A decrease in AQPs functionality is related to a decrease in sperm cells number and motility. Here we investigate the possible effect of human Papillomavirus (HPV) on both expression and function of AQPs in human sperm cells of patients undergoing infertility couple evaluation. Stopped-flow light-scattering experiments demonstrated that HPV infection heavily reduced water permeability of sperm cells in normospermic samples. Confocal immunofluorescence experiments showed a colocalization of HPV L1 protein with AQP8 (Pearson’s correlation coefficient of 0.61), confirmed by co-immunoprecipitation experiments. No interaction of HPV with AQP3 and AQP7 was observed. A 3D model simulation of L1 protein and AQP8 interaction was also performed. Present findings may suggest that HPV infection directly inhibits AQP8 functionality and probably makes sperm cells more sensitive to oxidative stress.Human sperm cells express different aquaporins (AQPs), AQP3, 7, 8, 11, which are localized both in the plasma membrane and in intracellular structures. Besides cell volume regulation and end stage of cytoplasm removal during sperm maturation, the role of AQPs extends also to reactive oxygen species (ROS) elimination. Moreover, oxidative stress has been shown to inhibit AQP-mediated H Persistent infection by High-Risk Human Papillomavirus (HR-HPV) is considered the main cause for human cervix, ano-genital, head, and neck cancer development . InflammIt is well known that HPV is responsible for inducing cancer, but growing evidence suggests that it also plays a role in causing transient male sub-fertility. As stated by Depuydt and coworkers, these are two sides of the same coin . Even thOther studies were unable to demonstrate that HPV infection is associated with poor sperm parameters. Brossfield et al. showed an increased velocity and progression of sperm exposed to L1 HPV-DNA fragments compared with controls . Rintala2O2 efflux through plasma membranes to the extracellular fluid has been considered an important ROS scavenging system [2O2, and for this reason were named peroxiporins: AQP3, AQP5, AQP8 and AQP9 [The mechanism underlying the possible damage of sperm functionality following HPV infection are still poorly understood. HR-HPV proteins modulate ROS concentration in host cells switching from a eustress to a distress condition . Oxidatig system ,22. Someand AQP9 ,27,28,29Sperm cells express two types of peroxiporins: AQP3, AQP8. The former is localized in the sperm tail membrane and in a few granules in the head and in the midpiece; the latter is found in the midpiece of spermatozoa, seemingly in the mitochondria and/or in the plasma membrane in the proximity of the midpiece ,31. SperThis study aims to elucidate whether HPV infection may damage sperm through AQP impairment. To this end, in human ejaculated sperm cells from normospermic and sub-fertile subjects with and without documented HPV-infections we determined: (1) water permeability features measured by a stopped-flow light-scattering method; (2) the interaction between AQPs and HPV capsid protein L1 by confocal fluorescence microscopy (3) the co-immunoprecipitation of HPV with AQP8; (4) a potential L1-AQP interaction mode by protein modelling and docking simulations.The results reported here provide evidence that HPV binds to AQP8 and inhibits sperm functions.Ninety-four male partners were recruited from infertile couples undergoing infertility evaluation at the Center for Reproductive Medicine, Fondazione IRCCS Policlinico San Matteo  after informed consent. The consent form was specifically designed for processing of the excess of semen volume collected for diagnostic analysis, in accordance with the Declaration of Helsinki, and with our local Ethical Committee guidelines on the use of residual biological material for research purposes.PR + NP ≥ 40%; PR ≥ 32%) [Each male produced the semen by masturbation after an abstinence of 2–7 days and semen samples were collected in a sterile plastic container confirmed to be non-toxic for spermatozoa. A routine semen analysis was performed within 1 h of collection, according to the methods described by the World Health Organization (WHO) . The sysR ≥ 32%) .group 1–67 were from subjects defined normospermic based on the following parameters: number of spermatozoa ≥ 15 × 106/mL, progressive spermatozoa ≥ 4.8 mil × 106/mL and physiological viability ≥ 58%); group 2–27 were from patients defined sub-fertile with at least one of the principal basal seminal parameters compromised (number of spermatozoa <15 × 106/mL or PR < 32%). In the present study, physiological morphology was not considered a parameter for discriminating between the two groups.Samples were divided into two groups on the basis of their characteristics: Samples were incubated at 37 °C until the analysis was performed. The analysis to assess volume, pH, fluidification, and viscosity was started within one hour from semen collection.Each semen sample was assessed for sperm motility and kinematics of movement using a disposable counting chamber . Sperm count was performed on undiluted specimens. The grid was on a cover glass. The number of spermatozoa counted in any strip of 10 squares of the grid indicated their concentration in millions/mL. No additional factors were necessary for the calculation. We counted at least 3 strips and the mean value was used. The chamber was 10 microns deep, which eliminates blurring and allows sperm to move freely. The applied sample was observed in one focal plane. The motility of each spermatozoon was graded as follows: PR, active motility; NP, all other patterns of motility with no progression; immotility (no movement) .To determine sperm morphology, each sample was analyzed by using Diff-Quik-stained slides . Restricted criteria by Kruger as indicated by the WHO manual were used to analyze at least 200 spermatozoa per sample . Samples were assessed for sperm viability by staining with 1% Eosin-Y in saline . Briefly, 50 μL semen samples were mixed with 2 drops of 1% Eosin-Y in a sterile test tube and a drop of semen-stain mixture was placed on a microscope slide. The smear was covered with a cover glass before drying and was immediately analyzed under the microscope. At least 200 spermatozoa were counted and classified as stained (dead) or unstained (viable).DNA extraction was performed on sperm samples (100–300 µL) using an automatic instrument  based on paramagnetic particles. 10 µL of the solution were used for PCR amplification of HPV sequences from the L1 region using SPF10 primers in a final reaction volume of 50 µL for 40 cycles.Positive and negative controls were introduced in each set of 12 reactions, including DNA from Siha and HeLa cell lines at a specified number of HPV copies, and blank reagents throughout all steps of the procedure.Concurrent amplification of human HLA-DPB1 gene was included in the assay as internal control for DNA adequacy.HPV type-specific sequences were detected by the line probe, INNO-LiPA HPV genotyping CE assay, version INNOLIPA HPV GENOTYPING EXTRA II , according to the manufacturer’s instructions. The EXTRA version of the assay allows the simultaneous and separate detection of 32 HPV types: 13 high-risk HPV types , 6 intermediate-risk HPV types , of 9 low-risk HPV types , and 4 unclassified HPV types . Hybridization patterns were automatically analyzed by the LiRAS system and checked by two independent readers.w the molar water volume, V0 the cell volume and A the cell surface area. Vo and A were obtained by Curry et al. [Osmotic water permeability of human sperm samples was measured by a stopped-flow light-scattering method as previously described ,34. The y et al. .Water transport was evaluated in sperm cells from normospermic and sub-fertile subjects, with or without HPV infection, following exposure to hypoosmotic medium (150 mOsm osmotic gradient).Immunolocalization of AQP3, 7, 8, and HPV was evaluated in human sperm samples, which were smeared on polylysine-coated slides, air dried and fixed in 4% paraformaldehyde in PBS for 30 min, and then washed with PBS. Antigen retrieval was performed by placing the slides in a Coplin jar containing retrieval buffer  and microwaving at high power level. When the solution started to boil (5 min) the oven was turned off. Two more cycles were repeated, and the jar was left at room temperature allowing slides to cool for at least 20 min. After washing with PBS, the slides were then blocked with 3% BSA in PBS at room temperature for 30 min. Double labeling experiments were performed by incubating the slides overnight (in the cold) with affinity pure anti-AQP3 or AQP7 or AQP8 primary antibodies  and with a prediluted mouse monoclonal anti-HPV antibody  that recognizes the major capsid L1 protein of the following HPV types: 6, 11, 16, 18, 31, 33, 42, 51, 52, 56, 58. In some experiments, other Anti-AQP8 and anti-HPV were used: Anti-AQP8 rabbit polyclonal IgG affinity pure  and Anti-HPV Type 16 L1 Monoclonal Antibody . Anti-AQP antibodies were used at the following dilutions in antibody diluent : AQP3, 1:300; AQP7 1:250; AQP8 1:250. After three 5 min washes with PBS, slides were incubated at room temperature with the fluorescent secondary antibody  and rhodamine red –X-conjugated affinity pure goat anti-rabbit IgG (H+L)  for 30 min.