A total of 376 women were included, with 242 (64.4%) exhibiting hr-HPV positivity. The predominant HPV genotypes were 16, 52 and 58. Factors associated with the immediate detection of HSIL+ pathology included a colposcopic impression of high-grade lesions, hr-HPV positivity, HPV 16 positivity, HPV 18 positivity, HPV 58 positivity, age less than 40 years, and biopsy of two or more pieces. However, only the first three factors were statistically significant in multivariate analysis. Among the 291 women who continued surveillance for 6 months or more, the median follow-up period was 41.8 months (interquartile range [IQR] 26.5–54.0). The prevalence of subsequent HSIL in women with hr-HPV positivity versus negativity was 3.6% versus 0.98%, respectively. The median time to the subsequent detection of SIL was 28.7 months (IQR 14.9–41.7). In conclusion, women with ASC-US in our study had a high proportion of hr-HPV positivity. Type-specific HPV testing could play a pivotal role in the development of specific management protocols for women with ASC-US.We conducted a prospective study to evaluate the prevalence of high-risk human papillomavirus (hr-HPV) positivity in women with atypical squamous cells of undetermined significance (ASC-US). Additionally, we assessed the association of hr-HPV positivity with the pathology of high-grade squamous intraepithelial lesions or worse (HSILClinical trial registration: https://thaiclinicaltrials.org, TCTR20161017002. The screening tests available include (i) high-risk human papillomavirus (hr-HPV) DNA/mRNA tests, (ii) cytology, (iii) co-testing, and (iv) visual inspection with acetic acid (VIA)3. In Thailand, cytology screening remains principal approach, yielding abnormal results in 1.5–4% of cases. The most frequently identified cytological abnormality is atypical squamous cells of undetermined significance (ASC-US)5.Cervical cancer ranks as the fourth most common cancer and the fourth leading cause of cancer-related death in women globally, with 56.8% of cervical cancer cases resulting in mortality6. In a prior report involving 1756 Thai women with ASC-US, 221 (12.6%) demonstrated immediate pathology of high-grade squamous intraepithelial lesion (HSIL) or adenocarcinoma in situ (AIS) or invasive cancer identified in 41 women (2.3%)7. Given such a high incidence of immediate HSIL or worse (HSIL+), the direct costs of colposcopy are borne by government public health security schemes, rending immediate colposcopy the primary triage method for Thai women with ASC-US.According to 2019 American Society for Colposcopy and Cervical Pathology (ASCCP) consensus on risk-based management, ASC-US with negative hr-HPV should be followed by a co-testing evaluation after three years. However, ASC-US cases with positive hr-HPV without genotype-specific strategy, necessitate as immediate colposcopy10. Geographic variation exists concerning the most common hr-HPV genotypes associated with HSIL+ pathology in women with ASC-US. Black women with ASC-US have significantly higher hr-HPV positivity rates and lower HPV 16 positivity than white women11. Two studies reported that the most common HPV types detected in women with ASC-US were types 16, 58, and 52, in descending order of prevalence13. Conversely, another study listed HPV types 16, 18, and 31 as the most common, in that order14. An additional study indicated that HPV types 16, 33, 58, and 51 were most associated with detected HSIL+15. The presence of specific hr-HPV types may significantly impact the triage of women with ASC-US during diagnostic colposcopy.Different types of hr-HPV exhibit varied oncogenicity. The two types accepted worldwide as the most oncogenic are HPV 16 and 18+ pathology among women with ASC-US cytology for each hr-HPV genotype, to identify factors that are associated with immediate HSIL+ pathology, and to investigate the prevalence of subsequent squamous intraepithelial lesion (SIL) pathology during follow-up and explore its associated factors.The aims of this study were to compare the proportions of HSILBefore this research begun, the Siriraj Institutional Review Board authorized the protocol . The work was also registered at the Thai Clinical Trials Registry (TCTR20161017002). Informed consent was obtained from all participants. Enrollment of study subjects occurred between March 2016 and March 2018. The authors confirm that all methods were performed in accordance with relevant guidelines and regulations. The inclusion criteria for this study included non-pregnant women aged 21 years or older presenting with ASC-US cytology. Participants were required to have no history of abnormal cervical cytology or treatment for cervical neoplasia, and to provide written informed consent before attending the colposcopy clinic.normal–lacking acetowhite epithelium or other abnormalities, low-grade—thin acetowhite lesion or rapidly fading acetowhitening or fine type of abnormal vessels, and high-grade—rapid appearance and dense acetowhitening, coarse type of abnormal vessels or sharp border. Up to four colposcopy-directed biopsies were obtained from all visible lesions, including those of the cervix, vagina, and vulva. If the squamocolumnar junction (SCJ) appeared normal, a random biopsy was performed at 12 o’clock. Endocervical curettage (ECC) was performed when the SCJ could not be fully visualized. Pathologic diagnoses were made by a gynecologic pathologist (SH), according to the 2012 classification of cervical pathology by the International Federation for Cervical Pathology and Colposcopy (IFCPC). Women with HSIL+ pathology underwent treatment following standard protocols. On the other hand, women diagnosed with LSIL pathology or less were scheduled for repeating cervical cytology every six months for two years or hr-HPV based-testing at one and two years. Subsequent routine screening was scheduled for women who showed all negative tests. During the follow-up period, the threshold for colposcopy was any abnormal cytology or positive hr-HPV.Colposcopy was performed within six weeks by gynecologic oncologists. At the beginning step of the colposcopy, a cervical smear was collected for hr-HPV DNA testing using a Wallach broom and preserved in PreservCyt solution . For the detection of L1 DNA, we utilized the Anyplex II hr-HPV Detection Kit . This kit has been validated by the European Commission and is capable of detecting 14 hr-HPV genotypes: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Colposcopic impressions were classified into three categories: + pathology, including HSIL, AIS, or cancer was calculated. Follow-up outcomes were collected until September 30, 2022, and participants who completed follow-up tests for six months or longer were analyzed for subsequent detection of SIL.Demographic data, results of the hr-HPV DNA testing, colposcopy findings, and pathology reports were collected. The proportion of HSIL16. With a delta error of 0.05, a total of 376 women were required for this study.The sample size calculation was based on the prevalence of hr-HPV detection in women with ASC-US, which stood as 56%+ pathology. The disease-free interval analysis was performed using the Kaplan–Meier method and Cox proportional hazards model. The results of associated factors are reported as odds ratios (OR) with corresponding 95% confidence interval (CIs). A P value of 0.05 or less was considered to be statistically significant.PASW Statistics, version 18 , was employed for all statistical analyses. The data are presented as number and percentage or median and interquartile range (IQR). The Chi-square test or Fisher’s exact test was used to compare categorical variables. Stepwise logistic regression analysis was conducted to identify variables independently associated with HSILIn total, we enrolled 376 women. Baseline demographic data, results of hr-HPV DNA testing, colposcopy impressions, and pathological report are presented in Table + pathology was found in 55 women (14.7%). No woman was diagnosed with AIS. In the case of the one woman who was diagnosed with SCCA, her conization specimen pathology revealed a depth of invasion of 1 mm without lymphovascular space invasion (LVSI). As a result, she underwent a hysterectomy, and the subsequent pathology report indicated no residual cancer. Among the 54 women diagnosed with HSIL pathology, treatment was successfully completed through cryotherapy, laser ablation, or loop electrosurgical excision procedure (LEEP). One of 54 women with HSIL pathology underwent subsequent hysterectomy and upper vaginectomy due to not-free conization margins and confirmed vaginal HSIL. Factors associated with immediate HSIL+ pathology by univariate analysis are presented in Table + pathology were a colposcopy impression of high-grade lesions , positive hr-HPV , and positive HPV 16 .Immediate HSILAmong the 291 women who continued follow-up for at least six months, the initial pathology was one woman was diagnosed with invasive cancer, 40 women with HSIL, 120 women with LSIL, and 130 women with inflammation/normal cervical pathology. The median follow-up time was 41.8 months (IQR 26.5–54.0). Subsequent detection of SIL pathology occurred in 22 women (7.6%), including 14 cases of LSIL and 8 cases of HSIL  of 98.9%18. Furthermore, hr-HPV testing is an economic strategy for classifying ASC-US with a colposcopy referral rate of 50.6%20. In addition to the ALTS study, the Kaiser Permanente Northern California (KPNC) research program conducted a similar investigation. In this cohort, the proportion of positive hr-HPV by HCII® in women with ASC-US was 48.4%, and immediate CIN 2+ was 7.7%. Among women with ASC-US, those positive for hr-HPV had a higher proportion of immediate CIN 2+ (14.8%) than those negative for hr-HPV (0.1%). Similarly, the proportion of immediate CIN 3+ in hr-HPV positive women was 4.9%, whereas it was 0.03% in hr-HPV negative women19.The randomized Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesion Triage Study (ALTS), which utilized hybrid capture® (HC) II in women with ASC-US, reported proportion of positive hr-HPV, immediate CIN 26. The alternative strategy suggests repeating cytology at one year. The WHO 2021 guidelines specifically recommend immediate colposcopy for women with ASC-US who test positive for hr-HPV3. None of the international guidelines have yet addresses the role of specific hr-HPV genotypes in triaging women with ASC-US cytology.Thus, from 2012 through to the most recent update of the 2019 ASCCP recommendations, two preferred management strategies for women with ASC-US cytology have emerged. The first strategy involves reflex hr-HPV testing, where a positive result leads to colposcopy, while a negative result permits HPV-based test in the next three years+ detection. Variations exist in the proportion of hr-HPV detection, which ranges from 32.0% to 85.3%, the proportion of initial HSIL+ pathology, and the diversity of hr-HPV genotyping across various studies of woman with ASC-US24. The ATHENA study, conducted with 1578 women with ASC-US using the cobas® test, found the proportion of positive hr-HPV to be 32.0% and the immediate CIN 2+ was 5.1% . No cases of AIS or invasive cancer were diagnosed16. Three other studies presented the proportion of positive hr-HPV in women with ASC-US as 33.7%, 41.0%, and 67.2%. The immediate CIN 2+/HSIL+ pathology was reported at 7.3%, 7.4%, and 23.1%25. The NPV of the hr-HPV test was reported to be 100%25. The current study showed a proportion of positive hr-HPV consistent with the range of previous studies, and the hr-HPV test demonstrated the capability to predict immediate HSIL+ with a sensitivity of 90.6%, an NPV of 96.3%, and a colposcopy referral rate of 64.4%.The higher the prevalence of hr-HPV infection, the higher the proportion of HSIL+ and varies