MARCHF8 promoter is highly activated by HPV oncoprotein E6-induced MYC/MAX transcriptional activation. The knockdown of MARCHF8 expression in human HPV-positive HNC cells restores cell surface expression of the tumor necrosis factor receptor superfamily (TNFRSF) death receptors, FAS, TRAIL-R1, and TRAIL-R2, and enhances apoptosis. MARCHF8 protein directly interacts with and ubiquitinates the TNFRSF death receptors. Further, MARCHF8 knockout in mouse oral cancer cells expressing HPV16 E6 and E7 augments cancer cell apoptosis and suppresses tumor growth in vivo. Our findings suggest that HPV inhibits host cell apoptosis by upregulating MARCHF8 and degrading TNFRSF death receptors in HPV-positive HNC cells.The membrane-associated RING-CH-type finger ubiquitin ligase MARCHF8 is a human homolog of the viral ubiquitin ligases Kaposi’s sarcoma herpesvirus K3 and K5 that promote host immune evasion. Previous studies have shown that MARCHF8 ubiquitinates several immune receptors, such as the major histocompatibility complex II and CD86. While human papillomavirus (HPV) does not encode any ubiquitin ligase, the viral oncoproteins E6 and E7 are known to regulate host ubiquitin ligases. Here, we report that MARCHF8 expression is upregulated in HPV-positive head and neck cancer (HNC) patients but not in HPV-negative HNC patients compared to normal individuals. The  Since host cell survival is essential for viruses to replicate persistently, many viruses have evolved to prevent host cell apoptosis. The human papillomavirus (HPV) oncoproteins are known to dysregulate proapoptotic proteins. However, our understanding of detailed mechanisms for HPV to inhibit apoptosis is limited. Here, we report that HPV induces expression of the membrane-associated ubiquitin ligase MARCHF8, which is upregulated in HPV-positive head and neck cancer. MARCHF8 ubiquitinates the tumor necrosis factor receptor superfamily (TNFRSF) death receptors, FAS, TRAIL-R1, and TRAIL-R2 for degradation. We further revealed that downregulation of the death receptors by MARCHF8 prevents cancer cell apoptosis and that knockout of MARCHF8 expression significantly inhibits in vivo tumor growth and enhances tumor-free survival of mice transplanted with mouse oral cancer cells expressing HPV16 E6 and E7. These results suggest that virus-induced degradation of death receptors leads to cancer cell survival in HPV-positive head and neck cancer. High-risk human papillomavirus (HPV) is associated with about 5% of all human cancers, including ~25% of head and neck cancers (HNCs) –3. HPV-pVirus-induced ubiquitination and degradation of death receptors is a potent mechanism for host cell survival and viral replication . SeveralMARCHF8 expression is upregulated in esophageal, colorectal, and gastric cancers  , we tested if E7 affects the extended MARCHF8 promoter region [1.5 kb]. The results showed that neither wildtype E7 nor E7 ΔDLYC induced promoter activity with up to the 1.5 kb upstream region . These results suggest that HPV16 E7 upregulates MARCHF8 expression through a different mechanism(s), which is independent from the immediate upstream promoter region of MARCHF8.To examine whether binding) ,34 and EMARCHF8 promoter regions (-60 to -10) in the context of HPV16 E6, we performed a DNA-protein pulldown assay using biotinylated 90 bp oligonucleotides containing the promoter sequence between -85 and +5. Our liquid chromatography-mass spectrometry identified 83 MARCHF8 promoter-binding proteins (S2 Table). Our in silico analysis using the Eukaryotic Promoter Database (epd.epfl.ch) predicted two enhancer boxes (E-box), the known binding sites of the MYC/MAX complex, between -60 and -10 in the MARCHF8 promoter . As predicted in our in-silico analysis , MYC-associated factor X (MAX), a member of the MYC family of transcription factors, was identified as a MARCHF8 promoter binding protein. The MYC/MAX heterodimer complex binds to the DNA sequence designated E-box (CACGTG). To determine if the E-boxes are necessary for MARCHF8 promoter activity, we generated a pGL4.2 reporter plasmid with a mutant 0.25 kb promoter region by deleting CG in either or both E-boxes  and determined the promoter activity. While CG deletion in only one of the two E boxes did not change any promoter activity, CG deletions in both E-boxes significantly abrogated the MARCHF8 promoter . To further validate the binding of MYC and MAX proteins to the E-boxes in the MARCHF8 promoter, MARCHF8 promoter binding proteins were pulled down using biotinylated 90 bp oligonucleotides (-85 to +5) containing the wildtype or mutant E-boxes. Then, MYC and MAX proteins were detected by western blotting. Consistent with its promoter activity , while the 90 bp oligonucleotide fragment with CG deletion in only one of the two E-boxes still binds to both MYC and MAX proteins , CG deletion in both E-boxes completely abrogates MYC and MAX binding to the MARCHF8 promoter . HPV E6 is known to interact with and stabilize the MYC/MAX complex to activate the hTERT promoter  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Denise A. GallowayAcademic EditorPLOS PathogensAlison McBrideSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens <span style="font-family: "Noto Sans", sans-serif; font-size: 12px;"> orcid.org/0000-0002-7699-2064 </span> ***********************The reviewers felt that the study is well done, clearly written and will contribute importantly to understanding virus/host interactions. However they had some concerns that need to be addressed.First, contrary to the title, the data show that both E6 and E7 can increase levels of MarchF8. This needs to be addressed and mutants of E6 should be use to better address the mechanism.Second, one reviewer points out that the difference in expression of MARCHF8 comparing HPV + versus HPV- in head and neck tumors is relatively small and could be explained by a number of other factors. These should be included in interpreting the results.Third, Multiple replicates of the MARCHF8/Fas/Ub (and other death receptor) complex pulldowns in Fig 5 should be quantified.Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: This work by Khalil et al . investigates the modulation of the cellular E3 Ub ligase MARCHF8 by human papillomaviruses. MARCHF8 downregulates surface levels of immune receptors including CD86, MHC-II, and TNF superfamily death receptors via Ub-dependent mechanisms, and HPV+ cells show decreased surface levels of many of these receptors. Using their previously published transcriptomics data from clinical samples, they show statistically significant upregulation of MARCHF8 in HPV+ but not HPV- head and neck cancers compared to healthy controls. Similar trends were seen in HPV+ versus HPV- head and neck SCC lines. They use promoter/reporter assays and find that E6 drives increased MARCHF8 expression by activation of Myc/Max which bind to E-boxes in the MARCHF8 promoter. Silencing of MARCHF8 restored surface levels of death receptors and led to enhanced apoptosis in response to ligands. KO of MARCHF8 in mouse cancer cells expressing E6/E7 caused increased apoptosis and decreased tumor growth in vivo. Overall conclusions are that HPV commandeers MARCHF8 to decrease surface expression of death receptors, thereby evading death receptor mediated apoptosis. These findings are highly significant for both the HPV life cycle (virology) and HPV-associated cancers.Overall this significant work is of high rigor. Multiple systems/models were used and appropriate controls were done. The data strongly supports their conclusion. Only one recommendation is outlined below.Reviewer #2: Khalil….Pyeon et al.“HPV E6 upregulates MARCHF8 ubiquitin ligase and inhibits apoptosis by degradingthe death receptors in head and neck cancer.”PPATHOGENS-D-22-0169.Herpesviruses express mutant analogues of the host-cell ubiquitin ligase MARCH8 that has been attributed as a negative regulator of cellular death-receptors. Inspired by these observations, the authors examined if MARCH8 might be involved in papillomavirus pathogenesis in head and neck cancers. This study is thus a hypothesis directed examination designed to elicit evidence that papillomavirus oncogenes confer traits of altered death receptor function via manipulation of MARCH8. While this is a potentially powerful approach, it is also subject to confirmation bias.The authors have examined previously acquired RNA expression data derived from microarray hybridizations for the expression of MARCHF8 ubiquitin ligase expression in head and neck cancers. Expression of MARCH8 was compared between multiple cancer samples that were either HPV+ or HPV- or to multiple samples of normal epithelium (site not otherwise specified). They observed modestly elevated expression of MARCH8 RNA in the HPV+ compared to the normal epithelium . Although the large number of samples allows for statistical significance, it is likely that most of the normal epithelial sample is comprised of differentiated cells that may have lower MARCH8 expression compared to proliferative basal cells---protein expression data for MARCH8 at the Protein Atlas is consistent with this in squamous epithelial samples . So it is unclear to me if the modest expression data difference between normal and HPV+ cancers is meaningful even if statistical significance is reached. There was no statistical difference between either the normal to HPV- sample sets or between the HPV+ and HPV- cancer sets. So Fig. 1A analysis is not strongly supportive. In Fig. 1B, similar RNA abundance analysis is carried out between HPV+, HPV- cancer cell lines, and tert-immortalized epithelial cells showing larger differences between tert-immortalized epithelium and HPV+ tumor cell lines and more modest differences between the tert and HPV- cell lines. But at the protein expression level, the differences between the tert-immortalized cells and the same cells expressing either E6, E7, or E6+E7 are modest, being about 2-2.5 fold elevated compared to vector, and although all are statistically elevated compared to vector-transduced cells, the E6, E7, and E6/E7 samples are quite similar to each other. This undermines the notion that the trait is conferred by E6 (vs E7) and undermines the title and subsequent experimental approaches of the study. Another way to demonstrate specificity would be the use of mutants of E6 and E7 which is not done. A mutant that blocks effects one trait  without changing another function  could be used to demonstrate specificity of action by E6 or E7 while also providing mechanistic insight. The comparison between HPV+ and HPV- cancers for MARCH8 expression is less compelling because we do not know if these two types of cancers even originate from the same epithelial cell type, meaning that other reasons, not contemplated by the authors, could account for the difference in MARCH8 expression between HPV+ and HPV- cancer cells.Therefore, if the authors propose that expression levels of MARCH8 are responsible for survival traits in E6/E7 expressing cells, then a careful dose response relationship must be established between MARCH8 expression levels and cell death traits in the range of expression alterations observed to be conferred by E6/E7. This is the key relationship. There should also be a demonstration of specificity of effect using intelligently selected mutants of E6 and or E7.Fig.2 demonstrates an effect between E-boxes and E6 induction of a synthetic promoter construct. Yet the effect is not compelling in that upon mutation of both E-boxes in the reporter plasmid, E6 activates the promoter 20X instead of 40X. That may be significant, but is it important? Given that they show E7 activates MARCH8 at the promoter level as well as E6 in TERT cells, I do not think Fig. 2 contributes to the arguments they are making.In Fig. 3, the SCC2 cell line seem non-supportive of their hypothesis for a role of E6 and E7 in that while MARCH8 levels are high  TRAILR1 levels are not reduced. The more informative experiment is the transduction of tert immortalized cells with viral oncoproteins and FACS analysis of death receptor expression. In this case, E6, E7, and E6/E7 all reduce surface expression. E6 does not reduce levels seen in cells also transduced by E7. This is inconsistent with their hypothesis of specificity of action of E6 vs E7 and supports a non-specific mechanism at work in oncogene expressing tert cells. Again, a careful mutant analysis might support a specific mechanism that is not obvious as yet.In Fig 4 the authors set up a crucial experiment, using shRNA to knock down MARCHF8 expression. It is a bit odd that all 5 shRNA knock down MARCHF8 to a similar degree, which has almost never been my experience with a set of 5 shRNA lentiviruses. This raises the possibility that the expression difference between scramble and the 5 shrna to MARCH8 is a difference