There is a close correlation between HPV infection and systemic immune status. The purpose of this study was to determine which lymphocytes in peripheral blood influence human papillomavirus (HPV) infection and to identify whether peripheral blood lymphocyte (PBL) subsets could be used as biomarkers to predict HPV clearance in the short term.This study involved 716 women undergoing colposcopy from 2019 to 2021. Logistic and Cox regression were used to analyze the association of PBLs with HPV infection and clearance. Using Cox regression, bidirectional stepwise regression and the Akaike information criterion (AIC), lymphocyte prediction models were developed, with the C-index assessing performance. ROC analysis determined optimal cutoff values, and their accuracy for HPV clearance risk stratification was evaluated via Kaplan‒Meier and time-dependent ROC. Bootstrap resampling validated the model and cutoff values.Lower CD4 + T cells were associated with a higher risk of HPV, high-risk HPV, HPV18 and HPV52 infections, with corresponding ORs (95% CI) of 1.58 (1.16–2.15), 1.71 (1.23–2.36), 2.37 (1.12–5.02), and 3.67 (1.78–7.54), respectively. PBL subsets mainly affect the natural clearance of HPV, but their impact on postoperative HPV outcomes is not significant (P > 0.05). Lower T-cell and CD8 + T-cell counts, as well as a higher NK cell count, are unfavorable factors for natural HPV clearance (P < 0.05). The optimal cutoff values determined by the PBL prognostic model  can effectively divide the population into high-risk and low-risk groups, accurately predicting the natural clearance of HPV. After internal validation with bootstrap resampling, the above conclusions still hold.CD4 + T cells were important determinants of HPV infection. T cells, NK cells, and CD8 + T cells can serve as potential biomarkers for predicting natural HPV clearance, which can aid in patient risk stratification, individualized treatment, and follow-up management.The online version contains supplementary material available at 10.1186/s12985-023-02039-6. Human papillomavirus (HPV) is a small, nonenveloped, highly hoster-specific double-chain looped DNA virus . To dateCervical epithelial cells are the primary targets of HPV infection. The HPV life cycle is closely related to epithelial differentiation, and no complete virus particles have been found in peripheral blood , 4. AlthAlthough local immunity has been recognized as a key factor in clearing cervical HPV infection , it is eThe systemic immune status can be reflected by changes in peripheral blood lymphocyte (PBL) subsets. Current research on the impact of PBLs on HPV infection and clearance is limited, with only a few studies reporting changes in PBL subset counts due to HPV infection –14. SpiwWe collected cross-sectional data from 716 women who underwent colposcopy and 12-month follow-up information from 164 HPV-positive patients. Our research objectives mainly include (1) investigating the relationship between PBLs and susceptibility to different HPV genotypes; (2) exploring whether PBLs affect the natural clearance of HPV in the short term; (3) examining whether PBLs influence postoperative HPV clearance in women with high-grade squamous intraepithelial lesions or worse (HSIL+); (4) identifying specific peripheral blood immune cells affecting HPV infection and clearance; and (5) establishing potential immunological marker thresholds to help more accurately predict HPV clearance. By investigating the above content, we aim to provide evidence-based references for rational patient risk stratification, individualized treatment, and follow-up.A retrospective cohort study evaluating the influence of PBL subsets on HPV infection as well as HPV clearance was conducted in Fuzhou, China. All subjects gave written informed consent to participate in the study. This study was approved by the Ethics Committee of Fujian Maternity and Child Health Hospital (2022KYLLR03050). The study population was recruited from women undergoing colposcopy in the Fujian Maternal and Child Health Hospital between January 2019 and December 2021. A total of 716 patients were enrolled. Inclusion criteria: Women who did not receive any dose of HPV vaccine; no condom use at least the past year; PBL subsets and cervical HPV genotyping were tested during the initial visit; clinical diagnoses were confirmed by pathology and immunohistochemical staining; Exclusion criteria: sexual partner greater than 1, history of smoking, abnormal immune function; abnormal white blood cell count and C-reactive protein, positive serological tests for hepatitis B, hepatitis C, syphilis, and HIV; presence of acute or chronic infection symptoms or signs; use of antibiotics, antiviral drugs, hormones, and immunotherapy drugs within the past month; having received or currently undergoing chemotherapy and radiation therapy; suffering from malignant tumors or serious heart, lung, liver, or kidney diseases. The correlation between PBL percentages and different types of HPV infection (mainly infection with a single HPV type) was analyzed. Follow-up information was collected  was used to identify 23 genotypes in exfoliated cervical cells, including 14 high-risk HPV genotypes , 4 possible high-risk HPV genotypes , and 5 low-risk HPV genotypes . Briefly, the extracted HPV DNA in a 25 µl reaction system was amplified with the PGMY09/PGMY11 primer, and 5 µL of the extracted HPV DNA served as a control. After amplification, the PCR products were immobilized onto a nitrocellulose membrane and hybridized with 23 different fixed type-specific probes. The results were recorded by directly visualizing blue spots on the membrane. All the detection procedures strictly followed the manufacturer’s instructions.Fresh peripheral blood samples (5 ml) were collected from patients using EDTA anticoagulant tubes. Twenty microliters of BD Multitest 6-color TBNK reagent  was placed into the bottom of a BD Trucount tube, and 50 µl of heparinized whole blood was added to the tube, mixed with vortex, and incubated for 15 min protected from light. Then, 450 µl of 1X BD FACS lysing solution was added and incubated again for 15 min protected from light. Finally, with a BD Canto II, we analyzed fresh blood samples for total Lym subset distributions, including T lymphocytes, B lymphocytes, NK cells, CD4 + T lymphocytes, and CD8 + T lymphocytes. All manipulations were carried out at room temperature (20℃-25℃).exp(β1 × 1 + β2 × 2 + …+βnXn), where “X” represents the percentage of lymphocytes, β represents the coefficients of lymphocytes in the model, and ho(t) represents the baseline hazard function. The optimal cutoff point for each model’s risk score was determined using ROC analysis, dividing the risk scores into high-risk and low-risk groups. Combining the optimal cutoff value with the lymphocyte model to predict HPV clearance.Univariate and multivariate analyses were performed using Cox proportional hazard regression to evaluate independent prognostic factors associated with HPV clearance under untreated and treated conditions. Bidirectional stepwise regression analysis and Akaike information criterion (AIC) were used to select the best variable combination for the lymphocyte model. Risk scores for each lymphocyte model were calculated based on the selected best variable combination using the formula Risk score = ho(t)* The predictive accuracy of univariate and multivariate Cox regression models was evaluated using the concordance index (C-index). Visualize the impact of changes in the lymphocyte model risk scores on HPV clearance through risk score distribution, risk status, and risk heatmap. Kaplan‒Meier curves and log-rank tests were used to compare the differences in HPV clearance rates between the high-risk and low-risk groups defined by the optimal cutoff value. Time-dependent receiver operating characteristic (ROC) curves were used to evaluate the accuracy of the optimal cutoff value in predicting natural HPV clearance. Subsequently, internal validation of the lymphocyte prognostic model was performed using bootstrap resampling . Bootstrap resampling is specifically applied to the following steps: determination of prognostic factors related to HPV clearance in univariate and multivariate Cox regression analyses, evaluation of the predictive performance of Cox models using the C-index, employment of bidirectional stepwise regression analysis and AIC method to select the best variable combination, validation of the generalizability of the optimal cutoff point, and evaluation of the accuracy of the optimal cutoff point in predicting natural HPV clearance using time-dependent ROC.https://hiplot.cn/). These statistical analyses were performed using R software (version 4.1.3).The Kolmogorov‒Smirnov test was used to assess whether the data followed a normal distribution. Independent sample t tests or Mann‒Whitney U tests were used to compare differences between two groups of continuous variables. Chi-square tests or Fisher’s exact tests were employed for comparisons of categorical variables between groups. Lymphocyte subsets were dichotomized using the median value. Chi-square tests were used to evaluate differences in lymphocyte subsets between the HPV-positive and HPV-negative groups. Binary logistic regression was utilized to assess the relationship between PBL subsets and different HPV genotypes (adjusted for age and gravidity). The above statistical analysis was performed by IBM SPSS Statistics 25. Univariate and multivariate Cox regression analyses, bidirectional stepwise regression, AIC calculation, risk score calculation, and log-rank tests were all performed using the survival package. The optimal cutoff value for the risk score was determined using the ggrisk package. Time-dependent ROC curves were plotted using the risksetROC package, and the area under the curve (AUC) was calculated for each curve. Bootstrap resampling was used for internal validation of the results of univariate and multivariate Cox regression analyses, bidirectional stepwise regression, AIC calculation, and the generalization ability of the optimal cutoff value. The boot package was used for bootstrap resampling. The plots were generated using the ggplot2 package and Hiplot (ORG) platform . Follow-up information was collected for 407 HPV-infected women. A total of 243 women lacking follow-up information were excluded. Table Patient characteristics at baseline are detailed in Table There were significant differences in PBL subsets between HPV-infected and uninfected women, which mainly reflected changes in the CD4 + T-lymph percentage and CD4/CD8 ratio. A decreased percentage of CD4 + T cells was associated with increased HPV, HR-HPV, HPV18, and HPV52 infections  vs. Bootstrap HR (95% CI): 1.09 (1.01–1.16) vs. 1.09 (1.01–1.18), C-index vs. Bootstrap C-index: 0.57 vs. 0.69) and NK cells (HR (95% CI) vs. Bootstrap HR (95% CI): 0.89 0.83–0.97) vs. 0.88 (0.81–0.97), C-index vs. Bootstrap C-index: 0.68 vs. 0.72) were found to have significant prognostic value for predicting natural HPV clearance Table . The C-i–0.97 vs.The results of bidirectional stepwise regression analysis indicate that T cells and NK cells can independently predict HPV clearance, while CD8 + T cells need adjustment for gravidity and parity to predict HPV clearance. These findings remain valid after internal validation using bootstrap resampling  Fig. a. In addRisk scores were calculated for three models , and optimal cutoff values for risk scores were determined using ROC analysis  to distinguish high-risk and low-risk patients. A higher risk score indicates a higher likelihood of natural HPV clearance. In the univariate models, the percentage of T cells or NK cells was linearly related to the risk score Fig. a-c, and We classified each