Human Papillomavirus is the main cause of cervical cancer, including squamous cell carcinoma of the oropharynx, anus, rectum, penis, vagina, and vulva. In recent years, considerable effort has been made to control HPV-induced diseases using either prophylactic or therapeutic approaches. A critical review of the literature about the therapeutic Human Papillomavirus vaccine was performed to analyze its efficacy in the treatment of female lower genital tract lesions and its possible perspective application in clinical practice. The most important medical databases were consulted, and all papers published from 2000 until 2021 were considered. We retrieved a group of seven papers, reporting the role of anti HPV therapeutic vaccines against the L2 protein in the order of their efficacy and safety in female lower genital tract disease. In addition, the immune response due to vaccine administration was evaluated. The development of therapeutic vaccines represents an interesting challenge for the treatment of HPV infection of the lower genital tract. Literature data underline that the L2 protein may be an interesting and promising target in the development of therapeutic HPV vaccines, but the possible strengths and the unclear longevity of L2 immune responses are factors to be considered before clinical use. In the last two decades, the incidence of cervical cancer in Western countries decreased by about one-third. However, this cancer represents the fourth most common cancer in the female population worldwide and about 90% of deaths from cervical cancer occurred in developing countries and the least developing countries largely due to the lack of prevention, diagnosis, and screening ,2.The main cause involved in cervical cancer development is Human Papillomavirus (HPV), which represents the most common sexually transmitted infection , and morLiterature data reported as the annual number of new cases of cervical cancer will increase up to 700,000, and the number of deaths are predicted to reach around 400,000 per year by 2030 . For thiHPV-associated cancers also include squamous cell carcinoma of the oropharynx, anus, rectum, penis, vagina, and vulva ,7. In reThe introduction of the prophylactic anti-HPV vaccine in clinical practice opened a new scenario in the panorama of HPV disease: in 2006, HPV vaccination was approved for the primary prevention of HPV-related diseases in young women, and subsequently, also in women up to 45 years of age ,9. ProphTo understand the significance of therapeutic vaccines and their possible role in the management of high-grade intraepithelial lesions or cancer, it is important to know the intrinsic characteristics of the HPV structure and its genome expression. Human Papillomavirus is a non-enveloped icosahedral virus with 72 capsomeres and a diameter of 55 nm. The genome is a circular double strand of DNA with a length of 8000 bp .Analyzing the expression of viral proteins in order of their expression, we can identify three different regions . Nine opThe modification of the cellular control mechanism caused the development of cervical lesions and the concomitant persistence of HPV infection . For thiThe L2 is formed by a sequence of 500 amino acids in length, with a molecular mass of 55 KDa . Each viHost proteins promote the cleavage and the phosphorylation of the L2 during virus assembly. This interaction promotes the assistance in capsid conformational change to receptor uptake. For this reason, the L2 is the most important protein to enable efficient virus entry in the host cell with DNA integration through vesicular trafficking and endosomal escape; nuclear transport of the L2 toward the nucleus, viral gene regulation, initial viral gene transcription, morphogenesis and viral capsid assembly ,21,22.For this reason, the L2 seems to be a perfect target of therapeutic vaccines, since the ending of several early processes could avoid the persistence and progression of HPV infection. Our critical review aims to analyze the possible role and efficacy of a therapeutic vaccine based on late capsid proteins (L2 in particular) in the treatment of HPV, genital lesions and their possible application in clinical practice, especially for the VIN  and VaIN , where the treatment is often difficult. Vin, VaIN, and AIN  occurred in young patients, where often, the extension of lesion and multifocal localization required excision surgery: for this reason, therapeutic vaccines against L2 capsid proteins could help the clinicians in tailored treatment.In order to perform a critical review, we consulted several international databases such as PubMed, Cochrane Database of Systematic Reviews, EMBASE, and Web of Science. We researched the following terms and their combinations: “Human Papillomavirus” AND therapeutic vaccines “HPV therapeutic vaccines”, “CIN AND therapeutic vaccines”, “cervical cancer and therapeutic vaccines”, and “late protein HPV therapeutic vaccines”. The research strategy is summarized in Indeed, two authors independently (A.G. and M.L.V.) analyzed reference lists of recognized papers in order to integrate them into the literature search program. We considered clinical trials regarding therapeutic vaccines based on the L2 protein. Exclusion criteria were the following: single case reports, book chapters, books, conference proceedings, and abstracts. In addition, publications written in a language other than English or in vitro experimental trials were excluded. Finally, we decided to not take into consideration trials regarding anti-HPV vaccines in experimental animal models in the “Results” section. The bibliographic research provided a preliminary set of papers, which were carefully analyzed by two authors (M.D. and M.T.) independently in order to avoid the risk of bias of selection, performance, detection, attrition, and reporting, according to the Cochrane Handbook for Systematic Reviews of Interventions ,25,26. FWe retrieved 58 papers from the preliminary bibliographic search, as we reported in In 2002, de Jong et al. evaluated the safety and immunological response of the HPV-16 L2E6E7 fusion protein (TA-CIN) vaccine, in a double-blind, randomized, and placebo-controlled, dose-escalating phase I trial, which involved 40 healthy volunteers . The population of study was randomized to undergo one of three different doses . The immunological profile revealed a significant anti HPV-16 L2E7E6 specific T-cell proliferation induced by TA-CIN in 8/11 patients treated with 533 micrograms of TA-CIN vaccine, which was evaluated with IFN-gamma ELISPOT. In addition, the author reported a significant humoral immunological response with IgG production in 8/32 subjects. All the patients enrolled showed no sign of serious or severe adverse events. Most of the events were reported within 7 days of vaccination. The most frequent adverse effects reported were the following: local injection site reaction and tenderness. Tenderness appeared when higher doses of TA-CIN were administered, while moderate tenderness was higher after vaccination with two high doses. Local reactions (redness and swelling) were a rare adverse effect. The most frequently systemic adverse moderate events were headache and fatigue, without a correlation with the dose of TA-CIN or the number of administrations .In 2003, Davidson et al. in a Phase II trial evaluated the immunological response and clinical features of patients with HPV-16 related high-grade Vulva Intraepithelial Neoplasia (HG VIN) treated previously with TA-HPV, which was based on modified HPV-16 and 18 E6–E7 proteins. In this trial, 18 women were enrolled and 10 demonstrated an increase in HPV-16 specific T-cell response. In one patient, there was a complete regression of vulvar lesions with histological confirmation and viral clearance by PCR evaluation. Eight patients reported a reduction in vulvar lesions of at least 50% of diameter, and in six of these women, the authors demonstrated a reduction in viral load; four patients showed a decrease in symptoms. Vaccine immunogenicity was confirmed by the increase in serological and cell-mediated immunity after vaccination. All 18 women reported vaccine-specific IgG, and in 17/18 patients, there was a >10-fold increase from the pre-vaccination titer. All patients showed an IFN-gamma ELISPOT response. Seven women reported vaccine-specific T cells after vaccination. In 14 of these women, an increase from the pre-vaccination level of T cells was reported. Regarding HPV-specific immunity, no responses were reported in any patient to the E6. One patient showed an increased E7 T cell response but with low level T cell responses to three different HPV-16 E7 peptides, three patients reported an increase in response but with low level T cell responses to two different HPV-16 E7 peptides, and three patients showed the same increased response but with low level T cell responses to one of the HPV-16 E7 peptides after vaccination. Serological responses were evaluated to HPV-16 and 18 E6 and E7 by ELISA. Two patients reported a clearly increased HPV-specific serological response detected at 4 weeks after vaccination, but IgG levels returned to the pre-vaccination level by 12 weeks post-vaccination. Clinical responding women had significantly more lesion-associated CD1a, CD4, and CD8-positive immune cells before vaccination than non-responders. The authors underlined that the therapeutic vaccine response may be influenced by the local immune response: indeed, responder patients reported a high level of CD4+ and CD8+ associated with lesions. Viral load was evaluated by real-time PCR. ELISPOT using HLA-A2 binding peptides was performed in order to investigate HPV-specific immunity. T cell proliferation was investigated with an HPV-16 L2E6E7 fusion protein. Antibodies were measured by ELISA. Lesion-infiltrating CD4+ and CD8+ immune cells response was evaluated by immunohistochemistry . In 2004, Smith et al. evaluated the immunogenicity of heterologous vaccination in 29 women with high-grade ano-genital intraepithelial neoplasia: each patient underwent three prime doses of TA-CIN (533 μg) i.m. at four weekly intervals, which was followed after 4 weeks with a single dose of TA-HPV by dermal scarification. In 17 patients, a TA-CIN-specific response after prime or prime-boost vaccination was reported. Ten of these 17 patients reported a definite vaccine induced response. Regarding the proliferative responses against the oncoproteins E6 and E7 using recombinant HPV-16 GST-E6 or GST-E7 proteins, there was only a significant increase in the response for GST-E6 at week 16. IFN-γ ELISPOT assays highlighted HPV-16 E6-specific T-cell responses in nine of 25 patients, which was induced by vaccination. In two women, HPV-16 E7-specific T cells were enhanced after all vaccinations. Three patients reported enhanced HPV-18 E6-specific T-cell activity post-vaccination at week 20. No significant changes were reported in serological responses to HPV-18 E6 or E7 after vaccination. The responses to HPV-16 E6 or E7 appeared transient: the level peaked at week 16, and it declined by weeks 20 and 24, which was in