that could be attributed to the scramble control. Expression of an shRNA escape MARCHF8 mRNA might resolve this possibility. The data does show that compared to scramble all 5 shRNA reduce MARCHF8 and increase death receptor expression as noted by the authors.In Fig. 5c, which band is Ub-Fas? Overall, Fig. 5 is supportive of MARCHF8 having a role in the ubiquitination of Fas, Trail’s and thereby negative regulation. This corroborates earlier results from others.Fig 6 supports the conclusion that when Marchf8 expression is manipulated by shRNA, apoptotic traits are altered as expected.Fig 7 utilizes a mouse expression system comparing “normal” mouse oral epithelium, the same cells transformed with HPV and the same cells transformed with HPV. Here the data is less convincing. Starting cells are compared to HPV-transformed  and HPV-non-transformed cells (oncogenes used in the transformation unspecified). So we have no idea what mechanisms might be involved. But what is clear is that both types of transformation events induced the expression of marchf8, with fas and trail-r1 alteration in the expected pattern. However, and unexpectedly, Trail-2 was not reduced in HPV- transformants but decreased in HPV transformants.In Fig. 8, marchf8 is knocked out using crispr and marchf8, fas, trail-r1 and trail-r2 expression follows the expected pattern. In Fig. 9 knockout of marchf8 reduces tumor growth rate in HPV expressing cancer cells, as one might expect from purported effects upon apoptosis, but also possibly from march8 effects upon other as yet undescribed effects upon other cellular transmembrane proteins.In summary, there is much to like about this study. The connections between marchf8 expression of the expression of TNF and fas receptors looks pretty solid in several experimental systems, but that work is not clearly novel to this study. The connections between the expression of E6 and the regulation of marchf8 seem poorly worked out to me. First, it looks clear that both E6 and E7 change the expression of marchf8 and this is ignored. The connection between E6 and myc and marchf8 looks tenuous. There is no E6 mutant analysis to support the specificity of E6 action, and one could make a strong case that this is a fairly non-specific induction of marchf8 related to transformation in the particular cells utilized.Reviewer #3: Overall, this well-conceived and well-executed work provides mechanistic details regarding how the HPV 16 E6 oncoprotein inhibits apoptosis in the virus’ host cells. As such, it advances the fields of HPV virology, virus/host interaction, the ubiquitin/proteasome pathway and cancer, and is anticipated to be a valuable addition to the literature.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: Multiple replicates of the MARCHF8/Fas/Ub (and other death receptor) complex pulldowns in Fig 5 should be quantified.Comment: Its puzzling that E7 causes increased steady state levels of MARCHF8 transcript but has no effect on MARCHF8 transcription in the promoter assays. Is E7 known to modulate RNA stability of other cellular transcripts? Could their be additional E7-responsive elements further than 1kb upstream of the MARCHF8 ORF? Might E7 have an effect if the promoter assays are done in primary keratinocytes?Reviewer #2: see above sectionReviewer #3: (No Response)**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: (No Response)Reviewer #2: The paper is well written and proof-read.Reviewer #3: • The manuscript demonstrates that following E6 expression, FAS, TRAIL-R1 and TRAIL-R2 are targets of MARCHF8 for ubiquitinylation and degradation. Are these proteins considered to be natural or canonical targets of MARCHF8, or is this enhanced turnover a specific consequence of the virus?• The authors state (line 51) that HPV is associated with about 25% of head and neck cancers, with no reference given. Estimates vary, but most recent estimates put the percentage of HN cancers that are HPV+ to be significantly higher than 25%.• The authors note that both E6 and E7 can upregulate expression of MARCHF8, but found that when using the plasmid reporter system, only E6 gave a positive signal. They then note that there may be an alternative mechanism associated with E7. It would have been helpful, perhaps in the discussion, to speculate on what some possible mechanisms might be.• When assessing apoptosis, the authors used FasL and anti-Fas to induce the process. It would have been interesting to see the impact of modulating MARCHF8 expression on apoptosis induced through other receptors (like TRAIL or TNF), or even through a pathway that routes through p53. This would have helped to further define the scope of this particular MARCHF8-dependent mechanism.• In the mouse oral cancer cells, while there is data provided on surface expression of Fas and TRAIL-R2, no data is given for TRAIL-R1. The total level of TRAIL-R1 is addressed.• The authors note the death domain carried by the target proteins. What other proteins carry this same domain, and is it anticipated that some or all of them might also be targets for MARCHF8?• Figure 10 is a helpful schematic. However, it is not referred to in the text.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: Reproducibility:https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at  18 Jan 2023AttachmentResponse-Khalil.pdfSubmitted filename: Click here for additional data file. 1 Feb 2023Dear Dr. Pyeon,We are pleased to inform you that your manuscript 'HPV upregulates MARCHF8 ubiquitin ligase and inhibits apoptosis by degrading the death receptors in head and neck cancer' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Denise A. GallowayAcademic EditorPLOS PathogensAlison McBrideSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens <span style="font-family: "Noto Sans", sans-serif; font-size: 12px;"> orcid.org/0000-0002-7699-2064 </span> ***********************************************************The authors have answered the reviewers concerns successfully.Reviewer Comments : 28 Feb 2023Dear Dr. Pyeon,We are delighted to inform you that your manuscript, "HPV upregulates MARCHF8 ubiquitin ligase and inhibits apoptosis by degrading the death receptors in head and neck cancer," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064


China has a high burden of human papillomaviruses (HPV)-related cancers, with the second highest incidence and mortality rate of cervical cancer worldwide.Financial constraints, regional restrictions and gender-specific vaccination policies have led to disparities in HPV vaccination rates across China.Public perceptions of the HPV vaccine vary widely, with factors such as lack of confidence, accessibility and awareness influencing vaccine uptake.Recommendations include increasing government funding for the HPV vaccine, expanding vaccine coverage, improving vaccine delivery yield, regulating injection pathways and enhancing scientific education on the HPV vaccine to raise awareness and increase vaccine uptake.Human papillomaviruses (HPV) are DNA viruses with a double strand of DNA that belong to the family Papovaviridae.4Globally, cervical cancer is the most common cancer associated with HR-HPVs and ranks fourth among women.21The policy adopted by China aligns with the WHO’s guidelines for standard HPV vaccination among those aged 9–14.China’s HPV vaccination programme was developed and launched later and more slowly than other developed countries. In China, the existing policy on HPV vaccinations only grants free vaccinations to girls aged 13–15 in select regions,29Currently, public health funding in China is relatively low.38As a class II vaccine with a commercial character, the HPV vaccine is available in China; however, the decision to receive the vaccine ultimately rests on the individual’s volition.Given that the recommended age for HPV vaccination falls within the category of minors, obtaining parental consent in addition to the individual’s permission is necessary.As for Chinese adult women, awareness of the HPV vaccine is mostly driven by socio-demographic factors. More specifically, individuals living economically advanced regions are inclined to pursue high-valent imported vaccines as they prioritise quality and effectiveness.et al,Even though different groups hold different views about the HPV vaccine, Loke The available evidence highlights the importance of increasing financial investment in immunisation, particularly in HPV vaccines, and enhancing the regulatory oversight by China’s drug authorities to monitor marketing approaches. In addition, it is essential to develop a comprehensive HPV vaccine promotion plan, especially launching educational programmes aimed at raising awareness and knowledge of HPV infection and vaccination in the community and rebuilding public confidence in domestically produced vaccines.To further improve the population’s public health, it is recommended that the government increase its expenditure. The implementation of national vaccine immunisation, which is one of the most cost-effective health interventions, should be prioritised. In view of China’s status as an LMIC, achieving universal HPV vaccination among school-aged men and women over time may be an uphill battle. To surmount this challenge, the authors advise that China take a cue from developed Western nations and prioritise the inclusion of women of the relevant age group in the free HPV programme during its initial stages of implementation. Furthermore, a reduced vaccination dose should be considered. In 2022, the Strategic Advisory Group of Experts on Immunization recommended girls and women aged 9–14 and 15–20 receive one or two doses and noted that a single dose is comparable to a two-dose schedule.18Alternatively, choosing the domestic HPV vaccine as the first choice is another feasible alternative. As it is half the price of the imported vaccine, which makes it more accessible to a larger population, even with limited funding. For certain provinces and cities that do not yet have sufficient funds to provide free vaccinations, innovative pricing and financing mechanisms can be used to alleviate the issue of high vaccine prices. For example, in Suzhou, China, parents can now pay for vaccines through their health insurance accounts, which addresses some parents’ vaccine hesitations.57With the widening of the age of 9vHPV vaccination to 9–45 years in China, more and more people are opting for this more protective vaccine. It has become evident that demand for the vaccine is surpassing the available supply. In this regard, Chinese biopharmaceutical companies should accelerate their research and development and clinical trials to mitigate the shortage of imported 9-valent vaccines. Chinese drug regulators can also choose to diversify their import channels by importing to regions such as Hong Kong and Macau, where demand has been relatively low. Meanwhile, regulating the high cost of HPV vaccines reservation fee and regulating the integrated service of hospital, community and school appointments and injections are recommended actions.Public health behaviour is contingent on both greater awareness of the need to prevent HPV infection and the cooperation of the government, medical institutions and educational institutions. The overall population’s rate of HPV vaccination cannot be raised unless people are educated about the significance and repercussions of HPV as well as the efficacy of the HPV vaccine.27The most comprehensive information on HPV vaccine, HR-HPVs related cancer prevention is provided to HCPs at provincial, municipal and county levels, which can effectively increase health literacy of different consultation audiences  and increase the vaccination rate. The education department should integrate health education related to HPV infection into the curriculum of middle and high schools and colleges. And health promotion activities can be conducted regularly in collaboration with HCPs. Previous studies in the USA have shown the beneficial effects of educational interventions delivered by healthcare providers on vaccine education and adherence.58 59On the other hand, exploring the online platform of sustainability services can facilitate access to professional answers whenever people have questions. In recent years, people have increasingly turned to the internet for quick answers, but without professional guidance. the information may be inaccurate. Developing a professional online inquiry platform that allows people to get answers from professionals would provide a valuable digital offering. Furthermore, HCPs can effectively disseminate information on HR-HPVs related cancers and the HPV vaccine by leveraging new media platforms such as TikTok, WeChat Public and Weibo, which are highly accessible and currently among the most effective channels for delivering health-related messages.


