A novel descriptor (Complex Correlation Measure (CCM)) for measuring the variability in the temporal structure of Poincaré plot has been developed to characterize or distinguish between Poincaré plots with similar shapes.CCM during atropine infusion, 70° head-up tilt and scopolamine administration in healthy human subjects. CCM quantifies the point-to-point variation of the signal rather than gross description of the Poincaré plot. The physiological relevance of CCM was demonstrated by comparing the changes in CCM values with autonomic perturbation during all phases of the experiment. The sensitivities of short term variability (SD1), long term variability (SD2) and variability in temporal structure (CCM) were analyzed by changing the temporal structure by shuffling the sequences of points of the Poincaré plot. Surrogate analysis was used to show CCM as a measure of changes in temporal structure rather than random noise and sensitivity of CCM with changes in parasympathetic activity.This study was designed to assess the changes in temporal structure of the Poincaré plot using CCM was found to be most sensitive to changes in temporal structure of the Poincaré plot as compared to SD1 and SD2. The values of all descriptors decreased with decrease in parasympathetic activity during atropine infusion and 70° head-up tilt phase. In contrast, values of all descriptors increased with increase in parasympathetic activity during scopolamine administration.CCM values with parasympathetic activity indicates that the temporal variability of Poincaré plot is modulated by the parasympathetic activity which correlates with changes in CCM values. CCM is more sensitive than SD1 and SD2 to changes of parasympathetic activity.The concordant reduction and enhancement in  Heart rate variability (HRV) is a powerful non-invasive method for analyzing the function of the autonomic nervous system. It is useful to understand the interplay between the sympathetic and parasympathetic autonomic nervous system, which serves to speed up and slow down the heart rate, respectively . HRV, thSD1) and long-term variability (SD2)) to quantify the information from the plot. The Poincaré plot of HRV signal is constructed by plotting consecutive points of RR interval time series . It is a representation of HRV signal on phase space or Cartesian plane  and heart rate dynamics [temporal structure (CCM) values]. Subtle differences in dynamics of HRV signal were detected by CCM in all phases of the study. These observations provide some novel information on the physiological relevance of CCM for Poincaré plot analysis: 1) The variability of temporal structure of Poincaré plot of HRV, quantified using CCM, correlates the parasympathetic activity 2) CCM is highly sensitive to changes in parasympathetic activity  as compared to SD1 and SD2. Although CCM captures temporal variation of Poincaré plot, it fails if the RR intervals are aligned on a line. However, existence of few zero area patterns does not affect the overall CCM value as it is measured using a moving window of three consecutive points. Further studies of CCM of HRV signal with changes in sympathetic activity may give the complete physiological explanation of CCM with respect to sympathovagal activity. Moreover, due to well published changes in autonomic regulation between men and women and in different age groups [CCM would be of interest in further studies.By using the quantitative Poincaré plot analysis of HRV signal, we observed that atropine infusion, 70° head-up tilt and scopolamine administration result in changes in heart rate variability [short term variation (e groups , and invThe authors declare that they have no competing interests.CKK conceived, derived and implemented the new descriptor, generated experimental results data and drafted the manuscript with supervision of AHK and MP. AHK, AV and MP contributed to the development of the new descriptor and participated in the discussion and interpretation of the results. All authors read and approved the final manuscript.


In conclusion, HBRT-H14 is applicable for CC screening with the advantage of genotyping for HPV 16 and HPV 18, which may help to improve triage management for women with negative cytology.The American Society for Colposcopy and Cervical Pathology (ASCCP) suggests that women ≥30 years old, with a negative cytopathological test but a positive high-risk (HR) human papillomavirus (HPV) test should undergo HPV 16 and HPV 18 genotyping. If this test is positive, immediate cervical pathology is required. Therefore, the aim of this study was to evaluate the effectiveness and clinical value of testing for 14 HR HPVs with HPV 16 and HPV 18 genotyping for cervical cancer (CC) screening. A total of 424 females from the China-Japan Friendship Hospital were selected and randomly divided into two groups (A and B). All participants underwent two different testing methods: the liquid-based cytology test (LCT) and a HPV DNA test. For the HPV DNA test, participants in group A underwent the hybrid capture II (HC-II) testing method while participants in group B were tested using the quantitative polymerase chain reaction  method. The sensitivity, specificity, positive predictive value and negative predictive value for the detection of cervical intraepithelial neoplasia (CIN) grade II or greater using HBRT-H14 were 96.30, 78.17, 23.21 and 99.68%, respectively. In Group B, compared with other HR HPV types, HPV 16 and HPV 18 infection led to the increased possibility of cervical lesions graded CIN II or higher . A significant difference in the rates of CC and CIN II or higher was observed among women who were i) infected with HPV 16 and/or HPV 18, ii) infected with other HR HPV types and iii) diagnosed as negative for HR HPV infection (χ It has been identified that ~95–100% of the incidences of cervical cancer (CC) are due to infection with the human papillomavirus (HPV) . There aBetween August 15, 2011 and November 10, 2011, a total of 424 females, with a mean age of 39.02±12.00 years , underwent CC screening and treatment in the China-Japan Friendship Hospital cervical cancer treatment center . None of the recruited patients had exhibited acute inflammation of the reproductive tract. Written informed consent was obtained from all patients. In group A, hybrid capture II (HC-II) and liquid-based cytology testing (LCT) were preformed; in group B, 14 patients underwent HR HPV with HPV 16 and HPV 18 genotyping using the quantitative polymerase chain reaction  test and LCT. For subjects who were HR HPV positive and had an LCT result that was at least as severe as atypical squamous cells-undetermined significance (ASC-US); HPV 16 and/or HPV 18 positive with negative cytological results; or had a cytological test result that was at least as severe as ASC-US and were HPV negative, colposcopies were performed.The thin-layer cytology specimens were obtained using SurePath™ , and an experienced gynecologist evaluated the histopathological slides. The Bethesda system (TBS) protocol, revised by the International Cancer Association in 2001, was used as the standard for cytological diagnosis . SquamouHBRT-H14 qPCR is a new PCR assay that detects 12 HR HPV genotypes  with simultaneous differentiation between HPV 16 and HPV 18. This method was based on the multiplex qPCR assay which utilizes 14 sets of specific primers and four probe sets, designed in the E region of the HPV genome, with varying fluorophores in order to obtain fluorescent detection signals. The channels of reporter dyes FAM, HEX, ROX and Cy5 represent 12 HR HPV genotypes, HPV 16, HPV 18 and the β-globin gene , respectively. Amplification was performed according to the manufacturer’s instructions .The HC-II test system  was used to detect HPV in the obtained samples  as previously described  underwent colposcopy testing and cervical biopsies. Multi-point biopsies were performed on the area of epithelial cells to which acetic acid had been applied and on the unstained iodine test point. For the normal transformation zone, four points 2 underwe in totalet al  reported2 test. P<0.05 was considered to indicate a statistically significant difference.Histopathological results were considered to be the gold standard. SPSS 16.0  was used to calculate the sensitivity, specificity, positive predictive value and negative predictive value of HBRT-H14 for CC screening. Comparisons among groups were made using the χ2=93.976, P=0.0001).Using the HBRT-H14 HPV test and the LCT screening method, 19 samples in 27 subjects with CIN II or above were HPV 16 positive according to the HPV DNA test, accounting for 70.37% (19/27), among which four samples were cytologically negative. One sample  was identified to be HPV 18 positive with other HR HPV infection; six subjects tested positive for other HR HPV (non-HPV 16 and non-HPV 18), accounting for 22.22% (6/27); and one sample  gave a negative HR HPV result . CIN II 2=2.09, P>0.05). The HPV 16 infection rates  also showed no statistically significant difference between the two groups . In HPV 16 positive subjects, the percentage of patients graded as CIN II or above was 25% (2/8) for group 1 and 65.38% (17/26) for group 2; the difference was deemed to be statistically significant . Due to the low detection rate of HPV 18, no statistical analysis was performed for this virus. The infection rates of other HR HPV (non-HPV 16 and non-HPV 18) infections were 15.00% (18/120) and 18.60% (56/301) in groups 1 and 2, respectively ; there was no statistically significant difference between the two groups. The rates of other HR HPV (non-HPV 16 and non-HPV 18) infection with CIN II or above are 11.11% (2/18) and 7.14% (4/56) in groups 1 and 2, respectively ; the difference between groups was considered to have no statistical significance.In order to examine whether the HBRT-H14 HPV test is only required in the CC screening of females ≥30 years old, the subjects were divided into two groups with 30 years of age as the borderline. 