Renal ischemia–reperfusion injury (IRI) is mainly responsible for acute kidney injury for which there is no effective therapy. Accumulating evidence has indicated the important role of mitophagy in mitochondrial homeostasis under stress. OGG1 (8‐oxoguanine DNA glycosylase) is known for functions in excision repair of nuclear and mitochondrial DNA. However, the role of OGG1 in renal IRI remains unclear. Herein, we identified OGG1, induced during IRI, as a key factor mediating hypoxia‐reoxygenation‐induced apoptosis in vitro and renal tissue damage in a renal IRI model. We demonstrated that OGG1 expression during IRI negatively regulates mitophagy by suppressing the PINK1/Parkin pathway, thereby aggravating renal ischemic injury. OGG1 knockout and pharmacological inhibition attenuated renal IRI, in part by activating mitophagy. Our results elucidated the damaging role of OGG1 activation in renal IRI, which is associated with the regulatory role of the PINK1/Parkin pathway in mitophagy. Schematic diagram of molecular model proposed in this study. OGG1 inhibits the clearance of damaged mitochondria under IR conditions, leading to injury of renal tubular epithelial cells. OGG1 expression is upregulated in renal tubular epithelial cells under IR conditions. OGG1 inhibits PINK1 transport to mitochondria, thereby inhibiting the activation of PINK1/Parkin mitophagy pathway, reducing the clearance of damaged mitochondria, and aggravating mitochondrial functional damage, including increased mitochondrial ROS production, decreased mitochondrial membrane potential, and decreased SOD and ATP levels. Further recruitment of Bax and downregulation of Bcl2 promote apoptosis and aggravate the injury of renal tubular epithelial cells induced by IR. All the secondary antibodies were purchased from Thermo Fisher Scientific. The specific inhibitor, Th5487, of OGG1 (HY‐125276) was purchased from Med Chem Express.2.22 at 37°C. To construct the BUMPTs renal hypoxia‐reoxygenation (HR) model, cells were cultured in serum‐free medium and exposed to hypoxia in a tri‐gas incubator  for 24 h followed by 4 h of reoxygenation (95% air and 5% CO2). To inhibit OGG1, 10 μM Th5487 or 0.1% dimethyl sulfoxide was used according to the indicated experimental scheme for the appropriate periods.The BUMPT  cell line was originally obtained from Dr. William Lieberthal and Dr. John Shwartz at Boston University. BUMPT cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) with 10% fetal bovine serum (Gibco), penicillin (100 U/mL), and streptomycin (100 μg/mL) in a humidified atmosphere of 5% CO2.3The mRFP‐GFP‐LC3 plasmid was encapsulated in adenovirus and obtained from Hanbio. The OGG1 short hairpin RNA (shRNA) oligonucleotides and its negative control encapsulated in lentivirus were synthesized by Genechem, and the cells were screened following incubation with 2.5 μg/mL puromycin  for 10 days. PINK1 siRNA oligonucleotides were synthesized by Sangon. BUMPT cells were transiently transfected with PINK1 siRNA and its negative control constructs using HiPerfect transfection reagent . The sequences of PINK1 siRNA and OGG1 shRNA oligonucleotides were as follows:PINK1 siRNA, 5′‐GCCTTGGGTTCAGCAAACA;OGG1 shRNA, 5′‐AAACTTTTTCCGGAATCTGTGG.2.4Eight‐ to ten‐week old male C57/BL6 mice were purchased and housed at the Center of Experimental Animals at Wuhan University. Heterozygous OGG1‐targeted allele using CRISPR/Cas9 to remove all exons of the OGG1 gene. C57BL/6J mice with homozygous OGG1‐targeted alleles were generated by interbreeding. All animal protocols were approved by the Animal Care and Use Committee of the Renmin Hospital of Wuhan University.To generate the mouse renal IR model, all mice  were first anaesthetised with phenobarbital sodium (60 mg/kg) and then the bilateral renal pedicels were clamped to induce 30 min of ischemia and 24 h of reperfusion. The same procedures were conducted in the sham group without renal pedicle clamping. Th5487 was intraperitoneally injected at 30 mg/kg in each mouse every day for 3 days before the IRI operation, and the mental state of the mice after injection was observed. The last injection was given 2 h before the IR model.Intercross heterozygous targeted mice were used to generate OGG1 KO homozygous targeted mice. Polymerase chain reaction (PCR) reactions were used to identify homozygous mouse genotypes. Homozygotes are one band with 662 bp. Heterozygotes are two bands with 662 and 834 bp and wild‐type (WT) allele is one band with 834 bp.PCR Primers 1 : Product size: 662 bp.F1: 5′‐GTACATTTTGGAGTCTAAGCACCG‐3′.R1: 5′‐CAATGTCAAATGTAATGCCAATCCC‐3′.PCR Primers 2 : Product size: 834 bp.F1: 5′‐GTACATTTTGGAGTCTAAGCACCG‐3′.R2: 5′‐ATGTCCCACCTGTAAACTGACTAAT‐3′.2.5BUMPT cells were seeded into 24‐well plates, incubated with the Cell Counting Kit reagent  for 1 h. Absorbance at 450 nm was measured using a multimode reader (Bio‐Rad).2.6Renal function was evaluated measuring the levels of serum creatinine  and blood urea nitrogen  using commercial kits purchased from Changchun Huili Biotech Corporation. Briefly, whole blood samples were placed at room temperature (15–25°C) for 2 h or at 4°C overnight and then separated in refrigerated centrifuge at 2–8°C for 1000 g/15 min. For SCR measurements, serum samples were added to a pre‐warmed reaction solution for 5 min. The absorbance at 546 nm was recorded at the fifth minute. For BUN measurements, serum samples were mixed with the reaction solution for 1 min. Absorbance at 340 nm was recorded in the second minute. Finally, SCR and BUN levels were calculated based on standard curves.2.7Kidney tissues were fixed with 4% paraformaldehyde for paraffin embedding and routine analysis. Kidney sections (4 μM) were stained with haematoxylin–eosin. Ten visual fields in the cortico‐medullary region of each section were observed under ×200 microscope. The percentage of damaged renal tubules in the selected fields was plotted to reflect the degree of tubular damage, which was characterized by brush boundary loss, tubule dilatation/flattening, tubule degeneration, tubule casting, vacuolization, and nuclear condensation.2.8Apoptosis was examined in tissues of the renal cortex using the Colorimetric TUNEL Apoptosis Assay Kit  and co‐stained with lotus tetragonolobus lectin  to visualize tubular damage and 4′,6‐diamidino‐2‐phenylindole (DAPI) to display nuclei. For quantification, four fields with 200 cells (recognized using DAPI staining) per field were observed for TUNEL‐positive cells under each experimental condition to evaluate the percentage of apoptosis. Apoptosis was detected by flow cytometry. Cell preparation was conducted using the Annexin V PE Apoptosis kit . In brief, cells were washed twice with cold PBS, resuspended in 1X binding buffer, and analysed using flow cytometry for 1 h.2.97 cells were pelleted by centrifuging the harvested cell suspension in a 2.0 mL microcentrifuge tube at ~850 × g for 2 min and then 800 μL of Mitochondria Isolation Reagent A was added. Then, 10 μL of Mitochondria Isolation Reagent B and Reagent C were added successively, mixed and centrifuged at 700 × g for 10 min at 4°C. The supernatant was transferred to a new 2.0 mL tube and centrifuged at 12,000 × g for 15 min at 4°C. Furthermore, 500 μL of Mitochondria Isolation Reagent C was added to the pellet and centrifuged at 12,000 × g for 5 min. Finally, we discarded the supernatant and the pellet containing the isolated mitochondria was mixed with 80 μL RIPA to extract mitochondrial proteins and perform immunoblotting.Mitochondria were extracted from cultured proximal tubular cells using a mitochondrial extraction kit . Briefly, 2 × 102.10Superoxide generation, MMP, and adenosine triphosphate (ATP) content were measured as previously described.2.11Paraffin‐embedded renal tissue sections were dewaxed at 65°C, and antigen repair was performed at 95°C in citrate buffer solution  for 1 h. After being incubated with 5% bovine serum albumin for 1 h, the cells were incubated with primary antibody at 4°C overnight and then fluorescence‐bound secondary antibody (Thermo Fisher Scientific) at 37°C for 2 h in the dark. DAPI (Antgene) was used for nuclear staining. For cell immunofluorescence, cover glass was added to the six‐well plate in advance, and the cells were plated until they grew to an appropriate density. The medium was then removed and the cells were fixed in 4% paraformaldehyde for 30 min. Finally, the subsequent operation is the same as that in tissue immunofluorescence. All images were captured using a fluorescence confocal microscope (Olympus). The fluorescence intensity values were analysed using the ImageJ software developed by National Institutes of Health. The immunohistochemical scoring criteria is the intensity score + the frequency score of positive cells. The intensity was scored as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. The frequency of positive cells was defined as follows: 0, less than 5%; 1, 5%–25%; 2, 26%–50%; 3, 51%–75%: and 4, greater than 75%.2.12Briefly, co‐immunoprecipitation was performed using an immunoprecipitation (IP/co‐IP) Kit (Thermo Fisher Scientific). For immunoblotting, total protein samples were separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Sigma), which were blocked with protein‐free rapid blocking buffer (Epizyme) for 30 min and incubated overnight at 4°C. HRP‐labelled goat anti‐rabbit/mouse IgG (H + L) antibody (Antgene) was used as the secondary antibody. Finally, the bands were detected by chemiluminescence (Bio‐Rad) and the grey values were analysed using ImageJ software. Statistical analysis was carried out on the ratio of grey value of each group of samples to that of internal parameters, and GraphPad software was used to draw the histogram.2.13Briefly, kidney tissues were dissected from renal ischemia‐reperfused mice and sham‐operated control mice for electron microscopy as previously described.2.14p < 0.05.We downloaded the IR dataset GSE192532 Data  from the2.15t‐test or one‐way analysis of variance. p < 0.05 was regarded as statistically significant difference.GraphPad Prism 6 developed by Dr. Harvey Motulsky was used for statistical analysis. All values are expressed as the mean ± SD. Differences between all groups were determined using Student's 33.1We first examined relevant IR datasets in the GEO database, identified DEGs in IR mice from the GSE192532 dataset, and mapped the volcano plot. Compared with the control, the volcano plot showed an increase in the expression of OGG1 after IR . Flow cytometry analysis of BUMPTs following PE and 7AAD staining showed increased tubular apoptosis after HR24h, which was suppressed by OGG1‐knockdown Figure . Consist3.5Mitochondrial dysfunction, including MMP collapse and mitochondrial permeability transition pore opening, are crucial factors contributing to IRI‐related damage that can result in cell death. MMP was measured using the probe JC‐1. When MMP depolarizes, JC‐1 exists as a monomer and emits green fluorescence. The results showed that MMP decreased, and apoptosis occurred after HR24h Figure . In addi3.6To further explore the protective mechanism of OGG1‐KO on renal IR, we downloaded the IR datasets (GSE192532 and GSE71647) and the OGG1 KO dataset (GSE35497) from the GEO database to perform VENN analysis and identify the intersection DEGs. The WENN analysis, described above, revealed 211 intersection DEGs between the three datasets that contained OGG1 and PINK1 Figure . We then3.7Next, we examined the function of PINK1 in HR‐induced mitophagy. PINK1 was knocked down using siRNA, and the knockdown efficiency was verified using immunoblotting Figure . As show3.8We continued to explore the regulatory function of OGG1 on the PINK1 mitophagy pathway in BUMPTs. As shown in Figure 3.9We investigated the role of OGG1 in regulating the PINK1/Parkin mitophagy pathway in renal IRI in vivo. As shown in Figure 4The oxidative stress response during IR is a key pathogenetic mechanism in renal IRIStudies on the role of OGG1 in IRI in various organs are limited. Lan et al.OGG1 is a DNA glycosylase enzyme with apurinic/apyrimidinic (AP) site lytic activity that removes ROS‐induced 8‐oxoG.In addition to its function in DNA damage repair, recent research has shown that OGG1 can interact with certain proteins to regulate inflammation, fibrosis and autophagy. For example, OGG1 inhibitors induce type I IFN release via the mtDNA‐cGAS‐Sting‐IRF4‐IFN‐β axis, thereby reducing progression of inflammation.DNA damage and repair are closely associated with autophagy.