HPV Infection Affects Human Sperm Functionality by Inhibition of Aquaporin-8.

Human sperm cells express different aquaporins (AQPs), AQP3, 7, 8, 11, which are localized both in the plasma membrane and in intracellular structures. Besides cell volume regulation and end stage of cytoplasm removal during sperm maturation, the role of AQPs extends also to reactive oxygen species (ROS) elimination. Moreover, oxidative stress has been shown to inhibit AQP-mediated H2O2 permeability. A decrease in AQPs functionality is related to a decrease in sperm cells number and motility. Here we investigate the possible effect of human Papillomavirus (HPV) on both expression and function of AQPs in human sperm cells of patients undergoing infertility couple evaluation. Stopped-flow light-scattering experiments demonstrated that HPV infection heavily reduced water permeability of sperm cells in normospermic samples. Confocal immunofluorescence experiments showed a colocalization of HPV L1 protein with AQP8 (Pearson's correlation coefficient of 0.61), confirmed by co-immunoprecipitation experiments. No interaction of HPV with AQP3 and AQP7 was observed. A 3D model simulation of L1 protein and AQP8 interaction was also performed. Present findings may suggest that HPV infection directly inhibits AQP8 functionality and probably makes sperm cells more sensitive to oxidative stress.

Outcome of Persistent Low-Grade Cervical Intraepithelial Neoplasia Treated With Loop Electrosurgical Excision Procedure.

OBJECTIVE: The aim of the study was to evaluate the outcome of persistent (≥2 years) low-grade cervical intraepithelial neoplasia (CIN 1) treated with loop electrosurgical excision procedure (LEEP). MATERIALS AND METHODS: A study of 252 subjects with persistent biopsy-confirmed CIN 1 diagnosed after low-grade squamous intraepithelial lesions or atypical squamous lesions of undetermined significance on Papanicolaou test and treated with LEEP. Post-LEEP follow-up cytological, colposcopic, and molecular diagnostic examinations were scheduled at 6 months, 1 year, and yearly thereafter. RESULTS: The 252 subjects enrolled had a total number of 1,008 visits per colposcopies (median = 3, range = 1-7) during a median post-LEEP follow-up of 25 months (range = 12-121). The cumulative incidence of CIN 2+ at 2 years and at 3 years of follow-up was 2.3% (4/176) and 5.5% (7/128), respectively, or 1.7 cases (95% CI = 1-2.8) per 100 woman-years. Low-grade cervical lesions during post-LEEP follow-up were diagnosed in 70 subjects (27.8%) or 10 cases (95% CI = 7.9-12.6) per 100 woman-years. Overall, persistent and multiple high-risk HPV infections during follow-up were associated with increased rates of CIN persistence or progression. CONCLUSIONS: Women with persistent CIN 1 after atypical squamous lesions of undetermined significance/low-grade squamous intraepithelial lesion treated with LEEP had a low rate of progression to CIN 2+ but remained at a high risk of low-grade cervical abnormalities during follow-up. This information should be taken into account when deciding on the treatment strategy and counseling women with persistent CIN 1.

Multiple Papillomavirus Infection and Size of Colposcopic Lesions Among Women With Cervical Intraepithelial Neoplasia.

OBJECTIVE: The aim of the study was to evaluate the association between the size of cervical lesions as detected by colposcopy and multiple human papillomavirus (HPV) infection in subjects with cervical intraepithelial neoplasia (CIN). METHODS: A case series of 898 subjects with CIN diagnosed by histopathology and infected by high-risk HPV. Human papillomavirus genotypes were identified using the INNO-LIPA genotyping system. RESULTS: The rates of CIN 1, CIN 2, and CIN 3+ lesions were 53.1% (477/898), 14.1% (127/898), and 32.7% (294/898), respectively. Among CIN lesions diagnosed by loop electrosurgical excision procedure or by cold-knife conization, the rates of multiple as compared with single HPV infections increased from 31.7% (59/186) in lesions covering 0% to 25% of the cervix to 39.2% (40/102), 41.9% (13/31), and 48.9% (45/92) in those covering 26% to 50%, 51% to 75%, and more than 75% of the cervix, respectively (χ for trend = 7.9; p = .005). In ordered logistic regression, after correction for confounders, odds ratios (ORs) of larger cervical lesions were higher in multiple as compared with single infections (OR = 1.82; 95% CI = 1.24-2.66; p = .002). This association was confirmed among subjects infected by HPV 16 (OR = 2.45; 95% CI = 1.14-5.26; p = .02) and in CIN 3+ lesions (OR = 2.43; 95% CI = 1.23-4.80; p = .01). CONCLUSIONS: Multiple high-risk HPV infection is associated with larger cervical lesions as detected by colposcopy. This association was confirmed among subjects infected by HPV 16 and in CIN 3+ lesions.