accordance with the single booster vaccination with E6 and E7 of TA-HPV at week 12. Indeed, the authors reported a significant response in the study group in both humoral and cellular immunological response, respectively; nevertheless, the study did not show a correlation between systemic HPV-16-related immunity and clinical outcomes. In fact, among 19 patients with stable disease, eight reported immunogenic responses, and in the four patients with disease progression, two did not show a T cell response, and two revealed an absence of both T cell and humoral responses .In the same year, Davidson et al. enrolled in a TA-CIN extension trial ten women affected by HPV-16-related VIN. These patients were treated primary with heterologous vaccination: prime boosters of TA-HPV (virus encoding HPV-16/18 and E7 vaccine) and subsequently with TA-CIN (HPV-16 L2E6E7 fusion protein vaccine). Three patients reported a clinical response, while six patients maintained stable disease. Two patients reported a partial or complete clinical response, which was confirmed by the reduction in lesion diameter of ≥50% following vaccination with TA-CIN. Six patients maintained stable disease, one patient reported significant symptomatic relief following vaccination, while one patent reported disease progression (increase in lesion diameter of ≥25% post-vaccination). Regarding serological responses, IgG levels did not demonstrate a significant change during the study. Nearly all (9/10) patients reported at least a two-fold increase in HPV-16 and/or 18 E6- and/or E7-specific IgG after vaccination with TA-CIN. In eight patients, an increased HPV-16 E7-specific IgG response was reported; in six women, an increased HPV-16 E6-specific IgG response was reported, and in one patient, an increased HPV-18 E6-specific IgG response after vaccination was not demonstrated. Regarding HPV-specific proliferative responses, all patients demonstrated a proliferative response. Analyzing HPV-specific ELISPOT responses, three patients reported a response to one or more HPV-16 E7 peptide before and after vaccination. Two patients reported an increase in HPV-specific peptide responses following vaccination. The results reported as the combination of TA-HPV and TA-CIN as heterologous vaccination may be an interesting combination in HPV-induced lesion treatment due to the immunogenicity conferred by the vaccine .In 2006, Fiander et al. reported in a multi-center Phase II trial the effectiveness of a prime boost regimen for the treatment of HPV-related diseases. In prime-boost vaccination, the authors evaluated heterologous HPV vaccines in the management of ano-genital intraepithelial neoplasia. In this study, patients were vaccinated with three doses of TA-CIN followed by one dose of a recombinant vaccine with virus-encoding HPV-16 and 18 E6/E7 (TA-HPV). Twenty-nine women were recruited for the study: 2 with VAIN 3, 27 with VIN3, and 2 patients with VIN3 also had anal intraepithelial neoplasia. A complete response was seen in 1 patient, partial response in 5, stable disease in 18, and progression in 5 of them. None of the patients developed invasive lesions .p = 0.03 and p = 0.03). The authors concluded that the modulation of the local and systemic immune response plays a role in the therapeutic vaccine effect [In 2010, Dayana et al. combined in a phase II clinical trial the TA CIN vaccine and Imiquimod  in 19 women to treat VIN2 and 3. The authors reported a complete regression of VIN in 6/19 subjects (32%) at week 10, in 11/19 patients 58% at week 20, and in 12/19 at week 52 (63%). At week 20 in responder patients, there was a significant increase in the response of CD4+ and CD8+  was approved, and it is currently ongoing. The trial is a randomized, multi-center, open-label pilot study. The first aim of this trial is to provide evidence for the safety and feasibility of the vaccine and the different immune responses when the dose is administered at different locations such as the thigh or arm in order to determine the site that can elicit the most potent immunological effect. Furthermore, the study aims to evaluate the level of circulating antibodies to HPV16 E6, E7, and L2, as well as the level of circulating T cells and the mononucleocyte response in the peripheral blood before and after vaccination (time frame: up to 4 years). In this pilot study, a single dose level (100 µg) is administered in order to evaluate the assessment for the safety and tolerability of administering the TA-CIN vaccine three times to either the arm versus the thigh of patients who have previously been treated for HPV-16-related cervical cancer in the past year with no evidence of disease recurrence. A total of 14 patients have been enrolled to demonstrate the safety of the TA-CIN vaccine as adjuvant therapy. The study design aims to evaluate the minimal or non-systemic toxicity of the administration of the vaccine. TA-CIN will be given as a single intramuscular injection every 4 weeks for a maximum of three times. The location of the injection (arm or thigh) is casual. Patients will be evaluated for safety and response to treatment during this period. The follow-up period includes the following: clinical visits are performed at different times after the administration to evaluate the clinical response .The development of therapeutic vaccines represents an interesting challenge for the treatment of HPV infection of the lower genital tract. Indeed, the development of therapeutic vaccines may constitute a valid clinical treatment option; nevertheless, there are no vaccines of this nature currently available in clinical practice ,14.Experimental animal models demonstrated that neutralizing antibodies represent the main goal of immunological protection because they were found in the microenvironment of microlesions in the cervical epithelium, which are the “door” of viral entrance to infect the basal lamina of cervical tissue . CurrentVaccines against the minor structural protein L2 demonstrated an improvement of efficacy because they conserved N-terminal epitopes, which may promote the increase in the level of cross-type neutralizing responses and protection against the skin and genital HPV infection, especially against the first 130 amino acids of the L2 peptide . As previously reported, the goal of the development of the next generation of HPV vaccines is to block the transcription and integration of viral genome into host cells. Since the L2 protein is not able to form VLPs as L1 proteins, the fusion of the L2 with early viral proteins  may promote the elicitation of a therapeutic immunological response . In ordeThe progression of HPV anogenital lesions and the development of high-grade pre-neoplastic lesions mirror a dysregulation of the immune system associated with an increase in E6 and E7 expression, with a consequent augmentation of HPV gene expression and loss of cell differentiation of the basal layer . During After HPV tissue attack, host cells activate the innate and adaptive immune systems in order to eliminate the infection. Macrophages, Langerhans cells, and the natural killer cells try to inhibit HPV proliferation and integration with Toll-like receptors (TLRs) expression. TLRs are able to recognize the viral components and promote the transcription of factor-like nuclear factor kappa B (NF-κB) and interferon response factor-3 (IRF3) to increase the proliferation of pro-inflammatory cytokines and antiviral cytokines  . Literature data supported the opinion that systemic T cell response against HPV infection can explain the different behavior of HPV lesions. Major histocompatibility complexes (MHC I–II) represent the basis of the acquired immunological system. MHC I exposes the antigen to cytotoxic T cells CD8+, while MHC II exposes the antigen to helper T cells CD4+. In particular, Th1 CD4+ T-cells are specific for HPV-16 E6, E7, and E2, because they are found in the peripheral blood of immunocompetent individuals with HPV confirmed infection . InteresFinally, literature data supported the hypothesis that the natural anti-HPV cellular response causes mild protection against HPV genotypes involved in lower genital tract infections, and the natural immunological response appears lower than the protection given by vaccine administration .Therapeutic vaccine technology is based on two fundamental points: the induction in the host of a T cell response against E6, E7 and L2 fusion proteins in order to improve effectiveness and to promote the lesion regression and the ability of an immunological response to identify the site of HPV infection .As we reported, the TA-CIN vaccine is able to promote an HPV-16 specific immunological response and high levels of anti HPV-16-neutralizing antibodies. Indeed, Gambhira et al., using the serum derived from previous clinical trials of Phase I and II, evaluated the power of the HPV E6E7L2 vaccine to induce the L2 specific response. The authors found a significant seroconversion and higher specific responses for HPV-16 and HPV-18 neutralizing antibodies, increasing vaccine dose in healthy volunteers. Therefore, the efficacy of the TA-CIN vaccine showed a reduction in high grade ano-genital intraepithelial lesions rather than in healthy volunteers .During the Phase I/II studies, a regression of the lesions and an important resolution of symptoms were found, and a strong response from CD4 + and CD8+ T cell produces the clearance of the virus, while in non-responders, there is a prevalence of T-Reg cells ,60,61,62® to induce a T cell and a humoral response against L2 and E6. The limits of these data are the frequent spontaneous rate of regression of genital warts [Therapeutic vaccines based on the fusion of viral protein technology are not only direct against high-grade cervical lesions or cervical cancer but also against genital warts. In this setting, clinical trials evaluated the tissue antigen–genital warts vaccine (TA-GW) based on a fusion protein of HPV-6 E7 and L2, and it was able to stimulate cellular and humoral immunity in order to promote the regression of existing genital warts and prevent their recurrence ,64. Two al warts ,66, but From a perspective point of view, it is interesting to analyze the feasible application of DNA vaccination conjugated with the current vaccine technology. Peng et al. in experimental animal models tested a DNA vaccine encoding for calreticulin in addition to E6, E7 and L2 of HPV-16 in order to study the efficacy in immunocompromised subjects with low CD4 T-cell levels. The safety and efficacy of the DNA vaccine may overcome the risk of administration of life vectors in immunodeficient subjects inducing an HPV cytotoxic T cell response. Interestingly, the T cell response promotes subcutaneous tumor regression and removes circulating tumor cells, and for this reason, DNA vaccines appear to offer several advantages ,68,69,70Finally, the L2 protein may be an interesting promising target in the development of a broad-spectrum HPV vaccine, but the possible strengths and the unclear longevity of the L2 immune responses are factors to be considered in clinical use.