This guideline provides evidence-based guidance on the risk-based management of cervical dysplasia in the colposcopy setting in the context of primary HPV-based screening and HPV testing in colposcopy. Colposcopy management of special populations is also discussed. The guideline was developed by a working group in collaboration with the Gynecologic Oncology Society of Canada (GOC), Society of Colposcopists of Canada (SCC) and the Canadian Partnership Against Cancer (CPAC). The literature informing these guidelines was obtained through a systematic review of the relevant literature via a multi-step search process led by information specialists. The literature was reviewed up to June 2021 with manual searches of relevant national guidelines and more recent publications. Quality of the evidence and strength of recommendations was assessed using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework. The intended users of this guideline include gynecologists, colposcopists, screening programs and healthcare facilities. Implementation of the recommendations is intended to promote equitable and standardized care for all people undergoing colposcopy in Canada. The risk-based approach aims to improve personalized care and reduce over-/under-treatment in colposcopy. In 2020, the World Health Organization (WHO) announced a global strategy towards the elimination of cervical cancer as a public health problem by reducing the incidence of cervical cancer to less than 4 per 100,000 . PrioritPrimary HPV screening is superior to cervical cytology in detecting pre-invasive disease and is the topic of a complimentary guideline . Once hiThe following guideline is presented in the context of primary HPV-based screening for cervical cancer, whereby a person’s HPV status is known on entry to colposcopy. The purpose of this guideline is to provide guidance on how to incorporate high-risk HPV (HR-HPV) testing and results within our current colposcopy framework with an emphasis on risk-based management. In Canada, each province and territory administers and delivers most health services independently and needs to consider their own populations and resources. This guideline is not meant to supersede local recommendations; rather, it is meant to provide evidence to advocate for equitable resources and standardized care for all people undergoing colposcopy in Canada.Guideline committee members were recruited to participate in the development of the document with consideration given to including colposcopists from a range of Canadian provinces and practice settings . The committee convened in the fall of 2020 and developed a series of objectives to guide the document.https://www.cadth.ca/strings-attached-cadths-database-search-filters (accessed on 10 May 2021). No date or language filters were applied to the search. The search strategy is reported in full in The literature informing the guideline was located using a multi-step search process led by an information specialist. First, a search for existing clinical practice guidelines related to colposcopy for cervical cancer was designed in Ovid MEDLINE All and executed on 12 May 2021. The search combined search terms for colposcopy, cervical cancer and the Canadian Agency for Drugs and Technology in Health guidelines filter for Ovid MEDLINE  framework; this is shown in Histopathology should be reported using the two-tiered terminology described by the LAST Project: LSIL for CIN1 and HSIL for CIN2/CIN3 .p16 immunohistochemistry may be used to upgrade CIN2 to CIN3. P16 should not be used to upgrade morphologically appearing CIN1 .Recommendations:The Lower Anogenital Squamous Terminology (LAST) Standardization Project for HPV-Associated Lesions, published in 2012, recommends harmonizing cytology and pathology into two-tier reporting . CIN 1 iGuidance for the use of p16 immunochemistry is published in the LAST project. In general, when the morphologic interpretation of hematoxylin and eosin (H & E) staining is suggestive of HSIL, then positive p16 immunochemistry is confirmatory [People with a positive HPV screening test should undergo HPV genotyping and reflex cytology before referral to colposcopy .People with HPV 16/18 should be referred to colposcopy .People with HPV ‘other’ ASCUS or LSIL should have HPV testing repeated at 12 and 24 months, only referred to colposcopy if they meet other criteria or have persistent HPV “other” at 24 months .People with HPV-positive HSIL, ASC-H, AGC, AIS or cytology suspicious for invasive cancer should be referred directly to colposcopy, regardless of HPV genotype .People with immunocompromise with any HR HPV should be referred to colposcopy .Recommendations:Primary HPV-based screening with reflex cytology allows for risk stratification based on immediate and future risk of histologic HSIL+. Large population-based studies, most notably data from the Kaiser Permanente Northern California (KPNC) cohort, have examined the risk of histologic HSIL based on combined HPV and cytology results, contributing to our understanding of these risk profiles ,11. BaseTabulated risks of various combinations of cytology and HR-HPV results are presented in The transformation zone should be assessed, and the type should be documented .International Federation of Cervical Pathology and Colposcopy (IFCPC) terminology is recommended for documenting colposcopic findings .Targeted biopsies of lesions are recommended. In the setting of HSIL, or positive HPV16/18, where colposcopic impression is normal, any area of acetowhitening, metaplasia or uncertainty should be biopsied .Endocervical curettage and endometrial biopsies are contraindicated in pregnancy .Endocervical curettage is recommended with: (i) a type 3 transformation zone, (ii) HSIL/ASC-H cytology when no lesion is identified, (iii) AGC/AIS cytology, (iv) when excisional treatment has positive margins and (v) in people over age 45 with HPV 16 .Endometrial sampling is recommended in those 35 years and older for all categories of AGC/AIS or atypical endometrial cells on cytology. Endometrial sampling is also indicated in those under 35 with increased risks of endometrial cancer  or atypical endometrial cells on cytology in those of any age .For pain management for routine exam and cervical biopsies, thorough pre-procedure counseling and non-pharmacologic methods are recommended. Oral analgesics may be considered. Topical and injected analgesics are not recommended for routine exam and biopsies of the cervix .Recommendations:When the cervix is first examined, cervical mucus is removed and gross examination is performed. Using a colposcope for visualization, 3–5% acetic acid is applied to the cervix, and the transformation zone is then visualized. The transformation zone type is noted  and any When abnormal colposcopic findings are identified, IFCPC recommends documenting both the location of the lesion, inside or outside the transformation zone and location of the lesion by clock position, and the size of the lesion, by the number of cervical quadrants the lesion covers and the size of the lesion in the percentage of the cervix affected. Further findings are categorized as grade 1 (minor) or grade 2 (major), suggestive of low- versus higher-grade lesions, respectively. Findings suspicious for invasion should also be described .Targeted biopsy of all discrete lesions is recommended. Studies have shown that taking two or more biopsies improves the sensitivity of the exam and increases the detection of histologic HSIL . ColposcEndocervical curettage (ECC) is contraindicated in pregnancy. ECC is generally not required with a type 1 transformation zone. ECC is recommended for type 3 transformation zones, HSIL/ASC-H cytology when no lesion is identified, AGC/AIS cytology and on post-treatment follow-up when excisional treatment has positive margins. Furthermore, ECC should be considered in people over age 45 with HPV 16 ,21. As pEndometrial sampling is contraindicated in pregnancy. Endometrial sampling is recommended in those 35 years and older for all categories of AGC/AIS cytology, as well as when atypical endometrial cells are found on cytology. Endometrial sampling is indicated in those under 35 with increased risks of endometrial hyperplasia or cancer  and when atypical endometrial cells are found on cytology in those of any age . Pain management should be a consideration during all colposcopy exams and procedures. Significant discomfort may impede the exam and can contribute to loss of follow-up. Pain during colposcopy is multi-factorial, influenced by physical factors, psychological factors and social factors . ThorougSeveral studies have shown that application of topical analgesics to the cervix prior to biopsy and manipulation are ineffective compared to placebo ,31,32,33After initial colposcopy assessment, those with normal or LSIL histology can be discharged from colposcopy .Where HSIL is identified on histology, excisional procedure is recommended .Recommendations:The majority of abnormal Pap tests are low-grade. Most of these will spontaneously resolve, especially in the young, previously vaccinated cohort . The purThe low-grade referral pathway  addresseAfter initial colposcopy assessment, if no histologic HSIL is identified, people can be discharged from colposcopy with recommendations for 12-month HPV-based screening with their primary care provider. This is in line with the 2019 ASCCP risk-based guidelines  and is bPeople with histologic HSIL identified at the time of the initial colposcopy assessment should undergo excisional treatment ; they suPeople with evidence of histologic HSIL should undergo an excisional procedure .In cases where no lesion is identified following referral for HPV-positive cytologic ASC-H/HSIL, review by an experienced cytopathologist should be considered .In cases where no cervical lesion is identified following referral for HPV-positive cytologic ASC-H/HSIL, VAIN must be ruled out by colposcopy .In cases of discordance, where no histologic HSIL is confirmed, management depends on transformation zone type and referral cytology .For a type 3 transformation zone, excisional procedure is recommended .For a type 1 or 2 transformation zone, the preferred management for ASC-H referral cytology is surveillance. For HSIL referral cytology, excisional procedure can be considered .If surveillance without treatment is undertaken, people should remain in colposcopy at 6-month intervals with HPV testing at annual intervals until HPV is negative on two consecutive tests and histology remains normal or LSIL .If HPV remains positive despite negative colposcopy, people should remain in colposcopy for surveillance at 12-month intervals until they meet the above criteria for discharge .If, during surveillance, there is evidence of cytologic or histologic HSIL, excisional procedure is recommended .Recommendations:The high-grade