® Gold antifade reagent with 4′,6-Diamidino-2-Phenylindole  and examined with a TCS SP5 II confocal microscopy system (Leica Microsystems) equipped with a DM IRBE inverted microscope (Leica Microsystems). Images were visualized and analyzed by LAS AF software (Leica Microsystems Application Suite Advanced Fluorescence). Control experiments were performed simultaneously using non-immune serum. Slides were then washed 3 × 5 min with PBS, mounted in ProLongg for 10 min at 4 °C and the supernatant (OS) was removed and kept. Protein content in the supernatant was measured as described below and 600 µg of protein lysate was incubated with 30 µL of anti- HPV Type 16 L1 Monoclonal Antibody (CAMVIR-1) for 20 min at room temperature (r.t.) with end-over-end rotation. The equilibrated spin column was loaded with the lysate and centrifuged at 1000× g for 1 min at r.t. The sample flow-through (FT) was collected and 100 µL of wash buffer added to the column . After centrifuging at 1000× g for a minute at r.t., 6 µL of Tris 1 M pH 8.0 were added to the collection tube and 60 μL Elution Buffer was added to the column and centrifuged again. Finally, the eluted solution (E) was collected for immunoblotting analysis.Co-immunoprecipitation (Co-IP) of AQP8 and HPV L1 protein was performed using Capturem IP & Co-IP Kit , according to the manufacturer’s instructions. Briefly, sperm cells were pelleted, resuspended in 400 µL of lysis buffer and 4 µL of protease inhibitors, and left on ice for 15 min. Cells were centrifuged at 17,000× Immunoblotting was carried out as previously described . BrieflyProtein content was determined with the Bradford method  with bovhttp://www.charmm-gui.org/) was used as a reference to filter out the 987 interaction modes clashing with the lipid bilayer. The remaining 17 poses were ranked according to ROSIE total energy score. The best scoring model was visualized by Pymol  and analyzed by PISA [AQP8 was modelled using the I-Tasser server  and the  by PISA .Q test or Student’s t test. To evaluate the significance of the differences of the means of the semen morphology parameters and of the osmotic water permeability, the Kruskal–Wallis test followed by Dunn’s multiple comparison test was used. All statistical tests were carried out using GraphPad Prism 4.00, 2003 .All data were expressed as mean ± S.E.M. or S.D. (Table of semen parameters). The significance of the differences of the means was evaluated by using one-way ANOVA followed by Newman–Keuls’s The semen characteristics of all subjects included in the study, either normospermic (n = 67) or sub-fertile (n = 27), who attended the clinic because of infertility, are presented in HPV typing of human sperm samples was done by SFP10-LIPA, a robust and sensitive method for the simultaneous detection of several HPV types. Results showed that 47 out of 94 sperm samples were HPV-infected: it was possible to identify the type in only 36 samples, whereas in 11 samples the types were unidentifiable by the test. Multiple HPV types were detected in 7 samples. Twenty-five samples were infected with HR, 6 with IR, 5 with LR and 2 with unclassified HPV types. The most common HR type was HPV 16 (n = 17) followed by HPV 31 and HPV 52 (n = 3), HPV 18 (n = 2) and HPV45, 51, 56 (n.1). Three IR HPV types were found: HPV 53 (n = 3), HPV 73 (n = 2) and HPV 66 (n = 1). The most common LR type was HPV 6 (n = 4) followed by HPV 44 and 81 (n = 1). HPV 67 and 89 (n = 1) were found as unclassified HPV types. Sperm cells act as functional osmometers displaying a rapid swelling when exposed to a hypotonic medium B–E. The p = 0.0171, Student’s t test for paired data).To test whether HPV infection was able to inhibit AQPs function, we measured the osmotic permeability of human sperm cells from both normospermic and sub-fertile subjects with and without HPV infection. HPV localization was determined to verify its possible interaction with AQP3, AQP7, and AQP8. To this end, colocalization experiments were performed. Double label immunofluorescence showed that HPV and AQP8 proteins colocalized in the midpiece of the spermatozoa, as per the resulting yellow fluorescence in merged images A of HPV Colocalization of AQP8 and L1 proteins was quantified using Pearson’s correlation coefficients and intensity profile plots by LAS AF software (Leica Microsystems Application Suite Advanced Fluorescence). Image analysis revealed a Pearson’s correlation coefficient of 0.612 . Figure In contrast, AQP3 and AQP7 staining did not show any overlap of the fluorescence signal with HPV, as per the absence of yellow fluorescence in merged images of AQP3 and 7 (red) and HPV (green) . In someTo confirm the AQP8 and L1 protein interaction, we performed co-immunoprecipitation with anti-L1 protein antibody followed by western blot. L1 protein pull-down resulted in the co-immunoprecipitation of AQP8 A. Bands Given the experimental evidence of colocalization between AQP8 and HPV L1, we tested a potential molecular interaction mode by molecular modelling and docking. A model for AQP8 was generated by I-Tasser; the top scoring model (C-score 0.85) was used as a base for tetramer generation by Z-Dock. The tetramer assembly with the highest similarity to available AQP structures was used for further analysis. Pentameric L1 docking onto AQP8 by ROSIE was followed by manual selection of the 1000 generated models versus a lipid-embedded AQ8 tetramer to exclude docking poses clashing with the lipid membrane. Seventeen models passed this selection criterium. The top-ranking pose  showed an interaction mode where the L1 pentamer is located on top of the AQP8 tetramer C. The in2O2 transport capacity in addition to that of water (peroxiporins) [Several AQPs have been found in human sperm and seem to be involved in volume regulation and, as recently reported, also in ROS elimination . In addiiporins) ,27,28,29iporins) ,31.2O2 efflux to the extracellular fluid can be considered a fundamental ROS removal system, especially in an oxidative stress status [Some studies suggest that HPV infection may have a deleterious effect on sperm quality (and function) possibly leading to male infertility ,16,17,44s status ,22.2O2 removal, we measured osmotic permeability to water, which is an index to that of H2O2 [To address the possible damaging effect of HPV infection in AQP-mediated H of H2O2 ,30. In f2O2 from the sperm cells or their mitochondria. Further investigation and more experiments will be needed to prove this hypothesis and reach a sound conclusion. Recently, in a study performed in the oviparous marine teleost, Sparus aurata, the effect of oxidative stress on the functionality of Aqp8b, an orthologue of human AQP8, was examined [2O2 efflux thus maintaining flagellar motility. Inhibition of Aqp8b led to an insufficient elimination of ROS and to a reduction of ATP synthesis which in turn caused a reduction of sperm motility until its progressive arrest. With all the precautions given that these results were obtained on fish sperms, we could speculate that in humans the inhibition of AQP-mediated H2O2 efflux by HPV could impair sperm function and/or cell number. In fact, according to our molecular modelling and docking simulation results, the L1 protein seems to have a favorite site of interaction in the region located midway of two adjacent monomers of AQP8, with the potential, in its pentameric form, to bind to multiple pore copies. Depending on AQP8 localization, we might speculate that during the infection phase, the whole virus could interact with the AQP8 located on the external surface of the plasma membrane and, during intracellular replication, monomeric or pentameric L1 produced ex novo could form complexes with intracellular  AQP8.The second purpose of this study was to elucidate the interaction between AQPs and HPV by confocal fluorescence microscope and by 3D structural simulation analysis. The results of double label immunofluorescence showed that L1, the major capsid protein of HPV, and AQP8 were highly colocalized in sperm . Apparenexamined . GiltheaAQP3, AQP7, and AQP8 null mice studies show a normal sperm production and a normal motility in response to physiological hypotonicity except for an increased vulnerability to hypotonic swelling occurring after entering the uterus observed in AQP3 deficient mice ,46,47,48The relevance of the peroxiporins (AQP3 and AQP8) in sperm cell functioning of AQP-null mice appears limited. Human male has a relatively low fertility and therefore may be more susceptible to toxic agents (in this case HPV infection and the resulting oxidative stress) for reproduction than male rodents . In thisHPV infection of sperms affects both AQPs expression and functionality. Alterations in water and hydrogen peroxide diffusion capacity are related to a direct interaction of viral L1 protein with AQP8. Thus, our data suggest that HPV reduces AQP8-mediated detoxification mechanism leading to sperm distress and sperm dysfunction.


In sperm, HPV prevalence was 14.8%/IUI cycle. Sperm samples containing HPV had a significantly higher DFI compared to HPV negative sperm samples . When HPV-virions were present in sperm, no clinical pregnancies were observed. More than 1 in 5 of samples with normal semen parameters  had an elevated DFI or was HPV positive. Sperm DFI is a robust predictor of clinical pregnancies in women receiving IUI with this sperm. When DFI exceeds 26%, clinical pregnancies are less likely and in vitro fertilization techniques should be considered.We wanted to determine the sperm DNA fragmentation index (DFI) cutoff for clinical pregnancies in women receiving intra-uterine insemination (IUI) with this sperm and to assess the contribution of Human Papillomavirus (HPV) infection on sperm DNA damage and its impact on clinical pregnancies. Prospective non-interventional multi-center study with 161 infertile couples going through 209 cycles of IUI in hospital fertility centers in Flanders, Belgium. Measurement of DFI and HPV DNA with type specific quantitative PCRs  in sperm before its use in IUI. Clinical pregnancy (CP) rate was used as the outcome to analyze the impact on fertility outcome and to calculated the clinical cutoff value for DFI. A DFI criterion value of 26% was obtained by receiver operating characteristic (ROC) curve analysis. Couples with a male DFI > 26% had significantly less CPs than couples with DFI below 26% (OR 0.0326; 95% CI 0.0019 to 0.5400;  Diagnosis of male infertility has mainly been based on the traditional semen parameters concentration, motility and morphology. It has, however, become increasingly clear that these standard sperm parameters recommended by the World Health Organization  are insuThe pathogenesis of HPV infection in the cervix can be divided in two pathways, a non-infectious, cell transforming, cancer-inducing pathway ,11 and aThe association between DNA damage