How an optimal level of human dopamine D4 receptor (hD4R) is maintained in synaptic membranes is not known. We show here that hD4R is ubiquitinated in primary neurons. We go on to show that ubiquitin is attached to hD4R through isopeptide and ester bonds. When lysine (Lys) residues of the hD4R are substituted with arginine (Arg) residues, cellular hD4R protein levels increase. A synergistic effect on hD4R levels is noted when cytoplasmic serine (Ser) and threonine (Thr) residues are mutated. Chloroquine, an inhibitor of lysosomal degradation, did not have an effect on hD4R protein levels. However, treatment with bortezomib, an inhibitor of the 20S proteasome, caused a dose-dependent increase in hD4R protein levels. The effect of bortezomib was attenuated in the receptor variants that lacked Lys or Ser/Thr residues, and the hD4R mutant that lacked 17 cytoplasmic Lys, Ser, and Thr residues was nearly insensitive to bortezomib treatment. We conclude that both isopeptide and ester bond ubiquitination regulate proteasomal degradation of hD4R. The hD4RhD4R contains a variable number of tandem repeats (VNTR) exon polymorphism in intracellular loop 3 (IC3). A 16-amino-acid, proline (Pro)-rich sequence is repeated two to 11 times in IC3 in human D4R proteins . Many stOptimal levels of hD4R are required for normal synaptic function, but how hD4R concentration is regulated in the membrane and the potential role of the cytoplasmic tandem repeat variants in the regulation process are not known. We have observed low hD4R protein levels in our protein expression system and noticed highly conserved ubiquitinatable residues on the cytoplasmic surface of the protein. We decided to explore potential ubiquitin-dependent degradation of the receptor. We hypothesize that both canonical, Lys-based isopeptide ubiquitination and non-canonical, Ser- and Thr-based ester bond ubiquitination regulate cellular degradation of hD4R.To test for hD4R ubiquitination, we utilized the four-repeat hD4R variant, which is the most common variant in the human population . We consA). Recent reports have also indicated that Ser and Thr residues can be ubiquitinated through ester bonds .We wanted to determine which amino acids in hD4R are modified by ubiquitin attachment. Ubiquitination canonically occurs through isopeptide bonds on Lys residues. There are only four Lys residues in the entire hD4R receptor , so we mFig. 2er bonds –15. TherB). We counted the number of PLA dots for WT hD4R-1D4 and each mutant. There was a statistically significant difference (p ≤ 0.001) between the average number of PLA dots per infected cell for WT hD4R-1D4 and the average number for KSTØ hD4R-1D4 . The difference in PLA dots per cell for WT versus KSTØ hD4R-1D4 confirms the specificity of PLA for detecting ubiquitinated receptor. KØ hD4R-1D4 and STØ hD4R-1D4 both had averages less than that of WT hD4R-1D4 but greater than that of KSTØ hD4R-1D4, indicating that isopeptide and ester bond ubiquitination both contribute to WT hD4R-1D4 ubiquitination . The fact that KØ hD4R-1D4 is statistically most similar to WT hD4R-1D4 and STØ hD4R-1D4 is statistically most similar to KSTØ hD4R-1D4 implies that ester bond ubiquitination may be the predominant form of ubiquitination for hD4R-1D4.To determine whether wild-type (WT) hD4R-1D4 was being ubiquitinated through isopeptide bonds, ester bonds, or both, we performed PLA on the KØ hD4R-1D4, STØ hD4R-1D4, and KSTØ hD4R-1D4 mutants. HEK293T cells were infected with baculovirus containing WT or mutant hD4R-1D4 cDNA . As withFig. 2D). We exploited the fact that ester bond ubiquitination is sensitive to base-catalyzed hydrolysis, whereas isopeptide bond ubiquitination is not (D).We also tested for hD4R-1D4 ubiquitination with an immunoprecipitation (IP) assay. We carried out IPs using the mutant hD4R-1D4 constructs expressed in HEK293T cells that also expressed FLAG-tagged ubiquitin. We performed the IPs with limiting quantities of the 1D4 mAb to ensure we were comparing equal amounts of mutant hD4R-1D4 protein. In the case of WT hD4R-1D4, there was a high molecular weight FLAG signal indicative of polyubiquitination of the receptor D. We expn is not . In the n is not D.Because the PLA and IP experiments indicate the presence of hD4R ubiquitination and a recent study also infers the presence of ester and isopeptide hD4R ubiquitination but does not report cell biological consequences of the modification , we wantA) were tested for function in an agonist-dependent (quinpirole) intracellular calcium release assay. The EC50 for the effect of quinpirole on KØ hD4R-1D4 was unchanged from that of WT hD4R-1D4. Both STØ hD4R-1D4 and KSTØ hD4R-1D4 remained functional, although they showed EC50 values that were shifted about 10-fold as compared with the EC50 for WT (B). These relatively modest differences in EC50 values can likely be explained by the influence of cytoplasmic substitutions on G protein coupling or the effect of G protein precoupling on agonist affinity or a potential difference in hD4R protein concentration in the plasma membrane between mutants . This trend held true when we transfected increasing amounts of receptor-specific plasmid. In fact, cells transfected with 0.625 μg of DNA encoding KSTØ had hD4R-1D4 protein levels 3 times greater than the hD4R-1D4 protein levels in cells transfected with 2.5 μg of DNA encoding WT receptor. These data support the hypothesis that both isopeptide bond and ester bond ubiquitination regulate hD4R protein levels post-translationally.Recent reports have shown that both isopeptide bond ubiquitination at Lys residues and ester bond ubiquitination at Ser and Thr residues can serve to regulate protein abundance , 10. To Fig. 3D). The protein level difference between KØ S239A/S245A hD4R-1D4 and S239A/S245A hD4R-1D4 was comparable with the difference in protein level between KØ hD4R-1D4 and WT hD4R-1D4 , indicating that isopeptide ubiquitin-dependent changes in hD4R protein levels do not require hD4R phosphorylation. The fact that S239A/S245A hD4R-1D4 protein levels are greater than WT hD4R-1D4 protein levels suggests that Ser-239 and/or Ser-245 may be subject to ester bond ubiquitination, which regulates in part the hD4R protein level. In other words, substitution of these two Ser residues removes potential sites of ubiquitination that may be promoting degradation of WT hD4R.Phosphorylation has been detected through mass spectrometry on Ser-239 and Ser-245, although no physiological role has been uncovered for these modifications , 21. CroFig. 3A). The protein levels for WT hD4R were reduced by about one-half after 1 h of incubation with cycloheximide. The KØ, STØ, and KSTØ hD4R-1D4 mutant proteins were all more stable than WT hD4R-1D4 with half-lives greater than 3 h. The increase in protein stability upon mutation of Lys, Ser, and Thr residues supports the hypothesis that WT hD4R protein is degraded in a ubiquitin-dependent manner.The increase in hD4R-1D4 protein levels upon mutation of ubiquitinatable Lys, Ser, and Thr residues led us to hypothesize that WT hD4R-1D4 protein is being degraded as a result of ubiquitination. To test the hypothesis that WT hD4R is degraded, we used cycloheximide to inhibit protein translation for 0–3 h in HEK293T cells expressing WT or mutant hD4R-1D4 A. The prm chloroquine was sufficient to inhibit the SDF1α-induced lysosomal degradation of the chemokine receptor CXCR4–1D4 but did not increase hD4R-1D4 protein levels (B) (m bortezomib (C). These results show that WT hD4R-1D4 is degraded proteasomally and is sensitive to bortezomib treatment.Screens with small molecule inhibitors that target various protein degradation pathways were carried out to evaluate how WT hD4R-1D4 was being degraded. Ubiquitinated GPCRs are canonically down-regulated by lysosomal degradation. However, chloroquine, an inhibitor of lysosomal degradation, did not have an effect on hD4R protein levels. A dose of 250 μn levels B 23). W. Wm chlovels (B) . There wrtezomib C. These C). These results support the hypothesis that isopeptide and ester bond ubiquitination both contribute to degradation of the WT hD4R receptor.A dose-response curve was then measured with bortezomib and KØ hD4R-1D4. There was also a dose-dependent increase in the level of KØ hD4R-1D4 with increasing concentrations of bortezomib. The total extent of the increase for KØ hD4R-1D4 was about half that of WT hD4R-1D4. STØ hD4R-1D4 displayed a dose-dependent increase in protein level as well, also totaling about half of that displayed by WT hD4R-1D4. The KSTØ hD4R-1D4 mutant showed very little increase in protein level with increasing concentrations of bortezomib, suggesting that the vast majority of hD4R-1D4 proteasomal degradation was dependent on Lys, Ser, and Thr residues in combination and not on other forms of ubiquitination such as thioester C. These B, C, and D). Second, treatments with lysosomal and proteasomal inhibitors (B and C) demonstrate that ubiquitin-dependent degradation of hD4R is proteasomal. Taken together, our data support a model of extensive proteasomal degradation of hD4R following ligand-independent ubiquitin.According to the canonical ubiquitination pathway, ligand-activated GPCRs at the plasma membrane are ubiquitinated and internalized, which promote their lysosomal degradation over recycling back to the cell surface . Our stuhibitors , B and CB), these plasma membrane receptors are not being lysosomally degraded after ubiquitination. The consequence of ubiquitination of plasma membrane hD4R is not clear but may be an example of proteasomal degradation of plasma membrane proteins  or a cuC and D, and 4C). Additionally, we have characterized a cytoplasmically Ser-less and Thr-less mutant, STØ hD4R-1D4 (A and 3A), that still retains protein function (B). We have confirmed, not just inferred, the identity of the non-Lys ubiquitination through quantifying the ubiquitination and proteasomal degradation of STØ hD4R-1D4 (C and 4C). Finally, we have made a functional Lys-less and cytoplasmically Ser-less and Thr-less mutant, KSTØ hD4R-1D4 (A and 3A), where ubiquitination and proteasomal degradation are substantially diminished and the protein half-life is substantially increased compared with WT hD4R-1D4 .In addition, we have detected a role for ester bond ubiquitination in hD4R degradation. Non-isopeptide  ubiquitination was first discovered in 2005 when Cadwell and Coscoy  reportedhD4R-1D4 A and 3A,function B. We havhD4R-1D4 C and 4C.hD4R-1D4 A and 3A,D).Before an ubiquitinated GPCR at the plasma membrane can be lysosomally degraded, it must be internalized. GPCR desensitization via internalization is canonically dependent upon agonist-induced phosphorylation of cytoplasmic Ser and Thr residues via G protein–coupled receptor kinases. Ser and Thr phosphorylation would be temporally mutually exclusive with Ser and Thr ubiquitination. Intriguingly, hD4R has been previously shown to be resistant to agonist-induced desensitization via the canonical pathway . The samFig. 