation of CD1A increased . Finally, methylation of NDN and DCC was significantly higher in males compared to females .Methylation of ge Table . HPV stats Table . TobaccoCCNA1, NDN, CD1A, and DCC, but hypomethylated in p16 as compared to HPV(-) tumors, regardless of clinical epidemiological traits (data not shown).Stratification by HPV status across these epidemiologic and epigenetic characteristics revealed HPV (+) tumors were hypermethylated across CD1A and NDN were significantly associated with OST in all HNSCC patients . The lowest quartile of methylation of CD1A (Q1) was associated with the lowest probability of OST (0.68 at 24 months) whereas higher methylation in Q2, Q3 and Q4 clustered above a probability of OST of 0.80 at 24 months. Methylation of the lowest quartile of NDN was associated with the lowest probability of OST. In terms of RPFT, there were significant associations with CCNA1, DCC and NDN methylation in all HNSCC patients. NDN revealed a trend of increasing probability of RPFT with increasing quartile of methylation. The lowest and highest quartiles of CCNA1 and DCC had the lowest probabilities of RPFT.Kaplan-Meier curves for the association between methylation, categorized into quartiles, and OST and RPFT across all HNSCC patients, are shown in Additional file CD1A in Q3 and Q4 were associated with better OST compared to the lower quartiles . Within HPV (-) patients, NDN and DCC were significantly associated with probability of OST. Low methylation of NDN in Q1 had the lowest probability of OST compared to methylation in all other quartiles . DCC methylation in Q2 was associated with the highest probability of OST . A similar pattern was observed for DCC methylation and RPFT, although with DCC, it was Q1 had much poorer probability of recurrence/persistence compared to other quartiles . . Patients with CD1A methylation in the 25th percentile had a 1.3 times higher hazard of a death event compared to those with methylation in the 75th percentile upon adjustment (95 % CI: 1.01-1.71). These results indicate that for all HNSCC patients, hypermethylation of NDN and CD1A are associated with better patient survival.Multivariable Cox proportional hazards models adjusting for site, stage, HPV status, age and comorbidity score showed significant associations of ts Table . TreatmeCD1A  and in RPFT for CD1A  and CCNA1 . In HPV (+) patients, CD1A methylation in the 25th percentile revealed a 3.34 times higher hazard of a death event than those in the 75th percentile (95 % CI: 1.88-5.93). In addition, patients had twice the hazard of a tumor recurrence or persistence if they were at the 25th percentile of CD1A methylation compared to the 75th percentile. These results suggest that hypermethylation of CD1A is associated with better survival and lower recurrence/persistence in HPV (+) patients specifically. Conversely, for CCNA1, HPV (+) patients had a 0.31 times lower hazard of a recurrent event or persistence comparing those in the 25th percentile to those in the 75th percentile of methylation, indicating that hypomethylation of this gene is associated with lower risk of recurrence/persistence in HPV(+) patients.To determine the extent to which HPV status may have played a role in these findings, stratified Cox models with interaction between methylation level and HPV status were used to measure associations separately within each group Table . DivergeNDN methylation in all patients and CD1A methylation in HPV (+) patients. These associations are significant at p-value < 0.004, the Bonferroni-adjusted p-value for multiple testing. Despite weaker associations, likely due to lower number of patients and events, there are indications that gene methylation is important in recurrence/persistence of HNSCC. Hypermethylation of NDN is associated with lower recurrence/persistence overall whereas hypermethylation of CD1A and hypomethylation of CCNA1 is associated with lower recurrence/persistence in HPV (+) patients only. These unique discoveries raise new questions about why these specific epigenetic changes differ among biologically distinct subsets of HNSCC patients (HPV + versus HPV -) and if these differences are linked to HPV status or other factors .Changes in methylation patterns are one of the most frequent events in human neoplasms. Epigenetic alterations have been increasingly recognized to play a role in the complex mechanisms of head and neck carcinogenesis , 21. ThiNDN is a maternally imprinted gene that has monoallelic expression. It encodes necdin, a protein that interacts with p53 to suppress growth and induce cell cycle arrest [NDN was associated with better survival in all HNSCC patients. Although the function of NDN is unknown, previous literature has implicated necdin to act as a “switch”, promoting quiescence under steady state conditions but bypassing p53 responses to promote proliferation or suppressing p53-induced apoptosis in a stressful state [e arrest . Althouge arrest , 26–30. ul state , 26, 44.CD1A encodes an immune protein responsible for presenting antigens by dendritic Langerhans cells to T lymphocytes, such as natural killer cells. Findings from our adjusted Cox model validated the difference seen in Kaplan-Meier survival curves across quartiles of CD1A methylation. Our analyses revealed hypermethylation was associated with better overall survival for all HNSCC patients and, for better survival and lower recurrence in HPV (+) patients specifically, even after adjusting for important clinical variables. Recent studies have highlighted important differences in immunologic status in patients’ peripheral blood and tumor microenvironment according to HPV status [V status , 46. PatCCNA1 was associated with lower hazard of recurrence/persistence in HPV (+) patients only. CCNA1 is a cell cycle regulator that binds to retinoblastoma, E2F transcription factor and p21 family proteins to promote cell cycle progression. This pathway plays a particularly important regulatory role in cell proliferation with HPV infection. Our results suggest that decreased methylation of this cell cycle regulator is protective against recurrence events. Our findings are consistent with previous findings in HPV (+) HNSCC patients [CCNA1 had no effect on protein or gene expression in HPV-positive HNSCC in spite of a strong correlation between CCNA1 overexpression and HPV positivity, suggesting that HPV may induce both promoter hypermethylation and overexpression [Hypomethylation of patients , 47, 48.pression , 50. ProCD1A is an immune regulatory gene and CCNA1 is involved in response pathways to HPV infection, it is plausible that these genes would play a larger role in more antigenic cancers with a viral etiology. NDN, which is involved in cell cycle regulation via p53 pathways, is likely to be important in regulation of altered cell cycle pathways contributing to the process of tumorigenesis [Considering igenesis . HoweverOur sensitive method of HPV detection has been previously validated with other commonly used assays to detect HPV . As expeCD1A, is indicative of comorbidity status, while several markers are differentially methylated according to HPV status, validating our previous study [NDN and CD1A compared to never and current smokers. Because total pack years is inversely associated with these genes, perhaps former smokers differ because duration of exposure and/or exposure at an early age is integral in the initiation of processes that permits carcinogenesis.Epidemiologic and clinical characteristics have generally been used to understand cancer phenotype, determine prognosis and inform treatment plans for patients. Epidemiologic factors such as smoking history, nutrition and comorbidity are well known significant prognostic factors for overall survival and indicate the importance of including such factors in studies of new molecular markers , 56, 57.us study . HoweverOur cohort shows the expected associations established in previous literature, such as the relationships between stage, site and HPV status with overall survival time, and the expected population characteristics of a HNSCC cohort established by previous studies, providing assurance that the new associations with survival demonstrated with this cohort are meaningful and can be extrapolated to the general HNSCC patient population. Identification of significant epigenetic markers of biologic tumor behavior and outcome should open new horizons for investigations and interventions directed at reversible gene alterations and potentially identify novel therapeutic targets.