geographically. The proportion of CIN 2+ in women with ASC-US who tested positive for hr-HPV was 13.3–35.6%23. ALTS reported that CIN 2+ in women with ASC-US who tested positive for hr-HPV was 26.2%, with the highest for HPV 16 positive women being 48.5%23. The ATHENA trial reported CIN 2+ in hr-HPV-positive women as 14.0%. They presented the risk of CIN 2+ in women positive for HPV 16, HPV 18, non-HPV 16/18, and negative for hr-HPV as 31.5%, 4.3%, 8.6%, and 0.8%, respectively17. A study of 1620 women with ASC-US found 75.3% to be positive for hr-HPV. And total immediate CIN 2+ prevalence was 33.6%, which broke down into positive hr-HPV 16/18 at 46.9% and hr-HPV non-16/18 at 30.3%21. Another study displayed a prevalence of CIN 2+ in ASC-US women who detected HPV 16, 18/45, another hr-HPV and negative hr-HPV at 31.6%, 10.9%, 6.4%, and 0.6%, respectively24. A retrospective study performed in China demonstrated that the common hr-HPV genotypes detected in ASC-US women were HPV 16, 52, 58, 18, 53/56, and 51, respectively. The proportion of HSIL+ was highest in women positive for HPV 16 (63.9%), followed by HPV 33, 51, 58, 52, and 18 , respectively15. Wang et al. reported HPV 52, 58, 16, 51, and 39 as the five most common hr-HPV genotypes detected in ASC-US women. They found the independent factor in predicting immediate CIN 2+ was HPV 16 . Other independent factors were HPV 58 , HPV 18 , HPV 52 , HPV 31 , and HPV 33 . The study also suggested using the HPV 16/18/31/33/52/58 model for triage in women with ASC-US to detect CIN 2+ with sensitivity, specificity, PPV, and NPV at 93.1%, 73.0%, 18.0%, and 99.4%, respectively, and a referral rate to colposcopy of 30.9%12. The current study found that the most common hr-HPV types in women with ASC-US were 16, 58, and 52, respectively. However, the highest proportion of HSIL+ was found in those infected with HPV types 16, 18, 58, and 59, respectively. These results, partially align with the ASCCP 2019 recommendations and suggest that hr-HPV genotyping should be integrated into triage management for women with ASC-US cytology.Each hr-HPV type delivers different risks for the development of CIN 2+/CIN 3+ in women with ASC-US who tested positive for hr-HPV, a study by Schiffman et al. suggested that the 3-year cumulative risk of CIN 3+ was 5.2%. Additionally, the risk was 16.0%, 7.4%, 7.0%, and 7.1% in women who tested positive for HPV 16, HPV 18, HPV 31, or HPV 33/58, respectively. Therefore, women who carrying these five genotypes should be referred for immediate colposcopy, whereas those carrying other hr-HPV types could wait a year before retesting26. The ALTS reported the 2-year cumulative rates of CIN 2 and CIN 3 in women with ASC-US as 6.7% and 8.8%, respectively27. Furthermore, when stratified by hr-HPV testing, the 2-year cumulative risk of CIN 3+ in women with ASC-US carrying hr-HPV was 15%, and this rate increased to 32.5% if they were carrying HPV 1623. The KPNC cohort reported that the 5-year risk for developing a CIN3+ pathology in women with ASC-US who tested positive for hr-HPV was 6.8%, compared to 0.4% in those who tested negative28. Thus, the surveillance interval for women with ASC-US should be specified based on the genotyping of hr-HPV prevalent in of the individual country. Based on the genotyping of hr-HPV and the timing of subsequent SIL detection reported in the current study, surveillance by cytology or hr-HPV testing at 12, 24, 36, and 48 months could be suitable.In terms of the cumulative risk of CIN2+ pathology. A study in New York of 2145 women with ASC-US who tested positive for the Aptima HPV mRNA test and underwent colposcopic directed biopsy found that the proportion of CIN 2+ was 8.8%. When considering age groups, the highest incidence of CIN 2+ was found in those younger than 25 years compared to the age group 50–59 years, with rated of 15.4% versus 4.8%, respectively29. The highest detection proportion of HSIL+ was in those aged ≤ 30 years (40.5%) and the lowest in the age group 51–60 years (21.7%). Invasive cancer was most often detected in those aged > 60 years (3.4%) and second most in the age group 51–60 years (2.7%). This is similar to the current study, which found HSIL+ to be more prevalent in individuals < 40 years and invasive cancer was detected more often in those older than 40 years15. On the contrary, the authors reported a positive trend with age groups of < 30 years, 30–44 years, and ≥ 45 years, in relation to the risk of non-HPV 16/18 CIN 3 30.Age has been previously reported as a significantly associated factor with an increased risk of developing CIN 2+ pathology to refer for colposcopy at the right time is necessary. Strategies for managing ASC-US with other secondary tests, such as E6/7 mRNA or p16/Ki67 dual stain, have been proposed to identify these women. The European Equivocal or Mildly Abnormal Pap Cytology Study (EEMAPS) reported that the proportion of positive p16/Ki-67 in ASC-US women who detected CIN 2+ was 20% compared to positive hr-HPV which was 69.5%. Moreover, p16/Ki-67 had a higher performance for CIN 2+ detection with a sensitivity and specificity of 92.2% (hr-HPV 90.9%) and 80.6% (hr-HPV 36.3%), respectively31. The Primary ASC-US LSIL Marker Study (PALMS) stated that p16/Ki-67 had a comparable sensitivity but higher specificity compared to HCII® for CIN 2+ detection in the ASC-US subgroup32. Zhu et al., compared the performance of three tests; the p16/Ki67, the hr-HPV DNA testing and the HPV E6/E7 RNA test, for detection CIN 2+ in women with ASC-US. They stated that the sensitivity was 98.2%, 98.2%, and 87.0%, respectively. Furthermore, the specificity was 82.5%, 17.5%, and 42.7%, respectively22. Further studies should be conducted to determine factors, such as, p16/Ki67 or DNA methylation, that could be added to the selection criteria to identify women with ASC-US for the highest benefit and the best cost-effective protocols for colposcopy.Optimal integrated screening and classification strategy for selected women at highest risk of having HSILThe strength of the current study is the prospective study that collected complete clinical data, colposcopy findings, hr-HPV testing, and pathology diagnosis were achieved within one month of the cytology examination date and have long-term follow-up data. The drawback of the current study is the fact that we did not measure the quantitative assay of hr-HPV or HPV E6/E7 mRNA testing, we did not evaluate inter- or intra-observer variation among the three colposcopists, and we did not perform cost-utility analysis.In conclusion, hr-HPV genotyping may play a role in the immediate management and follow-up schedule for women with ASC-US.


To compare the triage performance of HPV viral loads reflected by cycle threshold values (CtV) from two different HPV testing assays: the PCR based Cobas4800 and the isothermal amplification based AmpFire assay.We used the data from a sub-study of The Chinese Multi-Center Screening Trial and analyzed the data of the cases positive in both Cobas4800 and AmpFire assays with recorded CtV. Spearman’s correlation was applied to analyze the association between CtV from AmpFire and Cobas4800 assays, as well as the correlation between CtV and the histological lesion grades. The 50th percentile of CtV was used as the cutoff to construct triage algorithms for HPV-positive cases. McNemar’s test was used to analyze the differences in sensitivity and specificity for detecting CIN2 + and CIN3 + in different triage algorithms.Four hundred forty-six HPV positive women who had consistent HPV results from Cobas4800 and AmpFire in terms of the HPV genotype and reported Ct values were included in the analysis. The mean CtV of hrHPV tested by Cobas4800 and AmpFire were linear correlated. Direct association were showed between the severity of cervical lesions and the HPV viral loads reflected by CtV of hrHPV, HPV16, non-16/18 hrHPV and A9 group from both assays. HPV16/18 genotyping combined with low-CtV for non-16/18 hrHPV, especially A9 group, were demonstrated to be satisfactory in the sensitivity and specificity for detecting CIN2 + or CIN3 + .Ct value represented a good triage marker in both PCR-based and isothermal amplification HPV detection.The online version contains supplementary material available at 10.1186/s12879-023-08737-4. Cervical cancer is a significant global health challenge. As the fourth most common cancer among women, it created 604,000 new cases of cervical cancer and 342,000 new deaths worldwide in 2020 . The priHPV testing has been widely recognized as an effective primary screening method for cervical cancer, with high sensitivity and negative predictive value in detecting cervical lesions . HoweverCytology and HPV genotyping are the most common optional triage references used in cervical cancer screening programs . CurrentHrHPV viral load is another molecular indicator that may tell the status of cervical cancer development –11. SeveThe Cobas4800 assay is an FDA-approved real-time PCR-based HPV test that can provide both partial genotyping and cycle threshold values (CtV) for HPV-positive channels. The assay reports 14 high-risk HPV types in three channels: HPV16, HPV18, and a pooled 12 other hrHPV types . CtV from that assay represents the DNA amplification cycles required to make the fluorescence signal to reach a predetermined threshold. It is inversely correlated with the log amount of targeted DNA in the specimen .Our team has confirmed value of CtV from the Cobas4800 assay in positive triages by demonstrating that using HPV16/18 genotype and an appropriate cutoff of the Ct values for 12-hrHPV-in-pool as the triage protocol (the genotype/CtV protocol) could obtain a CIN2 + detection efficiency that was comparable to a protocol with combination of HPV16/18 genotypes and cytology (≥ ASCUSS) for non-16/18 hrHPV types [AmpFire is an isothermal, real-time fluorescent, and multiplex nucleic acid amplification method that allows detecting and genotyping 15 types of high-risk human papillomavirus (hrHPV) . One of In this study, we evaluated the performance of CtVs from AmpFire assays when making them as the reference for triage of HPV positive women through head-to-head comparison with the relevant CtVs from Cobas4800 HPV assay.This study was based on the data from a sub-study of The Chinese Multi-Center Screening Trial (CHIMUST) and its sub-study validating the effectiveness of AmpFire in detection of grade 2 and above cervical interepithelial neoplasia (CIN2 +).CHIMUST (Registration number: ChiCTR-EOC-16008456) was a multicenter population-based cross-sectional cervical cancer screening study led by our team. Ten thousand eight hundred and eight-five  women from 15 screening sites in 7 provinces in China were screened from August 2016 to January 2018. Enrolled women was those who were aged 30–59 of years, sexually exposed, non-pregnant, unscreened for at least 3 years, without history of hysterectomy or pelvic radiation, and consent in writing for participation. The trial was approved by the Ethics Committee of the Peking University Shenzhen Hospital (IRB:PUSH2016001) and the Institutional Review Board of Cleveland Clinic (USA) (IRB:15–1549) .Each participant provided a self-collected vaginal sample and a clinician-collected endocervical sample. The samples were split and tested for HPV using Cobas4800  and SeqHPV . In addition, the clinician-collected samples were processed for cytology testing using ThinPrep (Hologic) .Patients who tested positive for HPV on either assay (Cobas4800 and/or SeqHPV for self- and/or clinician-collected samples) were recalled for colposcopy, and colposcopy-directed and/or random biopsies were taken according to the Preventive Oncology International (POI) protocol . All hisThe sub-study was conducted later, with aim