−/− mouse livers, which may interfere with the results. To explore the association between OGG1 and PINK1 in ischemic renal injury, we isolated the mitochondria and cytoplasm to examine the protein levels of PINK1. We found that PINK1 expression increased in the mitochondria after HR24h, and this increase was further enhanced by OGG1 knockdown, suggesting that OGG1 may negatively regulate PINK1. This is in agreement with our results showing increased levels of the PINK1 mitophagy pathway proteins PINK1, Parkin, and LC3 in response to HR in the BUMPTs model and a further increase when OGG1 function was inhibited or OGG1 was knocked down (Figures The PINK1–PRKN/PARK2‐mediated mitophagy pathway may be the main mitophagy pathway in renal IRI. Figures  and 9A–F Figures . In addi Figures . The aboIn summary, this study provides substantial evidence indicating OGG1 activation in ischemic renal injury and alleviation of renal IRI by OGG1 inhibition or gene KO. Furthermore, mitophagy was induced in renal tubular cells during ischemic AKI and was regulated mainly by the PINK1/Parkin pathway. In addition, OGG1 can inhibit mitophagy by binding to PINK1 and regulating the transfer of PINK1 into mitochondria in IRI. The useful improvement effect of renal IRI of the OGG1 specific inhibitor Th5487 has been verified. Therefore, OGG1 appears to be a novel clinical target with therapeutic potential.Fan Zhao, Jiefu Zhu, and Xiongfei Wu contributed to the conception of the study. Lang Shi, Jiefu Zhu, Shengyu Dong, Yuzhen Li, and Jing Huang performed the experiments. Xiongfei Wu and Jiefu Zhu contributed significantly to the analysis and manuscript preparation. Fan Zhao and Jiefu Zhu performed the data analyses and wrote the manuscript. Fan Zhao, Yanwen Luo, Juan Wang, Halinuer Shadekejiang, Mingjiao Zhang, and Shengyu Dong helped perform the analysis with constructive discussions. Jiefu Zhu and Xiongfei Wu have reviewed the manuscript.The authors declare no conflict of interest.DATA S1. Supporting InformationClick here for additional data file.


Cervical cancer (CvC) is considered a preventable disease; however, in the Caribbean, it is still one of the fourth most common causes of death in women. Efforts to overcome obstacles to the treatment and control of this preventable disease are being made by several countries within the Caribbean. However, no health issue can be readily managed without first acquiring an understanding of the dynamics relating to its severity of impact reaching the target population, its clinical pathology, and the availability of treatment and/or preventative measures to control or halt its progression. To assess the status of CvC in the Caribbean, a review of the literature was conducted using PubMed. The Caribbean was defined in the review as comprising nations and islands whose coastlines are touched by the Caribbean Sea. This led to an assessment of the available literature on CvC for 33 Caribbean territories. The review showed a lack of published information on CvC and highlights the need for greater research. This also serves as a template for subsequent investigations. In 2020, CvC was listed as the fourth most common malignancy in women globally, as well as being widespread and fatal ,2. The rCvC is considered preventable, and as such, the necessity of an early diagnosis is considered the main course of management . This haHowever, it was observed that, among lower-income countries, there was a disparity in the availability and accessibility of screening and prevention programs . This diTo this end, a combination approach of primary and secondary treatment was considered most effective. This involved informed consent for the administration of the prophylactic HPV vaccine and the introduction of screening using HPV assays and Pap smears, as part of a female’s routine check-up, respectively . The datThere are three main HPV vaccines commercially available targeting HPV genotypes 16 and 18 -9, whichMethodologyThe PubMed search engine was selected to conduct a literature review on the information that is available from peer-reviewed publications on the topic of CvC, its contributing factors, development, progression, and prevention. This literature review was conducted within the last 63 years (1958 to October 2022) resulting from the key search words “cervical cancer in/and the Caribbean” on the PubMed database, which resulted in 379 records. Abstracts and full texts were reviewed for the inclusion eligibility of (1) all publications must discuss the Caribbean in general, or a specific Caribbean territory  were reported in 18 publications for 16 Caribbean territories Table . The ASIMortalityThere was limited data provided within the publications covering mortality Table . The ASMPatient demographicsAcross the 16 Caribbean territories, seven demographic factors were explored in the literature for CvC patients. These were age, marital status, ethnicity, income, highest level of education, socioeconomic status, and geographical residence Table . Two-thiIn addition to the deduction that women between the ages of 25 and 34 have the highest risk of contracting the HPV, it is believed that the target population's age has a significant impact on their level of knowledge and the actions they take in the direction of prevention and diagnosis . The oveMarital status was seen as an indicator of safer sexual practices and responsibility. Puerto Rico had the largest proportion of married people (80%) ,65, follThe participants' annual income was disclosed in the publications from the Dominican Republic and Cuba ,36,39; hMore research on how socioeconomic status affects outcomes to determine the impact of income and socioeconomic status on both the influence of accessibility and/or limitations was found to be crucial. Healthcare, treatment, and a more active lifestyle are all more readily available to people with higher incomes. Problems with health insurance and other financial constraints are the root causes of restrictions for those in lower socioeconomic classes. Similar to this, socioeconomic position denies access and privilege to some while imposing limitations and restrictions on others. Geographical influence and socioeconomic status are closely related because a person's social standing typically determines where he lives. Depravity, luxury, and riches are all influenced by one's employment status, income, social class, and fortune .Ethnicity was also shown to be associated with receiving an abnormal cervical smear, and as a result, it is thought to play a role in the development of CvC . Smits eClinicopathologyThe Bahamas, Curacao, the Dominican Republic, French Guiana, Guyana, Haiti, Jamaica, and Puerto Rico highlighted the need to investigate the impact co-morbidities had on the susceptibility of persons developing an HPV infection, on HPV-positive persons or persons undergoing treatment for CvC . The CarBehavioral risksThe age of first coitus is inversely proportionate to the probability of having a cervical neoplastic disease. The relationship is propagated by early marriages and childbearing . An actiGeneticsAn exceptionally wide variation of HPV types exists within the Caribbean: HPV-16, 18, 33, 42, 44, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, and 70 ,100. ThiHPV-16 and HPV-18 account for at least 70% of all invasive CvC cases, with HPV-16 being the most predominant . This coTreatment and controlCvC screening and vaccination are crucial in the control of CvC Table . The HPVIn tandem with the utilization of vaccination, efficiency in CvC screening leading to early detection of cytological abnormalities was identified to be imperative to furtive treatment and survival. Belize and Haiti offer all four screening methods mentioned in Table The only regions throughout the review that spoke of CvC treatment measures were the Bahamas, Belize, Guyana, the Netherlands Antilles, and Trinidad and Tobago Table . The trePubMed was found to be an excellent source of peer-reviewed publications on topics pertaining to CvC in the Caribbean. Although significant research has been conducted in the Caribbean, there still exists a substantial amount of data left to be garnered. There exist enormous gaps in clinicopathology and genetics, as well as a more detailed and specialized investigation specific to the behavioral risks and patient demographics is paramount. It was found that, even though the studies examined in the papers collected a sizable amount of data, a large portion of the data was not utilized. Thus, access to source data will allow for further valuable insights and contributions. In addition, to the measures indicated within this review, the disease burden of environmental impact was only remotely considered, as covered under behavioral risks by other studies, which identified the causative effect of smoking to be a potential cause of cervical carcinogenesis. Some countries, such as the Bahamas, Barbados, the Dominican Republic, Haiti, the Netherlands, and Trinidad and Tobago, are well on their way to mapping a sound course for the prevention and management of CvC by exploring multiple disease burden measures. However, they too require a more thorough examination of these measures, with a focus on causative agents and attitudes, not limited to HPV that must be evaluated and mitigated. Only then a pool of data from all territories can be created to equip the Caribbean region with the resources necessary to eliminate CvC.