sample into high- and low-risk groups using the optimal cutoff values for T-cell percentage, NK cell percentage, and CD8 + T-cell model risk score and performed internal validation using 1000 bootstrap resamples. The results showed that accurate prediction of natural HPV clearance could still be achieved after bootstrap resampling by using the optimal cutoff values for T-cell percentage (67.39%) (Bootstrap HR (95% CI): 1.09 1.01–1.14)), NK cell percentage (22.65%) (Bootstrap HR (95% CI): 0.91 (0.85–0.99)), and CD8 + T-cell model risk score (0.95) (Bootstrap HR (95% CI): 6.03(1.59–29.03)) as the threshold points (Table ), NK celOur study highlights the crucial role of PBL subsets in HPV infection and clearance. We found that changes in the composition of these lymphocyte subsets may influence an individual’s susceptibility to HPV infection and their ability to effectively clear the virus.By comparing the percentage of PBLs in HPV-infected versus HPV-uninfected women, we were surprised to find that the low CD4 + T-cell percentage was an important determinant of HPV infection. A lower CD4 cell percentage often promoted higher infection rates of HR-HPV, HPV18 and HPV52. In addition, the high prevalence of HPV is also related to a decrease in the CD4/CD8 ratio, which mainly increases the incidence of HR-HPV and HPV52 infection.CD4 + T cells are an integral part of successful cellular and humoral host immune defense. The activity of CD4 cells has a direct influence on the function and superiority of B cells and T cells . The CD4Previous studies have reported that women with lower CD4 + T-cell counts have a higher rate of HR-HPV infection and a higher HPV load , 18. WomIn the present study, we found that lower CD4 cell percentages and reduced CD4/CD8 ratios were important determinants of HR-HPV infection, particularly for HPV18 and HPV52. This finding has important implications for vaccination programs. When the CD4 + T-cell percentage or CD4/CD8 ratio is reduced, bivalent and quadrivalent HPV vaccines may not provide effective protection. For this group of people, the nine-valent HPV vaccine should be a better choice.As cervical cancer screening recommendations have evolved, an increasing number of HPV strains have been found in the early stage of infection. Normal and LSIL account for most HPV-infected women who are initially treated in outpatient departments. These patients have a relatively short duration of viral infection and mild symptoms, and the guidelines recommend follow-up observation without surgery . During T-cell percentage and NK cell percentage were identified as independent biomarkers for predicting natural HPV clearance within 12 months. However, there is a nonlinear relationship between CD8 + T cells and HPV clearance, which requires incorporating parity and gravidity into the model to better predict natural HPV clearance. A higher T lymphocyte percentage is associated with a greater likelihood of HPV clearance, while a lower T lymphocyte percentage is associated with a higher likelihood of persistent HPV infection. Conversely, increasing NK cell percentage is associated with a decreased likelihood of HPV clearance and an increased likelihood of persistent HPV infection. Using optimal cutoff values of 67.39% for T-cell percentage, 22.65% for NK cell percentage, and 0.95 for the CD8 + T-cell model risk score, high-risk and low-risk groups can be distinguished for predicting natural HPV clearance.In the early stages of infection, 90% of HPV can be cleared by the innate immune system alone . NK cellThe results from many studies have agreed that host CD8 + T-cell responses are required to eliminate HPV-infected cells. The regression rate of cervical precancerous lesions is closely related to the presence of intracellular granzyme B-positive cytotoxic T cells . The regTotal T cells also play a central role in the control of viral infections. Wang et al. reported that mouse papillomas were observed only in BALB/c or C57BL/6 mice whose T cells were removed with anti-CD3 antibodies, and they completely regressed within 8 weeks after removal was stopped. In BALB/c or C57BL/6 mice, neither CD4 + nor CD8 + T-cell removal alone was sufficient to form visible papillomas . Mice wiTo the best of our knowledge, our study is the first to discover that T cells, NK cells, and CD8 + T cells can serve as good indicators to predict natural HPV clearance in untreated women  at 12 months. Cutoff values of 67.39%, 22.65%, and 0.95 were identified as the optimal cutoffs for T-cell percentage, NK cell percentage, and CD8 + T-cell model risk score, respectively, demonstrating good performance in distinguishing high-risk and low-risk populations for natural HPV clearance. Some scholars have defined 12 months as a time point, after which patients with HPV infection are considered to have persistent infection, and their cervical lesions are more likely to progress. Our study can help identify this population early and recommend aggressive treatment to facilitate rapid clearance of HPV. However, there are certain limitations to our study. We were unable to perform a detailed and comprehensive assessment of PBL subsets. By detecting PBLs, we are unable to obtain specific information on changes in lymphocyte subtypes and can only obtain macroscopic changes in overall immune function. Furthermore, the sample population included in our follow-up cohort was small, and more multicenter prospective cohort studies are needed in the future to support our findings.In conclusion, we found that the percentage of PBL was associated with HPV infection as well as clearance. The lower CD4 + T-cell percentages and CD4/CD8 ratios were important determinants of HR-HPV infection. Low CD4 cell counts often facilitate HPV18 and HPV52 infection, while low CD4/CD8 ratios facilitate HPV 52 infection. People with reduced CD4 + T cells or a decreased CD4/CD8 ratio should not choose the bivalent or quadrivalent HPV vaccine. The nine-valent HPV vaccine could provide better coverage. Moreover, we found that T cells, NK cells, and CD8 + T cells can influence natural HPV clearance. T cells and NK cells can serve as independent biomarkers to predict HPV outcomes. CD8 + T cells need to be adjusted for parity and gravidity to predict natural clearance of HPV. Using a T-cell percentage of 67.39%, an NK cell percentage of 22.65%, and a CD8 + T-cell model risk score of 0.95 as the optimal cutoff value, we can effectively stratify risk populations and identify individuals at high risk for persistent HPV infection. These immunologic parameters will be helpful for patient risk stratification, individualized treatment and follow-up management.Below is the link to the electronic supplementary material.Additional file 1. Tables S1-S7.


Medical cannabis (MC) is used by Canadian Veterans to manage a wide range of health issues. However, there is little information comparing the reasons for MC use and its perceived effectiveness between Veterans and non-Veterans.We compared MC use among a convenience sample of Canadian Veterans and with non-Veteran controls, including demographics, reasons and patterns of use, and perceived effectiveness.www.MissionVav.com). The survey was also mentioned in a monthly newsletter from Veteran Affairs Canada. Self-reported effectiveness was evaluated using a 0 to 10 visual analogue scale .Between November and December 2021, Canadian Veterans using cannabis were invited to participate in a survey using a national press release, social media, and announcements on online platform dedicated to promoting health among Canadian Veterans and non-Veterans during the pandemic  males and 49 (31%) females. The mean age was 57 years (range 19 to 84). Among responders, 90 (63%) identified as Veterans. The most common reasons for MC use among Veterans included: insomnia (80%), anxiety (73%), and depression (52%). Veterans reported medical conditions such as chronic pain (88%) and arthritis (51%). Compared with non-Veterans, Veterans were significantly more likely to be male (83% vs. 49%), have a higher BMI (35.2 vs. 30.9), to report problems with sleep, anxiety, depression, and PTSD, and to use cannabis in edible form (51% vs. 22%). Self-reported mean effectiveness scores for MC were highest for PTSD (8.4), insomnia (8.2), anxiety (8.1), depression (8.0), and chronic pain (7.6).We found important differences in user characteristics and cannabis use patterns between Canadian Veterans and non-Veterans. Further controlled studies are required to validate these findings, but these data suggest that orally administered cannabis products may be worth further study. For many conditions, however, the evidence to support optimal clinical use is of poor quality and/or inconsistent. Canadian Veterans are disproportionately impacted by conditions for which medical cannabis (MC) is frequently used. The Life after Service Study demonstrated that Canadian Veterans, compared with Canadians who have not served, are more prone to suffer from a wide range of physical and mental health issues, including chronic pain, anxiety, post-traumatic stress disorder (PTSD), and insomnia.4Among Veterans, annual program expenditures for MC have been increasing rapidly. There are currently 617,800 Veterans in Canada and Veterans Affairs Canada has allocated $153,780,785 for MC to support 18,388 (3%) Veterans in the fiscal year 2021–2022. Program expenditures are expected to reach $195.2 million in the fiscal year 2022/2023.6 There is, however, clear evidence for the harms associated with cannabis use, including potential adverse effects on mental health and associations with respiratory, cardiovascular, and gastrointestinal events.Despite the uncertainty regarding the efficacy of MC, it is apparent that close to 20,000 Veterans, whose cannabis authorizations are paid for by Veterans Affairs Canada, continue to use this product to manage their medical conditions. MC is perceived a low-risk and safe product in comparison to many other drugs.7 CUD reports have grown significantly in the past decade, particularly among Veterans.9 The perceived net benefit of MC may be, in part, due to the fact that MC is often used to treat multiple conditions in the same individual.10 Accordingly, if users believe that they are treating multiple conditions, they may be more willing to accept the risk of an adverse secondary effect.A further concern for MC use is the risk of cannabis use disorder (CUD).11 In another recent study (7362 patients), MC was perceived to significantly reduce anxiety and depression.12Little research has been conducted to investigate patterns and perceived effectiveness of MC usage among those who use it regularly. One recently conducted study found that among 9766 older adults using MC, the primary reason was to alleviate pain. A majority of participants reported pain relief (73%), enhanced sleep (65%), and improved mood (53%). Further, 36% reported a reduction in the dosage of opioids.13Given that Canadian Veterans use MC for a wide range of symptoms, they are an appropriate group to evaluate for further understanding and optimizing the use of MC. They might also help to identify the target conditions that are most likely to respond to MC based on current usage and perceived benefits. It is also essential to understand whether the experience of Veterans is generalizable to other MC users. There has been relatively limited research on this issue, and only one study has explicitly evaluated the differences of the MC usage by Veterans versus non-Veterans, taking into account sex distinctions.The current study assessed MC use among a convenience sample of Canadians, comparing Veterans and non-Veterans to identify differences in user characteristics  reasons for use, usage patterns, perceived