to validate the performance of AmpFire in detection of CIN2 + and CIN3 + .The sub The Cobas4800 HPV (hereafter as Cobas) is a multi-PCR-based HPV assay that detects a total of 14 hrHPV types across three channels: HPV16, HPV18, and a pooled 12-HPV channel , along with a separate β-globin channel as a reference. The manufacturers have defined CtV-cutoffs for all the HPV channels to determine positive results, which are 40.5 for HPV16 and 40 for HPV18 and the pooled 12-HPV channels. When the CtV is equal to or lower than the cutoff, a positive result is obtained and the corresponding CtV is recorded. The CtV of negative channels is above the default CtV-cutoff and is not reported.The AmpFire assay  is an isothermal, real-time fluorescent, multiplex nucleic acid amplification method which has received regulatory approval from both the European Community and the Chinese Food and Drug Administration. The AmpFire Multiplex HPV assay is able to detect the presence of 15 high-risk HPV genotypes  and can simultaneously genotype HPV16 and 18 separately in a single reaction. As same as in Cobas, it detects the human β-globin gene as an inner control. This assay can directly detect HPV from non-processed samples with limited hands-on time and no need for DNA extraction and purification. The amplification reaction is incubated at 60 °C for 75 min, and fluorescence is recorded every minute from each channel . The Ct values for each amplification curve in all fluorescence channels are automatically reported. As the AmpFire assay is isothermal, there are no cycles, and the Ct value simply represents the reaction time in minutes. If the amplification curve for the β-globin gene is not exponential, the result is considered invalid .In order to make the results from the AmpFire to be match with those from Cobas, we just included the cases those were: 1) tested positive of hr-HPV on Cobas4800 for the clinician-collected samples in the primary screening; 2) tested positive of hrHPV on AmpFire assays for clinician-collected samples in the sub-study; 3) with the CtV reported, and 4) with matched results from Cobas and Ampfire in terms of at least one HPV genotype.When any case was reported by both assays to be the positive of non-16/18 hrHPV, the result from SeqHPV  will be referred to determine the exact types those were reported by Cobas and AmpFire in pool. Cases would be excluded if 1) the HPV type of a single-type HPV was not matched on Cobas and AmpFire; 2) if the non-16/18 hrHPV from Cobas and AmpFire were reported by SeqHPV having HPV16 and/or 18; and 3) if the non-16/18 hrHPV from Cobas and AmpFire were reported multi-type infections by SeqHPV. (in such cases we could not identify which type of hrHPV was the determinant of the CtV for non-16/18 hrHPV). With the above inclusive and exclusive criteria, cases that were positive of HPV53 only on AmpFire were not included and had no influence on the analysis.Cases with positive results for hrHPV from both the two assays with matched genotype information and identifiable genotype-related CtVs were included into the analysis . The analytic cases were classified into three groups in a hierarchy manner per the genotypes: HPV16, HPV18, and non-16/18 hrHPV. If not specifically indicated, HPV16 referred to a result that was positive for HPV16 only or multiple HPV types including HPV16; HPV18 referred to a result that was positive for HPV18 only or multiple types including HPV18 but excluding HPV16; while non-16/18 hrHPV refers to a result that was positive of single hrHPV type that are none or excluding HPV16 and/or 18. CtV analysis was also conducted through categorizing the non-16/18 hrHPV according the genotyping from SeqHPV, into three groups: HPV A9 group , HPV A7 group , and HPV A5/A6 group .The mean ± standard deviation was used to describe Ct values. Spearman's correlation coefficient was used to compare the association between CtV of hrHPV, HPV16, HPV18, non-16/18 hrHPV, and HPV A5/A6, A7, A9 group from the AmpFire and Cobas4800 assay. Spearman's correlation coefficient was also used to analyze the correlation between CtV from Cobas4800 or AmpFire and cervical lesion grades. Differences in CtV from Cobas4800 or AmpFire assay among histological lesion grades were compared by one-way ANOVA.To differentiate low-CtV positives from high-CtV positives, the CtV cutoff was determined primarily based on the 4% CIN3 + incident rate , receiveP < 0.05 was considered statistically significant.Data analysis was performed using SPSS v.26.0 software , and A total of 6,042 women were included in the sub-study of CHIMUST and had HPV test results from both Cobas and AmpFire with complete data on cytology and histology analysis , of whomp < 0.001  years. Twenty-two-point twenty percent  of them were positive for HPV16, 6.95% (31/446) were positive for HPV18, and 70.85% (316/446) were positive for non-HPV16/18 hrHPV of the hrHPV positives were reported as non-CIN, 16.14% (72/446) as CIN1, 17.04% (76/446) as CIN2 + , and 7.26% (34/446) as CIN3 +  . The CtVs of HPV16 in the CIN2 + and CIN3 + groups were significantly lower than those in both the non-CIN and CIN1 groups (p < 0.05). The CtVs of non-16/18 hrHPV in the CIN1 group were lower than those in the non-CIN group (p < 0.05), and the CtVs of A9 group in the CIN1 and CIN2 + cases were lower than those in the non-CIN cases (p < 0.05). No significant difference was found among different grades of lesion in terms of correlated CtVs of HPV18, A5/A6 group, and A7 group  and AmpFire (CtVAmpFire ≤ 22.6), 62.5% and 87.5% of CIN3 + cases in non-16/18 hrHPV group were detected “low-CtV” on Cobas4800 (CtVCobas ≤ 30) and AmpFire assay (CtVAmpFire ≤ 25.71), respectively, and 83.33% of CIN3 + cases in A9 group were detected the low-CtV on both Cobas (CtVCobas ≤ 32) and AmpFire (CtVAmpFire ≤ 26.48)  bear a disproportionate burden of the world cervical cancer incidence and mortality. Control of cervical cancer in LMICs presents unique challenges those are differ from high-income countries (HICs). According to a statistic report in 2020, less developed regions accounted for 88.1% of cervical cancer incidence and 91.4% of mortality .The AmpFire assay has proven to be equally accurate to Cobas4800 and SeqHPV in detection of CIN2 + or CIN3 + for both self- and clinician-collected samples . HoweverSelf-sampling has been proven to be an effective way to increase screening coverage in LMICs , 26. HowIn addition of HPV genotypes, HPV viral load from Cobas have been demonstrated to be a potential triage option –18. Give., who reported that the top three HPV genotypes (HPV16/33/31) with the highest CIN2 + /CIN3 + risk were all within the A9 group [.found that the viral load of some hrHPV genotypes  were positively correlated with the severity of cervical lesions but others were not [.also observed significant differences in viral loads of HPV16, 33, and 58 among different grades of cervical lesions, but no difference was observed in other genotypes [This analysis demonstrated the correlation between the CtVs of HPV16 and non-16/18 hrHPV and the severity of cervical lesions are reverse on both the Cobas4800 and AmpFire assays. Notably, when using Spearman correlation coefficient to express the relativity of the CtV and the severity of cervical lesions, the relativity of HPV-16 CtV with lesion severity is stronger than that of the CtV of non-16/18 hrHPV, which was consistent with prior analyses based on CHIMUST –31. ThosA9 group . Similarwere not . Duan et.who analyzed data from 1.7 million women across 68 population-based studies and showed that HPV16, 52, 58, 18, and 33 were the most prevalent subtypes in China [., the pooled data from 3045 women who tested positive for hrHPV with self-collected samples revealed that the three most prevalent genotypes were HPV52, HPV16, and HPV58. CIN2 + was found most frequently in HPV16 (31.23%), HPV33 (24.03%), HPV58 (18.41%), HPV31 (11.76%), HPV18 (7.75%), and HPV52 (7.30%) [Compared to the A5/A6 and A7 groups, the A9 group was proofed a higher prevalence and a higher risk of CIN2 + /CIN3 + . Our findings indicate that the A9 group that proportioned 52.2% (165/316) of the non-16/18 hrHPV types accounted for 73.3% (22/30) of the CIN2 + and 75% (6/8) of the CIN3 + cases resulted by the non-16/18 hrHPV. This is consistent with results from large population-based studies, such as Zhu et alin China . FurtherNoting the high prevalence of the A9 group in the Chinese population, our team in the prior work evaluated algorithms which use the A9 group from Cervista Assay (Cervista A9) for primary cervical cancer screening, and found it similarly sensitive for detection of CIN2 + and CIN3 + when compared to the full Cervista assay, but significantly reduced the colposcopy referral rate and cytology rate . In thisOur study evaluated data collected from a well-organized, population-based cervical screening program that involved 446 HPV-positive women from five provinces in China. Cervical exfoliated cell samples were obtained using a standardized sampling procedure, which makes the results applicable to the general population in various settings.However, the study also has limits. Firstly, although our results showed no significant difference in the sensitivity for detecting CIN2 + and CIN3 + between the triage algorithm combining HPV genotypes with viral load  and that combining HPV genotypes with cytology, the guideline-recommended algorithm, we cannot ignore the fact that part of the CIN3 + showed relative higher CtV, which could cause case missing when we adopt the genotype plus virus-load algorithm. In our study, there were 3 CIN3 + cases tested positive of non-16/18 hrHPV on Cobas HPV with high CtV. Among them one was reported high CtV by AmpFire HPV test as well. Some studies described that HPV integration into the genome of the cells were observed particularly in CIN3 and cervical cancer . CompletIn conclusion, the HPV viral loads reflected by CtVs for hrHPV, HPV16, non-16/18 hrHPV types, and A9 group were linearly correlated with the severity of cervical intraepithelial neoplasia in both Cobas and AmpFire. In comparison with the algorithm of “Genotype for HPV16/18 combined with Cytology for non-16/18 hrHPV types”, the algorithms of “Genotype for HPV16/18 combined with low-CtV for non-16/18 hrHPV” from either Cobas or AmpFire is equal sensitive but less specific in detection of CIN2 + and CIN3 + , and the algorithm of “Genotype for HPV16/18 combined with low-CtV for A9 group” is equal sensitive but significantly higher specific in detection of CIN2 + or CIN3 + . Both of the algorithms are satisfied enough to be a triage in cervical cancer screening services in lower-resource area where qualified cytology is not available.Additional file 1: Supplementary table 1. Ct values for specific HPV genotype among different grades of cervical lesions. Supplementary table 2. Distribution of CIN2+ and CIN3+ in low-Ct HPV positive and high-Ct HPV positive women according to different classification methods (n=446)