Arthrogryposis is a medical term used to describe congenital contractures which often affect multiple limbs. Distal arthrogryposis (DA) is one of the major categories of arthrogryposis that primarily affects the distal parts of the body, i.e., the hands and the legs. Although ten different types and several subtypes of DAs have been described, the genes associated with each of these DAs are yet to be characterized. Distal arthrogryposis type 10 (DA10) is a rare genetic disease, which is distinguished from the other arthrogryposis types by plantar flexion contractures resulting in toe-walking during infancy as well as variability in contractures of the hip, hamstring, elbow, wrist and finger joints with no ocular or neurological abnormalities. Symptoms of DA10 indicate impairment specifically in the musculoskeletal system. DA10 is still poorly studied.The objective of this study was to identify the candidate gene for DA10 by scrutinizing the protein-protein interaction (PPI) networks using in silico tools.TTN (the gene that codes for the protein Titin or TTN) follows the expression pattern similar to the other known DA associated genes and its expression is predominant in the skeletal and heart muscles. Titin also participates in biological pathways and processes relevant to arthrogryposes. TTN-related known skeletal muscle disorders follow the autosomal-dominant pattern of inheritance, which is a common characteristic of distal arthrogryposes as well.Among the genes that reside within the previously reported genomic coordinates  of the causative agent of DA10, only TTN appears to be the candidate gene for DA10. Our attempt to discover a potential candidate gene may eventually lead to an understanding of disease mechanism and possible treatment strategies, as well as demonstrate the suitability of PPI in the search for candidate genes.Based on the findings of the analyses and their correlation with previous reports, The online version contains supplementary material available at 10.1186/s43141-022-00405-5. ArthrogBamshad et al. described the most widely accepted clinical classification of AMC. According to this classification, AMC can be caused by amyoplasia, neurological abnormalities, or distal arthrogryposis. Amyoplasia is caused by impaired muscle growth leading to contractures that affect every symmetrical joint of the body. Internally rotated shoulders, extended elbows, flexed wrists, dislocated hips, equinovarus contractures in the feet, rigid fingers, and thumbs are common symptoms in these cases . Many paTen different types and several subtypes of DAs have been characterized and classified according to their proportion of shared features . However® (OMIM®) database [. These genes were used as input in NetworkAnalyst 3.0 [The list of genes that are known to be associated with different types and subtypes of DA Table  was retrdatabase . These glyst 3.0  to identlyst 3.0  and the lyst 3.0 . Based olyst 3.0 , PANTHERlyst 3.0 , Reactomlyst 3.0  and KEGGlyst 3.0  databaselyst 3.0 .Table 1LBased on genome-wide linkage analysis, a previous study reported that the candidate gene for DA10 resides on human chromosome 2 within the region between 179,700,000 and 188,500,000 bps in the chromosome assembly 38 (GRCh38)  . The lisThe functionally relevant genes to DA10  were used as input in NetworkAnalyst 3.0  to identMutations in different genes cause arthrogryposis Table . Even muThe same query genes were used as input to search protein-protein interactions and associated biological processes in the STRING interactome database Table . Among tTTN, appears to be very closely located to the predicted genomic coordinates of the DA10 candidate gene  that reside within 2q31.3–q32.1 encode miRNAs. Target genes of these miRNAs were retrieved from the miRDB [Three among these genes  to assess the most probable candidate gene for DA10. Input of TTN along with the 15 known AMCs and DAs associated genes generated a more extensive PPI network with 230 nodes  in the development of DA10.In this study, we used in silico tools and databases to identify the candidate gene for distal arthrogryposis type 10. Our exploration suggests the involvement of TTN is a large gene with 364 exons and multiple splice variants [TTN is expressed in the striated muscles– cardiac and skeletal muscles [TTN is not expressed in other tissues, non-muscular abnormalities are not supposed to occur in titinopathies. This is consistent with the results from a previous study on DA10 patients, which found all DA10 patients to have normal neurological responses, electromyographic patterns, and creatinine kinase levels [TTN-related skeletal muscle disorders follow the autosomal-dominant pattern of inheritance [variants . TTN is  muscles . Since Te levels . The majeritance , which ieritance .TTN can result in isolated cardiomyopathies, isolated skeletal muscle disorders, and combined cardiac-skeletal muscle diseases [The N2A isoform of TTN is predominantly present in the skeletal muscles, and this N2A isoform excludes some of the exons of predominant cardiac isoforms N2B and N2BA. That is why, mutations of diseases .TTN and manifests itself in the adult patient as weakening of the tibialis anterior muscle [TTN [TTN contributed to phenotypic severity among the TMD patients [Tibial muscular dystrophy (TMD) is caused by mutations in the r muscle . In Finncle [TTN . TMD patcle [TTN . Other fpatients . Similarpatients .TTN in congenital contractures. Fernández-Marmiesse et al have shown that a homozygous deletion of 5-bp in a prenatal exon of TTN (exon 197) causes a frameshift leading to a premature truncated protein, which results in congenital contractures [TTN, which is part of the fetal TTN isoform [TTN [There are reports on the involvement of ractures . Chervin isoform . Bryen eorm [TTN . These oThe biological pathways and processes associated with these 151 nodal proteins Table  are sharAs shown in Tables TTN and other DA-associated genes [TPM2 and PIEZO2, gene expression profiles of all other candidate genes of different distal arthrogryposis are restricted to specific organs  , 52. HowTTN resides within the previously reported genomic coordinates of the potential candidate gene of DA10. TTN is predominantly expressed in the skeletal and heart muscles and its expression follows a pattern similar to the other known DA-associated genes. TTN participates in biological pathways and processes relevant to arthrogryposis. Based on the findings of these in silico analyses and their correlation with previous reports, TTN appears to be the candidate gene for DA10.In this study, we scrutinized the protein-protein interaction (PPI) networks as well as the associated biological processes and pathways to identify the candidate gene for DA10. Additional file 1: Supplementary Table 1. List of interacting proteins in the network shown in Fig. Additional file 2: Supplementary Table 2. List of target genes of MIR4437, MIR548AE1 and MIR561 encoded miRNAs.Additional file 3: Supplementary Figure 1. The DA associated genes (in red) that participate in hypertrophic cardiomyopathy, dilated cardiomyopathy and/or adrenergic signaling in cardiomyocytes pathways. The other nodes that participate in these pathways are shown in blue. The rest of the interacting proteins are shown in green.Additional file 4: Supplementary Figure 2. Tissue specific expression profiles of TTN and genes known to be associated with DAs [with DAs .Additional file 5: Supplementary Figure 3. Tissue specific expression profiles of ITGA4, ITGAV, and NCKAP1 [d NCKAP1 .Additional file 6: Supplementary Figure 4. Tissue specific expression profiles of FBXO2 [of FBXO2 .


Nowadays, the striking numbers of infertile couples that turn to assisted reproductive technologies (ART) drive the research toward a more comprehensive understanding of the underlying causes. Male factors contribute to the inability to conceive in half of the cases, and it has been suggested that sexually transmitted infections could have a role in the onset of spermatozoa impairments. Since the impact of HPV infection on sperm quality and sperm DNA integrity is debated, we wanted to analyze its impact on conventional seminal parameters and the sperm DNA fragmentation index (DFI). Therefore, 117 semen samples of patients undergoing IVF were evaluated for the following characteristics: HPV DNA detection and sperm DNA fragmentation, concentration, motility, and morphology. The results showed a higher rate of HPV-negative patients (59.8% vs. 40.2%) and no HPV-related effect on DFI, sperm concentration, total sperm number, and total motility. Only progressive motility and morphology were found as significantly influenced by HPV positivity. Moreover, we observed a statistically significant difference in DFI when comparing high-risk HPV (HR-HPV) and low-risk HPV (LR-HPV) genotypes. Our data suggest that the presence of any HPV type, whatever the exact localization of the virions, can impair some sperm parameters, while HR-HPVs specifically affect the integrity of spermatozoa DNA. Infertility, defined as the inability to conceive after 12–24 months of unprotected sexual intercourse ,4. The etiology of male infertility is complex; to understand its causes, routine laboratory practice involves the basic examination of spermatozoa parameters, such as count, concentration, motility, and morphology  , is a crucial factor negatively affecting embryo development, implantation, and pregnancies in spontaneous and assisted reproduction . StudiesThe reasons behind spermatozoa genomic impairments could be several, from abnormalities in DNA packaging during spermatogenesis to aberrant apoptosis and exposition to oxidative stress. Environmental and pathological conditions, such as diseases, drugs, aging, and infections, are the main triggers   infection is the most widespread: it is estimated that about 80% of sexually active men and women will contract the virus at least once over their lifetime [More than 200 different HPV genotypes have been described to date, all characterized by a high tropism for skin and mucous membranes’ epithelia . Most ofAside from the full-blown role taken by HPV in the etiopathogenesis of malignant diseases, the detection of HPV DNA in semen, as well as in the ductus deferens, epididymis, testis, urethra, and external genitalia ,18,19,20In particular, studies have highlighted how HPV could be linked with lower spontaneous pregnancy rates , poor reThe negative effect of HPV infection on basic sperm parameters and the underlying mechanism of such impairment have been investigated in several studies ,30,31,32A possible correlation was observed between HPV sperm infection and cases of unexplained asthenozoospermia; but the literature data are still controversial and, in any case, the mechanism by which HPV virions could influence sperm motility is still unclear ,33,34. IAnother hypothesis identifies spermatozoa as carriers of HPV virions: it was shown that the L1 protein of the viral capsid can efficiently bind to syndecan-1, located on two sites of the sperm head equatorial region ,34,37. TOther authors suggested that an elevated reactive oxygen species’ (ROS) activity in HPV-positive seminal plasma could be linked to sperm membrane deterioration and, hence, to the exposition of sperm DNA to oxidative damage .To date, few studies have investigated DNA fragmentation in HPV-infected semen samples. Some authors observed an increase in DFI in HPV-positive patients, whereas other authors failed to find a correlation ,42,43,44The reason behind such discordant data could be a different localization of HPV virions in the urogenital male apparatus and in the elements that compose seminal fluid. In fact, the exact localization and origin of viral particles are still not clear: they could be located inside the sperm cell, in exfoliated urothelial epithelial cells, or in the prostatic fluid or, again, they can derive from partners or originate from non-dividing squamous epithelial cells that release the virions by an infectious virion-producing pathway ,46,47. Whatever the cause, HPV’s ability to infect spermatozoa and negatively affect their genomic integrity gains particular importance in assisted reproductive technology, where any natural selection of HPV-carrying sperm does not occur .To shed light on the controversial impact of HPV infection on IVF it is mandatory to carefully analyze the alteration of sperm parameters and DNA integrity. In this respect, we performed a retrospective observational study on semen samples of 117 male partners of HPV-infected women attending IVF procedures. For each sample, seminal parameters were evaluated, HPV was detected by conventional protocol, and a sperm chromatin dispersion (SCD) test was carried out to assess spermatozoa DFI.A retrospective observational study was performed on a sample of 117 male partners of HPV-positive women, with a mean age of 38.6 ± 8.1 (SD) years. Semen samples were collected at “D’Arena”, Clinical Analysis and Diagnostics Laboratory, Vallo della Lucania, Salerno, by masturbation after a recommended sexual abstinence period of 3–5 days. After