effectiveness, and sex-specific effects.www.MissionVav.com).Between November and December 2021, Canadian MC users, both Veterans and non-Veterans, were recruited to complete an online questionnaire. Respondents were invited to participate in the “CannCorps Study” in a number of ways. Canadian Veterans, family members, and friends were recruited via an online platform dedicated to promoting health among Canadian Veterans and their families  highlighted the survey, which was also mentioned in a monthly newsletter to Veterans from Veteran Affairs Canada. Finally, the general public was invited to participate in a press release to Canadian news agencies.The survey was available in English or French and took <10 min to complete on a computer, tablet, or smartphone. Participants were required to be at least 18 years of age and had used MC at least once in the past month or were considering use in the next month. Anonymity was maintained as no identifiers were collected and the email address of respondents was not recorded. The checklist for Reporting Results of Internet E-Surveys (CHERRIES) has been provided in Supplementary Material.A delegated review of the study was provided by member(s) of the McGill University Health Centre (MUHC) Research Ethics Board, more precisely its Cells, Tissues, Genetics, and Qualitative research panel. The research project was found to meet scientific and ethical standards for conduct at the MUHC.The survey questions focused primarily on MC use patterns, reasons for use, and the respondent's perception of efficacy of their specific MC therapy . SociodeAmong the 115 questions, 45 (39%) focused on the respondent's perception of effectiveness of MC, and MC use patterns . Participants were also questioned about their reasons for MC use, any concerns about MC use, whether they felt that cannabis use was out of control, and how difficult they found it to stop using.Questions were answered by yes/no, drop-down answer options, multiple-choice responses, open-ended responses, and rating scales. Self-reported effectiveness was evaluated using a 0 to 10 visual analogue scale .t-tests (for continuous variables) were used to compare the responses of Veterans versus non-Veterans. Non-parametric tests were used for highly skewed data. Linear and logistic regression models were performed to determine whether cannabis usage among Veterans was significantly different from non-Veterans after adjustment for age and sex. Analyses were also completed for males and females separately. All analyses were conducted using Python version 3.10.Descriptive statistics were used to summarize all responses. After stratifying the sample by Veteran status, chi-square tests  and independent p < 0.05) more likely to be male (83% vs. 49%).The survey was completed by 158 respondents. After excluding 10 outliers who provided unrealistic responses, 148 participants were selected for further analyses. Among respondents, 90 (63%) self-identified as Veterans and 58 (37%) as non-Veterans. There were 99 males and 49 females. The mean age was 57 years (range 19 to 84 years), 103 (73%) were married, 128 (82%) identified as Caucasian/White background, and 85 (60%) were retired. Compared with non-Veterans, Veterans were significantly , anxiety , and PTSD . Female Veterans vs. female non-Veterans were significantly more likely to use MC for conditions such as PTSD  and arthritis   .Perceived effectiveness of MC for both Veterans and non-Veterans was similar for the most common conditions and included: insomnia (8.3 vs. 8), PTSD (8.3 vs. 8.4), depression (8 vs. 8.2), anxiety (8 vs. 8.4), acute pain (7.8 vs. 7.8), chronic pain (7.5 vs. 7.8), and arthritis (7.4 vs. 8) . Perceivn = 91), followed by edibles  and vaporized cannabis  .The majority of respondents had used MC daily for at least 1 year, with oils identified as the most common mode of administration  . Both map < 0.01).Although not statistically significant, males tended to use MC in higher doses as compared with females . Male Veterans were more likely to use MC to treat a greater number of conditions, including arthritis and several mental health problems (p = 0.01), whereas there was no statistically significant association between number of conditions and CBD doses (p = 0.8) (The dose of THC was positively associated with the number of conditions being treated (p = 0.8) .p-values <0.001).In multivariable analyses, adjusting for age and sex, Veterans compared with non-Veterans were more likely to use MC for depression, anxiety, PTSD, sleeping problems, and arthritis and were more likely to use an edible mode of administration . One hundred and eleven (75%) respondents from both groups reported that their MC use was never out of control, and 95 (64%) respondents indicated that they never wished to stop the use of MC.Veterans were significantly less likely than non-Veterans to be concerned about the safety and adverse effects of MC use , traumatic brain injuries, or other service-related conditions.These specific conditions could potentially influence the reasons for seeking MC treatment and the effectiveness of its use compared with non-Veterans. In addition, there might be variations in the availability and quality of research, as well as clinical guidelines, for MC usage among Veterans compared with the general population. The unique circumstances and needs of Veterans could influence the focus and direction of research efforts, leading to potentially different recommendations and guidelines specific to this population. It is important to note that the extent of these differences may vary and are subject to further investigation and research.15 This prevalent use of and satisfaction with MC, often by self-administration, is in contrast to the limited evidence for substantial beneficial effects in the published literature.17Consistent with previous studies, these results indicate that MC is commonly used for a variety of physical and mental health complaints such as chronic pain, insomnia, anxiety, depression, PTSD, and arthritis.Despite the lack of strong scientific evidence demonstrating the effectiveness of MC compared with placebo, there was a strong consistent belief among participants that MC was effective for treating a wide range of physical and mental health conditions. Perceived effectiveness scores were particularly high for mental health problems, with average scores of 8 or greater.18–22 Whether this represents a placebo effect, regression to the mean, or true efficacy will require more scientific study. These data do provide guidance in targeting these studies to the management of chronic pain conditions and several mental health problems.These results are consistent with previous observational studies.24On average, MC was used by most respondents several times a day, which was in line with other studies suggesting that the MC provided symptom relief rather than modifying the disease pathophysiology.25–27 Although MC is reported to be used as a medicinal product, the poor attention to amount or content of the product may be bolstered by a perception of safety of cannabis as a recreational and natural product.A concerning observation of this study is that 60% of the respondents did not identify the strength or content (THC or CBD) of the cannabis product used. This lack of knowledge of product content by many users has been noted in previous studies.Alternately, lack of adequate knowledge by both patients and MC prescribers may also be a factor in poor knowledge of content. Further, we have observed limited concerns about safety and side effects of MC, especially from Veterans.28 These multiple conditions are more prevalent among Canadian Veterans compared with the non-Veteran populations.Consistent with other studies, one of the attractions of MC may be that it is used to treat multiple physical and mental health complaints.Cannabis sativa requires both study and understanding before any evidence-based recommendation can be made in this regard.This may help with mitigating polypharmacy burden as well as dealing with adherence to complicated pharmaceutical routines and their side effects. However, the complexity of effects of the large numbers of cannabinoid and non-cannabinoid molecules in the plant p = 0.09).Sex-based analyses are an important strength of this study. Although sex-based analyses are generally recognized as important tools for detecting disparities and providing insights for research and policy, this strategy has not always been used in research of Veteran populations. We did not observe any significant differences between males and females regarding their MC use patterns. Although males tend to use MC in greater doses, these differences were not statistically significant .30 There is, thus, an urgent need to address the health-related needs of female Veterans in all future studies.Considering that females make up roughly 14% of the ∼600,000 Veterans now residing in Canada,There are limitations to the current study that must be addressed. First, this was a convenience sample of respondents and the representativeness of these results to the population of users of MC remains to be confirmed. The response rate for this survey remains unknown as recruitment through links on social media or notices sent out by Veterans Affairs Canada does not provide an accurate denominator on how many MC users might have noticed the survey.Second, the sample size was modest, which may reduce the accuracy of the estimates provided. Third, it is possible that this sample of MC users was biased toward those who found it effective, whereas those who did not benefit did not reply to the survey.There remain many uncertainties around MC use, particularly among Veterans. Further study should address the specific molecular effects of herbal cannabis products, including various concentrations of THC and CBD, the contribution of other molecules such as terpenes and flavonoids to a therapeutic effect (termed the entourage effect), the interaction of cannabinoids with other medications, and the development of tolerance and importantly, adverse effects especially for long-term use. Ideal dosing as well as methods of administration of MC will also require attention.The responses from this sample of Canadians, both Veterans and non-Veterans, indicate the belief that MC is an effective therapy for both physical and mental health symptoms. While we identified some important differences in user characteristics and MC use patterns between Canadian Veterans and non-Veterans, daily dosage and the perceived effectiveness were similar.These preliminary results should be considered when developing additional studies on MC use and effectiveness. Larger studies are required to validate these findings, but this study suggests that orally administered cannabis products for the primary conditions identified in this sample may be worth further study.


