In France, there are recommendations and reimbursements for human papillomavirus (HPV) vaccination but no HPV vaccination programs. Therefore, vaccination is largely determined by parents’ initiative, which can lead to inequalities. The objective of this study was to determine the factors associated with poorer vaccination coverage rates in the most populated region of France.The data of this study were obtained from the National Health Insurance between 2011 and 2013. Correlations between vaccination initiation rate (at least 1 dose reimbursed) and socio-demographic/cultural factors were assessed using Pearson’s product-moment correlation coefficient. Multivariate analyses were performed using logistic regression.In total, 121,636 girls received at least one HPV vaccine dose. The vaccination rate for girls born from 1996 to 1999 was 18.7%. Disparities in vaccination coverage rates were observed between the 8 departments of the region, ranging from 12.9% to 22.6%. At the department level, unemployment, proportion of immigrants and foreigners, and coverage by CMU health insurance  were significantly inversely correlated with vaccination rates, whereas urban residence, medical density, income and use of medical services were not related to coverage. In the multivariate model, only the percentage of foreigners remained independently associated with lower vaccination coverage. At the individual level, the use of medical services was a strong driver of HPV vaccination initiation.We observed geographic disparities in HPV vaccination initiation coverage. Even if no clear factor was identified as a vaccination determinant, we observed a failure of vaccination only based on parents’ initiative. Therefore, an organized policy on HPV vaccination, such as school-based programs, can help improve coverage rates. Clinical trials have shown that prophylactic vaccination against human papillomavirus (HPV) prevents infection with HPV types 16 and 18  as well This study was approved and authorized by the French National Committee . No consent was given because the data were analyzed anonymously .This retrospective study was conducted with the French National Health Insurance Database . This national database includes reimbursements for the four main insurance that cover nearly 95% of the French population.In France, Cervarix and Gardasil are reimbursed by the National Health Insurance. Therefore, all administered doses are registered in the database. The reimbursement rate of HPV vaccines is 65% of their price (average market price: 109.60€ for Cervarix and 123€ for Gardasil).The data extracted from SNIRAM included information on the beneficiaries , the vaccine, if prescribed , and prescriber specialty.Data on the demographic characteristics of healthcare professionals according to their specialization and the region in which they practiced were obtained from the French National Medical Council . Land-usWe focused our study on the most populated region of France: Ile-de-France. In 2015, 1,496,933 girls aged 10 to 19 lived in this region. This region consists of 8 departments (Paris (75), Seine-et-Marne (77), Yvelines (78), Essone (91), Hauts-de-Seine (92), Seine-St-Denis (93), Val-de-Marne (94), and Val-d’Oise (95)) and 1300 cities, including the French capital of Paris. The departments of this region also exhibit important socioeconomic disparities. Therefore, Ile-de-France provided a good model to study the factors and barriers associated with HPV vaccination. All the factors previously described were obtained from the National Health Insurance for all women living in this region between January 2011 and December 2013.During the study period, the French guidelines recommended a 3-dose vaccine regimen to be administered to 11- to 14-year-old girls, with a catch-up vaccination until 19 years of age . There wThe vaccination rates (at least one dose reimbursed) were provided for girls born in 1996, 1997, 1998 and 1999, corresponding to the main target population (14 years old) for vaccination during the study period and the most relevant cohorts to compare vaccination rates between areas in the region. To avoid a loss of information regarding the doses received before or after the study period, we considered only patients  whose first dose was reimbursed after January 2012. The use of medical services  was assessed for girls born in 1999 (vaccinated or not), who constituted the most representative cohort of the target population (aged 14 years old in 2003) in our study. Comparisons of the patient’s characteristics and HPV vaccine series initiation between the 8 departments were performed with Pearson’s chi-square test, 2-tailed Fisher’s exact test or Student’s t-test. To understand the disparities of HPV vaccination (initiation) coverage between the 8 departments, we investigated the associations between the departments’ vaccination rates and several socioeconomic and cultural factors. Correlations were assessed using Pearson’s product-moment correlation coefficient. Multivariate analyses were performed using a logistic regression model. Differences were considered significant at p<0.05. All data analyses were performed using R software .From January 2011 to December 2013, 121,636 girls received at least one dose of HPV vaccine in the region of Ile-de-France. A total of 235,573 doses of vaccine were reimbursed. A total of 66,559 girls aged 14 years old were reimbursed for at least one dose between 2011 and 2013, yielding a HPV vaccine series initiation rate of 18.7%.The majority of the girls were vaccinated (initiation) at 14 years old, as recommended in France. In September 2012, the French authorities published new recommendations, with a target population aged from 11 to 14 years old. The age distribution changed in the studied population before and after the releases of the new age target recommendations . SeasonaOnly 49% of the girls who initiated the series completed the vaccination scheme with 3 reimbursed doses. The median delay between doses 1 and 2 was 75 days (range: 1–1063) and was 127 days between doses 2 and 3 (range: 1–1013). General practitioners (GP) were the main prescribers of HPV vaccines: 81% of the reimbursed doses (initiation as well as completion) were prescribed by GPs. We then explored the HPV vaccine series initiation rates of departments based on social welfare coverage. More than 95% of the French population have medical coverage, mostly through employment. Among the social welfare coverages, the Couverture Maladie Universelle (CMU) is a health insurance plan for those who are not otherwise covered through business or employment and who have a low income. With 7.3% of vaccinated girls covered by the CMU, we did not find any statistically significant differences (p = 0.98) between the rate of CMU recipients in the general population and the rate of CMU recipients in the vaccinated population (at least one dose reimbursed). However, when we explored the number of doses received by CMU recipients, we found that only 27% of the vaccinated girls completed the full vaccination scheme (3 doses), which was significantly lower than the rate in the non-CMU recipient population  . For thoFinally, we evaluated the global use of medical services of vaccinated and non-vaccinated girls in our study. Vaccinated girls (at least 1 dose) who were born in 1999 were reimbursed for a median of 48 pharmaceutical and medical procedures between 2011 and 2013. In contrast, non-vaccinated girls born in 1999 consumed a median of 29 procedures during the same period. We did not observe any differences in terms of medical consumption between departments .Disparities were observed between the 8 departments of the region in HPV vaccine series initiation coverage rates , which rWe first examined the geographic access to care in the 8 departments. We chose the medical density of GPs, as they were the main prescriber of HPV vaccinations. Although disparities did exist in medical demography in the Ile-de-France region, they were not associated with the differences observed in HPV vaccine series initiation rates  to 31,040€ in Hauts-de-Seine (department 92) in 2012. Despite these important differences between the departments, no association between income and vaccination rates (initiation) was found . Unemployment rate , immigration rate  and CMU recipient rate  were not found to have an independent impact on vaccination coverage (initiation).Our study included a large number of women in an unselected population. These data were based on the National Health Insurance reimbursements and reflect the real-life situation regarding HPV vaccination in the most populated region of France, assessing more than 6 million women. The comprehensiveness and objectivity of the data–not based on survey or questionnaires–provide rigorous and powerful results.We observed geographic disparities in this region in terms of vaccination initiation rates. In the United States (US), the National Health Interview Survey (NHIS), an annual, nationally representative, cross-sectional, multipurpose health survey, also revealed spatial variations in the HPV vaccination coverage, with higher rates in the West and North Central/Midwest of the US . The vacOur findings did not indicate lower vaccination initiation among populations with a high unemployment rate in multivariate analysis and therefore differed from those of previous studies, such as those of a large Danish cohort of vaccinated girls  born in 1996 and 1997 . FurtherWe found few socioeconomic factors influencing the vaccination coverage in the univariate analysis, and none of the studied factors were independently associated with vaccination rates in the multivariate analysis. Therefore, the disparities between departments must have multifactorial and complex drivers. Through these analyses, we observed that vaccination rates were inversely correlated with the prevalence of immigrants and foreigners in the 8 departments, in both the univariate and multivariate analyses for foreigners. However, the heterogeneity of the immigrant and foreign populations limits the understanding of the determinants of vaccination. In addition, we did not have access to data on ethnicity or religion. Indeed, ethnic studies in France are forbidden by law, unless special authorization is granted . The NatFurthermore, even in departments with higher vaccination rates in France, the HPV vaccination coverage was still very low. At the end of 2014, 17% of girls aged 16 had received 3 doses . This coThe negative publicity regarding HPV vaccine and the cultural origins of the disparities in vaccinations rates highlighted again the importance of providing clear information to patients. The literature shows that some ethnicities are correlated with having less knowledge and information on HPV and vaccination, such as Hispanic girls in the US , 30 or CGeographic disparities exist in HPV vaccination initiation coverage. Socioeconomic status could not explain these variations in our study. In France, the health care system and insurance provide good access to care, decreasing the socioeconomic inequalities. Therefore, more complex associations between both socioeconomics and probably cultural factors could explain the HPV vaccination coverage in our studied region. This underlines the need and importance of an appropriately informed population. Physicians, and especially general practitioners, have a pivotal role in providing the needed information, as they have a major influence on women’s vaccination choices. Even if no clear factor was identified as a vaccination determinant, we observed a failure of vaccination only based on parents’ initiative. To effectively reduce inequalities and improve vaccination coverage, an organized policy seems to be the most efficient option, such as school-based programs.