3It was previously observed by Van Craenenbroeck and co-workers  that hD4The hD4R contains a VNTR in IC3. It is important to note that the 16-amino-acid repeat sequence is not perfectly replicated in each repeat region. Although all repeat segments are Pro-rich, the amino acid identity between Pro residues is variable both within a given allele and among the alleles present in the human population. Intriguingly, despite the substantial differences in total amino acid number among hD4R variants, the ubiquitinatable residues  are nearly constant for the most common two-repeat, four-repeat, and seven-repeat alleles . We hypoBoth presynaptic and postsynaptic activation of D4R in the brain have numerous physiological consequences . For exaThe WT pcDNA3.1-hD4R-1D4 construct was described previously . Mutatio3VO4, and Benzonase at 4 °C for 1 h. Lysates were spun at 10,000 rpm at 4 °C for 10 min and then normalized for total protein using the DC Protein Assay (Bio-Rad). Lysates were analyzed by SDS-PAGE (using a 4–12% Bis-Tris gel) followed by immunoblotting on PVDF membrane with 1D4 Ab (m chloroquine diphosphate (Sigma) for the final 4 h and 200 nm SDF1α (PeproTech) for the final 3.5 h before harvesting. For proteasomal inhibition experiments, cells were grown in the presence of bortezomib  for the final 12 h before harvesting. For protein stability experiments, cells were incubated with 50 μg/ml cycloheximide  for the final 3 or 1 h before harvesting, or cycloheximide was added immediately before harvest (time = 0 h).HEK293T cells were transiently transfected with Lipofectamine and Plus reagents and grown for 48 h. For transfections with increasing amounts of receptor-specific plasmid, the total DNA level in each transfection was kept the same by supplementing with empty pcDNA3.1 plasmid. After harvesting, cell pellets were solubilized with radioimmune precipitation assay buffer with protease inhibitors, PMSF, NaF, Nah 1D4 Ab  and antiN-glycosidase F (New England Biolabs) and then split in half. One aliquot was incubated with 50 mm NaOH (pH 12.3) for 1 h at 37 °C. The other aliquot was incubated with an equivalent volume of PBS (pH 7.6) for 1 h at 37 °C. Samples were then analyzed by SDS-PAGE and immunoblotting as before with both 1D4 and anti-FLAG-M2 Abs (Sigma-Aldrich) and anti-mouse HRP secondary Ab.HEK293T cells were transfected as above with pcDNA3.1-hD4R-1D4 constructs and 3XFLAG-ubiquitin. After lysing and normalizing as above, cell lysates were incubated with limiting quantities of 1D4 Ab-coated protein G Dynabeads (Invitrogen 10003D) and incubated overnight. Beads were washed, and then the protein was specifically eluted with 1D5 peptide. Elutions were treated with peptide:d-lysine–coated 384-well plate. After 48 h, cells were incubated with FLIPR Calcium 4 Assay kit dye for 1 h in the tissue culture incubator. The fluorescence  of the wells was then recorded using Flex Station II 384 (Molecular Devices) while a dose curve of quinpirole was added to wells, in triplicate, from 0 to 100 nm (WT and KØ) or 0 to 500 nm (STØ and KSTØ). Normalized maximum-minimum fluorescence for each well was plotted for each dose curve, and a four-parameter fit was used to calculate EC50 for each construct. Dose curves were repeated three times on independently prepared samples, and a representative dose curve is shown.HEK293T cells were cotransfected with pcDNA3.1-hD4.4R-1D4 and qi5 using Lipofectamine 2000. Cells were transfected directly into a poly-d-lysine. The neurons were grown for 6 days in vitro in selective Neurobasal medium containing 2% B27 (Invitrogen) and 1% l-glutamine (Invitrogen) at 37 °C in a cell incubator  until the day of transfection.Cortices were dissected from C57Bl/6 16–17-day (E16.5) mouse embryos essentially as described previously , 34. ThePrimary neurons were transfected with 0.2 μg of pcDNA3.1-hD4R-1D4 using 0.6 μl of Lipofectamine 3000 and 0.4 μl of P3000. Growth medium was removed to keep a total volume of 70 μl during the initial stage of the transfection. After 4-h incubation, an excess of growth medium was re-added to the dishes. After 48 h, cells were washed twice in DPBS, fixed in 10% formalin for 10 min at room temperature, washed again with DPBS, and permeabilized with 0.4% CHAPSO for 10 min at room temperature. Cells were then processed following the manufacturer's instructions for DuoLink Detection Reagents Far Red (Sigma-Aldrich) using anti-1D4 and anti-ubiquitin (Abcam 7780) primary Abs. After PLA processing, cells were stained with phalloidin-Alexa Fluor 488 to visualize neuronal architecture. Samples were mounted in DuoLink mounting medium with DAPI (Sigma-Aldrich).For quantitative PLA, HEK293T cells were infected with pFBDM baculovirus prepared as described previously . Cells wConfocal images of PLA-treated neurons were acquired with a Nikon A1RSi point scanning confocal inverted microscope using a 60× oil immersion objective. Excitation lasers of 405, 488, and 640 nm were used. Images from single confocal planes are shown. Image processing  was done in NIS Elements. The brightness and intensity of the images were adjusted in Adobe Photoshop CS6 to enhance the possibility to observe fine structures on printouts, and the final figure was made in Adobe Illustrator CS6.For quantitative PLA in HEK293T cells, 20× magnification Z-stack images (every 1.5–2 μm) were captured using an FSX100 Olympus microscope. Exposure times were held constant while imaging samples within each experimental replicate. A total of four fields of view were obtained per sample per experiment.All image processing was done using ImageJ. Nuclei stained with DAPI were counted to obtain the total number of cells per image. Then the number of infected cells, identified as nuclei surrounded by a full ring of 1D4 immunofluorescence, were counted. The total number of PLA dots per field was determined by creating a maximum projection of the PLA channel, converting it to a 32-bit grayscale image, and recording the number of dots as the number of PLA signals.p value <0.05.The average number of PLA signals per cell of a control-infected plate was used to estimate background signal for each experiment. For each field to be quantified, the average number of background PLA dots per cell (averaged over the four images obtained from the control infection) was multiplied by the total number of cells in that field. This number was then subtracted from the total number of PLA dots in that field to remove background. The remaining dots were divided by the number of infected cells to account for variable infection efficiencies, resulting in the average number of PLA dots per infected cell per field. For each of the five biological replicates there were four fields per sample, resulting in a total of 20 data points per sample. Using Prism, a one-way ANOVA followed by Tukey's multiple comparison test was used to compare the mean of each group with that of every other group. Significance was determined by a J. C. P. and T. P. S. conceived the overall project. J. C. P. designed and performed HEK293T experiments. M. S. and J. C. P. designed and performed quantitative PLA in HEK293T cells. S. S.-W. and J. C. P. designed and performed mouse neuron experiments. M. A. K. performed cycloheximide and DNA titration experiments. T. P. S. and J. C. P. wrote the manuscript.


HIV and human papillomavirus (HPV) infections may have multiple interactions. We reviewed the strength of the evidence for the influence of HIV on HPV acquisition and clearance, and the influence of HPV on HIV acquisition.Observational studies suggest HPV incidence and clearance rate by HIV status (review 1) and of HIV incidence by HPV status (review 2). We pooled relative risk (RR) estimates across studies using random‐effect models. I2 statistics and subgroup analyses were used to quantify heterogeneity across estimates and explore the influence of participant and study characteristics including study quality. Publication bias was examined quantitatively with funnel plots and subgroup analysis, as well as qualitatively.We performed meta‐analytic systematic reviews of longitudinal studies of HPV incidence  and high‐risk HPV incidence  was approximately doubled among people living with HIV (PLHIV) whereas HPV clearance rate  was approximately halved. In review 2, 14 publications (11 independent studies) were included in the meta‐analysis. HIV incidence was almost doubled  in the presence of prevalent HPV infection. There was more evidence of publication bias in review 2, and somewhat greater risk of confounding in studies included in review 1. There was some evidence that adjustment for key confounders strengthened the associations for review 2. Misclassification bias by HIV/HPV exposure status could also have biased estimates toward the null.In review 1, 37 publications (25 independent studies) were included in the meta‐analysis. HIV and HPV interactions of clinical and public health relevance. HPV vaccination may directly benefit PLHIV, and help control both HPV and HIV at the population level in high prevalence settings. Our estimates of association are useful for mathematical modelling. Although observational studies can never perfectly control for residual confounding, the evidence presented here lends further support for the presence of biological interactions between HIV and HPV that have a strong plausibility.These results provide evidence for synergistic  High‐risk HPV types 16 and 18 account for about 70% of all cervical cancers and precancerous lesions, and a substantial fraction of anal, vulvar, vaginal and penile cancers The majority of individuals who are sexually active will acquire human papillomavirus (HPV) infection at some point in their lives, but most will develop the necessary immune response and clear the infection Current evidence suggests that HIV and HPV infections may interact in multiple ways Quantifying the magnitude of the biological interactions between HIV and HPV is important for many reasons. If HIV and HPV biologically interact together by increasing susceptibility to infection (for HIV and HPV), duration of infection (for HPV), and the severity and risk of developing disease (for HPV), it will facilitate HIV and HPV spread, and increase the burden of HPV infection and diseases among PLHIV. Presence of synergistic interactions would also mean that prevention efforts against one infection could provide indirect population‐level benefits for the other. For example, if HPV vaccination could prevent additional HIV infections and related deaths, this could be particularly relevant for high HIV prevalence countries in Sub‐Saharan Africa and strengthen the case for implementing HPV vaccination programmes in this region. Estimating the size of these interactions is important to parameterize mathematical models and understand how these biological interactions and the rollout of HIV interventions may influence future population‐level trends in HPV and HIV infections and related diseases, patterns of co‐morbidity, and HPV vaccination impact in high HIV prevalence settings.Here, we present a systematic and meta‐analytic review of all the evidence for the association between HIV exposure and subsequent risk of HPV acquisition and rate of HPV clearance. For completeness, we also updated and re‐analysed two previous meta‐analyses for the association between HPV exposure and subsequent HIV acquisition, which had a latest search date of 31st January 2012 2The systematic reviews and meta‐analyses were undertaken in accordance with Meta‐analysis of Observational Studies in Epidemiology (MOOSE)2.1PubMed and Embase databases were searched (up to 6th January 2017) to identify longitudinal studies investigating the association between HIV status and subsequent HPV acquisition and clearance (review 1) as well as the effect of HPV status on subsequent HIV acquisition (review 2). We screened publication titles, abstracts and articles for longitudinal studies using a combination of keywords and MeSH terms relating to HPV or cervical/anal/genital neoplasia, cancer, abnormalities or lesions and HIV. Bibliographies of relevant articles were examined for 2.2Publications were examined for eligibility to include prospective cohort studies, randomized controlled trials, and case–control studies nested within a cohort or trial, where the time sequence between HIV and HPV infections was determined, that were published in the English language, and that measured active genital HPV infection by detection of the virus in either cervical, penile or anal swabs or cervico‐vaginal lavage fluid using a test based on HPV DNA identification. We excluded studies that only measured HPV