Ogg1 −/− knockout progeny exhibited learning and memory deficits, which were enhanced by in utero exposure to a single low dose of ethanol (EtOH) in both Ogg1 +/+ and −/− progeny, but more so in Ogg1 −/− progeny. Herein, OGG1-deficient progeny exposed in utero to a single low dose of EtOH or its saline vehicle exhibited OGG1- and/or EtOH-dependent alterations in global histone methylation and acetylation, DNA methylation and gene expression (Tet1 (Tet Methylcytosine Dioxygenase 1), Nlgn3 (Neuroligin 3), Hdac2 (Histone Deacetylase 2), Reln (Reelin) and Esr1 (Estrogen Receptor 1)) in fetal brains, and behavioural changes in open field activity, social interaction and ultrasonic vocalization, but not prepulse inhibition. OGG1- and EtOH-dependent changes in Esr1 and Esr2 mRNA and protein levels were sex-dependent, as was the association of Esr1 gene expression with gene activation mark histone H3 lysine 4 trimethylation (H3K4me3) and gene repression mark histone H3 lysine 27 trimethylation (H3K27me3) measured via ChIP-qPCR. The OGG1-dependent changes in global epigenetic marks and gene/protein expression in fetal brains, and postnatal behavioural changes, observed in both saline- and EtOH-exposed progeny, suggest the involvement of epigenetic mechanisms in developmental disorders mediated by 8-oxoG and/or OGG1. Epigenetic effects of OGG1 may be involved in ESR1-mediated gene regulation, which may be altered by physiological and EtOH-enhanced levels of ROS formation, possibly contributing to sex-dependent developmental disorders observed in Ogg1 knockout mice. The OGG1- and EtOH-dependent associations provide a basis for more comprehensive mechanistic studies to determine the causal involvement of oxidative DNA damage and epigenetic changes in ROS-mediated neurodevelopmental disorders.Oxoguanine glycosylase 1 (OGG1) is widely known to repair the reactive oxygen species (ROS)-initiated DNA lesion 8-oxoguanine (8-oxoG), and more recently was shown to act as an epigenetic modifier. We have previously shown that saline-exposed  Ogg1 knockout mice, our lab has previously found that Ogg1 −/− progeny exposed in utero to saline exhibited learning and memory deficits, which were further enhanced by in utero exposure to a single low dose of EtOH on gestational day (GD) 17 in both Ogg1 +/+ and −/− littermates, with Ogg1 −/− progeny being most severely affected [Ogg1 +/+ littermates [Oxoguanine glycosylase 1 (OGG1) repairs 8-oxoguanine (8-oxoG), the major DNA lesion caused by reactive oxygen species (ROS) . 8-OxoG affected . We recetermates .Esr1 and (4) neurodevelopmental disorders using a battery of behavioural tests. We were particularly interested in estrogen receptors 1 and 2 , as one study has reported increased expression of ESR1 target genes in brains of untreated Ogg1 −/− young mice [Esr1 gene were analyzed via chromatin immunoprecipitation followed by quantitative polymerase chain reaction analysis (ChIP-qPCR) [Over 3% of Canadian children aged 5–14 are estimated to have a neurodevelopmental disorder , while tung mice , and we ung mice . These tung mice  within vIP-qPCR) . In compOgg1 knockout mice on a 129SV/C57BL/6J background strain were originally generated by Klungland and coworkers [Ogg1 mouse strain was identified as 58% C57BL/6 and 42% 129. Mice were housed in vented plastic cages  with ground corncob bedding . Mouse cages were maintained in a room with controlled light (14 h light–10 h dark cycle) and climate . Mice were provided with rodent chow  and water (U.V. sterilized reverse osmosis water acidified to a pH of 3 using HCl to minimize pathogen dissemination) ad libitum. Breeder pairs were set up by placing one sexually naïve Ogg1 +/− male with one sexually naïve Ogg1 +/− female per cage, which were left for their entire lifetime to generate progeny of all three genotypes  within the same litter. The number of animals used in each experiment is shown in each figure in parentheses or its legend.All animal protocols were approved by the institutional animal care committee in conformance with the guidelines established by the Canadian Council on Animal Care. Ogg1 heterozygous (+/−) knockout female mouse was mated overnight with one Ogg1 +/− male, and the presence of vaginal plug in the morning was designated as gestation day (GD) 1. GD 17 pregnant dams were given an intraperitoneal (i.p.) injection of a single dose of 2 g/kg EtOH (25% solution (v/v) in saline) or its saline vehicle. GD 17 lies within the sensitive time window for the development of brain function in mice, and we have shown in several mouse strains that in utero exposure on this day to a range of ROS-enhancing drugs reproducibly causes neurodevelopmental disorders similar to those observed in humans [Ogg1 females produces blood alcohol concentrations equivalent in humans to 4–6 drinks (based on women weighing 100–130 pounds) , and in Ogg1 mice causes neurodevelopmental disorders similar to those observed in human FASDs without maternal toxicity or fetal morphological abnormalities [One n humans ,4,7,20. malities . Fetusesg in a microcentrifuge. The supernatant or aqueous top phase was transferred to a new tube and 500 µL of 100% ethanol was added to the supernatant of each sample to precipitate DNA. The samples were spun at 15,700× g for 3 min, and the precipitate was washed with 500 µL of 70% ethanol, followed by another centrifugation at 15,700× g for 2 min. The supernatant was discarded, and the samples were air dried for 15–20 min. The pellets were resuspended with 100 µL ddH2O and were incubated at 4 °C overnight to solubilize DNA.DNA was isolated from a 1–2 mm tail snip of either fetuses or weaned progeny by heating the samples at 62 °C in 200 µL lysis solution  containing 80 µg of proteinase K (8 µL of 10 mg/mL) for 3 h. After the entire tissue was dissolved, 37 µL of 8 M potassium acetate solution was added to each sample, followed by freezing samples for >15 min (or overnight) at −20 °C. Samples were spun for 5 min at 15,700× Ogg1 using a PCR-based assay. Primers  used to amplify the 500-base pair (bp) band for the Ogg1 gene were Ogg1-sense (5′-ACTGCATCTGCTTAATGGCC-3′) (forward primer) and Ogg1-antisense (5′-CGAAGGTCAGCACTGAACAG-3′) (reverse primer). Primers used to amplify the 300 bp band for the neo-cassette responsible for disruption of the Ogg1 gene in the Ogg1 knockout mice were neo-sense (5′-CTGAATGAACTGCAGGACGA-3′) (forward primer) and neo-antisense (5′-CTCTTCGTCCAGATCATCCT-3′) (reverse primer). PCR reaction conditions were 2 μL genomic DNA, 2 μL per sample of 10× DreamTaq Green Buffer , 0.4 μL per sample of 10 mM deoxyribonucleotides (dNTP) , 0.3 μL per sample of each of the four 20 μM primers, 0.2 μL of DreamTaq DNA Polymerase  and 14.2 μL per sample of ddH2O for a final volume of 20 μL. Cycling conditions were 95 °C for 5 min and 35 cycles of 94 °C for 1 min, 55 °C for 1.5 min, 72 °C for 2 min and a final extension at 72 °C for 10 min.DNA was genotyped for the presence or absence of w/v) agarose gel in 1× TAE buffer  and 8 μg ethidium bromide. The agarose gel was run at 130 V for 40 min and then visualized and photographed under an ultraviolet light.DNA extracted from the tail snips of fetuses (as described above) was used to assess the sex of the fetus using a published protocol , with ~2Histones were isolated from fetal brain samples using a protocol adapted from Kim and Shukla 2006 . Briefly2, 10 mM Tris, 0.1 mM deferoxamine and 1% Triton X-100, pH 7.5. The entire homogenate was centrifuged at 1000× g for 15 min at 4 °C, and DNA was extracted from the nuclear pellets following the procedure in the kit. A 100-ng aliquot of DNA was used to measure the levels of 5-mC using an ELISA-based kit , while 150 ng of DNA was used to measure the 5-hmC levels using an ELISA-based kit . 5-mC and 5-hmC levels were measured in the same samples. The levels were quantified as suggested in the kit.DNA from the fetal brains was extracted using a DNA extraction kit , with minor modifications. Briefly, brains were homogenized using 2 mL of lysing buffer containing 320 mM sucrose, 5 mM MgClg for 15 min at 4 °C. In a new tube, isopropyl alcohol was added in a 1:1 ratio of isopropyl alcohol:RNA. Samples were incubated at room temperature for 10 min followed by centrifugation at 12,000× g for 10 min at 4 °C. The pellet was washed with 1 mL of 75% ethanol (in diethylpyrocarbonate (DEPC) water) followed by centrifugation at 7600× g for 5 min at 4 °C. The pellet was dissolved in 70 μL of DEPC water and its concentration was measured to be between 500 and 1000 ng/μL. The purity of RNA was determined by ensuring that the A260/A280 ratio was ~1.8–2.0 for pure RNA and the A260/A230 ratio was ~2.0. Before conversion to cDNA, samples containing 2 μg of RNA were treated with DNAse  at 37 °C for 30 min, followed by inactivation of enzyme at 75 °C for 10 min. RNA was then converted to cDNA using a High-Capacity cDNA Reverse Transcription Kit  and a 40 μL reaction was run under the following conditions: 25 °C for 10 min, 37 °C for 120 min and 85 °C for 5 min. Samples were then diluted to 10 ng/μL. About 12.5 ng of cDNA was used to run the RT-qPCR reaction. See A published protocol was adapted  with minFetal brains were homogenized using radioimmunoprecipitation assay (RIPA) buffer  containing protease inhibitor cocktail . The protein concentrations of the supernatants were determined with bicinchoninic acid (BCA) assay. About 75 μg of protein homogenate was loaded onto a 12.5% SDS PAGE gel. The gel was run at constant 150 V for 1 h in electrophoresis buffer . The protein was transferred onto a nitrocellulose membrane using 1× Transfer Buffer  at 100 V for 1 h 30 min. Membranes were then washed three times in Tris-buffered saline, 0.1% Tween-20 (TBST), followed by blocking using 5% non-fat milk blocking solution in TBST for 1 h at room temperature. Membranes were then washed again three times with TBST. The membranes were cut into three pieces to be able to incubate the blots with estrogen receptor 1  and estrogen receptor 2  in 5% TBST-milk and Gapdh  in 3% bovine serum albumin in TBST overnight in a cold room. The next day, membranes were incubated with secondary antibody for both ESR1 and ESR2  in TBST for 1 h at room temperature. All membranes (including Gapdh) were then washed three times for 5 min each with TBST, followed by a 10-min single wash with TBST. Membranes were then incubated with enhanced chemiluminescence stain  for 5 min and analyzed with a FluorChem8800 imager.w/v SDS, 2 mM EDTA) containing protease inhibitors  and sonicated at high intensity in 30 s on, 30 s off cycles for 15 min twice using a Bioruptor . An aliquot of sonicated chromatin was set aside to represent input fraction and to verify for sonication efficiency and stored at −80 °C. The sonicated chromatin was incubated with 30 μL Protein A agarose/salmon sperm DNA slurry , under gentle rotation on a tube rotator for 1 h at 4 °C, after which the sample was centrifuged to separate the beads. The supernatant was transferred to a new low DNA-binding tube (150 μL/immunoprecipitation). 