Slc4a11+/+ and Slc4a11−/− mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy.To establish conditionally immortal mouse corneal endothelial cell lines with genetically matched H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with Slc4a11+/+ and Slc4a11−/− C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time. Ion transport activities  were analyzed by intracellular pH measurement. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS).We intercrossed Slc4a11+/+ and −/−Slc4a11 mouse corneal endothelial cells (MCECs) remained proliferative through passage 49 and maintained similar active ion transport activity. As expected, proliferation was temperature sensitive and IFN-γ dependent. −/−Slc4a11 MCECs exhibited decreased proliferative capacity, reduced NH3:H+ transport, altered expression of glutaminolysis enzymes similar to the −/−Slc4a11 mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with Slc4a11+/+ MCECs.The immortalized 3:H+ transporter activity in Slc4a11−/− MCECs. Furthermore, −/−Slc4a11 MCECs recapitulate the glutaminolysis defects observed in −/−Slc4a11 mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents.This is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH SLC4A11 mutations are associated with congenital hereditary endothelial corneal dystrophy (CHED), Fuchs' endothelial corneal dystrophy, Harboyan syndrome (CHED plus perceptive deafness), and Peters anomaly.14 Up to 80 distinct mutations in 17 of the 19 exons of SLC4A11 have been identified in individuals with CHED,517 which is characterized at or soon after birth by bilateral diffuse corneal edema without other significant developmental abnormalities of the anterior segment.18 Histologically, the diffusely edematous corneal stroma accounts for the marked increase in corneal thickness observed clinically.19 In addition, CHED is associated with a uniform thickening of Descemet's membrane and vacuolization of corneal endothelium.20 The Slc4a11−/− C57BL/6 mouse recapitulates the human CHED-related defects, exhibiting a similar ground-glass diffuse corneal edema, increased corneal thickness, vacuolated corneal endothelial cells, and uniformly thickened Descemet's membrane.22 Furthermore, similar to human CHED,23 endothelial cell density in −/−Slc4a11 C57BL/6 mice is not significantly affected at early age.22 Based on these observations, both Han et al.22 and the authors of this study concluded that this Slc4a11−/− C57BL/6 mouse can serve as a good animal model for human CHED.+24–26 and can act as an NH3:H+ cotransporter.27 We recently demonstrated that this ammonia permeability is essential for human corneal endothelial cells, as these cells produce ammonia as a by-product of glutamine catabolism, which is required to maintain a high level of ATP production.21 Furthermore, we found that Slc4a11−/− mouse corneal endothelium has disrupted expression of enzymes involved in glutamine metabolism.21 However, only a very limited amount of corneal endothelial material can be obtained from mice, constraining further cellular, molecular, and metabolic studies.Recent functional studies of SLC4A11 indicate that it is permeable to H28 When cultured in vitro, corneal endothelial cells manifest limited passaging ability with rapid senescence and epithelial-to-mesenchymal (EMT) transition.29 This limited proliferative potential makes obtaining sufficient corneal endothelial sample a significant challenge for studying the molecular mechanisms that underlie corneal physiology and the pathophysiology in corneal endothelial diseases.Of neural crest origin, differentiated corneal endothelial cells are arrested in the G1 phase of the cell cycle, and studies in both mice and humans have revealed that these cells have very low proliferative potential in vivo.Slc4a11+/+ and Slc4a11−/− C57BL/6 mice22 with H-2Kb-tsA58 Immortomouse, which carries a temperature-sensitive mutant form of the simian virus-40 large T antigen.30 We used the resulting progeny to generate two conditionally immortal, genetically matched mouse corneal endothelial cell lines: Slc4a11+/+ mouse corneal endothelial cells (Slc4a11+/+ MCEC) and Slc4a11−/− mouse corneal endothelial cells (Slc4a11−/− MCEC). Here we report the successful establishment of these two genetically matched wild-type and Slc4a11 knockout MCECs. We examined proliferative properties, transport activity, and Slc4a11-related glutaminolysis activity. The resultant cells retain key endothelial transport function and the Slc4a11-deficient cells show altered glutamine metabolism consistent with what was observed in Slc4a11−/− mouse corneal endothelium.21 Overall, the generation of these cell lines establishes a valuable tool for studying aspects of corneal endothelial diseases that require a large number of cells.To overcome these limitations, we intercrossed the All mice were housed and maintained in specific pathogen-free conditions and used in the experiments in accordance with institutional guidelines and the current regulations of the National Institutes of Health, the US Department of Health and Human Services, the US Department of Agriculture, and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.H-2Kb-tsA58 transgenic Immortomouse  was intercrossed with Slc4a11+/+ and Slc4a11−/− C57BL/6 mice, respectively, to generate offspring bearing the Immortomouse transgene together with Slc4a11+/+ or Slc4a11−/−. Slc4a11−/− C57BL/6 mice have a targeted deletion of exons 9 to 13 of the murine Slc4a11 gene.31 The offspring were genotyped by PCR of genomic DNA from ear-punch sections following protocols using QIAamp DNA Mini Kit . Oligomer sequences (5′–3′) used for PCR were as follows: primer 1 (Immorto-F), AGT CCT CAC AGT CTG TTC ATG ATC; primer 2 (null-F), GAT CTG CCT GAG GTG TTA CTT G; primer 3 , GGA TGG CAT CAC TAG TCA TGA C; primer 4 (Slc4a11 wt-F), TCT GGA CTT CAA CGC CTT CT; primer 5 (Slc4a11 ko-F), GCC AAG GTA TGG AGA ACA CC; primer 6 , GCA CAA ACG TGA TGG AAA TG. Primers 1 and 3 were used to identify the SV40 transgene, Immorto (tsTAg). Primers 2 and 3 were used to identify the null allele. Primers 4 and 6 were used to identify the Slc4a11 wild-type allele, and primers 5 and 6 were used to identify Slc4a11 knockout allele. The first generations were bred, and the second-generation pups were genotyped by PCR to identify +/+Slc4a11 and −/−Slc4a11 animals that carried at least one copy of the SV40 tsTAg transgene to be used for tissue isolation.The MCECs were prepared from 12-week-old mice. Briefly, the globes were aseptically enucleated followed by cornea dissection and corneal endothelium peeling. Then corneal endothelial sheets with attached Descemet's membrane were placed in OptiMEM-I medium  supplemented with 8% heat-inactivated fetal bovine serum (FBS) , EGF 5 ng/mL , pituitary extract 100 μg/mL , calcium chloride 200 mg/L, 0.08% chondroitin sulfate , gentamicin 50 μg/mL , antibiotic/antimycotic solution diluted 1:100  and 44 units/mL IFN-γ . IFN-γ was used to stimulate the MHC promoter for tsTAg expression. Cells were further selected based on morphology via single-cell cloning for colonies with corneal endothelial hexagonal shape and contact inhibition. Cells were incubated at 33°C with 5% carbon dioxide.Slc4a11+/+ MCEC and Slc4a11−/− MCEC, were genotyped by PCR following protocols using QIAamp DNA Mini Kit (Qiagen). Primer sequences used were the same as the primers used for mouse genotyping listed above.The two lines, 2) or 37°C (5% CO2) in OptiMEM-I medium  supplemented with 8% heat-inactivated FBS , EGF 5 ng/mL , pituitary extract 100 μg/mL (Hyclone Laboratories), calcium chloride 200 mg/L, 0.08% chondroitin sulfate , gentamicin 50 μg/mL , antibiotic/antimycotic solution diluted 1:100  and with or without 44 units/mL IFN-γ . Culture at 33°C with IFN-γ is defined as permissive growth condition, whereas culture at 37°C without IFN-γ is defined as nonpermissive growth condition.MCECs were cultured at 33°C  attached to an inverted phase-contrast microscope.3 or 1 × 104/mL  in 24-well plates. Cells in four wells of 24-well plates were trypsinized to suspension and counted with a Cellometer Auto T4 . Doubling time was calculated using GraphPad Prism 6.1c .MCEC cells were seeded at 5 × 104/mL  in six 35-mm dishes and cultured with IFN-γ at 33°C to subconfluence. While one dish was subject to protein extraction as the sample for the permissive condition, the remaining five dishes were switched to 37°C without IFN-γ for the subsequent nonpermissive culture. In the following 5 days, one dish was subjected to protein extraction on each day  in the nonpermissive condition. Cells in each dish were washed two times with ice-cold PBS and suspended in 100 μL RIPA buffer  with Complete Protease Inhibitor Cocktail . Then the sample was sonicated and centrifuged for 20 minutes at 12000g at 4°C. Cell lysate samples (15 μL) were mixed with Protein Loading Buffer Blue (2X)  and a total volume of 30 μL was resolved on 1.5-mm-thick 10% SDS-polyacrylamide gels and wet-transferred to polyvinylidene difluoride membranes . Membranes were blocked with 5% nonfat milk in TBST  and probed with primary antibodies in the same buffer overnight at 4°C. The following primary antibodies were used: rabbit anti-SV40 T Ag 1:5000 ; and anti–glyceraldehyde 3-phosphate dehydrogenase antibody 1:1000 . Next, membranes were probed with secondary antibody  for an hour at room temperature. Bound secondary antibodies were detected using an enhanced chemiluminescence assay . Band densities with background subtraction were quantified using ImageJ .MCECs were seeded at 5 × 10i measurements were performed as described previously.32 Briefly, MCECs were cultured on poly-L-lysine and fibronectin precoated 25-mm diameter glass coverslips  for 2 to 3 days in semipermissive condition. Before each experiment, cells were incubated with 10 μM of pH-sensitive fluorescent dye BCECF-AM (2′7′-bis(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester, B1170; Thermo Fisher Scientific) in Ringer's solution for 30 minutes at room temperature, and washed in dye-free Ringer's solution for another 30 minutes. The Ringer's solution constitution is listed in 2 for bicarbonate-rich [BR] solutions) and adjusted to pH 7.5 with 1N NaOH at 37°C. Osmolarity of all solutions was adjusted to 295 to 300 mOsm with mannitol. Coverslips with subconfluent cells were mounted into a perfusion chamber, and the chamber was then placed on a stage warmer (37°C) of an inverted microscope . Solutions were kept at 37°C in a warming box, and the flow of the perfusate (∼0.5 mL/min) was achieved by gravity. Cells were imaged with an oil-immersion objective . BCECF fluorescence was excited alternately at 495 ± 10 nm and 440 ± 10 nm, and the emitted light was collected through a bandpass filter (520–550 nm). Fluorescence ratios (495/440) were obtained at 1 Hz, and converted into pHi using the high K+/nigericin calibration approach.33pH−ΔΔCt experimental design was used for relative quantification and normalized to mouse ACTB (β-actin) for differential expression levels of target genes.Total RNA from the MCEC cell lines  was extracted and purified using the RNeasy mini kit  with DNase digestion . Complementary DNA was generated with a High Capacity RNA-to-cDNA Kit  at 10 ng RNA/μL reverse transcription reaction concentration. Real-time quantitative PCR reactions were set up in triplicate using PowerUp SYBR Green Master Mix . Reactions were performed with murine gene primers listed in Slc4a11+/+ and Slc4a11−/− MCEC in 8% FBS OptiMEM-I in T-75 flasks seeded at 5 × 104/mL  were grown to confluence for 4 days at 33°C with IFN-γ then switched to serum-free OptiMEM-I supplemented with 200 mg/L CaCl2 and 0.08% chondroitin sulfate for 12 hours. Then the cells were incubated with serum-free Dulbecco's modified Eagle's medium (DMEM) conditional medium with 2.5 g/L U-13C6-D-glucose  + 4 mM L-glutamine and incubated for 12 hours. Serum-free DMEM conditional medium was made using DMEM  supplemented with CaCl2 200 mg/L, 0.08% chondroitin sulfate (Sigma-Aldrich Corp.), and the isotope-labeled glucose and nonlabeled glutamine (Sigma-Aldrich Corp.). After 12-hour incubation, isotope-labeled cells were washed three times with ice-cold 0.9% NaCl, quenched with 2 mL ice-cold 100% methanol, scraped off the plate and removed together with 2 mL quenching methanol into centrifuge tubes, vortexed