and diminished reproductive outcomes has led to the introduction of sperm DNA integrity testing into the clinical assessment of male infertility . AlthougIn this prospective multi-center study, we measured both the DNA fragmentation index (DFI) with the sperm chromatin structure assay (SCSA) and HPV DNA with type specific quantitative PCRs in sperm before its use in IUI. Biochemical and clinical pregnancy rates were used as outcome parameters to analyze the impact of these parameters on fertility outcome and to determine the clinical cutoff value for DFI.The study was based on a cohort of 161 consecutive subfertile couples of a prospective non-interventional multi-center study between November 2017 and November 2018. All patients were treated with IUI at the Genk Institute for Fertility Technology, ZOL Hospitals, Genk, Belgium and in the Algemeen Medisch Laboratorium (AML) Intermediate structure for human body material for inpatient obstetrics and gynecology centers, Sonic Healthcare, Antwerp, Belgium. From the 161 subfertile couples (209 cycles), 152 women were inseminated with partner sperm (190 cycles) and 9 women with donor sperm (19 cycles). Consent was obtained to use the sperm rest fractions for DNA fragmentation and HPV testing. The study protocol was reviewed by the Institutional Review Board of the University hospital of Antwerp . All participants signed consent that their rest material could be used for research purposes.Treponema pallidum (syphilis) were measured in all couples as prescribed by Belgian law before receiving any treatment. As part of the infertility workup, we also always tested for the presence of Trichomonas vaginalis, Chlamydia trachomatis and Neisseria gonorrhea by PCR [Included couples failed to conceive for at least 12 months. All patients underwent an infertility work-up prior to IUI treatment, as previously described . For eaca by PCR . Only coa by PCR .® 40/80, Nidacon, Sweden) or capacitation by removing the seminal plasma by washing with sperm preparation medium. Sperm antibodies were detected using the spermMar test kit for IgG and IgA . All rest sperm fractions of capacitations, instead of being discarded, were stored and transported at 4 °C to the laboratory . Twice a week, a DNA extraction was performed .Sperm samples were analyzed using the World Health Organization guidelines for semen analysis within one hour of production . Semen sβ-globin quantitative real-time PCR was utilized to assess the quality of the extracted DNA and to calculate the number of cells [HPV DNA testing was performed as described earlier . We chooof cells . The anaof cells . To calcThe flow cytometric SCSA, performed following the procedure, described previously , measureWe previously described how the patients were prepared for IUI  and how IUI procedures were performed . Out of p) values of <0.05 were considered statistically significant.The results were analyzed with MedCalc Statistical Software  . All IUIOne hundred and sixty-one consecutive infertile couples undergoing 209 IUI cycles were enrolled. Couples characteristics and data concerning capacitated sperm parameters are summarized in p < 0.0001). So a DFI cutoff of 26% was used to determine whether achieving a pregnancy would be very unlikely after IUI treatment  0.66 to 0.79; reatment .p = 0.017). In the latter group, the mean DFI was 13.3%, compared to 41.9% in the former (p < 0.0001). The data of the different sperm parameters according to low (≤26%) or high (>26%) DFI are summarized in p = 0.026). Percentage of progressive sperm  and normal sperm morphology  was also significantly higher in the group with low DFI. The mean age of men with a low DFI was lower than of men with a high DFI , lower IMC (p = 0.0086) and higher DFI (p = 0.0111) and HDS (p = 0.0025).The data of the different sperm parameters according to HPV positivity are summarized in p > 0.05), even when DFI% was below 26% .An overview of the detected HPV types is given in To further assess the impact of the different HPV types on IUI outcome, we plotted the HPV virion per spermatozoon ratio in function of DFI . The medWith routine WHO sperm assessment, the precise etiology of male factor infertility remains undefined in 30–50% of patients ,43. UnliIn this prospective study, we determined the sperm DFI cutoff for clinical pregnancies to be 26%. The significantly lower success rates after using sperm with high DNA fragmentation are in agreement with Bungum et al. (2007) who reported a 10 times increased chance in achieving a clinical pregnancy when DFI was lower than 30% . Additiop = 0.0016) [We previously reported a four times lower clinical pregnancy rate per IUI cycle (2.9%) in women inseminated with HPV positive sperm compared to women who were inseminated with sperm that tested negative for HPV  . The overall 14.8%) and high-risk HPV (9.6%) prevalence in partner sperm per IUI cycle of the current study is comparable to the high-risk HPV prevalence rate in a previously reported study of women receiving IUI treatment % and hig. BecauseHPV infections exert their deleterious effects on fertility through its virions. In previous work, we calculated that the presence of any HPV infection with 0.66 HPV virions or more per spermatozoon did not lead to clinical pregnancy in women undergoing IUI ,49, but A second explanation by which virions exert a negative effect on fertility is that infected spermatozoa can cause arrest in dividing embryonic cells by transferring HPV virions into blastocysts and oocytes . Here, nFinally, since HPV virions can also be produced locally in the infected cervical cells of the partner, these can also bind to spermatozoa on their way to the oocyte. So, as long as active virions are produced by either partner, the odds to get pregnant are jeopardized, and as a consequence, it is only when the infectious HPV lifecycle with production of virions ends in both partners, that the chances for pregnancy normalize. As HPV virion production in non-dividing cells is limited in time , and theIn 81 of the 209 IUI cycles (38.8%) with either the DFI > 26% and or HPV present, the pregnancy rate can be expected to be very low or even zero according to our results. In those cases, IUI should be avoided considering the price and burden associated with IUI which will differ substantially between countries. Studies to examine IVF and ICSI results in this specific DFI and/or HPV positive population are urgently needed to perform a good economic analysis considering the impact of DFI and HPV in artificial reproductive techniques (ART) programs. By including sperm DNA fragmentation analysis as a parameter in the routine diagnosis of male fertility patients in combination with HPV detection, it would be possible to avoid or to postpone unnecessary and unsuccessful IUI treatments, which would prevent a large burden of physical, financial and psychological stress caused by unsuccessful IUI trials. Although detection of infectious HPV virions could explain a substantial part of subfertility in our cohort, the elevated DFI is multifactorial and not exclusive to HPV infection, and can also be caused by other factors like oxidative stress . Importa


During a meeting in Munich, Germany, a presymptomatic attendee with severe acute respiratory syndrome coronavirus 2 infected at least 11 of 13 other participants. Although 5 participants had no or mild symptoms, 6 had typical coronavirus disease, without dyspnea. Our findings suggest hand shaking and face-to-face contact as possible modes of transmission. During the meeting, refreshments were served buffet style in the same room 4 times. In addition to 9.5 hours of discussions, the participants had dinner on February 20 in a nearby restaurant. Additional direct contacts between participants were handshakes during welcome and farewell with few short hugs without kisses. None of the participants, including the index patient (participant [Pt] 1), showed any signs of infection  before or during the meeting. No one wore a mask during the meeting. After the meeting, the index patient (Pt 1) shared a taxi with Pt 2, 4, and 9 for ≈45 min. We describe efficient spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulting from contact with a presymptomatic infected person during a scientific advisory board meeting held February 20–21, 2020, in Munich, Germany; the country had <20 diagnosed coronavirus disease (COVID-19) cases at the time. Eight dermatologists from 7 countries and 6 scientists from the same company attended the meeting at a hotel in central Munich. The meeting was held in a room  (>11 (85%) of the 13 other participants.National authorities contacted most meeting participants on February 26  and 27 ; Pt 14 was contacted by a coworker. Twelve participants, including Pt 1, were tested for SARS-CoV-2 by PCR; 2 were not, Pt 9 because he showed no signs of infection and Pt 6 because testing was not available at his location  at the time. Pt 6 later underwent ELISA testing, which showed IgA and IgG against the recombinant S1 domain of structural protein of SARS-CoV-2  . Thus, tAll participants were isolated either in a hospital or at home with or without their families, regardless of the outcome of the first PCR test. These measures resulted in the subsequent infection of 14 additional persons . Of the The index patient (Pt 1) was most likely infected by an outpatient he had examined in Milan, Italy, on February 18. The index patient reported that he had experienced no symptoms when attending the meeting. Probable transmission of SARS-CoV-2 from presymptomatic persons has been reported  via air flow, fomites, and hand shaking . The indOur findings indicate that hand shaking, aerosolization, and face-to-face contact may be relevant modes of transmission in this COVID-19 outbreak. Limitations include the lack of environmental samples and data about room ventilation and airflow patterns, as well as missing information about the infection status of Pt 9 and the inability to determine the actual impact of SARS-CoV-2 transmission from handshakes, droplets, and aerosolization.