Women having multiple sex partners are reportedly at an increased risk of HPV infection. However, the prevalence and risk factors of HPV infection in female sex workers (FSWs) vary considerably across racial/ethnic, socioeconomic, and geographic groups. This study aimed to determine the prevalence and risk factors of HPV infection in FSWs in Northeast China.A total of 309 FSWs identified and approached through a local police office and 1000 healthy subjects from a single factor undergoing annual gynecological examinations in Shenyang were recruited. A liquid-based ThinPrep Pap test and the Hybrid Capture II-based high-risk HPV DNA test, with or without a colposcopic examination, were performed on both FSWs and control subjects. Data on HPV infection and histological and cytological lesions of the cervix were obtained and analyzed. A questionnaire survey was administered to all 309 FSWs with their socio-demographic and behavioral information collected. The association of various socio-demographic and behavioral variables with HPV infection was assessed.P < 0.01), so were cervical lesions (P < 0.01). HPV prevalence in our sample of FSWs fell in the upper range of reported values in FSWs across different countries, and was similar to that for FSWs in the southeast Chinese city of Huzhou but higher than that for FSWs in southwest China, Guangxi, as compared with data from other studies within China. HPV infection in FSWs was significantly associated with the age at first sexual intercourse  and post-menopause  (P < 0.05).HPV was significantly more prevalent in FSWs (61.90 %) than in healthy control subjects (21.00 %) (FSWs are at a substantially high risk of HPV infection and cervical dysplasia development as compared with healthy control subjects in Shenyang, China. Age of first sexual intercourse and post-menopause are two independent risk factors for HPV infection in this special group of population. Intensified and coordinated efforts from government, public health sector, communities and families are needed to reduce the risk of HPV infection in this specific group of population. Cervical cancer is one of the most common causes of cancer death in women, claiming more than 270,000 lives annually worldwide . One of Genital infection with human papillomavirus (HPV), particularly the high-risk subtypes HPV-16 and HPV-18, is playing a major role in the etiopathogenesis of cervical cancer ; althougSince HPV is predominantly transmitted through sexual contact, HPV infection in women varies greatly with sexual activities and the number of sexual partners , 6. In aThis study was therefore conducted with two objectives: 1) to assess the prevalence of HPV infection and incidence of CIN in FSWs in Shenyang, China; and 2) to define putative risk factors for HPV infection in this unique group of population in Shenyang.The study was reviewed and approved by the Institutional Review Board of Liaoning Provincial Cancer Hospital, Shenyang, China and conducted in compliance with the ethical principles for medical research involving human subjects stated in the World Medical Association Declaration of Helsinki. Informed consent was obtained from all subjects.A total of 310 FSWs who were detained between October and December, 2013 for alleged engagement in illegal prostitution or sexual acts in public entertainment and leisure facilities  by police in Shenyang, China were identified and approached with the help from of a local police branch. They were all mentally healthy and non-pregnant without structural and anatomic malformations in the reproductive tract and without a history of pelvic irradiation. They had not been screened for cervical neoplasia within five years prior to their police detainment. After exclusion of one subject who refused to undergo gynecological examination, 309 FSWs were finally recruited to the study. As a representative sample of the general population, 1000 women who underwent free annual gynecologic examinations in December, 2013 in the Department of Gynecological Oncology, Liaoning Provincial Cancer Hospital were included for the purpose of normal control. They were employees from a single factory in Shenyany. No specific inclusion and exclusion criteria were applied to the selection of control subjects as a random sample of the general population.There was no difference in the cultural and racial/ethnic backgrounds between FSWs and control subjects; they were all Han Chinese.A liquid-based ThinPrep Pap test was performed as previously described  and highA standard risk factor assessment interview was conducted on 309 FSWs in person at the time of their police detainment. Data on their basic demographics, sexual activities and behaviors , reproductive history, menarche, sexually transmitted disease (STD) history, and smoking habit (past and present) were collected.P < 0.05. The statistical software SPSS 11.5  was used.Data on the prevalence rates of HPV and CIN in the two groups of subjects were analyzed by Pearson’s chi-square test. Putative risk factors for HPV infection were analyzed by univariate and multivariate logistic regression analyses. Differences were considered significant when P < 0.01). Cytological abnormalities, both ASCUS and ASCUS-H, were detected in a significantly higher proportion in FSWs than in control subjects (P < 0.01). In consistence with HPV infection and abnormal cytology, significantly higher occurrence of CIN 1 (P < 0.05), CIN 2 (P < 0.01) or CIN 3 (P < 0.01) was observed in FSWs than in control subjects.Presented in Table To assess the possible age dependency of HPV infection in FSWs, we stratified the 309 female sex workers into 5 different age groups. As shown in Table P < 0.001).To identify putative risk factors for HPV infection in FSWs, we first performed univariate regression analysis and the results are presented in Table We next performed multivariate logistic regression analysis. The results clearly revealed that HPV infection in FSWs was significantly associated with the age of first sexual intercourse and post-menopause while the other variables assessed had no effect Table .Table 4Rn = 200) in Peru [n = 369) in Philippines [n = 281) in northern Vietnam [N = 148) in South Korea [n = 288) in Australia [n = 254) in Thailand [n = 187) in Singapore [Routine HPV screening for women aged 21 and 65 years can reduce the chance of cervical cancer development . Various Vietnam , 47 % ofth Korea , 31.6 % ustralia , 22.9 % ingapore . Compareingapore , but wasIn consistency with HPV infection, cervical dysplasia, assessed either histologically or cytologically, was significantly more prevalent in FSWs than in healthy controls in this study Table . In the Numerous factors have been assessed for a putative link with HPV infection in FSWs in previous studies. However, inconsistent results have been obtained. In this study, we assessed various socio-demographic and behavioral variables, among which age of first sexual intercourse and menopause were the only two risk factors for HPV infection in FSWs in Shenyang.In agreement with our findings, young age  and menopause  have been suggested as independent risk factors for HPV infection in FSWs in previous studies , 24, 30.Education levels , 18 and The impact of cigarette smoking on HPV prevalence has been assessed in both men and women . HoweverThe benefit of condom use in protecting HPV infection in women is debatable . While aIn this study, HPV infection in FSWs in Shenyang was associated with all other socio-demographic, medical and behavioral variables including marital status, number of abortions, sexual disease history, use of contraception pills and other medications, age of puberty, and previous gynecologic and obstetric examinations. Nevertheless, these findings need to be further validated in studies with a large sample size.This study has several limitations. First, ThinPap test and cytological evaluation were performed only once on both FSWs and control subjects. This cannot totally rule out the possibility of miss the true abnormalities. Second, in evaluating the usefulness of condom use in protecting against HPV infection, only the information on ‘current condom use’ at the time of interview was collected. This might not represent a complete picture. A more accurate answer would have been obtained if condom use had been stratified into three scenarios: no condom use either in the past or at the present; condom use always; and condom use sometimes.HPV prevalence and cervical lesion incidence were substantially higher in FSWs than in healthy control subjects in Shenyang. Falling in the upper range of the reported values across different countries, HPV prevalence detected in FSWs in this study was similar to that for FSWs in southeast Huzhou but higher than that for FSWs in southwest Guangxi within China. Age of first sexual intercourse and menopause were two independent risk factors for HPV infection in our sample of FSWs. Public health endeavor is urgently needed to reduce the risk of HPV infection in FSWs in the northeast Chinese city of Shenyang.


Human Papillomavirus (HPV) genotyping is an important approach to fight cervical cancer due to the relevant information regarding risk stratification for diagnosis and the better understanding of the relationship of HPV with carcinogenesis. This paper proposed two new feature extraction techniques, i.e. ChaosCentroid and ChaosFrequency, for predicting HPV genotypes associated with the cancer. The additional diversified 12 HPV genotypes, i.e. types 6, 11, 16, 18, 31, 33, 35, 45, 52, 53, 58, and 66, were studied in this paper.In our proposed techniques, a partitioned Chaos Game Representation (CGR) is deployed to represent HPV genomes. ChaosCentroid captures the structure of sequences in terms of centroid of each sub-region with Euclidean distances among the centroids and the center of CGR as the relations of all sub-regions. ChaosFrequency extracts the statistical distribution of mono-, di-, or higher order nucleotides along HPV genomes and forms a matrix of frequency of dots in each sub-region. For performance evaluation, four different types of classifiers, i.e. Multi-layer Perceptron, Radial Basis Function, K-Nearest Neighbor, and Fuzzy K-Nearest Neighbor Techniques were deployed, and our best results from each classifier were compared with the NCBI genotyping tool.The experimental results obtained by four different classifiers are in the same trend. ChaosCentroid gave considerably higher performance than ChaosFrequency when the input length is one but it was moderately lower than ChaosFrequency when the input length is two. Both proposed techniques yielded almost or exactly the best performance when the input length is more than three. But there is no significance between our proposed techniques and the comparative alignment method.Our proposed alignment-free and scale-independent method can successfully transform HPV genomes with 7,000 - 10,000 base pairs into features of 1 - 11 dimensions. This signifies that our ChaosCentroid and ChaosFrequency can be served as the effective feature extraction techniques for predicting the HPV genotypes.The online version of this article (doi:10.1186/s12859-015-0493-4) contains supplementary material, which is available to authorized users. Human Papillomavirus (HPV) is a small double-stranded and most common sexually transmitting DNA virus. At present, more than one hundred types of Human papillomavirus have been identified. They are differentiated by the genetic sequence of the outer capsid protein L1. Approximately forty types can infect the mucosal epithelium. They are categorized according to their epidemiologic association with cervical cancer. Infection with low risk HPV types such as types 6 and 11 can cause benign or low-grade cervical cell abnormalities and genital warts. In contrast, high risk HPV types such as 16 and 18 act as carcinogens that can lead to the development of cervical cancer and other anogenital cancers.Cervical