thoroughly, and centrifuged at 8000g for 2 minutes. Supernatants were collected and the cell pellet was resuspended with 900 μL 90% methanol twice following vortex and centrifugation. Total supernatant was kept stationary at −20°C for 1 hour, then centrifuged at 15,000g for 5 minutes at 4°C. The supernatant was collected to new tubes and dried in a centrifugal evaporator at room temperature overnight. The level of each metabolite isotopologue was measured using GC-MS as reported previously.34 The fraction of each isotopologue and the contribution of 13C to the total carbon pool of each metabolite was calculated as previously reported.35 Briefly, the isotopologue distributions were corrected based on the natural abundance of elements, and the fraction is the contribution of each isotopologue to the total abundance of all the isotopologues. The contribution of 13C to the total carbon pool of each metabolite is the weighted average of all the labeled isotopologues according to the fraction distribution.t-test or paired t-test were used for two-group comparison. Two-way ANOVA was used for metabolite data.Statistical analysis was carried out using GraphPad Prime 6.0c . Student's Slc4a11+/+ MCECs, and Slc4a11−/− MCECs, were determined by PCR analysis of mouse or cell line genomic DNA. For the mice used, Slc4a11 wild-type alleles are present in Slc4a11+/+ mice (left two lanes) and in Slc4a11+/− mice (right two lanes), whereas Slc4a11 knockout alleles are present in Slc4a11−/− mice (middle two lanes) and in Slc4a11+/− mice. For Immorto (tsTAg) gene, all of the mice we used carry only one allele of the gene, and the other allele is detected as a null allele. We selected Slc4a11+/+Immorto+/− mice and Slc4a11−/−Immorto+/− mice for cell expansion. Genotypes of derived cell lines were confirmed by PCR of the cell line genomic DNA for Slc4a11 and Immorto (tsTAg) genes , in which the cells are elongated and lacking contact inhibition at high density. However, Slc4a11−/− MCECs had a more hexagonal shape closely resembling corneal endothelial primary culture and form a monolayer when confluent in the same IFN-γ (+) medium (Slc4a11+/+ MCECs. We define this 33°C IFN-γ (−) culture condition as “semipermissive.” Indeed, in the semipermissive condition, Slc4a11+/+ MCECs presented with a more hexagonal shape and cell monolayer was formed once confluent (Slc4a11−/− MCEC morphology in semipermissive culture was similar to that in permissive culture (P = 0.013) in the doubling time between Slc4a11+/+ MCECs (0.78 day) and Slc4a11−/− MCECs (0.79 day) in the permissive condition, Slc4a11−/− MCECs (doubling time: 0.93 day) grow significantly slower than Slc4a11+/+ MCECs (0.76 day) in the semipermissive condition (P < 0.0001)  medium C. We askonfluent C. In con culture C. In ter 0.0001) D.Immorto (tsTAg) gene. Given the dynamic SV40 large T antigen expression changes expected from IFN-γ withdrawal and change in culture temperature, we next analyzed the growth behavior of the MCECs under permissive  and subsequent nonpermissive conditions . In nonpermissive culture, in addition to the lack of IFN-γ stimulation on the HMC promoter, a shift to the 37°C incubation temperature induces degradation of the tsTAg mutant protein. As a result, MCEC proliferation gradually stopped once the culture was removed from the permissive condition into the nonpermissive condition , we chose to test the ion transporter properties of these cells in the semipermissive state.Removal of IFN-γ reduces stimulation of the HMC promoter constructed upstream of the ondition A. In addondition B. Both S T level A, 2B. ToSlc4a11+/+ and Slc4a11−/− MCECs revealed that these cells express mRNA encoding the essential transporters Na+-K+-ATPase, Na+/H+ exchanger , Na+-HCO3− cotransporter , H+-Lactate− cotransporters  (i monitoring of apparent proton fluxes indicate the activity of bicarbonate transporters (+/H+ exchangers (Slc4a11+/+ and Slc4a11−/− MCECs (see below).To verify that these MCECs still carry significant ion transport activities, we next examined the mRNA expression and transport activity of selected ion transporters. RT-PCR analysis of orter 4) A. Similasporters B, Na+/H+changers C, and lachangers D are pre3−] 28.5 mM) Ringer's. Due to simple diffusion of dissolved CO2 inward across the plasma membrane, the cell rapidly acidifies as CO2 reacts with intracellular H2O to form H2CO3 and further release one H+ as a weak acid. Then pHi slowly rises, which indicates the Na-HCO3− cotransporters are moving weak base HCO3− inward using Na+ inward transmembrane electrochemical gradient. We determined the initial slope of this pHi rise as an indirect measure of the apparent Na+-HCO3− cotransport activity. This assay revealed no significant difference between Slc4a11+/+ and Slc4a11−/− MCECs in apparent bicarbonate transporter activity . The overshoot after CO2/HCO3− removal is an additional indication that there was HCO3− accumulation inside the cell during BR perfusion.Sodium Bicarbonate Cotransporter: In +/H+ exchanger: In i recovers due to the activity of Na+/H+ exchangers and Slc4a11 proton permeability.25 Here, we measured the initial slope of this pHi recovery as an indirect measure of the apparent Na+/H+ exchanger activity, revealing that there is a borderline significant difference between Slc4a11+/+ and Slc4a11−/− MCECs  that can be attributed to Slc4a11 proton permeability.25Na+-lactate− cotransporter: Because we recently demonstrated that lactate transport is a significant component of the corneal endothelial pump,36 we next examined the lactate-dependent H+ fluxes in MCECs. Cells were initially perfused with BR Ringer's, and where indicated , 20 mM lactate  was applied twice for 60 seconds ,37 in which the lactate transmembrane inward gradient favors the inward movement of lactate and H+ together causing intracellular H+ accumulation. The acidification is less prominent in BR than in BF Ringer's because HCO3−/CO32− offers extra H+ buffering power. We measured the initial slope of the induced acidification on lactate perfusion as an indirect measure of the apparent H+-lactate cotransporter activity. Slc4a11+/+ and Slc4a11−/− MCECs in both BF and BR Ringer's .H3:H+ fluxes,27 and this result has been verified by two other independent groups.26 Furthermore, a recent report from a third group shows murine Slc4a11 mediates similar electrogenic transmembrane NH3:H+ fluxes.24 So we next set out to determine if the Slc4a11-mediated NH3:H+ flux is impaired in Slc4a11−/− MCECs  on NH4Cl application. Eventually the cell will reach an equilibrium where intracellular [NH3] equals extracellular [NH3]. Then a slow acidification occurs in the midphase (120–400 seconds) of the NH4Cl pulse, indicating there is an additional weak acid NH4+  flux entering the cell.38 In this case, Slc4a11 activity brings more NH3:H+ into the cell. Slc4a11+/+ MCECs compared with Slc4a11−/− MCECs, consistent with the additional NH3:H+ influx provided by Slc4a11. Then, when the NH4Cl was washed away by BF Ringer's, there is a pronounced and rapid acidification on NH4Cl removal (400–440 seconds)  that has entered the cell during the NH4Cl pulse.38 And even though NH4Cl was removed in this phase on the outside, there is NH4+ temporarily trapped intracellularly.38 The amount of NH4+ trapped is directly correlated with the extent of the acidification according to the Henderson-Hasselbalch equation.38Slc4a11+/+ MCECs. The pHi recovery (440–520 seconds) from this acid load is a phenomenon of the collective effect from Slc4a11-mediated NH3:H+ efflux and Na+/H+ exchanger-mediated H+ extrusion. We measured the initial rate of pHi recovery as a measure of apparent NH3:H+ efflux primarily attributed to Slc4a11 activity. We observed a rapid recovery in Slc4a11+/+ MCECs, but significantly slower recovery in Slc4a11−/− MCECs  between Slc4a11+/+ and Slc4a11−/− MCECs was used given there was no statistical significance between the two cell lines. 3:H+ efflux is 0.0044 ± 0.0015/s (n = 5) in Slc4a11+/+ MCECs, and significantly smaller 0.0005 ± 0.0003/s  in Slc4a11−/− MCECs.We recently reported that human SLC4A11 mediates electrogenic transmembrane NH/− MCECs A. Cells seconds) B. This i/− MCECs B. To morrecovery C, to obtSlc4a11−/− mouse corneal endothelium,21 although the kidney-type glutaminase 1 (Gls1) is upregulated 3-fold in Slc4a11−/− mouse corneal endothelium, the liver-type glutaminase 2 (Gls2) can no longer be detected in these cells. Therefore, we set out to determine if the Slc4a11−/− MCEC recapitulates this expression difference and manifests as functional differences in glutamine metabolism. Consistent with the observation in mouse corneal endothelial tissue in vivo, we observed an upregulation of Gls1, and a downregulation of Gls2 mRNA expression in Slc4a11−/− MCECs , fumarate, and malate carbon chains were labeled with U-13C-glucose-sourced carbon in Slc4a11+/+ MCECs in the presence of unlabeled glutamine , but also allows for the production of genetically matched cell lines directly from a transgenic mouse model: Slc4a11+/+ MCECs and Slc4a11−/− MCECs. The conditional immortalization approach provided another advantage that the expression of SV40 large T antigen can be eliminated by simple temperature maneuver and/or removal of IFN-γ. We found that the SV40 large T antigen declined and could be virtually eliminated (Here we report the generation of the first immortalized MCECs. The use of iminated B in the +/H+ exchanger, and lactate transport activities were not significantly changed by Slc4a11 knockout (Slc4a11−/− MCECs showed significantly less NH3:H+ flux relative to Slc4a11+/+ MCECs (3:H+ permeability provided by SLC4A11.27Analysis of ion transporter activity in the two lines of MCECs show that apparent bicarbonate transport, Naknockout . As expe/+ MCECs , consist3:H+ transport in Slc4a11−/− MCECs is likely the cause of observed changes in glutamine metabolism. Ammonia was reported to inhibit both the N-ethylmaleimide-sensitive and -insensitive fraction of glutaminase.40 In Slc4a11−/− MCECs, expression changes of glutaminolysis enzymes is consistent with our observation in Slc4a11−/− CHED mouse corneal endothelium tissue.21 Further analysis of TCA cycle intermediates in Slc4a11+/+ MCECs indicated that 50% of citrate and approximately 35% of other TCA cycle intermediates were derived from glucose, which is similar to what was found in an immortalized human corneal endothelial cell line.21 However, in Slc4a11−/− MCECs, we found a significantly increased fraction from glucose-sourced carbon, indicating reduced fraction from glutamine (39 this observed reduced flux of glutaminolysis suggests that the increased expression of Gls1 found in the Slc4a11−/− MCECs (The impaired ability to facilitate NHlutamine C, provid/− MCECs A may be SLC4A11 and murine Slc4a11 genes were reported to be a target of p53,41 a main cell metabolism regulator.42 Conversely, knock-down of Slc4a11 in bovine nucleus pulposus cells inhibits p53 activity by abolishing p53 phosphorylation.43 This suggests that the SLC4A11 transporter could indirectly regulate glutaminolysis enzyme expression via p53 phosphorylation, which is consistent with the finding that glutaminase GLS2 is a target of p53.44Interestingly, both human Slc4a11+/+ MCECs when tsTAg was induced by IFN-γ (45 it will be of interest to study if the SLC4A11 transporter plays a role in corneal endothelial cell proliferation, polarity formation, and EMT transformation.Additionally, we observed fibroblast-like morphology changes only in by IFN-γ C. Given +/+Slc4a11 MCECs and −/−Slc4a11 MCECs. These cells remain proliferative with reasonable endothelial morphology after prolonged culture, and present key active ion transport activities as expected from corneal endothelial cells. Slc4a11−/− MCECs recapitulate glutaminolysis enzyme expression changes as seen in Slc4a11−/− mouse corneal endothelial tissue. These cell lines allowed us to carry out further investigations using cellular-based approaches requiring a significantly larger sample volume than mouse tissue can achieve. Further analysis of TCA cycle intermediates suggests functionally impaired glutaminolysis in Slc4a11−/− MCECs. These Slc4a11+/+ MCEC and Slc4a11−/− MCEC cell lines provide an excellent tool for future cell-based experiments to study the pathophysiological changes resulting from loss of Slc4a11 function and for potential therapeutic pharmaceutical reagent screening.In summary, we successfully established two genetically matched conditionally immortal MCECs:


No study has estimated the potential impact of Human Papillomavirus (HPV) vaccination in Puerto Rico, a population with considerable burden of HPV-related morbidities. We evaluated the health and economic impacts of implementing a vaccination strategy for females and males in Puerto Rico, with the quadrivalent HPV (HPV4) vaccine, under different vaccination scenarios.We adapted a mathematical model which estimates the direct and indirect health benefits and costs of HPV4 vaccination in a dynamic population. The model compared three vaccination scenarios against screening only (no-vaccination) for three doses of HPV4 vaccine among individuals aged 11–15 years in Puerto Rico: 1) 34% for females and 13% for males (34%F/13%M), 2) 50% for females and 40% for males (50%F/40%M), and 3) 80% for female and 64% for male (80%F/64%M). Data specific to Puerto Rico was used. When not available, values from the United States were used. Input data consisted of demographic, behavioral, epidemiological, screening, and economic parameters.The model predicted decreases in: 1) HPV infection prevalence for females and males, 2) cervical intraepithelial neoplasia and cervical cancer incidence for females, 3) genital warts incidence for females and males, and 4) cervical cancer deaths among females, when various vaccination program scenarios were considered. In addition, when the vaccination percentage was increased in every scenario, the reduction was greater and began earlier. The analysis also evidenced an incremental cost effectiveness ratio (ICER) of $1,964 per quality–adjusted life year gained for the 80%F/64%M uptake scenario.HPV vaccine can prove its cost effectiveness and substantially reduce the burden and costs associated to various HPV-related conditions when targeted to the adequate population together with an organized HPV vaccination program. Human Papillomavirus (HPV) is a DNA virus with the ability to infect epithelial cells or mucous membranes in humans . Over 10Worldwide, cervical cancer is the second leading cause of cancer death in women between 15 and 44 years of age . HPV is On the other hand, genital warts are the leading STI in the United States (US), particularly among young adults . As geniResearch in the US, Mexico, United Kingdom, Colombia, and Italy has documented the economic impact of HPV-related morbidities and the cost effectiveness that HPV vaccination could have in these populations –17. It iThe importance of HPV in the etiology of cervical cancer and other HPV-related morbidities and the needs for preventive measures gave rise to the development of HPV vaccines . At presIn spite of the existence and approval of HPV vaccines, vaccination rates are low. In Puerto Rico, data from the 2014 National Immunization Survey–Teenshow that HPV-vaccination coverage rates for the 3 dose-schedule were estimated at 49.9% of females and 23.7% of males ages 13–17; this estimate is higher than for the US, where 39.7% of females and 21.6% of males of the same ages have completed the vaccination regimen . NonetheData on the public health and economic consequences of HPV vaccination in Puerto Rico is of great relevance to guide public policy and other prevention and control efforts for HPV related disease. It is imperative to compare the costs and benefits of introducing a vaccination plan along with the current screening program versus screening only (no-vaccination) plan, taking into consideration different vaccination coverage rates. Therefore, the aim of this analysis was to adapt a previously published economic model to evaluate the health and economic impacts in Puerto Rico of implementing an HPV4 vaccination strategy for females and males, 11–15 years of age, under different vaccination scenarios compared to a no-vaccination strategy. In addition, to estimate the HPV4 vaccine potential effect on HPV associated diseases and cervical cancer mortality, and to evaluate HPV4 vaccination cost-effectiveness utilizing a variety of vaccination scenarios from Puerto Rico’s healthcare point of view. This is one of the few instances where the model has been adapted to demonstrate the potential impact of increasing vaccination rates on the prevention of HPV-related disease.A previously published transmission dynamic model –31 for HA literature review was performed to attain the model parameter values, which included demographic, sexual behavior and screening variables. Population estimates came from the U.S. Census Bureau  and scre34% for females and 13% for males (34%F/13%M)50% for females and 40% for males (50%F/40%M)80% for female and 64% for male (80%F/64%M)The model assumed the following vaccine degree of protection:Against transient infection for females (any time DNA detection): HPV16: 76.0%; HPV18: 96.3%; HPV6: 76.1%; HPV11: 76.1%Against persistent infection for females: HPV16: 98.8%; HPV18: 98.4%○CIN: HPV16: 97.9%; HPV18: 100%; HPV6: 100%; HPV11: 100%○Genital warts for females: HPV6: 98.9%; HPV11: 100%○Genital warts for males: HPV6: 84.3%; HPV11: 90.9%Against individual diseases:Furthermore, the model assumed a lifelong protection and that the subjects adhered to the full three-dose vaccination regimen.The estimated HPV 6/11-related incidences of genital warts among females and males over a 100 year timespan, grouped by vaccination strategy, are shown in Figs Meanwhile, In the 50%F/40%M vaccination scenario, the estimated total direct cost avoided would be nearly $38 million in 25 years and $71.4 million in 50 years. The estimated costs avoided for genital warts in females goes from almost $13 million in 25 years to $20.6 million in 50 years; whereas in males it would be $16.2 million in 25 years and $26.6 million in 50 years. For CIN1, the estimated costs avoided in this scenario would be $1.2 million in 25 years and $2.6 million in 50 years. The estimated costs avoided for CIN2/3 would be $6 million in 25 years and $16.3 million in 50 years. For cervical cancer we see an estimated cost decrease of $1.5 million in the first 25 years and $5.3 million in 50 years.Finally, under a broader vaccination scenario of 80%F/64%M, the estimated total direct costs avoided would be nearly $90 million in 25 years and $162 million in 50 years. The estimated cost avoided for genital warts in females goes from $31.7 million in 25 years to $51.2 million in 50 years; whereas in males it goes from $38 million in 25 years to $62.7 million in 50 years. The CIN1 estimated cost reduction under this vaccination scenario is $2.7 million in 25 years and $5.8 million in 50 years. The CIN2/3 analysis shows an estimated cost reduction of $13 million in 25 years and $32.3 million in 50 years. Direct costs attributable to cervical cancer would have an estimated decrease of $3.4 million in 25 years and $10.5 million in 50 years.Consistent with previously conducted HPV vaccination health economic analyses, our data suggests that an HPV4 vaccination program can result in important public health and economic benefits in reducing HPV-related diseases over screening only, in the absence of vaccination. Additionally, increasing the vaccination coverage in females and males would greatly decrease HPV-related morbidity and mortality, resulting in greater HPV-related disease total direct cost avoidance in Puerto Rico. Although increasing vaccination rates would be expected to result in better outcomes, the model adaptation provides the means to estimate the magnitude of disease avoidance across the different scenarios within the modelled timeframe, hence providing additional information for the need to increase vaccination rates in a timely fashion.As the coverage rate increases, faster reductions in HPV-related disease incidences and costs are observed. At the 80%F/64%M vaccination coverage, the model shows that the vaccine program is cost-effective under the established threshold of $50,000 per QALY. The ICER of $1,960 per QALY is also consistent with the WHO  thresholGiven that cervical cancers have a more prolonged natural history, the greatest reductions in disease incidence within the first 25 years of the introduction of HPV4 are estimated to derive from the prevention of HPV 6/11- related genital warts. Real-world evidence of the impact of gender-neutral HPV vaccination has shown that, although the focus in some countries has been towards cervical cancer prevention among females, in others, the burden of HPV-associated cancers in comparable in males and females. All men have a high burden of HPV-associated diseases, a problem that is increased in industrialized countries but that could be diminished if they were immunized as well .Australia’s post-licensure monitor studies showed the first signs of a quadrivalent vaccine impact by reporting a sharp decrease in additional diagnoses of genital warts in young females within two years after vaccine, and a smaller decrease in new genital warts cases in young heterosexual men, suggesting indirect protection through herd immunity. Hariri has reported that emerging data from countries with high vaccination coverages, such as Denmark and Sweden, and those with lower coverages, such as the United States, Germany, and New Zealand, further strengthen the evidence of direct and indirect impacts of the HPV4 vaccine . A systeAmong study limitations is the exclusion of health benefits and cost offsets associated with other HPV related-diseases which may be a consequence of HPV 6, 11, 16 and 18 infections, whose inclusion may show an improvement on HPV4 cost-effectiveness. Also, only direct medical costs were included in the analysis, HPV associated labor productivity loss was not included, which represented for the US an estimate of $1 billion in cervical cancer .In Puerto Rico, labor productivity loss due to cancer deaths in 2004 was estimated to be around $64 million . Even whAnother limitation of the study is that data on the age-specific incidences of genital warts are under-reported in Puerto Rico, leading to inaccuracies in the model estimations. We therefore calibrated the model based on the annual age-specific incidences of genital warts in the US. In addition, data pertaining to the costs of follow-up care for incident episodes of HPV-related disease, including genital warts, were not publicly available. Therefore, these costs were estimated based on claims analysis from a private health insurance company (using ICD 9 and CPT codes). More accurate estimations of these costs represent an area for future research.HPV vaccination is covered by health insurance plans for females and males in Puerto Rico; however vaccination uptake is still suboptimal. Results show that increasing the vaccination coverage can lead to a reduction in the incidence and costs of HPV-related morbidity in Puerto Rico. Although these effects will not be seen immediately for cancer due to its natural history, the impact on anogenital warts would be observed faster. Notwithstanding, it is important to foster direct attention to the importance of the vaccination and in order to be fully protected, completing the vaccination schedule. As stated before, in Puerto Rico local legislation requires insurance companies to cover the vaccine but the vaccination itself is not mandatory according to the local vaccination list. Public health advocates and decision-makers can use the results of this model adaptation to develop strategies to increase vaccination rates in order to potentially avoid additional healthcare expenditures associated with HPV-related disease. Furthermore, this analysis should be used as a basis for future, more in depth analyses that further explore the burden of HPV-related diseases and the positive impacts that vaccination programs can have on them. That way the government can adopt a properly informed position regarding HPV vaccination. Our study supports putting in effect HPV4 vaccination for the prevention of HPV-related pre-cancerous lesions, cancers and genital warts in Puerto Rico. Continued surveillance of HPV-related conditions will help evaluate the long-term impact of vaccination and guide public health efforts.S1 Fig(XLSX)Click here for additional data file.S2 Fig(PPTX)Click here for additional data file.