We sincerely ask the national government to change their stance towards the HPV vaccine. We also strongly suggest that, in the meantime, local governments immediately begin to provide an appropriate information of cervical cancer and HPV vaccine to the targeted girls and their parents in a way similar to what Isumi City has now shown to be effective.In Japan, the governmental recommnendation of HPV vaccine has been suspended since June 2013, due to media reports of alleged adverse vaccination events. Although evidence of effectiveness and safety of the HPV vaccine has been universally demonstrated, and the medical and academic organizations across Japan have requested the resumption of the government’s recommendation, the Japanese government has not changed their official stance towards the HPV vaccine. Under the current suspension of the national government’s recommendation, one local government Isumi City started sending a leaflet containing information of cervical cancer and HPV vaccine, but not recommendation for the vaccine, to the tagted girls born in the fiscal year (FY) 2003. The cumulative vaccination rate of them reached 10.07% (14/139), which was significantly higher than that (0.00%) for girls born in FY 2002 who did not receive such a leaflet ( The cervical cancer screening rate in Japan has become extremely low, as compared to other developed countries over the same period. The screening rate for young women at the age of 20–24 is currently only around 10%, and even in females aged 25–29 and 30–39, the screening rate is only around 10–20% and around 10–30%, respectively2. Low inoculation rate of HPV vaccine is another issue to overcome to reduce cervical cancer incidence.The majority of cervical cancer cases can be easily preventable by a combination of HPV vaccination and routine cervical cancer screening. Despite this fact, we have previously shown that the age-adjusted incidence of cervical cancer in Japan has been steadily increasing since 20003. Females born between FY 1994 and FY 1999 have enjoyed rates of vaccination protection approaching 70%, although the vaccination was not free6. In April 2013, HPV vaccination became a part of the routine immunization program, meaning that thereafter females aged 12–16 could receive the vaccination for free. News stories related to cervical cancer prevention such as risk of developing cervical cancer, its etiology, and the effects of vaccination frequently appeared in the media until 2012, however, after March 2013 they were replaced with anti-vaccination contents7. Due to repeated media reports of alleged adverse vaccination events, the Ministry of Health, Labour and Welfare (MHLW) suspended its recommendation for the HPV vaccine in June 2013. For girls born in or after 2000 in Japan, who reached the age for vaccination after suspension of the governmental recommendation, their HPV vaccination rates have been declined, despite the vaccination was still free3. The cumulative vaccination rates for girls born in FY2000, FY2001, FY 2002 and FY2003 were reported to be 14.3%, 1.6%, 0.4% and 0.2%, respectively8. In Japan, parental consent is required for their daughter’s HPV vaccination, and our previous internet survey suggested that acceptance or refusal of the HPV vaccine was determined predominantly by the mother's perceptions of risk versus benefits, rather than the daughter's wishes in Japan9.Financial support from the Japanese government for HPV vaccination for girls aged 13–16 began in the fiscal year (FY: April–March) 201010 but also in Japan12, and safety of HPV vaccine has been also internationally and domestically demonstrated14. The medical and academic organizations across Japan have requested the resumption of the government’s recommendation;15 nevertheless the MHLW has not changed their official stance towards the HPV vaccine. Although the HPV vaccine is still regarded as a ‘routine vaccination’ in Japan, the national government still does not ‘officially recommend’ it. In addition, neither more potent 9-valent HPV vaccine nor immunization for boys have been approved yet in a national routine immunization program16. Moreover, seven years have now passed since the announcement of the governmental suspension of the HPV vaccine recommendation. We have already reported that Japanese mothers’ intentions to have their daughters vaccinated have been further eroded over this period17.Protective effect of HPV vaccine against cervical cancer or its precancerous lesion of the cervix has been shown not only from overseas1. A recent survey conducted by the MHLW found that only 5.6% of all local governments across the nation (97/1741), under the current suspension of the government’s recommendation, sent leaflets containing only information of HPV vaccine, but not recommendation for the vaccine, individually to the girls and their parents18. In the present study, we have analyzed the effectiveness of one type of leaflet that one local government, not on a national basis, has been sending to individuals of HPV vaccination age or to their parents under the governmental suspension of the HPV vaccine recommendation.Under these dire circumstances, we must find a way to re-disseminate HPV vaccination nationwide as a countermeasure against the recent increase of cervical cancer among young women19. On July 29, 2019, the Isumi City government started sending an informational/educational leaflet . We conducted the present study in accordance with relevant guidelines and regulations.Under the current suspension of the national government’s recommendation of HPV vaccine, providing an appropriate information of cervical cancer and HPV vaccine to the targeted girls and their parents from a local government was shown to be effective.When the Japanese government was actively promoting the vaccine, the cumulative rates of the first (of three) vaccination shots in the targeted girls born between FY 1994 and FY 1999 substantially increased from 58.60 to 94.67% Table . Howeverp = 0.003). Subsequently, the cumulative vaccination rate for girls born in FY 2003 in Isumi City reached 10.07% (14/139) by December 31, 2019, which was significantly higher than that (0.00%) for girls born in FY 2002 who did not receive such a leaflet (p < 0.001) , while it did not show significant difference from the rate 0.00% (0/148) for the girls born in FY 2002 (p = 0.34). Moreover, in 2019, 3 girls born in FY 2004–2007 received the HPV vaccine between April and July without information leaflet, and another 3 girls received it between August and December, however the cumulative vaccination rate did not increase significantly throughout the year .On the other hand, out of 489 girls born later in FY 2004–2007, who were also the targets of HPV vaccine but did not receive the individual notification/leaflet, only 6 girls had been vaccinated by December 31, 2019. This proportion 1.23% (6/489) for those born in FY 2004–2007 was significantly smaller than the rate 10.07% for the girls born in FY 2003 who received the leaflet (14/139)  regarded vaccine hesitancy, the reluctance or refusal to vaccinate despite the availability of vaccines, as one of the 10 threats to global health in 201923, unless the government's recommendations for HPV vaccination are reinstated. We sincerely ask the national government to change their stance towards the HPV vaccine. We also strongly suggest that, in the meantime, local governments could immediately begin, even if it is still under the current circumstances of the suspension of the governmental recommendation, to provide an appropriate information of cervical cancer and HPV vaccine to the targeted girls and their parents in such a way that Isumi City has now proven it useful. Up until now, there have not been any other reports to demonstrate the effectiveness of sending an information leaflet by local governments in Japan.Sending individually an information leaflet about cervical cancer and HPV vaccination from a local government to the targeted girls and their parents appeared to be effective for promoting HPV vaccination. By the first of FY2020, females born in FYs 2000, 2001, 2002 and 2003 were already over 12–16, the targeted ages for the vaccine. Future risk of cervical cancer for girls born in FY 2004 will significantly increase in FY 2020 when they become 16 years old, which is the last year for routine HPV vaccination17. Moreover, 45.0% of the targeted girls and 38.4% of thier mothers replied that they could not decide whether to receive the HPV vaccine because they did not have adequate knowledge about HPV vaccine well. In Japan, mothers play more important roles for decision of their daughters’ vaccination9. Fathers' participation in the mothers' decision-making did not increase the likelihood of HPV vaccination for their daughters, however, an educational leaflet was proved to be effective to enhance fathers’ enrollment for decision of their daughters’ vaccination24.A previous internet survey conducted by the MHLW found that 38.8% of the tagted girls had not heard of significance and safety of HPV vaccine at all, and that 33.8% of the mothers of the targeted girls wanted to get appropreate information at the consultation counter of a local government25. Improvement of the contents of leaflets by taking viewpoints of behavioral economics might further increase HPV vaccination rates21. There was a report that secondary acceptance of HPV vaccination by the parents of adolescents is more common than we might suppose26. We could improve vaccination rate more if we consider this possibility and repeat providing the leaflets. We suppose that this is the last strategy for re-dissemination of HPV vaccination in Japan while still under the suspension of the governmental recommendation We will no longer overlook this situation.We believe that providing an appropriate information of cervical cancer and HPV vaccine to the targeted girls and their parents, which can be conducted under the current circumstances, is a potentially effective tool to support decision-making of HPV vaccination for the targetted girls and their mothers, and it is educational as well for their knowledge and attitudes toward cervical cancer. It was demonstrated that effectiveness was enhanced when doctors explained about HPV vaccine using with informational leaflet, probably due to proper opportunities to understand the content well and ask questionsSupplementary Figure.


In general, human papillomavirus (HPV) vaccines have demonstrated efficacy in young women worldwide, but there is limited evidence on the efficacy of the quadrivalent HPV6/11/16/18 vaccine in adult women and no evidence of its effectiveness in Japanese adult women in particular. This study aims to evaluate the efficacy of the quadrivalent HPV6/11/16/18 vaccine for persistent HPV16/18 infection in Japanese women aged 27–45 years.This is an interventional, nonrandomized, non-double-blind prospective cohort study designed to compare the rates of persistent HPV16/18 infection between the vaccinated arm and unvaccinated arm. The subjects will consist of all women aged 27–45 years who have normal cytology results confirmed by cervical cancer screening from May 2019 to March 2021. The follow-up time is two years. The subjects will be divided into two groups: the vaccinated group and the unvaccinated group. The study will need to enroll 600 vaccinated participants  and 2200 unvaccinated participants (control arm).The findings of this trial (HAKUOH study) might provide the first local evidence on the subject and be significantly useful not only to medical academia but also to the Japanese Ministry of Health, Labour and Welfare. The findings could contribute to public health improvement by providing local supportive knowledge on the prevention of HPV infection through HPV vaccination in young adult women in Japan, where active recommendations have been suspended for a long time due to adverse effects.NCT04022148.Trial registration number: Registration began on December 1, 2019. In April 2013, the Ministry of Health, Labour and Welfare (MHLW) started a national vaccination program for women aged 12–16 years in Japan. However, human papillomavirus (HPV) vaccinations were reportedly associated with serious adverse events, and in June 2013, the MHLW announced the suspension of the active recommendation of HPV vaccination. Since then, the vaccination rate in Japan has declined drastically from approximately 70% in 2013 to 1% in 2018 [The very low vaccination rate in Japan has attracted great attention among different institutions. The Japanese Society of Obstetrics and Gynecology, Japanese Society of Gynecologic Oncology, and Japanese Association of Obstetricians and Gynecologists have advised the MHLW to re-implement their strong recommendation for HPV vaccination. The Global Advisory Committee on Vaccine Safety also stated no reason to suspend HPV vaccination or its active recommendation. However, their advice has not led to the MHLW reversing their decision to suspend the active recommendation , 3, and A previous study showed that the