4 μg antibody and 20 μL BSA was added to each immunoprecipitation tube  and the samples were incubated overnight at 4 °C. The next morning, 30 μL Protein A agarose/salmon sperm DNA slurry was added and samples were incubated at 4 °C for 2 h. The pellets were successively washed three times for 10 min each in 1 mL of Tris.Saline.EDTA (TSE I) buffer , twice in 1 mL of TSE II , twice in 1 mL of LiCL  and three times in 1 mL of Tris.EDTA (TE) buffer . Protein–DNA complexes were eluted in 110 μL elution buffer (1% SDS/TE) for 30 min, and the cross-links were reversed by overnight incubation at 65 °C (5 μL of 5 M NaCl and 1 μL of RNase A). DNA was purified using the Biobasic PCR Purification kit (Cat. BS664-250) and eluted in 40 μL TE buffer. CHIP DNA (5 μL) was amplified by PCR using primers listed in Chromatin was immunoprecipitated as previously described . BrieflyAfter weaning at 3 weeks of age, pups were handled for three consecutive days to accustom them to human exposure and reduce stress and anxiety. The following behavioural studies were completed in a single mouse cohort at the specified times: interaction-induced ultrasonic vocalization (USV) (3–4 weeks), open field activity (6 weeks), social interaction (7–8 weeks old), female-induced USV (4–5 months) and prepulse inhibition (~5 months) . At leasOgg1 genotype and treatment group.Interaction-induced USV was performed to assess social interactive behaviour at 3–4 weeks of age using lighting conditions of ~15–20 lux to minimize anxiety in the mice ,26,27. TLocomotor activity and anxiety were measured using an open field activity box at ~130 lux for 1 h at 6 weeks of age ,28,29,30Ogg1 wild-type untreated mouse was used as a “novel mouse”, was age and sex-matched and was used up to about four times throughout the experiment. The novel mouse was placed first in the arena and was allowed to habituate for 2 min prior to the addition of the test mouse. A 20 × 20 cm zone was defined around each of the inverted wire cup leaving a 5 cm gap in which the whole body of the test mouse must enter to interact with the novel mouse.Alterations in social behaviour were assessed at 7–8 weeks of age at ~6–8 lux ,26. The Ogg1 genotype and treatment group.This test was performed to analyze female-induced USV in male mice ,26. The A prepulse inhibition test was performed to measure abnormalities in sensorimotor gating or startle response in OGG1 mice (~5 months old) . The preOgg1 +/+ (p < 0.01) but not −/− fetal brains, which had returned to baseline at 24 h. However, at 24 h, H3K9ac levels were elevated in saline-exposed −/− vs. +/+ (p < 0.05) but not EtOH-exposed Ogg1 −/− vs. +/+ brains. H3K9me3 levels were increased by EtOH exposure in −/− but not +/+ brains at both 6 and 24 h (p < 0.01), whereas H3K27me3 levels were increased in only saline-exposed −/− vs. +/+ brains at only 24 h (p < 0.05).Global histone protein activation marks (H3K9ac and H3K4me3) and repressive marks (H3K9me3 and H3K27me3) were assessed via western blot in fetal brains exposed in utero to saline or EtOH . EtOH exp < 0.05), with no effect of EtOH observed. No differences were observed in 5-mC and 5-hmC levels at 1 or 6 h, and there were no OGG1- or EtOH-dependent changes in 5-hmC levels. The significance for both histone and DNA modifications is based on two-way ANOVA and a post hoc Tukey’s test.For DNA modifications B, at 24 Ogg1 −/− fetal brains, EtOH increased Tet1 expression  and decreased Nlgn3 expression  compared to saline  compared to saline, neither of which were altered in Ogg1 −/− progeny. See Expression levels of representative genes involved in learning and memory were measured via RT-qPCR. In o saline . In contOgg1 −/− young mice [Esr1 gene expression separated by sex  in comparison to saline-exposed Ogg1 −/− fetal brains, and among EtOH-exposed progeny, Esr1 expression was marginally increased in Ogg1 −/− fetal brains (p = 0.06) compared to Ogg1 +/+ littermates. When the data were separated by sex, Esr1 expression at 6 h was decreased in EtOH- vs. saline-exposed Ogg1 +/+ male fetal brains (p < 0.05), but not in Ogg1 −/− male brains nor in female fetal brains. At 24 h, although not significant, a similar trend for decreased mRNA was seen in male fetal brains. Esr2 gene expression at 6 h was decreased in EtOH vs. saline-exposed Ogg1 −/− (p < 0.05) but not in +/+ male brains nor in female fetal brains. No significant differences were observed in Esr2 levels at 24 h. When analyzed for Esr1/2 ratio, there was a decrease in its ratio at 6 h in EtOH-exposed Ogg1 +/+ male fetal brains but not −/− fetal brains (p < 0.05), with no differences observed in the females. The significance is based on two-way ANOVA and a post hoc Tukey’s test.Since one study has reported increased expression of ESR1-regulated genes  in brains of untreated ung mice , we anald by sex . In the Ogg1 −/− male fetal brains (p < 0.05), but not in Ogg1 +/+ male brains nor in female fetal brains. No differences in ESR1/ESR2 protein level ratios were seen. The significance is based on two-way ANOVA and a post hoc Tukey’s test.Protein levels were measured at 24 h post maternal treatment . No diffEsr1 gene were quantified via chromatin immunoprecipitation followed by quantitative PCR : −34 to 31 bp (p < 0.01), + 538 bp (p < 0.01), + 2.3 kbp (p < 0.01) and + 100.3 kbp (p < 0.05)) (Esr1 gene were observed (The association of histone modifications H3K27me3 (repression mark) and H3K4me3 (activation mark) within various regions of the tive PCR A. We cho< 0.05)) B. No OGGobserved c. See SuOgg1 −/− vs. +/+ females (p < 0.05) but not males when averaged over 1 h . Although similar trends were seen with in utero EtOH exposure, they were not significant. Analysis of centre zone activity revealed that saline-exposed Ogg1 +/− vs. +/+ male progeny showed greater time spent in the centre zone, but there was no Ogg1-dependent effect in saline-exposed females. In contrast, in utero EtOH exposure decreased the total time spent in the centre zone by Ogg1 +/− female progeny compared to +/− saline controls (p < 0.05), but had no effect on males. EtOH exposure also increased total time spent in the centre zone by Ogg1 −/− female progeny compared to both EtOH-exposed +/− and +/+ female littermates (p < 0.05). Interestingly, the increased time spent by EtOH-exposed Ogg1 −/− females was similar to that observed in saline-exposed female progeny of all Ogg1 genotypes. The significance is based on two-way ANOVA and a post hoc Tukey’s test.Increased hyperactivity was observed in saline-exposed over 1 h . When anOgg1 +/− progeny exhibited decreased velocity and track length in the social zone, with no differences in Ogg1 +/+ and −/− mice  compared to EtOH-exposed Ogg1 +/+ littermates (p < 0.05), with a similar trend observed in saline-exposed Ogg1 +/− mice  gene expression in Ogg1 −/− but not Ogg1 +/+ fetal brains may have contributed to altered methylation levels at the later time point, as TET proteins are involved in active demethylation of 5-mC to cytosine [Reln mRNA levels in Ogg1 +/+ but not Ogg1 −/− fetal brains, possibly because baseline Reln levels seemed to be already higher in saline-exposed Ogg1 −/− fetal brains, resulting in no further increase with EtOH exposure. EtOH appeared to transiently decrease neuroligin 3 (Nlgn3) regardless of Ogg1 genotype, although the decrease was significant only in Ogg1 −/− fetal brains. Neuroligin-3 (NLGN3) is an X-linked neuronal cell surface protein and a postsynaptic cell adhesion molecule involved in the formation and remodeling of the central nervous systems [Nlgn3 gene with EtOH exposure in embryo culture without altering its expression [Nlgn3 resulting in inhibition of synaptic transmission is associated with a model for autism spectrum disorders [In this study, the EtOH-mediated increase in Ten-eleven translocation methylcytosine dioxygenase 1 -mediated gene transcription ,46. For Ogg1 KO mice exhibited upregulation of Esr1 target genes [Esr1 and Esr2 mRNA levels and protein levels were quantified in fetal brains.ESRs are nuclear hormone receptors and transcription factors  that seret genes . We prevet genes . The preEsr1 gene expression. A marginal increase in Esr1 expression in EtOH-exposed Ogg1 −/− vs. +/+ fetal brain (sexes combined) was associated with a consistent trend for a decrease in gene-specific H3K27me3 marks measured via ChIP-qPCR in EtOH-exposed Ogg1 −/− vs. +/+ fetal brains across various regions of the Esr1 gene  in EtOH- vs. saline-exposed Ogg1 +/+ but not Ogg1 −/− brains. Analysis of ChIP-qPCR data by sex suggested that, although a trend for EtOH-mediated increase in H3K27me3 in Ogg1 +/+ fetal brains was observed in both males and females  to interact with 8-oxoG sites in genes, and CHD4 recruits repressive epigenetic modifiers complexes including DNA methyltransferases and histone H3K27 methyltransferases (such as EZH2 and G9a) to DNA damage sites, resulting in silencing of genes [Esr1 gene may act similarly to repress Esr1 gene transcription mediated by OGG1, as previously postulated [A decrease in /− cells . A decreof genes . Thus, Estulated .Esr1 mRNA levels and/or the time point analyzed. The protein levels were only measured using fetal brains extracted 24 h after maternal treatment due to loss of samples extracted 6 h later. However, the EtOH-mediated decrease in Esr2 mRNA and ESR2 protein levels in Ogg1 −/− (but not +/+) males  suggests that OGG1 may also sex-dependently regulate ESR2-mediated gene transcription, which has not been previously reported and merits further investigation.The absence of differences in ESR1 protein levels may have been due in part to limited differences in Ogg1 −/− mice (lighting conditions not provided) [Ogg1 −/− mice (sexes combined and tested at 20 lux) [Ogg1 −/− females when tested at ~50 lux [Previous studies that measured open field activity in untreated OGG1-deficient mice did not find differences at 3 months of age (sexes combined and tested at 20 lux)  or in 4-rovided) . However 20 lux) . Our own ~50 lux .Ogg1 −/− males. Unlike males, saline-exposed OGG1-deficient females travelled a greater distance compared to Ogg1 +/+ females, without exhibiting any differences in time spent in the centre zone, suggesting increased ambulatory activity without any effect on fear and anxiety in saline-exposed OGG1-deficient females. In contrast, with in utero EtOH exposure, although the total distance travelled in Ogg1 +/+ and −/− females was unaffected, the time spent in the centre zone was decreased in Ogg1 +/+ but not −/− females, suggesting that the EtOH-mediated increased fear and anxiety was OGG1-dependent.Herein, sex-dependent differences were observed in the saline-exposed group in our studies. This could be due to several factors that varied across studies , includiPrenatal exposure to EtOH has been shown to induce anxiety-like behaviour in rats and mice ,53. ESR Ogg1 mice, EtOH decreased velocity and track length in Ogg1 +/− mice, with a similar non-significant trend in Ogg1 −/− mice, but no effect in Ogg1 +/+ mice. In the USV test, both the saline- and EtOH-exposed Ogg1 +/− progeny exhibited similarly increased interaction-induced USVs compared to Ogg1 +/+ mice, which was significant for EtOH, suggesting that the increase was due to a heterozygous loss of OGG1 rather than EtOH. The mechanism may involve ESR-mediated gene regulation, as one study reported that Esr1/2 knockout male mice exhibited an abolition of sexual behaviour and decreased USVs in the presence of females, and a role for ESRs in regulating aggressive behaviour in male mice has been reported [Although no differences were observed in social interaction in saline-exposed reported . In addireported ,57, wherreported . These sOgg1 genotype on startle response in the prepulse inhibition test may be due to the relatively low dose of EtOH, given there was a non-significant trend of an EtOH-dependent decrease in prepulse inhibition in Ogg1 −/− but not +/+ males and a decrease in Ogg1 +/+ but not −/− females.The absence of a significant effect of EtOH or This study demonstrated OGG1-dependent effects of in utero exposure of the fetal brain to physiological ROS levels, or to a single low dose of EtOH on epigenetic marks and gene expression in fetal brains, and postnatal behaviour, potentially relevant to neurodevelopmental disorders including FASD. In particular, OGG1 alone or in association with 8-oxoG may play a role in estrogen receptor-mediated gene regulation, which may be further altered by the ROS-enhancing effect of in utero EtOH exposure, possibly contributing to sex-dependent differences in some behavioural disorders observed in OGG1 mice. The relatively modest effects of EtOH observed herein suggest that follow-up studies using a higher dose of EtOH will be necessary to fully understand the effect of EtOH on behavioural abnormalities in OGG1-deficient mice and the role of OGG1 in estrogen receptor-mediated gene regulation, among other pathways, at higher levels of oxidative stress, where OGG1 may work differently to regulate estrogen receptor-mediated gene expression. Given the OGG1-dependent alterations in some epigenetic marks and behavioural outcomes in saline-exposed progeny, it is likely that OGG1 epigenetically alters the regulation of additional genes contributing to developmental disorders, including disorders not involving xenobiotic exposure.