HPV infection is one of the most studied risk factors in cervical cancer—the second most common cancer site and cause of death due to cancer in the Philippines. However, there is a lack of population-based epidemiological data on cervical HPV infection in the Philippines. Local reports on co-infections with other lower genital tract pathogens, commonly reported globally, are also lacking, which emphasizes the need to increase efforts in targeting HPV prevalence, genotype, and distribution. Hence, we aim to determine the molecular epidemiology and natural history of HPV infection among reproductive-age Filipino women using a community-based prospective cohort design. Women from rural and urban centers will be screened until the target sample size of 110 HPV-positive women  is reached. Cervical and vaginal swabs will be collected from all screened participants. For HPV-positive patients, HPV genotypes will be determined. One hundred ten healthy controls will be selected from previously screened volunteers. The cases and controls will comprise the multi-omics subset of participants and will be followed up after 6 and 12 months for repeat HPV screening. Metagenomic and metabolomic analyses of the vaginal swabs will also be performed at baseline, after 6 months, and after 12 months. The results of this study will update the prevalence and genotypic distribution of cervical HPV infection among Filipino women, determine whether the current vaccines used for HPV vaccination programs capture the most prevalent high-risk HPV genotypes in the country, and identify vaginal community state types and bacterial taxa associated with the natural history of cervical HPV infection. The results of this study will be used as the basis for developing a biomarker that can help predict the risk of developing persistent cervical HPV infection in Filipino women. Cervical cancer is the second most common cancer site and the second most common cause of death due to cancer in the Philippines and globally . There wHuman papillomavirus infection is a necessary cause of cervical cancer  and is cNumerous epidemiological studies in several countries have confirmed that human papillomavirus strongly correlates with cervical neoplasia. HPV DNA was detected in almost 93% of cervical cancer patients, with no significant variations in HPV positivity among countries .One of the principal socioeconomic vulnerabilities cited for cervical cancer is poverty, thus explaining the lack of effective screening programs to detect and treat precancerous conditions and the inability to bear costs related to treatment, making cervical cancer incidence much higher in developing countries . This inHuman papillomavirus (HPV) infection is one of the most studied risk factors for cervical cancer . AlthougOf all high-risk HPVs, HPV-16 is the most prominent strain identified and associated with vaginal, vulval, anal, and penile cancers . HPV-16 Despite the risks, population-based epidemiological data on HPV in the general population in most parts of the Philippines remains unknown , which iDetermining the HPV genotype and distribution among the general population can help address the gap in the knowledge of the epidemiology of HPV in the Philippines ,25. The Papanicolaou smear testing leads to the early detection and treatment of precancerous cervical lesions in most developed countries with well-organized cytologic screening programs ,29,30,31Current screening strategies established in the country include pap smear testing and visual inspection with acetic acid (VIA) , which iMulti-omics is a field of scientific research that integrates various omics data into one. This approach combines two or more omics data sets that are then analyzed, visualized, and interpreted to determine different biological processes based on a given scope . Using aWhile it has already been established that HPV infection is a well-established risk factor for cervical cancer, some factors may contribute to cervical carcinogenesis . VaginalThe present study protocol aims to determine the factors associated with the natural history of cervical HPV infection in a cohort of women in rural and urban Philippines centers using an omics approach. Specifically, it aims to (1) describe the sociodemographic profile and reproductive health behavior of the study cohort; (2) determine the cervical HPV infection prevalence and genotype distribution in the study cohort; (3) characterize the vaginal microbiome and metabolomic profiles of HPV-positive and HPV-negative study participants; and (4) identify factors influencing the acquisition, clearance, and persistence of cervical HPV infection in the study cohort after 6 and 12 months at follow-up examinations.The DEFEAT HPV Study or the Determinants oF Acquisition, persistence, and clearance of oncogenic cervical Human Papillomavirus infection in a cohort of women in rural and urban Philippines using a multi-OMICs approach, will employ a community-based prospective cohort design. An initial cross-sectional study will identify age-matched HPV-positive and HPV-negative individuals, who will be invited to participate in the longitudinal study .The study will employ two non-probabilistic sampling techniques: convenience sampling for the selection of study sites and quota sampling for the recruitment of study participants. Patients will be recruited from the selected community centers and enrolled in the study if they satisfy the eligibility criteria. Recruitment and sample collection will continue until the desired number of participants is reached.http://sampsize.sourceforge.net/iface/index.html accessed on 1 January 2023) [The sample size was based on Casals-Pascual et al., 2020, regarding the number of participants required to find significant differences in alpha diversity in clinical microbiome studies . To findry 2023) .Recruitment of participants and subsequent sample collection will be performed in Likhaan Center for Women’s Health clinic in Tondo, Manila (urban site), and Naic, Cavite . These sites were selected based on good coordination between the principal investigator and the non-government organization, and the availability of trained local health personnel. Laboratory processing and molecular testing will be conducted at Virology Laboratory, Department of Medical Microbiology, College of Public Health, University of the Philippines Manila.The eligibility criteria for the initial cross-sectional survey will be the following: women aged 21 years or older will be eligible to participate if they are currently sexually active or seeking birth control and have an intact uterus. The eligibility criteria for the longitudinal study will be the following: women aged 21 years or older included in the initial cross-sectional survey who underwent HPV DNA PCR and Pap smear tests.The exclusion criteria will be verified during the physician’s pelvic exam, by means of medical record, and/or self-reported. Written informed consent will be obtained from all participants. Exclusion criteria for the study will be current pregnancy; menstruating during sample collection; taking antibiotics, antifungals, or antivirals currently or within the previous three months; treatment for cervical intraepithelial neoplasia within the previous 18 months; having a current referral for hysterectomy or had a total or radical hysterectomy for any indication; use of douching substances, vaginal applied medications and suppositories, feminine deodorant sprays, or vaginal lubricants within 48 h prior the visit; any skin condition in the genital area interfering with the study; sexual intercourse less than 48 h prior the visit; and having type I or type II diabetes, hepatitis, and HIV infection.Informed consent will be given to target participants during the survey. They will be given enough time to decide whether to participate in the study. The research team will provide and discuss the research details, so they will be fully aware of the nature of the study. Advantages and potential disadvantages will be discussed in full disclosure with the participants. The research team will also provide the following information during the process of securing consent: (1) significance of the study; (2) detailed flow on the processes involved in the testing and validation of the samples they will submit; (3) disposal protocol of the samples they will submit; and (4) referral plan in case they will have abnormal examination findings.Women receiving abnormal Pap smear results with positive PCR tests for oncogenic HPV will be referred to the rural health unit of their respective municipalities for appropriate management. These participants will be given follow-up diagnostic recommendations that range from repeat Pap smears at 6 and 12 months to colposcopy  to loop electrosurgical excision.To investigate the shifts in bacterial communities in the HPV states, we will group the study participants based on the following conditions: (1) Group 1.1 (HPV negative)—women who tested persistently negative in the detection of HPV types throughout 12 months ; (2) Group 1.2 (HPV acquisition)—women who were HPV negative at baseline and then became HPV positive during the follow-up period; (3) Group 2.1 (HPV clearance)—women who were HPV positive at baseline and then became HPV negative during the follow-up period; (4) Group 2.2 (HPV persistence)—women who were persistently HPV positive at baseline and during the follow-up periods. Patients will be asked to follow up for the study two times, 6 and 12 months after the first visit.An interviewer-administered questionnaire will be used in this study. The data collection tool will be pre-tested for content and face validity, and field interviewers will be trained before field deployment. The following data will be collected: (1) sociodemographic characteristics ; (2) medical history ; (3) awareness and practices of cervical cancer preventive behavior; (4) sexual and reproductive health behavior . A licensed physician will conduct standard gynecological examinations. These will be performed in a private room with the assistance of a nurse.A licensed physician will collect cervical and vaginal swabs. A sterilized speculum will be inserted without lubricant. The cervical and vaginal swabs will be collected by swabbing the cervix and the lateral walls of the mid vagina using an ENAT PM 2 mL L–shape Applicator  and an ENAT PM 2 mL Regular Applicator , respectively. The samples will then be immediately placed on ice and frozen at −80 °C within 1 h of collection. All samples will be subjected to a Pap smear test. A licensed pathologist will conduct a cytology report according to the Bethesda system. The cytology report will be categorized into normal and abnormal. Routine cervical cytology will include negative for intraepithelial lesion or malignancy (NILM) and inflammatory changes. Abnormal cervical cytology will consist of atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), and squamous cell carcinoma (SCC). Urine and stool samples will be collected and submitted for routine urinalysis and stool examination for the detection of parasitic infections. These tests will be performed by a licensed medical technologist and pathologist.