antibodies, which do not distinguish between current and past infection. No further exclusions were made on the basis of study quality, which was instead assessed in subgroup analyses and qualitatively. The main associations of interest were the relative risk (RR) of acquiring a new HPV infection or clearing an existing HPV infection (outcomes) by HIV serostatus (exposure) (review 1), and the RR of acquiring HIV infection (outcome) by HPV status  (exposure) (review 2). Given the large number of HPV types, we used the following standard HPV categories: (any) HPV, high‐risk HPV (HR‐HPV), low‐risk HPV (LR‐HPV) using the HPV groupings stated in the publications, and single HPV types included in the nonavalent vaccine (HPV‐6/11/16/18/31/33/45/52/58). Where available, estimates for the association between HIV and number of HPV types (dose–response for review 2), and estimates by CD4 cell count compared to HIV‐negative individuals (dose–response for review 1) were extracted. We did not include estimates which only compared HPV risk between PLHIV by CD4 levels, without using HIV negative as the comparator. Where possible, and if not otherwise reported, we derived estimates with HIV‐negative individuals as the comparison group.Studies measured HPV incidence and clearance in different ways, which we defined as follows. For HPV incidence, two situations were possible. An individual's HPV status could change from (1) no HPV DNA present  to HPV DNA subsequently being present (defined as “first HPV”); or from (2) DNA of one or more HPV types present to DNA of a new and different HPV type subsequently being present (defined as “new HPV”). For clearance of HPV two situations were possible. An individual's HPV status could change from (1) DNA of one or more HPV types present to no HPV DNA subsequently being present ; or from (2) DNA of a specific HPV type present to no DNA of that HPV type subsequently being present (defined as “clearance of any HPV type”). In addition, HPV clearance could be defined in the study on the basis of one HPV negative test or two successive negative tests.The association between HIV and HPV for grouped HPV types  could be measured in the study using one of three different units of analysis for HPV: individual‐level, type‐level, or visit‐level. At the individual‐level, only one occurrence for each HPV event was counted per individual during the entire follow‐up period. This meant that, for incident HR‐HPV, for example, an individual could acquire both HPV‐16 and HPV‐18 but this would only count as one event. By definition, clearance of all HPV types could only be measured at the individual‐level. When measured at the type‐level, an individual could have multiple events occurring at different times, for example, acquire multiple HPV types. The studies would count them and the rates would then be averaged over the group of HPV types (HR‐HPV or LR‐HPV) acquired. Note these units are equivalent for the incidence  of one HPV type as only one event is possible. At the visit‐level, which was less commonly used as the unit of analysis across studies, only one event could be counted per individual for each study interval, but events were then summed for all study intervals over the entire follow‐up period.Eligible studies were examined (KJL) to extract crude RR and adjusted aRR estimates  and 95% confidence intervals (95% CI), as well as information on participant characteristics  and study characteristics , including indicators of study quality such as frequency of visits/testing interval and key variables adjusted for, in a standardised form (comprehensive list in Table 2.3vs. HR‐HPV‐negative) to compensate for the few available estimates using HPV‐negative as the comparison. Estimates without 95% CI or which were numerically undefined (e.g. due to no unexposed cases) were excluded from the meta‐analysis. Statistical heterogeneity across study estimates was assessed with the I2 statistic. Subgroup analyses were used to explore the influence of participant and study characteristics and quality for the associations with HPV and HR‐HPV. The meta‐analyses were done in Stata (version 14) and forest plots produced in R.For each association, forest plots and pooled estimates were used to summarize independent RR estimates across studies. Pooled natural log RR estimates and 95% CI of log transformed study RR estimates were derived using a random‐effects model based on the inverse‐variance method 2.4Information on study quality from the data extraction, such as definitions of HPV incidence and clearance and unit of analysis, was assessed quantitatively using subgroup analyses by relevant study characteristics. In particular, we assessed potential for confounding, for example, in adjusted estimates using subgroup analysis by whether or not a key confounder  was adjusted for. We also compared pooled crude and adjusted estimates for the subset of studies which reported both types of estimates. Publication bias was assessed in three ways: first with funnel plots of crude study estimates as described in Appendix 33.1, of the effect of HIV status (exposure) on subsequent HPV acquisition and clearance (outcomes), we extracted data from a total of 41 publications reporting on 27 independent studies Of the 6430 potential publications identified from the PubMed and Embase searches, 553.2In review 1, 10 of the 27 studies were conducted in North America and 9 in Sub‐Saharan Africa , HR‐HPV (N = 10), LR‐HPV (N = 2), single HPV nonavalent vaccine type (N = 8) and by number of HPV types acquired (N = 2). Studies reported RR estimates of HPV clearance by HIV status for HPV (N = 15), HR‐HPV (N = 10), LR‐HPV (N = 3) and single HPV nonavalent vaccine type (N = 8). Eight studies compared HPV incidence or HPV clearance outcomes between HIV‐negative and PLHIV stratified by CD4 level (dose–response), six of which reported results of a statistical test for trend by CD4 level Table .In review 2, 10 of the 11 studies were conducted in Sub‐Saharan Africa (one study was from North America), all after 1996 with 1 after 2006, and 7 among females , HR‐HPV  and HPV‐16 , and slightly more equivocal increased risk of HPV‐18 , in PLHIV compared to HIV‐negative individuals Figure . Pooled In subgroup analyses by participant and study characteristics, the magnitude of the stratified pooled crude RR and adjusted aRR for incidence of HPV and HR‐HPV by HIV status were similar to unstratified pooled estimates , and remained statistically significant overall, with the exception of pooled RR for subgroups based on few study estimates, which were not always statistically significantly different from the null  and similarly for HR‐HPV  Figure . StatistIn subgroup analyses, stratified pooled crude RR and adjusted aRR for clearance of HPV and HR‐HPV by HIV status were similar in magnitude overall to unstratified pooled estimates, and remained statistically significant, with the exception of pooled RR for subgroups based on few estimates only, which were not all statistically significantly different from the null Table c,d.3.3.3p = 0.03, p < 0.01; Table Figure p = 0.04) but suggested that incident HR‐HPV infection risk declined with decreasing CD4 count p = 0.2p = 0.001No other associations comparing CD4 level ≤200 cells/μL and CD4 level >200 cells/μL were found , prevalent HR‐HPV , and prevalent LR‐HPV   Table . Pooled 3.5In review 1, 13 studies reported on the association between HIV and incident HPV infection for first HPV and 10 for new HPV  by definitions of HPV incidence and clearance and unit of analysis were similar in magnitude to unstratified pooled estimates, and the associations generally remained statistically significant overall Table . There wvs. 1.51, 95% CI: 1.25 to 1.83), and not for HR‐HPV  , but not statistically significantly so (95% CI overlapped)   Table , but thi9) Table . In subgd) Table . The sam5) Table . Our qua5) Table .3.6Our results indicate that the risk of subsequent HPV acquisition is approximately doubled in the presence of HIV infection, while the rate of HPV clearance is approximately halved. We updated previous meta‐analyses of the effect of HPV on subsequent HIV infection adding two new studies to earlier published reviews 3.7To the best of our knowledge, this is the first meta‐analytic review investigating all the evidence for the association between HIV and subsequent HPV infection (review 1), not just in women As with other systematic and meta‐analytic reviews of longitudinal studies of STI and HIV interactions, there are some limitations to our reviews that may affect our results in either direction Conversely, mathematical modelling to assess potential biases in STI and HIV interaction estimates for different study designs has suggested that whilst statistical adjustment can help reduce overestimation due to confounding, the magnitude of association could also be underestimated in the presence of misclassification of exposure Some studies reported multiple estimates of association for different HPV exposures (review 2) and outcomes (review 1) , which also provide a form of validation of consistency of results within a study, but may however increase the likelihood of finding spurious associations. We also differentiated between “first HPV”, defined as the acquisition of HPV DNA in those individuals without any HPV‐DNA present at baseline but who may have been exposed to HPV in the past (i.e. not naïve), and “new HPV”, defined as the acquisition of DNA of a new and different HPV type in those individuals who already had HPV DNA present at baseline. We also differentiated between clearance of “all” and “any” HPV. Definition of a clearance event itself  also varied between studies. However, we did not find evidence that these differences influenced our results.I2 statistic), was particularly high across estimates of HIV on HPV clearance, but results from subgroup analyses showed that pooled effect sizes remained similar across the subgroups examined.The studies in review 2 were concentrated in Sub‐Saharan Africa, which may limit the generalizability of our results to other settings. There were fewer study estimates for single HPV vaccine types and LR‐HPV associations (both reviews), HPV incidence and clearance by CD4 count level (review 1) and HIV acquisition by incidence and clearance of HPV (review 2). This could partly be due to publication bias, of which we found greater evidence for review 2 than review 1. This was observed in funnel plots, in subgroup analysis comparing reported estimates that were slightly higher that estimates that we calculated ourselves, and in our qualitative assessment of selective reporting of statistically significant associations. Nevertheless, we were able to successfully derive several new RR study estimates from available data (where the estimates themselves were not reported), which improved the precision of crude pooled estimates and reduced publication biases. It also meant that we were not able to derive adjusted estimates controlling for potential confounders, and as a proportion of studies fewer adjusted estimates were available for review 1 than 2. Heterogeneity . We found some evidence of a dose–response . Additional evidence of a biological dose–response also comes from studies comparing HPV acquisition and clearance by CD4 levels among PLHIV only, which was not the purpose of our study biologically plausibleanalogous to the interactions between HIV and HSV‐2 in terms of some similarity of biological mechanisms. In future, better evidence of these interactions could be obtained from discordant couple studies and/or studies with more frequent STI and HIV testing, using the best HIV tests with the shortest window period, and by adequately measuring potential sexual risk factors among participants and their partners In our review we have comprehensively summarized