INK4A, and Ki–67 by immunohistochemistry [IHC], and HPV E6/E7 ribonucleic acid (RNA) by in situ hybridization [ISH]). The risk scores for the prediction of cervical lesion progression (“risk biomolecules”) ranged from 2.56–2.60 in the normal and low–grade squamous intraepithelial lesion (LSIL) cases and from 3.54–3.62 in cases where precancerous lesions were predicted to progress. In Model 4, 23/86 (26.7%) normal and LSIL cases had biomolecule levels that suggested a risk of progression, while 5/86 (5.8%) cases were identified as precancerous lesions. Additionally, histologic grading with a single molecular biomarker did not identify 23 cases with risk, preventing close patient monitoring. These results suggest that biomarker level–based risk scores are useful for predicting the risk of cervical lesion progression and identifying precancerous lesion development. This multiple biomarker–based strategy may ultimately have utility for predicting cancer progression in other contexts.The current practice of determining histologic grade with a single molecular biomarker can facilitate differential diagnosis but cannot predict the risk of lesion progression. Cancer is caused by complex mechanisms, and no single biomarker can both make accurate diagnoses and predict progression risk. Modelling using multiple biomarkers can be used to derive scores for risk prediction. Mathematical models (MMs) may be capable of making predictions from biomarker data. Therefore, this study aimed to develop MM–based scores for predicting the risk of precancerous cervical lesion progression and identifying precancerous lesions in patients in northern Thailand by evaluating the expression of multiple biomarkers. The MMs (Models 1–5) were developed in the test sample set based on patient age range (five categories) and biomarker levels (cortactin, p16 E6 and E7, which interfere with cell cycle control and apoptosis and induce chromosomal instability [E6/E7 ribonucleic acid (RNA) have been developed; their sensitivity and specificity for detecting CIN 2 have been reported to be 71.4% and 75.8%, respectively [E6/E7 RNA may be more useful than HR–HPV DNA testing for diagnosing CIN 2+ and predicting disease progression [E6/E7 RNA testing has a specificity of 50% and a positive predictive value (PPV) of 62% for CIN 2+, both of which are higher than the corresponding values for HPV DNA testing (specificity of 18% and PPV of 52%). The higher specificity and PPV of HPV E6/E7 RNA testing are valuable in predicting insignificant HPV DNA infection among cases with borderline cytological findings [E6/E7 RNA in situ hybridization (ISH) method [INK4A/Ki–67 dual immunohistochemical staining on formalin-fixed paraffin-embedded (FFPE) cervical specimens correlated with E6/E7 RNA expression [E6/E7 RNA, combined with human protein biomarker assays, may facilitate the diagnosis of abnormal cervical lesions and predict their progression.Almost all cervical cancers and their precursor lesions, including squamous intraepithelial lesions (SIL) and cervical intraepithelial neoplasia (CIN), are caused by persistent infection with high–risk (HR) human papillomavirus (HPV) genotypes ,2,3,4,5.tability , is a haectively ,14,15,16gression . Real–tifindings ,20. Tran) method ,22. Addipression . Therefo® 2) are combined with assays for host protein biomarkers, such as p16INK4A and Ki–67, for the early detection of abnormal cervical lesions and the prediction of lesion grades; p16INK4A is a surrogate biomarker for HPV in women with invasive cervical cancer, and its expression is highly associated with pathological grading [INK4A and Ki–67 improves diagnostic accuracy [INK4A staining. Regardless of HPV status, diffuse p16INK4A immunostaining is a hallmark of high-grade squamous intraepithelial lesions [In developed countries, molecular techniques for HPV DNA detection  is located on chromosome 11q13. Cortactin recruits Arp2/3 complex proteins and binds to actin microfilaments. It also promotes lamellipodia and invadopodia formation, cell migration, endocytosis, cell mortality, and tumor invasiveness [CTTN gene and the resulting overexpression of cortactin have been observed in 15% of primary metastatic breast carcinomas and nearly 30% of HNSCCs [E6/E7 RNA in HR-HPV-associated cervical cancer and is a potential diagnostic biomarker studied by our group. Furthermore, the majority of these highly sensitive techniques have not yet been introduced to clinical practice.A preliminary study by our group found that the expression of HR-HPV siveness ,34. Cortsiveness ,36 at hisiveness , colorecsiveness . Amplifif HNSCCs ,38. Corthttps://breast.predict.nhs.uk/predict.html (accessed on 8 March 2023). Mathematical models (MMs) are also used to determine the likelihood of relapse and predict responses to chemotherapy among patients with breast cancer [Over the last two decades, a variety of machine learning techniques and feature selection algorithms have been widely applied to determine disease prognosis and predict certain conditions . These tt cancer , as wellt cancer ,45.In the comparison between histopathological method and modelling using multiple biomarkers, this study showed more advantages that can be used to derive scores for risk prediction, not only for the diagnosis of cervical lesion from similar biopsy samples. However, the current practice of histologic grading or using a single molecular biomarker can facilitate differential diagnosis.INK4A and Ki–67 were used as protein biomarker controls during immunohistochemical (IHC) staining. HPV E6/E7 RNA ISH was also used. The relationship between IHC staining and ISH data was evaluated in association with clinical characteristics, and MMs were developed to estimate risk scores using linear regression analysis. Receiver operating characteristic (ROC) curves and areas under the curve (AUC) were used to identify the best MMs. Risk scores from the model were then used to predict the risk of abnormal or precancerous cervical lesion progression and may have utility in other cancer contexts in the future.Our study investigated the expression of cortactin in FFPE cervical specimens with diverse lesion grades in combination with other related biomarkers. Biomarkers p1621−a P(1 − P)/d2″. The required number of participants was calculated from a mean ± SD of 52.9 ± 32.1% of HPV prevalence, a Z of 1.96 for the 95% confidence level, and a d of 0.05 [Three hundred and sixty-three FFPE cervical tissue samples were collected from women who underwent routine cervical cancer testing with a colposcopy at Phayao Hospital, Phayao, Thailand in 2012 (233 samples) and 2013 (130 samples). This work was approved by the Human Research Ethics Committee of the University of Phayao (2/015/59) and Phayao Hospital (HE–59–02–0008). The sample size was calculated according to the known prevalence of HPV in the community as follows: N (case/age group) = Z of 0.05 .All FFPE cervical tissues were reviewed by two pathologists, and the following histopathological grades were assigned: normal (211 cases), low-grade squamous intraepithelial lesion , HSIL (58 cases), and invasive cervical cancer . The 233 samples collected in 2012 were defined as the “test sample set” and the 130 samples collected in 2013 as the “confirmed sample set” . The tesThe selected areas of the FFPE cervical tissues were stained with hematoxylin and eosin and graded by a pathologist according to the World Health Organization criteria. Paraffin tissue blocks were made by removing 1.5 mm cores of the tissues and organized into TMAs .E6/E7 RNA chromogenic ISH was performed using the RNAscope 2.5 HD Detection Kit (BROWN) and Quick Guide for FFPE Tissues  with specific combinations of E6 or E7 probes to detect 18 different HR-HPV types when low copy target gene expression was anticipated (1–20 copies per cell). The FFPE sections (5 µm) were de–paraffinized through xylene and ethanol washes and treated as follows: pre–treatment 1 (endogenous hydrogen peroxide block solution) for 10 min at RT; pre–treatment 2 for 45 min at 105 °C; and pre–treatment 3 (protease digestion) for 30 min at 40 °C. After the treatments, the sections were rinsed with water. The tissues were hybridized in a hybridization solution with E6/E7 RNA chromogenic ISH probes in a moist chamber and without a cover slip for 2–3 h at 40 °C. Thereafter, the hybridized probe’s signal was amplified through the serial application of Amp 1 (pre–amplifier step), Amp 2 , Amp 3 (amplifier step), Amp 4 (label probe step), Amp 5, and Amp 6 ; this was followed by the washing steps. Horseradish peroxidase (HRP) activity was then evaluated through the application of 3, 3′–diaminobenzidine (DAB) for 10 min at RT. The sections were then counterstained with hematoxylin, cleared in xylene, and mounted with Permount. The expression signal data were recorded according to negative and positive staining. The internal controls used for the RNAscope chromogenic ISH were proprietary probes for human sequence ubiquitin C (positive control to demonstrate detectable RNA in the FFPE samples) and Bacillus subtilis (B. subtilis) dapB RNA targets (negative control). Ubiquitin C staining was scored to confirm the presence of the signal and its intensity. B. subtilis dapB staining was reviewed to confirm the absence of staining.INK4A, and Ki–67. Briefly, following de–paraffinization and re–hydration, the tissue sections on the slides were antigen-retrieved using a target retrieval solution  at 105 °C in an autoclave for 30 min. Rabbit monoclonal anti–cortactin antibody clone Ep1922Y , mouse monoclonal anti–human p16INK4A clone D25 , and Ki–67 monoclonal antibody clone 20Raj1  were applied at dilutions of 1:200, 1:100, and 1:100, respectively, in 1× phosphate-buffered saline for 60 min. This was followed by incubation with secondary detection antibodies using the Genemed Power–StainTM 1.0 Poly HRP DAB Kit for Mouse + Rabbit . Immunostaining results were evaluated using light microscopy with a 40× objective, and both Allred score  and positive/negative status were recorded.IHC staining was performed on the TMAs to determine the expression of cortactin, p16IHC staining patterns were scored in reference to the proportion of cells that stained: 0 = negative; 1 = rarely positive (<1%); 2 = focally positive (1–25%); 3 = variably positive (25–75%); and 4 = uniformly positive (>75%). In terms of the staining intensity, the IHC staining patterns were scored as follows: 0 = negative; 1 = weakly positive; 2 = moderately positive; and 3 = strongly positive. These scores were added to achieve an Allred scored (AS) ranging from 0 to 7 .The criteria for distinguishing positive and negative IHC statuses are shown in 1, X2, X3…Xn) consisted of the five age groups ; IHC results of p16INK4A, Ki–67, and cortactin; and E6/E7 RNA ISH results. The IHC positive/negative status was recorded as “2” (positive) or “1” (negative), whereas the AS of the staining intensity ranged from 0 to 7. The E6/E7 RNA expression in the chromogenic ISH was recorded as “2” (positive) or “1” (negative)  was the regression coefficient demonstrated as tolerance (0–1) and variance inflation factor . When the tolerance or VIF was near 1, a smaller association with the dependent variable (Y) was considered. When tolerance was near 0 or the VIF was high (>1), a high association with the dependent variable was considered. To develop the linear regression model, we used the test sample set (233 samples)  . B  . The expChi-square test (significance level: p < 0.05). The correlations between the cervical grade and the protein marker (AS) were evaluated using one-way ANOVA (significance level: p < 0.05). The MM was included in the regression analysis (significance level: p < 0.05), and the ROC curves and AUC were evaluated using SPSS.Statistical analysis was performed using SPSS version 16. The correlations between the cervical grade and the protein marker (positive/negative) were evaluated using the Pearson A total of 363 FFPE cervical tissue samples were studied. These samples were retrieved in 2012 (233 samples) and 2013 (130 samples) from women aged 19–95 years. E6/E7 RNA signals were mostly present in the cells  [Positive he cells . An incr lesions . Table 4. (2013) .INK4a and Ki–67 are shown in INK4a and Ki–67 were significantly associated with increasing severity grades of the cervical lesions, as shown in INK4A and Ki–67 for detecting LSIL+ and normal cases and for detecting HSIL+ and normal cases, respectively.The expression patterns