referral pathway  addresseIn cases of discordant cytology, histology and colposcopy findings, the presence and type of oncologic HPV infection, adequacy of colposcopy and transformation zone type need to be considered. For people referred with HPV-positive high-grade cytology, if colposcopy is adequate and no lesion is found, a detailed examination of the endocervix and vagina should be undertaken . Random For persons where high-grade discordance persists after cytopathology review , management depends on transformation zone type and referral cytology. All people with HPV-positive high-grade referral cytology (HSIL or ASC-H) with a type 3 transformation zone and otherwise negative colposcopy exam are recommended to undergo an excisional procedure. For those with HSIL referral cytology and a type 1 or 2 transformation zone, an excisional procedure can be considered, whereas close surveillance is preferred for ASC-H referral cytology. This is based on risk estimates that show the 1-year risk of CIN3+ is higher for discordance involving cytologic HSIL/histologic LSIL at 3.9% compared to cytologic ASC-H/histologic LSIL at 1.4% . Those wFor those who do not undergo an excisional procedure, close surveillance is required with colposcopy at 6-month intervals and HPV testing at annual intervals. If HSIL cytology persists or is identified at any follow-up visit, an excisional procedure is recommended. Once HPV is negative for two consecutive annual tests, and colposcopic impression/histology remain normal or LSIL, people can be discharged back to their primary care provider for repeat HPV-based screening in 12 months .In these cases, review by an expert cytopathologist should rule out CIN3 .Findings of CIN2 in a young person with a type 3 transformation zone or CIN2 identified on endocervical curettage should undergo excisional procedure .Findings of CIN2 in a young person with a type 1 or 2 transformation zone may be managed conservatively for childbearing considerations; surveillance should include colposcopy at 6-month intervals and HPV testing at annual intervals for 3 years to allow these young people to resolve their HPV infections .If CIN2 persists, continue colposcopy at 6-month intervals with HPV testing annually. People under 30 with persistent CIN2 > 36 months or CIN3 at any colposcopy visit should have an excisional procedure .People under the age of 30 with initial CIN2 who are managed conservatively can be discharged from colposcopy once histology is normal or LSIL and HPV is negative on two consecutive annual follow-up visits .People under the age of 30 with initial CIN2 who remain HPV-positive at annual follow-up should remain in colposcopy with HPV tests at annual intervals .Recommendations:There is controversy about the reporting of HSIL into distinct CIN2 versus CIN3 categories as there is low inter-rater reliability for CIN2 among pathologists . HoweverThe conservative management of the CIN2 pathway presented here  addresseFor those who are treated conservatively, colposcopy is recommended at 6-month intervals with HPV testing annually. This allows time for regression in this young cohort. At any point during follow-up, if CIN3 is found, an excisional procedure is required. Similarly, if histologic CIN2+ persists >36 months, an excisional treatment is recommended. If HPV remains positive with normal or LSIL histology, the person should remain in colposcopy including HPV testing at annual intervals. Once histology is normal or LSIL and HPV is negative on two consecutive annual follow-up visits, the person can be discharged from colposcopy to 12-month HPV-based screening with their primary care provider. Subsequent screening then follows the ‘Post-discharge follow-up for people with SIL not treated in colposcopy’ pathway .Recommended treatment for histological HSIL is an excision procedure with a LEEP .An ablative procedure with carbon dioxide laser is acceptable when used by trained and experienced colposcopists when specific criteria are met .Cryotherapy for HSIL is not recommended .Treatment should be performed in the clinic setting with local anesthesia plus a vasopressor to the cervix .Recommendations:Histologic HSIL is treated with either an ablative or excisional procedure. Excisional procedures offer a secondary diagnostic benefit as it allows for pathologic examination to assess for cancer and margin status. Excisional techniques include the loop electrosurgical excision procedure (LEEP), large loop excision of the transformation zone (LLETZ) and cold knife conization (CKC). Meta-analyses have shown similar oncologic outcomes, including recurrence rate, margin status and residual disease between LEEP and CKC ,53. HoweAblation with either carbon dioxide laser or thermoablation have also been used to treat HSIL. When ablation is used, the transformation zone is destroyed to a depth of 7 mm, as a SIL can exist to a depth of at least 4 mm in gland crypts and 7 mm is considered a suitable safety margin . AblativWith excisional procedures, the goal of achieving negative margins must be balanced with the risk of obstetrical complication. A meta-analysis suggests cervical dysplasia is a risk for preterm birth and excisional and ablative treatments increase that risk, with increasing depths of excision/ablation increasing obstetrical risks . Across Despite this knowledge, no study has been able to determine an optimal resection volume/length for excisional techniques. A 10 mm length seems to provide adequate margins while maintaining adequate cervical length for future pregnancies where risk of preterm birth increases with depth >10 mm . TreatmeThere are very few randomized controlled trials that directly compare treatment modalities. Most of the evidence comes from systematic reviews and meta-analyses with high heterogeneity. Minor and major adverse events from any treatment are low across the studies (<1%) . The mosAnalgesia is required for any ablative or excisional treatments of the lower genital tract. The most accepted technique is para-cervical block with local anesthetic and a vasopressor. The vasopressor reduces blood loss, reduces the threshold of toxicity from the local anesthetic and has been shown to reduce pain compared to local anesthetic alone. A systematic review and meta-analysis by Gajjar et al. confirmed that intracervical injection of local anesthetic with a vasopressor provided optimum analgesia for treatment, although data were scant, underpowered and showed a high degree of variability . OptimalIt is recommended that LEEP is performed in the clinic setting. The benefits of treatment in the outpatient clinic, as opposed to the operating room, include faster access to treatment, quicker recovery and overall cost-effectiveness. A LEEP in the operating room or under general anesthesia should only be carried out in extenuating circumstances . It is rAll people undergoing an excisional procedure in colposcopy should have an HPV test of cure and cytology at 6 months post treatment, as well as colposcopic assessment and endocervical curettage if endocervical margin is positive on excision specimen .People who are HPV-negative with normal, ASCUS or LSIL cytology and histology after treatment can be discharged from colposcopy .People who remain persistently HPV-positive with cytology and/or histology that is normal, ASCUS or LSIL, regardless of genotype, should remain in colposcopy at 12-month intervals until HPV is negative .After discharge from colposcopy, people treated for HSIL should have 12-month HPV-based screening with their primary care provider. If HPV-negative, they can resume HPV-based screening at 3-year intervals indefinitely .Recommendations: The post-treatment pathway  addresseA recent review of guidelines from seven countries  showed no agreement regarding timing of the test of cure and whether one or two post-treatment tests of cure are needed to decide who can be discharged from colposcopy  A study Based on recurrence risk, people treated in colposcopy who are HPV-negative with normal, ASCUS or LSIL cytology and histology at the 6-month post-treatment visit can be discharged from colposcopy. They should undergo HPV-based screening with their primary care provider at 12 months post-colposcopy; if HPV is negative, they can transition to HPV-based screening at 3-year intervals indefinitely. At any point, if HPV is positive, they should be re-referred to colposcopy for the usual indications. People who are HPV-positive on the 6-month test of cure should remain in colposcopy with HPV testing annually until negative. All people with HPV-positive AGC or AIS cytology should be referred directly to colposcopy, regardless of HPV genotype .At time of initial colposcopic assessment, all people with HPV-positive AGC or AIS should have endocervical curettage .Endometrial biopsy is recommended for all people >35 years old AND/OR risk factors for endometrial cancer AND/OR atypical endometrial cells on cytology .People referred with AGC-NOS cytology, in the absence of endometrial pathology and histologic HSIL or AIS, may be managed conservatively with colposcopy, cytology and HPV testing at annual intervals. If all negative on two consecutive visits, they can be discharged from colposcopy to HPV-based screening at 5-year intervals .All people AGC-N or AIS should undergo excisional procedure regardless of HPV status .Following the excisional procedure for AIS/AGC-N, if margins are positive, consider re-excision .Following excisional procedure for AIS/AGC-N, in the absence of cancer, surveillance with 6-monthly colposcopy, ECC and HPV testing is recommended; if surveillance is negative ×3 years in colposcopy , people can be discharged to HPV-based screening at 3-year intervals. If HPV is persistently positive or histology shows HSIL or persistent glandular abnormalities, people should stay in colposcopy and be managed per algorithms .Hysterectomy can be considered when post-treatment margins/ECC are persistently positive for AIS and/or fertility is not desired .Recommendations:Colposcopy is less reliable for the assessment of cervical glandular lesions than for squamous intraepithelial lesions. Most glandular lesions are recognized during evaluation of abnormal squamous cytology and small lesions that originate high in the endocervical canal can easily be missed . HoweverAlthough atypical glandular cells (AGC) make up <1% of cervical cytology samples, approximately one-third of these are associated with clinically significant pre-invasive or invasive disease ,77,78,79Risk estimates for HPV-positive glandular lesions are dependent on the cytology classification, as described by the Bethesda system in 2014 . A largeThe glandular referral pathway  addresseFor people referred to colposcopy for HPV-positive AGC-N who present with a type 3 transformation zone, a diagnostic excisional procedure is required to rule out