Markers of squamocolumnar junction cells in normal tonsils and oropharyngeal cancer with and without HPV infection.

HPV infection has been identified recently as the causative agent of a subset of squamous cell carcinomas arising in oropharyngeal tonsils. Factors influencing the susceptibility of tonsillar epithelium to HPV-induced oncogenesis are far from being elucidated. A 5-protein signature including cytokeratin (CK)7, anterior gradient (AGR)2, cluster differentiation (CD)63, matrix metalloproteinase (MMP)7, and guanine deaminase (GDA) has recently been found to identify a residual embryonic cell population in the squamocolumnar (SC) junction of the cervix, susceptible to HPV infection, and cancers originating from these cells. The expression of SC junction markers was investigated with immunohistochemistry in normal tonsils and in oropharyngeal carcinomas (OPC) fully characterised for HPV. All markers were constantly expressed in the reticulated epithelial cells of the tonsillar crypts, with variable diffusion and intensity; in OPC, positivity was observed in 36,5%, 29,2%, 39%, 17%, and 25% of cases with respectively AGR2, CK7, GDA, CD63, and MMP7 antibodies. No OPC was positive for all markers; 6 were completely negative. AGR2 and CK7 showed significant association with tumor- and HPV-related parameters. AGR2 expression was associated with tumor origin in the tongue base (p=0.013); CK7 was associated with non-keratinising morphology (p=0.013). p16 tumor cell expression was associated with AGR2 (p=0.021); transcriptionally active HPV infection was associated with AGR2 and CK7 (p=0.024 and 0.043). Expression of SC junction markers in tonsillar crypt cells might be related to the embryological development of tonsillar structures; their partial association with HPV oncogenic infection could help to identify HPV-susceptible cells and related OPC.

Identification of transcriptionally active HPV infection in formalin-fixed, paraffin-embedded biopsies of oropharyngeal carcinoma.

Human papillomavirus (HPV) oncogenic activity is the result of viral oncogene E6 and E7 expression in infected cells. Oncogene expression analysis is, however, not part of the routine diagnostic evaluation of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) since it requires fresh tumor tissue. We compared the diagnostic accuracy of several methods commonly employed for HPV characterization in OPSCC with the results of the newly available HPV E6/E7 mRNA in situ hybridization (ISH) on formalin-fixed, paraffin-embedded biopsy samples, in order to establish if the latter should be introduced in the diagnostic routine to increase accuracy when fresh tissue is not available. p16 immunostain, DNA ISH for high-risk HPV genotypes, SPF LiPA amplification and genotyping, and HPV16 E6 amplification were performed on 41 consecutive OPSCC samples. Twenty (48.7%) cases were positive by mRNA ISH; sensitivity and specificity were 100% and 90% for p16, 90% and 100% for DNA ISH, 70% and 76% for SPF10 LiPA, 90% and 76% for E6 amplification. A diagnostic algorithm considering p16 immunostain as first step followed by either high-risk HPV DNA ISH or HPV16 E6 amplification in p16-positive cases correctly characterized 90% of mRNA-positive and all mRNA-negative cases; combining the 3 tests correctly identified all cases. While no stand-alone test was sufficiently accurate for classifying HPV-associated OPSCC, the high sensitivity and specificity of the established combination of p16 immunostain, DNA ISH, and HPV16 DNA amplification suggests that the introduction of labour- and cost-intensive mRNA ISH, is not necessary in the diagnostic routine of oropharyngeal tumors.

Evaluation of the HPV typing INNO-LiPA EXTRA assay on formalin-fixed paraffin-embedded cervical biopsy samples.