of p16Five expression patterns of cortactin were observed according to their localization, staining, and intensity . PositivINK4A, Ki–67, and HPV E6/E7 RNA. The coefficients of the independent variables in the best five MMs are shown in p > 0.05); therefore, the normal cases were included with the LSIL cases in the confirmed sample set. In model 3, the risk score for the progression from LSIL was 2.60 (mean ± SD: 1.4843 ± 1.10780). The risk scores for the progression from HSIL (mean ± SD: 3.5374 ± 1.01427) and SCC (mean ± SD: 3.9516 ± 0.41838) ranged from 3.54 to 4.56 and from 3.95 to 4.37, respectively (p > 0.05). Interestingly, the predictive value of Models 1, 4, and 5 could significantly differentiate (1) the normal cases from HSIL–SCC (p < 0.001); (2) LSIL from HSIL–SCC (p < 0.001); and (3) HSIL from SCC (p < 0.05). The ROC curve and AUC of Models 3, 4, and 5 are shown in The MMs developed were simple linear regression models that included independent variables that consisted of age range (five categories) and the following biomarkers: cortactin, p16ectively . In modeModels 3, 4, and 5 were selected to assess the risk of abnormal cervical lesion progression and precancerous lesions in the confirmed sample set. The expected value (Y) was calculated in each case and compared with the risk score. When Y was equal to or lower than the risk score, the cervical lesions were suggested to have biomolecules characteristic of the baseline . When Y was higher than the risk score, the individuals were expected to be at risk of progression or to have “risk biomolecules.” Such individuals should be monitored. For example, when a histopathological LSIL case was evaluated by Model 3 and showed a predictive value of 1.59 (risk score: <2.60), the presence of LSIL with “baseline characteristic biomolecules” was suggested. However, when a histopathological LSIL case showed a predictive value of 3.02 (risk score: >2.60), it was suggested to be an LSIL case with present “risk biomolecules”. For the prediction of precancerous lesions in the normal and LSIL cases, the cases were predicted to have precancerous lesions when Y was higher than the risk score for HSIL (risk score: >3.54).p = 0.000) in terms of predicting the histopathological normal and LSIL cases with risk biomolecules and precancerous lesions  and HSIL groups , while Models 3 and 5 predicted the cases with precancerous lesions in 3/86 (3.5%) cases in the LSIL group and 20/34 (58.8%) cases in the HSIL group. As shown in  lesions . In the  lesions , the AUC lesions , the AUCAs previously reported, atypical cervical cells slowly grow and progress to precancerous lesions over a period of 10–20 years. Patients with these abnormal cells need to be monitored closely to prevent cervical cancer. In low–income countries, including Thailand, it is difficult to monitor these patients since they are usually lost in the follow–up. In this study, we were able to collect data from the initial presentations of our patients; however, we were unable to obtain follow–up results. Some of the patients might be at risk of developing cervical cancer.INK4A and Ki–67 expression as risk factors for cervical cancer. However, to date, no biomarkers have accurately predicted the progression of abnormal cervical cells and the development of precancerous lesions. The present study aimed to develop MMs and risk scores using a new biomarker, cortactin, combined with p16INK4A, Ki–67, and HPV mRNA. We intended to identify the best MM and risk score to predict the progression from normal cervical tissues to LSILs and HSILs and the risk of developing cervical precancerous lesions. We found that the sensitivity, specificity, PPV, and NPV of p16INK4A/Ki–67 were 68%, 69%, 61%, and 74% for detecting LSIL+ and 92%, 68%, 91%, and 96% for detecting HSIL+, respectively. These results are comparable to those of other studies. Li and colleagues found that the sensitivity, specificity, PPV, and NPV of p16INK4A/Ki–67 FFPE were 94%, 88%, 69%, and 98% for CIN 2+ detection, respectively, and 84%, 96%, 88%, and 96%, respectively, for CIN 3+ detection [INK4A and Ki–67 was strongly associated with disease progression [Many studies have reported the clinical significance of p16etection ,52. Amongression .INK4A [Cortactin can promote cell migration, cell mortality, and tumor invasiveness in melanoma, colorectal cancer, and glioblastoma ,34, and INK4A ,58 in ceINK4A . HoweverINK4A . TherefoE6 and E7 correlates with the progression to high-grade lesions [E6/E7 RNA was combined with assays of biomarkers of human DNA, RNA, or protein for the diagnosis and prediction of abnormal cervical lesions. The sensitivity and specificity of HPV E6/E7 RNA for detecting high-grade cytology (CIN 2) were 71.4% and 75.8% [E6/E7 RNA was 82.5% (77.3–86.8) for detecting CIN 2+ and 39.6% (34.0–45.5) for detecting CIN 3+ [E6/E7 RNA were 88% and 54% for predicting LSIL+, and they were 93% and 44% for predicting HSIL+, respectively , as well as for reducing patients’ anxieties and frequencies of follow up [An increased expression of HR-HPV  lesions  and even lesions ,14,15,16nd 75.8% ,14,15,16g CIN 3+ . Herein,ectively . The preith LSIL . Moreoveollow up ,61.http://www.predict.nhs.uk/predict.html (accessed on 8 March 2023), which uses MMs to determine the likelihood of relapse and to predict responses to chemotherapy for breast cancer [Several prediction models are currently widely used in clinical practice, including the model for breast cancer incidence, the Adjuvant Online Decision Aid ,44,62 ant cancer . Three oThe mean time for abnormal cervical cell progression from LSIL to HSIL with 8/45 (18%) oncogenic HPV types was 73.3 months (95% CI: 64.8–81.8 months). For non-oncogenic HPV , the mean time was 91.3 months (95% CI: 85.1–97.4 months), while for the 2/44 (5%) cases negative for HPV, the mean time was 83.5 months (95% CI: 78.0–89.1 months) . In ModeOur model also suggested that 13–14/34 (38–41%) cases of HSIL with “risk biomolecules” (3.95–4.23) might progress to cervical cancer. This is in broad agreement with the findings by Austin (2020), wherein they determined that only around 30% of CIN 3 lesions would progress to cervical cancer in 30 years . HoweverWu et al. (2021) validated a prediction model in two cohorts in China with a follow-up duration of 3 years. In the first cohort, 42 cases were diagnosed as CIN 2+, with thirty-seven cases predicted to progress and five cases to not progress. In the second cohort, 28 cases were diagnosed as CIN 2+, with 11 cases predicted to progress and 17 cases to not progress . AlthougINK4A, Ki–67, cortactin, and HPV E6/E7 RNA, can provide a risk score for predicting the progression of abnormal cervical cells and the development of precancerous lesions in patients with normal histology and LSILs. For example, the relevant equation (Model 4) was Y = 0.535 + 0.387 (Ki–67AS) + 0.142(p16INK4A AS) + 0.530(cortactinP/N) + 0.506(RNA E6/E7P/N) − 0.786 (Ki–67P/N). These results suggest that monitoring patients with MM–based analyses of multiple biomarkers could help physicians design optimal therapeutic strategies and help predict cancer progression in the future.MM-based analysis of the expression levels of multiple biomarkers, including p16


For women living with HIV (WLHIV), the burden of persistent HPV infection, cervical pre-cancerous lesions and cancer have been demonstrated to be higher than among HIV-negative women. As Ghana and other lower-middle-income countries (LMIC) work toward developing national cervical cancer programmes, it is essential that local scientific evidence be provided to guide policy decisions, especially for such special populations. The objective of this study was to determine the distribution of high-risk HPV genotype and related factors among WLHIV and its implication for the prevention of cervical cancer prevention efforts.A cross-sectional study was conducted at the Cape Coast Teaching Hospital in Ghana. WLHIV, aged 25–65 years, who met the eligibility criteria were recruited through a simple random sampling method. An interviewer-administered questionnaire was used to gather socio-demographic, behavioural, clinical and other pertinent information. The AmpFire HPV detection system  had a higher likelihood of being co-infected.In all, 330 study participants, with mean age of 47.2 years (SD ± 10.7), were involved. Most  had HIV viral loads < 1000 copies/ml and 41.2% (n = 136) had ever heard of cervical screening. The overall hr-HPV prevalence was 42.7%  and the five commonest hr-HPV types among screen positives were HPV59 (50.4%), HPV18 (30.5%), HPV35 (26.2%), HPV58 (17%) and HPV45 (14.9%). Most infected women  had multiple hr-HPV infections, with about 57.4% (n = 81) having 2–5 h-HPV types, while 2.8% (n = 4) had more than five hr-HPV types. A total of 37.6% (n = 53) had HPV16 and/or18, while 66.0% (n = 93) had the hr-HPV genotypes covered by the nonavalent vaccine. Women with HIV viral load ≥ 1000copies/ml (AOR = 5.58, 95% CI 2.89–10.78, This study found out that the prevalence of hr-HPV still remains high in women with HIV, with a notable occurrence of multiple infections and infection with genotypes 16 and/or18. Additionally, an association was established between hr-HPV and infection HIV viral load.. Therefore, comprehensive HIV care for these women should include awareness of cervical cancer, consideration of vaccination and implementation of screening and follow-up protocols. National programmes in LMIC, such as Ghana, should consider using HPV-based screen-triage-treat approach with partial genotyping. Cervical Cancer remains a major public health problem globally, with a global incidence rate of 3.1 per 100,000 women  in 2018.The main aetiologic factor, persistent infection with sexually transmitted high-risk Human papillomavirus (hr-HPV), has been identified and the contribution of various genotypes has been a focus of several studies in different geographical locations , 9. HPV Studies have shown that the burden of cervical pre-cancerous lesions and cancer for Women living with HIV (WLHIV), is higher, compared to HIV-negative women –6 WLHIV A study conducted in Europe found that hr-HPV screening reduced the incidence of invasive cervical cancer (ICC) by about 60–70%, compared with cytology and allows for longer screening intervals . Such fiGlobally, there is a gap in specific protocols for cervical cancer prevention for WLHIV. Questions remain on the best approach, in terms of the age at which to begin screening, the possible role of HPV vaccination, frequency of screening, the screening tests and many more. These questions arise as the evidence accumulated from studies among WLHIV shows that the epidemiology of HPV and cervical cancer is definitely modified by co-infection with HIV , 23 and Efforts to eliminate cervical cancer require a combination of approaches that includes awareness creation, implementing screening measures and vaccination. Apart from studies that have investigated the prevalence of HPV among Ghanaians, other studies have focused on the awareness and knowledge of women with and without HIV , 28. GenAs Ghana, through the Ghana Health Service (GHS), works toward developing a cervical cancer programme, it is essential that scientific evidence be provided to guide policy decisions on women, particularly high-risk populations. The siloed approach to HIV, HPV and cervical cancer research has not been very impactful, hence there is a need for a more comprehensive and multidisciplinary approach, which will offer a more nuanced understanding and showcase how progress can be sustainable. This study aimed at determining the distribution of high-risk HPV genotype and related factors among WHIV and its implication for the prevention of cervical cancer in a LMIC such as Ghana.A hospital-based analytical cross-sectional study was conducted at the HIV clinic of Cape Coast Teaching Hospital (CCTH), in Ghana fromNovember, 2020 to April, 2021. CCTHis a tertiary hospital, which serves the Central and the Western regions of Ghana and well as the south-western part of the country. It is a 400-bed capacity hospital and provides specialist care across more than 20 departments and units, covering a range of medical and surgical specialties. It has a well-structured HIV/AIDS clinic that operates every week of the year. The facility has the capacity to offer a wide range of services for the prevention, screening and management of cervical cancer, but unfortunately, these services have remained