a glandular lesion not identified at colposcopy. A systematic review by Schnatz et al. found the risk of AIS and malignancy following AGC-N cytology was 13% and 21%, respectively, compared to 2.9% and 5.2% for AGC-NOS . TherefoAll people with cytology suggestive of adenocarcinoma in situ (AIS) should undergo a diagnostic excisional procedure to rule out adenocarcinoma, even when definitive hysterectomy is planned ,42,74. THistorically, CKC was considered superior to LEEP for AIS. However, more recent retrospective reviews have shown equivocal rates of residual disease and recurrence between these treatment modalities ,84. ManyAdherence to long-term surveillance following conservative management for AIS is crucial; this needs to be considered when determining best management for people with AIS. Following an excisional procedure for AIS with negative margins, when conservative management is undertaken, follow-up colposcopy with HPV testing and ECC should be carried out at 6 and 18 months, then annually for 3 further years in colposcopy. After treatment, HPV status is highly predictive of recurrent AIS. A study by Costa et al. found that HPV testing significantly predicted persistence/clearance of AIS at 6-month follow-up . In thisAll people discharged from colposcopy should have HPV-based screening at 12 months with their primary care provider .Subsequent management depends on referral cytology and HPV status.People referred with low-grade cytology who are HPV-negative on 12-month screening with their primary care provider may transition to routine HPV-based screening at 5-year intervals .People referred with low-grade cytology who are HPV-positive (regardless of genotype) at 12-month screening with their primary care provider should be re-referred to colposcopy for the usual indications .People referred with high-grade cytology (untreated) should have two negative annual HPV tests in colposcopy with colposcopic findings that are normal or LSIL before they can be discharged to 12-month HPV-based screening with their primary care provider . If HPV remains negative at 12-month post-colposcopy screening, they may transition to HPV-based screening at 3-year intervals indefinitely. If HPV is positive at 12-month post-colposcopy screening, they should be re-referred to colposcopy for the usual indications .Recommendations:The post-discharge pathway  addressePeople referred to colposcopy with HPV-positive low-grade cytology, where no histologic HSIL was found at time of colposcopy, have a low risk of subsequent HSIL within the next three years . HoweverRegarding the choice of post-colposcopy screening test, a study by Katki et al. found that for people referred with HPV-positive ASCUS or LSIL cytology, in whom no HSIL was identified at colposcopy, a single negative HPV/cytology co-test post-colposcopy reduced the 5-year risk of HSIL+ to 1.1%, compared to a single negative HPV test (2.0%) or a single negative cytology test (6.4%). A second negative HPV/cytology result in this context did not significantly reduce the risk any further . Guido eBased on these data, all people seen in colposcopy for HPV-positive low-grade cytology, in which no high-grade cytology was found at the initial visit, can be discharged to HPV-based screening with their primary care provider at 12 months post-colposcopy. If HPV is negative, they can resume routine HPV-based screening at 5-year intervals. If HPV is positive on post-discharge screening, they should be re-referred to colposcopy for the usual indications.Despite the absence of histologic HSIL on colposcopic-directed biopsy, people who are HPV-positive with high-grade cytology at time of referral remain at higher risk of HSIL, sufficient to warrant ongoing colposcopy per the ASCCP 2019 risk-based thresholds ,90. All Those under 25 should not have screening with HPV testing or cytology .If screening occurs and high-grade cytologic abnormalities are identified, indications for colposcopy remain the same, regardless of age .When a CIN2 lesion is confirmed, and CIN3 is ruled out, conservative management may be undertaken when childbearing considerations outweigh the risk of invasive disease .Recommendations:Studies have shown the highest prevalence of HPV infection is in those under the age of 25, and this population also has the highest rate of spontaneous clearance of HPV ,92. InvaRecommendationsRisk-based threshold for entry to colposcopy are the same, regardless of pregnancy .Pregnant people should be evaluated by an experienced colposcopist .Pregnant people who are HR-HPV-positive with reflex normal or low-grade referral cytology (ASCUS or LSIL) should have HPV-based screening repeated 3 months post-partum ; pregnant people who are HR-HPV positive with reflex high-grade or glandular cytology  should be seen in colposcopy within 4 weeks .Endometrial biopsy and endocervical curettage are contraindicated in pregnancy. Cervical biopsies are indicated when there is a concern for HSIL or cancer; adverse obstetrical outcomes of cervical biopsies are rare .Excisional procedures for biopsy-proven HSIL or AIS in pregnancy can be delayed until 8–12 weeks post-partum .Biopsy-proven carcinoma in pregnancy should be referred urgently to gynecologic oncology .Pregnant people should be screened according to provincial guidelines. Risk-based indications for colposcopy are the same, regardless of pregnancy. Pregnant people who are HR-HPV-positive with reflex normal or low-grade referral cytology (ASCUS or LSIL) should have HPV-based screening repeated 3 months post-partum; pregnant people who are HR-HPV-positive with reflex high-grade or glandular cytology  should be seen in colposcopy within 4 weeks.The pregnant cervix undergoes changes in appearance that may complicate colposcopic diagnosis; therefore, pregnant individuals should be evaluated by an experienced colopsocopist ,95. PregEndometrial biopsy and endocervical curettage are contraindicated in pregnancy as they may negatively impact the pregnancy. Cervical biopsies are indicated when there is a concern for HSIL or cancer ,99,100. Colposcopy is recommended for all immunocompromised people who are HPV-positive, regardless of HPV genotype .Recommendations:In immunocompromised people of any age, colposcopy is recommended for all cytology results if HPV-positive . ImmunocMenopausal people have higher rates of cervix cancer and unsatisfactory colposcopy. Consider ECC and larger excisions when indicated .Consider pre-treatment with vaginal estrogen for 6 weeks prior to colposcopy to increase the rates of a satisfactory exam in postmenopausal people .Recommendations:Colposcopy can be challenging in menopause as the transformation zone recedes up the endocervical canal (type 3 transformation zone). Abnormal lesions are more likely to involve the cervical canal and vagina . On systColposcopy providers should be aware of the barriers to access cervical cancer screening and colposcopy, including geographical, socioeconomic, cultural, physical, psychological, provider-related and system-related barriers .Colposcopy providers are encouraged to seek additional training in cultural safety and trauma-informed care .Every effort should be made to facilitate access to care for individuals from historically underserved populations, including people with mobility restrictions, obesity, members of the transgender community, immigrants, Indigenous peoples, people from rural communities and those with mental health disorders .Recommendations:Barriers to accessing cervical cancer screening and colposcopy can be geographical, socioeconomic, cultural, physical, psychological, provider-related and system-related. Data support inequitable access to primary cervical screening for wheelchair users , sex wor


Treponema pallidum (HR: 4.02 (95% CI: 1.06–15.24)) and a history of anal HSIL or SCCA (HR: 21.52 (95% CI: 1.59–291.6)) were risk factors for oropharyngeal HPV infection, whereas ART duration (8.8 vs. 7.4 years) was a protective factor (HR: 0.989 (95% CI: 0.98–0.99)). Conclusions: The prevalence of HPV infection and dysplasia was low in the oropharyngeal mucosae. A higher exposure to ART was protective against oral HPV infection.Background: The main objectives were to describe the prevalence of HPV, its genotypes and HPV-associated dysplastic lesions in the oropharyngeal mucosa of PLHIV and related factors. Material and Methods: This cross-sectional prospective study consecutively enrolled PLHIV attending our specialist outpatient units. At visit, HIV-related clinical and analytical variables were gathered, and oropharyngeal mucosa exudates were taken to detect HPV and other STIs by polymerase chain reaction. Samples were also taken from the anal canal of all participants and from the genital mucosa of the women for HPV detection/genotyping and cytological study. Results: The 300 participants had a mean age of 45.1 years; 78.7% were MSM and 21.3% women; 25.3% had a history of AIDS; 99.7% were taking ART; and 27.3% had received an HPV vaccine. HPV infection prevalence in the oropharynx was 13%, with genotype 16 being the most frequent (2.3%), and none had dysplasia. Simultaneous infection with  The survival of people living with human immunodeficiency virus (HIV) (PLHIV) has improved since the introduction of antiretroviral therapy (ART) ,2,3,4. HThis prospective cross-sectional study consecutively enrolled 300 persons living with confirmed HIV attended by infectious disease units in the “Hospital Universitario Virgen de las Nieves” (HUVN) and “Hospital Universitario San Cecilio” of Granada and “Complejo Hospitalario” of Jaen and included in a screening program for HPV infection-related anal and cervical dysplasia lesions.Study inclusion criteria were age ≥18 years and participation in the anal and/or genital cancer screening program of the HUVN. The exclusion criteria included being a pregnant WLHIV.During the first visit, data were gathered on sex, age and nationality; number of different sexual partners over past 12 months (NP12m); total number of sexual partners since onset of sexual relations (NPT); time in months since the onset of sexual relations; utilization of condoms and percentage utilization ; employment situation (active or retired); educational level ; smoker or ex-smoker (packs/year); alcohol consumption (standard drink units (SDUs)); intravenous drug user (IDU) or ex-IDU; HIV acquisition mechanism ; HPV vaccination status ; months with HIV diagnosis; CDC-classified HIV stage; on-ART or ART-naïve patient; duration of ART (in months); number of ART regimen before first visit; current ART line and months under treatment with this line; virological failure; polypharmacy; presence/history of other infections (chronic liver disease or infection by hepatitis B virus (HBV), chronic liver disease or infection by hepatitis C virus (HCV), and syphilis); HBV vaccination status; presence