2=93.976, P=0.0001). In cytologically negative subjects diagnosed with ASC-US and LSIL, the differences between the various types of HPV associated with the CC were deemed to be statistically significant .Of the 379 subjects considered to be normal or inflamed by the cervical cytological method, 18, 5, and 56 subjects, respectively, were tested to be HPV 16, HPV 18 and other HR HPV (non-HPV 16 and non-HPV 18) positive. Histological results following the cervical biopsy were that samples from four of the HPV 16 positive patients, one of the HPV 18 positive patients and none of the patients positive for other HR HPV were graded as CIN II or above. In the nine subjects diagnosed with ASC-US using the cervical cytological method, two were HPV 16 positive and were graded CIN II according to the cervical biopsy; the other seven subjects were both HPV 16 and HPV 18 negative, and evaluated as having inflammation according to the biopsy. Using the cervical cytological test, 17 subjects were diagnosed with LSIL, of which two were HPV 16 positive and graded CIN II by biopsy, and 11 subjects were other HR HPV (non-HPV 16 and non-HPV 18) positive, among which four were graded CIN II. The remaining four subjects were HR HPV negative and one was confirmed to have inflammation, three were confirmed to have CIN I according to the biopsy results . A gradiAnalysis of 14 HR HPVs with the 16/18 genotyping real-time PCR kit is based on a multiplex qPCR assay utilizing specific sets of amplification primers with varying fluorophores in order to obtain fluorescent detection signals. qPCR is capable of dispensing with the problems associated with electrophoresis and the probability of contamination from traditional PCR . PCR ampIn this study, three β-globin negative samples were reported among 424 samples (0.7%), rendering these three subjects invalid. It was possible that the HPV copy number was too low to be amplified due to too few cells being obtained. False negative results in HPV DNA detection are likely to increase if this quality monitoring option is lacking. Thus it is critical to include quality monitoring in HPV detection.In the natural history of HPV infection, the majority of infections are temporary, particularly among young women. Only a small proportion are persistently infected by HR HPV, and usually CC develops following 10 years of continuous infection . Thus, tet al (Song et al  consideret al ,11.et al (The guidelines from the ASCCP and those of the Spanish CC screening and prevention programs suggest that cytologically negative but HR HPV positive subjects should continue to undergo HPV 16 and HPV 18 genotyping . If the et al .


Intratypic variations of HPV-18 are known to differ in the persistence of the infection, frequency of carcinogenesis and the progression of precursor lesions to advanced cervical cancer. This study was designed to analyze sequence variations of HPV-18 isolates in order to discover novel HPV-18 variants and to evaluate the variations among infected women in southwest China. Cervical biopsies from 56 HPV-18-positive women with cervical neoplasia were assayed by PCR amplification and sequencing of all eight genes  of the HPV-18 genome. The most frequently observed variation was a C to G transversion at nucleotide 287 of E6, which was found in 48.2% of samples. Analysis of E7 revealed only one specimen as having sequence variations. In addition, we have identified several novel variations: A551C in E6, G6906A in L1, and C4915T and C5147A in L2. The mutations in E6 and L2 are silent, while the E7 mutation results in a single amino acid change. This study complements and expands on previous descriptions of HPV-18 variants. The sequence variation data presented here provides a foundation for future research on HPV-induced oncogenesis and may prove valuable for developing diagnostic probes and in the design of HPV vaccines for targeted populations. Cervical cancer is the second leading cause of cancer-related deaths in women worldwide HPV is a double-stranded circular DNA virus with a genome size of about 8000 bp that encodes early proteins  and late proteins  In addition to the diversity among types of HPV, there are many natural intratypic variants, some of which contain alterations that lead to changes in the amino acid (AA) residues of functional and/or antigenic domains. Some of these changes have been shown to influence the persistence of the infection, morbidity of carcinogenesis, and the progression of precursor lesions to cancer The study was approved by the Education and Research Committee and the Ethics Committee of Sichuan University . Written informed consent was obtained from each patient, who granted us permission to use the data obtained in subsequent studies.th People’s Hospital of Chongqing. Medical background information was recorded from all patients.Specimens were obtained from the cervical biopsies of 56 women with cervical cancer who tested positive for HPV-18 at the Cancer Hospital of Sichuan Province and the 4DNA was extracted using a commercial kit (U-gene DNA kit) according to manufacturer’s instructions .DNA was extracted and stored in a designated area free from potential DNA contaminants.PCR reagents were mixed under a biosafety-hood in a designated room that was free from potential DNA contaminants. Amplification of the integrated HPV genes was performed with primers that were designed according to the HPV-18 reference sequence published in GenBank (accession number NC_001357) .2, 400 µmol/L dNTPs, 3 U Taq DNA polymerase and 4 pmol primers.The PCR amplification was performed under the following conditions. An initial 5 min denaturation step at 95°C was followed by 35 amplification cycles, with each cycle including a 45 s denaturation step at 94°C, a 45 s annealing step at 55°C, and a 60 s elongation step at 72°C. Amplification was complete after a final 10 min elongation step at 72°C. Each 50 µl PCR reaction contained 4 mmol/L MgClAmplicons were visualized on 1.0% agarose gels stained with ethidium bromide under UV transillumination (data not shown).PCR products were automatically sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s instructions. The sequences were subsequently analyzed by NCBI Blast and DNAMAN version 5.2.2. HPV-18 DNA nucleotide positions were numbered according to the HPV-18 reference sequence (NC_001357).All data were confirmed by repeating PCR amplification and sequence analysis at least twice.In this study, nucleotide (nt) sequences of E1 (nt 914 to 2887), E2 (nt 2817 to 3914), E4 (nt 3418 to 3684), E5 (nt 3936 to 4157), E6 (nt 105 to 581), E7 (nt 590 to 907), L1 (nt 5430 to 7136) and L2 (nt 4244 to 5632) were determined. Sequence variations were identified in 33 for the 56 specimens assayed . In 23 oAs shown in DNA sequence analysis of the HPV-18 E2 region revealed seven variations: a T to G transversion at nt2856, G to C transversion at nt2857 and C to G transversion at nt2858, leading to a C14A AA substitution ; a G to T transversion at nt2859, leading to a V15L AA substitution  ; a C to DNA sequence analysis of the HPV-18 E6 region revealed two variations that did not lead to AA changes. The most frequent variation was a C to G transversion at nt287  , with a DNA sequence analysis of the HPV-18 E7 region revealed two variations, both of which occurred only once and in the same specimen: a C to T transition at nt640, which did not result in an AA change , and an DNA sequence analysis of the HPV-18 L1 region revealed six variations: a G to A transition at nt5503, leading to a R25Q AA substitution  ; a C to DNA sequence analysis of HPV-18 L2 region revealed two variations that did not lead to AA changes: a C to T transition at nt4915   and a C No variations were detected in the HPV-18 E4 and E5 regions.A study by Angulo et al. Among the early gene products, the E1 viral protein plays a critical role in controlling viral replication and load, and it requires the interaction with the E2 protein to bind to the LCR 6GGT) The E2 protein contains three distinct domains: a transactivation domain (TAD), an unstructured hinge domain, and a DNA binding domain (DBD) Mutagenesis analysis of the E2 TAD indicated that most of the conserved AAs in this domain are required for both transcriptional activation and viral DNA replication E1 shares the sequence variations at nt2856–2859 with E2 in TAD, through which E2 interacts with the E1 helicase E6 and E7 encode the main viral proteins involved in human epithelial cell immortalization and transformation. The oncoproteins are able to increase the expression of genes related to DNA synthesis and coordinate cell cycle induction, thereby increasing the productive life cycle of HPV The E7 protein interacts with another cellular tumor suppressor, pRb, and disrupts its normal function in regulating the cell cycle, thereby stimulating expression of many E2F-regulated transcripts Changes in the L1/L2 regions of the HPV genome may be important in determining the infectious potential of different variants, as well as in defining epitopes relevant to vaccine design In the L1 coding region, one A to G transition at nt5503 and four C to G transversions were found. These five variations were also observed in a study by Arias-Pulido et al. Like L1, the L2 protein plays an important role in the initial steps of papillomavirus infection Interestingly, no sequence variation was found in E4 and E5, in contrast to a report by Arias-Pulido et al. In summary, this study reports sequence variations in the E1, E2, E6, E7, L1 and L2 genes of HPV-18 isolates from southwest China. The sequence variations in the genes examined may contribute to HPV oncogenesis. Our data will provide a solid foundation for further biological and clinical studies, and may also be employed in epidemiological studies where sequence variations are used as markers for monitoring HPV infections in target populations. Elucidating the functional and pathological differences between intratypic variants of HPV, as well as determining the molecular basis for their propensity to induce different histological types of cervical cancer, are important areas of future investigation. Further studies of HPV sequence variation will also need to include different geographic regions with distinct and isolated human populations.