While factors associated with receipt of human papillomavirus (HPV) vaccination have been well characterized, less is known about the characteristics associated with parents’ intent to have their adolescent children vaccinated. This study aimed to examine factors associated with parental intention toward HPV vaccination.We analyzed data on 10,354 adolescents aged 13 to 17 years from the 2014 National Immunization Survey–Teen. Weighted multivariable logistic regression was used to examine associations between sociodemographic characteristics of mothers and adolescents, as well as a health care provider recommendation with parents’ intention to have their children receive HPV vaccine.Among unvaccinated adolescents, Hispanic ethnicity , mothers with less than a high school diploma , and having a health care provider recommend the vaccine  were significantly associated with parents’ intention to have their adolescent child vaccinated within the next 12 months. In addition, non-Hispanic black race was a significant predictor of parents’ intent to vaccinate for boys .Maternal education and Hispanic ethnicity were the strongest predictors of parental intent to vaccinate against HPV, followed by provider recommendation. As HPV vaccination rates in the United States remain below the Healthy People 2020 goal, messages may need to be targeted based on maternal education, race/ethnicity, and provider recommendation. Nearly all sexually active adults in the United States will acquire human papillomavirus (HPV) infection during their lifetime . If infeThe Advisory Committee on Immunization Practices (ACIP) recommends routine HPV vaccination for US adolescents aged 11 and 12 years , with caWhile factors associated with receipt of HPV vaccination are well characterized, less is known about characteristics associated with parental intent to have their children vaccinated against HPV. This understanding is necessary when considering progression across the stages of health behavior change, as described by the Transtheoretical Model . Intent The NIS-Teen is an annual cross-sectional survey that uses 2 phases of data collection to obtain vaccination information from a national probability sample of parents and guardians of US adolescents ,17. In tNIS-Teen 2014 captured vaccination information for 38,703 eligible adolescent boys and girls aged 13 to 17 years in United States. Of these, 21,057 adolescents  had adequate provider-verified vaccination records. Our study was limited to parents of 10,354 adolescents  with adequate provider-verified vaccination records who had not received any dose of HPV vaccine and whose parents were subsequently asked about their intention to have their child vaccinated against HPV in the 12 months following the survey.The primary outcome of interest was intention to receive HPV vaccine, as ascertained from the survey question “How likely is it that teen will receive HPV shots in next 12 months?” Response options included “very likely,” “somewhat likely,” “not too likely,” “not likely at all,” and “not sure/don’t know.” For the purpose of this analysis, intention to receive HPV vaccine was dichotomized into “likely” and “not likely.” The 574 respondents (parents of 386 [7.6%] boys and 188 [6.1%] girls) who answered “not sure/don’t know” were excluded; however, a subanalysis of these parents  was also conducted.Adolescent sociodemographic covariates were sex, race/ethnicity , and poverty status. Poverty status (above poverty or below poverty) was assessed by comparing respondents’ household income to the US Census poverty level . Because2 tests, stratified by the adolescent’s sex. Weighted multivariate binary logistic regression analyses were performed to examine the association between adolescents’ sociodemographic characteristics and the parents’ intention to have them receive HPV vaccine in the next 12 months, stratified by the adolescents’ sex. To test for differences in associations between boys and girls, 2-tailed z tests were performed by using the estimate and standard errors of the logistic regression coefficients (log odds ratios). This method is equivalent to testing for interactions within a single model but keeps the individual models more interpretable. A second multivariate logistic regression was conducted to examine factors associated with the parental answer, “not sure/don’t know” (about future HPV vaccination) versus all other categories of intent . All analyses were performed in SAS version 9.4  and adjusted with weights as provided in the NIS-Teen 2014 data set to be representative of the US population. The level of statistical significance for all analyses was P < .05. To preserve sample size and reduce potential bias, missing values for explanatory variables were treated as a separate category in the logistic regression models but were not interpreted in the results.Differences in characteristics of adolescents were evaluated by using χP < .001). Approximately 7% of all survey respondents responded “not sure/don’t know” for whether they would vaccinate their adolescents for HPV in the next 12 months (data not shown).Among unvaccinated boys, variables independently predictive of intent to vaccinate against HPV were non-Hispanic black race/ethnicity  and Hispanic race/ethnicity  compared with non-Hispanic whites; mothers with less than a high school diploma  or high school diploma or general equivalency degree  compared with college graduates; never married mothers  compared with married mothers; and a provider recommendation for HPV vaccine  . Among u2 tests indicated that, compared with those who expressed their intention about their likelihood to vaccinate their adolescents, parents who answered “not sure/don’t know” were significantly different based on adolescent’s race/ethnicity (P = .001), poverty status (P = .001), mother’s education (P = .003), and provider recommendation for HPV vaccine (P = .001).Characteristics of adolescents whose parents reported that they were “not sure/don’t know” if they would vaccinate their children in the next 12 months are presented in This secondary analysis of NIS-Teen 2014 data found that among parents of US adolescents aged 13 to 17 years, maternal education emerged as the strongest predictor of parental intent to obtain HPV vaccination for their children. Although provider recommendation was also a significant predictor of intent to vaccinate, the effect was not as strong as maternal education and was stronger for boys than girls. This differs from what is known about actual receipt of HPV vaccination, where provider recommendation is well established as the strongest predictor of actual vaccine uptake . For exaThe lower intent to have their child vaccinated among college-educated mothers and parents of non-Hispanic white adolescents may be counterintuitive, because people from racial/ethnic minority groups and those with fewer economic resources often have challenges with access to medical care and uptake of medical services . HoweverOur findings indicated some effect size differences among boys and girls in which parental intent to vaccinate was stronger for boys than for girls in the same category. For example, mothers with some college education were more likely to intend to vaccinate their sons and less likely to intend to vaccinate their daughters than mothers who were college graduates. Research suggests that parenting roles may be more protective of adolescent daughters than adolescent sons. This may be due in part to parents’ concerns about the unique risks for adolescent girls  . SeveralThe importance of provider recommendation for HPV vaccination is consistent with earlier research that suggests this to be the strongest predictor of HPV vaccine receipt ,23. Our Approximately 7% of all survey respondents responded “not sure/don’t know” for whether they would vaccinate their adolescents for HPV in the next 12 months. Our analysis indicated that parents of adolescent boys and non-Hispanic blacks and parents living below the poverty line were more likely to remain undecided about their intention to have their children vaccinated. This may be attributed to the lower awareness of HPV and HPV vaccine among non-Hispanic black women and those living below the poverty line . While tA strength of this study is that it used a provider-verified vaccination history of a large, nationally representative sample of US adolescents. This reduces the potential for recall bias and social desirability bias with respect to parents reporting their child’s HPV vaccine status. However, this study also has a several limitations. Because of the cross-sectional nature of the data, causality cannot be determined, and temporal interpretations are limited. Second, although statistical adjustments were made to account for nonresponse and households without telephones, a possibility of nonresponse bias still exists. Our analysis was limited to the information included in NIS-Teen; therefore, we were not able to account for other factors associated with parental intention for HPV vaccination, such as personal or family history of gynecological cancer and sexually transmitted diseases, which have been shown to be positively associated with parental intention toward adolescents’ vaccination . Lastly,This study highlights several differences in parental intention toward HPV vaccination based on adolescents’ sociodemographic characteristics and the receipt of provider recommendation for HPV vaccination. Interventions should consider maternal education attainment, and studies should seek to understand why adolescents whose mothers have a college degree are not likely to intend to have their children vaccinated. Additionally, health care providers should actively engage in discussions with parents about HPV and strongly recommend the vaccine to all eligible patients concurrently with other routinely administered vaccinations to dispel any potential negative assumptions or opinions regarding HPV vaccination, especially among girls. Tailored efforts could be made to providers who serve different communities, primarily those who serve non-Hispanic whites, particularly those from high-income households or with mothers with college degrees. Additional studies are needed to examine provider–patient interactions in these settings to better understand how to effectively frame those efforts.