On 12 November 2021, after a stagnation of 8.5 years, Japan decided to resume proactive recommendations for HPV vaccines. However, it is not known how the public reacted to the resumption of proactive recommendations for HPV vaccines, which are key elements in health communication that promote HPV vaccination. This study aimed to capture discussions on HPV vaccination on Twitter and to identify user characteristics, sentiments, discussed themes and their potential reach before and after 12 November 2021, when the Ministry of Health, Labour, and Welfare’s decision to resume proactive recommendation for HPV vaccines was first reported by the media in Japan. This study conducted a content analysis of tweets related to HPV vaccines posted on 11 November and 12 November 2021. Tweets were coded in terms of user characteristics, sentiments  and themes. Reach was measured by the number of retweets, likes and followers. A total of 3623 tweets were identified. The results showed that approximately 50% of health professionals and researchers tweeted neutral content about HPV vaccines. The most frequently discussed theme was the safety and side effects of HPV vaccines, which was accompanied by mainly negative sentiments. Although health professionals and researchers are influential on Twitter, half of them tweeted neutrally about HPV vaccines. Influential professionals, such as health professionals and researchers, are expected to disseminate accurate information to correct misinformation and recommend HPV vaccination on Twitter to overcome the HPV vaccination crisis, which is characterized by the low vaccination rates. Monitoring the spread of information about HPV vaccines, the users who disseminate, sentiments and themes discussed on Twitter, can be useful for understanding the public perception of HPV vaccines.Most Twitter users expressed neutral sentiments about HPV vaccines after news reports of Japan’s resumption of proactive recommendations. After the news reports, the most discussed theme was safety concerns regarding HPV vaccines, which were mainly tweeted negatively.Although health professionals and researchers gained more followers, approximately half of them tweeted neutrally about HPV vaccines. They are expected to disseminate accurate information and recommend HPV vaccination on Twitter.Human papillomavirus (HPV) is the most common sexually transmitted infection worldwide . DespiteJapan reported one of the lowest HPV vaccine coverages among high-income countries . In 2013Twitter has been studied as a health research tool  who tweet about the HPV vaccine, and are there any differences in user characteristics before and after the news reports on the resumption of proactive recommendations for HPV vaccines?RQ2: Is there any difference in the sentiment of the tweets before and after the news reports, and any difference in reach (i.e. the sum of followers) by sentiment after the news reports?RQ3: Does the sentiment of tweets differ between users on Twitter? Is there any difference in reach (i.e. the sum of followers) by the type of users after the news reports?RQ4: What are the most frequently discussed themes on Twitter regarding the HPV vaccine before and after the news reports and is there any difference in sentiment?The first author (MT) applied and was approved for academic research on the Twitter application programming interface (API) through a developer account . Tweet dwakuchin’ and ‘yobouseshu’ in the search, which correspond to the following English terms: ‘vaccine’, ‘vaccination’ and ‘immunization’. The Japanese refer to cervical cancer and HPV vaccines as ‘shikyu keigan’ and ‘sikyu keigan wakuchin’. We applied a language filter to retain only Japanese tweets and used the keywords (((HPV OR ‘sikyu keigan’) AND (‘wakuchin’ OR ‘yobouseshu’ OR ‘yobou wakuchin’)) OR Gardasil OR Cervarix OR Silgard) on Twitter and ‘HPV wakuchin’ on Google Trends based on previous studies  of the total sample of tweets was randomly selected, and TN coded them to assess inter-rater reliability for all categories. All data coding was performed using Microsoft Excel .For RQ1, we manually classified users according to their usernames and self-descriptions, consistent with previous studies .A total of 3623 tweets were extracted, of which 349 were tweeted on 11 November 2021 and 3274 were tweeted on 12 November 2021. A total of 271 and 2657 unique users tweeted on 11 and 12 November, respectively. The number of tweets on 12 November was 9.38 times the number on 11 November.The total potential reach of these tweets (the sum of followers) was the highest at 36 169 806 users for neutral sentiment, followed by 9 978 113 users for positive sentiment and 1 478 480 users for negative sentiment.The total potential reach among users (the sum of followers) was the highest for reputable news sources at 25 481 438, followed by 14 644 538 for general public and 3 996 638 for health professionals.N = 3 430 984) of all health professionals’ followers, with 49.1% tweeting positively, 45.3% tweeting neutrally and 5.7% tweeting negatively regarding HPV vaccines. The top 12 researcher influencers had 87.6% (N = 715 001) of all researchers’ followers, with 50.0% tweeting positively, 33.3% tweeting neutrally and 16.7% tweeting negatively regarding HPV vaccines. The top 13 politician influencers had 94.8% (N = 1 035 150) of all politicians’ followers, with 38.5% tweeting positively, 61.5% tweeting neutrally and 0% tweeting negatively regarding HPV vaccines.This content analysis presented an overview of the user characteristics, sentiments and themes of public discussions about HPV vaccines, and their potential reach on Twitter before and after the news reports of the resumption of proactive recommendations for HPV vaccines. The tweet volume of the HPV vaccine increased 9.4-fold after the news reports, indicating the magnitude of public interest.Regarding user characteristics, general public who could not be classified into any specific category accounted for approximately 80% of users. Health professionals accounted for approximately 5% on both days which was lower than expected. Previous studies found that approximately 10% of English tweets related to HPV vaccines . This alThe sentiment analysis showed that negative tweets were 27.8% before the news of the resumption of recommendation, which was similar to previous studies (e.g. 25–43%)  among health professionals, researchers and politicians accounted for most of the followers. The median number of retweets and likes increased as the number of followers increased, suggesting that interaction and reach of Twitter discussions are strongly dependent on those influencers. Only half of the self-defined health professionals, researchers and politicians were positive towards HPV vaccines, whereas more than 40% (including influencers) were neutral. These results show a striking contrast with the results of a study reporting that 95.3% of advocacy users, including health professionals and research institutions, tweeted positively about HPV vaccines in English . Negative sentiment dominated 42.6% of the tweets with the theme of ‘safety and side effects’. Side effects were the most common theme in Japanese online information on HPV vaccines in 2016  tweeted neutrally about HPV vaccines. The most frequently discussed theme was concerned with the safety and side effects of HPV vaccines; it was mostly mentioned from a negative perspective. Influential professionals, including health professionals and researchers, are expected to provide peer-to-peer discussions and disseminate accurate information through tweets to correct misinformation and recommend HPV vaccination on Twitter and to contribute to overcome HPV vaccination crisis.daad153_suppl_Supplementary_Appendixs_1-2Click here for additional data file.