cancer is the second most common cancer significantly causing morbidity and mortality in women worldwide . PersistPapilloCheck®;, PCR-RFLP, HPV genome sequencing, INNO-LiPA, LinearArray®; HPV Genotyping Test, etc. These methods detect the HPV genotypes from some regions of genomes. Even though these HPV genotyping tests are beneficial and employed for HPV diagnosis in patients nowadays, they have some limitations. To illustrate this aspect, the HPV genotypes are hardly detected in cases of inadequate samples or low amplification signals of some genotypes. Contamination with previously amplified material can lead to false positive results. Furthermore, mistaken classifications can be occurred through cross-reactivity among similar types in the tests based on hybridization [HPV genotyping is necessary for managing effective medical treatment strategies to patients with persistent infection and for evaluating prevention strategies to individual patients to be immunized with type-specific HPV vaccines . Currentdization .To avoid these problems, some computational methods for identifying HPV types were developed -16. SincWhile classifying the HPV into high and low risk types is the urgent aspect for diagnosis of the cancer as claimed by many researchers, the study on how to predict specific genotypes of the virus has not significantly focused. In fact, the identification of HPV genotypes infecting the patients is more essential than a rough classification of HPV risk types. To clarify this issue, HPV genotyping can provide more information regarding risk stratification. With the persistent infection, the risk of a precancerous lesion is in between 10% to 15% with HPV types 16 and 18 but below 3% for all other high risk types combined . FurtherChaos Game Representation (CGR) was proposed as a unique and scale-independent representation for genomic sequences by Jeffrey . It is aSingular value decomposition (SVD) is a matrix factorization technique with various applications. For instance, it can be used to solve underdetermined and overdetermined systems of linear equations, find inverse and the pseudo-inverse matrices, compute the matrix condition number and calculate the vector system orthogonality and orthogonal complement . SVD is Due to the significance of HPV genotyping, the objective of this paper is to predict the HPV genotypes from their genomes, which is similar to the conventional methods of genome detection in clinical laboratories. The remaining sections of this paper are organized as follows. Section “As realized by a series recent publications -58 in reHPV genome data from genotypes were collected and their features were extracted by our proposed feature extraction techniques, i.e. ChaosCentroid and ChaosFrequency, as inputs for classification. These features were divided into the training and testing sets by a 2-fold cross validation technique. Four different classification models were deployed to train and test the experimental data sets. Then, the prediction performance from the obtained results were evaluated and compared with other methods. Our proposed method consists of the following four main procedures, i.e. data collection, feature extraction, prediction, and performance evaluation.To remove the homologous sequences from the benchmark data sets, a cut-off threshold of 25% was imposed in ,61 to exLinearArray®; HPV Genotyping Test. This HPV genotyping is a widely used qualitative test developed by Roche Molecular Diagnostics for detecting HPV genotypes associated with cervical cancer. The test can detect 37 high and low risk HPV genotypes, including those considered as a significant risk factor for HSIL progression to cervical cancer. To challenge the prediction, only HPV genotypes having genome diversity were concentrated in this experiment. Some of 37 genotypes containing few genomes were excluded. For this reason, only HPV genotypes 6, 11, 16, 18, 31, 33, 35, 45, 52, 53, 58 and 66 were involved. The genomes of these HPV genotypes were collected from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The data set contains Human Papillomavirus genomes of 12 genotypes, including high, possible high, and low risk types. For each HPV genotype, the number of genomes as well as the minimum and maximum lengths are shown in Table HPV genotypes collected in this experiment are those important genotypes detectable by All viral genomes in this HPV data set were previously published and are publicly available on GenBank or NCBI databases. In addition, the genome names, NCBI access numbers, and HPV genotypes of all genomes in the HPV data set are properly cited in Additional file xi and yi be the coordinates of nucleotide ηi at the ith position in the nucleotide sequence. Algorithm 1 illustrates how to construct a CGR for capturing a given nucleotide sequence.The following techniques, i.e. ChaosCentroid and ChaosFrequency, were proposed to extract the features from the chaos game representation of HPV genomes. To identify each genotype, the relations among subsets of HPV genomes must be clarified. These relations are actually the local features. Since the CGR captures the information of the whole genome data, extracting the global features from the CGR may not be efficient enough to distinguish the HPV genotypes. The local features hidden in various sub-regions of CGR must be more contemplated. In this work, we concentrate on extracting the local features rather than global features. The difference between ChaosCentroid and ChaosFrequency are the feature representation. HPV genomes contain A, C, G, and T nucleotides. Prior to the discussion of ChaosCentroid and ChaosFrequency, the detail of how to construct CGR is the following. Let n×n and n∈R+. Its center is also located at the coordinates . Each corner of this square represents the same nucleotide as that of the original CGR. After Algorithm 1, CGR can be viewed as an image of distributed dots. Figure ChaosCentroid and the second one is called ChaosFrequency. The detail of each feature is the following.A CGR can be viewed as a square whose corners are at coordinates -1,-1), , , and  representing nucleotides A, C, G, and T, respectively. Note that the size of CGR according to the coordinates of A, C, G, and T nucleotides is equal to 2×2 units. However, this unit size of original CGR is not appropriate for discussing our algorithm. Therefore, the geometrical structure and the physical size of our CGR are re-defined as follows. The size of CGR square is set to ,-1,  is set to 0.4 for ChaosCentroid and 0.1 for ChaosFrequency. The same network structure of multi-layer perceptron was adopted for this RBF network. The determination in Equation  test and jackknife test, jackknife test was deemed the most objective that can always yield a unique result for a given benchmark data set, as elucidated in  and demoIn this experiment, we adopted Equation 11 of  to formuN+ is the total number of HPV genomes of the investigated genotype whereas N− the total number of HPV genomes of the other genotypes that are not investigated whereas N+ is the total number of HPV genomes of genotype 6, while N− is the total number of the genomes of the other genotypes, excluding genotype 6.where According to Equation However, the set of metrics in Equation The value of variable The results of the HPV genotype prediction gained by ChaosCentroid and by ChaosFrequency feature extraction with the predicting system based on multi-layer perceptron neural network are summarized in Tables 7 and 8, respectively, of Additional file % of sensitivity and 100% of specificity in all genotypes, excepting genotype 16. It implies that the features of all genomes extracted by ChaosFrequency are totally predicted to genotype 16. In contrast, ChaosCentroid can obtain high performance metrics, including accuracy, sensitivity, specificity, and Matthew’s Correlation Coefficient in almost all genotypes. This is because a centroid is computed from the coordinates of every dots. It is obvious that different HPV genotypes must have different distribution of dots and centroids. So, predicting HPV genotypes with high performance from these centroids is possible. But in case of ChaosFrequency, the probability of distribution of every HPV genotype is equal. This makes the feature of each HPV genotype indistinguishable.When However, when the value of The results of the HPV genotype prediction gained by ChaosCentroid and by ChaosFrequency feature extraction with the predicting system based on radial basis function network are summarized in Tables 9 and 10, respectively, of Additional file The results of the HPV genotype prediction gained by ChaosCentroid and by ChaosFrequency feature extraction with the predicting system based on k-nearest neighbor technique are summarized in Tables 11 and 12, respectively, of Additional file k.The results of the HPV genotype prediction gained by ChaosCentroid and by ChaosFrequency feature extraction with the predicting system based on fuzzy k-nearest neighbor technique are summarized in Tables 13 and 14, respectively, of Additional file NCBI viral genotyping tool  is a webThe experimental results have shown that all methods, excepting ChaosCentroid with radial basis function network, can achieve the best performance of the four metrics, including accuracy, sensitivity, specificity, and Matthew’s Correlation Coefficient, in predicting the HPV genotypes of the data set. It demonstrated that both of the proposed techniques and the NCBI genotyping tool can be used to predict the genotypes of HPV genomes. Even though there is no significance between the proposed techniques and the NCBI genotyping tool, some issues should be considered.The NCBI genotyping tool provides a reliable method based on homology searching sequence alignment procedure. The limitation of alignment is that it is difficult to identify or classify the protein or DNA sequences in the case that they does not have a significant sequence homology. Besides, the alignment with multiple sequences will take time consuming and only one query sequence at a time can be processed by this tool. So, this method is not appropriate for large scale tasks.In contrast, the proposed techniques, i.e. ChaosCentroid and ChaosFrequency, are based on Chaos game representation, which provides a unique and scale-independent representation of DNA sequences through the statistical distribution of mono-, di-, tri-, or higher order nucleotides along DNA sequences. An advantage of CGR over alignment is that it has the potential to reveal the evolutionary and/or functional relationships between the sequences having no significant homology, as elucidated in . FurtherSince user-friendly and publicly accessible web-servers represent the future direction for developing practically more useful models, simulated methods, or predictors -80, we mThis paper proposed two new feature extraction techniques, i.e. ChaosCentroid and ChaosFrequency, based on chaos game representation and singular value decomposition for predicting HPV genotypes from nucleotide sequences in HPV genomes. Both extracting techniques concentrate on the local information among nucleotides. For the sub-regions in CGR, ChaosCentroid pays attention to capture the structures of the sequences in a form of centroids, while ChaosFrequency focuses on capture the distribution of sub-sequences in a form of frequencies. Four different predicting systems, i.e. multi-layer perceptron neural network, radial basis function network, K-nearest neighbor technique, and fuzzy K-nearest neighbor technique, were deployed. From the experiment, we found that the features extracted by our proposed feature extraction techniques are significant and independent of the predicting systems. The comparative results demonstrated no significance between our proposed techniques and the NCBI viral genotyping tool. In addition, local information is more important than global information in order to achieve high performance of prediction.