A protocol for cervical cancer screening among sexually active women 25 to 65 years of age was introduced in 2006 in Catalonia, Spain to increase coverage and to recommend a 3-year-interval between screening cytology. In addition, Human Papillomavirus (HPV) was offered as a triage test for women with a diagnosis of atypical squamous cells of undetermined significance (ASC-US). HPV testing was recommended within 3 months of ASC-US diagnosis. According to protocol, HPV negative women were referred to regular screening including a cytological exam every 3 years while HPV positive women were referred to colposcopy and closer follow-up. We evaluated the implementation of the protocol and the prediction of HPV testing as a triage tool for cervical intraepithelial lesions grade two or worse (CIN2+) in women with a cytological diagnosis of ASC-US.During 2007-08 a total of 611 women from five reference laboratories in Catalonia with a novel diagnosis of ASC-US were referred for high risk HPV (hrHPV) triage using high risk Hybrid Capture version 2. Using routine record linkage data, women were followed for 3 years to evaluate hrHPV testing efficacy for predicting CIN2+ cases. Logistic regression analysis was used to estimate the odds ratio for CIN2 +.p < 0.01).Among the 611 women diagnosed with ASC-US, 493 (80.7%) had at least one follow-up visit during the study period. hrHPV was detected in 48.3% of the women at study entry (mean age 35.2 years). hrHPV positivity decreased with increasing age from 72.6% among women younger than 25 years to 31.6% in women older than 54 years  were identified and all but one had a hrHPV positive test at study entry. Sensitivity to detect CIN2+ of hrHPV was 97.2%  = 85.5-99.9) and specificity was 68.3% (95%CI = 63.1-73.2). The odds ratio for CIN2+ was 45.3 (95% CI: 6.2-333.0), when among ASC-US hrHPV positive women were compared to ASC-US hrHPV negative women.Triage of ASC-US with hrHPV testing showed a high sensitivity for the detection of CIN2+ and a high negative predictive value after 3 years of follow-up. The results of this study are in line with the current guidelines for triage of women with ASC-US in the target age range of 25-65. Non adherence to guidelines will lead to unnecessary medical interventions. Further investigation is needed to improve specificity of ASC-US triage. Invasive cervical cancer (ICC) is the third most common tumor in women worldwide. Persistent infection with high risk Human Papillomavirus types (hrHPV) is considered a necessary cause for the development of ICC ,2.Cytology based cervical cancer (CC) screening has been fundamental in decreasing the incidence and mortality of cervical cancer in those countries with high screening coverage rates . One of Catalonia is a region in the North-East part of Spain with a population of 2,802,504 million women aged 25 and above in 2008. The incidence of ICC in 2008 was 7 per 100,000 representing 2.7% of all cancer cases in the region . As in oIn 2006, HPV DNA testing for high risk types using Hybrid Capture version 2 (hrHC2) was introduced on a regular basis for the triage of ASC-US. The main aim of the present study was to evaluate, for the first time in Spain, the value of hrHPV as a predictor marker of CIN2+ lesions.Public health in Catalonia is free of direct charge for the population and provides full access to medical care and cancer screening interventions. About 70% of the gynaecological care in the population of Catalonia is carried out within the Public Sector.In Catalonia, from June 2006 to October 2007 a new screening protocol for CC in sexually active women aged 25-65 years was implemented to increase coverage and to reach a 3 years interval between screening cytologies . YoungerPrimary care doctors and midwives were regularly required to evaluate the adequate compliance of women to the general screening recommendations detailed in the protocol.As per protocol, follow-up of women with a screening cytological result of ASC-US in the absence of liquid-based cytology included recommendation for another cervical cells sample to evaluate the presence of high risk hrHPV within a 3 months period from the initial result. Follow-up after a 3 year interval was recommended for those women with negative hrHPV test results. For those women with positive hrHPV test result referral to colposcopy and biopsy (if appropriate) were recommended.A total of 37,711 hrHPV tests were performed within 2007-2008 in the context of the population based screening protocol in Catalonia. Of these, 5,861 hrHPV tests were among women with a diagnosis of ASC-US. Among all participant centres, eight pre-defined geographical areas were selected for protocol monitoring: Baix Llobregat litoral, Osona, Bages, Berguedà, Solsonès, Sant Martí and Ciutat Vella districts of city of Barcelona, Gironès - Plà d'Estany and Tarragonès. Participating centers were consistent with the distribution of the urban/rural balance of the population in whole Catalonia.HPV tests were performed in five referral laboratories. All of them followed an external quality control (QC) for the performance of hrHC2 testing. The Catalan Institute of Oncology was in charge of coordinating the QC. The QC analysis resulted in very high performance in all the laboratories (data not shown). The overall project was approved by the ethical committee of the Catalan Institute of Oncology.-1, which corresponds to 1.0 relative light unit (RLU/CO). Cervical cytologies were taken with conventional Pap smears or with liquid based cervical cytology and processed and read at the routine pathology centres. All the cytology results followed the 2001 Bethesda System [HPV DNA testing was conducted by five reference laboratories: Josep Trueta Hospital, Hospital Consortium of Vic, University Hospital of Tarragona Joan XXIII, Hospital del Mar, Bellvitge University Hospital. HPV detection was performed using hrHC2 tests  which detect 13 high risk oncogenic HPV types . An HPV sample was considered positive if it attained or exceeded the Federal Drug Administration (FDA)-approved threshold of 1.0 pg HPV DNA mla System  and theyFor the histological diagnosis, the protocol guidelines recommended the use of CIN classification .All histological, cytological and molecular data of cases diagnosed with ASC-US were collected and evaluated to its final worse diagnosis using a record linkage approach based on the medical record number of the women. Once the information was available, all patient data were anonymised. Those women with negative hrHPV test after ASC-US diagnosis should be recommended a follow up visit in a 3 year interval. When a woman had any abnormal consecutive pathology test result after the initial ASC-US, the final diagnosis was the worst during the period and censored at the time of treatment, if any. In these later cases the assignment to a final diagnosis was based on the result of the biopsy before treatment. For those women with initial HPV positive test results, a final diagnosis of 'normal' was reported in patients with 1) two negative consecutive cytological samples, 2) two negative consecutive cervical biopsies, or 3) one negative cytological sample and one negative cervical biopsy within a 1 year interval or a second hrHPV negative test result. For those women with hrHPV negative results, a final diagnosis of "normal" was reported if at least one test result was negative at the end of follow up period. When both cytological and histological specimens were collected at the same time, the worst histological abnormality result was reported as final diagnosis.t-test for continuous variables and a chi square test for categorical variables. Finally, a logistic regression analysis adjusted by age and centre was performed in order to calculate the odds ratio of developing a CIN2+ lesion as a function of hrHPV status at study entry. The analysis was performed as a cross-sectional evaluation at the end of the first round of screening.Data analyses were carried out using SPSS version 17.0. Age was grouped into five categories: < 25, 25-34, 35-44, and 45-54 and ≥ 54 years old. The relative light units/cut off (RLU/CO) was divided into nine categories: 0-0.5, 0.6-0.99, 1-1.99, 2-49, 50-99, 100-499, 500-999, 1000-1999, ≥ 2000. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) were estimated together with exact 95% CI, one or two sided as appropriate. When appropriate, comparison between categories was performed with a During 2007 and 2008, 65,378 women from the study population were screened with conventional cytology and a total of 69,952 Pap smears were obtained. Out of them, 95% were negative for cervical intraepithelial lesion or malignancy, 2% were reported as ASC-US/atypical glandular cells of undetermined significance (AGUS), 0.08% were reported as atypical squamous cells-cannot exclude HSIL (ASC-H), 2% were reported as LSIL, 0.3% were reported as HSIL and 0.03% were suspected of cancer. 