Chlamydia trachomatis (CT), Neisseria gonorrheae (NG), Trichomonas vaginalis (TV), Mycoplasma genitalium (MG), and Human alphaherpesvirus 2 (HSV-2). The Kappa test evaluated the concordance between anal and genital infections. The overall prevalence of anal HPV infection was 31.3% in HIV-uninfected and 97.6% in HIV-infected women. The most frequent anal high-risk HPV (hrHPV) types were HPV18 and HPV16 in HIV-uninfected women and HPV51, HPV59, HPV31, and HPV58 in HIV-infected women. Anal HPV75 Betapapillomavirus was also identified. Anal non-HPV STIs were identified in 13.0% of all participants. The concordance analysis was fair for CT, MG, and HSV-2, almost perfect agreement for NG, moderate for HPV, and variable for the most frequent anal hrHPV types. Thus, a high prevalence of anal HPV infection with moderate and fair concordance between anal and genital HPV and non-HPV STIs was observed in our study.The aim of this study was to classify the diversity of anal HPV and non-HPV sexually transmitted infections (STIs) and compare the concordance between anal and genital infections in HIV-infected and uninfected women living in the Tapajós region, Amazon, Brazil. A cross-sectional study was performed with 112 HIV-uninfected and 41 HIV-infected nonindigenous women. Anal and cervical scrapings were collected and analyzed for HPV,  Data on sexually transmitted infections (STIs) are predominantly related to genital infections. Studies on the anal human papillomavirus (HPV) and non-HPV STIs remain scarce. Cervical cancer screening might contribute to the prevention of anal cancer in women as a secondary prevention measure for anal cancer when stratified to HIV-uninfected women older than 45 years, those with high-risk HPV (hrHPV) cervical infection, or HIV-infected women . ChangesWomen were invited to participate in the study after signing a written informed consent document in strict compliance with the Brazilian ethical guidelines involving research on human subjects (protocol numbers 2.013.163 and 2.121.697). An epidemiological interview was then conducted.This study was supported by previous studies in which we evaluated the acceptability of cervico-vaginal self-collection, HPV and other STI testing, and sociodemographic data of 153 nonindigenous women, 112 HIV-uninfected and 41 HIV-infected, living in the Tapajós region, fully described elsewhere ,9,10. FoThinPrep Solution. Anal and cervical sample collection and processing, as well as the transportation of the biological samples according to international rules (IATA) for laboratory analyses, are described elsewhere [Anal scraping was performed using a brush introduced approximately 1.95 inches into the anal canal, rotated 680° five times, and later preserved in lsewhere .DNA extraction from the anal samples was performed with the QIAamp DNA Mini Kit  according to the manufacturer’s protocol and was quantified by spectrophotometry on a NanoDrop . Nested PCR was performed to increase the specificity of HPV DNA detection, with PGMY09 and PGMY11 primers for the first round and GP5+ and GP6+ primers for the second round, as previously described , in an a® SV Gel and PCR Clean-up System . HPV genotyping and multiple HPV types were performed according to previously published protocols [Betapapillomavirus was identified through nucleotide sequencing analysis and confirmed at the International Agency for Research on Cancer in Lyon, France, by type-specific multiplex genotyping (TS-MPG) assays combining multiplex polymerase chain reaction (PCR) and bead-based Luminex technology , as described elsewhere [Nested PCR products were purified with a Wizardrotocols ,11. HPV rotocols . The onllsewhere ,14.Chlamydia trachomatis (CT), Neisseria gonorrheae (NG), Trichomonas vaginalis (TV), Mycoplasma genitalium (MG), and Human alphaherpesvirus 2 (HSV-2 or herpes simplex virus 2). Duplex real-time PCR was performed for CT and NG, and three real-time PCRs were performed independently to detect TV, MG, and HSV-2. The molecular detection of other non-HPV STIs was performed as previously described [In this study, the non-HPV STIs assessed were escribed ,10. All p-value ≤ 0.05 was used to compare the proportions between the groups. A Mann–Whitney test with 95% CI and p-value ≤ 0.05 was performed to evaluate the associations among median HIV viral load, CD4+ T-cell count, and anal hrHPV infection. Pearson’s correlation test with 95% CI and p-value ≤ 0.05 was performed to evaluate the association of the quantitative proportion of the CD4+ T-cell count and the number of distinct hrHPV types. A Kappa test with 95% CI and p-value ≤ 0.05 was performed to evaluate the concordance between anal and genital HPV and other STIs  [Descriptive statistics of the qualitative variables were determined by frequency distribution. The Chi-square test at 95% CI and d HSV-2)  isolatedd HSV-2) ,9,10. Wen = 112) and HIV-infected women (n = 41). The mean age of all participants was 36.9 (±12.9) years. Most of the HIV-infected women were single (p = 0.006), had an average of 4.3 gestations (p = 0.050), started sex at the age of 17 years or below (p = 0.052), had more than four sexual partners in life (p = 0.012), and regularly used condoms (p = 0.010) (p = 0.457) did not significantly differ between the two groups  women agreed to participate in the study, and their anal and cervical scrapings (clinician-collected samples) were collected on the same day. The participants were divided into two groups: HIV-uninfected women (= 0.010) . The prao groups .Alphapapillomavirus  and Betapapillomavirus  are presented in The overall prevalence of anal HPV infection was 31.3% 35/112) among HIV-uninfected women and 97.6% (40/41) among HIV-infected women. A high diversity of anal HPV genotypes was identified in HIV-uninfected and HIV-infected women participating in the study . Anal HP among HIn = 112) and HIV-infected women (n = 41) were further analyzed according to the presence or absence of anal hrHPV, based on sociodemographic characteristics. Among HIV-uninfected women, most of those with anal hrHPV had more than four lifetime sexual partners (p = 0.040) and cervical HPV (p = 0.045), whereas among HIV-infected women, only cervical hrHPV was significantly associated with anal hrHPV (p ≤ 0.001). The practice of anal sex was not significant for the occurrence of anal hrHPV in both groups of women infected and not with HIV  had single anal HPV infections, and 3.0% (1/35) had multiple anal HPV infections. In HIV-infected women, multiple anal HPV infections were much more prevalent, at 85.0% (34/40), and single anal HPV infections accounted for 15.0% (6/40).Alphapapillomavirus. Based on the oncogenic risk classification, the HPV samples were grouped into high-risk, “probable” or “possible” carcinogenic, and low-risk groups (p < 0.001).Anal HPV types found in the participants were predominantly of the genus k groups . Anal hrn = 6) and HPV16 (n = 5) were the most frequent anal hrHPV types in HIV-uninfected women, representing 73.3% (11/15) of the total anal hrHPV types genotyped in this group. The most frequent anal hrHPV types in HIV-infected women were HPV51 (n = 12), HPV59 (n = 10), HPV31 (n = 9), and HPV58 (n = 9).The overall prevalence of hrHPV was 42.9% (15/35) among HIV-uninfected women and 75.0% (30/40) among HIV-infected women. HPV18 (p = 0.110), and median CD4+ counts (Mann–Whitney test p = 0.828). Pearson’s correlation test shows that HIV-infected women with lower CD4+ counts do not carry a significantly higher number of distinct anal hrHPV types .All HIV-infected women were on combination antiretroviral therapy (cART). There was no significant association between anal hrHPV infection, median HIV viral load  of HIV-uninfected women and 7.3% (3/41) of HIV-infected women . The pre/112 of HGenital HPV infection and other STIs observed in the groups of women included in the present study have already been published by our group ,9,10. ThBetapillomavirus genus, were detected  and HIV-infected (97.6%) women living in the Tapajós region, higher than the national prevalence of anal HPV infection (25.68%) . Moreovedetected .Our earlier analyses revealed that this cohort of women had a high prevalence of cervical HPV infection, hrHPV, and other STIs ,9,10. HoIn this study, a high overall prevalence of anal HPV infection was identified in HIV-uninfected and HIV-infected women. However, the prevalence of anal hrHPV infection, despite the practice of anal sex, did not differ significantly among the participants . FurtherNo significant association was found between anal hrHPV infection and median HIV viral load or median CD4+ count. Although lower CD4+ counts did not lead to a significantly higher number of distinct anal hrHPV types, anal hrHPV infections were significantly more prevalent in HIV-infected women than in HIV-uninfected women (Chi-square test p < 0.001). Persistent hrHPV infection is directly associated with cancer development due to the accumulation of alterations in numerous cellular processes, including DNA repair, angiogenesis, or apoptosis caused by the viral oncogenes E6 and E7 ,24. In aOur study has some limitations. The cross-sectional epidemiological design does not allow the temporal relationship between exposure and effect to be established, making it difficult to establish a causal relationship. The impediment to performing the Kappa test for concordance analysis for TV and the difficulty in interpreting the results of non-HPV STIs may have occurred due to the low prevalence of other STIs, potentially influenced by the sample size. In addition, as a limitation for performing the Kappa test for concordance analyses, we categorized HPV types as high- and low-risk and the most frequent anal hrHPV types, rather than HPV genotypes, due to the great variability of HPV types observed in both groups, which would reduce the statistical power of the comparative analyses. This is a limitation of this type of comparative study when there is a great diversity of identified HPV genotypes.This high prevalence of anal HPV and hrHPV infections and the high diversity and multiplicity of HPV types identified in HIV-uninfected and HIV-infected women in the Tapajós region add to the growing body of evidence published in the scientific literature that the high prevalence and long-term persistence of HPV infection is a hereditary characteristic of the prehistoric people of the Amazon ,28,29. TRecent studies have suggested that hrHPV infection is a sufficient cause of anal intraepithelial lesions and anal cancer ,24,30. HThe early onset of sexual activity (sex at the age of 17 years or under), a high number of pregnancies (3.4 gestations on average), the number of sexual partners , and the nonuse of condoms are possibly the main risk factors related to the high prevalence and diversity of anal HPV types identified in the two groups of women included in the present study. Social factors, such as difficult access to communities and lack of access to basic public health services, have a higher impact on the consequences of long-term persistent anal HPV infection for women. Cultural and behavioral factors, such as low frequency of condom usage and limited hygienic conditions, might explain recurrent infections associated with high HPV prevalence and diversity . In addiIn agreement with the literature ,7,33, HPThe most frequent anal hrHPV types identified in HIV-infected women included in our study were HPV51, HPV59, HPV31, and HPV58, which are HPV types not covered by the 4-valent or 2-valent HPV vaccines available in Brazil. HPV16, followed by HPV18, are the types most related to anal cancer; however, HPV31, HPV33, HPV45, HPV52, and HPV58 were extracted from the nonHPV16 fraction and are possibly associated with anal cancer . This stThe Tapajós region, Amazon, located in northern Brazil, has the highest cervical cancer incidence and mortality rates among women in a national ranking. It is necessary to consider reviewing and updating public health policies on cervical cancer screening and furthering the fight against HIV/AIDS and other STIs. New technologies are warranted for screening and achieving early diagnoses, such as alternative self-collection samples for HPV and non-HPV STIs and DNA testing based on nucleic acid amplification assays (NAATs). Altogether, these approaches might enable a better assessment of cervical screening results to stratify anal cancer risk in high-risk groups, irrespective of HIV status, and to expand cancer screening services to populations in limited-resource settings and remote geographical regions.Overall, the prevalence of anal HPV infection is high in HIV-uninfected and HIV-infected women living in the Tapajós region of the Amazon. We identified a much higher prevalence rate of anal HPV and hrHPV than those reported in the literature for the Brazilian population, with different types of hrHPV not included in the 4-valent HPV vaccine. There was a greater diversity and multiplicity of anal HPV types, particularly in HIV-infected women. Moderate and fair concordance between anal and genital HPV infection and non-HPV STIs were identified, respectively. We encourage public health care officials to consider stratifying anal cancer risk in high-risk groups, irrespective of HIV status, when assessing cervical screening results, particularly in regions where there is a high prevalence of anal HPV and hrHPV infection with high diversity and multiplicity of types.