all the available observational evidence on HIV and HPV interactions. Our review was designed to minimize and assess the possibility of confounding and bias affecting our findings. We restricted our analysis to longitudinal studies of the associations, and carried out extensive sensitivity analyses, comparing adjusted and unadjusted estimates in multiple ways, extracting information on the comparability of exposed and unexposed groups, and exploring the influence of possible selective reporting of non‐significant results. The likelihood of confounding explaining the associations in not the only consideration when assessing the plausibility for causation. For example, our results meet many of the 9 Bradford Hill criteria for causality 4Our study provides an evidence base for multiple biological interactions between HIV and HPV. These interactions have a number of important clinical, epidemiological and public health implications. In particular, the excess burden of HPV in PLHIV has implications for the clinical management of PLHIV requiring more frequent screening, follow‐up and management of precancerous lesions due to HPV. HPV vaccination, which has been proven to be safe and immunogenic among PLHIV, may confer particular benefit to this group In addition, interventions such as HPV vaccination could in theory have additional indirect benefits on HIV/AIDS, even if the relative risk of HIV acquisition due to HPV is modest. Given the burden of HPV and HIV and abundance of co‐infections, HPV vaccination could prevent a non‐negligible number of AIDS deaths particularly in Sub‐Saharan Africa. If reducing HPV provides benefits for HIV, this would optimize HPV programmes, particularly in the context of combination prevention, providing additional incentives for programme implementation. Our comprehensive review can help inform decisions on HPV vaccination in women and men, including MSM. Mathematical modelling studies are needed to understand the potential impact of these multiple interactions on HIV and HPV trends and in the context of intervention rollout for high HIV prevalence settings.MB reports an unrestricted grant from Merck in the past 3 years in relation to herpes zoster (completed). MD declares a role as a consultant to GlaxoSmithKline (GSK) in the past 3 years in relation to herpes zoster vaccine. PM has received funding from GlaxoSmithKline (GSK) and in kind donation from QIAGEN for research projects on HPV vaccines or HPV testing. KJL, MMR, PMB and MCB declare no conflicts of interest.MCB designed and supervised the study and provided technical guidance. The literature search was done by KJL. KJL and MMR extracted the data and performed the analysis. KJL produced the first draft of the manuscript, which was subsequently revised and edited by MCB and MMR. All authors contributed to the interpretation of results, contributed technical expertise, commented on the drafts, and approved the final version.Appendix S1. Additional supporting information.Figure S1. Forest plots of the crude relative risk (RR) by HIV status of: (S1a) incident LR‐HPV infection; (S1b) incident HPV‐6/HPV‐11 infection; (S1c) clearance of LR‐HPV.Figure S2. Forest plots of the crude and adjusted relative risk (RR) by HIV status of: (S2a) incident HPV‐16/HPV‐18 infection by CD4 cell count; (S2b) clearance of HPV‐16/HPV‐18/HPV‐31/HPV‐45 by CD4 cell count.Figure S3. Funnel plot of the crude estimates for HIV on incident HPV.Figure S4. Funnel plot of the crude estimates for HIV on incident HR‐HPV.Figure S5. Funnel plot of the crude estimates for HIV on clearance of HPV.Figure S6. Funnel plot of the crude estimates for HIV on clearance of HR‐HPV.Figure S7. Funnel plot of the crude estimates for prevalent HPV on HIV.Figure S8. Funnel plot of the crude estimates for prevalent HR‐HPV on HIV.Table S1. Summary of the 41 publications identified reporting on the effect of HIV infection on HPV acquisition and clearance (review 1).Table S2. Summary of the 15 publications identified reporting on the effect of HPV infection or clearance on HIV acquisition (review 2).Table S3. Additional study characteristics used to assess quality of studies from publications included in the review of longitudinal studies of the effect of HIV on HPV acquisition and clearance (review 1).Table S4. Additional study characteristics used to assess quality of studies from publications included in the review of longitudinal studies of the effect of HPV infection and clearance on HIV (review 2).Table S5. Subgroup analyses of the association between prior exposure to HPV and subsequent HIV infection by participant and study characteristics (review 2).Click here for additional data file.


Large-size data on type-specific HPV prevalence in Southwest China are required to estimate the cervical cancer burden in the country and to prepare for HPV-based cervical screening program and further HPV vaccination of China. This HPV study is a pooled analysis of data from five years in Chongqing of China, which is cross-sectional in design using data collecting.n = 10542) were triaged by cytology, colposcopy or histological diagnosis. Among HPV-positive women, 43.8% had normal, 22.5% had ASCUS, 0.2% had LSIL, 12.6% had HSIL and 6.0% had ICC. The most common HPV genotypes were HPV16, 58 and 18 in ASCUS, HPV16, 18 and 58 in LSIL, HPV16, 58 and 33 in HSIL, and HPV16, 58 and 18 in ICC. The prevalence of Group 1/2A HPV types increased with increasing CIN grade and accounted for 96.05% of the CIN 3+ lesions, while HPV16 accounted for 71.1%. HPV-positive women steadily increased with age, peaking at 31–40 years.The positivity of HPV was 26.2% (10542/40311), single type was 25.7% (10360/40311), multiple type was 8.2% (3306/40311), high-risk HPV was 30.9% (12490/40311), and low-risk HPV was 2.9%(1169/40311). The most common genotypes were HPV16,52,58 and 18. HPV-positive women (The type-specific prevalence rate of HPV 16 and HPV 18 were a little lower than the mean of international meta-analyses. Single HPV genotype infection was predominantly detected in different groups of cervical lesions in Chongqing, and HPV16, 52, 58 were the priority HPV types. The HPV genotyping study was found to be valuable for planning further preventive program for cervical cancer. Cervical cancer is the second most common female cancer worldwide, which results in >250000 deaths annually . The infMore than 100 different HPV genotypes have been identified, of which, 40 infect the genital tract . In geneHPV DNA testing has emerged as a worthful complementary method to conventional Papanicolaou (Pap) staining for enhancing the effect of cervical cancer screening. It has played an indispensable role in determining suitable clinical management schemes for cervical cancer screening and subsequent HPV vaccination plan –12.Cervical cancer could be effectively controlled by primary and secondary prevention like cervical screening and prophylactic HPV vaccination. However, to date there are no large-size data on type-specific HPV prevalence in Southwest China women, especially in Chongqing, making it difficult to design a national HPV vaccination strategy. Because government validation for the HPV vaccines will likely be upcoming after the fulfillment of ongoing clinical studies, an extensive understanding of type-specific HPV prevalence among Chinese women is crucial. Data on the type-specific prevalence of carcinogenic HPV types are required to supervise the effect of vaccination and to formulate HPV-based cervical screening strategies of Chinese women across the country when the vaccines are taken. The purpose of the study was to provide valuable data about the type-specific prevalence of HPV infection and its connection to findings in histology and cytology triage.A total of 40311 (ranging from 18 to 75 years) women who participated in cervical cancer screening were included continuously from August 2009 to April 2014 by the laboratory. The participants were enrolled according to the following selection criteria: non-pregnant, sexually active, without miscarriage in past 6 months. Moreover, we excluded participants who have a history of total hysterectomy, or were being treated with vaginal medicine at that time or will have surgery for cervical diseases in 6 months. Sexual activity was defined as any sexual intercourse that included vaginal penetration. Girls or adolescents with no history of sexual activity, i.e. vaginal , anal or oral intercourse, were considered not sexually active. All women had undergone cervical cancer screening in our Health Check Center or at the Department of Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University. All cervical samples of the participants were gained by professional gynecologists via cytobrush and resuspended in 20 ml of liquid-based cytology medium (Tellgen Life Science Co.Ltd.). Cervical samples were continuously accessed for cervical cytology and the existence of HPV DNA.In short, 50 μl of the liquid-based cytology sample was moved to Eppendorf tube. The Eppendorf tube was centrifuged and the supernatant was discarded. Then, 200 μl of the denaturing reagent  was added in the Eppendorf tube to extract genomic DNA. Then the tube was incubated at 100 °C for 10 min. The solution was centrifuged and the supernatant was moved to another Eppendorf tube. The nucleic acid concentration was calculated by examining the A260/A280 ratio (1.8–2.0).Cervical exfoliated specimens were examined for HPV DNA using Tellgen plex™ xMAP™ HPV DNA Test assay (Tellgen Life Science Co. Ltd.). The following HPV types were detected by the assay: low-risk HPV (LR-HPV): HPV6, 11, 40/42/44, and 61/73; high-risk HPV (HR-HPV):HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 82, 83 and 26/55.On the basis of the manufacturer’s instructions, PCR was performed in 20 μl reaction mixture per person containing 100 ng of DNA, 10 μl PCR premixed solution, 0.8 μl thermus aquaticus (Taq) and 5 μl primer mixture. Amplification was carried out by initial denaturation at 95 °C for 5 min followed by 5 cycles of denaturation at 95 °C for 30s, annealing at 58 °C for 30s and elongation at 72 °C for 30s. In total, 40 cycles were conducted.Twenty-two microliters of hybridization reagent (containing 5000 microspheres labeled with every type-specific probe) and 3 μl of the denatured biotin-labeled PCR solution were added into 96-wellmicrotiterplates (Tellgen Life Science Co. Ltd.) for every well. The mixture was incubated in 99 °C for 5 min and maintained at 48 °C for 30 min for hybridization. Seventy-five microliters of Streptavidin-R-Phycoerythrin was added into the plates after hybridization. Reactions were performed at 48 °C for 15 min. The last, the mixture was tested by a Luminex 200 analyzer instrument .Cervical slides were prepared using a liquid-based cytology method, the ThinPrep system (Cytyc Corporation). Cytological classifications of disease grade were made in conformity to the Bethesda 2001 criteria. The slides were evaluated for cervical cytology by three academic cytopathologists of the First Affiliated Hospital of Chongqing Medical University. A diagnosis was assigned to each case as having negative for intraepithelial lesion or malignancy (NILM) or having an epithelial cell abnormality such as atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intra epithelial lesion (HSIL) and invasive cervical cancer (ICC).Women underwent colposcopy with the Preventive Oncology International, Inc. (POI) micro-biopsy protocol of directed and random biopsies if they met one of the following criteria: (1) had LSIL or greater diagnoses on cytology; (2) were from a rural site testing positive for HPV infection by HC2 by either self or direct collection; or (3) were from an urban site and had ASC-US/HPV-positive or LSIL or greater cytological findings.The results had been obtained by three academic Gynecology doctors of the First Affiliated Hospital of Chongqing Medical University. A diagnosis was assigned to each case as having NO Lesion, cervical intraepithelial neoplasia grade 1 (CIN 1), CIN 2, CIN 3.