BACKGROUND: The HPV genotyping line probe assay INNO-LiPA EXTRA allows the detection of a wider spectrum of viral types compared to the earlier V2 version of the assay. Its performance in formalin-fixed paraffin-embedded tissues is unknown. OBJECTIVES: To test the EXTRA assay in HPV genotyping of paired cervical scrapings and corresponding FFPE biopsy specimens. STUDY DESIGN: Paired samples from 188 women with abnormal cytology were examined using the INNO-LiPA HPV genotyping assay, version EXTRA. The assay can simultaneously detect 18 high-risk, 7 low-risk, and 2 unclassified HPV types. Kappa statistics were used to measure interrater agreement between groups. RESULTS: The evaluation of paired cervical scraping and biopsy samples gave a 100% overall agreement for HPV status and of 72.9% (kappa 0.6554) for the number of infecting HPVs. 392 out of 507 individual HPV types were concordant, corresponding to a positive agreement rate of 87.2% (95% CI 84.1-90.3). As to the individual genotypes, the agreement was absolute for HPV 45, 68, 73 (kappa 1), excellent for HPV 6, 11, 16, 18, 31, 35, 39, 44, 51, 52, 53, 54, 56, 69/71 and 82 (kappa 0.7796-0.9714), good for HPV26, 33, 43, 58, 66 and 74 (kappa 0.6768-0.7449), and poor for HPV 59 and 40. These agreement values were comparable to those obtained with the V2 assay. CONCLUSIONS: The EXTRA assay provided excellent performance in HPV typing on FFPE samples comparable to the earlier version of the test despite higher complexity and increased coverage of types.

Multiple human papillomavirus infection with or without type 16 and risk of cervical intraepithelial neoplasia among women with cervical cytological abnormalities.

PURPOSE: To evaluate the impact of multiple human papillomavirus (HPV) infections on the risk of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) in subjects with cervical cytological abnormalities. METHODS: A cross-sectional study of 3,842 women attending a colposcopy service was carried out. Genotyping of 18 high-risk, seven low-risk, and two undefined-risk HPVs was carried out by the INNO-LiPA genotyping system. RESULTS: The final colposcopic/pathological diagnoses were as follows: 1,933 (50.3 %) subjects were negative; 1,041 (27.1 %) CIN1; 280 (7.3 %) CIN2; 520 (13.5 %) CIN3; and 68 (1.8 %) invasive cervical cancer. The prevalence of HPV infection was 75.8 % (2,911/3,842), whereas multiple HPVs were detected in 34.5 % of HPV-positive subjects (2,255/3,842). The adjusted risks of CIN3+ in the group with multiple compared to the group with single infection were 2.31 (95 % CI = 1.54-3.47), among HPV16-positive women, and 3.25 (95 % CI = 2.29-4.61, p = 0.21 compared with HPV16-positive subjects), in HPV16-negative subjects. Out of a total of 1,285 subjects with mild lesions, followed up for a median of 16.1 months (interquartile range = 8.9-36.8), the rate of progression to CIN2-3 was 0.6 % (5/541) among subjects negative or with low-risk HPVs, 1.7 % (8/463) among those with single high-risk HPV, and 5 % (14/281, p < 0.001 compared with HPV-negative/low-risk HPV and p = 0.038 compared with single high-risk HPV) among those with multiple high-risk HPVs. CONCLUSIONS: Among women with cervical cytological abnormalities, infection by multiple high-risk HPVs increased the risk of CIN3+ in both HPV16-positive and HPV16-negative subjects. These findings suggest a potential synergistic interaction between high-risk HPVs, favoring the progression of CIN lesions.

Diagnostic accuracy of colposcopy in relation to human papillomavirus genotypes and multiple infection.

OBJECTIVE: The aim of this study is to evaluate the diagnostic accuracy of colposcopy for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) in relation to the detection of human papillomavirus (HPV) type 16 and multiple HPV infection. METHODS: A cohort study of 2526 subjects attending a colposcopic service because of cytological abnormalities. HPV genotypes were identified using the INNO-LIPA genotyping system. RESULTS: The final colposcopic/pathological diagnoses were as follows: 1282 (50.8%) negative, 709 (28.1%) CIN1, 169 (6.7%) CIN2, 318 (12.6%) CIN3 and 48 (1.9%) invasive cervical cancer, respectively. Among women with ASCUS/LSIL, assuming any colposcopic abnormality as a cut-off, there were no significant differences in the sensitivities (83.8%, 95% CI=76-89.6 as compared to 84.1%, 95% CI=73.2-91.1, p=0.9) and ROC curves (0.61, 95% CI=0.58-0.65 as compared to 0.59, 95% CI=0.54-0.64, p=0.5) in the detection of CIN3+ lesions between subjects with single and multiple high-risk infection, and between subjects infected by HPV16 (83.1%, 95% CI=73.7-89.7, ROC=0.59, 95% CI=0.54-063) or other high-risk HPVs (84.7%, 95% CI=75.6-90.8, ROC=0.62, 95% CI=0.58-0.66, p=0.8 and p=0.6 compared to HPV16). After correction for confounders, the odds ratios of CIN3+ associated with any abnormal colposcopic findings were 2.47 (95%CI=1.44-4.23, p=0.001) among HPV16 positive, 3.34 (95% CI=2.16-5.42, p<0.001) among other high-risk HPVs and 1.3 (95% CI=0.72-2.48, p=0.36) among subjects with negative/low-risk HPVs. CONCLUSION: In routine clinical practice, multiple infection or HPV16 positivity did not affect colposcopic accuracy in the diagnosis of CIN3+ lesions. The sensitivity of colposcopy was poor among subjects who were uninfected or infected by low-risk HPV genotypes.