We examined a large series of biopsy-proven invasive cervical cancers with surgical staging and HPV re-testing to estimate the relevance of HPV-negative cervical cancers in a Caucasian population.We prospectively collected smears from 371 patients with a biopsy-proven diagnosis of cervical cancer for HC2 testing of high-risk HPV (HR-HPV). In HC2-negative cases, smears and paraffin embedded tissue blocks underwent additional HPV genotyping.HC2 tests showed 31/371 cases (8.8%) had negative findings. Surgical staging showed that 21/31 HC2-negative cases (68%) were not cervical cancer. Overall, 340/350 cases of primary cervical cancer confirmed by surgical staging tested HC2 positive (97.2%). Non-high-risk HPV subtypes were detected in five cases  and high-risk subtypes in four patients with HC2-negative cervical cancer (two HPV 16 and two HPV-18). The remaining case, a primary undifferentiated carcinoma of the uterine cervix, tested negative for HPV-DNA with all tests.The main explanation for HPV-negative cervical cancer was a false diagnosis, followed by cancers associated with non-HR-HPV types, and false-negative HR-HPV results. Truly HPV negative seem to be very rare in Caucasian populations. Retrospective analyses without surgical staging may overestimate the proportion of HPV negative cervical cancers. Patients with cervical cancer who did not undergo surgery or HC2 testing were not eligible. The Ethics Committee of Lower Saxony was consulted, but stated formal ethical approval was not required because the study was based only on anonymous routine clinical data.Cervical swabs using a brush were taken from all participants. HPV testing was done routinely with hybrid capture 2 (HC2) according to the standard protocol recommended by the manufacturer . HC2 detects high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. The threshold for a positive HC2 result was 1 RLU (relative light unit).All participants underwent surgical staging in our department. In stage Ia1 cases, excision of the transformation zone with clear margins was considered sufficient, while stage Ia2 cases underwent at least excisional treatment and sentinel lymph node biopsy or hysterectomy with complete pelvic lymph-node extirpation. Stage Ib-IIa cases were typically treated by radical hysterectomy with lymphadenectomy either by laparotomy or laparoscopy, and more advanced stages underwent exploratory laparotomy/laparoscopy with exploration of the abdominal cavity and removal of pelvic and para-aortal lymph nodes.A diagnosis of primary cervical cancer was based on surgical staging with exclusion of malignancies of other origin. If necessary, immunohistochemistry was used to confirm sarcoma, lymphoma, or cancers of other origin. Distinguishing between endometrial cancer and endometrioid cervical cancer is difficult and, in advanced stages, sometimes arbitrary; therefore, for this analysis only, endometrioid cancers with exclusive or dominant infiltration of the uterine cervix that spared at least parts of the uterine cavity were defined as cervical cancers. Patients were excluded from further analysis if a positive diagnosis of cervical cancer was not confirmed by surgical staging.Primary cervical cancers that were confirmed by surgical staging but tested negative for HR-HPV by HC2 underwent further HPV-testing. Paraffin embedded tissue blocks were deparaffinized and DNA was extracted automatically using the QIAsymphony DSP DNA Mini Kit . SPF10 HPV genotyping was performed using the INNO-LiPA HPV Genotyping Extra® test , which identifies 20 HPV genotypes classified as Group 1, 2A and 2B carcinogens  and eight low-risk HPV or intermediate-risk genotypes . LiPA Extra is a line blot assay based on SPF-10-PCR as described previously The study endpoints for patients with a negative HC2 test and confirmation of invasive cervical cancer after surgical staging were histology , HPV status on additional tests, and specific HPV subtype. The objective of the study was to determine whether any patients had a definitive HPV-negative invasive cervical carcinoma. All data are reported descriptively for individual patients.3The analysis included 371 cases with an initial diagnosis of primary cervical cancer and a HC2 test result . The medSurgical staging of the remaining 23 HC2-negative cases confirmed the diagnosis of primary cervical cancers in 10 cases, while 13 cases were reclassified as malignancies of other origins. Specifically, two cases with cervical cancer, diagnosed at another center prior to referral, were reclassified as B-cell lymphomas on the final specimens.Overall 340 of 350 confirmed primary cervical cancers were HPV positive and 10 were HPV negative. Additional HPV tests identified HPV infection in 9 of 10 cases , comprisHistologic assessment of 350 confirmed primary cervical cancers showed that 275 (78.6%) were classified as squamous cell carcinoma (SCC), 48 (13.7%) as adenocarcinoma (AC), 11 (3.1%) as adenosquamous carcinoma (ASC), and 16 (4.6%) as other types, including neuroendocrine small cell cancer (n = 4), undifferentiated cancer, and mixed types. Cases of adenocarcinoma were classified mainly as not otherwise specified (NOS), mucinous, or endocervical type, while there were only a few cases of clear cell adenocarcinoma (n = 2), villoglandular (n = 2), endometrioid (n = 3), or mucinous intestinal types (n = 1). SCC accounted for 7 of 10 cases of HC2-negative primary cervical cancer . The sin4The aim of this study was to characterize the histopathology of women with putative HPV-negative cervical cancer using highly sensitive HPV testing and rigorous histological assessment following surgical staging. The results showed that true HPV-negative primary cervical cancers are very rare.Our results are in accordance with The Cancer Genome Atlas Research Network (CGARN) ‘Integrated Genomic and Molecular Characterization of Cervical Cancer Study’, which used next-generation sequencing to characterize primary cervical cancers Previous studies of HPV prevalence among patients with cervical cancer have generally reported higher rates of HPV-negative tumors than we showed in this study In our study, additional PCR-based testing of tumors from patients with confirmed primary cervical cancer after surgical staging showed that most samples with an initial HC2-negative test result (9/10) were HPV positive. Retesting showed that HC2-negative, HPV-positive results can be explained either by the failure of the initial test procedure to detect HR-HPV subtypes or by infection with other HPV subtypes not identified by the standard HC2 test. All established and validated HPV screening tests are designed to detect HR-HPV and will therefore miss the 1‒2% of cases associated with non-HR-HPV. This is an accepted gap of current routine HPV tests While the limitations of routine HC2 tests are well known, this study highlights the possibility of an erroneous diagnosis of primary cervical cancer based solely on biopsy. While the role of surgical staging remains controversial, this procedure was useful in this research context to fully characterize carcinomas invading the cervix. This study showed that HC2-negative tumors diagnosed as cervical cancer may originate from other primary sites. While secondary malignancies rarely occur in the uterine cervix, and mostly result from direct spread of other uterine tumors to the cervix, several primary solid tumors, including ovary, breast, stomach, gallbladder, pancreas, and lung, may to metastasize to the uterine cervix This study has limitations inherent to all retrospective analyses of data collected from a single department. Nevertheless, all subjects were enrolled and evaluated according to a standard protocol. The median age of subjects included in the analysis was slightly younger than the overall average for cervical cancers overall (approximately 50 years). This minor difference can be explained by the exclusion of women with distant metastases at presentation and those with severe co-morbidity who were not able to undergo surgical staging; in addition, cancer cases from the colposcopy unit tend to be younger than the overall population of patients with cervical cancer. The study was initiated in 1999 and therefore includes subjects from a pre-vaccination era. The impact of HPV vaccination on the prevalence of HPV-negative cervical carcinomas is not yet known.5This study confirms the almost universal involvement of HPV infection in primary cervical cancer. HPV-negative tumors occurring in the uterine cervix are histologically distinct from true HPV-positive cervical cancer.


The aims of this study were to detect HPV E6/E7 mRNA expression in women with high-risk genotypes  analysing its relationship with tissue pathology and 2) 2-year follow-up of E6/E7 mRNA tested group.Our samples were genotyped and classified by pathologists according to Bethesda system. After RNA extraction, E6/E7 oncogene mRNA detection was performed by NucliSens® EasyQ® HPV v1 Test (bioMérieux).The results of the present study showed that E6/E7 mRNA positivity rate was 68.29 % in women tested once and 69.56 % in women tested twice. According to tissue pathology, all samples with high-grade lesions were positive for mRNA. Among women with low-grade lesions varied over the years from 89.28 to 84 % in women tested once and from 77.77 to 70 % in tested twice. Among women without lesion, positivity rate maintained in women tested once (from 50 to 41.38 %) and decreased in tested twice, from 63.63 to 44.44 %. Regarding lesion evolution, mRNA positivity was higher in women with lesion progression (53.13 %) and in women with positive results in two tested samples (83.33 %).HPV E6/E7 mRNA detection may be an effective screening test and biomarker for cervical cancer in women infected with these five genotypes. Nonetheless, further studies are needed to standardize as routine triage test. Human papillomavirus (HPV) infection is responsible of cervical cancer, which is the second most frequent cancer affecting woman worldwide –3.More than 100 genotypes have been described; 12 of them are classified as high-risk genotypes (HR-HPV) due to their high oncogenic potential . Among tHPV viral oncoprotein expression differs among infection state  , 11. HPVTo date, cervical cancer screening is based on cytology and this has been a powerful tool to reduce the incidence and mortality of this type of cancer , 15, butThe present study was designed to 1) study HPV E6/E7 mRNA expression and analyse its relationship with tissue pathology in order to test mRNA as an effective biomarker for cervical cancer and 2) 2-year follow-up of a group of women and test E6/E7 mRNA expression.Samples from women which were referred from Consultation of Sexually Transmitted Diseases and Gynaecological consultation at Basurto University Hospital  were analyzed due to possible HPV infection during the last 7 years (2007–2014). All patients gave written and informed consent prior to their inclusion in the study.The samples were categorized by pathologists according to Bethesda system: 1) negative (no lesion was found) 2) Atypical squamous cells of undetermined significance (ASCUS) 3) Low-grade squamous intraepithelial lesion (LSIL) and 4) High-grade squamous intraepithelial lesion (HSIL).Samples were analyzed with Cobas® HPV Test  that detects 14 high-risk HPV DNA genotypes, 16 and 18 separately and other 12 high-risk HPV genotypes altogether. Samples that were positive for other HR were genotyped with Linear Array HPV Genotyping Test kit (Roche Molecular Diagnostics), a line-blot assay that detects 37 genotypes .RNA was extracted from 200 μl of samples by NucliSENS Lysis Buffer and NucliSENS® miniMAG®  according to manufacturer’s instructions.E6/E7 oncogene mRNA was detected by NucliSens® EasyQ® HPV v1 Test (bioMérieux) following manufacturer’s instructions. This test detects the E6/E7 oncogene expression of five HR-HPV genotypes . The quality of extracted RNA was monitored by an internal control: human U1 small nuclear ribonucleoprotein specific protein A (U1A). If neither U1A nor mRNA were detected, the test result was analyzed as invalid. Invalid runs were repeated once again.In the present study, E6/E7 mRNA expression was analyzed in a total of 128 samples (corresponding to 105 women). Eighty-two women’s samples were analyzed once and in 23 women mRNA expression was studied twice . All these women were positive for HPV DNA (DNA positivity rate was 100 %).HPV-16 was the most prevalent genotype , followed by 18 , 45 , 31  and 33 . Nevertheless, regarding oncogene expression, HPV-33 was the most expressed , followed by 18 , 16 , 31  and 45 .Eighty-two women samples were tested for E6/E7 mRNA oncogene expression only once. The mean age was 33.62 ± 10.57 years (range 17–63). Most of them were infected with multiple HPV infection genotypes (54/82) whereas 34.15 % of them had single HPV genotype infection.More frequently detected genotype was 16 (67.01 %) followed by 18, 45, 31 and 33 .The E6/E7 mRNA positivity rate was 68.29 % (56/82). The mean age of E6/E7 positive and negative women was similar (33.53 ± 11.12 vs 33.8 ± 9.62 years). HPV 16 E6/E7 mRNA