liquefaction at room temperature, sperm concentration, motility, and normal morphology were assessed according to World Health Organization guidelines for semen analyses [C. trachomatis, N. gonorrhoeae, U. parvum, U. urealyticum, M. hominis, and M. genitalium; no seropositivity for human immunodeficiency virus type 1 or 2, human T-cell lymphotropic virus type 1 or 2, hepatitis B or C virus, and T. pallidum; no genetic disease such as cystic fibrosis; and no inflammatory disorders such as varicoceles. The study was performed according to The Code of Ethics of the World Medical Association (Declaration of Helsinki) [Two groups of patients were defined: an HPV-negative group composed of 70 patients and an HPV-positive group composed of 47 patients. The inclusion criteria were as follows: no previous known genital infections; no current infection of elsinki) .Informed consent was obtained from all subjects. No funding or financial compensation was provided to the participants.DNA was extracted from samples using the Qiamp DNA mini kit . After liquefaction at room temperature, following the manufacturer’s protocol, 200 μL of semen samples were processed and elution was performed in 200 μL. The isolated DNA was quantified and analyzed with the spectrophotometer to determine its concentration and purity.After DNA extraction, both HPVs’ genome detection and genotyping were carried out using AMPLIQUALITY HPV-TYPE EXPRESS v3.0 , which is based on the combined use of a PCR and a reverse hybridization assay. The amplification was performed according to the manufacturer’s instructions. After PCR, denatured amplicons were hybridized with specific probes for 40 HPV genotypes. The assay covered all currently known HR-HPV genotypes and probable HR-HPV genotypes , HPV-69, HPV-70, HPV-73, HPV-82) as well as several low-risk types (LR-HPV) . The distinction between high-risk and low-risk genotypes is based on the International Agency for Research on Cancer (IARC) classification .6/mL to prevent sperm DNA halos from overlapping. If the sperm concentration was lower than 10 × 106/mL, dilution was not performed. Following the assessment of conventional semen parameters, samples were diluted in phosphate buffered saline solution 1× pH 7.4  at the concentration of 5–10 × 10® kit  or median and interquartile interval ). t-test was used to compare the mean of unpaired samples. When the distribution of samples was not normal, the Mann–Whitney test was used. Differences between groups were analyzed using the chi-square test or Fisher’s exact test for categorical variables. All tests with p < 0.05 were considered significant. All data were analyzed with Matlab statistical toolbox version 2008  for 32-bit Windows.A test for normal distribution was performed by the Shapiro–Wilk test. A Of the 117 semen samples from male partners of HPV-positive women of infertile couples, 70 were HPV negative (59.8%) and 47 (40.2%) were HPV positive. Patients with HPV semen infection had a mean age of 38.6 ± 8.1 (SD) years, not different from the non-infected subjects. p-value = 0.32) and no significative differences between the HPV-negative vs. HPV-positive groups in seminal parameters: sperm concentration, total sperm number, and both progressive and total motility. We also observed a significantly lower rate of normal forms in HPV-positive samples in comparison to HPV-negative samples (p < 0.0001). In addition, HPV positivity was significantly associated with head defects (p = 0.0047), neck and midpiece defects (p = 0.0002), and tail defects (p = 0.0033). The relationship between HPV and its negative effect on sperm morphology remains weakly understood, but it could be due to the presence of HPV virions located in the different semen fractions.p = 0.0254), immotile spermatozoa (p = 0.0372), and normal forms (p < 0.0001). We observed statistically significant differences in progressive motility , which showed a significant increase in the high-risk group.On the contrary, progressive motility and morphology, which varied in relation to HPV positivity, did not show any genotype-related alteration. This data confirmed that the viral presence alone, regardless of the type, produced alterations in the aforementioned parameters.Millions of people worldwide encounter difficulties at the time of conceiving; in about half of the pregnancy-seeking couples, male infertility is recognized as a determining factor . To understand the causes underlying this condition, clinical practice involves the analysis of general quality semen parameters, such as concentration, count, viability, motility, and morphology. However, evidence suggests that the abnormalities of the mentioned elements are not fully indicative of negative fertility outcomes in populations of first-pregnancy planners, especially when assisted reproduction procedures are involved. On the contrary, DFI, an indicator of sperm DNA damage, has been strongly correlated with male infertility issues ,7 , testicular conditions , and pathological status .Sexually transmitted pathogens, specifically Chlamydia trachomatis and Mycoplasma, as well as hepatitis B antigens, have been indicated as agents of spermatozoa DNA fragmentation ,63,64. OGutiérrez et al.  and KaspResults from in vitro studies showed the effects of HPV on DFI. Specifically, Connelly et al.  assessedOur investigation pointed out how HPV positivity does not modify DFI rates, except when comparing HR and LR genotypes. In fact, we found how infections with at least one HR-HPV showed a substantial increase in genomic damage compared to LR-only infections. As far as we know, hardly any study has analyzed the specific influence of HR- or LR-HPV on semen quality and DNA integrity. In particular, Boeri et al.  describeThese findings indicate that it would be appropriate, especially for LR types, to consider the heterogeneity of HPV infections regarding the exact localization of viral particles. Indeed, at the beginning of the infection, HPV virions that originate from the partner are probably mainly localized in seminal plasma and have a limited effect on spermatozoa. Then, when the HPV virions are produced by the infected epithelial cells, they start to be detected also in the spermatozoa fraction . VirionsTherefore, we propose to use a protocol that can easily reveal the presence of viral particles in the different fractions that compose seminal fluid. In our previous study , we highThis valid method, recently included in the ESHRE guideline for MAR  in patients with viral infection/disease  (p. 113)Since it is possible to extract DNA from seminal plasma, sperms, and epithelial cells separately ,65, in oBased on our findings, we can conclude that HPV semen infection, regardless of HPV type and exact location of the virions, significantly impairs spermatozoa’s progressive motility, morphology, and immotile sperm rate. Conversely, the increased rate of DFI was a genotype-related alteration since it was only observed in HR-HPV-infected samples when compared to LR-HPV-infected ones. This is a crucial result as, to the best of our knowledge, the different influences of HR and LR genotypes on semen parameters and sperm genomic integrity have hardly been analyzed. Therefore, our work can explain the conflicting data reported in the literature about HPV and sperm DNA damage. Furthermore, since the effect of HPV infection on seminal parameters could be influenced by the exact localization of viral particles in different semen fractions, when it comes to LR-HPV infection, it would be advisable to perform the differential lysis protocol before testing the DFI. This approach would allow the identification of spermatozoa HPV infection, which could have the highest negative impact on genomic integrity and male fertility. In this study, there were some results, such as the comparison between HPV-positive low- and high-risk patients, where the subgroups analyzed were relatively small, especially the low-risk group. Therefore, in this case, they should be interpreted as preliminary results and should encourage future research.


The infection rate of human papillomavirus (HPV) is high in the coastal regions of China. However, the infection rate among high-risk genotypes of women in Putian City is unknown. Therefore, this study aimed to analyse the epidemiology of high-risk HPV infection among women in Putian and provide a reference for the diagnosis, treatment and vaccination of cervical cancer in this region.The data used were obtained from the Chinese government’s public health program . A total of 40,693 female cervical cell exfoliation samples screened for high-risk HPV at the Affiliated Hospital of Putian University from July 2020 to December 2021 were enrolled. DNA was extracted using a fully automatic extractor. Then, 14 high-risk genotypes of HPV were detected by polymerase chain reaction. The characteristics of HPV infection, distribution of high-risk genotypes, infection types and thinprep cytologic test (TCT) classification at different age groups were analysed.χ2 = 74.03, P < 0.01). The infection rates of high-risk HPV52, HPV58 and HPV16 were in the top three and increased with age. Single infection was dominant (84.7%), followed by double infections (12.7%). The cervical cytology of 3899 HPV-positive people can be classified into negative for intraepithelial lesion and malignancy , atypical squamous cells of undetermined significance , atypical squamous cells—cannot exclude high-grade squamous intraepithelial lesion , low-grade squamous intraepithelial lesion  and high-grade squamous intraepithelial lesion . HPV16 infection rate increased with increasing severity of cervical cytology , whereas the infection rates of HPV52  and HPV58  showed opposite trends.Among the 40,693 samples, 3899 were infected with HPV, with an infection rate of 9.6%. Accordingly, HPV infection rates gradually increased with age, and statistically significant differences were observed among age groups  will lay the foundation for better cancer outcomes in the future. Cervical cancer ranks fourth among malignant tumours in women worldwide. The prevalence rate is increasing annually and has affected young people. In 2018, the World Health Organization (WHO) launched a call to accelerate the elimination of cervical cancer as a global public health problem. In recent years, regional differences have been observed in cervical cancer screening. Specifically, the screening coverage, testing methods and post-screening management differ among high-, middle- and low-income countries. This situation reflects complex factors related to the economic strength of the government, organised population-based programs, monitoring and quality assurance measures and female participation rates . In 2009Human papillomavirus (HPV) is a double-stranded DNA virus (without an envelope) of the papillomavirusidae . It is hCervical cancer screening methods include conventional cytology, liquid-based cytology, visual inspection with acetic acid, nucleic acid testing and cytology based on Romanovsky-Giemsa staining. However, HPV nucleic acid testing, especially high-risk HPV, has shown greater advantages in improving the detection rate of cervical intraepithelial neoplasia (CIN) 2+ and reducing the incidence of cervical cancer –13. HighThe epidemiological characteristics of cervical cancer vary remarkably across different geographical locations. Approximately 90% of the world’s cervical cancer occurs in countries without effective HPV screening and vaccination . HoweverThe data for this study were obtained from the Chinese government’s public health program , under which regular tests are conducted every year. The present survey was conducted in Putian City, Fujian province. A total of 40,693 females who were screened for high-risk HPV at the Affiliated Hospital of Putian University from July 2020 to December 2021 were enrolled. The age range was 30–70 years (50.1 ± 7.9). The inclusion criteria are as follows: no history of hysterectomy or cervical surgery, no severe endocrine or autoimmune diseases, abstinence before the examination, no vaginal administration and non-pregnancy. The exclusion criteria are as follows: incomplete information, history of cervical conization and hysterectomy, history of cervical treatment in the last 3 months, acute reproductive tract inflammation, elimination of secondary re-examination data and exclusion of common gynaecological diseases . All tests were performed with the informed consent of the enrolled women. This study was approved by the Ethics Committee of the Affiliated Hospital of Putian University (202224).Cervical samples were obtained from women by using a cytobrush according to the instructions, and the samples were used for genomic DNA extraction. The samples of cervical exfoliated cells were thoroughly vortexed and mixed. Then, 350 µl of liquid was absorbed into the nucleic acid extraction reagent. HPV DNA was extracted using a nucleic acid extractor. DNA (5 µl) was mixed well with amplification reagent (20 µl). After capping the amplification tube and quick centrifugation, the PCR amplification reaction was carried out via real-time fluorescence quantitative PCR. All procedures were performed strictly according to the kit’s instructions.Specimens that were positive for HPV genotyping were directly used for TCT. Two experienced cytopathologists independently diagnosed the liquid cytology specimens. A third cell pathologist reviewed the sample when the diagnosis of the two cytopathologists differed. According to Bethesda’s system, cytological results were classified into negative for intraepithelial lesion and malignancy (NILM), atypical squamous cells of undetermined significance (ASC-US), atypical squamous cells—cannot exclude high-grade squamous intraepithelial lesion (ASC-H), low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL) .