613 ASC-US cases that had a concomitant hrHPV testing were included in the present study . Two cases were excluded: one with history of vaginal cancer and the other with endometrial cancer. The final sample size for the analyses was of 611 women. All the hrHPV tests were performed within a maximum period of 6 months after ASC-US diagnosis. The 68 cases of Tarragonès were included despite of being diagnosed in 2008 because follow-up was available for the majority of the women (81%).p = 0.02).The mean age of ASC-US cases was 35.4 years (standard deviation (SD) = 11.3) ranging from 15 to 79 years (Table INK immunochemistry positive test result suggestive of an HPV driven lesion. No sample was available for further testing.Table one sided = 82.9) for CIN3+ (16/16). The specificity of hrHC2 for the detection of histologically confirmed CIN2+ was of 68.3% (CI = 63.1-73.2) (108/341), excluding intermediate diagnoses such as HPV positivity or LSIL. The NPV of hrHPV test for CIN2+ was of 99.6% (CIone sided = 98.2) (254/255).The sensitivity of hrHC2 for the detection of histologically confirmed CIN2+ was of 97.2% (CI = 85.5-99.9) (35/36); and of 100% . The odds ratio of CIN2+ was 45.3 (95% CI = 6.2-333.0) when ASC-US hrHPV positive women were compared to that of ASC-US hrHPV negative women.hrHPV status at study entry was the only statistical significant variable associated to the risk of CIN2+ .Among hrHPV positive cases, the lag time from ASC-US to CIN2+ was shorter and statistically significant in comparison with the lag time between ASC-US and other milder diagnostics .The results of this study confirm that among women with a new diagnosis of ASC-US, hrHPV DNA testing clearly defines the group of women with an underlying high risk of CIN2+ with a PPV of 14.3% and a global NPV of 99.6% after a 3 years follow up period. hrHPV testing among women with ASC-US identified a high number of CIN2+ (37/493) cases conferring a risk of CIN2+ 45 times higher than the one observed in ASC-US negative women. Sensitivity for CIN2+ was of 97.3% and sensitivity for CIN3+ was of 100%. Specificity for CIN2+ was of 68.3%.ASC-US is generally described to be more common in young women than in postmenopausal women ,18. In tThe majority of women with hrHPV negative values (91.4%) did not develop any pathology at the end of the first round of screening. According to previous studies ,20,21, hOur data are consistent with other studies using hrHC2 as a triage test -26. Del The limitations of the present study are that the follow-up window was slightly over a 3 years period - from January 2007 up to February 2011. Although information from 80.7% of the women was available, it is possible that appointments for the remaining ones were fixed later than the 3 years period. An additional limitation is that colposcopies among hrHPV negative women were not randomly carried out. However, a relevant proportion of these patients obtained negative colposcopy results for CIN2+ suggesting that verification bias was not affecting our results. Our data was restricted to information generated within the public health system serving over 70% of the gynaecological care. Therefore, we cannot extrapolate our data to women being attended in the private sector as they may differ in terms of risk factors from HPV acquisition or cervical cancer.In this study we used the information from five sentinel centres with available record linkage system and no detailed medical records were available. We did not review the pathology diagnosis of the initial cytology of ASC-US nor the follow-up diagnosis by cytology, biopsy or hrHPV testing. Therefore, discrepancies between pathologists in classifying ASC-US cannot be excluded.A strength of our study was that information was retrieved from a public health system in which a common protocol was introduced and monitored for its adequacy and performance across the different laboratories and medical centers. The protocol was homogenously introduced and monitored and coordinated irrespective of the clinical decisions taken.HPV DNA testing as a triage test for ASC-US has been shown to predict CIN2+ lesions and to provide very high negative predictive value among those cases that followed the protocol guidelines. This study supports our current national recommendations for ASC-US triage in women older than 25 years of age. Guidelines for younger women entering into screening protocols need to be re-evaluated. Non adherence to guidelines will lead to unnecessary medical interventions and psycho-social effects, such as anxiety. Further research is needed to improve specificity of ASC-US triage.CC: Cervical cancer; HPV: Human Papillomavirus; hrHPV: High risk HPV types; ASC-US: Atypical squamous cells of undetermined significance; HC2: Hybrid Capture System; hrHC2: Hybrid Capture version 2 for high risk types detection; RLU/CO: Relative light unit; CIN2+: High grade cervical intraepithelial lesions grade 2 or worse; ASC-H: Atypical squamous cells cannot exclude a high grade squamous intraepithelial lesion; LSIL: Low-grade squamous intraepithelial; HSIL: High-grade squamous intraepithelial lesion; CIN2: High grade cervical intraepithelial lesions grade 2; CIN3: High grade cervical intraepithelial lesions grade 3; AGC: Atypical glandular cells; AIS: Endocervical adenocarcinoma in situ.Silvia de Sanjose received occasional travel fund to conferences/symposia/meetings by either GlaxoSmithKline, Sanofi Pasteur MSD, Merck & Co. or Qiagen.F. Xavier Bosch is member of the advisory board of GlaxoSmithKline, Merck Sharp & Dohme, and Sanofi Pasteur MSD and of the speakers bureau of GlaxoSmithKline. He received occasional travel fund to conferences/symposia/meetings by either GlaxoSmithKline, Sanofi Pasteur MSD, Merck & Co. or Qiagen.Belen Lloveras has received occasional travel fund to conferences/symposia/meetings are occasionally granted by either Qiagen or RocheAll other authors refer no conflict of interest.RI and SDS drafted the manuscript and performed the statistical analysis. All authors contributed to the collection and quality of data and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/25/prepub


Cervical cancer incidence and mortality rates are high among women from Appalachia, yet data do not exist on human papillomavirus (HPV) prevalence among these women. We examined the prevalence of genital HPV among Appalachian women and identified correlates of HPV detection.We report data from a case-control study conducted between January 2006 and December 2008 as part of the Community Awareness, Resources, and Education (CARE) Project. We examined HPV prevalence among 1116 women  from Appalachian Ohio. Analyses used multivariable logistic regression to identify correlates of HPV detection.The prevalence of HPV was 43.1% for any HPV type, 33.5% for high-risk HPV types, 23.4% for low-risk HPV types, and 12.5% for vaccine-preventable HPV types. Detection of any HPV type was more common among women who were ages 18–26 , current smokers , had at least five male sexual partners during their lifetime , or had multiple male sexual partners during the last year . Similar correlates were identified for detection of a high-risk HPV type.HPV was prevalent among Appalachian women, with many women having a high-risk HPV type detected. Results may help explain the high cervical cancer rates observed among Appalachian women and can help inform future cervical cancer prevention efforts in this geographic region. Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) in the United States (U.S.) A high-risk population that has not yet been examined in terms of HPV prevalence is women living in the Appalachian region of the U.S. Appalachia consists of the entire state of West Virginia and portions of 12 other states that extend from New York to Mississippi There may be several reasons for the current cervical cancer disparities among women from Appalachia. Risky sexual behavior that likely increases the chance of HPV infection is common among these women The Institutional Review Boards of The Ohio State University, the University of Michigan, and the Centers for Disease Control and Prevention (CDC) approved this study.This study was part of the Community Awareness, Resources, and Education (CARE) Project, one of eight Centers for Population Health and Health Disparities (P50) funded by the National Institutes of Health (NIH). We report on one component of the CARE Project, a case-control study that focused on the determinants of abnormal cervical cytology among women living in Appalachian Ohio. A secondary aim of this study was to examine HPV prevalence among these women, the focus of this report. Appalachian Ohio is a 32 county region (29 at the time of this study) located in the southern and eastern sections of Ohio. Cervical cancer incidence and mortality rates are 12% and 21% higher, respectively, in Appalachian Ohio counties compared to the non-Appalachian counties in Ohio Women included in this study were from 17 participating health clinics located throughout the Appalachian Ohio region. Women were eligible to participate if they were: 1) age 18 or older; 2) a resident of an Appalachian Ohio county; 3) had an intact uterine cervix and corpus; 4) were not pregnant; 5) did not have a history of cervical cancer; and 6) provided written informed consent. Women scheduled for a routine Papanicolaou (Pap) test on a day that a study nurse was at one of 17 clinics were asked to participate in the study. Women completed a self-administered questionnaire following consent and prior to undergoing their Pap test. During the visit, the provider took an additional research cytology sample and obtained a cervical sample in specimen transport medium  for HPV testing.Women with abnormal cervical cytology at study entry were considered cases for the case-control study that aimed to identify the determinants of abnormal cervical cytology. Women were classified as having abnormal cervical cytology according to the 2001 Bethesda System for Reporting Pap Smear Results The STM sample was stored at –70°C until shipped to the Centers for Disease Control and Prevention (CDC). At CDC, the samples were thawed and 150 µL of the 1.0 mL sample were extracted with the MagNA Pure DNA kit III , with 10 µL of extract used in the Research Use Only (RUO) Linear Array (LA) HPV Genotyping Test . The LA assay detects 37 HPV types  and includes ß-globin as an endogenous positive control. Samples positive for the XR probe on the LA HPV strip that were also positive for HPV 33, 35, and 58 were tested with an HPV 52 quantitative PCR assay to confirm or exclude the presence of HPV 52 Based on HPV testing results, we examined detection of: 1) any HPV type; 2) a high-risk HPV type ; 3) a low-risk HPV type ; and 4) a vaccine-preventable HPV type  and whether they had ever been pregnant. We collected information on women’s cervical cancer screening histories and determined if they had a Pap test within the three years prior to study entry and whether they ever had a previous abnormal Pap test. Women indicated if they had ever been told by a doctor that they had an HPV infection . Women also indicated their age at first sexual intercourse, number of male sexual partners during their lifetime, number of male sexual partners within the last year, if any of their male partners had been diagnosed with an STI, and if they had any female sexual partners during their lifetime. We collected data on several demographic characteristics, including age, race/ethnicity, marital status, education, and annual household income. Age was categorized as 18–26 years old (within the recommended age range for HPV vaccination p<0.05) into a multivariable logistic regression model. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were obtained from these multivariable logistic regression models. All logistic regression models included health clinic as a random effect since women were clustered within health clinics. Data were analyzed using SAS Version 9.2  and all statistical tests were two-tailed, using a critical value of α = 0.05.We first calculated descriptive statistics in examining the prevalence of HPV among participants. We then used logistic regression to identify correlates of two outcomes: detection of any HPV type and detection of a high-risk HPV type  Most women were less than 40 years old (67.8%), non-Hispanic white (93.9%), had at least a high school degree (88.2%), and reported a household income of $50,000 or less (82.3%). Less thThe overall prevalence of any HPV type, as measured by HPV DNA detection in the LA assay, was 43.1% . Using tIn multivariable analyses, detection of any HPV type was more common among women ages 18-26 compared to women ages 40 or older . CurrentMultivariable correlates of high-risk HPV detection were highly similar to those for detection of any HPV type . DetectiAppalachia is a geographic area with high cervical cancer incidence and mortality rates The design of our study should be considered when interpreting these results. Our data are from a case-control study that was designed to identify determinants of abnormal cervical cytology, so one-fourth of our sample had an abnormal Pap test upon study entry. This is higher than the prevalence of abnormal cytology among women from studies that were not case-control studies . These findings coincide with those from previous studies examining HPV prevalence among women in the U.S. Study strengths include a large sample size and inclusion of women from throughout the Appalachian Ohio region. Our study also has several limitations. We were not able to examine the persistence or clearance of HPV infection. The study included women presenting to health clinics, which may limit the generalizability of findings since these women may have greater access to Pap testing compared to others from Appalachian Ohio. This may be important since Pap testing within recommended guidelines tends to be less common among women from Appalachian Ohio compared to women from non-Appalachian Ohio HPV was prevalent among Appalachian women and many had a high-risk HPV type detected. These data not only provide the first insight into the prevalence of HPV among Appalachian women but may also help explain the existing cervical cancer disparities within this geographic region. Results can help inform future cervical cancer prevention efforts in Appalachia, specifically those designed to increase HPV vaccine uptake or women’s awareness of and knowledge about HPV and cervical cancer.The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agencies.


Objective. To determine the frequency of highrisk (HR) HPV and intraepithelial neoplasia following ASCUS papcytology screens in menopausal women. StudyDesign. Following IRB approval, we performed aretrospective review of all cases of ASCUS pap tests, HPV results,and relevant clinical-pathologic data in women age 50 or over fromNovember 2005 to January 2007 within a tertiary care center. Statistical analyses were performed in EXCEL. Results. 344 patients were analyzed for a totalof 367 screening pap tests. 25.29% (87/344) patients were HRHPV positive, with greater percentages of HR HPV cases occurringin women age 65–74. Within HR HPV cases, 79.3% (69/87)underwent colposcopy. 27.5% (19/69) biopsy proven lesions werediscovered, including cervical, vulvar or vaginal . Within thenegative HR HPV group 3.1% (8/257) patients were diagnosedwith dysplasia or carcinoma. Within both HR HPV positive andnegative groups, patients with no prior history of lower genitaltract lesions or cancer were identified. Conclusion. Reflex HPV testing plays an importantrole in ASCUS triage in menopausal women. Pap test screening andHPV testing should not be limited to women of reproductive age asthey may aid in the diagnosis of intraepithelial neoplasia inwomen of older age. Cervical cancer remains the fourth leading cause of cancer mortality in women worldwide today . It is eMost studies on cervical cancer screening have focused on premenopausal women, with little information provided on the perimenopausal or postmenopausal (PMP) population. However, these women remain at increased risk for cervical cancer given that there is a secondary peak in prevalence of high-risk HPV subtypes in this older population , and thaFurthermore, there is very limited data available on HPV infection and lower genital tract disease in menopausal women. Given that high-risk (HR) HPV reflex testing has increased the detection of Cervical Intraepithelial Neoplasia (CIN) based on the US National Cancer Institute ASCUS-LSIL Triage Study , we seekThis single-institution study was conducted after approval by and following guidelines set forth by the Institutional Review Board. Pap cytology tests were obtained by obstetrician-gynecologists, internal medicine primary care physicians, as well as gynecologic-oncologists. Tests were done primarily at a tertiary care center, with a small percentage of cases received from local affiliated community health centers.Digene Hybrid Capture II System . The Sure Path  system was used to process all cytology specimens. Follow-up colposcopy and biopsy results were obtained from the electronic medical record. These exams were performed by both general obstetrician-gynecologists, nurse practitioners supervised by obstetrician gynecologists, and gynecologic-oncologists. Additionally, demographic information including age, race, age of menopause, past medical history, and past gynecologic history was collected. Using a pathology database from November of 2005 to January 2007, all cases of ASCUS in women aged 50 or over were retrospectively identified. Starting in November 2005, all cases of ASCUS pap results automatically underwent reflex high-risk (HR) HPV testing using the Data was entered in Excel. Basic distribution statistics were performed in Excel.344 patients were analyzed, with a total of 367 ASCUS cases. There were 22 patients who were screened twice and one who was screened three times during this interval. The mean age of menopause was 49.5 (SD 22.5) years. The mean age of subjects was 59.8 (SD 8.7) years. 17.4% (60/344) of patients were past smokers, and 11.04% (38/344) currently smoke. Overall, 46.5% (160/344) are currently married. 12.2% (42/344) are currently divorced and single, and 10.5% (36/344) are divorced or widowed with a new sexual partner at the time of the exam. Hypertension was the most frequently reported medical condition, present in 33.1% (114/344) of all cases. Conditions that may affect HPV-related diseases, such as, lupus, hepatitis, and postorgan transplantation occurred infrequently. In review of past gynecologic history, 42.7% (147/344) of patients never had an abnormal pap smear. 44.8% (154/344) of patients had been previously diagnosed with ASCUS. 15.7% (54/344) had history of CIN, and 3.5% (12/344) had history of cervical carcinoma in situ (CIS) and squamous cell carcinoma (SCC). 8.4% (29/344) had history of VIN. 14.0% (48/344) had prior hysterectomies, for either malignant or benign indications. In total, 20.3% (70/344) had a documented history of CIN, VIN, or VAIN.The majority of ASCUS cases occurred in women age 50–60, in which 56% of patients were less than 60 years old . With inOf these ASCUS samples, 25.3% (87/344) patients were HR HPV positive. This included all patients who on first pass were HR HPV positive, as well as one patient who was screened twice in this interval, who was first negative for HR HPV, and then positive. The greatest percent of HPV positive cases by age occurred within the age group of 65–74 group . Of patients who underwent repeated screens during our study period: 5 patients were negative on both screens (of which 1 patient was diagnosed with VAIN I). 12 patients were positive on both screens . 1 patient was positive on 3 screens. 3 patients were positive on first screen, then converted to negative (of which 1 patient was diagnosed with VAIN III). 1 patient was negative initially, and then positive on repeat screen. Of the HPV positive patients, 79.3% (69/87) underwent colposcopy. 27.5% (19/69) of biopsy proven lesions were discovered, including CIN, VAIN, and VIN. Biopsy results revealed 7 patients with CIN I, 2 patients with CIN II, 2 patients with CIN III, 5 patients with VAIN and 1 patient with VIN. Of the HPV positive patients who did not initially receive colposcopy, further analysis showed that one patient underwent hysterectomy with no abnormal findings; 4 patients underwent colposcopy which were all negative; 3 patients underwent vaginal exam only (given prior hysterectomy) which were negative. 