Untypable human papillomavirus infection and risk of cervical intraepithelial neoplasia among women with abnormal cervical cytology.

The risk of cervical intraepithelial neoplasia and/or invasive cervical cancer associated with untypable human papillomavirus (HPV) infections has been not investigated fully. HPV infection caused by 18 high-risk and 7 low-risk genotypes as detected by the INNO-LIPA genotyping system, was investigated in 4,258 women with abnormal Pap smear referred to a colposcopic service. The prevalence of HPV infection was 76.1%. Rates of cervical intraepithelial neoplasia grade 3+ were 0.88% (9/1,017) in HPV-negative subjects, 1.8% (7/380) in subjects with untypable HPV infection, 3.2% (11/343) in subjects with single/multiple low-risk types, 28.3% (201/709) in subjects with multiple low and high-risk types, 15.2% (162/1,069) in subjects with single high-risk types, and 31.2% (229/733) in those with multiple high-risk types. Compared to women without any HPV infection, the odds ratios of cervical intraepithelial neoplasia grade 2+ or grade 3+ in subjects with untypable or low-risk HPV genotypes were 5.73 (95% CI = 2.79-11.78) and 12.4 (95% CI = 6.31-24.5, P = 0.014 compared to untypable) and 3.1 (95% CI = 1.11-8.16) and 7.1 (95% CI = 2.9-17.2, P = 0.07 compared to untypable), respectively. In the subgroup of subjects with cervical intraepithelial neoplasia grade 1 or negative colposcopy/biopsy, the progression to cervical intraepithelial neoplasia grade 2+ at follow-up (median 25 months, range 6-70) was 2% (14/684), 3.4% (7/205), and 5.6% (11/195, P = 0.04 compared to negative) among negative, untypable, and low-risk HPV infection, respectively. The risk of cervical intraepithelial neoplasia associated with untypable HPV infection was higher than that recorded among uninfected women, but lower than the risk associated with low- or high-risk HPV genotypes.

Oral HPV infection and persistence in patients with head and neck cancer.

OBJECTIVE: To investigate the presence and persistence of human papillomavirus (HPV) infection in the oral mucosa of patients with head and neck squamous cell carcinoma (HNSCC), and its correlation with prognosis. STUDY DESIGN: HPV infection was characterized in tumors and pre and posttreatment oral scrapings in 51 patients with HNSCC and matched controls using the SPF10 LiPA Extra assay. p16INK4A immunostain and in situ hybridization for high-risk HPV genotypes recognized transcriptionally active infection in tumor samples. The risk of infection was compared in patients and controls. The association of pretreatment HPV status with recurrence and survival and with posttreatment HPV persistence was assessed. RESULTS: Oral HPV infection risk was significantly higher in patients with HNSCC than in controls (P < .001). Oral HPV infection was associated with infection in the first posttreatment scrapings (P = .015), but did not affect recurrence or prognosis. CONCLUSION: Oral HPV infection is frequent in patients with HNSCC and has no prognostic implications, suggesting that posttreatment polymerase chain reaction monitoring on oral cells is not effective to monitor patient recurrence risk.

Exfoliated cells of the oral mucosa for HPV typing by SPF10 in head and neck cancer.