and history of other sexually transmitted infections (STIs) in anal, female genital and oropharyngeal mucosae; presence and history of condylomas in any localization and their treatment; and history of anal, cervical/vaginal/vulvar and/or oropharyngeal dysplasia and its degree.N. gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis).Analytical variables considered were nadir CD4+ T cell count, CD4 and CD8 lymphocyte counts and viral load at diagnosis and at baseline. At the same visit, cotton swabs impregnated with physiological saline were used to take anal canal and female genital samples for HPV detection and genotyping using the polymerase chain reaction (PCR) technique  and the ThinPrep Pap Test  for cytology. Both samples were analyzed in the hospital pathology laboratory by the same pathologist who validated the HPV PCR results and cytology study. A cotton swab was introduced into the oral cavity and rotated over the internal and external surfaces of the upper and lower lips, the gingival region and the anterior and posterior surfaces of the tongue and the oropharynx; it was then placed in an individual vial with physiological solution. Oropharyngeal mucosa samples were sent to the Microbiology Department for DNA extraction and PCR study of HPV and other STIs  or high-grade (HSIL) anal squamous intraepithelial lesions (ASILs), atypical squamous cells of uncertain significance (ASCUS) or uncertain lesions whose high grade cannot be ruled out (ASCUS-H). The Reagan classification was used to categorize cases of ASCUS, LSIL or HSIL as dysplasia, with ASCUS and LSIL being low grade and HSIL high grade .After rectal inspection and digital anorectal examination, all participants underwent high-resolution anoscopy (HRA) with a Carl Zeiss 150 fc © colposcope . In brief, 5 mL acetic acid was instilled through a transparent disposable anoscope and left in place for around 3 min before its removal for examination of the mucosa; 5% Lugol’s iodine was then instilled for 1 min before repetition of the anoscopic examination. Samples were selected from quadrants of apparently normal mucosa and from areas with Lugol-negative aceto-white lesions. The biopsies were performed with an endoscopic retrograde cholangiopancreatography (ERCP) catheter.The histological classification was based on the Lower Anogenital Squamous Terminology (LAST) Standardization Project for HPV, categorizing lesions as LSIL (AIN1/condyloma), HSIL  or invasive anal squamous cell carcinoma (ASCC) .Genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51–53, 56, 58, 59, 66, 68, 73 and 82 were considered high-risk (HPV-HR) and genotypes 6, 11, 34, 40, 42–44, 54, 55, 57, 61, 70–72, 81, 83, 84 and 89 low-risk (HPV-LR). Genotypes 39, 45, 59 and 68 were classified as subspecies of HPV 18 and genotypes 31, 33, 35, 52, 58 and 67 as subspecies of HPV 16 [Women with abnormal cervical cytology or genital HPV infection were referred to the Gynecology Department for evaluation.All participants underwent oropharyngeal mucosa inspection before the sampling for HPV PCR, and those with a positive oropharyngeal HPV PCR and/or the presence of symptoms ) and/or visible lesions in the oropharyngeal mucosa were referred to the Otorhinolaryngology Department for examination.The category HSIL-plus includes high-grade lesions (HSILs) and invasive ASCC.The sample size was calculated using Ene 2.0 statistical software. It was estimated that at least 123 PLHIV were needed to expect a frequency of 25% of oropharyngeal high-risk HPV prevalence in PLHIV, considering previously published global data, with a precision of 5% and a confidence interval of 95% .Descriptive analysis: The means, standard deviations, medians and percentiles were calculated for quantitative variables and the absolute and relative frequencies for qualitative variables. The prevalence of HPV infection was calculated with 95% CI.A bivariate analysis was conducted on the relationship between possible risk factors for HPV infection and the presence of dysplasia in oropharyngeal mucosa. The Kolmogorov–Smirnov test was used to check the normality of variable distribution. Student’s t-test for independent samples was used for quantitative variables when normally distributed; the Mann–Whitney U test was used when non-normally distributed; and the Wilcoxon test was applied for related quantitative variables. Qualitative variables were analyzed with Pearson’s chi-square test or, when application criteria were not met, Fisher’s test. Finally, a multivariate logistic regression analysis was performed using a stepwise procedure, entering variables found to be significant in bivariate analyses and other factors considered relevant in the literature . SPSS 21.0  was used for data analyses, and the level of significance was 0.05 in all tests.All participants gave their informed consent to participate in the study, which was approved by the Research Ethics Committee of the hospital . All data were treated in compliance with national data protection legislation .The study included 300 PLHIV  with a mean age of 45.1 years; 87% held Spanish nationality. They had a median of 1 sexual partner over the previous 12 months and a median of 20.5 sexual partners since the onset of sexual relations; 2.7% used condoms during oral sex, 34.3% during vaginal sex and 34.7% during anal sex; and 35.9% of participants were active smokers. HPV vaccination had been received by 31% , and 27.3% had completed the vaccine regimen; 1.7% had chronic HBV infection and 6.3% chronic HCV infection; and 64.3% were vaccinated against HBV. At the first visit (baseline), 5% had active syphilis and 4% anogenital condylomas/warts. Among participants, 95.6% had acquired HIV by sexual pathway (78.7% were MSM). The median time since HIV diagnosis was 15 years (IQR: 8.5–24.9), 25.3% were in AIDS stage at diagnosis and 99.7% had been receiving ART for a median of 13.5 years (IQR: 7.7–19.5).One MSM-LHIV (0.3%) had had a history of HPV-associated oropharyngeal epidermoid carcinoma five years earlier; 25% of women had a history of genital dysplasia (12.5% CIN1 and 18.8% CIN 2/3); none had a history of cervical squamous carcinoma, while 82% of PLHIV had a history of anal dysplasia , as shown in N. gonorrhoeae in 20 (90.9%) and Chlamydia trachomatis in 2 (9.1). Among all participants, 39 (13.0%) had HPV infection in oropharyngeal mucosa, 29 (9.7%) with HR serotypes, 15 (5%) with LR serotypes and 5 (1.7%) with simultaneous infection. The most frequently isolated genotypes in patients with oropharyngeal HPV were HR genotypes 16 (2.3%) and 68 (1.7%) and LR genotype 6 (1%). The examination of all participants by the otorhinolaryngologist detected no symptoms or visible lesions in the oropharyngeal area, and no cases of dysplasia were observed in the oropharyngeal mucosa. The presence of STIs other than HPV in the oropharyngeal area was observed in 22 (7.4%) participants: Among the 64 female participants, 31 (50%) had genital HPV infection: 14 (25%) with HR serotypes, 17 (30.4%) with LR serotypes, 7 (12.5%) with HPV coinfection and 3 (5.2%) with simultaneous oropharyngeal and genital HPV infection. The most frequently isolated genotypes in genital mucosa were LR genotypes 62/81 (8.9%) and HR genotypes 16 and 42 (7.2% for both).Cervical cytology was normal in 55 women (88.7%), ASCUS in 4 (1.3%) and LSIL in 3 (1.0%). Gynecological evaluation detected CIN1 in one woman (0.7%). Among the 300 PLHIV, 238 (82.6%) had HPV infection of the anal mucosa: 189 (67.3%) with HR serotypes, 168 (59.8%) LR serotypes and 122 (43.8%) with coinfection. Simultaneous HPV infection in oropharyngeal and anal mucosa was observed in 25 (8.9%). The most frequent genotypes in anal mucosa were LR 44/55 and 62/81  and HR 16 and 68 (14.7% for both).Anal cytology was normal in 118 participants (39.6%), LSIL in 110 (36.9%), HSIL in 8 (2.7%) and ASCUS in 33 (11.1%). HRA and biopsy results revealed LSIL (AIN1) in 84 (29.2%) (19 (22.3%) in women and 65 (77.4%) HSH) and HSIL plus (AIN2/AIN3/Carcinoma in situ) in 5 (1.7%) (1 (20%) in women and 4 (80%) MSM). Treponema pallidum  and a history of anal HSIL-plus  were associated with oropharyngeal HPV infection. In the multivariate analysis, simultaneous infection by Treponema pallidum (HR 4.02 (95% CI: 1.06–15.24); p = 0.04) and a history of HSIL-plus ; p = 0.02) emerged as risk factors, while a longer duration of ART (8.8 vs. 7.4 years (HR: 0.989 (95% CI: 0.98–0.99)); p = 0.034) emerged as a protective factor. In bivariate analyses, simultaneous infection by HPV infection of the oropharyngeal mucosa was infrequent in this cohort of PLHIV and less prevalent than anal or cervical HPV infections. Genotype 16 was the most frequently isolated genotype (2.3%), habitually as a monoinfection with no associated dysplasia. The most frequent genotypes in female genital mucosa were LR, including genotype 44/55, while the most frequent in the anus were HR-HPV, with a predominance of genotypes 16 and 68. A South American study of eight PLHIV with HPV-associated oropharyngeal lesions found that patients had multiple HPV infections, largely involving HR genotypes 16, 52 and 56 . In a EuAmong the mucosae examined in the present investigation, the highest prevalence and degree of dysplasia was observed in anal mucosa samples, with almost 30% diagnosed by HRA with LSIL/AIN1 and 1.7% with HSIL. No participant presented oropharyngeal dysplasia, and only 1.7% of the women had CIN1. The risk of HPV-related anal cancer is higher in PLHIV than in the general population . Its greIn the present study, the prevalence of HPV was up to 6-fold higher in anal versus oral mucosae. A recent study of 103 MSM-LHIV described a prevalence of oral HPV infection of 14% and no associated dysplastic lesions, similar to the present findings, with an anal HPV infection prevalence of 88.3% and an HSIL prevalence of 24.3% .Treponema pallidum and a history of HSIL-plus in the anal canal emerged as risk factors for oropharyngeal HPV infection. Simultaneous infection with HPV and other STIs is frequent in PLHIV because they share common risk factors and acquisition mechanisms [In this cohort of PLHIV, concomitant infection by chanisms . Studieschanisms . Anotherchanisms .A longer time with ART proved to be a protective factor against oropharyngeal HPV infection in the present study, in line with previous findings of a lower oral mucosal infection rate after 12 months of ART . This isThe main limitations of this study are derived from its design and the specific population, hampering the extrapolation of data to other types of populations. Its strengths include the largest sample size to date for this type of study, the methodology and systematic analysis applied and its novelty: it is one of the few prospective investigations of the characteristics of HPV infection in the oropharyngeal mucosa of PLHIV.Treponema pallidum and a history of anal cancer precursor lesions or ASCC emerged as risk factors for this infection, while greater exposure to ART was a protective factor.HPV infection has a low prevalence in the oropharyngeal mucosa of PLHIV in our setting, with a predominance of high-risk genotypes; it is not associated with dysplasia. Simultaneous infection by