3 patients had no followup and are presumed to be lost to followup. The remaining patients all had at least repeat pap exams. Within the negative HR HPV group, 11.7% (30/257) patients underwent colposcopy. While current guidelines do not recommend colposcopy in HPV-negative women, in 2005, some women were still being treated based on older recommendations for colposcopy for atypical pap smears. Three percent of patients (8/257) were diagnosed with CIN, VIN, VAIN, or carcinoma. Findings included: one with CIN 1, one with VAIN II, one with VAIN III, one with newly diagnosed vaginal SCC, one with vulvar CIS, one with vulvar SCC, and 2 with newly diagnosed endometrial cancer. Overall, 7.8% of patients with ASCUS had positive colposcopic findings (19 HPV-positive plus 8 HPV-negative colposcopy biopsy proven cases/344). HPV triage allowed identification of 19/27 (70.3%) of the dysplastic or cancer cases.Despite the common assumption that peri- and postmenopausal (PMP) women are at low risk for lower genital tract disease, we found that 25% of ASCUS patients who were age 50 years or older had high-risk HPV. Within each five-year age bracket starting at 50, the greatest percentage of HR HPV occurred within women aged 60–70s. Furthermore, several of these women with biopsy proven diagnoses of lower genital tract disease had no prior history of HPV-related gynecologic disease. We presume that these cases are due to newly acquired infections. Without continued surveillance and gynecologic care, many PMP women may suffer from undiagnosed lower genital tract disease. Furthermore, 27% of HR HPV-positive patients were found to have a biopsy proven abnormality, including CIN, VIN, VAIN, or cancer. Even in the HPV-negative group, 3% of patients had biopsy proven disease, including two who have never had lower genital tract disease. They were newly diagnosed with endometrial cancer. Since we could not accurately differentiate women with a remote history of lower genital tract disease from those with more recent abnormalities, we did not exclude patients undergoing follow-up surveillance for recent abnormalities. Therefore, we reported all findings seen at this tertiary care center. Although these rates of lower genital tract abnormalities may be higher than that of the general PMP population, use of the pap cytology screening and HPV reflex testing helped identify a large proportion of PMP women with lower genital tract disease.P = .48 [ Our results are consistent with other reports regarding the presence of lower genital tract disease, including HPV-related disease in PMP women. In a recent study of HR HPV A SCUS outcomes in PMP women age 45 years and older, 25.4% of ASCUS samples were HR HPV positive, similar to ours. Of these women, 30.9% revealed persistent LSIL/CIN1 and 3.68% were diagnosed with HSIL/CIN2or 3, with one patient diagnosed with squamous cell carcinoma (0.74%) [P = .48 . ContrarAlthough guidelines for cervical cancer screening remain quite variable, our findings suggest that a significant proportion of PMP have new or persistent lower genital tract disease. Screening recommendations by various health organizations have been summarized in The strengths of this study include that it was a single-institution study, with a designated gynecologic pathology department, diagnosing both cytologic as well as histologic specimens. The samples were aggregated from both community clinics affiliated with the institution as well as gynecologic-oncology subspecialty departments. The use of reflex testing was standardized during this time interval. Limitations of the study include the relatively small number of cases. Given that the occurrence of HPV-related genital disease is relatively uncommon, the frequencies reported in this study could be falsely elevated due to the referral base to a gynecologic oncology department, specialty colposcopy clinic, and gynecologic-pathology department within a large tertiary care center. Secondly, the actual incidence of HR HPV in this population is unknown. To our knowledge, the rate of lower genital tract disease in this older population remains undefined. In summary, PMP women remain susceptible to HPV related lower genital tract disease. Reflex HPV testing identified a significant fraction of patients with lower genital tract disease. Despite their increased age, these women should not be disregarded and should continue to undergo routine gynecologic exams, pap cytology testing, and reflex HPV testing in the appropriate clinical context. Reflex HPV remains a useful modality for identifying several treatable lower genital tract diseases in this older population.


Understanding the biological significance of HPV-DNA semen infection could permit us to explain most of the idiopathic male and couple infertility, leading to a better management of infertile men and a better timing for sperm banking storage before ART cycles.Recent evidences identify Human Papillomavirus (HPV) sperm infection as a possible cause of male and couple infertility. It acts through different mechanisms at various steps of human conception and early gestational development. We performed a systematic review to assess the role of HPV semen infection on male and couple infertility. Analysis of available and eligible data does not permit us to fund clear evidences about clinical impact of HPV infection on fertility, although sperm parameters impairment is the most widely recognized effect. Regarding biomolecular implications, the available data are often conflicting. More studies are required to define the role of HPV sperm infection in clinical practice. The great majority of evidences are obtained by  Human Papillomavirus (HPV) infection is one of the main sexually transmitted diseases worldwide . The mosHPV infection is also related to anogenital warts and different neoplasia, such as vaginal, vulvar, penile, anal, oral cavity, head, and neck cancers , 4, 6. CGreat progresses have been reached in understanding the pathologic mechanisms of HPV infection. Both effective screening programs  and interventions (HPV vaccination) have been developed in order to reduce HPV related disease in women –12.Nevertheless few data are available on male infection , 13–15.Recent findings underlined the role of HPV semen infection in male and couple infertility, focusing mainly on its high prevalence among 18–40 years old men , 17.The exact localization of HPV in the spermatozoa is not well defined, even if recent studies demonstrated that the virus binds two distinct sites along the equator of the spermatozoa's head , 19.Many Authors hypothesized that HPV can modify spermatic parameters causing sperm motility reduction, seminal pH alterations, and spermatozoa DNA fragmentation , 20–22. in vitro studies demonstrated that spermatozoa can carry HPV-DNA and transfer it to oocytes, it is still not clear if in vivo the HPV-infected sperm is able to fertilize oocyte and to transfer the viral genome  [In 2010 Skoczynski et al. compared the prevalence of HPV infection in placentas from term deliveries and spontaneous abortions. The comparison between the two groups showed no differences in terms of HPV-DNA detection since it was found in 24.4% of placentas at term (12.8% of HPV 16/18 types) and in 17.7% of miscarriages (11.8% of HPV 16/18) [ : n.s.] .P < 0.01), 40% versus 13.7% in case of female infection, and 100% versus 15.9% in case of both partners infections (P < 0.001) , while spermatozoa infection was detected in 72% of infertile men. Despite the mean sperm motility results significantly reduced in all infected men compared to noninfected ones ( < 0.05) .Considering sperm-washing procedure (in order to remove HPV-DNA from the sperm surface) Brossfield et al. showed that all the applied techniques did not positively influence the spermatic HPV-related motility and that, between them, the centrifuge wash technique results were the best ones .P < 0.01) [On the other hand Foresta et al. observed that only Ficoll and swim-up procedure were useful to reduce the infection in 30% and 26% of sample, respectively ( < 0.01) .P < 0.05). Authors reported that direct swim-up reduces up to 24% the HPV sperm infection, while modified swim-up is able to remove completely HPV-DNA from spermatozoa (P < 0.01). Anyway, Authors underlined how modified swim-up results were responsible for a slight decrease of sperm motility, viability, and DNA integrity [P < 0.01). The evidences that men with HPV-DNA infection associated to positive sperm-Mar test are affected by 24 months lower spermatozoa motility than negative ones lead the authors to propose the positive sperm-Mar test as predictor tool for future progressive spermatozoa' motility [Garolla et al. demonstrated the negative effects of HPV infection reporting that only progressive sperm motility was significantly reduced in infected semen samples  [Later, both Connelly et al. and Lee et al. demonstrated increased apoptotic phenomena in sperm cells exposed to E6/E7 genes of HPV-DNA types 16 and 18 ( < 0.05) , 22.Chan et al. demonstrated in a mouse experimental model the ability of HPV-DNA infected sperm to transmit its genome to blastocysts . CabreraForesta et al., using the hamster egg-human sperm penetration test (HEPT), demonstrated the sperm ability to transfer both the capsid protein L1 and E6/E7 viral genes to oocytes with a subsequent gene expression by transfected blastocysts .P < 0.05). The ability of HPV-DNA type 16 to induce DNA fragmentation and subsequent trophoblastic death was also confirmed by You et al. in 2002 [Calinisan et al., transfecting blastocysts with the E6-E7 region of types 16, 18, 31, and 33, detected the presence of DNA fragmentation only in the subgroup of blastocysts infected by HPV-DNA type 16 . Simultaneously, authors reported that the invasion ability of transfected trophoblast progressively decreased from day 3 to day 15 (25.2–57.6% lower than negative controls) (P < 0.001) [Gomez et al. showed that the apoptosis rate in transfected trophoblastic cells was 3-fold (2.4–3.7) and 5.8-fold (5.6–5.9) greater at 3 and 12 days, respectively, if compared to negative controls (< 0.001) . in vitro experiments and murine models which may not reflect the real in vivo human situations. All these data should be validated by large in vivo perspective studies but unfortunately the literature lacks information about this field because of ethical policies concerning human reproduction experiments.The limitations of all these studies were certainly related to the artificial conditions linked to theIt is widely proven that sexually transmitted infections (STD) represent a possible cause of male infertility since they can induce urethral stickiness, epididymis inflammation, and orchitis up to testicular failure . The impThe high prevalence of male HPV detection raised strong interest for its possible consequences on male fertility. HPV infection natural history in men is still mostly unknown. Recent findings suggest that the incidence of genital HPV infection is 38.4 per 1000 male/months (95% CI 34.3–43.0). The mean duration of male infection results of 7–52 months are (6.80–8.61) for any HPV type and of 12–19 months (7.16–18.17) are for hrHPV 16 . in vitro studies showed a negative influence of HPV upon several aspects of male fertility. However, only few studies investigated the in vivo effects of HPV on human reproduction phases. In 1997 Hermonat et al. reported the correlation between HPV infection and spontaneous miscarriages in first trimester of pregnancy. The higher percentage of HPV-DNA detection, if compared to voluntary abortions, paved the way to consider HPV as one of the possible etiologic agents responsible for early pregnancy loss [Allncy loss . This hyncy loss . in vitro fertilization techniques, this aspect seems to be of crucial importance. The first prospective study on this topic reported a significant pregnancy loss increase in couples undergone ARTs and male partner with semen HPV infection (miscarriage rate of 100% when both partners results are infected) [Nowadays, in spontaneous conception, the rate of early miscarriages and major birth defects does not seem greater in HPV-exposed couples than in unexposed ones , 29. Hownfected) .So we can conclude that the role of HPV infection in spontaneous abortion is still not clear and its association with adverse pregnancy outcomes is not certainly demonstrated. Further perspective longitudinal studies are necessary to better understand the possible roles of HPV infection in early miscarriage and in other adverse pregnancy outcomes.However, if the evidences of Perino et al. will be confirmed, a major care on HPV status of couples attempting ART procedures should be necessary. Thus, HPV male vaccination could represent a possible strategy for male fertility preservation and for ART success rate improvement.The molecular mechanisms by which HPV could impair sperm quality and fertilized oocytes development has been neither completely demonstrated nor clarified.From our analysis, HPV presence in spermatozoa may be associated with an impairment of sperm parameters. Lai et al. firstly demonstrated that HPV presence in sperm could affect spermatozoa's motility  although it seems to be not statistically significant. A possible association between HPV semen infection and asthenozoospermia has also been described . These fThe slight motility increase described by Connelly et al. in HPV exposed spermatozoa might be explained considering that the observation was performed after two hours of incubation. This fact suggests that HPV-DNA requires a suitable interval time to determine molecular changes on sperm motility apparatus. The authors reported a mean reduction of lateral head amplitude even if the virus seems not able to decrease oocytes sperm fertilization ability .Rintala et al. in HPV positive semen detected only a pH change without any impairment of other parameters .For many years semen pH impairment was considered the most important mechanism explaining fertility decrease due to asymptomatic genital infections, especially the bacterial ones.In our opinion pH impairment represents only one of the several factors that may influence male fertility, since the aetiological mechanism could involve many other factors such as mean sperm motility, presence of ASA on the spermatozoa surface, and qualitative semen parameters impairment till asthenozoospermia , 32–34.Since several cases of reported idiopathic asthenozoospermia do not present any known risk factor except for the positivity to HPV-DNA, semen washing procedures could represent a way to improve spermatozoa quality before ART procedures , 32, 34.Starting from these considerations, many studies proposed different semen washing methods to eliminate HPV-DNA sperm infection, but all proposed techniques failed in the scope, except for the modified swim-up technique. This last one contemplates enzymatic treatment (Heparinase-III), apparently able to completely remove HPV-DNA from sperm cells although with a deterioration in semen quality.Nowadays all the available data suggest that the classic procedures cannot eliminate HPV sperm infection and that HPV-DNA semen screening can be considered only as an epidemiological investigation helping to define the best timing (regression of semen infection) to start ART cycles.It is well demonstrated both in men and women that HPV infection is generally transient and only few patients are subject to persistent infection. Our suggestion to test the sperm for HPV before sperm banking and ART cycles could represent the last option for the clinicians to try to improve the semen quality both for fresh use and for frozen preservation. Certainly, the vaccine option could have useful results for both male and female patients presenting poor reproductive outcomes till the couple infertility . in vitro evidences showed that HPV infected spermatozoa maintains the ability to fertilize oocytes and to express viral genome in the product of conception. This suspect seems to be confirmed by a higher rate of in vitro blastocysts and trophoblastic HPV-related apoptosis probably responsible of in vivo fertility rate reduction of infected couples.The HPV-DNA presence in sperm and the related modification induced by the infection seem to play a role in physiopathology of unexplained male infertility. However,Evidences reported by Chan et al. , Lai et  in vivo human models it was found that HPV genomes are expressed in fertilized oocytes, blastocysts, and trophoblastic cells [In both experimental murine andic cells , 25, 37.ic cells –40.In vitro experiments showed that HPV transfected blastocysts and trophoblastic cells were affected by a reduction in decidua invasion capacity, potentially responsible for a failure of maternal uterine wall invasion by the extravillous trophoblastic cells, subsequent placental dysfunction, and adverse pregnancy outcomes  [arriage) . in vitro artificial and experimental conditions. Further validation in in vivo studies could be therefore needed.Nowadays both the exact mechanism and timing through which the HPV infection modifies trophoblastic genes expression and increases cell death have not yet been understood. All the considered studies have important limits which should not be underestimated, linked to theThe actual high prevalence of HPV sperm infection represents a large scale problem for the sperm donors banks , 49.HPV screening for all semen samples before sperm banking should be considered as a real option of semen storage for future ART cycles.The rationale is that, when possible, the semen banking should be postponed until HPV infection resolution. The main limitations in this field are linked to the poor knowledge of HPV male infection natural history and of the most adequate interval time to postpone the storage.Moreover, neither evidences are available about long term effects of previous HPV sperm infection nor which parameters could recover after the infection-linked damages.In absence of effective and safety sperm washing procedures able to eliminate the infection, HPV male vaccination should be considered as a possible strategy for the prevention of HPV semen impairment and for the improvement of couple fertility outcomes , 50, 51.Male vaccination could represent also a reliable option for couples undergoing ART cycles because fertility of female partner results yet partially compromised: this could solve the problem linked to sperm banking, avoiding the potential HPV negative effect on sperm quality .The biggest concern of Public Health Programs and clinical practitioners about the cost-effectiveness of HPV male vaccination is linked to the several biases affecting available data. For example, a recent study by Kim and Goldie concluded that the HPV vaccination of all 12-year-old boys would not be cost effective . All reaMost of analysed data suggested that the HPV sperm infection could be responsible for a decreased fertility rate through different mechanisms acting at various steps of the human embryo development. in vitro studies evidences. Improvement in the knowledge of HPV-DNA sperm infection mechanisms, timing, and link to fertility impairment could explain most of the actual “idiopathic” male and couple infertility.The still debated clinical features related to HPV-DNA sperm infection are the increased risk of early miscarriage and the higher incidence of unexplained male infertility (related to sperm parameters impairment). HPV-DNA sperm test should be realized in semen donors and before ART cycles, despite the limitations linked toThe cost-effectiveness analysis related to fertility improvement in HPV vaccinated male requires further evaluation in the next future.The implications related to possible achievement of herd immunity after a mass population vaccination program could overcome the existing doubts, explaining and resolving the unclear aspects in this field.