was detected in 71.42 % of positive samples (40/56), 18 in 10.71 % (6/56), 33 in 3.57 % (2/56), 16 plus 33 multiple infection in 3.57 % (2/56), and the following genotypes were only detected in 1.78 % of positive samples (1/56) .The E6/E7 mRNA results were analyzed regarding their cytology status. There were 77 women with cytology and E6/E7 mRNA test. Pathogenicity was divided into three groups: 1) normal or negative (no lesion was found), 2) ASCUS and LSIL and 3) HSIL. Among E6/E7 mRNA positive samples, 47.89 % belonged to women with ASCUS or LSIL . HPV single infection was detected in 52.17 % (12/23) and multiple HPV infection in 47.83 % (11/23). The most detected genotype was 16 (73.47 %) followed by 18 (14.29 %) and 31, 33 and 45 genotypes (4.08 % of incidence each one).The E6/E7 mRNA positivity rate was 69.56 % (32/46) and the oncogene expression was mostly detected in HPV-16 in 68.75 % of samples (22/32), followed by 18 in 15.63 % (5/32), 16 plus 31 multiple infection in 6.26 % (2/32), and the followings genotypes in 3.12 % (1/32): 33; 16 plus 18 and 16, 31 plus 33.According to E6/E7 mRNA positivity rate, 55.56 % had some type of lesion (38.89 % ASCUS or LSIL and 16.67 % HSIL) in samples taken first   or had a previous negative result in mRNA but then mRNA was detected (16.67 %) Fig. . OtherwiSingle HPV infection was detected in 40 of 105 analyzed women and 65 women were infected with multiple HPV infection. Among women with single HPV infection E6/E7 mRNA positivity rate was 67.50 % whereas that rate was 70.77 % in women with multiple HPV infection. The 60 % of women with single HPV infection had normal cytology results whereas the 40 % showed some lesion (high grade or low grade lesion). On the other hand, the 58.33 % of women with multiple HPV infection had some lesion and only the 41.66 % has normal cytology results. Among women with multiple HPV infection and positive for E6/E7 mRNA the 19.56 % were expressing more than one genotype oncogenes.Human papillomavirus is closely related with cervical cancer. HPV infections usually show a regression in the next 2 years, but few patients infected with HR-HPV genotypes are at risk of developing cervical cancer , 22. DurDNA positivity rate was less valuable in clinical use than mRNA as it has been observed in previous studies , 23, 24.Among mRNA positive women HPV 16 single infection transcript was the most recurrent, though, the main majority of women tested had multiple HPV infection. HPV 16 tended to express more than other genotypes, which may be connected with the fact that it is considered the most carcinogenic genotype . NeverthIt was proved that E6/E7 mRNA positivity rate was higher in women with high-grade lesion than in samples with low-grade lesion or without lesion , 19, 26.According to E6/E7 mRNA as cervical disease biomarker, mRNA positivity increased over time in specimens with lesions in women tested once and in tested twice, namely, E6/E7 mRNA positive samples showed a malignancy progress during the next years. These results were expected since E6 and E7 oncogene are over expressed in precancerous lesions and it has been proved how these oncogenes and cervical lesion severity increases at the same time . TherefoNevertheless, in women who were tested twice, there was no difference in mRNA negativity between samples tested first and specimens tested one or two years later, so it seemed that there were a significant number of specimens that had minor cervical lesions but were negative for E6/E7 mRNA. This event could be related with the fact that low-grade cervical lesions normally disappears in a few months without treatment . Even soFinally, E6/E7 mRNA could be a good prognosis marker since it was observed that women who were positive for E6/E7 mRNA had more probabilities to develop lesion progression. Besides, women in which samples mRNA was negative had more possibilities to remain the same pathological grade or have infection clearance. These findings are in concordance with previous studies where E6/E7 mRNA was considered a short-term prognostic factor for high-grade lesions . NeverthE6 and E7 oncogene mRNA detection seems to be a useful instrument as a triage test in women infected with HPV-16, -18, -31, -33 or -45 genotypes. Moreover, it could be a promising biomarker since it has been proved its relationship with lesion grade, making possible the reduction of HPV positive women referred to colposcopy. Even so, further studies are needed to use NucliSens® EasyQ® HPV v1 Test as standard screening method in HPV positive women.


High risk human papillomavirus (HR HPV) infects cells at the squamocolumnar junction (SCJ) of the cervix, causing cancer. Cytokeratin (CK)7 is an SCJ marker, and stains cervical neoplasia. CK19 is a binding partner of CK7 and expressed in cervical cancer. Despite this possible association between CK7/CK19 and cervical cancer, not much is known about the mechanism of CK7/CK19 involvement in HR HPV-mediated cervical carcinogenesis.We analyzed the expression pattern of CK7, CK19, and p16 by using immunohistochemistry and HPV infection by in situ hybridization in 25 cases of high grade cervical intraepithelial neoplasia (CIN3) and in 30 cases of squamous cell carcinoma (SCC).CK19, p16, and HPV expression was positive in all CIN3 and SCC cases. CK7 expression was positive in all CIN3 cases and in 20/30 (66%) SCCs. Each protein showed diffuse or patchy staining with topographic distinction. Patchy staining of CK7 and episomal HPV DNA overlapped in the upper layer of CIN3 and central portion of an invasive nest in the SCC, whereas patchy CK19 staining and integrated HPV DNA were usually noted in the lower layer of CIN3 and the periphery of the SCC nest. The p16 staining pattern coincided with that of CK19 in a subset of SCC.These results suggest that CK7 may be more related with viral episomal replication and CK19 with viral integration, contributing to viral replication and malignant transformation in HR HPV infected cells. In addition, coordinate CK7/CK19 staining may be used as a valuable marker for predicting physical status of HR HPV and E7 oncoprotein level in cervical tumor.The online version of this article (doi:10.1186/s13000-017-0609-4) contains supplementary material, which is available to authorized users. More than 200 human papillomavirus (HPV) types have different epithelial tropisms and high risk (HR) HPV infects the cervical epithelium, causing cancer . DiscretCK19 pairs with CK7 in simple epithelia and with CK8/18 in stratified squamous epithelial cells [HPV DNA exists as episomal or integrated state in cervical neoplasia –13. In CDuring HPV life cycle, the viral replication and the expression of viral genes are closely tied to the differentiation program of host epithelial cell . In thisThe list of CIN and SCC was obtained from archives in the Department of Pathology at Nowon Eulji Medical Center, Eulji University. We retrieved 25 CIN3 and 30 SCC cases collected by cone biopsy, loop electrosurgical excision procedure (LEEP), or hysterectomy. The age of all CIN3 and SCC patients ranged from 27 to 79 years and the mean was 48 years. The stage of 30 SCC patients was classified into 11 cases of stage Ia, 10 stage Ib, 5 stage II, 3 stage III, and 1 stage IV according to the International Federation of Gynecology and Obstetrics staging. Ten non-neoplastic cervical specimens from the uteri removed for non-cervical pathologic conditions were also included. After reviewing all hematoxylin and eosin (HE) stained slides from each case, representative blocks for each case were selected. In two cases of CIN3 (LEEP) and one case of SCC (LEEP), immunohistochemical evaluation was not available in the slides made from initially selected blocks by repeat cutting due to technical reasons and other blocks for the same case were used for immunohistochemistry. This study was performed with the approval of the Institutional Review Board of Nowon Eulji Medical Center.Immunohistochemical staining was performed using Dako Autostainer . Four micron tissue sections were cut from selected blocks and positioned on poly-L-lysine coated slides. After deparaffinization and rehydration, antigen retrieval was performed using citrate buffer (pH 6.0) at 121 °C for 10 min. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 5 min. The primary antibodies used in this study were CK7 , CK19 , and p16 (Dako p16INK4a kit). Color was developed with diaminobenzidine, and the slides were counterstained with hematoxylin.CK7 and CK19 staining was interpreted as positive when approximately five to six contiguous cells showed cytoplasmic and/or membranous block staining . With p1In situ hybridization (ISH) was performed using the INFORM HPV III Family 16 Probe (B) , according to the manufacturer’s recommendations. The probe cocktail has demonstrated affinity for the following HR HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66. The HPV signal patterns were classified as the episomal or integrated form, found within the nuclei of cervical epithelial cells. The episomal HPV was defined as large globular dense nuclear staining. The integrated HPV was defined as discrete dot-like signals or scattered tiny particles in the nuclei.In the non-neoplastic cervix Fig. , CK7 staThe results of immunohistochemistry for CK7, CK19, and p16 in CIN3 are shown in Fig. HPV was detected in all CIN3 cases including 11 (44%) cases with combined episomal and integrated form and 14 (56%) cases with integrated form. Episomal HPV DNA was usually seen in the upper layer corresponding with CK7 staining area Fig.  and o, wThe results of immunohistochemistry for CK7, CK19, and p16 in SCC are shown in Fig. The expression of p16 was also weaker in SCC compared with CIN3, with even one patchy stained case  and integrated form in 16 (53%) of 30 SCC cases. The cases with integrated HPV DNA revealed no topographic distinction. In SCC#19, an episomal form was seen in the entire tumor nest but more frequently in central portion of the nest, overlapping with the expression of CK7 Fig. . HoweverIn this study, we found consistent expression of CK19, p16, and HR HPV in CIN3 and SCC cases. However, CK7 expression was lost in one-third of SCC cases, although its expression was positive in all CIN3 cases. CK7 and CK19 showed topographic distinction in patchy stained CIN3 and SCC cases. In addition, CK7 and CK19 staining appeared to be more linked with episomal and integrated forms of HPV, respectively. The expression of CK7 or CK19 has been intensely studied in HPV mediated oropharyngeal SCC as well as cervical neoplasia , 19, 20.In SiHa cells, HPV16 E7 mRNA interacts with the 6-mer peptide SEQIKA of CK7 and E7 mRNA translation is inhibited . In contIn CIN1, CK7 staining is predominantly strong in the surface epithelial cells , 6. As tCK19 expression extends upward from the basal to superficial layer (bottom up expression) as CIN grade is increasing , 9. p16 In the present study, CK19 and CK7 were both stained at the cells of the transformation zone and SCJ of the non-neoplastic cervix, although staining intensity of CK19 was weaker than that of CK7 in SCJ. In normal human organs, co-expression of CK7 and CK19 occurs in hepatic progenitor cells, which are capable of differentiating into hepatocytes and cholangiocytes . If SCJ In cervical cancer, HR HPV is mainly detected in integrated form , 11, 29,There are still questions left unresolved in this study. The first is how the cells with stem cell character were simultaneously present in the upper layer and the lower layer of CIN3 showing CK7 and CK19 positivity, respectively as shown in CIN3#12. One possible explanation would be concurrent neoplastic transformation of CK7 positive cells in the upper layer derived from SCJ and CK19 positive basal stem cells in the lower layer infected by different or multiple HR HPV , 33. CINThe second question is why CK7 expression was not observed in 10 of 30 cases of SCC whereas CK19 expression was retained even with a patchy pattern in all SCC cases. This might be related to a basically different expression level of CK7 and CK19 depending on the location in the cervix in which SCC arises. As CK7 was not detected in the ectocervix distant to SCJ, SCC developed from this area would show only CK19 positivity. However, it remains to be explored why CK7 and CK19 did not co-localize to the same tumor cell populations despite their pairing property.The last one is why the expression of CK19 as well as CK7 was lower or weaker in SCC compared to CIN3. Changes of CK7 and CK19 expression level in invasive cancer compared with CIN3 might be related to migration capacity of cancer cells  or asymmIn conclusion, our results support earlier studies in which CK7 might be a predictive marker for HPV infection and CIN3 progress. In addition, consistent expression of CK19 and p16, accompanied by HPV integration in CIN3 and SCC tissues supports the idea that CK19 may promote E7 oncoprotein production, contributing carcinogenic events. We suggest that coordinate CK7/CK19 staining may be used as a valuable marker for predicting the physical status of HR HPV and E7 level in cervical tumor.