χ2 test was applied to compare the infection rate of different age groups and the infection rate of high-risk genotypes according to additional TCT classifications. Differences were considered statistically significant if the difference was P < 0.05.Data were analysed using GraphPad Prism 7.0 software. An interval of 95% confidence (95% CI) was calculated for HPV prevalence. The χ2 = 74.03, P < 0.01; Table In this study, 40,693 cases were analysed. A total of 3899 cases were HPV positive, and the overall HPV infection rate was 9.6% , in which ASC-US, ASC-H, LSIL and HSIL accounted for 6.6%, 1.4%, 3.2% and 0.8%, respectively Table . In NILMχ2trend = 43.64, P < 0.01), while the infection rates of HPV52  and HPV58  showed the opposite trend  in HPV-positive people gradually increased, thus confirming the high risk of cervical cancer in middle-aged and older women. Interestingly, the proportion of high-risk genotypes differed in different stages of cervical cytological abnormalities. For example, HPV16 ranked first in HSIL and only ranked third in non-HSIL. The HPV52 phenotype was opposite to that of HPV16. HPV16, HPV52 and HPV58 were the top three types of CIN in China . This stThis study retrospectively analysed the characteristics of high-risk HPV infection, genotype distribution and cervical cytology in 40,693 women in Putian, but this study has some shortcomings. First, the data were derived from the screening results in the past 2 years, and long-term dynamic observations are lacking. Moreover, considering the limited sample size, the results might not accurately reflect the epidemiological characteristics of HPV in the whole region. Therefore, in a follow-up study, the sample size will be expanded, and the monitoring time will be extended. Second, the HPV detection kit could detect 14 high-risk genotypes. Although it contained the standard typing of the Asian population, not enough types of typing were used, and a risk of missed detection was involved. Third, the women included in this study were aged 30–70. However, a few investigations have focused on HPV infection in people under 30 years old, especially those under 20 years old, which is not conducive to the epidemiological study of HPV in the whole female population.In summary, regional HPV screening was conducted to provide valuable clinical guidance for cervical cancer prevention and vaccine selection in this region. Vaccination can effectively prevent HPV infection. It is also an effective first-class measure , 46. Biv


Through the initial base excision repair (BER) pathway, 8-oxoG is recognized and excised, and subsequently, other proteins are recruited to complete the repair. OGG1 is primarily located in the cytoplasm and can enter the nucleus and mitochondria to repair damaged DNA or to exert epigenetic regulation of gene transcription. OGG1 is involved in a wide range of physiological processes, such as DNA repair, oxidative stress, inflammation, fibrosis, and autophagy. In recent years, studies have found that OGG1 plays an important role in the progression of kidney diseases through repairing DNA, inducing inflammation, regulating autophagy and other transcriptional regulation, and governing protein interactions and functions during disease and injury. In particular, the epigenetic effects of OGG1 in kidney disease have gradually attracted widespread attention. This study reviews the structure and biological functions of OGG1 and the regulatory mechanism of OGG1 in kidney disease. In addition, the possibility of OGG1 as a potential therapeutic target in kidney disease is discussed. Renal inflammatory diseases are a group of diseases that occur in the kidney and involve the inflammation of cells, including obstructions, tumors, metabolism, heredity, or injury . Acute kROS, the most common cell damage factor in AKI, can be generated either externally or through the cellular environment from the metabolism of cells themselves . GuanineκB/RelA binding to cis-elements and thereby induce the rapid expression of chemokines/cytokines and inflammatory cell accumulation in airways [β1-induced cell transformation through interacting with Smad7, activates SMAD2/3, and promotes fibrosis by interacting with the TGF-β/Smad axis [OGG1 can bind to promoter regions rich in 8-O guanine, causing changes in DNA conformation, the recruitment of transcription factors, and the activation of downstream gene transcription . The mec airways . OGG1 almad axis . Recent mad axis . In hypemad axis .β1-induced cell transformation [OGG1 has been extensively studied in tumors, but less commonly in renal tumors. OGG1 is a common mutation site in renal tumors such as renal clear cell carcinoma . Chronicormation .β-slice and is found only in the OGG1 enzyme. Since OGG2 and AGOG enzymes can cleave 8-oxoG from DNA in the absence of the A domain, this suggests that the N-terminal domain has other functions that are not specifically enzymatic digestive functions. In addition, human OGG1 is present as two major alternative splice isomers, hOGG1α and hOGG1β [α isoform is expressed in the cytoplasm, nucleus, and mitochondria, whereas the human OGG1β isoform is only expressed in the mitochondria. The absence of mitochondrial translocation signals in the A domain of the hOGG1 N-terminus seems to prevent its localization to mitochondria [OGGs belong to the helix-hairpin-helix superfamily of base excision repair DNA glycosylases . The held hOGG1β , which hchondria  and suppchondria . Therefoin vitro experiments, with an increase in APE1 [κB signaling pathway and induce downstream inflammatory gene expression [OGG1 is a DNA glycosylase enzyme with apurinic/apyrimidinic (AP) site lytic activity which removes ROS-induced 8-oxoG . OGG1 ca in APE1 , OGG1 AP in APE1  reduced  in APE1 . OGG1 bipression , 35.OGG1 is mainly located in the cytoplasm and can enter the nucleus and mitochondria to repair DNA and activate multiple transcriptional pathways to regulate cellular homeostasis during cell injury. OGG1 regulates homeostasis through a variety of pathways, including DNA repair , oxidatiThe main types of DNA damage  include 2O2 exposure, OGG1 was recruited to the KRAS promoter and further recruited the downstream nuclear factors MAZ and hnRNPA1, which are critical for transcription, reflecting the transcriptional regulation of OGG1 in the folding and stability of DNA promoter regions [in vitro and in cells, thereby controlling the interaction between OGG1 and RECQL4 after DNA repair by maintaining RECQL4 in a low-acetylated state. The stimulation of human α-OGG1 catalytic activity by AP endonuclease-APE1 was proposed to enhance turnover and bypass of AP lyase activity [α-OGG1 activity was also observed in the presence of the scaffold protein XRCC1 [α-OGG1 also physically interacts with PARP1 to stimulate polyADP-ribosylation [OGG1-deficient cells exhibit enhanced spontaneous mutagenesis , 47. Upr regions . During  regions . Genome- regions . Additioactivity . Owing tin XRCC1 . Human αsylation . This suOGG1 knockout mice are viable, fertile, and do not show marked pathological defects in adulthood. However, a third study reported significantly higher lung tumorigenesis in OGG1 knockout mice at 18 months after birth than in WT mice. Furthermore, OGG1-/- mice accumulate 8-oxoG in the liver nucleus and mitochondrial DNA in an age-dependent manner . Strong Abnormal base excision repair pathways of OGG1 and OGG1 polymorphisms are associated with a variety of tumors in humans . The OGGα-OGG1 protected the mitochondrial network from fragmentation after exposure to menadione. Targeting the human mutant protein MTS-OGG1-R229Q to the mitochondria results in decreased mtDNA integrity and cellular survival after exposure to oxidative agents when compared to the wild-type MTS-OGG1, and catalytically inactive α-OGG1 mutants did not preserve the mitochondrial morphology in the cells exposed to oxidative stress [The most important factor in maintaining the balance of mitochondrial homeostasis is the stable repair of mitochondrial DNA (mtDNA). OGG1, a key enzyme involved in mtDNA repair, is important for mitochondrial homeostasis. OGG1 decreases mitochondrial fragmentation and improves mitochondrial function in H9C2 cells under oxidative stress conditions . The leve stress . In addie stress . Mitochoe stress . Nrf2 cae stress .Under oxidative stress, the repair of 8-oxoG by the BER enzymes OGG1 and APE1 perturbs the structural equilibrium of the VEGF promoter DNA sequence between duplex and G-quadruplex structures, resulting in epigenetic modifications of gene expression . BindingIn addition to targeting DNA, oxidative stress can also affect proteins such as OGG1. Studies  have shoα-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. NF-κB is an important inflammatory transcription factor that enables downstream NLRP3 signaling and TNF-α expression, further promoting the release of IL-18, IL-1β, and other inflammatory cytokines [κB [κB/RelA binding to cis-elements and facilitates the recruitment of specificity protein 1, transcription initiation factor II-D, and p-RNA polymerase II, resulting in the rapid expression of chemokines/cytokines and the accumulation of inflammatory cells in mouse airways [OGG1 plays an important regulatory role in the immune inflammatory response. OGG1-deficient mice had good survival and showed significant resistance to acute and systemic inflammation. Visnes et al.  developeytokines . This deines [κB  and proi airways . OGG1-/- airways . Biochem airways   and one transition (GC to AT) with a base substitution. All three of these substitutions led to amino acid changes in the human OGG1 protein.The ion site . Chevillion site  identifiOGG1 promoter. In the kidneys of patients with tuberous sclerosis, loss of a protein encoded by tuberous sclerosis complex 2 (Tsc2) can downregulate OGG1 protein expression by regulating transcription factor AP4 binding to the OGG1 promoter, thereby increasing the incidence of tumors.In addition, the incidence of multiorgan tumors observed at 34 weeks in OGG1 knockout mice showed that the incidence of colon adenomas and total colon tumors showed a trend with a sharp increase, with bladder hyperplasia and an increased rate of tubular hyperplasia also observed . RapamycOGG1 has been widely studied in brain and lung ischemia-reperfusion injury but less so in renal ischemia-reperfusion injury. In a renal ischemia-reperfusion experiment in rats , high-peκB pathway to activate the inflammatory response is not weakened. Inhibition of OGG1 with TH5487 interferes with OGG1 incision activity, resulting in fewer DNA double-strand breaks in cells exposed to oxidative stress and reducing the DNA mutation rate caused by OGG1 exposure to AP sites, which may reduce the incidence of cancer in cases of oxidative stress damage [The DNA repair function of OGG1 is decreased in ischemia-reperfusion injury, yet the function of inducing the NF-s damage . In addis damage .Staphylococcus aureus is an opportunistic pathogen and is one of the leading causes of life-threatening sepsis [Staphylococcus aureus sepsis induces early renal mtDNA damage, upregulates the expression of inflammatory factors, such as TNF-α, IL-10, and Ngal mRNA, and activates OGG1 [α and the activation of inflammation [H. pylori infection in mouse models [g sepsis . Staphyltes OGG1 . OGG1, aammation , 130. Maammation . Furthere models . These rOGG1 and XRCC1 involved in base excision repair are related to DNA damage [β1-induced cell transformation [OGG1 gene, which modulates the level of 8-oxoG in the leukocytes of CKD patients [OGG1 seems to be as important as the effect of base excision and repair [Chronic kidney disease (CKD) is a multifactorial chronic disease characterized by genetic abnormalities. Genome-wide association studies  have ideA damage . OGG1 prormation . Anotherpatients . OGG1 had repair . How OGGPrevious studies have shown that OGG1 inhibits tumorigenesis via base excision and repair. In recent years, studies have found that OGG1 participates in the regulation of renal inflammatory diseases by upregulating certain inflammatory cytokines, inducing oxidative stress and inflammation, promoting fibrosis, and regulating autophagy. The powerful anti-inflammatory effect of the OGG1 specific inhibitor TH5487 has been verified. As an emerging target, OGG1 has new prospects for the clinical treatment of inflammatory diseases of the kidney.