SLC4A11 gene, protein, and its mutations in the pathophysiology and clinical presentation of CHED. Individuals with CHED have mutations in SLC4A11 which encodes a transmembrane protein in the SLC4 family of bicarbonate transporters. The expression of SLC4A11 in the corneal endothelium and inner ear patterns the deficits seen in CHED with corneal edema and hearing loss (Harboyan syndrome). slc4a11-null-mouse models recapitulate the CHED disease phenotype, thus establishing a functional role for SLC4A11 in CHED. However, the transport function of SLC4A11 remains unsettled. Some of the roles that have been attributed to SLC4A11 include H+ and NH4+ permeation, electrogenic Na+-H+ exchange, and water transport. Future studies of the consequences of SLC4A11 dysfunction as well as further understanding of corneal endothelial ion transport will help clarify the involvement of SLC4A11 in the pathophysiology of CHED.Congenital hereditary endothelial dystrophy (CHED) is a rare autosomal recessive disorder of the corneal endothelium characterized by nonprogressive bilateral corneal edema and opacification present at birth. Here we review the current knowledge on the role of the  Congenital hereditary endothelial dystrophy (CHED) is a rare disorder of the corneal endothelium with an early onset of corneal edema. Original classifications of CHED described two forms . CHED1 w SLC4A11 gene as the genetic basis of CHED [ SLC4A11 encodes a membrane protein that was originally termed “BTR1” (Bicarbonate Transporter Related protein 1): a name that reflects its membership in the SLC4 family of bicarbonate transporting membrane proteins [3− transport is not among the numerous molecular actions that have been attributed to SLC4A11 [ SLC4A11 have been described in more than one hundred individuals with CHED , is expressed in a wide variety of cell types including corneal endothelium, spiral ligament fibrocytes of the inner ear, and various renal epithelia [Thepithelia , 21, 29. slc4a11 have also replicated the corneal defects seen in humans [Disease of the corneal endothelium is the hallmark of CHED with corneal edema and opacification presenting at birth or shortly thereafter. The degree of opacification varies from mild to severe with a bluish-gray ground glass appearance . The opan humans –31. Thesn humans , 31. In n humans , 31. slc4a11, even in the strain of mouse that did not exhibit any CHED-like ocular signs [In the mouse inner-ear, slc4a11 is expressed in the vestibular labyrinth and in fibrocytes underlying the stria vascularis , 30. Thear signs , 30. slc4a11 have a urine concentrating defect resulting in decreased urine osmolarity and corresponding decrease in all urine electrolyte concentrations [Slc4a11 is expressed in mouse kidney and has been detected in the proximal tubules, the thin descending limb of the loop of Henle, and the collecting ducts , 35. Twotrations , 31. Inttrations .There are several case reports of glaucoma presenting in patients with CHED. However, to date, there are no reports on the expression of SLC4A11 in the trabecular meshwork and aqueous outflow pathways of the eye. Review of published cases suggests a tenuous association. One series reported three cases of glaucoma with CHED . The fir+ and H+ (or OH−) channel [+ and/or H+/OH− transport mediated by SLC4A11 has subsequently been observed in other mammalian cell lines [4+ permeability in HEK293 cells [+-coupled borate cotransporter (therefore renamed “NaBC1”) [SLC4A11 dysfunction clearly plays a key role in CHED pathogenesis, but little is known of its normal function. Several molecular actions have been assigned to SLC4A11, yet which of these are of physiological or pathophysiological importance remains to be determined . The fir channel . Na+ andll lines , 31 and ll lines . SLC4A1193 cells . An init“NaBC1”)  is contr“NaBC1”) , 21, 31. in vivo action is uncertain. If SLC4A11 functions like an aquaporin to mediate H2O flux, then it could promote transcellular (stroma to aqueous humor) water flux in concert with aquaporin 1 [+-H+/OH− transport, then it could support the “pump” mechanism by modulating intracellular pH, volume, or membrane potential [Immunohistochemical studies reveal the presence of SLC4A11 in the corneal endothelium of humans, rats, and mice , 28, 29.aporin 1 . If SLC4otential , 20. SLC4A11 mutations (or mice with a disrupted slc4a11 gene) exhibit contrasting corneal endothelial phenotypes which may help advance our understanding of SLC4A11 dysfunction in CHED. In humans, the corneal endothelial monolayer is present and dystrophic or is absent with only rare fibrotic-appearing, atrophic endothelial cells present [ slc4a11-null mice have a corneal endothelial monolayer of cells present [Humans harboring present , 40–42.  present , 31. HowThe embryology of corneal endothelium and Descemet's membrane formation also supports the argument of disruption of endothelial cell function. The dysfunction occurs postnatally. The first wave of migration of neural crest cells from the rim of the invaginating optic cup forms the corneal endothelial monolayer and trabecular meshwork . Two fac per se (and thence loss of docking sites for dependent interacting-proteins), cell stress due to the accumulation of misfolded transport protein, and maladaptive compensatory changes in the expression of other gene products. Many of the CHED mutations recapitulated in heterologous systems are predicted to generate a misfolded SLC4A11 protein and have been shown to accumulate in intracellular compartments when expressed in cultured cells [2O and H+/OH− transport in model systems without any deleterious effect upon SLC4A11 protein expression [ slc4a11-null mouse that is predicted to express no misfolded slc4a11 protein product [There are numerous ways a defective membrane transport protein could contribute to a disease state, the most obvious being loss of transport function. Others include loss of proteined cells . As mentpression , 21. Mor product . Thus, w SLC4A11. Mouse models of CHED with deficits in slc4a11 expression recapitulate many of the features of the disease and will allow us to better understand its development and pathophysiology. The functional role of SLC4A11 in the corneal endothelium is unsettled yet it likely impacts corneal endothelial ion transport as part of the “pump” mechanism to maintain corneal clarity.Current advances in genetic testing for individuals with CHED have narrowed the spectrum of disease to mutations in a single gene,


There are inconsistencies in the literature on reproductive and genital health determinants of human papillomavirus (HPV) infection, the primary cause of cervical cancer. We examined these factors in the Ludwig-McGill Cohort Study, a longitudinal, repeated-measurements investigation on the natural history of HPV infection.We analyzed a cohort subset of 1867 women with one complete year of follow-up. We calculated odds ratios (OR) and 95 % confidence intervals (CI) for reproductive and genital health characteristics from questionnaire and laboratory data in relation to 1-year period prevalence of HPV infection. Two outcomes were measured; the first based on phylogenetic grouping of HPV types based on tissue tropism and oncogenicity  and the second based on transient or persistent HPV infections.Lifetime  and current  condom use and use of contraceptive injections  were associated with increased risk of HPV infection. Intrauterine device use was protective . These factors were not associated with persistence of HPV infection. Tampon use, previous gynecologic infections and cervical inflammation were associated with an overall increased risk of HPV infection.Cervical HPV infection was associated with reproductive and genital health factors. Further studies are necessary to confirm the low to moderate associations observed.The online version of this article (doi:10.1186/s12879-016-1446-x) contains supplementary material, which is available to authorized users. Cervical cancer is the fourth most common cancer in women worldwide . DespiteChlamydia trachomatis [Condom use has been inconsistently associated with a decreased risk of HPV infection , 6. Use chomatis , 13, humchomatis , 15, andchomatis . DespiteThis study focuses on the role of reproductive and genital health on cervical HPV infections using data from a large longitudinal study conducted to investigate the natural history of HPV infection.The design and methods of the Ludwig-McGill Cohort Study have been previously described . The stuQuestionnaires were administered by one of two study nurses  at each of the four visits in the first year and once per year afterwards. Baseline questionnaires collected detailed information on sexual and reproductive histories, and behavioural characteristics including smoking and hygiene habits. Cervical specimens were taken at each visit for conventional Pap cytology and molecular HPV testing. Ectocervical and endocervical cells were collected using an Accelon biosampler . Pap smears were read and graded at the Jewish General Hospital .Presence of HPV DNA was detected using polymerase chain reaction to amplify a highly conserved segment of the L1 viral gene using PGMY consensus primers , 19. TypAside from questions with predetermined answer choices, responses to open-ended questions were grouped into categorical variables, including comments disclosed to the administering nurses, but not explicitly asked . Second, we categorized HPV infections into mutually exclusive transient or persistent infections in the first year. Transient HPV infections were those involving one positive HPV test result followed by two subsequent negative results. Persistent infections were two or more positive HPV test results of the