® SYBR® Green, 0.625 μL of each 10 μM primer, and 4 μL of deionized distilled water, in a total volume of 12.5 μL. PCR will be conducted with initial denaturation of 95 °C for 15 min, followed by 35 cycles of denaturation at 95 °C for 20 s, annealing at 56 °C for 1 min, extension at 72 °C for 1 min, and final extension of 72 °C for 4 min. All samples that present positive for β-actin will be used for the detection of HPV genotyping [Genomic DNA will be extracted from cervical and vaginal swabs using the Qiagen DNA Extraction Kit according to the manufacturer’s procedure. Extracted DNA in aqueous solution will be quantified (ratios of 260/280 and 260/230) using NanoDrop 2000 Spectrophotometer . Sample adequacy will be evaluated by means of the amplification of the human β-actin gene using 1 μL of genomic DNA, 6.25 μL of 2x QuantiTectnotyping .The extracted genomic DNA from the cervical swabs will be subjected to HPV genotyping using Anyplex II HPV28 Detection Kit  following the manufacturer’s instructions. This test can detect the HPV DNA of 28 anogenital HPV types ,59.The genomic DNA extracted from the vaginal swabs will be used to detect lower genital tract infections . AnyplexTM II STI-7 Detection (V1.1)  will be used following the manufacturer’s instructions. This kit is a cost-effective and rapid diagnostic tool for detecting multiple sexually transmitted infections ,61.Bacterial vaginosis (BV) will be assessed using Amsel criteria  and the The protocol for metagenomic sequencing will be adapted from a previous study on the association of the cervical microbial community and HPV infection status in Korean women . The rawv/v) solution [Briefly, cervicovaginal samples (50 μL) will be extracted using a 500 μL methanol. The aqueous supernatant will be vacuum dried and reconstituted using water:acetonitrile , and mean values will be used to summarize and analyze the collected data. Descriptive analysis will be performed by obtaining quantitative variables’ means and standard deviations. Proportions and frequencies will be reported for qualitative variables. The chi-square test will be used to test the categorical variables, and the Kruskal–Wallis test will be employed to compare cohort characteristics based on HPV infection status. One-way analysis of variance with Tukey’s test will be used to assess differences in cohort characteristics based on the natural history of HPV infection, i.e., acquisition, clearance, or persistence of the infection.p < 0.05.The multivariable logistic analysis will be performed after adjusting for age, menopausal status, oral contraceptive use, and smoking habit. A logistic model with a robust variance will be estimated to compare those with HPV persistence to those with HPV clearance and HPV negativity. Hazard ratios (HRs) and 95% confidence intervals (CIs) will be obtained from the models as measures of association. The statistical analyses will be performed with SAS 9.4, and R version 3.3.1 with ggplot2 packages will be used for visualization. The results will be considered statistically significant if This study was approved by the University of the Philippines Manila Research Ethics Board (UPMREB 2021-0645-01) on 25 January 2022. Informed consent will be obtained from the participants invited to participate in the study by one of the investigators or the research assistant. The informed consent form will be in both English and Filipino. If the patient is illiterate, the researcher/person taking consent will read out the informed consent form in the presence of a literate witness. Participation in the study will be voluntary, with the option to opt-out without reason and any penalty or issue. Assurance of privacy and confidentiality and declaration of risks, benefits, compensation, and conflicts of interest will be stated in the informed consent. The study will only proceed after the signing of the certificate of informed consent in the said form by the participants. All procedures performed in this study will follow the ethical standards of the institutional research committee and with the Helsinki Declaration and its later amendments.C. trachomatis, N. gonorrhoeae, M. genitalium, U. parvum., U. urealyticum, T. vaginalis, and bacterial vaginosis) among study participants.The primary outcomes of this study include the prevalence and genotype distribution of HPV infection among women in urban and rural communities in the Philippines. This study will also document the persistence or clearance of HPV infection among HPV-positive study participants and the incidence of HPV acquisition and negativity among initially HPV-negative participants after 6 and 12 months. This study will also determine the prevalence of reproductive tract infections  to immunize individuals to prevent the development of HPV and cervical cancer . CurrentThe lack of updated epidemiological information on HPV infection and cervical cancer in the country emphasizes the need for more research in this field. Despite recent efforts, morbidity and mortality from HPV infection and cervical cancer remain high ,21,24. MThe molecular assessment of the characteristics of HPV genotypes specific to the Philippines remains scarce. There is also limited information on the potential risk factors associated with persistent cervical HPV infection. It may require further validation to help determine the predisposed susceptibility of Filipino women to infection and the possible means of immunization that can be discovered . FurtherOnly about 50–60% of patients diagnosed with cervical cancer receive some form of treatment , which eOther projects are also in place in the country to intensify the fight against HPV and cervical cancer, such as the Scale up Cervical Cancer Elimination with Secondary prevention Strategy (SUCCESS) project, which has been recently launched by the Department of Health in partnership with Jhpiego and funded by Unitaid on 12 April 2021 . The proAmong the current limitations foreseen by the researchers in this project are its selection criteria, which include urban and rural areas in the Philippines. With this, the results of the study may not be generalizable to the whole country and can only account for a representative sample of the total population. Furthermore, the follow-up period of the study is only limited to 12 months.Regardless of the limitations, the DEFEAT HPV study provides a novel perspective on assessing the prevalence of HPV among Filipino women at high risk of contracting HPV and thereby developing cervical cancer. This presents an excellent opportunity not only to improve HPV screening coverage in the country but also to upscale current efforts toward reaching the 2030 target of the World Health Organization to accelerate the elimination of cervical cancer as a public health problem  .In conclusion, the results of this study will be used to update the current prevalence and genotypic distribution of cervical HPV infection in the reproductive-age Filipino women population. With the use of a multi-omics approach, this study will improve our understanding of the epidemiology of cervical HPV infection in the Philippines. This is because multi-omics could provide researchers with a greater understanding of the flow of information regarding HPV, as it assesses the original cause of the disease  to determine its functional consequences and interactions during development. This study can provide new information that can be used to guide public health programs and clinical practice guidelines on cervical HPV infection in the Philippines.


The Research Topic that accompanies this editorial, encompasses 5 articles that mainly focus on sex disparities in head and neck cancer.Preissner et al. report a retrospective analysis of over 140000 cases of head and neck cancers, with a subgroup of 62775 cases of OPC, defined as cancer of the base of the tongue, tonsil and oropharynx. The authors compared the prognosis between cancers of these anatomic locations of men and women with or without HPV infection. They found that female compared to male sex has a greater impact on prognosis of HPV-positive cancers in specific anatomic locations, such as base of the tongue and tonsils. This might be interpreted by a lower consumption of tobacco and alcohol by women, which is an adverse prognostic factor in HPV-positive disease.Human PapillomaVirus (HPV) has been recognized as a significant contributing factor in squamous cell cancers of the head and neck (SCCHN), particularly of the oropharynx. HPV-related oropharyngeal carcinoma (OPC) has emerged as a distinct disease entity with different pathogenesis, molecular profile and prognosis. More specifically, patients with HPV-positive disease tend to be affected at a younger age, present with extensive nodal involvement albeit with smaller tumors and are characterized by improved clinical outcomes . In the Tagliabue et al. sought to evaluate differences in survival and prognosis based on sex/gender in a cohort of patients with oral cancer that were treated in a single center in Italy. The authors collected and analyzed demographic and clinical data including hemoglobin levels and neutrophil to lymphocyte ratio from 576 patients, 230 women and 346 men. Interestingly, men were found to have better clinical outcomes that women in early-stage tumors, and low hemoglobulin rates were an independent prognostic factor for women in early-stage disease. No differences were found in stages III-IV, possibly due to poor survival associated with advanced stage disease.In the same context, squamous cell carcinoma of the oral cavity represents a malignancy more frequently encountered in men that has been traditionally etiologically linked to tobacco and alcohol consumption. Indeed, according to Global Cancer Statistics, men present with an age-standardized rate of 6.0, whereas the corresponding number for women is 2.3  as a short course neoadjuvant treatment in resectable SCCHN. Erlotinib was administered at two different doses depending on smoking status; more specifically, current smokers received erlotinib at an increased dose of 300 mg. In addition, patients were subjected to positron emission tomography/computed tomography (PET/CT) scan at baseline, 4-7 days post initiation of erlotinib and before surgery. Interestingly, among 19 patients who participated, 11 were responders. In addition, the percentage change in early and post-treatment PET/CT scan as compared to pre-treatment CT scan was significantly associated with radiologic response on post-treatment CTs. This study highlights the importance of window-of opportunity studies, the need for further research on targeted treatments in SCCHN and the utility of radiomics as a biomarker in response to treatment.Current studies on SCCHN mainly focus on immunotherapy and combinatorial approaches. However, there is a lack of significant research on targeted therapies. Since the approval of cetuximab as a radiosensitizer in locally advanced SCCHN  and as cCao et al. sought to investigate differences in proteomics in PTMC tumor tissues with or without LNM. In addition, differences of protein profiles based on whether tumor tissues were derived from men or women were evaluated. Using a direct data-independent acquisition (DIA) proteomics approach to detect differentially expressed proteins (DEPs), the authors found that in tumors with LNM, DEPs were primarily enriched in categories associated with mitochondrial dysfunction and showed downregulated expression of specific proteins with well-known associations to tumor metastasis. Moreover, the proteomics profiles of men and women with PTMC and LNM were largely distinct. More specifically, compared to female PTMC patients with LNM, the DEPs in the cancerous tissues of male PTMC patients with LNM were mostly enriched in the categories of extracellular matrix (ECM) structural constituent, basement membrane (BM) and collagen-containing ECM. In addition, using bioinformatics to analyze the DEPs between male LNM and female LNM, it was revealed that male LNM show enrichment in the GR signaling, GP6 signaling, CDK5 signaling, VEGF signaling, and mTOR signaling pathway. Finally, upregulation of two DEPs, MMRN2 and NID2 was found in male LNM tissues compared to female LNM tissues and correlated with poor prognosis.On the other hand, papillary thyroid microcarcinoma (PTMC), defined as a papillary thyroid carcinoma measuring ≤ 10 mm in maximum diameter, is traditionally a tumor with excellent prognosis and indolent behavior. However, several tumor characteristics such as lymph node metastasis (LNM) are considered high-risk and require more aggressive treatment, such as surgery and radioactive iodine therapy. In conclusion, the present Research Topic has included several important articles that add new information to current knowledge regarding differences in biology of tumors and prognosis between men and women with head and neck cancer.PE: Conceptualization, Data curation, Resources, Writing – original draft, Writing – review & editing. IK: Resources, Writing – review & editing. AP: Conceptualization, Supervision, Writing – review & editing.