We examined self‐report data on four mental health outcomes: probable posttraumatic stress disorder (PTSD), depression, psychological distress, and overall mental health functioning. Weighted adjusted odds ratios (aORs) between each outcome and cohort were estimated, controlling for covariates in four models: cohort plus sociodemographic variables (Model 1), Model 1 plus physical health variables (Model 2), Model 2 plus potentially traumatic events , and Model 3 plus other military service variables (Model 4). Mental health outcome prevalence was highest for VTs versus other cohorts, with the largest aOR, 2.88, for PTSD, 95% CI , p < .001 . Physical health and PTEs contributed most to observed effects; other service variables contributed least to aORs overall. Mental health dysfunction persists among VTs years after the war's end. The present results reaffirm previous findings and highlight the need for continued mental health surveillance in VTs.Mental health data from the 2016–2017 Vietnam Era Health Retrospective Observational Study (VE‐HEROeS) were analyzed by cohort, represented by United States Vietnam theater veterans (VTs) who served in Vietnam, Cambodia, and Laos; nontheater veterans (NTs) without theater service; and age‐ and sex‐matched nonveterans (NVs) without military service. The exposure of interest was Vietnam theater service. Surveys mailed to random samples of veterans ( The year 1973 marked the end of American troop involvement in the Vietnam War. But for some individuals who served during this conflict, the war never really came to an end , conducted with United States veterans and civilians between 1984 and 1988  was completed using NVVRS data , which was conducted to provide a broad view of the health of this older veteran population. This national, large‐scale epidemiological study was the first comprehensive health survey of United States Vietnam War veterans conducted in over 30 years. Veterans were identified using a sampling frame obtained from a well‐established database developed by the U.S. Department of Veterans Affairs (VA) and multiple other federal data sources, including the Department of Defense (DoD). VE‐HEROeS data allows researchers to compare three cohorts: (a) Vietnam theater veterans, categorized as individuals deployed to Vietnam, Cambodia, and/or Laos between February 28, 1961, and May 7, 1975, during the Vietnam War; (b) nontheater veterans, categorized as servicemembers who served elsewhere during that period; and (c) sex‐ and age‐matched United States nonveterans. Nonveteran, civilian controls were included to identify the unique effects of service in theater on health status via comparisons to theater and nontheater veterans.The objective of the present analysis was to report on the mental health of aging Vietnam theater veterans relative to demographically comparable nontheater veterans and nonveterans. We hypothesized that psychological issues would still be prevalent nearly 50 years after the end of the war, specifically among individuals who served in the Vietnam theater, that would underscore the need for continued mental health surveillance of this population. The present analysis also included an evaluation of the relative importance of physical health, exposure to traumatic events, and other service variables on the association between cohort and mental health outcome, as well as the statistical interactions that exist among mental health outcomes, race/ethnicity, and cohort.n = 9,870,000) was derived from the VA's U.S. Veterans Eligibility Trends and Statistics (USVETS) database. Stratified random sampling was applied to the veteran sampling frame based on period of service  to generate the veteran sample . Samples of 6,000 theater, nontheater, and nonveteran respondents were estimated to provide 80% power at a two‐sided significance level to detect a 20% difference in odds ratios (ORs) of 1.2 or greater for cohort  comparisons. For the veteran sample, a 40% response rate was assumed, allowing for missing or incorrect addresses and deaths. Deceased veterans and those with missing address information were removed from the veteran sample .The cross‐sectional 2016–2017 VE‐HEROeS was a mail survey of the health of Vietnam War theater and nontheater veterans in the United States, defined by the U.S. Congress as individuals who served in the United States military between February 28, 1961, and May 7, 1975, as well as a sex‐ and age‐matched sample of nonveterans without United States military service  was derived after removing invalid or incomplete data and ineligible individuals .The civilian  sampling frame was developed in two phases that consisted of a random selection of 300,000 United States residential household addresses sourced from a commercial vendor (Phase I), after which a screener questionnaire was mailed to these households (Phase II) to identify members born before 1958 with no military service. Screener questionnaires from 43,198 households were returned, from which the nonveteran sampling frame  and 67.0%  for nonveterans and were computed based on the number of respondents (S1), eligible nonrespondents (S2), known ineligible respondents (S3), individuals with unknown eligibility (S4), and an estimate (e) of the proportion of cases of unknown eligibility that were actually eligible, calculated as RR = S1/(S1+S2+e*S4), where e = (S1+S2)/(S1+S2+S3)] . Scores of 50 or higher typically indicate average or better‐than‐average mental health–related quality of life. In this analysis, we assigned a value of 1 to indicate a below‐average score and a value of 0 to indicate an average or above‐average score. In a large subsample of VE‐HEROeS respondents , the SF‐8TM overall demonstrated excellent internal consistency, Cronbach's α = .94  of the Short‐Form Health Survey  was used to assess current probable PTSD. The PC‐PTSD‐5 has demonstrated high diagnostic accuracy  question that was based on a provider‐identified diagnosis: “Have you ever been told by a doctor or other health professional that you had [condition]?” CDC, . The VE‐SE = .08; specificity = .96, SE = .02; Kessler et al., Past‐month psychological distress, broadly defined as mental disorders such as general anxiety and mood disorders , was used to estimate physical health functioning. The PCS was assessed as a continuous variable from the same eight SF‐8TM subscales underlying the MCS. The PCS has demonstrated good test–retest reliability (r = .73) and discriminant validity based on the SF‐36® veteran version  and criterion validity  was calculated as the quotient of height (kilograms) and weight (meters squared). Data regarding lifetime cigarette smoking were obtained from 2013 NHIS questions CDC, .1The variable “cohort” comprised theater veterans, defined as individuals who served in Vietnam, Cambodia, or Laos; nontheater veterans, categorized as veterans who served during the war but not in the Vietnam theater; and nonveterans, defined as civilians in the United States who had not served in the military. Focus was placed on comparisons between theater veterans and the other two cohorts because of the recognition that Vietnam theater veterans sustained considerable health risks as part of their “Vietnam Experience”  were evaluated.Sociodemographic variables were age at the time of survey administration, sex, educational attainment, marital status, income, and race/ethnicity. For the latter, race was categorized as White, Black, and “other race.” Non‐Hispanic White and non‐Hispanic Black were used to categorize non‐Hispanic single‐race individuals whereas non‐Hispanic “other race” was used to categorize other races of singular origin  and multiracial non‐Hispanic individuals. Hispanic ethnicity included participants who only reported themselves as being of Hispanic, Latino, or Spanish origin, or multiracial individuals who identified themselves as being of Hispanic, Latino, or Spanish origin.n = 23,396, 99.7% male) consisted of 6,735 theater veterans, 12,131 nontheater veterans, 4,530 nonveterans. Women (n = 750) were not excluded because of the poststratification weight adjustments that were made expressly for theater veteran comparisons. Sensitivity analysis based on a domain analysis by sex revealed little or no difference in regression statistics when women were excluded  procedures for complex survey designs. Statistics were weighted except for counts. Survey weights were adjusted for the complex survey design and nonresponse and poststratified using the sex and age distributions of the veteran sampling frame. To compare theater veterans to nontheater veterans and nonveterans, the nontheater and nonveteran weights were poststratified further to match the weighted age and sex distributions of theater survey respondents. Weights used in the present analysis were designed to provide prevalence estimates for Vietnam theater veterans and not for nontheater veterans or nonveterans. Jackknife repeated replication (JRR) was used to estimate standard errors based on the complex survey design. Hot deck imputation was applied to replace missing data for any one of 16 key survey questions from the total set of 82 survey items, with the estimated percentage of missing data ranging from 0.0 to 7.0%. Listwise deletion was still applied because non–key questions could include missing data. The analytical sample  and relative risks, the latter computed using Stata/MP  and unstandardized beta values with 95% confidence intervals were computed. Age and sex were not included in the models because we adjusted for them in the weights. Multicollinearity was tested, and tolerances (unweighted) above 0.40 were deemed satisfactory , Model 2 included the covariates from Model 1 plus the physical health variables , Model 3 included the Model 2 covariates plus PTEs, and Model 4 included the Model 3 covariates plus military service characteristics . The first‐order interaction of Cohort x Race/Ethnicity was examined. Adjusted odds ratios . SF‐8TM PCS was lowest in theater veterans  versus other cohorts, p < .001. As shown in Figure TM subscale scores emerged by cohort such that theater veteran scores were lowest, p < .001. The mean number of PTE types experienced was highest for theater veterans, p < .001 . On average, theater veterans scored 1.23 points lower on the MCS compared with nontheater veterans after controlling for other independent variables, B = −1.23, 95% CI , p < .001. The effects attenuated from Model 1 to Model 4 across outcomes. The smallest decreases occurred upon the addition of other military service variables, whereas larger decreases in effects came from the addition of physical health variables and PTEs.As shown in Table OR = 3.23, 95% CI ; depression, aOR = 2.08, 95% CI ; psychological distress, aOR = 2.86, 95% CI ; and lower MCS, B = −4.17, 95% CI , compared with White participants, p < .001. Weaker, but still significant, effects emerged for Black theater veterans versus White theater veterans for PTSD, aOR = 1.41, 95% CI , p = .022; depression, aOR = 1.45, 95% CI , p = .003; and MCS, B = −2.57, 95% CI , p < .001. Among nontheater veterans, PTSD and lower MCS were evident mostly among Black and Hispanic individuals, and PTSD was elevated among Black versus Hispanic nonveterans, aOR = 4.06, 95% CI , p = .042.As shown in Table Although almost 50 years have passed since the official end of the Vietnam War in 1975, Vietnam theater veterans still report poor mental health. Adverse mental health status at this point in participants’ life trajectory was still higher for theater veterans versus nontheater veterans and nonveterans, paralleling patterns originally reported by the NVVRS in the 1980s .In previous studies of Vietnam‐era service members, theater veterans exposed to high amounts of war zone stress have been shown to report poorer physical health than nontheater veterans or nonveterans  and lifetime generalized anxiety disorder (GAD) among Hispanic male theater veterans 22.4%) relative to their White counterparts  estimate  for NVVLS theater veterans (Marmar et al., In VE‐HEROeS, the prevalence of probable current PTSD was 18.2% in theater veterans. The VE‐HEROeS estimate was higher than the PTSD Checklist for In a sample of U.S. Army personnel with PTSD, a mean baseline adjusted K6 score of 10.33 was found (Olmstead et al., For some theater veterans, memories of military service and later‐life events, such as the loss of family or fellow soldiers, retirement, socioeconomic concerns, weakening physical health, or cognitive decline, may have caused an emergence of late‐onset stress symptoms in older age (Davison et al., Several study limitations should be discussed. The results do not reflect individuals who were homeless or incarcerated and may have had a higher risk of poor mental health nor do they reflect the deceased, leaving individuals in better health to be surveyed. The VE‐HEROeS’ research design did not include oversampling minorities or women. With regard to women specifically, the findings assessed even before poststratification weight adjustments were made would not have been representative of the approximately 5,000–11,000 women veterans who served in the Vietnam theater Earley, . The oveThe present study had several important strengths. Large samples of veterans and nonveteran controls provided the necessary statistical power to determine significant differences. Samples were obtained from a national frame that was generated from USVETS, an established and multisourced database of socioeconomic and benefit characteristics of all United States veterans. The veteran samples reflect a range of military service characteristics, and weights were designed to minimize bias when estimating population means and proportions for characteristics that described the theater veteran population. We examined topics with limited previous research coverage, namely the effects of military service on later‐life mental health in Vietnam theater veterans (Marini et al., In conclusion, the VE‐HEROeS adds updated information about the mental health of aging Vietnam theater veterans. The patterns of mental health disorders reported in the present analysis have persisted over decades. Overall, this study reaffirmed results from previous investigations of the mental health of Vietnam theater veterans and underscored the need for continued surveillance of the complex health effects related to involvement in the Vietnam War.VA supports efforts to provide limited, restricted access to research data under written agreements consistent with commitments made to protecting subjects’ privacy and confidentiality and subject to resource availability. Contact the corresponding author for further information.Supplementary Table S1: Comparisons of Effects: Model Estimates, With/Without WomenClick here for additional data file.Supplementary Table S2: Mental Health Outcomes – Relative RisksClick here for additional data file.