The prevalence and role of human papillomavirus (HPV) in the aetiology of oesophageal squamous cell carcinoma is uncertain. Based on the presence of HPV in the oral cavity and its causal association with squamous cell carcinoma of the oropharynx, we hypothesised that HPV is more strongly associated with proximal than distal oesophageal squamous cell carcinoma.INK4a expression. Multivariable logistic regression was used to calculate odds ratios and 95% confidence intervals.A population-based study comparing HPV infection in relation to tumour site in patients diagnosed with oesophageal squamous cell carcinomas in the Stockholm County in 1999–2006. Multiplex polymerase chain reaction genotyping (PCR) with Luminex was conducted on pre-treatment endoscopic biopsies to identify type specify HPV. Carcinogenic activity of HPV was assessed by p16INK4a expression was similarly common (24% and 16%) in the HPV-positive and HPV-negative groups.Among 204 patients, 20 (10%) had tumours harbouring HPV DNA, almost all (90%) of HPV high-risk type, mainly HPV16. Tumours containing HPV were not overrepresented in the upper compared to the middle or lower third of the oesophagus . P16This study found a limited presence of HPV in oesophageal squamous cell carcinoma of uncertain oncogenic relevance and did not demonstrate that HPV was more strongly associated with proximal than distal tumours. The introduction of preventive vaccines against human papillomavirus (HPV) has increased the incentive to clarify possible causal links between HPV and non-cervical carcinomas, for example oesophageal squamous cell carcinoma cancer. Oesophageal cancer is the 8th most common type of cancer and the 6th most common cause of death from cancer, globally, This population-based study included patients diagnosed with oesophageal squamous cell carcinoma in the Stockholm County in Sweden during the period 1999 to 2006. The patients were identified through the Swedish Cancer Register, a nationwide register initiated in 1958 with a completeness of 96% for all cancers Detection of HPV in the tumour biopsies was conducted as follows. Formalin-fixated paraffin embedded biopsies were sectioned into 4×15 µm slices on glass, and macro-dissected to make sure that the section contained at least 70% tumour cells. To minimise contamination, blanks were added after every 5th sample and treated in the same way as regular samples during slicing and micro-dissection. DNA extraction was performed using the High Pure RNA Paraffin Kit (Roche's) without DNAse. PCR was performed by adding 10 µl of DNA sample and 40 µl of reaction mixture, containing broad-spectrum GP5+/6+ primers (BGP5+/BGP6+), to the Qiagen Multiplex PCR Master Mix  INK4a, a surrogate marker for HPV activity, INK4a  and a standard streptavidin-biotin peroxidase method. One pathologist (EA) examined and graded all samples according to predetermined conditions, and samples were categorised as negative if no of the tumour cells were staining for p16 INK4a, weakly and unspecifically stained if a minority and ungrouped tumour cells were stained, and positive if there was a homogeneous staining of a clear majority of the tumour cells were stained for p16 INK4a. The slides showing weak and unspecific staining were included in the p16 INK4a negative category in the analysis.To assess biological activity of HPV in the tumours, p16Fisher's exact test was used to identify predictors for HPV-positive tumours. A p-value below 0.05 was considered statistically significant. Unconditional logistic regression was used to calculate relative risk between HPV infection and risk of various tumour sites in the oesophagus, expressed as odds ratios (OR) with 95% confidence intervals (CI). Using multivariable modelling, ORs were adjusted for age , sex, and tumour differentiation . Any missing data was grouped into a separate category in these analyses.The study was approved by the Regional Ethics Review Board in Stockholm.Among 348 new cases of oesophageal squamous cell carcinoma in the Stockholm County between 1999 and 2006, 67 (19%) were excluded because of; tumour misclassification , diagnosis based on autopsy results , or failure to identify any endoscopic biopsy . Among 281 (81%) eligible patients, 77 (27%) were classified as non-participants; 51 (18%) because of lack of tumour material and 26 (9%) because of tumour material being unavailable. Finally, 204 patients (73%) remained for the present study. Characteristics of the participants, non-participants and excluded patients are presented in INK4a expression was evaluated in 17/20 (85%) HPV positive cases  and in 113 HPV negative cases . The prevalence of p16INK4a expression in the HPV-positive (24%) and HPV-negative (16%) tumours was not statistically significantly different  DNA in oesophageal squamous cell carcinoma in a Swedish population, and does not provide support for the hypothesis of an increased prevalence of squamous cell carcinomas infected with HPV in the proximal compared to the distal oesophagus. Furthermore, the p16Strengths of the present study include its population-based design, the high number of patients included compared to other studies in the same area, the valid assessment of the exposure and outcome, and the ability to adjust for potential confounding factors. The use of oesophageal squamous cell carcinoma patients as both case and control subjects ensured similar risk factor profiles, such as for example smoking and alcohol use, and thus reduced confounding. The use of endoscopic biopsies in all participants circumvented biased detection of HPV infection between tumour sites. The biopsies were taken prior to any preoperative treatment, which ensured that the studied tumour material was not affected by e.g. chemotherapy or radiotherapy. Finally, the detection of HPV DNA using Luminex on PCR product is an established method with a high sensitivity. INK4a, which has previously been used as a surrogate marker for HPV in cervical cancer INK4a immunohistochemistry. Another concern was selection bias introduced by patients from certain hospitals more often being excluded.Limitations include a low statistical power, which was largely due to insufficient DNA material in some biopsies and a low prevalence of HPV infection in the tumours. Furthermore, due to the limited size of the tumour biopsies, we could not assay for HPV E6 and E7 mRNA as marker for active HPV in tumour cells, instead we analysed for p16 This study did not reveal any higher occurrence of HPV infection in the tumour location of the upper third of the oesophagus as hypothesised. Instead, most tumours positive for HPV DNA were located in the middle third of the oesophagus. This might merely be an effect of chance, or speculatively this might be a true finding. The middle part of the oesophagus, the transition zone, holds no significant contraction amplitude when measuring peristaltic contractions, To our knowledge, this is the first study comparing the association between HPV infection and the development of squamous cell carcinoma in different locations in the oesophagus as the primary endpoint. Previous studies show diversity in frequency of tumours containing HPV DNA. A review article from 2002 looking at frequency of HPV DNA in oesophageal tumours showed great diversity of between 0 and 70% of tumours containing HPV DNA. INK4a expression. The low portion (24%) of overexpression of p16INK4a in the HPV positive cancer biopsies indicate that HPV may not be involved in tumour progression in oesophageal cancer to a large extent. These results are in line with a recent case-control study of 222 patients with oesophageal squamous cell carcinoma. INK4a. Although 90% of the HPV positive tumours harboured high-risk HPV types (and 5% putative high-risk types), we found no correlation between the subset of tumours containing HPV DNA and p16 In conclusion, this population-based study from Sweden found limited presence of HPV with uncertain oncogenic relevance in oesophageal squamous cell carcinoma and could not demonstrate that HPV was more likely associated to proximal than distal squamous cell carcinomas of the oesophagus.The presence of human papillomavirus (HPV) in oesophageal squamous cell carcinoma was not correlated to the tumour location within the oesophagus