same HPV group, with no more than one negative result between two positive test results. HPV test results that did not fall into these definitions were excluded.a priori  and empirical confounders. The latter, identified using a 5 % change-in-estimate strategy, included race, marital status, education, age at first intercourse, age at menarche, number of pregnancies, smoking, alcohol drinking and years since last Pap smear.We estimated odds ratios (ORs) and 95 % confidence intervals (CIs) for HPV outcomes using logistic regression models, adjusting for n = 1267) was used in these restricted analyses. The restricted analyses, while compromising on precision, were intended to increase the validity of observed associations and were thus used to confirm associations seen in the non-restricted estimates. Adjusted ORs were calculated using the non-restricted crude estimate. All data were analyzed using Stata 12.0 .For the analysis of 1-year period prevalence, we compared women with a specific group infection  to a floating control group of those who did not have that group infection. We called this the non-restricted analysis. Since concurrent and cumulative multiple-type infections were common in the cohort, we conducted separate analyses restricted to women who only harboured infections with HPV types from a single phylogenetic group . A fixed control group that included only HPV-negative women  and current  condom use was significantly associated with an increased prevalence of Subgenus 3 HPV infections. Current condom use was also significantly associated with transient HPV infections . OC use was not significantly associated with any HPV outcome analyzed. Use of IUDs was protective across all HPV outcomes with significant effects in Subgenus 1 , Subgenus 2  and transient  infections. Tubal sterilization had a predominantly protective, although insignificant, effect across all outcomes. Of the open-ended responses, use of contraceptive injections increased the prevalence of all HPV outcomes, with Subgenus 1 , Subgenus 2  and transient  HPV infections being statistically significant. Natural and external products were not significantly associated with any HPV outcome, although there were not enough observations to calculate Subgenus 1 ORs.Menstrual cloth use had a protective effect for Subgenus 2, transient and persistent HPV infections with a significant effect on Subgenus 1 infections . Conversely, all point estimates for hygienic tampon use were above the null with Subgenus 2 infections approaching borderline significance and transient infections being statistically significant . Frequent douching was not associated with most HPV outcomes, although there was a significantly protective effect on Subgenus 3 infections . There was also no effect of the types of douching products used, with the exception of “unknown” products and Subgenus 3 HPV infections . Lastly, genital discomfort, recent discomfort and reported pain/bleeding were not associated with any HPV infection outcome.In general, no significant associations or trends were observed with the use of gynecologic products and some categories lacked observations. Of note, both former  and current  antibiotic use were associated with persistent HPV infections, while current antibiotic use  and former antifungal use  were significantly associated with Subgenus 1 infections. No significant trends were observed with the use of homemade gynecologic products, other than use of “unknown” homemade products and Subgenus 3 infections  and persistent HPV infections . Due to low numbers of women using these products, the CIs were very wide, indicating low precision in the estimates.From information volunteered by the participant and not explicitly asked in questionnaires, there were no significant associations between cervical ectropion and any HPV infection outcome. A self-report of gynecologic surgery was associated with an increased prevalence of Subgenus 1 HPV infections . Information on previous gynecologic infections or treatments volunteered by the participant was also associated with a significant increase in prevalence of Subgenus 1 HPV infections  with no association with other HPV outcomes. When previous history of gynecologic infections was explicitly asked, there were no significant associations between non-HPV-related infections and any HPV infection outcome. There was, however, a significant increase between previous HPV-related infections and Subgenus 1 , Subgenus 2 , transient  and persistent  HPV infections. Using qualitative notes from the cytology reports, there were no significant associations with bacterial or fungal infections and any HPV infection. General inflammation was positively associated with Subgenus 1 , Subgenus 2 , transient  and persistent  HPV infections.This study provides novel insights on reproductive health and vaginal hygiene factors in HPV infection. While some of these factors seemed to be associated with adverse or protective effects regarding cervical HPV infection, they largely did not play an appreciable role on the risk of infection in the Ludwig-McGill cohort study population.Previous studies  have demIUD use was associated with an overall protective effect, consistent with other studies , 25. WhiContraceptive injection use was associated with an increased prevalence of Subgenus 1, Subgenus 2 and transient HPV infections. While previous studies have shown an increased association with cervical cancer , 26, fewUse of menstrual cloths had a slightly protective effect on HPV infections, while use of hygienic tampons had an adverse effect. Use of menstrual cloths has not previously been studied in association with HPV infection and there does not appear to be a biological mechanism for this protective effect. Very limited evidence exists for an association between tampon use and risk of HPV infection. Some studies show no association , while sChlamydia trachomatis, Neisseria gonorrhoeae, HSV-1 and 2, HIV [Gynecologic product use served as a proxy for vaginal health conditions. Our results suggest that bacterial infections and use of products to control these infections may play a role in increasing susceptibility to HPV infection. BV has been positively associated with HPV infections , which id 2, HIV  and possPrevious gynecologic infections were associated with increased prevalence of Subgenus 1 infections, which we hypothesize to be related to BV. Self-reports of HPV-related infections were significantly associated with all outcomes, except for Subgenus 3 infections. Women who reported a previous HPV-related infection could have an increased risk of re-infection due to sexual behaviours or, just as plausibly, redetection of a latent HPV infection. General inflammation noted in cytology results was significantly associated with all HPV outcomes other than Subgenus 3 infections. A possible link between inflammation and cervical carcinogenesis might exist. There are indications that chronic inflammation or inflammation from other infections may play a role in increasing risk of HPV infection , possiblSurprisingly, cervical ectropion was not associated with any HPV outcome. The location of the transformation zone can affect potential exposure to HPV infection and risk of cervical cancer . Our resIn restricting our analysis to only women with one full year of follow-up, we may have introduced a selection bias. However, we believe that this enhanced internal validity and efficiency in defining the HPV outcome variables. Furthermore, losses to follow-up in the first year did not play a significant role as preliminary analyses of the non-restricted data yielded similar results. We classified type-specific HPV outcomes into groups based on phylogenetic relatedness to guide the statistical search for predictors on the basis of viral behavior and tropism. We were unable to study individual type-specific infections due to lack of precision.Several limitations need to be underscored. Due to the low-income setting of the study clinics where patients were recruited from, the study population was very specific, with respect to cultural and social norms. Secondly, outside of qualitative observations from cytology reports, exposure measurements were based on self-reported responses. As this study pertains to sexual histories and behaviour, there is a possible social desirability bias where women, especially those with riskier sexual behaviours, would tend to answer questions on sensitive topics in a socially acceptable manner. Additionally, poor recall, especially on lifetime exposures, might be at play. While we anticipate that any possible exposure misclassification would be non-differential to outcome status, women with a previous history of gynecologic infections may be more conscious of the products they have previously used. In addition, the Ludwig-McGill cohort study was not designed to investigate measures of vaginal health and hygiene as a primary objective. This post hoc analysis considered exposure measures that predominantly served as proxies for vaginal conditions. Because several of these conditions are often asymptomatic or characterized by mild symptoms, women may not have been aware of these conditions. Lastly, there could still be residual confounding due to unmeasured variables, particularly related to male behaviors.This study presents detailed analyses on the effects of vaginal health and hygiene factors, some of which seemed to be associated with risk of HPV infection differentially by subgenus grouping based on propensity to colonize different lower genital tract tissue subsites. Further research using hypothesis-driven designs with more accurate measures of exposure is necessary to examine the observed associations.