Integration of the HR-HPV DNA into the host genome plays a crucial role in cervical carcinoma. An alteration of the pRb pathways by the E7 proteins is one of the mechanisms that’s account for the transforming capacity of high-risk papillomavirus. For the proper understanding of the underline mechanism of the progression of the disease, the present study investigate the correlation of concentration of host pRb protein, viral E7 oncoprotein and viral load in early and advanced stages of cervical carcinoma. It was found that the viral load in early stages (stage I and II) was less (log The HPV genome is non-enveloped, double-stranded DNA, approximately 8 kb in size, with eight open reading frames consisting of six early  and two late (L1 and L2) genes that encode “early” (E) or “late” (L) proteins3. The E6 and E7 proteins have transforming properties that can convert normal cells into cancerous ones. Based on the High and Low-risk of causing cancer, more than 111 human papillomaviruses (HPV) types have been described. Among them, around 14 are classified as high risk including HPV-16, HPV -18, HPV-31, HPV-33, and HPV-45. The low risks include HPV-6 and HPV-11 which causes genital warts4.Cervical Cancer originates within the cells that line the cervix. It is primarily caused by the sequel to long-term, unresolved infection by certain genotypes of the human papillomavirus. The high-risk HPV-16 and HPV-18 subtype infections are the main types of HPV that are involved in the initiation and progression of the cervical carcinoma5. Various studies indicate that HPV infection alone is not capable of transforming a normal epithelial cell to malignant one. The majority of HPV infections are asymptomatic and self-limiting hence could be naturally cleared in 70–90% of individuals with HPV infection. Hence genetic alterations in the genome of host cell seem to be required for the development and progression of cervical carcinoma.HPV infection is essential but not sufficient for tumor induction6. High-risk types, most notably HPV16 and HPV18, are the aetiological agent of cervical cancer. More than 35 HPV types are found in the genital tract, and HPV16 accounts for 50% to 60% of the cervical cancer cases in most countries including the United States and Europe, followed by HPV18 (10–12%) and HPVs 31 and45 (4–5% each)7. In a south Indian study, 87.8% high-risk HPVs positivity, with HPV-16 (66.7%), HPV-18 (19.4%), and other high-risk HPV types (1.7%), among HPV-positive cervical carcinoma patients was reported8.HPV is probably the most common sexually transmitted disease agent. It is estimated that the worldwide age-standardized prevalence of current HPV infection is 10.5% in women; the prevalence varies about 20-fold between different regions, from 1.4% in Spain to 25.6% in Nigeria11 This might be due to the interruption of the HPV-16 E2 open reading frame, leading to cellular immortalization by increasing the level of mRNAs of viral oncogenes E6 and E711. The E2 gene is the most frequent site of disruption of viral DNA12. Host genome integration is believed to occur at some specific integration target sequences, the so-called genomic hotspots, where regions of microhomology  between viral and human genome sequences are found and are correlated with frequent fragile14 and transcriptionally active sites of the genome15.The E7 oncoproteins principally act through protein–protein interactions to change key pathways that regulate the progression of the cell cycle and cell proliferation by inactivating the retinoblastoma pocket proteins, suggesting that viral protein interactions with host cell proteins play a crucial role in regulating cell cycle progression in cervical carcinomas. The present study is designed to evaluate the risk of cervical cancer progression by quantifying E7 and pRb protein in the early and advanced clinical stages of cervical cancer and correlating it with the viral load. The analysis of clinically validated biomarkers such as HPV viral load, E7 proteins and cellular pRb profile in different stages of cervical cancer will help in detection of specific biomarkers for the screening of different stages of cervical cancer and a better understanding of the underline mechanism.HPV-16 integration into the host genome is a crucial step in the progression of cervical cancer which provides selective growth advantage compared to cells having HPV-16 viral particles as episomesThe cervical cancer patients recruited for the study were women who were attending Gynaecology OPD of MNJ cancer hospital Hyderabad. A total of 105 HPV-16 positive cervical cancer tissue samples and 30 HPV negative non-cancerous samples from patients who are suffering from some gynecological problems not related to cervical cancer were collected after taking approval from the institutional ethical committee of Maulana Azad National Urdu University Hyderabad and MNJ cancer hospital Hyderabad. All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained from all the subjects and/or their legal guardians. The study subjects were aged 18–70.A portion of the collected biopsy tissue samples was formalin-fixed, paraffin-embedded, and used for histopathological examination and the other portion was stored in PBS vials at − 80 °C for viral load determination and western blot analysis. Stages of the cancerous cases were confirmed through histopathology using FIGO classification.Genomic DNA was isolated from the histopathologically confirmed biopsy tissue samples of cervical cancer patients and controlled using salting out manual method. The HPV16/18 L1 amplicons were PCR-amplified from positive control genomic DNA using Taq polymerase and standard PCR conditions for cloning of HPV16 and 18 L1 amplicons. Both HPV16/18 L1 amplicons were cloned into plasmid vectors according to obtain the plasmids, pHPV16L1, and pHPV18L1, respectively. Purified plasmids were used to derive standard curves in the real-time PCR assay for the detection and quantification of HPV16 and 18 L1 amplicons.Real-time PCR reactions were performed using the Sybr Green master mix after standardizing the PCR conditions. For standard curves, real-time PCR was performed on purified plasmids, pHPV16 and pHPV18.TMBCA Protein Assay Kit, Cat No 23225 and 23227) using bovine serum albumin as standard. Total proteins were separated by polyacrylamide gel electrophoresis using 10% resolving and 5% stacking gel and transferred to a nitrocellulose membrane.Frozen Tissue biopsy samples were lysed using a protein lysis buffer. Total protein was measured by Bicinchoninic Acid (BCA) method using a BCA protein assay Kit (ratio 50:1), according to the manufacturer’s instructions , and mouse anti-human papillomavirus 16/18 (E7) monoclonal antibody (1:100 dilution: Thermo Fisher Scientific (Cat. No. 28-0006 and Cat. No CF811359 respectively) were incubated with the Nitrocellulose membrane (NC) overnight at 4 °C. Antibodies against Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for the target proteins and incubated under the same conditions . After incubation with the primary antibodies, the membrane was washed with PBST and the membrane was then incubated with goat anti-mouse secondary antibody  for one hour. The membranes were washed with PBST and the protein bands on the membrane were detected using enhanced chemiluminescence substrate  for one minute according to the manufacturer’s instructions and the bands were visualised in ChemiDoc (Bio-Red). The bands on the membrane were analyzed by densitometric analysis using IMAGE J Software. The NC membrane was cut horizontally prior to hybridization with primary antibodies according to the molecular weight of 3 different  proteins to quantify from a single blot, and incubated separately with specific primary and its corresponding secondary antibodies for images development.The present study was undertaken after approval from the institutional ethical committee of Maulana Azad National Urdu University Hyderabad and MNJ cancer hospital Hyderabad. Written informed consent was obtained from all the subjects and/or their legal guardians.One hundred and five cervical cancer patients with at least one positive marker of cervical abnormalities were recruited for the study. All patients were south Indian aged 30–90. According to histological findings, 07 patients were diagnosed with stage I, 53 patients were diagnosed with stage II, 30 patients were diagnosed with stage III and 15 patients were diagnosed with stage IV. In cervical carcinoma, all samples are HPV-16 positive including 8 samples co-infected with HPV-18.10 value) was evaluated in 105 cervical cancer patients in different stages. We observed that the median HPV16 viral load increases with the lesion grade. In Stage I, viral load ranged from 240 copies/µg of DNA  to 460 copies/µg of DNA  with a median viral load of log10 value 2.6. In stage II, viral load ranged from 122 copies/µg of DNA  to6624665 copies/µg of DNA)  with a median viral load of log10 value 4.4.In stage III, viral load ranged from 659 copies/µg of DNA  to 26,213,401 copies/µg of DNA)  with a median viral load of 4.9 and in Stage IV, viral load ranged from 788 copies/µg of DNA  to198908450 copies/µg of DNA  with a median viral load of 5.2 were detected. Figure p value < 0.001) as shown in Table The number of HPV16 viral genomes  per ng of cellular DNA , stage I and stage II , stage II versus Stage IV  and stage I and stage IV .Tukey's multiple comparisons test was used to compare HPV16 viral load among different stages of cervical cancer tissue samples, A significant difference in viral load was found between stage I and stage III . Also, a high and significant viral load was found in stage III versus stage I  and stage IV versus stage I cervical cancer cases . No significant difference in HPV16 viral load was found between stage III and IV  as shown in Fig. Mann–Whitney U-Test, a nonparametric form of T-test was used to check the difference in viral load in different stages of cervical cancer. HPV16 viral load in stage II was high and statistically significant in comparison to stage I cervical cancer cases  while two samples showed normal expression. In stage II, 43/53 (81.03%) samples had no significant difference in pRb expression when compared with stage I , but a significant difference was found when compared to normal non-cancerous tissue samples  while ten samples showed normal expression. In higher stages, the expression of pRb was significantly less in 24/30 (80%) cases in stage III  and 13/15 (86%) in stage IV  when compared to normal non-cancerous cases. Also, a less statistically significant difference was found in the expression of pRb of stage III  and stage IV cervical cancer cases  compared to normal non-cancerous cases. Rest 8 samples of higher stages shows normal expression.The retinoblastoma protein (pRb) is a nuclear phosphoprotein that plays an essential role in regulating the cell cycle. HPV E7 protein has been proposed to interact with host pRb tumor suppressor protein, thus abrogating its function. The expression levels of pRb in different stages of cervical cancer cases were measured relative to normal non-cancerous tissue samples. In stage I, 5/7 (71.4%) samples showed significantly high expression of pRb , stage I versus III (mean difference 0.2519), p-value (< 0.0001), and between normal control versus stage IV (mean difference 0.1952), p-value (< 0.0001) and the lowest mean value were found between normal control versus stage II (mean difference − 0.094), p-value (0.004), Normal control versus stage I (mean difference − 0.1507), p-value (0.0090) and between stage III versus stage IV (mean difference 0.094), p-value (0.097).The pairwise Tukey multiple comparisons test that was used to check the mean difference and level of significance of retinoblastoma tumor suppressor protein (pRb) between normal control and different stages of cervical cancer cases Table . The higp value < 0.001), however in late stage III and stage IV, the expression of pRb is less and statistically significant compared to normal non-cancerous tissue samples  as shown in Figs. Based on Mann–Whitney U-Test, it was observed that the expression of pRb tumor suppressor protein varies significantly within different stages of cervical cancer compared to normal non-cancerous tissue samples. Stage I and II show a high and significant difference in pRb expression compared to normal non-cancerous tissue samples  with reference to GAPDH protein. However, in advanced stage III and stage IV, the expression of E7 is statistically significant high compared to pRb expression  with reference to GAPDH protein.  