χ2 test was performed to evaluate the significance of differences between designated groups. All analyses were two-sided and interpreted as being significant at P ≤ 0.05.All analyses were carried out using SPSS 17.0 statistical software. Type-specific prevalence of HPV infection and their exact binomial 95% confidence intervals (CI) were calculated for women overall and stratified by age as well as by cytologic and histologic findings. Pearson’s In all, 40311 women were recruited HPV screening in Chongqing during 2009–2014. And 10542  were positive for HPV. The average age of the study population was 46.2 years (range 18–75 years). The prevalence of HPV exhibited reached a peak at 31–40 years and 41–50 years, with a positive proportion of 9.4 and 8.0%, respectively , 25.9% of these participants were HPV-positive. The HPV infection ratio of the current smoker was 28.8%. As shown in Table Twenty-five different HPV types were identified in the study population. There were 20 high-risk HPV genotypes  and five low-risk genotypes   and 13.22% low-risk types (Group 3).In order to analyze the correlation between prevalent genotypes of HPV infection and cervical lesions, using the selection criteria (as Method part shown), a part of women were chosen for cervical biopsy or colposcopy as mentioned above.Among the 10542 HPV-positive women, 493 had colposcopy data. The estimated attribution for each cytological grade is shown in Table As shown as Table The estimated attribution for each cytological grade is shown in Table A large majority of the Group 1/2A HPV types accounted for 80%, 100%, 87%, 95% in ASCUS, LSIL, HSIL, ICC, respectively. However, the infections ratio of Group 2B was 5.5%, 0, 2.3%, 5.2% in ASCUS, LSIL, HSIL, ICC, respectively. The proportion of infections with Group 3 was 5.5%, 0, 2.3%, 2.6% in ASCUS, LSIL, HSIL, ICC, respectively.P = 0.207).Figure Among the 271 cytology abnormalities, we compared the data for colposcopy and histological diagnosis. In the ASCUS-positive group, histological diagnosis showed 140 with no lesion, two CIN 1 and three CIN 2, while colposcopy indicated 130 with no lesion, six CIN 1 and nine CIN 2. In the HSIL-positive group, histological diagnosis showed 56 CIN 1, 20 CIN 2 and eight CIN 3, while colposcopy indicated 45 CIN 1 and 39 CIN 2. However, histological diagnosis showed four CIN 1, eight CIN 2 and 25 CIN 3, while colposcopy indicated six CIN 1, nine CIN 2 and 22 CIN 3 in the ICC-positive group. The data showed that cytological diagnosis was consistent with colposcopy and histological diagnosis Fig. .HPV infection is the most common sexually transmitted disease, which is connected with various clinical status, ranging from asymptomatic infection to malignant cervical disease. Our study was a large-scale report which demonstrated the type-specific HPV prevalence in Chongqing, and its relation to the cervical disease burden. The three most prevalent carcinogenic HPV types in the Chongqing screening population were HPV16 (0.7%), 52 (0.5%) and 58 (0.4%). The prevalence of HPV16 in our study was slightly lower than that reported in the women with normal cytology of Western and Northern European countries , 14. RegHPV 18 ranks second genotype in Europe about general population , but shoHigh-risk HPV infection is the principal factor in cervical cancer. Distribution of HPV genotypes showed increased prevalence of high-risk HPV in direct relation to the severity of cervical cytopathology . In the Among the population with CIN 2 and 3, HPV 16, 31, 33, 18 and 58 were the most common genotypes . In ChonThe age peak of HPV prevalence was 31–40 years, which was different from 45 years onwards reported in Latin America, the Caribbean, and most countries across Africa . A peak The results regarding the effect of smoking on HPV infection have been contradictory. Some studies revealed that smoking was associated with an increased trend of HPV prevalence , but notIn the present study, our results showed that women having three or more sexual partners had significantly higher risk of HPV infection. The results was consisted with other studies , revealiSimilar to other studies , the preThis is the first study to identify Initial impact of HPV infection in non-vaccinated women of Chongqing. We report the results of a population-based HPV screening of 40311 women in Chongqing Province of China. Our studies would demonstrate the understanding of the epidemiology of HPV infection. The data will assist in developing a putative screening program, that the HPV types would be included in the HPV vaccine in future to provide protection against those high-risk types of HPV with high prevalence identified in our study. However, there are several limitations in this study, one of which is the absence of some risk factors, such as gravidity, number of abortion, condom use and education. And a population-based research about the correlation between these risk factors with cervical cancer will be the direction of our further study. Moreover, larger and prospective studies about the mechanism are also needed to validate our findings.Our results give a broad summary of the distribution of HPV type in Chongqing Province in women with different grades of cervical lesions. The most common HPV types were 16, 52, 58 and 18. Further, large, multicenter prospective studies are needed to fully evaluate the varied HPV pattern in Chinese women, and to provide substantial information directly useful for planning HPV preventive vaccination in China.


Approximately 18% of all human cancers have a viral etiology, and human papillomavirus (HPV) has been identified as one of the most prevalent viruses that plays causative role in nearly all cervical cancers and, in addition, in subset of head and neck, anal, penile and vulvar cancers. The recent introduction of next generation sequencing (NGS) and other omics approaches have resulted in comprehensive knowledge on the pathogenesis of HPV-driven tumors. Specifically, these approaches have provided detailed information on genomic HPV integration sites, disrupted genes and pathways, and common and distinct genetic and epigenetic alterations in different human HPV-associated cancers. This review focuses on HPV integration sites, its concomitantly disrupted genes and pathways and its functional consequences in both cervical and head and neck cancers. Integration of NGS data with other omics and clinical data is crucial to better understand the pathophysiology of each individual malignancy and, based on this, to select targets and to design effective personalized treatment options. During the past three decades, it has become clear that viruses may have important player in the development of various human malignancies , 2. It hAdvances in the next-generation sequencing (NGS) technologies, including whole genome sequencing (WGS), whole exome sequencing (WES), RNA sequencing (RNA-seq), miRNA sequencing (miRNA-seq), whole genome bisulfite sequencing (WGBS), its corresponding analytical tools and ‘omics’ techniques, have provided comprehensive novel genomic and epigenomic information on HPV-associated cancers. NGS approaches have e.g. successfully been used for the identification and characterization of viral integration sites into the human genome, the identification of disrupted genes including oncogenes, tumor suppressor genes, and DNA repair genes, the detection of HPV-prone genomic instability and altered cellular pathways. In this review, we will focus on HPV-associated cancers, HPV integration sites within the human genome, and its concomitant disruption at the genomic, transcriptomic and epigenomic levels.Almost ~610,000 human cancers are the consequence of HPV infection every year in worldwide . HPV infE1, E2, E4, E5, E6, and E7) and late (L) region encompassing two ORFs (L1 and L2)  DNA was isolated from genital warts (HPV6) and subsequently cloned by Gissmann and de Villiers in the late 1970's . Shortlypression . HR HPVspression . In earlpression , 53. Thepression , 55. Thepression  and 71.2pression , 58. In pression , 59. Conpression , 60. Dur cancers  and HNSClization , 55.E6 and E7 are found to be amplified as viral-host fusion genes. The E6 and E7 proteins inactivate the p53 and Rb tumor suppressor proteins, respectively  as well as in HPV-negative cases   , 34. Eve%] , 34.  cancers .BARX2 and IRX4 have been found to be hyper-methylated and, consequently, expressed at lower levels in HPV integration-positive HNSCCs compared to integration-negative cases, whereas SIM2 and CTSE genes have been found to be hypo-methylated and, consequently, expressed at higher levels in HPV integration-negative HNSCCs compared to integration-positive cases [IRX4 has been found to be hyper-methylated and expressed at a lower level in HPV integration-positive oropharyngeal tumors compared to non-integrated tumors [In contrast to the genomic changes mentioned above, HPV-positive HNSCCs appear to exhibit distinct epigenetic profiles compared to HPV-negative cases , 82, 89.ve cases . Similard tumors .CCND1, CCND2 and CCNA1 were found to be up-regulated in HPV-negative HNSCC, while CDKN2A, CCNE2, CCNB1 and MCMs were found to be over-expressed in HPV-positive cases. In addition, multiple genes that regulate DNA replication and cell proliferation, including PCNA, E2Fs, CDC2, and CDC7, were found to be significantly up-regulated in HPV-positive HNSCCs and cervical cancers compared to HPV-negative HNSCCs. Interestingly, three testis-specific genes SYCP2, TCAM1 and STAG3, were found to be over-expressed in HPV-positive HNSCCs and cervical cancers. Normally, these genes are not expressed in somatic cells [In concordance with the epigenetic profiles described above, gene expression array studies have revealed similarities and differences between HPV-positive and HPV-negative cancers, as well as similarities between HPV-positive HNSCCs and cervical cancers , 85, 91.miR15a (4.19 fold) and miR-20b (3.42 fold) has been found to be higher in HPV-positive HNSCCs than in HPV-negative cases, while the expression of miR-139-3p (2.26 fold) and miR-145 (2.19 fold), among others, has been found to be lower in HPV-positive than in HPV-negative HNSCCs. Also, the expression of miR-145 has been found to be lower in HPV-positive cervical cancers [in vitro [miR-9 (1q23.2), miR-15b (3q25.32), miR-28-5p (3q27.3), miR-100 and miR-125b (11q24.1) have been found to be associated with recurrent chromosomal alterations [MiR-9 is a potential oncogene, and its over-expression has been associated with increased copy numbers of chromosome 1q and increases cell viability, anchorage-independent growth and migration in vitro [miR-34s was found to be deregulated after HPV infection by the viral oncoprotein E6 [miR-20b, miR-9, miR-9*, miR-492, miR-545, miR-591, miR-422a, miR-142-3p, miR-383,miR-520g, miR-101, miR-381, miR146a, miR34a, miR412, miR-155, miR-554, miR380-5p, miR-16, miR-219, miR-29c, let-7g, miR-30e-5p and miR-139 is significantly higher in HPV-positive OSCCs than in HPV-negative cases, whereas the expression of miR-193b, miR-107, miR-23a, miR-365, miR-452, miR-198, miR-196a, miR-181d, miR-324-5p, miR-550, miR-487b, miR-599, miR-382, miR-221 and miR-22 is lower in HPV-positive versus HPV-negative OSCC cases [miR-34 has been found to be down-regulated in cervical cancers [Based on microRNA (miRNA) profiling, HPV-positive and -negative HNSCCs can be clustered into two distinct subgroups, whereas significant similarities between HPV-positive HNSCC and cervical cancers have reported , 71, 91. cancers  and thisin vitro . Notablyerations . MiR-9 iin vitro . In concCC cases . Interes cancers , suggestCCND1 is frequently observed in HPV-negative cases, whereas up-regulation of E2F1 is common in HPV-positive cases. In contrast, mutation and/or amplification of PIK3CA are a common feature of both HPV-positive and -negative HNSCCs and cervical cancers [Recent efforts of The Cancer Genome Atlas (TCGA) network together with other NGS and ‘omics’ studies in virus-associated tumors have provided detailed information on the etiology of HPV-driven cancers. It is important to stress that integration of NGS data with other ‘omics’ data is crucial to better understand the pathophysiology of each individual disease and, based on this, to select targets and effective personalized treatment options. HPV-positive and -negative cases may exhibit both similar and different genomic alterations, whereas the epigenomic and transcriptomic profiles may be distinct between these groups. For example, the expression of cell cycle regulatory and testis-specific genes distinguishes HPV-positive from HPV-negative HNSCCs. Similarly, over-expression of  cancers . These d