TMC6 and TMC8. Genetic variation in the TMC6/TMC8 region has been linked to beta-type HPV infection and squamous cell carcinoma of the skin, cervical cancer, HPV persistence and progression to cervical cancer. Here, we have tested the hypothesis that the common TMC8 SNP rs7208422 is associated with high-risk HPV infection and risk of head and neck squamous cell carcinoma (HNSCC). Seropositivity to the HPV L1 protein  was measured in 514 cases and 452 population-based controls. Genotype was significantly associated with seropositivity to HPV18 L1  and borderline significantly associated with HPV16 L1 . There was a consistent inverse association between TMC8 genotype and infection with other HPV types, including statistically significant associations for HPV31 and HPV52. Consistent with these results, the variant T genotype was associated with a reduced risk of HNSCC , even among subjects seronegative for all HPV types . Our data indicate that common genetic variation in TMC8 is associated with high-risk HPV infection and HNSCC etiology.HPV infection is a causal agent in many epithelial cancers, yet our understanding of genetic susceptibility to HPV infection and resultant cancer risk is limited. Epidermodysplasia Verruciformis is a rare condition of extreme susceptibility to cutaneous HPV infection primarily attributable to mutations in  TMC6 and TMC8 confer extreme susceptibility to infection with cutaneous (beta-type) HPV. These genes encode EVER1 and EVER2 which form a heterodimer that interacts with the zinc transporter molecule ZnT-1 to control zinc levels within the cell [TMC8 was associated with seropositivity to cutaneous (beta) HPV and increased risk of squamous cell skin cancer (SCC) [TMC6 and TMC8 genes could be important factors for host protection from papillomaviruses. While the complex of EVER proteins is known to regulate zinc homeostasis in keratinocytes, it has recently been demonstrated that the TMC6/8 genes are also expressed at high levels in T-lymphocytes [TMC6/8 genes may impact HPV infection and cancer risk either directly by modulating HPV infection in epithelial cells, or indirectly through immune surveillance.Null mutations in phocytes ,4. ThereTMC8 has been associated with beta-HPV infection, it is also possible that TMC8 genetic variation contributes to high-risk HPV infection and attendant cancer risk . In fact, work by Wang et al [TMC6/TMC8 gene region as being associated with HPV16 persistence. A more recent study by Castro et al [TMC8 is associated with seropositivity to high-risk HPV types and risk of HNSCC.Although common genetic variation in ng et al  using dero et al ), has noStudy protocols and materials for recruitment of cases and controls were approved by the Institutional Review Boards at the nine medical facilities and Brown University. Written informed consent was obtained from all study subjects.in situ, lip, salivary gland, or nasooropharyngeal cancer or recurrent HNSCC were excluded. The cancer registry was queried to insure that all eligible cases in the area were identified. HNSCC cases included diagnosis codes 141–146, 148, 149, and 161 according to International Classification of Disease, Ninth Revision (ICD-9). Cases were further classified by site into oral (ICD-9 codes 143–145), oropharygeal , and laryngeal (ICD-9 code 161) disease. Controls were frequency-matched to cases on age (± 3 years), gender, and town of residence identified from Massachusetts town books through systematic random selection. The town book includes all the residents more than 17 years of age listed by street address and precincts. A potential control of the same gender and age as the case was sought from this list, alternating the search direction through the book starting at the case address.This population-based case-control study was conducted in Greater Boston, MA between December 1999 and December 2003 and has been previously described . Cases wE. coli bacteria. Glutathione cross-linked to casein was coupled to fluorescence-labeled polystyrene beads  and GST fusion proteins were in situ affinity-purified on the beads directly in a one-step procedure. Each fusion protein was bound to a spectrally distinct bead set. Fusion protein-loaded bead sets were mixed and incubated with human sera. Antibodies bound to the beads via the viral antigens were detected with biotinylated goat-anti-human IgG (H+L) secondary antibody and streptavidin-R-phycoerythrin. A Luminex 100 analyzer  was used to identify the internal color of the individual beads and to quantify their reporter fluorescence (expressed as median fluorescence intensity (MFI) of at least 100 beads per set per serum).Venous blood samples were obtained from cases and controls. Serum was separated within 12–24 hr of blood drawing and samples were stored at -80°C. To detect antibodies against HPV, a glutathione S-transferase (GST) capture enzyme-linked immunosorbent assay (ELISA) was used, in combination with fluorescent bead technology, as described in detail previously ,10,11,12MFI values were dichotomized as antibody positive or negative. A standardized cut-off for HPV16, 18, 31, 33, 35, 45, 52 and 58 L1 antibodies was defined earlier  and applTMC8 gene (rs7208422), resulting in a change from asparagine to isoleucine. DNA was extracted from buffy coat using Qiagen genomic DNA extraction kits. Genotyping of the TMC8 polymorphism was done using the Taqman allelic discrimination technique , as previously published [We collected data on a (A > T) coding SNP in exon 8, codon 306 of the  gene rs708422, reTMC8 genotype and HPV serology data. To estimate the associations between TMC8 genotype and HNSCC, we calculated odds ratios (ORs) and 95% confidence intervals (CIs) from unconditional logistic regression models. Models included the study matching factors gender and age, however the third matching parameter, town of residence, was not included in these unconditional regression models given the number of towns represented. Risk models were also adjusted for smoking (pack-years) and drinking (average drinks per week). We additionally performed the analysis stratified by tumor site as well as by HPV16 serology status. To examine the relationship between TMC8 genotype and HPV serologic markers, we used logistic regression with positive serology as the response variable and TMC8 genotype as an independent variable. All statistical analyzes were performed using SAS9.1, and all statistical tests were two-sided.Data analysis was restricted to Caucasian subjects with both Among the 775 subjects, 361 were cases with HNSCC  and 414 were control subjects . There wTMC8 genotype was associated with HPV18 seropositivity , with similar non-significant trends observed for HPV16 , HPV31 , and HPV52 . We further examined the association between genotype and serology among oropharyngeal cancer cases; similar OR’s were observed, but with statistical imprecision (data not shown).We evaluated the association between genotype and HPV serology ; given tTMC8 genotype was also associated with a reduced risk of all types of HNSCC . These findings were consistent across anatomic sites of HNSCC ; this relationship remained for those negative for all HPV types . Subgroup analysis restricted to those seropositive was not possible given the low prevalence of seropositivity among controls . Gaud et al have demonstrated that the TMC8 polymorphism alters the ability of EVER2 to interact with TRADD, thereby altering apoptosis potential [TMC8 influences HPV susceptibility by two different mechanisms, dependent on the presence of absence of the E5 protein. In vitro experiments will be necessary to definitively answer these questions.Previously, we reported that this same genetic variation in nfection . At firsnfection . As Larznfection . It is potential . We hypoTMC8 polymorphism modulates this biology, impacting T-cell surveillance functions and cancer risk. Additional epidemiologic studies of non-viral mediated cancers will help elucidate whether TMC8 is a generalized cancer susceptibility factor.While EVER2 expression in keratinocytes has received considerable attention, given its role in the inherited syndrome Epidermodysplasia Verruciformis, recent work demonstrates that EVER2 expression in T lymphocytes exceeds that found in keratinocytes. In T-cells this high expression of EVER2 is rapidly down-regulated upon TCR stimulation, leading to an increase in intracellular Zn levels. We hypothesize that the TMC8 genotype and susceptibility to infection, this potential limitation in sensitivity would bias our results to the null. Another limitation of these data is that the serologic response we assess is not necessarily reflective of an infection at the tumor site, but might instead reflect a high-risk infection at a site other than the tumor. However, the low seroprevalence of high-risk HPV among non-diseased individuals  [TMC6/TMC8 gene were associated with cervical cancer, however this study lacked measures on HPV . Clearly a comprehensive evaluation of the extent, character and phenotype of variation in the TMC6/TMC8 gene region will serve to more fully delineate our understanding of genetic susceptibility to HPV infection. Finally, given our very strong associations between TMC8 genotype and HNSCC in those without evidence of HPV infection it will be important to establish whether the TMC8 gene is a generalized cancer susceptibility factor.Of interest, a previous study of cervical cancer by Wang et al, using a GWAS approach, identified a rare SNP in the