Firearms have become an increasingly common method of suicide among women Veterans, yet this population has rarely been a focus in firearm suicide prevention research. Limited knowledge is available regarding the preferences, experiences, or needs of women Veterans with respect to firearm lethal means counseling (LMC), an evidence-based suicide prevention strategy. Understanding is necessary to optimize delivery for this population.Our sample included forty women Veterans with lifetime suicidal ideation or suicide attempt(s) and firearm access following military separation, all enrolled in the Veterans Health Administration. Participants were interviewed regarding their perspectives, experiences, and preferences for firearm LMC. Data were analyzed using a mixed inductive-deductive thematic analysis.Women Veterans’ firearm and firearm LMC perspectives were shaped by their military service histories and identity, military sexual trauma, spouses/partners, children, rurality, and experiences with suicidal ideation and attempts. Half reported they had not engaged in firearm LMC previously. For those who had, positive aspects included a trusting, caring relationship, direct communication of rationale for questions, and discussion of exceptions to confidentiality. Negative aspects included conversations that felt impersonal, not sufficiently comprehensive, and Veterans’ fears regarding implications of disclosure, which impeded conversations. Women Veterans’ preferences for future firearm LMC encompassed providers communicating why such conversations are important, how they should be framed , what they should entail , whom should initiate  and where they should occur .This study is the first to examine women Veterans’ experiences with, and preferences for, firearm LMC. Detailed inquiry of the nuances of how, where, why, and by whom firearms are stored and used may help to facilitate firearm LMC with women Veterans. In 2020, the sex- and age-adjusted suicide rate among United States (U.S.) Veterans was 57.3% higher than that of non-Veteran adults within the U.S. . The magAmong women Veterans, use of firearms as a method of suicide increased by 11.2% from 2001 to 2020 –an increase not observed to the same extent for Veteran men (4.8% increase) or non-Veteran women (2.1% decrease)  ; and abln = 3,655). We also sent mailings (n = 220) to women Veterans who previously participated in research with our center who consented to be contacted about future research. Additional recruitment methods included posting information about the study through electronic monitors in VHA facility waiting areas, at community events, informing healthcare providers about the study, and snowball recruitment . We used a purposeful sampling approach to ensure diversity in participants’ demographics , and geographic characteristics . Specifically, we monitored participant characteristics as study enrollment progressed, in order to identify gaps within the sample. Accordingly, later mailings emphasized specific Veteran characteristics  to facilitate recruitment of a diverse sample. Lastly, to ensure that the sample included those whose firearm access was through personal ownership, as well as those who resided in homes with firearm(s) that other household members owned, ownership was queried during screening and considered when scheduling. Forty participants completed qualitative interviews (30 to 90 minutes in duration) between April 2021 and January 2022. Most participants were recruited through CDW  or repository  mailings, and a few through word-of-mouth . Recruitment continued until we reached data saturation .All study procedures were approved by the Colorado Multiple Institutional Review Board (Protocol 19–2055) and the VA Eastern Colorado Healthcare System Research and Development Committee. Informed consent was obtained prior to initiating study procedures, via oral affirmation of consent by trained study staff. Appointments were conducted via phone or Microsoft Teams. After participants provided informed consent to participate, the University of Washington Risk Assessment Protocol (UWRAP ) was admFollowing the interview, an abbreviated version of the SITBI  was admiA bracketing exercise was first conducted among all study staff who participated in coding , 40. FolTo characterize the sample, descriptives are provided. The sample tended to be middle-aged . Additional sample descriptives regarding demographics, military service, and VHA use are provided in Most participants had experienced lifetime trauma and sexual violence , includiThree broad themes were identified: (1) women Veterans’ backgrounds, personal experiences, and beliefs shaped their perspectives on firearms and firearm LMC; (2) previous experiences with LMC were viewed as positive and collaborative or negative and with stigmatizing interactions that resulted in uncertainty and fear; and, (3) participants’ preferences for firearm LMC included what, when, why, how, and with whom to discuss firearms within the context of LMC with healthcare providers. Together, these themes provide a holistic, integrated appraisal of women Veterans’ perspectives, experiences, and preferences regarding firearm LMC.Women Veterans described factors that shaped and influenced their perspectives on firearms and firearm LMC. For many women Veterans in our sample, military service was a formative event in their lives, and their experiences as Service members influenced their current beliefs regarding firearms. One woman explained how her years of military service had oriented her to “be this sort of protective figure with a gun, where you’re willing to give up your life and protect others with this lethal means,” and that “when you’ve been a Soldier, it’s such a part of you, having a gun.” This identity, however, was held in tension: many participants acknowledged that this familiarity with firearms in a protector role could yield destructive results. One participant stated that firearms can be “a threat to yourself and other people, too… it’s a really complex thing.”As noted previously, the majority of participants (85%) screened positive for MST, and many described how their MST experiences had shaped their current use of firearms  and perspectives on firearms and firearm LMC. For example, when asked about her reasons for maintaining access to a firearm, a Veteran who had experienced MST stated, “If I’m clinging to my firearm, that’s my protection, that’s my safety, that’s my safety blanket. I think I’m gonna fear discussing it because you might take it away from me.” In contrast, a few women who had experienced MST described not wanting any access to firearms due to the psychological impact of MST and the resultant suicide risk, although this was not common. Recalling her previous experiences of MST and IPV, one Veteran stated, “It’s been helpful for me not to be around guns…. not owning a gun has saved my life these last 10 years. Because there’s been times where I was so sad that, if I’d had a gun, if I would have been weak for that split second, it would take all my strength to not matter.”Notably, among women who had experienced MST, associated feelings of betrayal and mistrust had a profound impact on their willingness to discuss firearms with their healthcare providers. As one Veteran described, survivors of MST are “automatically going to be more distrustful of the system because they’ve not gotten the proper care they deserve, they been lied to, manipulated, cheated on… one hundred percent they are going to be distrustful of the whole system.” This sense of institutional betrayal led many women in our sample to be distrustful of discussing firearms with VA providers. Drawing parallels between previous feelings of “constantly battling” and feeling as though “her career may be on the line” after seeking care while in the service, one Veteran explained how this manifested as continued apprehensions of disclosing her firearm access to her VA clinician. “I already had my own fears of what was really in my head with the job that I did [in the military]…But now I have the fear of my clinician breaking confidentiality and this becoming bigger than I was prepared for.” One participant who also described being reticent to discuss firearms with her healthcare providers stated that she was distrustful of the military, and by extension the VA, because “they’re not there to serve me, they’re there to serve the government.”“He said they [the firearms] were safe, she [their daughter] couldn’t get up there [and access the firearms], so I should just shut up and leave it alone and quit poking at it.”Women Veterans also described their relationships with their spouse or partner as another factor that influenced their firearm access, including how and where firearms were stored, as well as if firearms were stored locked. In some instances, this was a collaborative process, in which both parties had similar perspectives and beliefs about firearms in the home. For example, one Veteran described a conversation with her partner about her reasons for locking her partner’s firearm: “I explained to her that I am bipolar, that I am impulsive, and I explained my reasons [for locking up her firearm]. And she accepted them. She understands that, in order to be safe in this house, I need to feel safe in this house.” For some women, however, this process was imbalanced, whereby their partner or spouse had exclusive control about firearms in the home. “My ex did everything without consenting me and made all of his decisions independently of what I thought [about the firearms],” one Veteran stated. Another woman who had experienced IPV recalled her husband’s response to her objections regarding how he had been storing his firearms, given that they had a young child in their home: with whom and where they lived, also shaped their perspectives on firearms and firearm LMC. In addition to whether they lived with a partner, many women conveyed how the presence of children  in their home changed how they thought about, stored, and used firearms, as well as their willingness to engage in firearm LMC. In describing the need for secure firearm storage, one woman noted that she had been “adamant about that because we had small children…I just didn’t want any accidents. That was my worst nightmare.” She described allowing her husband to have firearms in the home “as long as you can promise me our children will never shoot each other or shoot themselves by accident…my biggest fear was one of my children getting ahold of a gun.” Consequently, all firearms were locked securely in a safe.Closely related to the aforementioned findings, women Veterans’ living space, including For some women living in rural areas, firearm ownership was culturally normative: “You’re just like everyone else in the rural area–if you live in the woods, you have a firearm…it’s almost automatic where I’m from to have a firearm. And to not have a firearm, you feel almost naked.” Many women Veterans noted that growing up in a “gun culture” or being around others who valued firearms in similar ways also shaped their beliefs regarding firearms. Some women explained that they grew up in “gun totin’ families,” that “you don’t go into my family’s house and ask them if they have a gun–you ask them, how many they got?”Finally, women Veterans described how their personal histories of suicidal thoughts or attempts had shaped their perspectives of firearms and firearm LMC. Due to such experiences, many were cognizant of the danger firearms might pose to themselves and expressed that they would be willing to engage in firearm LMC. For example, one woman had previously believed that restricting her firearm access was “ridiculous.” While hospitalized for a suicide attempt, however, a conversation with a healthcare provider had completely changed her thinking, despite her earlier belief: “It really wasn’t until a healthcare provider at the mental health hospital said, ‘You know what, you really don’t need to have a gun in your house’…I realized–I think that’s correct, and it really changed my thinking.” Some other women Veterans expressed having similar beliefs after a suicidal crisis or suicide attempt. One expressed that she did not trust herself to have a firearm because “it’s too easy. Too spontaneous.” Another explained that not having a firearm accessible is “just logical. If I’m sad enough that I wanna die, and that’s the quickest way to die, why would I keep it near me?”any conversation with a healthcare provider regarding firearms, despite all being in VHA care and having a history of suicidal ideation and/or attempt(s). Women who had experienced such conversations described positive or negative experiences discussing firearms with healthcare providers, detailing factors that had facilitated or hindered previous attempts to engage in firearm LMC. Many of the factors described here, while specifically framed around firearm LMC, are nonetheless largely applicable to general clinical interactions women Veterans have experienced when receiving VHA care. In relaying these experiences, women Veterans revealed