In addition to liquid-based cytology (LBC) and HR HPV testing, p16/ki-67 dual-staining is another method for cervical cancer screening. The combination of any two methods can improve the accuracy of screening, but some cervical lesions are still missed or misdiagnosed. In this retrospective study, the significance of LBC, HR HPV testing and especially p16/ki-67 dual-staining in cervical lesion screening was evaluated with reference to histological diagnosis. At the same time, we tried to explore the value of p16/ki-67 dual-staining combined with LBC and HR HPV testing (triple detection) in improving the diagnostic specificity of CIN2+ and reducing the missed diagnosis of CIN2+ lesions. We found that p16/ki-67 dual-staining was valuable in identifying cervical CIN2+ lesions and reducing the missed diagnosis of CIN2+ in HPV negative patients. More than 96% of CIN2+ patients were positive for two or three tests of triple detection. Whole positive triple detection can effectively predict high grade cervical lesions. In conclusion, the triple detection can distinguish almost all cervical CIN2+ lesions. Our data put forward and highlight the feasibility and significance of triple detection in cervical lesion screening. Cervical cancer is one of the most common female malignant tumors worldwide, and human papillomavirus (HPV) infection is essential cause. Cervical cancer screening is valuable to early find out uterine precancerous lesions. Timely treatment of these lesions may prevent or avoid the occurrence of cervical cancer. At present, cervical cancer screening mainly adopts three strategies, including liquid-based cytology (LBC), HPV testing with partial genotyping, as well as the combined application of the above two methods.Each method has its own advantages and disadvantages. Cytology screening is one of the earliest and most widely used methods, which is characterized by high specificity and relatively low sensitivity. When diagnosed by LBC, about 4-8% atypical squamous cells of undetermined significance (ASCUS) and 12-15% low-grade squamous intraepithelial lesions (LSIL) are grade 2 or more severe cervical intraepithelial neoplasia (CIN2+) lesions, which need to be triaged –5. In adIn order to overcome the shortcomings of HPV and cytology screening, find precancerous lesions as early as possible and reduce the referral rate of colposcopy, it is necessary to find other biomarkers with high sensitivity and specificity. p16/ki-67 dual-staining cytology was reported to be an alternative method in cervical cancer screening –13. p16 Although different screening methods or method combinations have been used to early identify cervical epithelial lesions or cervical cancer, some patients are still missed or misdiagnosed. In this retrospective study, we evaluated the significance of p16/ki-67 dual-staining in cervical cancer screening and the value of triple detection  in improving the specificity of CIN2+ diagnosis and reducing the missed diagnosis of CIN2+ lesions by comparing the coincidence of three examinations, including LBC, HPV testing and p16/ki-67 dual-staining, with histological diagnosis.This retrospective study involved 806 patients selected form the Department of Pathology, the First Affiliated Hospital, Sun Yet-sen University from January 2015 to December 2020 Figure 1in situ (AIS), squamous cell carcinoma (SCC), and adenocarcinoma (ADC). Except for NILM, other cervical lesions were defined as ASCUS+ or positive. After liquid-based cytology, the residual cytological materials were used for HR HPV testing and p16/ki-67 immunostaining.Thin-layer cytology slides were prepared with SurePath Pap Test  and stained by the Papanicolaou method. Cervical cytology was independently interpreted by cytopathologists and classified according to the Bethesda 2015 classification system  and 435 patients by Hybrid Capture 2 (HC2) HPV Test , respectively. Cobas HPV Test is able to detect HPV16 and HPV18 individually and other 12 pooled HR HPV genotypes . HC2 system can detect 13 pooled HR HPV genotypes , and a relative light unit of 1 (1.0 pg/mL) was used as the cut off for HR HPV positivity. All procedures were carried out in accordance with the manufacturer’s instructions.Immunostaining for p16 and ki-67 expression was performed on cytological specimens using the CINtec Plus kit and VENTANA BenchMark XT automated slide stainers  according to manufacturer’s instructions. The primary antibody cocktail comprises a mouse monoclonal antibody (clone E6H4) against human p16 protein and a rabbit monoclonal antibody (clone 274-11 AC3) against human ki-67 protein. Horseradish peroxidase-mediated conversion of 3,3-diaminobenzidine (DAB) and alkaline phosphatase-mediated conversion of Fast Red resulted in brown cytoplasmic/nuclear staining at p16 antigen sites and red nuclear staining at ki-67 antigen sites, respectively. p16/ki-67 dual-staining cells showed brown cytoplasm signals for p16 expression and dark red to red brown nuclear signals for the co-expression of p16 and ki-67 in the same cell. The presence of one or more p16/ki-67 dual-staining cervical epithelial cells was defined as a positive result, regardless of cell morphology Figure 2in situ (AIS) and adenocarcinoma (ADC) were referred to as CIN2+; CIN3, SCC, AIS and ADC were referred to as CIN3+; and Negative  and CIN 1 were referred to as CIN2-.Tissue samples were collected by colposcopy and hysterectomy and processed according to standard histological procedures. The sections were independently diagnosed by the pathologists according to the classification of the 2014 WHO guidelines for cervical histopathology . Final hP values were from 2-sided tests and results were deemed statistically significant at P < 0.05.Chi square of trend for proportion was calculated to test linear associations between screening methods and increasing severity of cytological and histological diagnoses. Associations between p16/ki-67 expression and HR HPV positivity were examined using logistic regression models. Sensitivity [true positive/], specificity [true negative/], positive predictive value  and negative predictive value  were calculated for 2 different endpoints, CIN2+ and CIN3+. Estimates were provided with their 95% confidence intervals (95% CI). In addition, area under ROC curve (AUC) and referral rates to colposcopy based on test positivity were calculated. McNemar tests were used to compare paired matching data such as sensitivities, specificities, PPV and NPV between different screening methods. Analyses were performed in R, version 3.3.1. All A total of 806 women were enrolled in our study. They all completed LBC, HR HPV testing and p16/ki-67 dual-staining, which were confirmed by histological diagnosis. Their average age was 42.4 ± 10.6 years, ranging from 19 to 81 years, with a median of 41 years. They were respectively diagnosed as the Negative, CIN1, CIN2, CIN3, AIS, SCC and ADC in histology or categorized as NILM, ASCUS, AGC NOS, LSIL, ASC-H, AGC FN, HSIL, AIS, SCC and ADC in cytology Table 1.Ptrend < 0.001), and with severity of the histological lesions from 15.3% (33/216) in Negative to 91.3% (63/69) in patients with cancers (SCC+ADC) (Ptrend < 0.001).We first analyzed the positive rates of p16/ki-67 dual-staining and HR HPV in histology or cytology categories and the positive rates of LBC in histology category Table 1.p < 0.001) (p=0.002 and p=0.042). To analyze the correlation between HPV genotypes and p16/ki-67 dual-staining, 371 cases with partial HPV genotyping were grouped into HPV16/18 positive, other 12 HR HPV types positive, and HR HPV negative. The positive rates of p16/ki-67 dual-staining in HPV16/18 positive  and other 12 HR HPV types positive  were significantly higher than those in HR HPV negative (p < 0.001). When the cases were stratified by histology, the positive rate of p16/ki-67 dual-staining in CIN2- group was significantly different (p=0.006 and p=0.020), but there was no significant difference in CIN2+ group (p=0.300 and p=0.254)  Table 2.p=0.780, p=0.254 and p=0.366) and PPV of p16/ki-67 (p=0.186). The specificity and PPV of p16/ki-67 alone was 81.7% and 76.8%, respectively, which was higher than 41.8% and 54.3% in cytology (p < 0.001) and 26.3% and 47.3% in HR HPV (p < 0.001). The NPV of cytology was 95.2%, which was the highest among various methods . Among all analytical methods, AUC of p16/ki-67 alone or triple detection was the highest. The change trend of these parameters also existed in CIN3+ patients.We calculated the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under ROC curve (AUC) of p16/ki-67, HR HPV and cytology as well as three different combinations of these methods in screening for CIN2+ or CIN3+ Table 3.P>0.05, We further analyzed the relationship between HPV genotypes and severity of cervical squamous or glandular epithelial lesions as well as the positive rate of p16/ki-67 Table 4.p < 0.001). Obviously, the positive rate in HPV negative CIN2+ cases is higher than that in HPV negative CIN2- cases.Of a total 806 patients, 147 were HPV negative, including 124 CIN2- and 23 CIN2+ Table 2.When the triple detection was used to screen CIN2+ and CIN3+, the cases positive for all three methods was 80.0% (268/355) and 82.1% (197/240), respectively Table 5.When reviewing the patients’ medical history, we found that 8 patients were positive for HR HPV and p16/ki-67 dual-staining Table 6.Accumulating evidence demonstrated that p16/ki-67 dual-staining cytology showed a high sensitivity and specificity in identifying high grade cervical lesions . In the Compared with HR HPV negative samples, the positive rate of p16/ki-67 dual-staining in HR HPV positive samples is higher, which is reasonable. However, we noted that the positive rate of p16/ki-67 dual-staining was relatively high in HR HPV negative CIN2+ cases 16/23, Table 2.In clinical practice, cervical cytological specimens are usually screened first by LBC or HR HPV testing. Due to the low sensitivity of cytological method and low specificity of HPV primary screening, consequently, p16/ki-67 dual-staining was widely adopted in the stratification of HPV+, HPV+/NILM, ASCUS and LSIL , 25, 29.Although the triple detection is simple and valuable in screening high grade cervical lesions, it is mainly based on a retrospective study. Since most participants are patients with high grade cervical lesions, the subject bias may have an impact on the true meaning of the results. In addition, the sample size of this study was not too large. Before promoting this strategy, a comprehensive evaluation carried out in the screening population is needed.