Using EdgeR, differentially expressed genes (DEG) were identified. Immune subset analysis was performed using Functional Analysis of Individual RNA-Seq/ Microarray Expression (FAIME) and immune gene RNA transcript count analysis. In total, 1,634 DEGs were identified, with a dominant immune signature observed in HPV(+) tumors. After normalizing the expression profiles to account for differences in B- and T-cell number, 437 significantly DEGs remained. A B-cell associated signature distinguished HPV(+) from HPV(−) tumors, and included the DEGs CD200, GGA2, ADAM28, STAG3, SPIB, VCAM1, BCL2 and ICOSLG; the immune signal relative to T-cells was qualitatively similar between TILs of both tumor cohorts. Our findings were validated and confirmed in two independent cohorts using TCGA data and tumor-infiltrating B-cells from additional HPV(+) HNSCC patients. A B-cell associated signal segregated tumors relative to HPV status. Our data suggests that the role of B-cells in the adaptive immune response to HPV(+) HNSCC requires re-assessment.Human papilloma virus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) has a better prognosis than it's HPV negative (HPV(−)) counterpart. This may be due to the higher numbers of tumor-infiltrating lymphocytes (TILs) in HPV positive (HPV(+)) tumors. RNA-Sequencing (RNA-Seq) was used to evaluate whether the differences in clinical behaviour simply reflect a numerical difference in TILs or whether there is a fundamental behavioural difference between TILs in these two settings. Thirty-nine HNSCC tumors were scored for TIL density by immunohistochemistry. After the removal of 16 TIL Head and neck squamous cell carcinoma (HNSCC) accounts for 6% of all cancers, with ~650,000 new cases reported and 350,000 HNSCC-related deaths per year worldwide , 2. Hist+ GZMA+ PRF1+ T-cells  as previously described [Frozen tumor sections taken immediately adjacent to the tissue analysed by RNA-Seq were stained with hematoxylin and eosin (H&E); tumors were assessed as TIL high (TILescribed  (Supplemescribed  (SupplemTwo HNSCC patient cohorts consisting of 137 HPV(+) and 407 HPV(−) tumors were analysed retrospectively for survival relative to TIL density and HPV status. The primary endpoint was death from HNSCC, i.e., disease-specific survival, as previously described [CD19 (B-cells) and CD4 and CD8A (T-cells) were used as the covariates. Unsupervised clustering of tumors was performed following variance stabilizing transformation of trimmed mean of M-values (TMM) normalized data. DEGs between HPV(+) and HPV(−) tumors were identified with a false discovery rate (FDR) corrected p-value of <0.05  and a fold change of >2 or <-2. A detailed description of the RNA-Seq data analysis performed can be found in the Supplementary Methods S3.RNA-Seq  was performed using the HiSeq 2000 platform  (Supplementary Methods S2). RNA-Seq data have been deposited in the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (NCBI) under accession number GSE72536. RNA-Seq data was mapped using human genome (hg19) and TopHat (version 2.0.9), counted with HTSeq-count (version 0.5.4)  and diffGGA2), ADAM metallopeptidase domain 28 (ADAM28), CD200, Spi-B transcriptional factor (SPIB), stromal antigen 3 (STAG3), Vascular Cell Adhesion Molecule 1 (VCAM1), Inducible T-Cell Co-Stimulator Ligand (ICOSLG) and B-Cell CLL/Lymphoma 2 (BCL2) and adhere to the MIQE guidelines for RT-qPCR (Supplementary Methods S4) [STAG3 and CD200 was validated in the original tumor RNA, HPV(+) n=8 and HPV(−) n=8 patient tumor samples. B-cells (CD19+) isolated from an independent cohort of HPV(+) tumors (n=6) using a BD FACSAria™ sorter  were assessed for gene expression of ADAM28, BCL2, CD200, GGA2, ICOSLG, SPIB, STAG3, and VCAM1. RT-qPCR analysis was performed using the comparative Ct (cycle threshold) method (2-ΔΔCt) using Actin as the control gene and is defined as a normalized relative gene expression compared to a the control gene [Real-time quantitative reverse transcription PCR (RT-qPCR) assays were performed using Taqman® probes for golgi-associated, gamma adaptin ear containing, ARF binding protein 2 (hods S4) . Gene exrol gene .q-value <0.05) were identified with ConsensusPathDB [GO terms associated with biological processes and biological pathways that were significantly over-represented for DEGs  [+ and CD8+ T-cells  between the HPV(+) and HPV(−) cohorts.The distribution and proportions of TILs were assessed at both the tumor sample and group level. At the sample level, the gene expression (RNA-Seq) and surface protein expression (IHC) of  (FAIME)  was adaphttp://gdac.broadinstitute.org/runs/stddata_2015_11_01/data/HNSC/20151101/), the RNA-Seq methodology and processing have been described by TCGA [HNSCC RNA-Seq data (TCGA HNSC HiSeqV2 2015-02-24) was obtained from The Cancer Genome Atlas (TCGA) Genome Data Analysis Center (GDAC) Firehose website (


The histological study did not reveal evident signs of infection caused by HPV on vestibular papillae, besides perinuclear halos. HPV DNA was detected on vestibular papillae in 27%. Our results confirmed a scarce correlation between vestibular papillae and HPV. Thus, we consider papillomatosis of the vulvar vestibule, in most cases, to be equivalent to physiological papillomatosis and, therefore, should not be treated.The main goal of this study was to investigate, through a biomolecular study, the correlation between papillomatosis of the vulvar vestibule and human papillomavirus (HPV) infection, as well as to establish the necessity of treatment. A total of 44 female adolescents between 12 and 18 years of age were selected through a prospective study with a confirmed diagnosis of papillomatosis of the vulvar vestibule. Vulvar biopsies were obtained for the histological and biomolecular detection of HPV DNA through polymerase chain reaction (PCR). Twenty (45%) adolescents were virgins (group A), the other 24 (55%) were sexually active. The virgin adolescents (group A) and 12 sexually active adolescents (group B) did not show cytological and/or colposcopic alteration, suggesting infection by HPV either on the cervix or vagina. These were compared with 12 other sexually active adolescents who showed cervicovaginal infection caused by HPV (group C). Fisher exact test was applied for statistical analysis of the results, considering alpha equal or less than 0.05. There was no statistically significant difference in relation to HPV DNA through PCR among virgin and sexually active adolescents in group B, however, both differed from those in group C (A + B × C:


Human papilloma virus (HPV) is one of the major public health problems and the main causes of cervical cancer. The prevalence HPV infection in developing countries with low financial resources is high. This study aimed to determine the relative frequency of HPV genotypes and its sociodemographic characteristics in women referred to a general hospital in Tehran, Iran from 2014-2015.This cross-sectional study was performed in 400 women with Pap smear samples, referring to to a general hospital in Tehran, Iran from 2014-2015. The detection of 28 HPV genotypes was performed by using the Multiplex PCR technique. The sociodemographic survey was conducted for each HPV positive woman.  HPV-positive infection was detected in 155 (38.75%) women aged 17-85 years. HPV 16 (19.1%) was the most prevalent type, followed by HPV 39 (12.5%) and HPV 18 (8.9%). The highest rate of HPV infection was observed at the age of 36 years (7.7%). The level of education and economic situation of each woman were showed most of HPV-positive women had a high school diploma (34.6%) and average economic situation . 60.9% of these women were a housewife, and 67.3% lived in the capital . Determination of HPV genotype and risk factor related to HPV infection in each geographical region can lead to the production of effective vaccines against the HPV virus. It can also be useful for disease management and high sensitivity diagnosis of cervical intraepithelial neoplasia.  Human papilloma virus (HPV) is one of the major public health problems and the main causes of cervical cancer . It is tet al the global prevalence of HPV was 11.7%. The HPV prevalence was 24% in Sub-Saharan Africa, 21.4% in Eastern Europe, 16.1% in Latin America  according to the manufacturer’s instruction and then multiplex polymerase-chain-reaction (PCR) tests for HPV DNA were performed.Multiplex PCR testHPV genotyping was performed using the HPV-HCR Genotype-Eph kit (AmpliSens(R), Russia).The kit is based on simultaneous amplifying in one tube (multiplex-PCR) of four types of HPV DNA and allows the user to detect infections and co-infections of high-risk HPV genotypes. DNA from each HPV genotype was used as a positive control and distilled water in place of template DNA run at the same conditions as a negative control. oC for 15 min, followed by 42 cycles, including 1 sec denaturation at 95oC, 3 sec annealing at 63oC and 40 sec chain elongation at 72oC and a final elongation at 72oC for 1 min and cooling at 4oC. PCR products were visualized on 1.5% agarose gel by ethidium bromide staining  with the following condition: The amplification was carried out with initial enzyme activation at 95staining , 19. A participant was considered HPV-positive if the test results by PCR or genotyping were positive. Samples that were negative for HPV DNA were amplified with primers for the cKi-ras gene to ensure the integrity of the samples. Samples in which neither HPV DNA nor the cKi-ras gene was amplified were considered inadequate for analysis and were excluded.Ethical considerationInformed consent was obtained from all women enrolled, and the study was approved by the Ethics Committee of the Iran University of Medical Sciences (Ref. number: IR.IUMS.REC.1391-17028).Statistical analysisData analysis, which was obtained from Multiplex PCR test and the questionnaires were entered into an Excel spreadsheet and then data were performed using Statistical Package for the Social Sciences, version 18.0, SPSS Inc, Chicago, Illinois, USA (SPSS) software. All quantitative variables were evaluated by the mean±SEM and type-specific prevalence of HPV were summarized using frequency distributions.Demographic and Sociodemographic CharacteristicsThe mean±SD age of participants in this study was 42.58±12.21 yr old (ranging from 17-89 yr old). Among 400 women included in this analysis, overall HPV prevalence was 38.75% [positive HPV (n=155) and negative HPV (n=245)]. The mean age of HPV-positive women was 40.47 yr old (ranging from 20-80 yr old). Also, the mean age of menarche and marriage among the HPV-infected women were 13.28±1.57 and 19.38±4.96 yr, respectively and 70.4% of them had not experienced menopause until this study was underway. The mean age of menopause among HPV-positive women was 47.29±6.93 yr. 22.4% of HPV-positive women had never been used any contraception method . 