was found between stage I pRb and stage I HPV E7 − 0.053) and stage II pRb and stage II HPV E7. Also, expression of HPV E7 between stage I and III (− 0.18) and stage I and IV (-0.15) were found significantly negatively correlated  is one the prime cause of cervical cancer. High-risk HPV-16/18 is involved in the multistep pathogenesis of cervical carcinoma. Along with High-risk HPV-16/18, the progression of cervical cancer can be influenced by the host immune system and other carcinogens20. In our study, we included only HPV-positive cases as the main aim of our study was the determination of viral load and its association with the progression of the disease. The number of HPV16viral genomes  per ng of cellular DNA  was evaluated in 105 cervical cancer patients in different stages. The association between the viral load and the different phases of disease is still debated. We found a higher HPV16 viral load associated with advance stages of cervical cancer. The HPV-16 viral load increased with the severity of the disease, with significant differences between early and late-stage carcinoma. Most studies have observed that higher HPV viral loads are associated with augmented abnormal cytological or histological Diagnosis28. Although some studies did not observe a positive association between the viral load and the severity of the disease30. The differences in results may be due to different methods used to measure and analyze viral loads and also the range of viral load in different patients is enormous. The studies with the most comparable reporting to ours were that of34 who found increased HPV16 viral load with the increased severity of the disease.The etiopathologic association of high-risk HPV infection with squamous cell carcinoma of the uterine cervix has been well documented in several studies36. The functional state of pRb depends on the state of its phosphorylation, which occurs in a cell cycle-dependent manner. The hypophosphorylated form of pRb functions as a cell cycle regulator. It is also known that several adenoviruses and other DNA viruses proteins including the SV40 large tumor antigen and HPV E7 protein bind to the hypophosphorylated form of pRb41. The binding of viral oncoproteins to hypophosphorylated form pRb is thought to functionally inactivate its tumor-suppressor activity. The expression of pRb has not been intensively studied in human cervical cancers along with the HPV status.The known mechanism of loss of normal Rb function is either due to mutation in Rb gene or binding of HPV-E7 protein to pRb-E2F complex which removes cell cycle restriction. HPV-16 E7 protein interacts with and functionally inactivates the tumor suppressor protein pRb and destabilizes the Rb-E2F complex resulting in the release of an active E2F transcription factor. The free E2F stimulates the cell to enter into the S phase of the cycle. It is reported that the E7 protein is found predominantly in the nucleus and in small amounts in the cytoplasm of the cervical cancer cell line CaSki and cervical carcinoma cells, suggesting an important role of E7 in the development of cervical carcinoma48. However, such studies were not able to distinguish between different cell types within the tissues analyzed. Some immunohistochemical studies on pRb in various human cancer and normal tissues including the uterine cervix shows that pRb is expressed in mature and differentiated cells50. According to a study conducted by51, pRb expression is relatively less in CIN III and Squamous Cell Carcinoma compared to the normal squamous epithelium and squamous metaplasia.Several studies in different cancerous and normal tissues employing both protein and mRNA analysis, have indicated that the Rb gene is ubiquitously expressedp-value < 0.001).Also, pRb expression of the early stage of cervical cancer tissue samples was found high than the pRb expression of normal noncancerous tissue samples . In contrast to previous reports  in contrast to advanced stages (stage III and IV) of cervical cancer where pRb expression is very less but the expression level of HPV E7 is found significantly high then pRb . Low expression of E7 protein in the early stage of cervical cancer possibly results from the host's factor preventing the E7 gene expression at transcriptional, translational, and/or posttranslational levels.In our study, an inverse relation between pRb and E7 protein both in the early and advanced stages of cervical cancer was observed. In the early stage, pRb is overexpressed and E7 expression is less , the pRb detection could be very low to undetectable as we found in advanced cervical carcinoma samples. Other types of human cancers are also associated with altered pRb expression patterns58. Invasive cancers of early clinical stages express high pRb levels59. However, late clinical stages of invasive cancers express low to the undetectable level of pRb61.The viral load may also be another important factor in determining the expression levels of E7 protein in different stages of cervical carcinoma as a high viral load was found and associated with a high grade of the cervical lesion. The advanced stages are associated with a high viral load compared to the early stages. The variable levels of E7 oncoprotein expression in different cells might contribute to the heterogeneity. Changes in pRb expression in individual cells could be observed as they undergo G0/middle G1 phases of the cycleIn conclusion, the HPV viral load tends to be higher in women with high-grade cancer diagnoses, especially for HPV16. The HPV16 viral load increased with the severity of the disease, with significant differences between early and late-stage carcinoma. There is an inverse relationship between the expression of HPV 16 E7 and pRb protein in early (stage I and II) and advanced stages (stage III and IV) of cervical cancer. There is a positive correlation between viral load and expression of HPV E7 oncoproteins in early and advanced stages of cervical cancer and a high level of HPV E7 expression correlates with reduced pRb levels in different stages of cervical cancer. Therefore, a combination of HPV viral load and expression status of its E7 proteins along with host pRb expression may be used as a potential diagnostic biomarker for screening and characterization of the early stages of cervical carcinoma and management of the disease.Supplementary Figures.


TTN expression significantly correlated with the clinical stage , node metastasis , histological type , race  and TP53 mutation , considering The Cancer Genome Atlas database. However, for patients who had higher TTN expression, the overall survival was remarkably shorter than patients who had low TTN expression. Furthermore, TTN was lowly expressed in four CRC cell lines. TTN overexpression facilitated CRC cells in terms of the proliferation, metastasis and invasion. Based on gene set enrichment analysis, the ERB pathway might be responsible for TTN‐related CRC. Besides, TTN was involved in the response to azacitidine. Overall, TTN might serve as a potential novel therapeutic target for treating and overcoming chemotherapy resistance in CRC.Colorectal cancer (CRC) is identified as a primary cause of death around the world. The current chemotherapies are not cost‐effective. Therefore, finding novel potential therapeutic target is urgent. Titin (TTN) is a muscle protein that is critical in hypertrophic cardiomyopathy. However, its role in CRC is not well understood. The study focused on exploring the possible role of TTN in CRC carcinogenesis. TTN mRNA and protein expression levels presented an obvious downregulation in CRC tissue samples, relative to normal control ( TTN contains an N‐terminal I‐band and a C‐terminal A‐band. The A‐band possesses kinase activity and participates in active contraction. The I‐band contains two regions of the tandem immunoglobulin domain, which can bind proteins implicated in protein quality control pathways.TTN gene‐related lncRNA TTN‐AS1 was found to regulate the transcriptional activity of relevant genes by targeting miR‐16‐5p and the tumour suppressor gene PTEN, resulting in decreased sensitivity of tumour cells to sorafenib. The resistance significantly diminished upon depletion of TTN‐AS1.Titin is a muscle protein that is encoded by the 22.1CRC tissues and normal controls came from two cohorts receiving surgery at the Affiliated Hospital of Qingdao University. Samples of 19 CRC patients and 19 adjacent normal controls collected between 2016 and 2018 were included in Cohort 1. Samples of 32 CRC patients and 32 normal controls collected between 2017 and 2020 were included in Cohort 2. Clinicopathological features, including gender, age, TNM stage, tumour size and degree of differentiation, were recorded for the study cohorts. Our study has obtained the approval of the Ethics Committee of the Affiliated Hospital of Qingdao University, and obtained all participants' informed consent.2.2HCT116, LoVo, HT‐29 and SW620 CRC cell lines were provided by the Cell Bank of the Chinese Academic of Sciences. HCT116, LoVo, HT‐29 and SW620 CRC cell lines were cultured in DMEM/F‐12 (Hyclone) with 10% FBS (Gibco) and 1% penicillin–streptomycin (Hyclone). Mycoplasma testing has been carried out for the cell lines used.2.32O2) at room temperature (RT). The sections underwent one night of incubation at 4°C using rabbit polyclonal antibody against TTN . Later, the sections underwent the treatment of horseradish peroxidase conjugated anti‐rabbit IgG antibody (1:500) for 30 min at RT. The non‐specific binding and antibody detection were blocked by the DAB Detection kit. Tissue sections then received haematoxylin counterstaining. A light microscope served for the detection of immunostained sections. Image‐Pro Plus (version 6.0) served for the semiquantitative analysis. In addition, The Cancer Genome Atlas (TCGA) database assisted in validating the association of TTN expression with CRC clinical features.Twenty percent formaldehyde‐fixed tissue samples underwent paraffin embedding, followed by being cut into thin sections (5 μm) and rehydrated. Then heat‐induced antigen retrieval was conducted in citrate buffer or Tris/EDTA buffer. Tissue sections received 10 min of blocking in 3% hydrogen peroxide  served for extracting the total RNA from cell lines and colon samples. PrimerScript TM RT reagent Kit with gDNA Eraser (TAKARA) served for the synthesis of first‐strand cDNA following the protocol of the manufacturer. The primers for TTN included, F: 5′‐GCGTAAGACTCAGGCATCCA‐3′ and R: 5′‐AGTAGAGGTTGTCAGCGTTGT‐3′. We conducted qPCR under the conditions of 30 min of predenaturation at 95°C, 5 s of 40 cycles of denaturation at 95°C, annealing and 34 s of elongation at 60°C. Target gene mRNA expression was amplified and analysed with ABI Prism 7300 SDS Software using GAPDH as an internal standard. The 22.5Guide RNA (gRNA) for site‐specific gene editing mediated by CRISPRa was used to trans‐activate TTN expression. The plasmids lenti dCAS‐VP64‐puro, lenti sgRNA(TTN)‐MPH‐Blast were used for CRISPR‐Cas9 activation. Subcloning PCR‐amplified Single‐guide RNA (sgRNA) of TTN was sub‐cloned into the H7284 plenti‐U6‐gRNA‐2x(wt+f6)MS2‐CMV‐MCP‐P65‐HSF1‐IRES‐Blast vector (OBiO Technology) using the forward primer: 5′‐CCGGGTATGTGACACTACCAAAT‐3′ and reverse primer: 5′‐AAACATTTGGTAGTGTCACATAC‐3′, so as to generate the TTN expression construct. The LoVo cell line was transduced by lenti dCAS‐VP64‐puro and lenti sgRNA(TTN)‐MPH‐Blast. Stable cell lines with overexpressed TTN were selected using puromycin (2 μg/mL) and blasticidin (10 μg/mL). qPCR served for the detection of the mRNA expression level of TTN after transfection.2.63 cells/well). 