HPV infection rate will increase for every 1-year delay in resuming the active recommendation . This fiAdult women are not covered under the national immunization program of Japan due to cost-effectiveness concerns, which might be attributed to the high prevalence of persistent HPV16/18 infection in this population. In this study, we will focus on women with normal cytology results because we verified that approximately 99% of the women with normal cytology results have HPV16/18 negative results, as described in our previous study  . This stThis is an interventional, nonrandomized, non-double-blind prospective cohort study that aims to compare the rate of persistent HPV16/18 infection between the vaccinated arm and unvaccinated arm, as shown in Fig. The subjects will consist of all women aged 27–45 years who have normal cytology results confirmed by cervical cancer screening programs from May 2019 to March 2021. All women will have Japanese nationality and live in the Fukui Prefecture of Japan. In the Japanese cervical cancer screening program, the government covers the cost of cytology tests. Women are screened every 2 years. The uptake rate for cervical cancer screening in Japan is approximately 40%, with no regional differences. The data collection began in December 2019. The follow-up time will be 2 years because in the Japanese cervical cancer screening program, women are screened every 2 years. The inclusion criteria are as follows: normal cytology results confirmed by cervical cancer screening from May 2019 to March 2020, aged 27–45 years, an intact uterus, and willingness to undergo the HPV-DNA test (cobas4800) within 12 months. The exclusion criteria are as follows: pregnancy, treatment or follow-up evaluation for CIN within the previous 12 months, and previous administration of the HPV vaccine. The subjects will complete the questionnaire about sexual behavior and whether or not they were previously tested for HPV. The subjects will be divided into two groups: the vaccinated group and the unvaccinated group. In the experimental arm comprises women who have been vaccinated three times in 1 year after NILM confirmation by cytology. These women will participate in the cervical cancer screening program after 2 years. We will obtain a residual part of the sample for cytology, and the sample will be used for the HPV test. In women with HPV16/18-positive cells, p16 and Ki67 expression will be analyzed to judge persistent infection. The control arm will comprise women who participate only in the cervical cancer screening program after 2 years. We will obtain a residual part of the sample for cytology, and the sample will be used for the HPV test. In women with HPV16/18-positive cells, p16 and Ki67 expression will be analyzed to judge persistent infection. Subjects in 11 hospitals/clinics who consent to be vaccinated will be enrolled in the vaccinated group and will receive the quadrivalent HPV6/11/16/18 vaccine (Gardasil®) at day 1, month 2, and month 6. Subjects who refuse to be vaccinated will be enrolled in the unvaccinated group.The primary efficacy end point is the incidence of persistent HPV16/18-related infection over 2 years. The secondary efficacy end point is twofold: the incidence of persistent HPV16/18-related infection over 2 years by age group (27–30/31–35/36–40/41–45 years) and the consent rate for vaccination by age group (27–30/31–35/36–40/41–45 years). Women in both arms will receive routine cytology screening and an HPV-DNA test  2 years after the first visit. Among women with HPV16/18-positive cells in both groups, p16 and Ki67 expression will be analyzed using the CINtec® PLUS kit (Roche Diagnostics). Persistent infection is defined as cases in which there are HPV16/18-positive cells overexpressing p16 and Ki67 [P-values of < 0.05 will be considered statistically significant. All statistical analyses will be carried out using SPSS version 25.Data for HPV16/18 infection, cervical lesions and potential confounders  from all subjects will be included in the analysis. The association among all data will be analyzed. The chi-squared test will be used to compare the incidence of persistent infection between the vaccinated and unvaccinated groups. Based on the assumed incidence rate of persistent infection among adult women (0.9 per 100 person-years) and an assumed risk reduction of 80% [Previous studies have confirmed the prophylactic effect of HPV vaccination on cervical cancer in females aged 9–26 years. The FUTURE study showed that the quadrivalent HPV6/11/16/18 vaccine reduced the incidences of HPV16- and HPV18-related cervical precancers and cervical cancer in women aged 16–26 years . The stuAs mentioned above, adult women are not covered under the national immunization program of Japan due to cost-effectiveness concerns related to high HPV16/18 prevalence. In this study, we will enroll only women with normal cytology results because our previous study (FCCS study) revealed that women from Fukui Prefecture with normal cytology results were basically negative for HPV16/18 .As the Japanese cervical cancer screening program uses only cytology without HPV-DNA testing, there are no available data on the detection of HPV16/18 DNA. However, the FCCS study has shown that the HPV16/18 infection rates were significantly lower among those with normal cytology results than among those with abnormal cytology results (1.0% vs. 22.0%). Therefore, the present study will target women with normal cytology results .While young adult women aged 27–45 years are not covered under the national immunization program, they have the chance to visit gynecologists periodically to undergo cervical cancer screening provided through the national program. This regular visit is a suitable opportunity for vaccine recommendation, as normal cytology results still indicate susceptibility to HPV infection, and during the visit, the necessity of vaccination can be conveyed by the physician directly to the patient, not to her parents or guardians.A possible limitation of the study is that the study design involves a nonrandomized, and not randomized, intervention. If the study were to adopt a randomized intervention design, we speculate that the participation of the patients cannot be assured under current circumstances in Japan, where the MHLW has suspended its strong recommendation for HPV vaccination.The findings of this trial might provide robust local evidence supporting HPV vaccination among young adult women aged 27–45 years who show negative cytology results. By utilizing the data from this study, we intend to develop a platform covering both cervical cancer screening and HPV vaccine recommendations for those proven to have negative cytology results. This new platform might serve as an effective means to drive HPV vaccination among those not covered under the national immunization program of Japan and raise questions about the rigid state of the 5-year national immunization program. This study could provide local supportive evidence for preventing HPV infection through HPV vaccination in young adult women and thus contribute to public health improvement in Japan, where active recommendation of HPV vaccination has been suspended by the MHLW for a long time.


INK4a, is used in clinical practice to determine the high-risk human papillomavirus (HPV) status of oropharyngeal squamous cell carcinomas (OPSCC). With a specificity of 83%, this will misclassify some patients compared with direct HPV testing. Patients who are p16INK4a-positive but HPV DNA-negative, or RNA-negative, may be unsuitable for treatment de-escalation aimed at reducing treatment-related side effects. We aimed to identify cost-effective serum markers to improve decision making for patients at risk of misclassification by p16INK4a alone.The surrogate immunohistochemical marker, p16INK4a/HPV DNA and E6/E7 mRNA. Serum protein expressions were compared between groups of patients according to HPV status, using the unpaired t-test with a Benjamini-Hochberg correction. ROC curves (AUC) were calculated with SPSS (v25).Serum proteins from pre-treatment samples of 36 patients with OPSCC were identified and quantified using label-free mass spectrometry-based proteomics. HPV-status was determined using p16Of 174 serum proteins identified, complement component C7 (C7), apolipoprotein F (ApoF) and galectin-3-Binding Protein (LGALS3BP) significantly differed between HPV-positive and -negative tumors (AUC ranging from 0.84–0.87). ApoF levels were more than twice as high in the E6/E7 mRNA HPV-positive group than HPV-negative.Serum C7, ApoF and LGALS3BP levels discriminate between HPV-positive and HPV-negative OPSCC. Further studies are needed to validate these host immunity-related proteins as markers for HPV-associated OPSCC. Human papilloma virus (HPV) is an established aetiological factor for oropharyngeal squamous cell carcinoma (OPSCC), with nearly 90% of HPV-positive OPSCCs being caused by the high-risk genotype HPV16 , and it INK4a as a surrogate immunohistochemical marker for HPV status [INK4a testing has a sensitivity of 94% [INK4a, is tumor suppressor gene through the inhibition of the catalytic activity of the cyclin D1/CDK4/6 complex. The cyclin D1/CDK4/6 complex phosphorylates the retinoblastoma protein (pRb), resulting in the release of the transcription factor E2F and the initiation of cell-cycle progression. Conversely, the pRb/E2F repressor complex inhibits transcription of several genes, including CDKN2A [INK4a overexpression, and it accumulates within the cells [INK4a has a specificity of 83% [Ink4a in head and neck carcinomas has been explained by mutation or amplification of CDKN2A or by mutations in RB1 or histone H3 lysine 36 methyltransferase genes [The difference in the tumor behavior is reflected in the latest WHO classification of head and neck tumors, in which HPV-positive and HPV-negative OPSCCs are classified as distinct clinical entities, using the overexpression of p16V status , 14. Accy of 94% . CDKN2A,he cells . Despitey of 83%  as it isy of 83% . Frequense genes .INK4a and HPV DNA detection methods, alone and combined, and suggested that p16INK4a immunohistochemistry (IHC) combined with HPV DNA PCR testing has an enhanced specificity (96%) while maintaining high sensitivity (93%) [INK4a positivity also provided the strongest diagnostic accuracy as well as prognostic value in OPSCC [INK4a surrogate marker is used in the clinic to determine whether the OPSCC is HPV-positive or HPV-negative. However, there is a specific subgroup of OPSCCs, which are p16INK4a -positive but negative for HPV DNA, and these have been reported to have distinctive clinical and morphological features, and a worse prognosis and increased risk of distant metastases than those positive for both p16Ink4a and HPV DNA [The accepted gold standard method for the detection of HPV with an aetiological role in OPSCC is testing for E6/E7 mRNA, based on the assumption that E6 and E7 oncogenes are persistently transcribed in HPV-associated carcinomas . Howeverty (93%) . Mena etin OPSCC . Current HPV DNA , 23.At present, there is a consistent misclassification of some patients with OPSCC, as no method for determining the HPV status of an OPSCC is completely accurate. Therefore, we set about identifying serum proteins that could be simply and cost-effectively tested, to improve the detection of mis-classified patients.INK4a, DNA and E6/E7 mRNA statuses: E6/E7 mRNA -positive vs -negative, and p16INK4a/DNA -positive vs -negative. The aim of this was to identify serum proteins that could discriminate between the p16INK4a+ OPSCCs that are HPV-associated, and those that are p16INK4a+ but HPV-independent.To this end, we have used a label-free quantitative mass spectrometry methodology to identify the serum proteome in patients with OPSCC, comparing the protein expression levels between patients according to p16Fig 1.The overall workflow is shown in S1 Table explains which data files were used in this paper.It is work noting that the serum proteomics data is from an overarching serum proteomics mass spectrometry project and overlaps with another cohort that has previously been published by Tuhkuri et al. , TuhkuriThe data from HPV DNA detection, p16 and RNA scope on these patients has also been published elsewhere by Randen-Brady et al.   samples, were collected from 36 patients with both of these samples available, diagnosed with OPSCC between the years 2012 and 2016 at the Department of Otorhinolaryngology–Head and Neck Surgery, Helsinki University Hospital, Helsinki, Finland. Clinical data were collected from patient records. The UICC  8fication  was usedAfter being allowed to clot at room temperature, blood samples were centrifuged at 4°C at 1000xg for 10 minutes in order to separate the serum. Sera were aliquoted and stored at -70°C, until they were all assayed at the same time.The study plan was approved by the Helsinki University Hospital’s institutional Research Ethics Board (DNr. 51/13/03/02/2013). Informed written consent was obtained from all patients. The study was performed in