87.2% oSocioeconomic analysis of HPV-positive women was performed. This means that level of education and economic situation of each were evaluated. Most of HPV-positive women had a high school diploma (34.6%) and average economic situation (67.9%). 60.9% of these women were a housewife, and 67.3% lived in the capital. A survey of the HPV-positive women showed that 96.8% of them were not smokers and 93.6% were not the hookah smokers . Twenty-It has been proven persistent HPV infection can lead to cervical cancer and intraepithelial neoplasia , 20. SerAccording to our results, 155/400 (38.75%) studied women were positive and infected by one or more types of HPV. Also, the most prevalent types of HPV were HPV16 (20.2%). 22 types of HPV were observed, including HPV 15, 16, 17, 18, 31, 33, 35, 39, 40, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 70, 73 and 75.The prevalence of HPV types was changeable in different geographic regions, but HPV 16 were reported as the most prevalent and cancerous type among the other types of Human papillomavirus . Accordiet al reported that HPV 18, 45, 31, and 33 were respectively the most common types of HPV after HPV 16  followed by age 46 yr (5.8%). In the study was performed by Dunne thirties . It coul.7% follothirties , 33. et al have reported that age was one of the significant risk factors for acquisition high-risk HPV infection and high-risk HPV infection was decreased with advancing women age  in HPV positive women. In all risk factors investigated in this study HPV16 was the most prevalent type. HPV genotyping and detection of risk factors associated with HPV acquisition in each region can lead to develop an effective screening program and increase the level of awareness of community members. It can also be useful for disease management and high sensitivity diagnosis of cervical intraepithelial neoplasia. Further studies should be done to investigate the HPV genotyping of the other region of Iran or the other part of the world.


Several studies have addressed the impact of viral infections on male infertility. However, it is still unknown whether human papillomavirus (HPV) can alter seminal parameters. The aim of this study was to determine the prevalence of HPV in the semen of male partners of couples seeking fertility evaluation. Additionally, we assessed the possibility that HPV infections affect seminal parameters. A total of 229 semen samples were collected from men in the Sperm Analysis Section of São Camilo Laboratory of Maringá, Brazil, between October 2015 and March 2016. Basic seminal parameters were analyzed, and HPV was detected and genotyped by polymerase chain reaction. HPV DNA was detected in 16.6% of samples. Of these, 10.5% had single type HPV infections, 6.1% had multiple HPV infections, 5.7% had exclusively high-risk HPV, and 6.1% had exclusively low-risk HPV. Samples positive for single and multiple types of HPV were associated with abnormal viscosity, and samples positive for multiple HPV types were also associated with hypospermia, higher pH, and increased leukocyte numbers. These findings suggest that the male partners of infertile couples with seminal HPV infections may have prostate disturbances indicative of glandular dysfunction, which may influence fertility. Human papillomavirus (HPV) is one of the most common viral sexually transmitted diseases in both males and females worldwide . Low-risOnce it became established that the virus is primarily transmitted through direct epithelial contact , little So far, conflicting results have been reported on the effects of HPV infection on semen quality , 11–17.  Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, and Neisseria gonorrhoeae by multiplex polymerase chain reaction assay performed by us [From October 2015 until March 2016, 297 males were attended in the Sperm Analysis Section of São Camilo Laboratory, Maringá . Eligible men were 18 years or older and had semen analysis requested by their physician as part of a fertility evaluation, after failing to conceive with their partner after one year of unprotected intercourse. Participants who met the following criteria were excluded: symptoms of genitourinary infections, antibiotic treatment within the previous three months, reproductive system abnormalities , a history of vasectomy, and infertility therapy in the preceding year. Additionally, subjects who tested positive fored by us  were excμL was transferred to tubes containing 1.0 mL of sterile 0.9% NaCl solution and immediately stored at −80°C until genomic DNA could be extracted.Prior to semen collection for analysis, men were asked to abstain from sexual intercourse or masturbation for 3–5 days. Before semen collection, subjects were instructed to wash their hands with soap, and all samples were collected after urinating and washing the glans penis and coronal sulcus with soap and water. Semen was collected by masturbation and ejaculation directly into standard sterile containers that had previously been shown not to have any cytotoxic effects on human spermatozoa. Participants avoided contact with the interior wall of the sterile container to prevent sample contamination. Freshly collected semen was immediately incubated at 37°C for 15–60 min for liquefaction. Afterwards, samples were homogenized and 300 6/mL for sperm concentration; 32% for progressive motility ; 58% for sperm vitality ; 4% for sperm morphology ; and <1.0 for leukocytes. The terms used to describe samples with values lying outside the reference range were hypospermia for seminal volume; oligozoospermia for sperm concentration; asthenozoospermia for progressive motility; necrozoospermia for sperm vitality; teratozoospermia for sperm morphology; and leukocytospermia [Detection of basic seminal parameters was performed according to criteria published by the World Health Organization . The folospermia .β-globin gene using the primers GH20 and PC04. Two types of controls were used in the reaction: a sample without DNA (negative control) and an HPV-positive sample (positive control). Final amplified products were loaded onto a 1.0% agarose gel stained with 150 ng/μL ethidium bromide and subjected to electrophoresis in a horizontal tank at 110 V for 45 min in 0.5x TBE buffer . A 100 bp marker  was used as a size standard. The amplified DNA fragments were visualized on a transilluminator with UV light and then photographed. HPV was genotyped by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, in which amplified DNA was cleaved with restriction enzymes to generate DNA fragments of different molecular sizes. Aliquots of each amplified product were subjected to digestion with the restriction enzyme HpyCH4V  as described by Santiago et al. [To remove any polymerase chain reaction (PCR) inhibitors from the semen, samples were incubated for 15 min with proteinase K in phosphate buffered saline and then centrifuged. DNA was extracted using an AxyPrep™ Body Fluid Viral DNA/RNA Miniprep Kit  according to the manufacturer's instructions. The quality and quantity of purified DNA were determined by spectrophotometry on a NanoDrop 2000 Spectrophotometer . HPV was detected by PCR using the primers MY09 and MY11 . This reo et al. . To betto et al. . The reso et al. . A totalo et al. –25.p values <0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism 6.0 .Seminal parameters were compared between HPV-positive and HPV-negative groups, HR HPV-positive and HR HPV-negative groups, LR HPV-positive and LR HPV-negative groups, and multiply infected HPV-positive and multiply infected HPV-negative groups using Chi-square tests. The means of age and seminal parameters were compared in the total study population and in strata of HPV presence as follows: HPV-negative; mutually exclusive HPV-positive subgroups  by one-way analysis of variance (ANOVA). Two-sided After written consent, a total of 229 participants who passed the inclusion and exclusion criteria of the study provided one semen sample each . ParticiThe HPV infection rates in semen samples are summarized in p = 0.0005), and samples with multiple HPV infections were associated with hypospermia (p = 0.01) and abnormal seminal viscosity (p = 0.0002). Interestingly, semen samples negative for multiple HPV infections were associated with normal seminal viscosity (p = 0.03). Still, samples that were HR HPV-positive had a statistically borderline association with abnormal seminal viscosity (p = 0.057).p = 0.0003 and p < 0.0001, resp.).To our knowledge, this is the first study in Brazil and Latin America to detect and genotype HPV in a large cohort of male partners of couples seeking fertility evaluation and address the possibility that HPV infection may affect seminal parameters. The results of this study reveal that HPV infection in semen is rather common in male partners of infertile couples. Furthermore, we demonstrated an association between HPV seminal infections, particularly infections involving multiple HPV types, and reduction in seminal volume, abnormal viscosity, and elevation of seminal pH. These changes could potentially play important roles in male subfertility and/or infertility.The possibility that HPV infection could affect seminal parameters and thus fertility is a highly debated topic in the field of human reproduction , 4, 26. HPV16 was the most prevalent genotype detected in semen samples infected with both single and multiple types, which is in accordance with other recent studies , 10. HowThe high prevalence of HPV DNA and more specifically HPV16 in the semen of asymptomatic males in our study supports previous evidence that semen functions as a transport medium for HPV from the male genital tract to the female cervix/uterus  and highMale fertility depends on the two equally important major components of semen: healthy spermatozoa  and the composition of seminal fluid, which is important for sperm function. There was no significant association between HPV infection and sperm concentration, motility, or vitality, as recently described by Luttmer et al. . HoweverSecretory products from the seminal vesicles and prostate are crucial for sperm motility, viability, and chromatin stability, but they are also important for semen coagulation and liquefaction. Seminal vesicles secrete the major fraction of the ejaculate (≈60%) . They prLimitations of our study include the absence of data on mixed antiglobulin reaction testing, which was not tested in each primary semen analysis but performed only upon physician's request. Secondly, as our study did not include analysis of medical records, it does not include data on the female partner's characteristics, such as age, hormone levels, tubes, pelvic status, and additional male partner characteristics such as clinical analysis of the prostate and seminal vesicles. Still, semen results were taken from only one semen analysis per subject. Finally, our study did not include normal fertile subjects (control group). However, we believe that our results may encourage further studies to evaluate the influence of HPV infection on changes in seminal parameters.This study is the first to reveal a significant association between HPV-positive semen and hypospermia, abnormal seminal viscosity, higher mean pH, and higher mean numbers of leukocytes, particularly in semen samples positive for multiple HPV infections. Our data support a model where seminal HPV infections lead to changes in prostate markers that are indicative of glandular dysfunction and could change the proportion of fluids secreted from the prostate and the seminal vesicles. Thus, HPV seminal infections could play an important role in male infertility. These results deserve further attention and additional studies should be conducted with larger cohorts.