0, 24, 48, 72 and 96 h of treatment was followed by the addition of 10 μL CCK‐8 solution into each well. The plates received 2 h of incubation at 37°C. An automatic microplate reader  served for reading the absorbance at 450 nm.Cell counting kit‐8 (CCK‐8) served for detecting the cell viability. We plated LoVo, LoVo‐H7281+vector and LoVo‐H7281 cells in 96‐well plates  at 0, 24, 48, 72, 96 and 120 h. Image‐pro plus 6.0 software was used for the analysis of the images. Next, a transwell assay served for the evaluation of the cell invasion. Briefly, cells seeded in a matrigel invasion chamber (Corning) received 48 h of culturing at 37°C. Invaded cells received crystal violet staining (Beyotime). A microscope illustrated related images.2.8t‐test or two‐way anova tests served for comparing the means between two or three groups. The Kaplan–Meier method assisted in drawing survival curves. A p < 0.05 reported statistical significance.Data were in the form of mean ± standard deviation. SPSS 20 (IBM) served for all statistical analyses. Student's 33.1n = 19) compared to adjacent normal tissues (n = 19). Representative images of the IHC experiment are shown in Figure p = 0.008) . Similarly, TTN presented an obvious downregulation in CRC tissues, relative to normal controls   Figure . To veri) Figure . TTN exp) Figure . Accordi3.2p < 0.05; normal vs. Stage 4, p < 0.05), node metastasis , histological type , race  and TP53 mutation    Figure . In summ) Figure .3.3p < 0.001)   Figure . This re) Figure . A trans) Figure .3.4p value, and te value  served for analysing the underlying pathways of TTN involved in the development of CRC, finding that six pathways, namely, histone modifications, response to azacitidine, mRNA splicing, ERB pathway, VDR pathway, regulation of TP53 activity through acetylation, were significantly differentially enriched in low‐expressed TTN phenotype in terms of normalized enrichment score (NES), nominal e Figure ; Table 24Over the last decades, although various approaches have been employed in CRC treatment,Manipulating the expression of TTN in tumour cells revealed the positive impact of TTN overexpression on colon cancer cells in terms of the proliferation, invasion and metastasis, proving the involvement of TTN in colon cancer progression. Low TTN expression may indicate a poor prognosis. We further verified the impact of TTN expression on prognosis through survival analysis, finding the lower OS in group with high expression relative to group with low expression, which was consistent with cellular results.However, the result of CRC sample based on IHC seemed contrasted with the functional experiments of the LoVo cell. The results from IHC showed TTN was downregulated in CRC tissue, while over‐expressed TTN promoted CRC cell proliferation and migration in vitro. The possible explanation for the above results is that TTN was mainly observed to express in inflammatory cells based on IHC results. While the normal colon tissue has higher proportion of inflammatory cells than CRC tissues, higher TTN expression in the normal tissues was showed in IHC results compared to tumour tissues. In fact, TTN might be a potential oncogene in the development of CRC. Therefore, in tissues with high tumour heterogeneity, single‐cell sequencing can be conducted to avoid the interference caused by inflammatory cells in the tumour microenvironment. These results also indicate the potential impact of inflammation on CRC development, which is consistent with previous studies.In the GSEA analysis results, we identified six pathways that play important roles in the CRC carcinogenesis or chemoresistance. Histone modifications play a crucial role in regulating gene expression and chromatin structure. Aberrant histone modifications, such as histone methylation and acetylation, can lead to dysregulated gene expression patterns, including the activation of oncogenes and the silencing of tumour suppressor genes.Overall, the present study provides evidence for the role of TTN in CRC and confirms that inflammation remarkably impacts CRC development. Furthermore, GSEA was used to analyse the possible mechanisms of TTN‐induced colon cancer and found that the ERB pathway was associated with TTN in colon cancer based on bioinformatics analysis. Besides, TTN was also associated with response to azacitidine, indicating its potential role in improving chemotherapy resistance. To sum up, TTN critically impacts CRC occurrence and development through the ERB pathway and its high expression in cancer cells indicates a poor prognosis of colon cancer. Therefore, TTN might be a novel therapeutic target specific to CRC.5According to the results in the study, TTN may remarkably impact colon cancer process through the ERB pathway and high expression of TTN indicates a poor prognosis of colon cancer patients. Importantly, TTN can indicate the prognosis of CRC and serve as a new therapeutic target.Hongyun Wei: Formal analysis (lead); funding acquisition (lead); methodology (lead); project administration (lead); software (lead); writing – original draft (lead). Keyu Ren: Software ; writing – review and editing (supporting). Qian Zhang: Formal analysis ; methodology ; writing – original draft (supporting). Yanchun Jin: Writing – original draft (supporting). Bin Cao: Data curation (lead); validation (lead); writing – review and editing . Zibin Tian: Conceptualization ; data curation (supporting); writing – review and editing (supporting). Tao Mao: Validation ; writing – review and editing . Linlin Ren: Conceptualization (lead); data curation (supporting); funding acquisition ; project administration ; supervision (lead); validation (supporting); writing – review and editing (supporting).The work was completed with the support of the National Natural Science Foundation of China  and the Natural Science Foundation of Shandong Province  to Linlin Ren, Shandong medical and health science and technology development plan project (Grant number 202003030357) and Postdoctoral Science Foundation of China under Grant RZ2100002858 to Hongyun Wei.The authors declare no conflict of interest.


Q test was used to assess heterogeneity and the I2 index was employed for calculating the variation in the pooled estimations. The analysis was based on data from 27 case–control studies, which in total yielded 1607 tumour tissue samples of prostate and 1515 control samples ). According to the data obtained, there was high risk of prostate cancer by HPV infection in both cases. HPV was found in prostate cancer in 25.8% of cases, while in normal tissue samples the virus was detected in 9.2% of cases and in 17.4% with BPH as a control. In particular, more studies on the association of HPV and prostate cancer are needed to prove the role of HPV in the development of prostate cancer. In addition to the controversial question of whether HPV infection is associated with prostate cancer risk, it is worth considering whether the samples used as a control have an impact on the results. The impact of HPV in prostate tumour tissue samples on outcome should also be investigated.The involvement of human papillomavirus (HPV) in the prostate carcinogenesis is a controversial issue. The presented meta-analysis was carried out to systematize the currently available research results regarding this question. The meta-analysis includes case–control studies from 1991 to 2022, which were collected from publicly available bibliometric databases. The meta-analysis was performed using Meta-Essentials_1.5 software. We used Begg’s and Egger’s methods to assess publication bias. Cochran’s  According to GLOBOCAN estimates, prostate cancer is the second most common cancer, the fifth leading cause of death among men, and the most frequently diagnosed cancer in more than half of the countries in the world. The highest incidence rates are observed in Northern and Western Europe, the Caribbean, Australia/New Zealand, North America and South Africa, while the lowest rates are in Asia and North Africa2. Such variability in morbidity and mortality in different countries can be explained not only by socioeconomic status and different health policies, but also by the prevalence of prostate-specific antigen screening, which is used to detect indolent cases in a particular country3. Despite the fact that in Russia the incidence of prostate cancer is at a relatively low level compared to other countries, the mortality rate ranks second after lung cancer2.Prostate cancer is a major public health problem, being the most common malignancy of solid organs in men worldwide4. The presence of any affected family member approximately doubles the risk of cancer5. According to a study by Grill et al., having one first-degree relative younger than 60 years of age at the time of diagnosis increases the risk of prostate cancer by 2.5 times (1.6 times for first-degree relatives aged ≥ 60 years)6. The genetics underlying family predisposition and hereditary cancer syndromes associated with prostate cancer are complex. Nyberg et al. identified one high-risk gene specific for prostate cancer in which men with an inherited HOXB13 mutation were at increased risk of prostate cancer throughout their lives7. There are also data in the literature about other genes that are responsible for predisposition to prostate cancer, such as BRCA1, BRCA2 and the MSH2 gene associated with Lynch syndrome9. Ethnicity also plays an important role in the prostate cancer etiology. Black men in the United States and the Caribbean have the highest incidence rate in the world, while Asian men, according to the literature, have the lowest risk of developing prostate cancer13. Recent studies have shown that genetic predisposition may be a potential risk factor for prostate cancer in men of African descent15.Relatively little is known about the etiology of this common disease. The established risk factors are limited to age, family history, and genetic predisposition. Up to 20% of men with prostate cancer have a family history of the disease16. These viruses modify tumour cell behavior by inhibiting tumour suppressor signaling, blocking apoptosis pathways, promoting metastasis and triggering angiogenesis. Human papillomavirus is one particularly important oncogenic virus. We have previously carried out 2 meta-analyses that assessed the association of HPV infection with endometrial and ovarian cancer. Our previous meta-analysis did not find an association of HPV infection with the risk of endometrial cancer. It has been suggested that the reason for the low persistence of HPV in endometrial cancer and the lack of association of the virus with the risk of this type of cancer is due to the deeper layer of the endometrium, as it is involved in the repair of the uterine mucosa after rejection