The Carrageenan-gel Against Transmission of Cervical Human papillomavirus trial’s interim analysis (n = 277) demonstrated a 36% protective effect of carrageenan against incident HPV infections. Herein, we report the trial’s final results.Carrageenan demonstrated potent anti-HPV (human papillomavirus) activity In this exploratory phase IIB randomised, placebo-controlled trial, we recruited healthy women aged ≥18 years primarily from health service clinics at two Canadian Universities in Montreal. Participants were randomised (1:1) by the study coordinator (using computer-assisted block randomisation with randomly variable block sizes up to a block size of eight) to a carrageenan-based or placebo gel to be self-applied every other day for the first month and before/after intercourse. Participants, study nurses, and laboratory technicians (HPV testing and genotyping) were blinded to group assignment. At each visit , participants provided questionnaire data and a self-collected vaginal sample . The primary outcome was type-specific HPV incidence (occurring at any follow-up visit). Intention-to-treat analyses for incidence were conducted using Cox proportional hazards regression models, including participants with ≥2 visits. Safety analyses included all participants randomised. This trial is registered with the ISRCTN registry, ISRCTN96104919.Between Jan 16, 2013 and Sept 30, 2020, 461 participants (enrolled) were randomly assigned to the carrageenan (n = 227) or placebo (n = 234) groups. Incidence and safety analyses included 429 and 461 participants, respectively. We found 51.9% (108/208) of participants in carrageenan and 66.5% (147/221) in placebo arm acquired ≥1 HPV type . Adverse events were reported by 34.8% (79/227) and 39.7% (93/234) of participants in carrageenan and placebo arm (p = 0.27), respectively.Consistent with the interim analysis, use of a carrageenan-based gel compared to placebo resulted in a 37% reduction in risk of incident genital HPV infections in women with no increase in adverse events. A carrageenan-based gel may complement HPV vaccination.10.13039/501100000024Canadian Institutes of Health Research, CarraShield Labs Inc. We searched PubMed using the keywords “carrageenan” and “human papillomavirus” or “HPV” from inception to January 9th, 2023, without applying language or date restrictions, to identify 23 experimental research articles that reported on carrageenan’s anti-HPV activity . Results were consistent with a protective effect of carrageenan in 1) pre-clinical studies (n = 18), 2) a post-hoc analysis showing lower HPV prevalence in the carrageenan group among the most compliant, 3) an observational study showing carrageenan may accelerate clearance of existing HPV infection, and 4) an interim analysis of the CATCH study demonstrating a 36% protective effect of carrageenan against incident HPV infection(s). Conversely, a clinical trial conducted in men who have sex with men did not demonstrate a protective effect of carrageenan on incidence, or clearance of anal HPV infections, and reported more adverse events in the carrageenan relative to the placebo arm.The CATCH study is the first clinical trial designed to assess the efficacy of a carrageenan-based gel in reducing the risk of incident and prevalent HPV infections in women. Results were consistent when considering HPV subgenera and type-specific analyses. The addition of carrageenan to a lubricant gel does not appear to impact gel tolerability.The results of the CATCH trial indicate that carrageenan-based gels could complement HPV vaccination in protecting against HPV-related diseases. Our findings of the clinical efficacy of carrageenan may encourage future research in this area. It would be important to further examine adherence by looking at determinants of adherence, explore the possibility of the addition of carrageenan to condoms, assess the impact of a carrageenan-based gel on anal HPV infections in women, and continue research in the area of multi-purpose prevention technology for agents against HPV, human immunodeficiency virus, and other sexually transmitted infections.The most common sexually transmitted infection (STI) agent worldwide is human papillomavirus (HPV), which is a necessary cause of cervical cancer.While HPV vaccination is a highly effective intervention for primary prevention of HPV, the development, implementation, and maintenance of HPV vaccination programs across different settings are not without their challenges, including the negative impact of the COVID-19 pandemic derailing the uptake of HPV vaccines, temporary HPV vaccine shortage, cold chain requirements, and vaccine hesitancy.in vitro and in vivo,Carrageenan, an anionic polymer derived from red algae, previously showed promise as a potent anti-HPV inhibitor The CATCH trial is an exploratory phase IIB block-randomised, placebo-controlled trial. The full details of the study protocol  2010 checklist.Eligible women to participate were those aged 18 years and older, living in Montreal and planning to remain in Montreal for at least the next year, who had vaginal sex with a male partner during the past 3 months and expect do so again in the next 3 months  will change), not currently in a relationship that has lasted longer than 6 months , willing to follow study instructions and attend follow-up visits for 12 months, who understand French or English, and who use a medically acceptable method of contraception and intend to use it for the duration of the trial. Women were considered ineligible based on the following exclusion criteria: have had a hysterectomy, have a history of cervical lesions/cancer or genital warts, pregnant or planning to immediately become pregnant, currently breast-feeding, had a recent (within the last 6 weeks) pregnancy, abortion, or genital surgery, have human immunodeficiency virus (HIV) infection, have a known allergy or hypersensitivity to vaginal lubricants and have allergy to any of the ingredients of the study product or placebo, have participated in any research studies (past 3 months) related to HPV or cervical cancer, or have participated in any research studies (past 3 months) that require taking medications or supplements, undergo medical tests or procedures, or undertake dietary or exercise regimens. Prior to randomisation, participants gave written informed consent , which was administered by the study nurse.Participants were randomised (1:1) to a carrageenan-based or placebo gel by the study coordinator, using computer-assisted block randomisation with randomly variable block sizes (up to a block size of 8). The intervention and placebo gels were packaged in identical containers, except for the product code label; four different codes were used for the intervention gel and an additional four for the control. The participant, study nurses, and laboratory technicians (HPV testing and genotyping) were blinded to group assignment.Participants were instructed to apply the study gel every other day for the first month as well as before  each vaginal intercourse. Approximately 5–10 mL of the gel was to be applied to the vagina, penis, and/or condom. STI protection methods  were encouraged. Participants were asked to refrain from intercourse at least 48 h prior to their follow-up visits. Both study gels are commercially available (Divine9 and Divine), made by CarraShield Labs Inc . The study gels are identical; both are water-based, clear, odourless, tasteless, and of a similar viscosity. The distinguishing feature is the inclusion of carrageenan in the intervention gel.At enrollment and each follow-up visit, participants completed a computer-assisted questionnaire on sociodemographic characteristics, smoking and alcohol use, medical and sexual history (only at enrollment), sexual activity and study gel use since last visit (only at follow-up), and condom and lubricant use (both enrollment and follow-up). In addition, a daily online calendar included questions on sexual behaviour, gel use, and adverse events (AEs). Participants could enter and modify calendar information for the seven previous days, which required them to log in at least once a week with a username and password. Additional details are provided in A self-collected vaginal sample was obtained at each study visit. Samples were tested using Linear Array  for detection and genotyping of 36 HPV types.The primary outcome was presence of a newly detected vaginal HPV infection that was not detected at enrollment, and the secondary outcomes were HPV type-specific clearance of infection(s) detected at enrollment  and adherence  to the intervention measured using data from the daily online calendar. Adherent participants were defined as those whose cumulative adherence to gel use was as recommended in >50% of all intercourse acts prior to failure/censoring . We alsoThe calculation of sample size was informed by two cohort studies conducted in Montreal based on a cumulative incidence proportion of infection at 1 year of 19.5% and a clearance rate of 38% after 1 year. Assuming an expected effect size of 50%, we calculated a sample size of 463 for incidence and 388 for clearance, which accounted for 10% loss to follow-up, a 2-sided hypothesis with 80% power and type I error of 0.05.Based on oncogenic risk and tissue tropism, we grouped HPV types into subgenera; low-risk types in subgenus 1: HPVs 6, 11, 40, 42, 44, and 54; high-risk types in subgenus 2: HPVs 16, 18, 26, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69, 70, 73, and 82; and commensal HPVs in subgenus 3: HPVs 61, 62, 71, 72, 81, 83, 84, and 89.Adherence to the intervention was defined as the number of gel uses divided by the number of vaginal intercourses in the seven days prior to the study visit (from follow-up survey data) and between consecutive study visits . We compared adherence between groups and study visits. Due to (non-significant) imbalances in baseline HPV vaccination status between arms and discrepancies in reporting of vaccination status between screening (pre-enrollment) and the baseline survey (at enrollment), we validated participants’ vaccination status by either confirming their status via email or (in the case of no email reply) by using the date of vaccination reported in their baseline or follow-up survey .Cox proportional hazards regression models were used to evaluate the efficacy of carrageenan in reducing vaginal HPV incidence/detection and prevalence, according to an intention-to-treat (ITT) approach. The analysed population consisted of a subset of the ITT