The Copenhagen Self-Sampling Initiative (CSi) has shown how human papillomavirus (HPV)-based self-sampling can be used to increase screening participation among 23,632 nonattenders in the Capital Region of Denmark. In this study, we describe HPV prevalence and genotype frequency in 4,824 self-samples as determined by three HPV assays  and compare the results with those for physician-taken follow-up samples. The HPV self-sample findings were also compared to the findings for a reference population of 3,347 routinely screened women from the Horizon study, which had been undertaken in the same screening laboratory. Nonattenders had an HPV prevalence of 11.3% as determined by the CLART assay, which was lower than that for women from the Horizon study (18.5%). One-third of the CSi women who tested HPV positive by self-sampling tested HPV negative on the physician-taken follow-up sample. The CLART and Onclarity assays agreed on 64%  of the HPV-positive self-taken samples. When the HC2 assay results were added into a three-way comparison, the level of agreement decreased to 27% . Our findings suggest that further validation of HPV assays on self-taken samples is needed for optimal HPV detection and correct clinical management of HPV-positive women. Human papillomavirus (HPV)-based self-taken samples can be an alternative to cervical cytology screening for women who do not attend screening (nonattenders) , 2. A me4–The pilot implementation of “The Copenhagen Self-Sampling Initiative” (CSi) was initiated to investigate whether self-sampling by nonattenders was a viable option for increasing participation in screening. Another aim was to compare three HPV assays on self-taken samples. By use of an opt-in strategy, 20% of 23,632 invited nonattenders returned self-taken samples . All samThe frequencies of different HPV genotypes among self-sampling nonattenders are infrequently reported in the literature, mainly because the majority of the studies used the HC2 assay, which does not allow one to distinguish between the HPV genotypes detected. In this study, we not only employed assays with extended or full genotyping but also compared the findings to those of the Danish Horizon study, a previous study using physician-taken samples from routinely screened women, where the CLART assay was also used in that study.One of the major outcomes of the Horizon study was that the frequencies of ≥CIN2 detection were fairly similar for the assays compared, but the assays detected different HPV infections in the same samples . On sampIn this study, we compared the detection of HPV infections by three assays  and the subsequent cytology results for women with detected infections. The findings were compared to those for routine screening samples from the Horizon study , 16.High-risk HPV (HR-HPV) infections were detected in 769 (15.9%) self-taken samples, based on a positive test result on any assay . The Onclarity assay showed the highest prevalence (14.1%), followed by the CLART and HC2 assays  . The HPVHPV16 was the genotype most frequently observed both for routinely screened women and in self-sampling, constituting 25.4% and 24.3%, respectively, of the HR-HPV types (data not tabulated), and with a detected prevalence of 2.9% in self-sampling. The second most frequent HR-HPV genotype in self-sampling was HPV51 (2.0%), followed by HPV31 (1.6%) . In the Of 736 HR-HPV infections detected by the Onclarity or CLART assay in the CSi study, a total of 471 infections  were detected by both assays ; Fig. 1AThe relatively low agreement was not substantially increased when genotype HPV66 was included on the CLART assay so that it would match the genotypes targeted by the Onclarity assay. The levels of agreement were highest for HPV16, HPV31, and HPV52 , whereas the lowest level of agreement was observed for HPV45 (40%) .When the HC2 assay was added into the comparison, with 964 valid test results, the level of positive agreement was 27% . In pairP = 0.67) (data not reported).Almost two-thirds of women with HPV-positive test results were also found HPV positive in the follow-up sample (69.1% for the CLART assay and 63.7% for the Onclarity assay) . Women wTC) values of the internal controls included in the Onclarity assay, as well as the TC values for positive samples, allowed a quantitative evaluation of sample quality. Mean TC values on self-taken samples were approximately 2 cycles lower than those on physician-taken samples for all three human beta-globin (HBB) control wells , indicating a larger concentration by volume of human material in self-taken samples than in physician-taken samples   (The use of the cycle threshold ( samples . Discrep cycles) . Concord cycles) .Among self-sampling attenders, the prevalence of HR-HPV was between 11% (by the CLART assay) and 14% (by the Onclarity assay), and about one-third of HPV-positive women had abnormal cytology upon follow-up. Agreement in the detection of HR-HPV between the CLART and Onclarity assays was observed in approximately two-thirds of cases overall, and in three-fourths of cases when HPV16, HPV31, or HPV52 infections were involved. About one-third of the women whose self-taken samples tested HPV positive were found to be HPV negative on the physician-taken follow-up sample, and this was particularly the case when the signal strength in the self-taken sample was close to the manufacturer-set cutoff, indicating small amounts of viral input material. The detected HR-HPV prevalences, overall and genotype specific, were lower than those in a routinely screened population from the same uptake area, although among the infected women, the proportion with abnormal cytology was higher.Both the CSi and the Horizon study were population-based studies undertaken in a routine screening laboratory. Our laboratory is the sole provider of cervical screening for this part of Denmark; it has a long-standing cervical screening program with a ∼75% coverage rate . Hence, A limitation of the comparisons between the HC2, Onclarity, and CLART assays in the CSi may be that BD's Cervical Brush Diluent (CBD) medium, which was used to resuspend the self-sampling brush heads prior to analysis, has not been used for self-sampling previously, although it has been used extensively on physician-taken screening samples . The CLAThis is one of the first studies comparing different HPV assays in terms of their ability to detect HR-HPV in self-taken samples. Besides, this is an unsupervised self-sampling setting, which is the most relevant setting for a routine rollout of this service to screening nonattenders.In line with previously published experience on physician-taken samples , 22, we 4–For screening purposes, the relevant endpoint is histological confirmation of precancerous high-grade cervical lesions especially; this is beyond the scope of the current analysis and will be reported in detail separately.TC values) for real-time PCR and a higher proportion of samples above the cutoff for HPV positivity. The TC values on positive self-taken samples of women whose physician-taken samples were also HPV positive tended to be lower than those for women whose physician-taken samples were HPV negative. Finally, stronger signals were found when all three assays confirmed the HPV-positive test result than when only one assay detected the infection. In line with our research on routinely screened women from the Horizon study, this could also suggest that women whose HPV infections had higher signal strengths may have been more likely to harbor high-grade CIN  or every 5 years (at the ages of 50 to 65 years). The design of the CSi study has been explained elsewhere . BrieflyThe Horizon study was undertaken in the same screening laboratory in 2011 1416, 23–2823–All returned brushes were preprocessed and were analyzed for HR-HPV. Brush heads were removed from the holders and were placed in empty 5-ml Eppendorf tubes . Three milliliters of BD's Cervical Brush Diluent (CBD) was added. Brush heads were removed after 15 min, and samples were vortexed to achieve homogeneous mixtures. Each sample was aliquoted into four empty tubes using the following schedule: 450 μl was transferred to an Eppendorf tube for DNA extraction and CLART analysis, 1,000 μl to a tube for Onclarity testing, and 500 μl to a tube for HC2 analysis , while all residual material was stored at −20°C for backup.For the CLART assay, two 200-μl aliquots were transferred directly to a MagNA Pure 96 system  for DNA purification. The two 100-μl purified DNA outputs were then pooled. The assay provided full genotyping of 35 HPV types, without quantitative measurement of signal strengths. For clinical management, HR-HPV types were defined as HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -68, corresponding to the latest classification of genotypes as carcinogenic (group 1) or probably carcinogenic (group 2A) by the International Agency for Research on Cancer (IARC) . Low-risTC) cutoff for HPV positivity is 34.2.For the Onclarity assay, the 1,000-μl aliquot was prewarmed for cell lysis. The sample was loaded into the Viper Lt real-time PCR system . The Onclarity assay detects six HR-HPV genotypes individually , whereas another seven HR-HPV genotypes and one low-risk HPV genotype are detected in three combinations . The standard cycle threshold , and the results were read automatically on a luminometer. The threshold for positive test results was 1 relative light unit (RLU)/cutoff (CO). Due to budgetary restrictions, HPV testing by the HC2 assay was completed only on the first 1,008 consecutive self-samples.Women's screening histories since January 2000 were retrieved from the nationwide Pathology Database (Patobank), which has been virtually complete for the entire country since the mid-2000s.The invitations for self-sampling were sent between May 2014 and April 2015. For the current analysis, we retrieved the screening activities until November 2015, which allowed for delays in returning the brush , the labWomen were categorized as “intermittently screened” if they had a cytology sample within the last 10 years before the self-sampling invitation and as “long-term unscreened” if they had no sample for ≥10 years. The analyses using this categorization were restricted to women ≥34 years old, i.e., those who had been eligible for screening for ≥10 years.TC values and adjustment for age (data not shown). Hence, we included all test results in the analyses.Due to hardware failure and technical issues with the Viper Lt system, some of the samples were stored at −20°C before testing with the Onclarity assay. No significant differences were seen when HPV prevalences determined for frozen and fresh samples were compared, including a comparison of the respective As in the Horizon study , positivTC values) of the Onclarity assay. The TC medians were stratified according to the median follow-up time (27 days) for women who went for follow-up.For the comparison of positive HPV test results in self-taken samples and physician-taken follow-up samples, we calculated medians and interquartile ranges (IQR) for the signal strengths  for proportions and for prevalence agreement were calculated on the assumption that they were binomially distributed, whereas the 95% CIs for mean differences in Stata SE 13.1 and Microsoft Excel 2010 were used for data analyses.The CSi was a time-limited pilot implementation initiative mandated by the Danish Health Authority. The Horizon study was undertaken as a quality development study in accordance with the Committee Act under The National Committee on Health Research Ethics . In both cases, therefore, formal ethical approval was not required. Linkage to the Patobank was approved by the Danish Data Protection Agency under notification numbers AHH-2015-084-04139 and AHH-2015-080-04109 for the CSi and Horizon studies, respectively.