that many aspects that constitute a positive or negative firearm LMC experience closely mirror what would make for a positive or negative clinical experience generally. Detailing these past experiences can reveal ways to better engage women Veterans in future firearm LMC discussions.As stated previously, only half of women Veterans in this sample could recall ever having Women Veterans who had participated in LMC and had negative experiences with firearm LMC described aspects that made such experiences negative. For example, many felt it was not comprehensive enough. Many were offput by the impersonal “checklist” approach to these conversations, where providers “ran through the motions,” rapidly working through a standard set of basic questions regarding firearms, such as simple “yes” or “no” questions regarding if they owned a firearm or if it was stored loaded and/or locked. Many women described experiences discussing firearms with their healthcare providers that were not perceived as productive or positive, describing apprehensions and fears about disclosing information about their firearms which then impeded LMC conversations. For example, numerous women described how they had been (and still would be) apprehensive and unwilling to engage with a provider in discussing firearms and their mental health out of fear that it would result in involuntary hospitalization. “You keep those things secret,” one Veteran explained, not wanting to disclose her suicidal ideation and firearm access to any healthcare provider, stating how “even if I had access, I’d tell ‘em I didn’t have access.” “You don’t tell people that or else they put you in a mental hospital. You don’t discuss it because the moment you do, you’re institutionalized.” Many also expressed concerns about having their firearms seized if a provider deemed them to be a threat to themselves or others. One woman explained that she didn’t disclose whether she owned a firearm during a mental health crisis because “the only thing running through your head in a crisis is ‘these people are going to call the police on me, they’re going to send someone to take my guns and take away my rights, they’re gonna put me in the hospital, they’re gonna hold me against my will.’” While many women described concerns regarding the implications of truthfully disclosing their firearm access, which resulted in them not disclosing their firearm status, none described experiencing any adverse actions, such as seizure of firearms, as a result of discussing their firearm access.Hesitations regarding engaging in firearm LMC were borne from feeling that their providers were cold, distant, and impersonal, lacking a personal touch that conveyed that they could be trusted. Many women felt that they were not heard during healthcare visits and that they were treated as “just another number, another science project, a case study… that human factor wasn’t in the room…I’m not gonna sit here and be vulnerable and bleed all over the floor and you just sit there and stare at me.” Another woman expressed that, if she felt unheard, “I’m getting frustrated with them, I’m just going to take the easiest way out. Just tell me what you’ve got to say, and I want to get out of here.” Such feelings further disinclined women Veterans from engaging in firearm LMC. Perhaps more detrimental, many women left encounters that had entailed discussing firearms feeling as though the provider perceived them to be irresponsible or dangerous with their firearms: “They also suggested that [I was irresponsible], asking me why I have it [the firearm] in the first place…I didn’t like it. I didn’t like the fact that they were questioning me about why I have it.”why they were asking firearm questions, and noting exceptions to confidentiality assuaged their fears and promoted trust. “Be upfront and just impartial, like ‘Hey, we have to ask you these questions. We’re disclosing that if you say X, Y, Z, we’re going to have to report it,’ kind of stuff,” one Veteran explained. “Just a nonbiased, nonjudgmental meeting, the facts… As long as [they] knows that they’re not gonna take it away just cuz they’re honest about it.” One woman explained, while “there are some individuals who think you are trying to take their gun away from them,” such conversations are necessary and need to be direct: “‘Do you have access to firearms?’ And just open up that conversation.”Positive LMC experiences were predicated on perceived trust, rapport, mutual understanding, a genuine connection, and the perception that the healthcare provider was discussing firearms with them because they cared about the Veteran, rather than simply as part of routinely “running through the motions.” Providers being attentive, thoughtful, and demonstrating compassion and genuine care for Veterans’ concerns also fostered positive experiences discussing firearms. For example, this could include conversations feeling more like “talking to a trusted friend rather than a clinician,” where a provider “doesn’t just jump right into the conversation…they start simple with ‘how are you feeling today?’” Veterans, particularly those who had experienced MST, also preferred providers who were cognizant of their needs, such as “maintaining some distance, he didn’t make any sudden moves, he respected my bodily autonomy.” Attention to these needs engendered trust and rapport, leading many Veterans to feel more at ease and willing to discuss firearms. Veterans described how providers being upfront and direct about their intentions, communicating clearly Women Veterans described their preferences for how providers should engage Veterans in firearm LMC in the future. As described earlier, many of these preferences reflected their personal backgrounds, previous LMC experiences, and current firearm beliefs. Importantly, echoing earlier sentiments, while many of these preferences were focused on firearm LMC, many also related to other, more general aspects of clinical care. In presenting their firearm LMC preferences, women Veterans again made a link to their preferences for future engagement with VHA broadly. In particular, an overarching notion of feeling welcome and respected within VHA appeared paramount to engagingt in VHA services and firearm LMC specifically. That is, once engaged in VHA services with a trusted provider and rapport present, meaningful conversations surrounding firearm LMC could occur. Understanding these preferences–even those that are more pertinent to general VHA care–is vital, as meeting these preferences can further encourage women Veterans to seek VHA care and thus, subsequently engage in firearm LMC.Why are (or Aren’t) LMC conversations important? Women Veterans provided their thoughts on why (or why not) firearm discussions are important to have. Many participants expressed that firearm access ought to be limited during mental health crises. One explained that “people that are suicidal, they are less likely to act on it if they don’t have the means, the method, or the motivation. So if someone is having a difficult time in their life and they don’t have access to a firearm, that takes away a lethal means.” Limiting access during times of elevated suicide risk was seen as valid and necessary for suicide prevention, albeit complex.Some women Veterans expressed concern that if firearm access is restricted, many will find alternate means instead. A Veteran wondered “are they going to take the kitchen knives too? I mean, there are a lot of different ways. You could just buy a bottle of sleeping pills or something at the store and do it that way.” Some also questioned whether suicide could be prevented outright, arguing that a motivated individual could not be stopped. “None of those things–the lock box, the firearm lock–none of those things will prevent it,” one woman said. “They won’t prevent it. It will just slow it down by a matter of minutes or however long it takes them to remember where they hid the key. But again, if that’s how you’re feeling, there’s nothing that will prevent it.”Furthermore, some Veterans expressed concerns about the political and constitutional implications of firearm LMC, worried that depriving someone of their rights and property required a measured approach. This was truly “a sticky question,” one participant stated, explaining how “I don’t want to ever say if you’re a female Veteran and you’ve identified yourself as a victim of MST or PTSD or anything else, then you can no longer own a gun. That’s not okay with me. I don’t want it to ever be an additional stigma or negative because you sought help and now you can’t protect yourself.” Reiterating this point, another Veteran believed that while “sometimes people reach a certain mental point, they need help to protect them from themselves, but at the same time, I also don’t believe in confiscating people’s firearms either.”How should LMC conversations be discussed and framed? Participants then described how conversations about firearms should begin and how provider framing and tone matter. Women Veterans felt that it was best if providers made firearm conversations routine and normal, calmly and dispassionately asking about firearm access. Participants noted that making these discussions just another part of a broader assessment could normalize them, whether through part of a basic questionnaire or a quick conversation with their providers. Another piece of this normalization comes from framing the conversation around safety in general, not specific to firearms, but encompassing other means and expanding the scope to include the safety of others around the Veteran. A participant stated that providers should “preface it with the concerns of safety. You know, ‘For safety concerns, we like to ask if you have access to any firearms. And it’s not only the safety for you as a Veteran, but the safety of those around you.’” “I think if there was a better way of communicating, to say ‘It’s alright to not be alright,” one Veteran began explaining, “‘We’re gonna get you the resources you need, but in the meantime, we’re going to create a safety plan and that safety plan is gonna be a temporary removal of your access to firearms and your access to other weapons, bows and arrows, knives, whatever.’”Furthermore, participants also noted a need for providers to address IPV when attempting to discern firearms access among women Veterans. They explained a need for providers to be attuned to body language, posture, and other non-verbal clues that may indicate their patient may currently be experiencing IPV. Veterans described how providers ought to “watch their body language and see how they react. If they look down, if they’re visibly tense, if they just shrink back, if they look away from you, if they break eye contact–there may be some issues around guns in the house and they’re afraid to say something.” Discerning these non-verbal cues can be especially vital in instances in which a Veteran is accompanied to clinical visits with a partner, or may not feel empowered to broach the subject themselves. Having these skills would thus enable providers to effectively engage women Veterans in discussing firearms, as well as to understand ways in which firearms may be used to further enact IPV.What should be discussed in LMC conversations? Women Veterans expressed that firearm access should be assessed by not only discerning if the Veteran has access to a firearm, but also their storage practices  and the function of ownership . Women Veterans recommended a series of additional, deeper probes that they felt more comprehensively and holistically appraised firearm access as important to help limit firearm access, when appropriate, and prevent firearm suicide. One woman Veteran suggested that, in addition to generally asking about firearm access, providers ought to ask specifically about the Veteran’s sense of safety being in a home with a firearm. “I think something that’s important to ask is not just ‘Do you have guns?’”, one Veteran explained, advocating for providers to go deeper, recommending that they also ask: “‘Does anyone else in the household have guns? Does your significant other have a gun? Has he or she ever threatened you with a gun or tried to intimidate you?’ Because no one ever asks that, they just ask ‘Do you have access to firearms?’”Women Veterans also expressed that it is important for providers to assuage women Veterans’ fears that disclosing firearm ownership would trigger seizure of their firearms, involuntary institutionalization, or permanently marked VHA medical records. Describing this latter concern, one Veteran was cautious regarding whom she discussed firearms with, indicating that “the only notes that go in my file are those of the [non-VHA] psychiatrist; the VA providers cannot see my notes from the Vet Center. So that provides me with another layer of trust that they’re not [accessible], I can be the conduit of my records, I have control of that piece of it.” Further, many women Veterans described that, in order to discuss firearm access with their healthcare providers, they wanted “guarantees that it would be confidential and that there would be no impact on my future ability to buy firearms, my future ability to use firearms, my career” and “assurances that it wouldn’t affect their future ability to hold a security clearance or work in police work…I think that would be crucial…” Such concerns are consistent with other, similarly charged and potentially sensitive clinical issues, and underscore the need for precise framing and language on the part of clinicians to educate and assuage these fears.Who should initiate LMC conversations? In terms of who should initiate LMC conversations in clinical settings, women Veterans placed great emphasis on this being any healthcare provider with whom they had a trusting, longstanding relationship. “I would only have concerns if I didn’t trust the person,” one Veteran explained when asked what would make her more willing to talk to a healthcare provider about her firearms. “You can tell if somebody really cares about you or not, or if they’re just asking questions because they have to be there.” Further–and perhaps related–women Veterans also felt that healthcare providers who understood military culture, terminology, hierarchy, and had military experience were better equipped to understand the needs of Veterans and more able to discuss firearms. Explaining this need for shared language and understanding, one participant conveyed the following about working alongside other women Veterans in group therapy, “[women Veterans] weren’t picky about whether or not it was a man or a woman. They were picky about the fact that the people they were talking to didn’t have a background in the society, the military society, the chain of command, what you learn in the military and the state of mind.” She continued to emphasize how not having this competency can hinder engaging with Veterans because if “they don’t know the terminology and then they get thrown in a group and somebody is using terminology they don’t understand, and they ask what it is, and that will shut the group down in a heartbeat.”However, some women Veterans expressed a preference to engage in LMC with women providers. For example, when asked who she would feel more comfortable discussing firearms with, a Veteran stated: “I would probably prefer a psychologist–specifically a female versus a male.” Similarly, another woman expressed how, given the impact of prior trauma on her decisions to own and increase access to firearms, “Having a female therapist, especially when it comes to any kind of sexual assault or abuse, it’s just easier to talk to a woman than a man because typically, it’s the man who is doing the abusing.”Where should LMC conversations occur? While some Veterans suggested that trusting, caring, and respectful relationships can occur anywhere, many women Veterans were quick to express some hesitations about discussing firearms with providers in VA settings. Some, particularly those who had experienced MST, expressed that the physical space within VA clinics made them less likely to be engaged in VA healthcare: “I experienced military sexual trauma, so it was hard being in the VA, being in a setting that [was] primarily male and knowing that one in three women, and actually, it’s more like one in two women have experienced sexual harassment or even sexual assault. And sitting next to a man and wondering, well, are you someone that raped a woman or beat her up or made disparaging comments? I still wonder if the guy I’m sitting next to or standing next to in line has done that.” One participant shared her anxieties about VA facilities, noting the lack of “privacy and atmosphere, and if you’re walking into a sterile environment, it just feels very clinical, people are psychoanalyzing you. Are they really interested in helping you, or are you just a lab experiment?”Additionally, many participants described being dismayed regarding their perception of the lack of women-specific groups, spaces, or specialty care available, and accordingly, appeared to be disinclined from engaging in LMC in these settings. One Veteran conveyed her frustration and discomfort with mixed-gender support groups: “For a long time, I didn’t want to go to groups with other male Veterans because my story and my stress was so different.” Women described being more willing to engage in firearm discussions in groups comprised solely of other women Veterans because “there’s a shared experience, and it’s sort of like a sisterhood…if one of your sisters is in danger, you want to be there for her and prevent her from doing something that you can’t undo.” Similarly, another stated that “instead of giving me the locks or offering the firearm locks, get me into a group with other women Veterans.” A third woman echoed this, noting how such groups “help as far as talking about firearms because I don’t know what other women do…I think having support, like a peer support is just as important as having the mental health therapist.” Thus, the ability to engage in firearm LMC with fellow women Veterans, in a setting that felt safe and was specific to women Veterans, was a preference of some women Veterans for engaging in LMC in VA healthcare settings.When should LMC conversations occur? Regarding when LMC discussions should occur, women Veterans again stressed the need for them to feel natural and routine, not something for a provider to “make a big deal out of.” Veterans were generally agreeable to receiving questions about firearm access when posed alongside assessing for suicide risk or in the context of general mental healthcare. Importantly, this does come into tension with a concern raised earlier by women Veterans: while it is necessary for LMC discussions to feel routine and normal, such conversations need to feel compassionate and borne from genuinely caring about the individual and her well-being. If suicide prevention conversations about firearms become too common, they can lose their impact and again feel like a “checklist” approach that many felt lacked precision and care. One woman Veteran described how this occurs, stating that “I know you guys are trying to help, but it’s become a joke among Veterans that if you go to the VA, they’re going to ask you if you’re suicidal regardless of what you’re there for.”A summary of findings , derived from participants’ statements, as well as our suggestions regarding clinical implications of these findings, can be found in Firearm-related suicide among women Veterans is a profound public health concern. Optimizing firearm LMC for women Veterans requires understanding and consideration of gender-specific needs, experiences, and preferences. With that in mind, we conducted the largest qualitative study of women Veterans to date to explore how understanding such factors can facilitate gender-sensitive firearm LMC with this population. Our findings underscore the unique backgrounds and lived experiences of women Veterans that influence their firearm and firearm LMC perspectives and preferences. Women Veterans described their experiences discussing firearms with healthcare providers, detailed preferences for how firearm discussions ought–and ought not–be broached, and revealed ways in which firearm LMC can be optimized for at-risk women Veterans. Curating effective and satisfactory LMC strategies requires attention to women Veterans’ individual histories, previous experiences with firearm LMC, perceptions of VHA facilities , and stated preferences.Our findings suggest that, to optimize firearm LMC discussions with women Veterans, providers should work with their women Veteran patients to explore their histories, with a focus on experiences that may impact Veterans’ perspectives of firearms and willingness to engage in firearm conversations. Injuries and traumas sustained during service, including those related to MST as well as IPV, comprised some of the experiences that impacted women Veterans’ perspectives on firearms and firearm LMC. For example, woman Veterans who had experienced MST tended to have firearms for personal protection, tended to prefer women providers for firearm LMC, and described betrayal and mistrust as MST-related concerns that had deterred them from discussing firearms with healthcare providers. Acknowledging these prior experiences  and their potential impact on women Veterans’ willingness and comfort with discussing firearm access and implementing different means of firearm storage when their suicide risk is elevated may enable providers to better understand women Veterans’ reasons for having firearms or storing them unsafely, and accordingly, may facilitate tailoring firearm LMC within a trauma-informed framework. While women Veterans are routinely screened for both MST and IPV within VHA, such experiences are vastly underreported –47. Thusif a Veteran owns a firearm, but the firearm’s use, meaning, purpose, and function, providers can gain insights into what storage recommendations or changes may be best for the Veteran, as well as potential barriers and facilitators to doing so. Willingness to adopt certain storage methods may be different for a woman Veteran whose firearm ownership serves as an important reminder of her military service, compared to a woman Veteran who maintains access to unlocked, loaded firearms as a means of protection in the context of prior IPV or MST. Providers can work with women Veterans to consider different in-home or out-of-home storage options that work best for their own personal circumstances  and preferences. To improve Veterans’ willingness to disclose their firearm access, providers can also re-frame these discussions by carefully explaining their rationale for asking such questions and describing how such information will be used at the beginning of such conversations.Furthermore, recognizing the influence of women Veterans’ cultural backgrounds and upbringings is relevant to firearm LMC. By assessing not just We identified an important tension in themes that is relevant for implementing firearm LMC interventions with women Veterans. While some women Veterans indicated that making LMC discussions routine, even through standard questionnaires, might serve to normalize conversations and make them more acceptable, others preferred that such discussions be delivered in a personalized fashion, rather than through a “checklist” approach, or, as one perceived, as “running through the motions.” Women Veterans also desired that LMC conversations be initiated by a clinician who is trusted and within the context of a long-standing relationship. This is consistent with prior research in which Veterans have underscored the import of trust to firearm conversations , as wellwho owns the firearm, if the Veteran feels safe in the home with this person and the firearm, if they have any say or agency in how the firearm is stored and used, and if they have been threatened or intimidated with it. Such questions are especially pertinent to women Veterans given knowledge of the high proportion of women Veterans who live in households with a firearm that is not their own  [Only half of women Veterans in our sample indicated that they had engaged in a firearm LMC discussion with a healthcare provider, suggesting a need for increased firearm LMC. Moreover, our findings suggest that the purview of firearm LMC questions with women Veterans needs to be enhanced. Instead of solely asking standard firearm LMC questions, providers could additionally ask more broadly rtner’s) , 55. Doirtner’s) . Furtherrtner’s) , which mAdditional training and education for healthcare providers can facilitate this broader line of inquiry, as well as continued (or increased) use of firearm LMC with women Veterans. Such training is needed given what is known about healthcare providers’ lack of knowledge and familiarity with firearms and storage devices , 58, 59,Lastly, when describing their preferences for firearm LMC, women Veterans discussed some concerns about firearm LMC occurring in VHA facilities, due to perceived lack of women-specific spaces and concerns about sexual harassment. They also described a desire for more women-specific support groups and programming. These concerns and preferences have been noted in previous studies of women Veterans , 69–71; There are important limitations to note. First, although qualitative studies generally are not intended to be generalizable , given oPreventing firearm suicide remains paramount, and continued firearm LMC represents an important effort to prevent suicide. Optimizing this practice requires a gender-sensitive approach that includes broader, comprehensive assessment of women Veterans’ reasons for owning, using, and storing firearms. Understanding the unique backgrounds, personal preferences, and past experiences of women Veterans, including with respect to trauma and interpersonal violence, can enable healthcare providers to work collaboratively with their women Veteran patients to facilitate firearm LMS during times of elevated suicide risk; thereby reducing the potential for firearm injuries and deaths in this population.S1 Appendix(DOCX)Click here for additional data file.