of the surface layer17. Analysis of the association of HPV with the risk of ovarian cancer showed that HPV was found in 28.6% of cases of ovarian cancer, while in the control group samples the virus was detected in 8.8%. However, a geographical heterogeneity was found in the prevalence of HPV in ovarian cancer, with a higher risk in those from the Asian continent18. According to the literature, HPV, which is best known for its ability to cause cervical cancer in women, may be associated with the prostate cancer pathogenesis. Evidence from genetic and clinical studies suggests that there may be a causal relationship between prostate cancer and human papillomavirus1. For example, Whitaker et al. concluded in their study that the identification of HPV-associated koilocytes in prostate cancer samples is an indication of HPV infection and the potential oncogenic effect of the virus on prostate cancer19. Medel-Flores et al. obtained significant results of HPV and prostate cancer association; they also found koilocytes in all in situ HPV-PCR-positive specimens20. Also interesting is a study by Glenn et al. in which authors showed that high-risk HPV is present in benign prostate tissues prior to the development of HPV-positive prostate cancer. Their study observed higher expression of HPV E7 oncoproteins in benign prostate tissues compared to advanced prostate cancer that subsequently developed in the same patients. Their observation suggests that HPV oncogenic activity is an early phenomenon in most prostate oncogenesis21. But, in addition to studies that have found an association between HPV and prostate cancer, there are others that have not confirmed a role for HPV infection in prostate carcinoma23. Nahand et al. didn’t find significant correlation between the presence of HPV and prostate cancer, but they observed a significant relationship between the viral proteins expression and cellular factors involved in the malignant progression of prostate cancer. The authors concluded that it is likely that HPV infection potentially modulates prostate tumour cell behavior by influencing inflammation, angiogenesis, apoptosis mechanisms, and suppression of antitumour immunity, which in turn contributes to oncogenesis24.Only 10–15% of all human cancers are caused by virusesFrom the above, we can conclude that the presented scientific results are highly controversial contradictory and require systematization. Thus, our aim is to systematize the available data regarding HPV and prostate cancer, which will help in deciding on the prospects for further research on the association of HPV with this type of cancer.All included studies were case–control studies. Tumour prostate tissue samples were the mandatory study material.Strict consistency with localisation and diagnosis. Diagnoses must be confirmed morphologically.The presence and type of HPV must be determined by PCR only.PCR examination of normal tissue control samples is mandatory.Tissue or paraffin blocks or frozen tissue must be used.The following modified participants, interventions, comparators, outcomes, and studies approach (PICOs) guided eligibility screening of studies for inclusion in our study  to August 21, 2022. We also carried out a ‘snowball’ search to identify additional studies by searching the reference lists of publications eligible for full-text review and using Google Scholar to identify and screen studies citing them Fig.  Table. TWe independently reviewed titles and abstracts of the first records and discussed inconsistencies until consensus was obtained. If necessary, N.V.L. was consulted to make the final decision. I.A.T. and M.K.I. extracted all relevant information from the studies, including: author, publication date, number of HPV positive and negative patients, HPV subtypes.Risk of bias was evaluated with a Joanna Briggs Institute (JBI) checklist for case control studies  tool was used to assess the quality of evidence for the primary outcome. The assessment was based on five parameters: risk of bias, inconsistency (or heterogeneity) between studies, indirectness, imprecision (risk of random errors), and publication bias. The evidence for each item was rated as high, moderate, low, or very low .Two meta-analyses were carried out that differed between samples from the control group. In 1 case, normal prostate tissue samples were used as the control group and in 2 case, benign prostatic hyperplasia samples were used as the control group. The meta-analysis was performed using Meta-Essentials_1.5 softwareThe meta-analysis includes case–control studies from 1991 to 2022, which were collected from publicly available bibliometric databases. We found 271 records in databases searching. After duplicates removal, 234 records were screened, from which we reviewed 73 full-text documents, and finally included 27 articles. These 27 case–control studies include 1607 prostate tumour tissue samples and 1443 control samples   of prostate cancer risk in HPV infection was 3.07 1.80–5.21) (wherein relative risk RR (RR 95 CI) 1.17 (1.09–1.26), p = 0.0000012), the differences were statistically significant and p = 7.37e−6. The sample heterogeneity index 1  of prostate cancer risk in HPV infection was 1.94 1.43–2.63) (wherein relative risk RR (RR 95 CI) 1.01 (1.00–1.02), p = 0.220), the differences were statistically significant and p = 0.0005. The sample heterogeneity index was –2.63 19.According to the available literature data, the frequency of HPV prevalence in benign prostatic hyperplasia (BPH) ranges from 0 to 65%Based on the meta-analysis results, we can conclude that the risk of prostate cancer by HPV infection is high in both cases OR 3.07, 95% CI 1.80–5.21 if normal prostate tissue is taken as a control and OR 1.94, 95% CI 1.43–2.63 if BPH is taken as a control. However, in meta-analysis using a risk ratio, a very different picture is observed. The association of HPV and prostate cancer risk is significant in the studies when normal prostate tissue is used as a control  compared to studies when BPH is used as a control . When using BPH as a control, HPV was detected in 10.8% of pre-tumour samples, whereas in normal tissue samples it was detected in 9.2% of cases. The HPV prevalence in prostate cancer varies up to 25.8% with normal prostate tissue as a control and up to 17.4% with BPH as a control. In addition to the controversial question of whether HPV infection is associated with prostate cancer risk, it is worth considering whether the samples used as a control have an impact on the results.50. In their opinion, the results may differ depending on where the tissue sample was taken. However, judging from the studies in this meta-analysis, where both normal prostate tissue and BPH were taken as controls37, it can be concluded that with a clear quantitative advantage of BPH samples compared to normal tissue, the HPV detection percentage in BPH is higher. No significant association between HPV and BPH was found in these studies. According to the literature, many past authors expressed the opinion that HPV probably doesn’t play a causal role in the prostate cancer development because there is an equal prevalence of high-risk HPV in benign and malignant prostate tissues51. One reason for the low HPV definition may be that the prostate gland, as a derivative of the urethra, is composed of half glandular cells, 1/4 fibrous and 1/4 muscular fibres. However, it is still unclear whether HPV infection can cause morphological changes in the glandular epithelium17. It is also interesting to note that DNA detection could determine the current infection status only if the pathogen had infected the tissue50. Because there is a “hit and run” phenomenon of HPV, previously described by other researchers, in which HPV-infected cells temporarily acquire a complete or incomplete viral genome in the early stages of cancer development, but the virus becomes undetectable in the later stages of cancer21. This contrasts with the causal role of HPV in the development of cervical cancer, in which HPV is necessary for both initiation and maintenance of tumourigenesis. This obvious involvement of HPV in the early stage of prostate tumourigenesis could explain the immensely viral load of HPV in fully developed prostate cancer52.The first research data on the association of human papillomavirus with prostate cancer appeared as early as 1990, when McNicol and Dodd identified HPV types 16 and 18 in prostate tumour tissue. Since then, an intensive study of the HPV prevalence in prostate cancer began. In a 2017 meta-analysis, the authors draw attention to the fact that most studies use BPH as a control51. There is a higher prevalence of HPV E7 oncoprotein expression in benign prostate tissues compared to subsequent prostate cancer specimens from the same patient. Thus, the much higher prevalence of HPV oncoprotein E7 expression in benign prostate tissues compared to subsequent prostate cancer in the same patients suggests that HPV oncogenic activity is an early phenomenon in prostate tumourigenesis21. Furthermore, one of the key markers of tumour progression is the determination of HPV genome physical forms. Khatami et al. in their study, besides detecting HPV prevalence, determined the physical status of the HPV genome in HPV-positive samples and found that 50% of samples had HPV in an integrated form, which is a key event in the malignization process. In addition, the integrated HPV DNA is characterized by overexpression of E6 and E7 genes, as the break of the ring DNA occurs in the localization area of E1 and E2 genes. The authors also concluded that HPV infection may play a role in tumour progression by enhancing resistance to apoptosis in human prostate tumour cells52. In cervical cancer and HSIL, the integrated form of HPV occurs much less frequently, in 26.5% and 22.9% of cases, respectively, but also plays a negative role in the survival of cervical cancer patients and the risk of neoplasia malignization54. Because of the possible role of HPV in prostate cancer, it can be argued that it is becoming increasingly important to intensify existing HPV vaccination strategies. Studies based on high carcinogenic risk HPV typing and virological indicators such as viral load and viral physical status are needed to further analyse the association of HPV with prostate cancer risk and its impact on disease prognosis.It is also interesting to note the results of a retrospective cohort study where the authors showed that high-risk HPVs, predominantly HPV 16 and 18, are typically present in benign prostate tissues 1–10 years before developing HPV-positive prostate cancer. Similar results were obtained in another meta-analysis; HPV-16 infection was significantly associated with an increased risk of prostate cancer (OR 1.38), whereas HPV-18 was not (OR 1.05)Despite the heterogeneity of the current literature on methods for detecting HPV in patients with prostate cancer, HPV infection should be considered as a potential risk factor for the development of prostate cancer. The present meta-analysis shows a significant prevalence of HPV infection in prostate tumour tissue samples and the association of HPV with prostate cancer when normal prostate tissue was used as a control. This makes further studies in this direction promising. In particular, more studies on the association of HPV and prostate cancer are needed to prove the role of HPV in the development of prostate cancer. This kind of study should be carried out with the inclusion of normal prostate tissue as a control instead of BPH. There is also a need to investigate the effect of HPV in the prostate tumour tissue samples on the outcome of the disease.Supplementary Information.