HPV infection in the superficial cells of the oral mucosa could reflect the presence of HPV in head and neck cancer cells. Due mostly to the use of heterogeneous analytical methods, discordant data exist in the literature regarding the agreement between the presence of HPV in non-neoplastic oral mucosa and in tumour tissue from the same patient. The presence of HPV DNA and viral types were compared in paired cytological and biopsy samples from 56 patients with head and neck neoplastic and preneoplastic lesions using the highly sensitive SPF10 LiPA Extra assay, which has been validated recently for formalin-fixed paraffin-embedded tissue using paired cervical cytology and biopsy samples. Kappa statistics were used to measure the inter-rater agreement. The overall agreement with respect to HPV infection was 96.43% (kappa=0.8367). For 76.79% of subjects (kappa=0.6937), the same number of HPV types was detected in cytological and biopsy specimens. The overall positive typing agreement was 90.90%, comprising 130 out of 143 individual HPV type analyses. The agreement shown was good for HPV 18, 44, 45, 54 and 66 (kappa=0.6585-0. 7321), excellent for HPV 6, 16, 40, and 54 (kappa=0.8108-0.8679), and absolute for HPV 11, 31, 33, 35, 39, 51, 52, 53, 59, 74, and 69-71 (kappa=1.0000). The high sensitivity of the SPF10 LiPA and its excellent performance both for recognising HPV infection and for identifying the viral types present in tumour tissue and in oral exfoliated cells make it a useful method for the assessment of HPV infection in patients with head and neck cancer. The excellent agreement for HPV infection and genotyping in paired samples suggests that oral exfoliated cells can be used for HPV detection in the head and neck region.

Time trends of human papillomavirus type distribution in Italian women with cervical intraepithelial neoplasia (CIN).

OBJECTIVE: It is assumed that the circulation of HPV types in a population is stable over time although there are limited historical data to support this view. The existence of possible cohort effects in the circulation of HPV types has major implications for vaccination strategies and risk assessment in HPV-infected women. We analysed archival biopsy samples of cervical intraepithelial neoplasia (CIN) to study the distribution of HPV types in Northern Italy over the years 1985-2007. METHODS: DNA from formalin-fixed paraffin-embedded cervical biopsies from the years 1985-87 (67 samples) and 1995-97 (92 samples) was HPV-typed by the SPF-(10) Lipa assay. Cases were compared with 159 control biopsies from the years 2005-07 matched by patient age and CIN grade. Quantitative PCR was used to compare titres of HPV sequences in DNA extracted from biopsies of the three periods. Type-specific PCR was used to confirm HPV51 and 52 typing by SPF-(10) Lipa. RESULTS: HPV51, 52, 53, 56, 58, and 66 were markedly under-represented or undetectable in samples from past periods whereas they represented 5.7-30.8% of present infections. Frequency of multiple HPV infections and high-risk infections (p=0.0001) also increased in recent years. The main changes occurred over the last decade. Infections by HPV16, 18, were three times more frequent 20 years ago than today (p=0.012). Loss of amplifiable HPV sequences over prolonged storage was not observed. Type-specific PCR confirmed all HPV51 and 52 infections. CONCLUSIONS: Secular trends in the distribution of HPV types among women with CIN may occur in specific populations.

A combined histologic and molecular approach identifies three groups of gastric cancer with different prognosis.

The limited prognostic value of currently used histologic classifications of gastric cancer and their failure to account for the complexity of the disease as revealed by more recent investigations prompted a combined reinvestigation of histologic, molecular, and clinicopathologic patterns in 294 extensively sampled, invasive gastric cancers representing all main histotypes and stages of the disease and followed for a median of 150 months. Among histologic parameters tested, only cellular atypia, angio-lympho- or neuroinvasion, Ki67 proliferation index, expansile/infiltrative type growth, and T8 cell-rich high lymphoid intra-/peritumor response (HLR) proved to be stage-independent predictors of patient survival. Among molecular tests, p53 gene exon 7 (loop 3) and 8 (loop-sheet-helix motif and S-10 band), but not p53 protein overexpression, TP53 LOH or 18qLOH, were found to worsen prognosis. Microsatellite DNA instability was a favorable prognostic factor when coupled with HLR. Patient survival analysis of the main histotypes and their subtypes confirmed the favorable prognosis of HLR, well-differentiated tubular, muconodular, and low grade diffuse desmoplastic cancers, and highlighted the worse prognosis of anaplastic and infiltrative-lymphoinvasive mucinous cancers compared to ordinary cohesive and diffuse cancers. Distinct roles of individual morphologic and molecular factors in tumor progression of the different histotypes have been recognized. The combination of survival-predictive histotypes and individual histologic or molecular parameters allowed us to develop a classification of all gastric cancers into three grades of increasing malignancy which proved to be of high prognostic value.

Clustering patterns of human papillomavirus genotypes in multiple infections.