HPV infection is the major pathogenic factor underlying cervical cancer and precancerous lesions. The cervical HPV infection rates in gynaecological outpatients from Hangzhou, China, were studied in the period from January 2011 to December 2015.Exfoliated cervical cells were harvested from gynaecological outpatients in Hangzhou from January 2011 to December 2015. Twenty-one HPV subtypes were detected using flow-through hybridization. The HPV infection rates in various disease groups were compared using the Chi-square test. The infection rates of different HPV subtypes in different calendar years and in different age groups were analysed using the linear-by-linear association test and gamma value.P > 0.05). However, the 15–24-year-old age group showed the highest HPV infection rate, and significant differences (linear-by-linear association test) were detected among the different age groups (P < 0.05). The HPV infection rates exhibited an upward trend in the 15–24-year-old and >24–34-year-old groups over the past five years. There were significant differences in the HPV infection rates among the disease groups (P < 0.05).A total of 43,804 patients were recruited, of whom 9752 (22.3%) were infected with HPV. The top five among the 21 HPV subtypes detected in terms of infection rates were HPV-16, −52, −58, −53 and −18. No significant differences (linear-by-linear association test) were found in the HPV infection rates when compared over the studied years (HPV-16, −52 and −58 were the major HPV infection subtypes in Hangzhou, China. The 15–24-year-old age group had a relatively high HPV infection rate with an upward trend over the past five years and thus represented a population susceptible to HPV infection. Human papilloma virus (HPV) contains a circular double-stranded DNA genome that exhibits strict organization and host specificity. More than 200 HPV genotypes have been characterized to date, and more than 40 HPV types can infect the genital area and result in genital lesions. HPV genotypes are classified as low-risk and high-risk types based on their potential to cause cancer , 2.The incidence of cervical cancer worldwide ranks second in female malignancies and is exhibiting a trend towards younger ages . MethodsThe latest research showed that HPV-52, −16 and −58 were the HPV subtypes with the highest infection frequencies in China [Clinical specimens were collected from 43,804 gynaecological outpatients at the First Affiliated Hospital of Zhejiang Chinese Medical University and the First Affiliated Hospital of the Medical School of Zhejiang University from January 2011 to December 2015. A woman was considered eligible to enter the study if she a) had current or past sexual activity, b) was not pregnant at the time of enrolment, c) had never been screened or treated for cervical cancer, d) had not undergone a total uterus or cervix resection, e) agreed to undergo an HPV test and f) agreed to participate in the present study. This study was conducted in accordance with the Declaration of Helsinki and a protocol approved by the First Affiliated Hospital of Zhejiang Chinese Medical University and the First Affiliated Hospital of the Medical School of Zhejiang University . There were 5001 cases in 2011, 6410 cases in 2012, 7863 cases in 2013, 11,402 cases in 2014 and 13,128 cases in 2015. The recruited patients were 17–88 years old, with a median age of 44 years. In accordance with a prior overseas study, [The opening of the cervix was exposed using a vaginal dilator. Excess secretions at the opening of the cervix were wiped away using a cotton swab. A cervical brush was inserted into the opening of the cervix and rotated clockwise 3–5 times to acquire exfoliated cervical cells. The cells were placed into a sample tube containing cell preservation solution, stored in a refrigerator at 4 °C and analysed within 3 days of collection.Samples that tested positive for β-globin were analysed by PCR amplification of HPV DNA. HPV-positive samples were confirmed by PCR with universal L1 primer MY09/11 and GP5/6 systems. DNA from HeLa and Caski cell lines was used as positive controls, and mixtures without sample DNA were used as negative controls. HPV genotypes were determined using an HPV GenoArray Test Kit , according to the manufacturer’s instructions , 9. GenoThe test was conducted in four steps as follows: (1) HPV DNA extraction, (2) PCR amplification, (3) flow-through hybridization  and (4) result interpretation. Positive test results appeared as bluish violet dots. Samples that were positive for only one HPV subtype were defined as single HPV infections, and samples that were positive for more than one HPV subtype were defined as multiple HPV infections.P-values were two-sided, and statistical significance was defined as P < 0.05.The data were analysed using SPSS 22.0 statistical software. The positive infection rate is expressed as a percentage. For multiple infection patients, the HPV-positive rate was calculated repeatedly for each genotype. The infection rates were compared based on disease group using the Chi-square test. The linear-by-linear association test and gamma value for trends were used to evaluate changes in HPV prevalence by calendar year and by age group. P > 0.05) , and the rate of multiple infections was 5.2% . Among the 21 HPV subtypes detected, the infection rate for the low-risk subtypes was 4.5% , and the infection rate for the high-risk subtypes was 20.1% . Regarding the infection rate, the top five HPV subtypes were HPV-16 , HPV-52 , HPV-58 , HPV-53  and HPV-18 ; the top three low-risk HPV subtypes were HPV-8304 , HPV-6  and HPV-11 . The HPV-positive rate in 2011 was 22.7% (1133/5001), with single infections in 16.5% (823/5001) and multiple infections in 6.2% (310/5001) of the subjects. The HPV-positive rate in 2012 was 21.6% (1383/6410), with single infections in 15.8% (1015/6410) and multiple infections in 5.7-% (368/6410) of the subjects. The HPV-positive rate in 2013 was 22.2% (1745/7863), with single infections in 16.4% (1291/7863) and multiple infections in 5.8% (454/7863) of the subjects. The HPV-positive rate in 2014 was 22.6% , with single infections in 16.2%  and multiple infections in 6.4%  of the subjects. The HPV-positive rate in 2015 was 22.2% , with single infections in 15.4%  and multiple infections in 6.9%  of the subjects . Additionally, significant differences (linear-by-linear association test)were found among the age groups in the infection rates for the 11 high-risk subtypes  and the four low-risk subtypes  , 23.5% (2155/9159), 21.3% , 20.6%  and 24.1% (1629/6750), respectively. The highest infection rate appeared in the 15–24-year-old group, and significant differences (linear-by-linear association test) were found among the age groups (P < 0.05) and in the single and multiple infection rates among the HPV-positive patients (P < 0.05) (Table The HPV infection rates of the different disease groups were as follows: 97.2% 889/915) in the cervical cancer group, 88.6% 287/324) in the CIN3 group, 52.7% (143/270) in the CIN2 group, 50.8% (978/1924) in the CIN1 group and 18.5%  in the other diseases group. Significant differences were found in the HPV infection rates among disease groups  15,189 accreditation. The results showed that the average HPV infection rate was 22.3% in general gynaecological outpatients from Hangzhou over the past five years. This value was close to the HPV infection rate of 22.80% in gynaecological outpatients from the same region reported by Liu et al. , 13 and The present study showed that the infection rate of the high-risk HPV subtypes was 20.1% in Hangzhou, which was 4.43 times the infection rate found for the low-risk HPV subtypes in this region (4.5%). This ratio is higher than the previously reported ratio of high-risk/low-risk HPV infection rates in China . The curCervical cancer has long plagued the majority of women’s health care in China, particularly for rural women; every year, there are approximately 130,000 new cervical cancer cases . The besAge is an important factor associated with HPV infection. One point of view is that young women have frequent sex. Reports in the literature suggest that young women are more prone to have multiple partners ; additioIn the present study, the HPV infection rate varied in different disease groups. The highest HPV infection rate appeared in the cervical cancer group (97.2%). This rate was close to the rate reported in the literature of 99.7% . WhetherThe present study evaluated HPV infection in gynaecological outpatients in Hangzhou, China, from 2011 to 2015. This study was an investigation of HPV infection with the largest sample size in this region to date. The results showed that HPV-16, −52 and −58 were the dominant HPV infection subtypes in Hangzhou, which provided objective evidence for the application of preventative vaccines in this region. Moreover, we found that the HPV infection rate progressively increased every year in the age groups below 34 years. HPV infection exhibited a trend towards younger patients, and the 15–24-year-old group was a population susceptible to HPV infection. These findings provide objective evidence for the selection of this age group for vaccination in the study region.