population, as it was not possible to include participants without follow-up visits in the analysis. The proportional hazards assumption was assessed based on Schoenfeld residuals and suggested no violation of the assumption (p = 0.39). Start time was set to the date of the baseline visit when participants were randomised and the first sample was collected. The end time was the date the participant was either censored (due to a participant completing participation or withdrawing from the study) or had the outcome. Missing HPV data for follow-up visits were censored in incidence and clearance analyses. Incidence analyses, performed by any HPV positivity  and subgenera , included participants who had at least 1 follow-up visit. We performed exploratory sub-group analyses to compare the intervention effects according to main baseline characteristics and cumulative adherence to gel use prior to failure (new detection of an HPV type) or censoring and whether participants were enrolled prior or after amendment to gel usage instructions, i.e., gel use before intercourse vs. before and after intercourse.We performed additional post-hoc sensitivity analyses to account for missing outcome data over follow-up. First, we performed a complete-case analysis restricting to participants with valid HPV results at all study visits. Second, we applied single imputation methods involving 1) a best-worst analysis which assumed all participants with missing HPV data in the carrageenan arm were HPV-negative over follow-up and those in the placebo arm had a newly detected HPV infection, and 2) a worst-best analysis which assumed all participants with missing HPV data in the carrageenan arm had a newly detected HPV infection and those in the placebo arm were HPV-negative over follow-up.Participants were included in clearance analyses if they had at least two study visits and were HPV-positive at baseline. We considered two definitions of clearance: liberal (one HPV-negative visit following at least one HPV-positive visit) and conservative (two consecutive HPV-negative visits following at least one HPV-positive visit). We considered time to 1) clearance of all baseline HPV infections and 2) first clearance of any baseline HPV infection.Our primary analyses for incidence and clearance were also done at the HPV-level, where the unit of analysis was each individual HPV type. The Cox proportional hazards models were stratified by HPV type and clustered by participant. For incidence analyses, participants were considered at risk for any HPV type absent at baseline, where each participant could contribute up to 36 observations, each corresponding to an HPV type. For HPV-level clearance analyses, participants were considered at risk for clearing any of their baseline HPV infections.daily calendar, 2) follow-up survey administered at each follow-up visit, 3) adverse event module (nurse report) during follow-up visits, and/or 4) reporting of an adverse event during adverse event follow-up. AEs reported in the daily calendar were graded  by participants.To assess safety of the study gels, we tabulated self-reported AEs and/or reactions that were recorded by means of the 1) Data management and analyses were done in SAS V9.4 and Stata version 17, respectively. The trial was registered (ISRCTN96104919) and approved by Health Canada (authorisation file number 169160). A data safety and monitoring board reviewed and recommended publication of the previously published interim analysis,The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. All authors had access to the dataset and had final responsibility for the decision to submit for publication.Recruitment took place between January 16th, 2013 and October 30th, 2020. As shown in Baseline characteristics of study participants are shown in Follow-up characteristics of the participants are shown in As shown in Overall, there was a similar proportion of AEs reported in each arm: 34.8% (79/227) in the carrageenan and 39.7% (93/234) in the placebo arm . Those mNo differences were observed in sub-group analyses by baseline characteristics  and cumulative adherence to gel use; amendment to the gel use instruction (before intercourse only vs. before and after intercourse) appeared to result in a more protective effect compared to gel use before .in vitro from a cervicovaginal lavage using combination products of MIV-150, zinc acetate dihydrate, and carrageenan (20 participants),Clinical studies, and trials in particular, on the efficacy of carrageenan-based gels against HPV infection are scarce . The CATAdherence to the intervention was balanced by arm. We would expect to see that more compliant participants would have increased protection against incident HPV infections. Another trial, assessing Carraguard®’s anti-HPV activity post-hoc, found a protective effect of carrageenan against prevalent HPV infections but only among adherent users .The assessment of gel safety, comparing Divine9 (intervention) to Divine (placebo), found 37% of participants reported AEs. A large trial (N = 6202), comparing Carraguard® (a carrageenan-based gel) to methylcellulose with up to 2 years of follow-up, reported that 23% of participants experienced AEs.While the study gels were approved by Health Canada and have received 510(k) clearance by the United Stated Food and Drug Administration (FDA), they are hyperosmolal and therefore do not meet the current WHO recommendation for osmolality (<1200 mOsM/kg).The trial was not without limitations. First, gel use was self-reported, which may be subject to social desirability bias if participants tended to overreport adherence to gel use. This could possibly lead to misclassification of exposure and would be expected to bias the estimate towards the null in adherence analyses. Second, the proportion of AEs experienced by participants in the carrageenan arm might have been underestimated. Comparing participants in carrageenan to placebo arm, there were fewer participants randomised (227 vs. 234), more participants who discontinued participation after their first visit (19/227 vs. 13/234), and fewer participants who reported AEs (19/227 vs. 9/234), leading to fewer participants available to report AEs in the carrageenan arm. While AEs were balanced between arms, this does not exclude the possibility that the study gels could have caused more irritation than would be expected from other common lubricants or methylcellulose, which have been used in other microbicide trials. Thirdly, the placebo gel, while identical to the intervention gel except for the addition of carrageenan, may not be a true placebo, as it could have anti-HPV activity that would bias the estimate towards the null. Fourth, the true effect of gel use may be closer to the null due to the chance that carrageenan could inhibit detectability in a small proportion of samples. Fifth, although there was considerable loss to follow-up, the median follow-up time of participants who withdrew was 3.2 months, and 40.2% (82/204) participants who withdrew experienced the outcome (not shown). Therefore, we expect any bias from loss to follow-up to be minimal. Sixth, there were imbalances between groups at baseline, however, adjusting for these imbalances and for adherence did not change the point estimate significantly. Additional limitations inherent in RCTs include measurement bias in ITT estimates, as well as selection bias introduced 1) in per-protocol estimates, 2) by the use of a hazard ratio, and 3) due to missing outcome data. We attempted to overcome the aforementioned limitations by incorporating sensitivity analyses, including best-worst/worst-best scenarios and IPW. The results of which were consistent with a protective effect.The greatest strength of the trial is that it is the first phase IIB randomised controlled trial designed to assess the efficacy of a carrageenan-based gel on incident and prevalent HPV infections. A strength of having two identical gels, with the exception of the addition of carrageenan, is that the protective effect observed can be attributed to the presence of carrageenan. An additional strength is the collection of detailed information over time, in particular the implementation of a daily calendar to obtain details of gel use, sexual activity, and adverse events, which lead to the collection of 95,327 observations. HPV positivity was assessed by detecting 36 different HPV types using the Linear Array, which allowed us to assess genotype-specific HPV incidence among HPV-positive women.,It has been hypothesised that carrageenan works by binding directly to the HPV capsid, thereby preventing HPV from binding to heparan sulfate proteoglycan (HSPG), which is a key step in the infection process on the basement membrane.Multiple mechanisms may be responsible for the anti-HPV activity demonstrated in the CATCH study.This research supports current investigations on the use and utility of carrageenan co-formulated with other anti-microbials (such as griffithsin and carrageenan) in multi-prevention technologies,Despite the high osmolality of the study gels, carrageenan was protective against incident HPV infections in women. Given that carrageenan-based gels have not demonstrated efficacy in preventing anal HPV infections in men, it would be necessary to conduct a gel tolerability study with the reformulated carrageenan-based gel at the correct osmolality in both women and men that include measurements of anal HPV.In conclusion, carrageenan compared to placebo gel use was associated with a reduction in incident HPV infections. Topical microbicides against HPV, such as carrageenan-based gels, may have utility as a complement to HPV vaccination among sexually active women.The authors contributed to: conceptualisation ; data curation ; formal analysis (CL); funding acquisition ; investigation , methodology ; project administration ; resources ; supervision ; validation ; visualisation (CL); writing—original draft (CL); and writing—reviewing and editing . CL and SBP directly assessed and verified the underlying data. All authors had full access to the study data and had the final responsibility to decide to submit for publication.https://doi.org/10.5683/SP3/0DS6FP). Following publication, the anonymised study data and a codebook will be made also available. The study protocol and accompanying documents, such as the informed consent form were previously published.The sample code used to generate the results are available in the McGill Dataverse repository . FC reports grants from Réseau FRQS-SIDA during the conduct of the study and grants to his institution for HPV-related work from Merck Sharp and Dome, Roche Diagnostics and Becton Dickinson, outside of the submitted work. JET is an employee of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc, Rahway, NJ, USA. P-PT, ANB, SBP, and CL have nothing relevant to this article to declare.