accordance with the latest version of the Declaration of Helsinki.INK4a was detected immunohistochemically on FFPE tissue samples on individual slides as previously described [INK4a . If over 70% of tumor cells were strongly immunopositive for p16INK4a, the tumor was defined as p16INK4a positive [p16escribed , 27. Bripositive . As a poAn HPV PCR assay was used to detect HPV DNA in the tissue samples. Firstly, DNA was extracted from 4 to 10 μm diameter sections cut from formalin fixed paraffin embedded (FFPE) tissue sections using the high salt method as described earlier . For HPVIn situ hybridization (ISH) for high risk HPV E6/E7 mRNA was performed manually using the RNAscope® 2.5 HD Reagent kit  according to the manufacturer´s protocol as described also in detail in our recent papers , 31. FivThese methods have been described previously in detail  and alsoS1 Table explains which data files were used.This was performed as previously described in detail  and alsoThe data analysis has been described previously in detail . BrieflyThe following comparisons between the samples’ protein abundances were performed:E6/E7 mRNA+ (n = 13) vs E6/E7 mRNA- (n = 18)INK4a-/DNA- (n = 16) vs p16INK4a+/DNA+ (n = 11)p16INK4a+/DNA- (n = 5) vs p16INK4a-/DNA- (n = 16)p16INK4a-/DNA+ (n = 4) vs p16INK4a-/DNA- (n = 16)p16INK4a-/DNA+ (n = 4) vs p16INK4a+/DNA+ (n = 11)p16INK4a+/DNA- (n = 5) vs p16INK4a+/DNA+ (n = 11)p16The data were imported into EZinfo 3.0.3.0 , a statistical package that works with Progenesis QI for proteomics. The quantified proteins in the comparisons were compared using an unpaired t-test on a protein-by-protein basis, with a p-value of 0.05 being the cut-off for statistical significance. The Benjamini-Hochberg multiple testing correction was performed, with an FDR cut-off of 0.1. ROC curves were calculated using SPSS (version 25). All samples that were positive for HPV DNA contained HPV16 DNA, except for one sample that was HPV18 positive; this tumor was p16INK4a- but E6/E7 mRNA+.Serum from 36 patients with newly diagnosed OPSCC was analyzed. Clinical parameters are shown in S1 Fig. HPV DNA results are shown in S3 Table.Representative images of p16+ and p16-, E6/E7 mRNA+ and E6/E7 mRNA- are shown in Table 1 shows the significant proteins after multiple testing correction, between HPV-positive and HPV-negative as tested using E6/E7 mRNA and p16INK4a/DNA, respectively. The other comparisons, which included samples with discordant p16INK4a/DNA yielded no significant proteins. S4 Table details all of the identified proteins for each comparison.A total of 174 serum proteins with 2 or more unique peptides were identified and quantified by mass spectrometry. E, we aimed to identify serum proteins that could be easily and economically implemented to make a differential diagnosis between HPV-associated and HPV-independent OPSCCs. This approach would improve the detection of patients whose OPSCCs are mis-classified with regards to HPV status by p16INK4a testing. For this, we compared the proteomic differences using different proxies for HPV: E6/E7 mRNA, and p16INK4a combined with HPV DNA. We identified three proteins  and complement component C7 (C7) that differentiate between HPV+ and HPV- tumors. C7 was the only protein that differentiated between HPV+ and HPV- tumors using either p16INK4a/DNA or E6/E7 mRNA as HPV detection methods.In this study, using nUPLC-UDMSper se.ApoF is a sialoglycoprotein, also known as “lipid transfer inhibitor protein”, and is an inhibitor of cholesterol ester transferase that binds to LDL, and regulates cholesterol transport . Lipid mINK4a/DNA proxy (Table 1). C7 is a critical component of the terminal pathway of complement activation. C7, along with complement components 6, 8, and 9, is involved in membrane attack complex (MAC) formation [We identified significantly different expression of Complement component C7 using either the E6/E7 mRNA proxy or the p16ormation . Sublytiormation . These pormation .Thus, similarly as with ApoF, these increased serum concentrations of C7 may result from local upregulation of complement genes in primary tumor sites, mainly by endothelial cells and cells participating in immune responses i.e. the integral component of both the premetastatic tumor niche the tumor microenvironment. Elevated serum levels of C7 have been linked to a better prognosis in ovarian and non-small cell lung cancer (NSCLC). Additionally, overexpression of C7 was shown to inhibit the colony formation of NSCLC cells, which indicates that C7 might be a potential tumor suppressor .α, IFN-β, IFN-γ, TNF-α, poly(I:C), dsRNA, and dsDNA”, as thoroughly reviewed by Loimaranta et al. [LGALS3BP is a secreted glycoprotein that was originally identified during investigations into different malignancies. Furthermore, the expression of LGALS3BP is upregulated in both chronic and acute viral infections, perhaps due to its induction by a variety of molecules that “either mimic or are characteristic for an ongoing inflammation and microbial infection, such as IFN-a et al. . As one a et al. . Thus, ia et al. –49.INK4a and HPV statuses, to identify possible serum biomarkers that can improve classification of patients who would be misclassified when using p16INK4a or HPV DNA testing.The main strength of the study is that, to our knowledge, this the first time that the serum proteomes have been assessed and compared in OPSCC patients with different p16per se. However, it remains to seen whether these proteins are specific enough for HPV or whether they are reflecting also other viral associated malignancies.Importantly, all the identified three proteins could be linked also immunity and inflammatory responses also of importance for HPV Limitations of this study include small group sizes. As a consequence of this, and insufficient smoking data, we were unable to compare the proteomic signatures of those with a <10 year smoking and >10 year smoking history, and different disease stages, which are known to affect the prognosis of these tumors , 51.We have identified three interesting serum proteins with the potential to improve stratification of OPSCC patients. Serum levels of C7, APOF and LGALS3BP differentiate between HPV-positive and HPV-negative OPSCC. Further research is warranted to confirm these findings on a larger cohort.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S1 Fig(EPS)Click here for additional data file.


Since the introduction of HPV vaccines, several studies have been conducted in different countries to assess HPV knowledge and vaccine acceptance. The aim of this study was to perform a systematic literature review to summarize results and identify factors associated with HPV knowledge and vaccine acceptance in adolescents and their parents and to compile the measurement tools used in the published research studies performed in European countries where HPV is licensed.A systematic literature review was conducted for studies published between January 1st 2006 and December 31st 2017.Seventy non-interventional studies performed in 16 European countries met the inclusion criteria. Thirty-eight of them reported data on HPV knowledge and 40 reported data on HPV vaccine acceptance. Further, 51.8% of adolescents (range 0% to 98.6%) and 64.4% of parents (range 1.7% to 99.3%) knew about HPV infection. Insufficient information and safety concerns were the main barriers to vaccination acceptance.HPV knowledge and vaccine acceptance are still modest and vary widely between studies across EU countries. Coordinated efforts should be made to provide the relevant population with information for informed decision-making about HPV vaccination. Human papillomavirus [HPV] infection is one of the major causes of infection-related cancer worldwide and is the causal factor in other diseases such as genital warts or recurrent respiratory papillomatosis . More thApproximately, 80% of sexually active individuals will be infected by HPV during their lifetime . Most ofCurrently, there are three licensed HPV vaccines in the European Union (EU): a bivalent, including HPV types 16 and 18, approved in 2006; a tetravalent, including HPV types 6, 11, 16, and 18, authorized in 2006; and a nonavalent vaccine, including HPV 6, 11, 16, 18, 31, 33, 45, 52 and 58 that was licensed in 2015 . More reDespite these advances, coverage rates of HPV vaccination programs differ widely between countries . To addrSince most HPV vaccination programs target mainly young adolescents, parents have the authority to take most decisions about vaccination. Therefore, the success of HPV vaccination programs will largely depend on parental decision-making . A comprThe concept of knowledge commonly includes the awareness about “who, where, and when” one should be vaccinated and self-estimated sufficiency of information about vaccination or satisfaction with information on vaccination . While aSince the introduction of HPV vaccines, several studies have been conducted in different countries to assess HPV knowledge and vaccine acceptance.At the end of 2017, two systematic reviews explored factors related to the uptake of vaccination programs in the EU , 17. TheThe aim of this study was to perform a systematic literature review to identify factors associated with HPV knowledge and vaccine acceptance in adolescents and their parents, to summarize the results for both outcomes, and to compile the measurement tools, items, and questionnaires used in published research studies performed in European countries where HPV vaccines are licensed.We reviewed all the scientific literature published between January 1st, 2006 and December 31st, 2017 to identify studies evaluating parental and/or adolescent HPV knowledge and/or acceptance of HPV vaccination. Our search was limited to studies targeting populations from European countries where HPV vaccines were licensed when this protocol was written to ensure homogeneity as far as common regulation  among the included countries and common vaccination recommendations  .Population: only studies performed in parents of children of any age under 19 years or an adolescent population defined as individuals aged 9–18 years old living in a European country where HPV vaccines were licensed were included.Intervention: not applicable.Comparator: results regarding HPV knowledge and acceptability of HPV vaccination were recalled, whenever possible by sex of the respondent, sex of the target child, and country.Outcomes: a study was regarded as measuring knowledge of HPV if it assessed a set of true/false, yes/no or any other format of questions that could be translated into a scoring system showing the knowledge and understanding of how the virus is spread, what conditions result from HPV infections and how HPV can be prevented. A study was regarded as measuring acceptance of HPV vaccination whenever it somehow evaluated a positive or negative intention or willingness toward vaccinating children (girls or boys) or oneself (in the case of adolescents) in the future (vaccine intention); or having consented or not to vaccination their children or oneself (in the case of adolescents) in the past. In addition, drivers for accepting HPV vaccination and barriers and reasons to refuse it were recorded and analyzed.Study design: quantitative survey research studies published as original articles were included. Reviews, editorials, and gray literature  were not included in the search.Time: only studies published in English during the last 11 years were included (2006–2017).Studies whose primary outcomes were HPV knowledge and/or acceptance of HPV vaccination were included. Following PICOTS, we defined the following criteria for study selection:The search strategy used appropriate keywords, medical subject heading, and free-text terms for the following concepts: “human papillomavirus AND [survey OR questionnaire OR assessment] AND [knowledge OR acceptance OR attitudes] AND vaccine.”A combination of text words and MeSH Terms was defined with a medical librarian and after several preliminary manual test searches.The following principal sources of electronic reference libraries were searched to access the available data: The Cochrane Library, Medline through PubMed, EMBASE, and Popline. An exploratory search in Google and Google Scholar was also made to avoid any publication bias.We also included the World Bank Library Network, although we did not obtain relevant publications from this source.The list of search queries used per bibliographic source is provided in the The titles of all studies identified were screened independently by two reviewers and duplicates were removed. Titles were screened for inclusion and abstracts were further reviewed based on eligibility criteria. Any disagreements on selection of studies between the two primary reviewers were resolved by an expert committee comprised of four expert pediatricians in HPV and vaccination . Following retrieval of the full texts of all the studies that met the inclusion/exclusion criteria, data confirming these criteria were extracted from each study by the two reviewers on a standardized