Indigenous women from the Amazon regions have some of the highest rates of cervical cancer in the world. This study evaluated cervical cytology and human papillomavirus (HPV) in native women that differ by lifestyle and interaction with western society. Yanomami women are isolated deep in the Amazon with a hunter/gatherer lifestyle. Macuxi and Wapishana women live in proximity to western society.To select a representative group of women from each district, random cluster sampling was used, considering each registered village as a cluster. Cervical samples were collected for cytology and HPV detection and typing by PCR amplification and next generation sequencing. The study was approved by the National IRB and by tribal leaders.664 native women were enrolled from 13 indigenous villages (76% participation rate). Yanomami women had higher rates of abnormal cytology  and prevalent HR-HPV . Yanomami women >35y of age were significantly more likely to have HR-HPV, whereas women ≤35y did not significantly differ between groups. Prevalence of HPV was significantly different amongst geographically clustered Yanomami women (p<0.004). The most prevalent HPV types in the entire group were HPV31 (8.7%), HPV16 (5.9%) and HPV18 (4.4%).Isolated endogenous Yanomami women were more likely to be HPV+ and rates increased with age. Study of HPV in isolated hunter-gather peoples suggests that long-term persistence is a characteristic of prehistoric humans and patterns reflecting decreased prevalence with age in western society represents recent change. These studies have implications for cervical cancer prevention and viral-host relationships. Among the nearly 500,000 new cases of cervical cancer (CC) estimated annually worldwide, 80% occur in middle or low income countries such as Brazil, where 1Despite improving the health of indigenous populations being identified as a priority for Healthcare Organizations, the heaCurrently most isolated indigenous groups in the Americas are inhabitants of the Amazonian rainforest, with high rates of cervical cancer and little knowledge of the HPV within these groups. A study by Ong et al. investigDifferent populations, especially isolated indigenous people, may exhibit distinct characteristics of HPV infections, related to their ancestry, lifestyle and sexual behavior. Moreover, knowledge about the epidemiology of HPV is necessary to understand the etiologic role of different HPV types and variants in these populations in order to develop effective preventative strategies against CC. Isolated indigenous groups (Yanomami) and groups with permanent interaction with the surrounding society (Macuxi and Wapishana) inhabit the extreme north of the Brazilian Amazon, presenting an ecological difference that can be used to investigate the influence of isolation and prehistoric living conditions on HPV diversity and associated cervical disease. To date, the prevalence and diversity of cervical HPV in these groups have not been studied. The objective of this investigation was to evaluate the prevalence of cervical intraepithelial lesions and HPV in indigenous groups from the Amazon region, before the vaccination against HPV.The study was approved by the IRB of the Tropical Medicine Foundation of the Universidade do Estado do Amazonas. As indigenous peoples are considered vulnerable by the Brazilian government, the study was also reviewed and approved and by the National Committee for Ethics in Research involving Human Beings in Brasília—DF . Tribal leaders agreed and formally authorized the execution of the study, but some questions were not approved by them  and were excluded from the questionnaire . The Yanomami people have no written language, but only spoken language. So they can not be considered illiterate. Consequently, the study considered the use of fingerprints in the consent form, and this item was fully approved by both IRB. The female participants were widely informed about the study aims and methods, and signed (or fingerprinted) the consent form. Assistance from professional translators was used as necessary. All samples were coded and did not have individual identifying information.A cross-sectional study was performed, which consisted of selecting a random sample of indigenous women in remote areas of the northern Amazon region of Brazil, to collect biological samples , between February and July 2013. The study was designed to evaluate the prevalence of cervical intraepithelial lesions by cytology, and HPV infection by PCR amplification and next generation sequencing (NGS) in a pre-HPV vaccination population comparing two groups of women defined below.Due to cultural, ethnic and anthropomorphic factors, the Federal Government of Brazil has divided the indigenous groups from northern Amazonian region into two geographic regions : the Eastern Indigenous District (composed mainly by Macuxi and Wapishana groups), and the Yanomami Indigenous Disctrict (Yanomami and Yekuana groups) .Karib linguistic affiliation and the Pemon ethnic group. These groups are characterized by having very tenuous ethnic borders with non-indigenous society, 3332, 3This study is the first random sampling of a difficult to access group of women and revealed a high prevalence of HPV in indigenous groups from northern Brazilian Amazonian region, reaching 45.9% in Yanomami women. Despite differences in laboratory methods, this seems to be a higher prevalence than those usually reported in Brazil. Studies on the HPV prevalence are generally concentrated in the more developed regions in the country (south and southeast), ranging from 10.4 to 24.5% in asymptomatic (non-indigenous) women –36. Two This study was performed in a non-vaccinated population. A high genotypic diversity of HPV in both indigenous groups was observed, mainly in Yanomami group. While Rocha et al. found 14The above results classify indigenous women, particularly the Yanomami women, at high risk for HPV infection and cervical cancer. Our study also revealed a significant difference in prevalence of cervical lesions between the two ethnic groups studied, which differed in 2 centuries of interaction with the surrounding society, and that have been coexisting geographically but historically and socially separated. The impact of environmental and socio-cultural changes that take place in virtually all indigenous communities on women's health have pointed to an epidemiologic transition in American indigenous peoples, due to increasing contact with western society and lifestyles, 44. In 34. Difficulties of women’s understanding and acceptance of the screening, infrastructure deficiencies and logistical barriers related to access difficulties may explain the low inclusion of Yanomami women to complete and effective prevention programs. The higher prevalence of Yanomami women who had never had a cytological examination reported in the present study corroborate this notion.Relevant characteristics and cultural practices of reproductive health of Yanomami women were highlighted by Early et al. and may A topic that deserves attention is the relation between HPV and age in these groups. The prevalence of HPV in the Eastern indigenous District presented an L-shaped curve, similar to that described for non-indigenous Brazilian women. HoweverThis study has limitations. First, the cross-sectional design does not allow the use of temporality and causality criterion, since outcome and risk factors were measured synchronously, so bias and reverse causality could not be eliminated. Second, most Yanomami women did not know the Christian calendar, and they informed their age based on lunar cycles, making this data relatively imprecise, mainly for the older participants, requiring caution in this analysis. However, we have analysed age data using the standard IARC age categories, what strengthen the credibility of the results. Third, data from indigenous census were used to adjust the sample size calculation and to calculate the participation rate. Despite being the best data available, these data can be inaccurate, due to the isolation of the Yanomami tribes.Despite the identification of 8 novel HPV genomes, only 1 was identified in the most isolated ethnic group. These types were rare, and we were unable to identify novel endemic papillomaviruses in the Yanomami women that are supposed to have been socially isolated from non-indigenous populations for at least 12,000 years. It seems more likely that most, if not all, HPV types evolved thousands of years ago in prehuman primates and took genomic forms similar to those existing today. Nevertheless, future studies of the HPV variants in these women should better disclose their origins.Indigenous women from the extreme north of the Brazilian Amazon, especially the Yanomami ethnic group, have increased risk of CC. The high prevalence of HR-HPV, in addition to their geographic isolation and subsequent lower adherence to screening (and treatment) of intraepithelial lesions may contribute to this vulnerability. Yanomami women have relatively higher prevalence of HPV16/18/31 compared to Macushi and Wapishana women, the most oncongenic types. An increasing prevalence and persistence of HPV with age was observed in Yanomami women, which may represent a hunter-gatherer pattern. There was a lower relative and absolute prevalence of HPV16 in relation to that described in non-indigenous women from Brazil, and the opposite was observed for HPV18. Despite the identification of 8 potential novel HPV genomes, and we were unable to demonstrate the presence of novel endemic papillomaviruses unique to the Yanomami women.S1 Fig(TIFF)Click here for additional data file.