largely uncoordinated and opportunistic, which is reflective of the national situation.Women living with HIV (WLHIV), who were registered to receive care at the HIV clinic constituted the study population. Eligible participants were WLHIV between the ages of 25 and 65 yearswith the exclusion of those, who were pregnant, had undergone total hysterectomy, local treatment for cervical lesions or had never engaged in peno-vaginal sexual intercourse.The prevalence of hr-HPV among WLHIV aged ≥ 18 years, in Cape Coast was 60.6%  and baseThe study participants were interviewed by trained research assistants  using a pre-tested structured questionnaire that included their socio-demographic characteristics, their knowledge of HPV and cervical cancer, their reproductive health and other characteristics. Additional clinical characteristics relating to the HIV care, including viral load, were obtained from the HIV clinic records. The women were then trained to take their cervico-vaginal samples by themselves using a brush. The specimen was then placed in a labelled tube, sealed, and transported to the testing laboratory within 48 h without any transport medium. A screen, triage and treat approach, as recommended by WHO, was followed.HPV DNA testing with genotyping was used as the primary screening test for all participants. All screen-negatives were counselled to rescreen with HPV DNA test in 3 years. Screen-positives were stratified into 2 groups based on the genotypes of the high-risk HPV found: those with HPV genotype 16 or 18 and those with other high-risk HPV. Those in the first group were immediately assessed, after application of acetic acid 5% on the cervix, for eligibility for ablative therapy, based on the visibility and position of the transformation zone and the location and size of the lesion, if the patient has a visible lesion. Those eligible for ablation were treated with thermocoagulation, using the Liger Medical HTU-110 ThermoCoagulator. Those ineligible for ablation, received loop electrosurgical excision procedure (LEEP), using the Liger Medical ESU-110 electrosurgical generator, and the specimen sent for histopathology evaluation. If the histopathology evaluation indicates a high-grade lesion, adenocarcinoma in situ (AIS) or cancer, the client is referred for further evaluation and treatment. If the histopathology evaluation indicates a low-grade lesion, such clients, as well as those who received ablative treatment will be reevaluated in one year.The screen-positives for other high-risk HPV were triaged, using visual inspection with acetic acid (VIA), if a client is 45 years old or younger, and cytology (Pap test), if the client is older than 45 years. This segregation was based on the changes in location of the transformation zone with age. All VIA and cytology screen-negatives were counselled to repeat the HPV test in one year. If they tested negative again, they move routine interval screening schedule. Should a client test positive or found to have a lesion on the cervix, suspicious of cancer on VIA, tests positive for a high-grade lesion on Pap test or tests positive again for other high-risk HPV on repeat screening, she undergoes colposcopy and biopsy and the specimen sent for histopathology evaluation. Subsequently, she undergoes further evaluation and treatment, depending on the findings of the colposcopy and histopathology report.AmpFire HPV detection system  was used. It is an isothermal PCR assay that individually detects15 high risk HPV , 33–44 tAll data for this study were entered using a Computer-Assisted Personal Interview (CAPI) developed by the Centre for Data Archiving, Management, Analysis and Advocacy (C-DAMAA) at the Department of Economics, University of Cape Coast. This ensured that all the controls were in place to ensure data quality and the data is hosted on a secure server and accessible only to the assigned administrator. Data were, subsequently received from the administrator and exported to STATA 16.0 for statistical analysis.All categorical sociodemographic variables were described using frequencies and percentages. Means and corresponding standard deviations were used to describe sociodemographic variables that were continuous and normally distributed. High-risk HPV prevalence and genotypes among HIV/HPV co-infected women were analysed using frequencies, percentages, and charts.Seventeen questions were used to assess participants’ knowledge of cervical cancer. Each correct response to a question was scored one  and zerop values < 0.1 were used to construct both bivariate and multivariable logistic regressions with HIV/HPV status as the dependent variable. Age and viral load were considered as a priori variables. Hence, irrespective of whether p values of age and viral load were less than 0.1, they were considered in both the bivariate and multivariable logistic regressions. Similar method was used to construct another bivariate and multivariate logistic regressions with HPV 16 and/or 18 genotype status of study participants as dependent variable and socio-demographic, knowledge and behavioural variables as independent variables.Chi-square tests were used to examine the association between HIV/HPV co-infection status of study participants and socio-demographic, knowledge and behavioural variables. All variables with p values < 0.05 were considered statistically significant.Bivariate and multivariable regression with the main outcome as women’s cervical cancer Knowledge (insufficient or sufficient knowledge) were also performed. Chi-squared tests were initially performed to examine the association between cervical cancer knowledge and women’s sociodemographic and behavioural characteristics. The variables that were associated using the chi-squared tests were used to construct both the bivariate and multivariable logistic regressions. All statistical tests were two-sided and A total of 330 study participants’ results are presented. The mean age was 47.2 years SD ± 10.7). Most of the participants have current partners (37.6%) and 57.9% are involved in unskilled work. The median number of pregnancies was 4 (IQR 2–5) and most (51.2%) had 3–6 lifetime sexual partners and the mean age at first sex was 18.7 years (SD ± 3.3). Among those who had been sexually active in the past 3 months (N = 172) consistent condom usage rate was 77.9%. Most (69.1%) of the women had their HIV viral loads < 1000 copies/ml as against 30.9% whose viral loads were ≥ 1000 copies/ml. See Table 0.7. MostThe overall hr-HPV prevalence was 42.7%  of the study population  and those with HIV viral load ≥ 1000 copies/ml  had a higher likelihood of being co-infected.Table p value = 0.049). Women who had 1–3 , 4–6  and 7 or more pregnancies  were respectively 91%, 93% and 95% less likely to have HPV 16 and/or 18 genotype as compared to those who had no pregnancy.Table p < 0.001). Women with primary education were 62% less likely to have sufficient knowledge about cervical cancer as compared to those without any education . Women with secondary education had an increased likelihood of having sufficient knowledge of cervical cancer as compared to those without formal education .Table HPV-based cervical cancer screening is recommended globally for both women living with or without HIV. This has implications for LMIC and others with resource limitations, but it also has the potential to address some of the barriers and challenges which have negatively impacted cervical precancer screening efforts in poor resource settings. For WLHIV the need to work toward integrating cervical precancer screening into their routine care cannot be overemphasized. Castle et al. clearly make the case for a comprehensive HIV care and how that would impact not only the attainment of the global HIV elimination targets but also targets for cervical cancer elimination . This stThe socio-demographic characteristics of WLHIV revealed that the mean age of patients was 47.2 years. In relation to cervical precancer screening this would mean that a significant proportion of WLHIV in our setting are outside the WHO’s set priority age group for screening which is 25–45 years , 48, andThe median number of pregnancies among the participants was 4, while 51.2% of the participants had 3–6 lifetime sexual partners, and the mean age at first sex was 18.7 years. Among those who admitted being sexually active within the past 3 months, consistent use of condoms was 77.9%. These are all factors recognised to be associated with the risk of HPV acquisition, and persistence, therefore the risk of cervical cancer , 38. WhiThe median duration of living with HIV since confirmed diagnosis was 4 years and the majority (69.1%) of the women had their HIV viral loads < 1000 copies/ml as against 30.9% whose viral loads were ≥ 1000 copies/ml. This study found that WLHIV with higher viral loads had higher odds of having hr-HPV infection. There has been many findings including from a recent systematic review and meta-analysis concerning the role of ART, CD4 count and viral load levels and HPV infection and cervical cancer risk which suggests that sustained viral suppression from ART can have a positive impact on CC prevention , 42, 61.Our current hr-HPV prevalence (43.7%) is lower than what we reported in a previous study 65.6%) that relied on another testing method (Anyplex-II HPV 28) and involved a cohort of WLHIV who had more evidence of immunosuppression . It is w.6% that Inasmuch as the algorithm used for this study conforms to WHO recommendations, its use in a LMIC like Ghana, is fraught with numerous challenges. The first amongst them is the health illiteracy level of the population, which fosters people’s desire to dwell on cultural and religious beliefs and misconceptions and negatively influence the assimilation of cervical cancer prevention education and the utilization of available services. The second challenge is the existence of scanty number of facilities with structured cervical cancer prevention programmes, which is a reflection of the non-existence of national programmes in many LMICs like Ghana. As a result, existing prevention programmes are largely opportunistic and unsustainable. The limited number of laboratories, capable of performing HPV DNA test, pose a big challenge to accessibility; there are no more than four of such facilities in Ghana and these are mostly centered in the national Capital. Coupled with this is the cost of testing, which is beyond the reach of many. Lastly, in an environment, where there is no proper address system and poverty is rife, the proportion of loss to follow-up is great, as client-tracing becomes an arduous task and many clients, who need to make visits for follow-up are unable to do so due to financial constraints. These challenges, among others, pose a serious threat to the quest to roll out effective, comprehensive and sustainable cervical cancer prevention programmes in LMIC like Ghana.Concerning genotypes, hr-HPVtypes 52 and 16 are not as prevalent in this study as in our previous study though even in this study they still feature prominently beyond the top 5 common hr-HPVs . In thisThough phylogenetically linked to other common hr-HPVs, the high prevalence of hr-HPV types such as 35, and 59 among others may warrant their inclusion in subsequent vaccines for our region of the world , 68, 69.This study was a single facility-based cross -sectional study and we recognise that larger national studies among WLHIV are needed to guide the national guidelines. While acknowledging this limitation, we believe that it has contributed to the growing body of evidence that supports the use of a self-collected HPV-based screen-triage-treat approach, as part of comprehensive HIV care in settings like Ghana.The prevalence of hr-HPV among WLHIVremains high in this study with high rates of multiple infections and infection with genotypes 16and/or18. An association was found between HIV viral load and hr-HPV infection. The study also identified knowledge/awareness gap and other potential areas for intervention to reduce the incidence of cervical cancer among WLHIV in Ghana, such as increasing awareness of cervical cancer, the possible role of vaccination, screening and follow up.The study also suggests that an HPV-based screen-triage-treat approach with partial genotyping must be considered as part of national programmes for LMICs like Ghana. Comprehensive HIV care for these women should include education on cervical cancer and its prevention.