abstraction sheet. Any disagreement on the final selection of studies to be included in the review was resolved by the expert committee.Study quality was assessed by two independent reviewers using the Mixed Method Assessment Tool (MMAT) developed by Pluye et al.  see Sup to identData extraction was performed by the lead reviewer using a bespoke form. An additional reviewer checked a random sample of 15% of the data records to detect possible extraction errors.The form’s suitability was assessed by performing a pilot extraction of three studies selected on the basis of the diversity of their content and design. Both the lead reviewer and the additional reviewer performed the pilot extraction independently. Results and completion difficulties were subsequently compared to improve the questionnaire accordingly. The final data extraction form used is included in the Items were classified under headings and subheadings based on their conceptual meaning. Main headings were knowledge about HPV, knowledge about HPV vaccine, acceptability of vaccines in general, and acceptability of HPV vaccine. The complete list of subheadings defined under each heading is shown in first column of Table For each individual item reported by the studies included, the literal item text, the reported number, and percentage of participants choosing the response answer that implies knowledge  and/or acceptability , respectively was recorded in a database.For each heading and subheading, the number of items reporting data, sum of the studies’ sample size, sum of the number of participants answering each item as previously defined, computed pooled percentage , arithmetic mean of the percentages of the items included in the heading, minimum reported percentage, and maximum reported percentage for each conceptual heading in the original study were obtained or calculated.p values (depending on availability) reported in the included studies that presented a statistical association with HPV knowledge and/or with the acceptance of HPV vaccination. Total number of studies studying each factor is reported and whether the analysis of the relationship was multivariate or bivariate.To collect the information on factors associated with HPV knowledge and/or HPV vaccine acceptance, reviewers extracted the data of odds ratio, beta coefficients, or A total of 2118 publications were identified: 609 were retrieved from PubMed, 206 from Cochrane Library, 1124 from EMBASE, 141 from the World Bank Library, and 38 from Popline. The exploratory search in Google and Google Scholar did not yield any new peer-reviewed publication not already included.Duplicates were excluded and 1666 publications were retained for title and abstract-based screening. Of these, 1516 were excluded according to selection criteria. Only 150 were selected for full-text review. Eleven of them were excluded because the aim of the study was not consistent with the objective of this systematic review, ten were not original papers providing primary data, one was conducted in a country where HPV vaccines were not commercialized, 18 did not provide segregated data for the target population of this review or were conducted in a different target population, 31 studies were performed in non-European countries, four were published in a language other than English, and three were additional duplicates.Thus, 72 studies that met the inclusion criteria were finally included, although two had to be excluded after quality evaluation with MMAT. Ultimately, 70 publications were included for further analysis. See PRISMA flow diagram Fig. , list ofAll 70 publications were non-interventional studies, 14 were qualitative studies and were therefore not included in the quantitative synthesis, although they were used to guide the conceptual framework to classify the items of quantitative studies under headings and subheadings. Items used across studies to measure this knowledge were divided into two main headings: knowledge of HPV and knowledge of HPV vaccine. We also included a third heading for “other” information, such as knowledge of sexually transmitted infections (STI). Items related to information source were also classified see .Thirty-eight publications reported data about HPV knowledge and 40 about HPV vaccine acceptance. Of them, 20 and 37 studies, respectively, reported quantitative results about factors influencing HPV knowledge and HPV vaccine acceptance.In terms of geographical distribution, publications included in this review were produced in 16 European countries, the UK having most publications, 20 (28.6%), followed by Italy, ten (14.3%), and Sweden, eight (11.4%). There was only one pan-European study involving four countries: France, the UK, Germany, and Italy.Twenty-nine studies were conducted on adolescents aged between 9 and 21 years ; 36 were conducted in parents of adolescents (12 in mothers only and 24 in in both parents). A summary of the characteristics of studies included in the systematic review is provided in Table Thirty-eight studies reported data results on HPV knowledge. Table Between 1.1%  and 94.5Studies performed in Finland were the ones showing the highest overall knowledge of HPV among parents; around 79% of Finnish respondents answered correctly to items related to HPV knowledge, followed by studies in the UK and Germany . The lowest percentage was in The Netherlands (37.9%). Among adolescents, Belgium studies showed the highest percentage of HPV knowledge (93%), followed by Italian’s (66.6%), and the lowest was in Sweden (10.9%). HPV vaccination knowledge varied also between 6.2 and 90.6% in parents, being highest in the UK and lowest in the Netherlands studies; and between 10.1 and 87.1% in adolescents, being highest in Belgium and lowest in Latvia  (ranging from 2.0%  to 27.5%Forty studies reported data on HPV vaccine acceptance. Table The percentage of adolescents with positive intention to be vaccinated against HPV was 64.3% (pooled percentage), ranging from 45.6%  to 79.5%The percentage of parents with positive intention to vaccinate their children against HPV was 59.2% (pooled percentage) ranging from 32.2%  to 65.6%By countries, studies conducted in Nordic countries like Sweden and Iceland presented the highest percentage of vaccine acceptance in adolescents and parents, respectively Fig. .Fig. 3IBetween 7.7%  and 32.5Among parents, between 7.9%  and 68.1In contrast, 7.7%  to 43.0%The main sociodemographic factors associated with HPV knowledge in adolescents and their parents identified in this analysis were female gender (nine studies out of ten studying this factor), extent of higher education (five studies out of six), and higher income groups (one study out of two). Regardless of the investigators’ questions about HPV knowledge, the percentage of female adolescents knowing about HPV is consistently higher than that of boys: ranging from 16.4%  to 92.8%Adolescent’s age at first sexual intercourse, age of respondent parents, and religion were also identified in several publications as being related with HPV knowledge, although results are discrepant.Additionally, being vaccinated against HPV or having a vaccinated older sister were also positively associated with levels of HPV knowledge.A complete list of factors associated with HPV knowledge is included in the Up to 80 factors presented a statistically significant association with HPV vaccine acceptance in at least one of the studies included in this systematic review: 21 were sociodemographic or family characteristics, 37 factors were drivers, and 22 were barriers to vaccine acceptance.Within demographic factors, female gender and younger age of respondent parent, female gender of the adolescent, higher household income, and previous childhood vaccinations are the ones most consistently associated with HPV vaccine acceptance.Drivers associated with HPV vaccine acceptance in the studies were belief in vaccine efficacy (eight studies out of eight studying this factor), existing awareness of HPV (six studies out of six), belief that HPV vaccine prevents cervical cancer (six studies out of six), susceptibility to HPV infection (four studies out of five), receiving information from the doctor (four studies out of four), desire to fit in social norms (four studies out of four), perception of disease severity (three studies out of three), and intention to do Pap test (three studies out of three).The most frequently identified barriers to HPV vaccine acceptance were doubts about HPV vaccine safety profile (12 studies out of 12) followed by the belief that the vaccine will impact sexual behavior (six studies out of six), low perceived susceptibility to HPV infection (three studies out of three), and doubts about HPV vaccine efficacy (three studies out of three).A complete summary of the factors associated with parental and adolescent HPV knowledge and vaccine acceptance is included in the A complete list of items used in these questionnaires, including 38 and 40 questionnaires to assess HPV knowledge and vaccine acceptance respectively, is provided in the This review shows that HPV knowledge and vaccine acceptance vary widely across different studies and countries. In general, figures are still modest and lower in comparison with other routine vaccines. Safety concerns are still the main barrier to vaccination, and lack of HPV and vaccine knowledge has been identified, which is even greater for male vaccination. In contrast, main drivers to vaccination are perception of efficacy of HPV vaccine and social responsibility.To our knowledge, this is the first systematic literature review to summarize factors influencing HPV knowledge and vaccine acceptance among adolescents and their parents since the marketing authorization of HPV vaccines in Europe and complementary to a recent systematic review on HPV vaccine hesitancy in Europe . AdditioVaccination acceptance is critical to ensuring the success of national immunization programs. Previous knowledge has been already identified as a known prerequisite for informed decision-making and vaccine acceptance . HPV vacRecognition of the social and economic impact of the entire HPV burden of disease is still inadequate : recurreOur results show that HPV knowledge are still moderate and vary widely between European countries and the populations interviewed. These results are substantially aligned with findings from previous publications: according to Loke et al. , the perIn our study, percentages of HPV vaccination acceptance are also quite modest and lower than for vaccines in general. The percentages of parents and adolescents that intended to vaccinate or receive the vaccination were 59.2% and 64.3%, respectively.As previously described, our results showed that female gender and having higher education impact HPV knowledge and vaccine acceptance positively. Main drivers for HPV vaccination included the perception of HPV severity and impact and the belief that HPV vaccines are effective. This was also identified in previous publications  and furtMore than 11 years after authorization, HPV vaccines have proven to be effective –83 and tSo far, 28 countries have extended their HPV national immunization programs to include boys and prevent them from suffering HPV-related diseases . HoweverA recent review  has showAlso, we found that HPV knowledge and vaccine acceptance varied by several patients’ characteristics . These The items compiled in the different questionnaires could be useful for countries and investigators who intend to assess HPV knowledge and vaccine acceptance in their countries to make their results comparable with existing published data and to assess temporal trends or factors that influence the variations in HPV knowledge and vaccine acceptance.Main limitations of this review stem from the variability across the studies included and, particularly from the lack of coincidence of the items used in each study to measure HPV knowledge and vaccine acceptance in adolescents and their parents. Also, it is difficult to assess a temporal trend in HPV knowledge and vaccine acceptance, as the questionnaires used and target populations differed widely between studies. Other limitations are heterogeneous sampling methods, sample sizes, population included, year of the study, and HPV vaccine implementation in the country. Also, this study is focused only in studies conducted in Europe and results cannot be extrapolated worldwide. This systematic review excluded all non-English-language publications. However, since most relevant research is commonly published in international journals in English, we do not expect to have missed any relevant study. Nevertheless, pooled results should be interpreted with caution.Concerted efforts should be made to conduct multifaceted and tailored interventions to the population providing balanced information for decision-making on HPV vaccination. Increasing HPV vaccination uptake in males and females could dramatically change the epidemiology of HPV-related diseases and their consequences in countries.Additional file 1. SM-1- Search strategy.