P < 0.05 and P < 0.0001). Great variation in HPV prevalence was observed between study populations of different geographical locations. This review demonstrates an association between spontaneous abortion, spontaneous preterm delivery, and the presence of HPV in both the cervix and the placenta. However, a reliable conclusion is difficult to draw due to the limited number of studies conducted on material from pregnancies with adverse outcome and the risk of residual confounding.Based on the current literature, we aimed to provide an overview on Human Papillomavirus prevalence in normal pregnancies and pregnancies with adverse outcome. We conducted a systematic literature search in PubMed and Embase. Data extracted from the articles and used for analysis included HPV prevalence, pregnancy outcome, geographical location, investigated tissue types, and HPV detection methods. The overall HPV prevalence in normal full-term pregnancies was found to be 17.5%  for cervix, 8.3%  for placental tissue, 5.7%  for amniotic fluid, and 10.9%  for umbilical cord blood. Summary estimates for HPV prevalence of spontaneous abortions and spontaneous preterm deliveries, in cervix  and placenta , were identified to be higher compared to normal full-term pregnancies ( Intrauterine infection by bacteria is well established as a pathway leading to spontaneous abortion and spontaneous preterm birth , 2. OthePregnancy has previously proven to be a state of mild immunosuppression due to the decrease in the number of natural killer cells , possiblIt is therefore of great interest to examine how widespread HPV infections are among pregnant women and whether or not there is an association between HPV infection and spontaneous abortion or spontaneous preterm delivery. Moreover, nowadays there exists a successful vaccination to prevent infection and disease caused by infection with HPV-6, HPV-11, HPV-16, and HPV-18. Thus, there might be a chance to minimize the risk for pregnancy complications by applying the same or a modified version of vaccination. Our group studies the impact of placental HPV infection on spontaneous abortion and preterm delivery. In this context the aim of this study is to provide an overview of the existing literature by doing a systematic review on HPV prevalence in pregnancy. We focused on pregnancies with adverse outcome and included a discussion of possible factors influencing or explaining the reported differences in HPV detection rates.PRISMA and MOOSE guidelines were used where applicable .A systematic literature search was conducted in the PubMed and Embase databases and search terms were used as follows: (1) “Human papillomavirus AND pregnancy”; (2) “Human papillomavirus AND preterm delivery”; (3) “Human papillomavirus AND preterm birth”; (4) “Human papillomavirus AND abortion”. The search was restricted to articles in English, on humans and published between January 1995 and October 2014. The search was carried out on October 28, 2014. in vitro fertilization studies were excluded.In order to identify relevant articles for whole-paper revision, duplicates were removed and titles and abstracts were screened based on the following exclusion criteria: studies investigating cell lines only, HPV vaccines, or sperm-related aspects, as well as guideline articles, general articles describing public health, and literature reviews. The remaining articles were assigned to subsequent whole-paper revision. These articles were systematically reviewed in accordance with the inclusion and exclusion criteria. The inclusion criteria were set to the following: studies on asymptomatic healthy pregnant women and women experiencing a spontaneous preterm delivery or spontaneous abortion; investigation of HPV infection within cervix, placenta, amniotic fluid, or umbilical cord blood; HPV detection test directly linkable to index pregnancy. Studies restricted to nonpregnant women or HPV positive women only, case reports, follow-up and association studies, andStudies including women with history of HPV-related lesions were not excluded, as this will be the case in any normal study population. There was no restriction for studies of different geographical origins, the time point of sample collection, methods of sample collection, and HPV testing method. Information on the latter was included in P values were two-sided and P < 0.05 was considered significant.Data, which includes information about the investigated tissue types, country where the study was performed, sample size, sampling time point, HPV prevalence, and HPV detection method, was extracted and analyzed using MatLab (version R2011b). Data from all studies was pooled and women were grouped according to their pregnancy outcome or the pregnancy status at time point of sample collection. An overall HPV prevalence was calculated including 95% confidence interval (CI) in normal pregnancies and in pregnancies with adverse outcome. This was done for various tissue types, geographical origins, time points of sample collection, and HPV detection methods used. Studies from Mexico and Brazil have been grouped into “Latin America.” Statistical significance between two proportions was tested using “Two-sample test of proportions.” All n = 4639) [n = 7116) [n = 1681) [n = 1034) [The initial PubMed database search resulted in the identification of a total of 650 articles . After r = 4639) , 66, 67, = 7116) , 57, 61, = 1681) , 65, 68, = 1034) , 50, 54.P < 0.05, P < 0.001, and P < 0.0001, resp.).HPV prevalence in healthy pregnant women giving birth at term was investigated in 38 of the 45 included studies and provided data on 13 757 pregnant women. HPV prevalence appears to be highly dependent on the tissue type tested . In all HPV prevalence in pregnancies with adverse outcome, including spontaneous abortion and spontaneous preterm delivery, was investigated in 10 of the 45 included studies and provided data on 681 pregnancies. Three studies were investigating cervical HPV infection and seven studies looking at HPV prevalence in placental tissue. Details are given in P < 0.0001) and spontaneous abortions  (P < 0.0001) and 24.9% (P < 0.0001) of cases, respectively  . This is < 0.05) . An even < 0.05) , whereasectively .Information on geographical distribution of the investigated studies was collected to see if ethnicity might be a contributing factor to the great variation in HPV prevalence observed. The goal of the investigation was to determine whether women from different countries may be differentially exposed to HPV and some may thereby have a higher risk for possible HPV-induced pregnancy deficiencies.P < 0.0001) HPV prevalence compared to European and Asian women , meaning that HPV prevalence in pregnant women is dependent on geographical or ethnical parameters, with pregnant women from USA and Latin America having the highest HPV prevalence reported. The same tendency can be observed in placenta, amniotic fluid, and umbilical cord blood, but numbers were insufficient for proper analyses.an women . Latin AVariation of HPV prevalence between studies conducted on different continents can also be observed in studies investigating pregnancies with adverse outcome , but a pP < 0.05, P < 0.01, P < 0.001, and P < 0.0001 resp.). Not significant was the difference between the proportions for the first trimester and at birth samples (P = 0.12), the second trimester and the third trimester, respectively, and the postpartum period (P = 0.07 and P = 0.39), and the third trimester and the postpartum period (P = 0.14).There are multiple factors that may influence HPV prevalence. For the present analysis, data on the time point of sample collection and the HPV detection methods used were collected. P < 0.05, P < 0.01, and P < 0.0001, resp.). Not significant was the difference between proportions for PCR and DNA chip (P = 0.25). The HPV detection method used most widely was PCR with n = 9674 followed by DNA chip with n = 5461.Also the HPV detection methods used may influence the found HPV prevalence. P < 0.05 and P < 0.0001), in both placenta  and cervix . Beyond the geographical location, the time point of sample collection in pregnancy as well as the HPV detection methods used may influence the results on HPV prevalence.In this quantitative analysis on the prevalence of HPV infection in normal pregnancies and pregnancies with adverse outcome, 45 studies were included and data on 14 470 pregnant women were analyzed and summarized. HPV prevalence in normal pregnancies was found to vary between tissue types and study populations of different geographical locations. The highest HPV prevalence could be reported in cervix  and in the population from Latin America ) and USA ). In comparison to HPV prevalence found in normal pregnancies, spontaneous abortions and spontaneous preterm deliveries were found to have higher HPV positive detection rates (The present work has some weaknesses. First, heterogeneity between studies is a problem. Due to the limited number of studies conducted within the field of HPV infections and adverse pregnancy outcomes, inclusion criteria were set relatively widely. This results in a higher number of pregnancies to analyze but possibly more heterogeneous study groups and thereby inflict restricted options to compare directly between studies. The possibility of forming totally homogenous study groups is limited by the study quality and information given, and data on patient-level is missing in most, if not all, of the included studies, which makes controlling for all potential biases difficult. HPV prevalence in pregnant women has been reported to be higher compared to nonpregnant women , 68, 69. in utero is therefore well protected. Nevertheless, multiple studies indicate that viral infection can impair trophoblast function, potentially contributing to pregnancy loss or abnormal implantation [An infection with HPV during pregnancy has been associated with risk for spontaneous abortion and spontaneous preterm delivery as well as placental abnormalities –21. Our antation , 77. Furantation .As previously mentioned, the wide range of HPV prevalence found is related to several factors including study design, geographic and demographic characteristics, choice of detection method, and risk factor profiles, such as maternal age, gestational age, and history of cesarean section , 70, 79.The time point of sample collection during pregnancy is thought to have an influence on HPV prevalence as differences in HPV prevalence regarding the trimester tested have been published –83. Our Finally, we compared HPV prevalence with respect to choice of HPV detection methods, since studies have shown that HPV DNA detectable and the HPV-genotypes identified largely depend on this . Our anaFunctional studies investigating HPV infection in relation to pregnancy are limited. Following the detection of HPV in placental tissue , 18, 62 Based on the present quantitative analyses it can be concluded that HPV prevalence is higher in pregnancies with adverse outcome, such as spontaneous abortion or spontaneous preterm delivery, compared to women experiencing a normal full-term pregnancy. HPV infection may therefore be constituted as a risk for the present pregnancy.HPV prevalence has been shown to be dependent on the tissue type tested and the geographical location of the study population analyzed. However, the number of studies investigating HPV infection on material from spontaneous abortions and spontaneous preterm deliveries is very limited and study groups are heterogeneous which makes a reliable conclusion difficult. It can be stated that study design is important, the selection of proper controls is essential, and, for a valuable comparison between studies, similarity in samples/patients needs to be controlled as strictly as possible. Furthermore, one should keep in mind that the simple detection of a virus never is equal to a real causative role in the adverse outcome of a pregnancy or diseases in general. It is therefore inevitable to study the viral activity and cellular localization to be able to conclude on the impact for a given situation. Therefore we recommend including an investigation of the molecular mechanism of HPV infections in material of pregnancies with adverse outcome and inviting researchers to conduct new studies to clarify HPV's impact on spontaneous abortion and spontaneous preterm delivery. As a consequence of the aforementioned limitations our study group has initiated a prospective study addressing the complexity of HPV in pregnancy.