Many human papillomavirus (HPV) infections are sustained by multiple viral genotypes whose effect on the risk of cervical intraepithelial neoplasia (CIN) is unknown. The study investigated whether specific HPV types or species may affect the likelihood of multiple infections and have a clustered distribution in a consecutive series of 681 women with a histological diagnosis of CIN. HPV typing was performed by the SPF(10)-LIPA assay; associations were evaluated by loglinear analysis of multiple contingency tables after stratification by age and CIN grade. HPV prevalence was 99.4% with a 72.1% rate of coinfection. The risk of coinfection was higher for types 6, 11, 16, 18, 31, 33, 51, 52, 56. Significant interactions were found for species A7-A9-A10, A6-A9 and A7-A10. Coinfection by types 31-35-56, 16-51-52, 16-18 and 51-52 was more frequent than expected. Interactions between viral species and HPV 16-18 were maintained among CIN1, whereas interactions of 16-51-52 and 31-51-56 were significant only in CIN> or =2. Interactions between species and types were lost among women younger than 32 years. Significant clustering of HPV types and species occurs among women with CIN. This has implications for the assessment of the oncogenic potential and the prevention of HPV infections.

Validation of the SPF10 LiPA human papillomavirus typing assay using formalin-fixed paraffin-embedded cervical biopsy samples.

Lower levels of performance of human papillomavirus (HPV) typing assays in studies using formalin-fixed paraffin-embedded (FFPE) tissue compared to those using exfoliated cervical cells have been reported. The interpretation of current studies is limited by bias in inclusion criteria, sample matching, and methods of cell collection. We aimed to validate FFPE tissue for typing by the use of the SPF(10) LiPA assay, comparing cervical scrapings to punch and cone biopsy specimens. We examined 165 paired cervical scraping and FFPE punch biopsy samples, and 66 paired FFPE punch and cone biopsy samples. HPV typing was performed using the SPF(10) LiPA assay. Kappa statistics were used to measure interrater agreement. The overall agreement with respect to HPV status was 100%. For 74.5% of subjects (kappa = 0.6147), the same numbers of HPV types were detected in scraping and biopsy specimens. The overall positive typing agreement was 95.4% (range, 93.4 to 97.3) for 441 out of 484 individual HPV type analyses. Agreement was good for HPV-39, -42, -43, and -70 (kappa = 0.6506 to 0.7166), excellent for HPV-6, -16, -18, -31, -33, -35, -40, -51, -52, -56, -58, and -66 (kappa = 0.8499 to 0.9665), and absolute for HPV-11, -44, -45, -53, and -68. In 43.9% of cases (kappa = 0.247), the same numbers of HPV types were found in punch and cone biopsy specimens. Overall positive agreement for typing was 86.8% (range, 82.5 to 91.1) for 204 out of 266 individual HPV type analyses. More infections by HPV-18, -33, -51, and -52 were detected in cone specimens. HPV typing by SPF(10) LiPA performed equally well for cervical scraping specimens and standard pathological material. Some viral types are preferentially detected in cone specimens, likely reflecting better sampling of diseased epithelium and endocervix tissue.

Cervical infections by multiple human papillomavirus (HPV) genotypes: Prevalence and impact on the risk of precancerous epithelial lesions.

A large proportion of human papillomavirus (HPV) infections is sustained by multiple genotypes. The effect of multiple infections on the risk of cervical intraepithelial neoplasia (CIN) and the potential efficacy of vaccine on these infections are controversial. We performed viral typing by SFP(10)-LIPA on a consecutive series of 1,323 women undergoing colposcopy, 69% of whom had cervical biopsy, and correlated CIN severity with the type and number of HPVs. Overall prevalence of HPV-DNA was 68.9%, 97.3% in CIN1, and 98.1% in CIN>/=2. HPV positivity correlated with younger age (35.9 vs. 37.3 years, P = 0.026) and history of CIN (P < 0.001). Multiple types were detected in 44.2% of cases, including 63.1% CIN1 and 80.8% CIN>/=2. Twenty-three different types were detected, HPV-16, 31 and 52 being the most frequent. Infections by HPV-6, 11, 16, or 18 occurred in 59.4% of CIN1 and 71.3% of CIN>/=2. Number of viral types and class of oncogenic risk were linearly correlated with CIN severity (P < 0.0001) by univariate and multivariate analyses controlling for age and history of CIN. The effect of the number of HPV types was maintained after exclusion from the model of infections by HPV-6, 11, 16, and 18. Frequency, distribution, and clinical correlates of multiple HPV infections highlight the importance of assessing individual types in the management and the prediction of outcome of women with abnormal baseline cytology and point to potential limitations in current vaccine strategies.