We propose an economical cervical screening research and implementation strategy designed to take into account the typically slow natural history of cervical cancer and the severe but hopefully temporary impact of COVID-19. The commentary introduces the practical validation of some critical components of the strategy, described in three manuscripts detailing recent project results in Asia and Africa.The main phases of a cervical screening program are 1) primary screening of women in the general population, 2) triage testing of the small minority of women that screen positive to determine need for treatment, and 3) treatment of triage-positive women thought to be at highest risk of precancer or even cancer. In each phase, attention must now be paid to safety in relation to SARS-CoV-2 transmission. The new imperatives of the COVID-19 pandemic support self-sampled HPV testing as the primary cervical screening method. Most women can be reassured for several years by a negative test performed on a self-sample collected at home, without need of clinic visit and speculum examination. The advent of relatively inexpensive, rapid and accurate HPV DNA testing makes it possible to return screening results from self-sampling very soon after specimen collection, minimizing loss to follow-up. Partial HPV typing provides important risk stratification useful for triage of HPV-positive women. A second “triage” test is often useful to guide management. In lower-resource settings, visual inspection with acetic acid (VIA) is still proposed but it is inaccurate and poorly reproducible, misclassifying the risk stratification gained by primary HPV testing. A deep-learning based approach to recognizing cervical precancer, adaptable to a smartphone camera, is being validated to improve VIA performance. The advent and approval of thermal ablation permits quick, affordable and safe, immediate treatment at the triage clinic of the majority of HPV-positive, triage-positive women.Overall, only a small percentage of women in cervical screening programs need to attend the hospital clinic for a surgical procedure, particularly when screening is targeted to the optimal age range for detection of precancer rather than older ages with decreased visual screening performance and higher risks of hard-to-treat outcomes including invasive cancer. Cervical cancer remains a leading female malignancy. Highly efficacious vaccines against human papillomavirus (HPV) and accurate screening methods have been developed; however, in practice, global preventive efforts have lagged behind scientific advances. The COVID-19 (coronavirus disease 2019) pandemic will unavoidably decrease the resources that health systems can devote to cervical cancer prevention. The pandemic, if it persists, threatens to increase morbidity and mortality from many diseases including cervical cancer, due to disruption of clinical and preventive services, and because of delay in implementation of new health initiatives [It is important not to abandon the recent WHO  call for elimination of cervical cancer. At the same time, given the potential threat of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) spread, and limited preventive resources that are now even more strained, those of us devoted to cervical cancer prevention must adapt. The goal of cervical screening is to identify the few women with cervical precancer that can be treated to prevent cancer, while minimizing harm to the great majority. Even an important malignancy like cervical cancer is uncommon, and invasive disease typically takes decades to develop. Less than 5% of women in most populations are at risk of cervical cancer in their lifetime; at a single point in time, only 1% or less of women have precancer.In this commentary, we propose a cervical screening research and implementation strategy designed to take into account the typically slow natural history of cervical cancer and the severe but hopefully temporary impact of COVID-19. In three companion manuscripts –4, we hiThe main phases of a cervical screening program are 1) primary screening of women in the general population, 2) triage testing of the small minority of women that screen positive to determine need for treatment, and 3) treatment of triage-positive women thought to be at highest risk of precancer or even cancer. In each phase, attention must now be paid to safety in relation to SARS-CoV-2 transmission.The new imperatives of the COVID-19 pandemic support self-sampled HPV testing as the primary cervical screening method. HPV DNA testing is the most sensitive cervical screening test, which thereby confers the greatest and longest reassurance when the test is negative . A negatSelf-sampling to obtain the specimen for HPV testing yields reassurance equivalent to clinician sampling, and has proven to be quite practical and teachable by simple graphics or animated video presentation, as demonstrated by the accompanying manuscript from Nigeria . A sealeThe advent of relatively inexpensive, rapid and accurate HPV DNA testing, as validated in an accompanying article from the prominent research team in Shenzhen, China , makes iHPV testing is sensitive but not specific because most infections will not cause precancer; fewer still will lead to cancer. The risk is dependent on HPV type. Typing of individual HPV types can be incorporated with minimal additional cost into self-sampled HPV testing, providing important risk stratification useful for triage of HPV-positive women. The types of HPV defined as carcinogens vary quite substantially in risk. HPV16 is uniquely carcinogenic. HPV18 and HPV45 are important causes of cervical cancer but tend for reasons that are still not completely understood to cause relatively few diagnosed cases of precancer. The types of HPV related to HPV16, namely, HPV31, HPV33, HPV35, HPV52, and HPV58 are conceptually worth distinguishing from the lower risk, minimally carcinogenic types  . Typing Because even the highest-risk HPV types are common in aggregate at screening ages in high-prevalence settings , a second “triage” test is useful to guide management. In high-resource settings, reflex cervical cytology or the novel dual-stain immunocytochemical test  are currAutomated visual evaluation (AVE) is under development as a triage diagnostic technology . This isThe advent and approval of thermal ablation permits quick, affordable and safe, immediate treatment at the triage clinic of the majority of HPV-positive, triage-positive women. As a part of AVE, deep-learning algorithms are in development that will assist health workers in the judgment of whether a lesion can be ablated or whether referral for excision is required. For those women needing an excisional large-loop excision of the transformation zone (LLETZ) procedure, mobile battery-operated LLETZ units and innovative electrode designs are now available to permit safer local performance, and possible distancing of those treatments from hospitals devoted to COVID-19 care. Overall, only a small percentage of women in cervical screening programs need to attend the hospital clinic for a surgical procedure Fig. , particuIt is imperative to keep in mind that it is the treatment of screen-detected precancer and not the screening of precancer that prevents the development of cancer. In this context, AVE and other screening methods must be backed up by a tracking system to minimize loss to follow up from screening to triage to treatment, particularly in low-resource settings without any existing electronic medical records systems. The establishment of a routine screening program, with repeated surveillance for HPV negative and low-risk HPV positive women, is yet another challenge in low-resource settings that is likely to persist even past the COVID pandemic. A risk threshold for treatment based on a combination of HPV types, AVE score, and other triage tests needs to be chosen very carefully in such settings given the risk of exposure to COVID, availability of resources, and the reality of once or twice in a lifetime access to screening for many women relying on the public sector.The COVID-19 pandemic motivates the transition away from reliance on speculum examinations for screening. It might logically be the right time to move away from such methods including the venerable Pap test, in favor of reliable and accurate HPV-based technologies based on self-sampling. Affordability remains, however, a real and important constraint. The affordability of screening is made more challenging by new safety considerations that limit the social crowding implicit in high-throughput lower-cost approaches. The need for such measures will ease as the pandemic eventually ends, but the current emergency mandates that we push past the inertia of current practices. Primary screening is “elective”, in that the great majority of women are not at immediate risk. In restarting existing screening efforts, it makes sense to prioritize women already known to have precancer in need of treatment, paying intermediate attention to those with lesser abnormalities, and to consider very cautiously for now the net benefit of routine screening for low-risk women previously screened HPV-negative.


Although diesel airborne particulate matter (PM2.5) has been known to play a role in many human diseases, there is no direct evidence that therapeutic drugs or proteins can diminish PM2.5‐induced diseases. Nevertheless, studies examining the negative control mechanisms of PM2.5‐induced diseases are critical to develop novel therapeutic medications. In this study, the consensus PDZ peptide of ZO‐1 inhibited PM2.5‐induced inflammatory cell infiltration, pro‐inflammatory cytokine gene expression, and TEER in bronchoalveolar lavage (BAL) fluid and AM cells. Our data indicated that the PDZ domain in ZO‐1 is critical for regulation of the PM2.5‐induced inflammatory microenvironment. Therefore, the PDZ peptide may be a potential therapeutic candidate during PM‐induced respiratory diseases. The PM2.5 concentration in Korea is 2.4‐fold greater than the 10 µg/m3 annual guideline for PM2.5 recommended by the World Health Organization, and approaches the recommended 25 µg/m3 24‐hour mean.Air pollution is a global concern and is becoming increasingly severe in Korea. The annual average airborne 2.5‐µm particulate matter (PM2.5) concentration in Korea was 24.0 µg/m2The National Institute of Standards and Technology  SRM 1650b standard diesel PM (PM2.5), with diameters below 2.5 mm, was purchased from MERCK (NIST‐1650b).3IL‐6, IL‐1α, IL‐1β, and TNFα gene expression in BEAS‐2B cells  2 and regulator of G protein signaling 12 (RGS12) gene expression in a time‐dependent manner ; Data curation (lead); Writing‐original draft (lead). Tae‐Jin Lee: Conceptualization (lead); Methodology (lead); Writing‐original draft (lead). Ji Wook Kim: Methodology . Yu Som Shin: Methodology . Ju Deok Kim: Methodology . Sung Won Ryu: Methodology . Siejeong Ryu: Methodology ; Supervision . Yung Hyun Choi: Methodology ; Supervision . Cheol Hong Kim: Data curation ; Validation . EunAe You: Methodology . SangMyung Rhee: Methodology . Kyoung Seob Song: Conceptualization (lead); Writing‐review & editing (lead).