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital, Sun Yet-sen University. The patients/participants provided their written informed consent to participate in this study.LY and SC performed study concept and design, and writing, review and revision of the paper; XC, XL, LF, HM, TT, and LW performed experiments and provided analysis and interpretation of data, and statistical analysis; All authors read and approved the final paper.This study was funded by the National Natural Science Foundation of China (No. 31670788 and No. 81172485) and Open Fund of Guangdong Key Laboratory of Pharmaceutical Functional Genes (No. 2020B1212060031 and No.2017B030314021).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Our data do not support a primary role of oncoviruses in melanoma carcinogenesis; however, the finding of HPV and EBV DNA in a considerable fraction of mucosal and OMs suggests that these viruses may act as cofactors in the development of extra‐CMs.The role of human oncoviruses in melanoma has been poorly investigated. The aim of this study was to investigate the association between oncoviruses and melanomas searching for human papillomavirus (HPV), Epstein Barr virus (EBV), and human herpesvirus 8DNA in melanoma specimens. Formalin‐fixed and paraffin‐embedded tissue specimens of cutaneous, mucosal, and ocular melanomas (OM) were selected from the Pathology Departments of the Galliera Hospital (Genoa) and the University Hospitals of Turin and Cagliari. Cutaneous and mucosal nevi have been collected as controls. The oncoviruses search has been performed with different polymerase chain reaction reagent kits. Fifty‐four melanomas  and 26 nevi  specimens were selected. The detection rate for one of the investigated oncoviruses was 17% in mucosal, 20% in ocular, and 0% in cutaneous melanomas (CMs). Despite the differences between groups seeming remarkable, there was no statistical significance ( Cutaneous melanoma (CM), which arises in non‐glabrous skin, is the most common subtype; mucosal melanoma (MM), the rarest subtype, arises from melanocytes of the mucosal membranes; ocular melanoma (OM) develops from the melanocytes in the uveal tract of the eye.The etiology of melanoma is multifactorial, reflecting the interaction between host‐related and environmental risk factors.With regard to environmental factors, the exposure to ultraviolet (UV) radiation is the main risk factor for CM.As for MM, since it mostly arises on surfaces which are not exposed to sun, this well‐known risk factor for CM is unlikely to be involved. Exposure to formaldehyde for sinonasal MM and cigarette smoking for oral MM have been suggested as risk factors.For OM, besides the host susceptibility factors, two professional occupations, welding, and cooking have been associated with tumor development for the exposure to cooking oil fumes, containing carcinogenic agents such as polycyclic aromatic hydrocarbons, and to intense artificial sources of optical radiation including UV, visible, and infrared light.The role of other environmental factors, such as the human oncoviruses, in the pathogenesis of melanoma has been poorly investigated. Oncoviruses include DNA and RNA viruses that usually are a necessary but not sufficient conditions for developing cancers.Only a few studies have focused on the involvement of human papillomaviruses (HPVs) in melanoma showing contradictory results.The aim of this study was to investigate the association between oncoviruses and melanomas of the skin, mucosal membranes, and eye searching for the presence of high risk (HR)‐HPVs, EBV, and HHV‐8 DNA in melanoma tissue specimens. Archive materials of formalin‐fixed and paraffin‐embedded (FFPE) melanomas were examined using different polymerase chain reaction (PCR) reagent kits. The same tests have been performed in cutaneous and mucosal nevi, as control samples.2A retrospective data search was conducted in the electronic archives of the Pathology Departments of three Hospitals: Galliera Hospital (Genoa) and University Hospitals of Turin and Cagliari.FFPE tissue specimens of MMs and OMs diagnosed between January 1, 2000, and December 31, 2018, were selected. An analogous number of CMs and cutaneous and mucosal nevi diagnosed between January 1, 2018, and December 31, 2020, has been collected from the Galliera Hospital, Genoa.From each FFPE block, two sections of 5 × 10 μm were cut, deparaffinized with xylene, and rehydrated through a graded series of distilled water‐ethanol solutions. QIAamp DNA FFPE Tissue Kit (QIAGEN) was used for DNA extraction. DNA was quantified with a NanoDrop 3300 fluorospectrometer using the RiboGreen dye diluted in TE buffer (Thermo Fisher Scientific).One of the two section specimens obtained from each melanoma sample have been analyzed for the search of HPV DNA in the Pathology Department of the Galliera Hospital, according to the manufacturer's instruction. The other section specimens were sent to the Microbiology Laboratory of the University of Milano‐Bicocca, Milan, Italy, where the analysis for the search for other oncoviruses has been performed.2.1HPV detection and genotyping were performed using the Xpert HPV Assay. Xpert includes reagents for the simultaneous detection of 13 HR‐HPV types  and 1 possible HR‐HPV type (HPV66), a human reference gene (hydroxymethylbilane synthase), and an internal Probe Check Control (PCC). The 14 targeted HPV types are detected in five fluorescent channels: fluorescent channel 1 (HPV16), 2 (HPV18 and −45) (“HPV18/45”), 3 , 4 (HPV pool P4: HPV51 and HPV59) and 5 . The human reference gene (fluorescent channel 6) verifies specimen adequacy. The PCC verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability. In total, the assay utilizes six fluorescent channels for the detection of individual types of HPV, groups of HPVs, and the human reference gene. Each fluorescent channel has its own cutoff parameters for target detection/validity. If a sufficient signal is detected by the human reference gene (if the sample has sufficient cellularity), the assay results are reported as an overall “positive” if any type of targeted HPV is detected, or “negative.” Detailed technical descriptions of the assay have been reported elsewhere.Tissue sections were retrospectively analyzed by quantitative real‐time PCR assays also for DNA search of EBV  and HHV‐8 , according to the manufacturer's instructions.2.2p ≤ 0.05.Fischer's exact test was used to analyze the differences in the virus detection rate between melanomas and nevi and between cutaneous and extra‐CMs. The results were considered statistically significant at 3Overall, 54 melanoma specimens  and 26 nevi were selected. Clinical, histopathological, and virological features of the studied specimens were described in Tables p = 0.54); likewise, the HPV detection rate in extra‐CMs  was higher than in CMs (0%) but the difference was not statistically significant (p = 0.54).The HPV detection rate in all melanoma specimens  resulted higher than in nevi (0%) but the difference was not statistically significant (p = 0.51); similarly, the EBV detection rate in extra‐CMs  was higher than in CMs (0%) but the difference was not statistically significant (p = 0.51).The EBV detection rate in all melanomas  resulted higher than in nevi (0%) but the difference was not statistically significant (p > 0.5).Considering only the samples suitable for the investigation of both HPV and EBV, the detection rate of one of these oncoviruses was 17% in MMs (3/18 cases), 20% in OMs (1/5 cases), and 0% in CMs (0/13 cases). Despite the remarkable difference in the oncovirus detection rate between groups (in favor of MMs and OMs compared to CMs), these differences were not statistically significant ; indeed, HPV16 and HPV18, the most common genotypes in all HPV‐related cancers worldwide,The HPV detection rate in our series (6%) is comparable to that found by Miracco et al. (7%).The presence of HPV in mucosal and cutaneous tissues is not surprising given the mode of the virus transmission by direct contact but the detection of HPV in the choroid melanoma tissue Table , never dThe role of HPV in melanoma development could be either direct through infection of melanocytes or indirect, acting as a cofactor with UV radiation in sun‐exposed mucosal membranes (such as the conjunctiva). The oncogenic properties of the HR‐HPVs reside in the transforming activities of the viral oncoproteins E6 and E7 by interfering with cell cycle regulatory proteins and the immortalization of the infected cells.In our work, besides HR‐HPVs, the presence of EBV and HHV‐8 DNA has been investigated in melanomas and nevi specimens Tables  and 2.We detected EBV DNA in two extra‐CM specimens. Despite the difference in EBV detection rate between extracutaneous and CM specimens was not statistically significant, the EBV detection in a non negligible fraction of extra‐CMs (9%) may suggest a possible role of this virus in melanoma carcinogenesis. After the primary infection, EBV infection establishes a life‐long latency, predominantly in B cells, with occasional reactivation and shedding of progeny viruses. Since the lymphoid system is its “natural niche,” EBV is a risk factor for the development of B and T cell lymphoproliferative disorders.Lastly, in our series, we reported the first case of melanoma that harbored both HR‐HPV and EBV DNA. This co‐infection has been described in other human cancers ,In conclusion, the detection of oncoviruses DNA in a fraction of extra‐CMs (17% of MMs and 20% of OMs) does not indicate necessarily an association between these infections and tumors; however, it cannot be excluded that these viruses may act as cofactors, probably in association with other risk conditions, in the development of melanoma in uncommon body sites.Notably, our work describes for the first time several new findings in the context of oncoviruses and melanoma: the detection of “uncommon” HR‐HPV genotypes in melanoma, differently from the frequently reported HPV16/18 genotypes; the presence of HPV‐EBV DNA in choroid melanoma tissue, supporting the hypothesis that these infections can spread from the skin/mucosal membranes via the bloodstream to other tissues and, finally, the presence of EBV DNA in MM.Further studies are needed to better determine the role of HPV and EBV infection in melanoma, possibly by assessing their viral load to quantify virus activity.Giulia Ciccarese conceived the presented idea and wrote the manuscript with the support of Francesco Drago, Franco Rongioletti, and Carlo Tomasini. Davide Santinelli collected the data with support of Laura Pizzatti, Laura Atzori, and Luca Pilloni. Angela Pastorino, Francesco Broccolo, and Alice Urbani carried out the laboratory analysis. Aurora Parodi, Francesco Drago, and Franco Rongioletti helped supervise the project. all authors discussed the results and contributed to the final manuscript.The authors declare no conflict of interest.