E6/E7 was expressed in the placentae from pregnancies with PET, other pregnancy complications  and diabetes mellitus), and uncomplicated pregnancies. Placental tissues collected from two geographical locations were subjected to RNAscope analyses of high- and low- risk E6/E7, and individual HPV types identified using an HPV array. High-risk E6/E7 expression was increased in both PET cohorts, (81% and 86% of patients positive for high-risk HPV DNA compared to 13% of control patients). Various HPV types were identified. Although HPV 18 was the most frequent in all cohorts, the majority of individuals had multiple HPV types (55% of the PET compared to 25% of the control cohort). Further evidence that E6 and E7 is present early when placental pathology underlying preeclampsia is established, is provided with the finding of high-risk E6/E7 in the first-trimester placenta anchoring trophoblast. In conclusion, E6/E7 expression and multiple HPV types were frequent in placentae from preeclampsia-complicated pregnancies. Whether HPV is causative of pregnancy complications is uncertain. E6 and E7 affect functions underling preeclampsia (PET) in cultured trophoblasts, but whether E6 and E7 is produced in the placenta is uncertain. Here, we investigated whether  Human papillomavirus (HPV) is a prevalent infection amongst women of reproductive age . During E6/E7 expression. One investigation of HPV 16-positive placentae found no evidence that E6/E7 was expressed [E6/E7 was expressed in placental trophoblasts using assays capable of detecting many HPV types in placentae from PET-associated pregnancies and first-trimester pregnancies to determine whether HPV gene expression was active in the placenta.For HPV to have a causative effect on placental cell function, HPV gene expression is required. Despite findings showing that E6 and E7 of high-risk HPV can affect key trophoblast functions in vitro, there is a lack of evidence of placental xpressed . Understxpressed . Here, wE6/E7 from high-risk HPV was expressed in HPV DNA-positive placentae, and whether E6/E7 was more frequent in PET complicated pregnancies was investigated using RNAscope in five cohorts of placentae from New Zealand and two cohorts from Japan  contained cases selected based on a positive high-risk HPV DNA result from in situ hybridization, and included placentae with FGR or diabetes (both complications had a higher frequency of high-risk HPV DNA from a previous study), and a fourth cohort without any complications (high-risk HPV control) [E6/E7 in placentae from a different geographical location and included a cohort of placentae complicated with hypertensive disorders in pregnancy (HDP) and a control cohort without complications. The HPV status of both Japanese cohorts was determined as part of this study. Whether om Japan . Since hE6/E7 expression, sections from all placentae were tested for RNA quality using UBC as a positive control via RNAscope assay. This was performed as RNA quality can be compromised in the placenta due to difficulties in standardizing sampling and gene expression differences are affected by the delivery method and term of labor [E6/E7 expression . In the FGR and diabetic cohorts, E6/E7 expression did not significantly differ from that of the HPV-positive control cohort. In the Japanese HDP cohort 6 out of 7 cases (86%) with high-risk HPV DNA had high-risk E6/E7 expression .pression . None ofE6/E7 expression in the PET cohort, an analysis of cells positive for HPV (HPV L1 protein) and p53 found reduced p53 staining in HPV-positive cells in the decidua basalis of the PET cohort compared to the HPV-positive control cohort .An analysis of low-risk ses 33%, . Two HDPE6/E7 was also expressed in placentae from PET-complicated pregnancies. In summary, low-risk E6/E7 expression in the PET placentae was due to the presence of a different HPV type, individual HPV types were typed using a PCR and hybridization-based array method capable of detecting 30 HPV types . Individual HPV types were tested in the New Zealand cohorts used in the E6/E7 analyses. The HPV typing array was successful in identifying the HPV type in most high-risk HPV DNA-positive cases identified by in situ hybridization .HPV type 18 was the most frequently found type in the PET cohort . The presence of HPV 18 in the placenta was confirmed in five cases using an HPV 18-specific probe . All casE6/E7 expression in the PET cohort was not due to the presence of a different HPV type. However, this study suggests the presence of multiple HPV types may be relevant to PET. Ten cases determined as HPV negative by in situ hybridization were included as controls; the more expansive HPV array typing method confirmed that all HPV-negative cases were negative . There wE6/E7 were also expressed in early gestation when the foundations for PET are established, it would suggest an effect of E6/E7 on PET. To determine if E6/E7 was expressed in first-trimester placentae, 32 cases  had sufficient decidua basalis and chorionic villus tissue and good or moderate RNA quality necessary to assess high-risk E6/E7 expression. The same cases were also typed for the presence of high-risk HPV DNA using in situ hybridization. Consequently, 46% of cases were positive for high-risk HPV DNA; fourteen cases displayed HPV DNA in the decidua basalis tissue in both the endometrial epithelium and endometrial stromal cells/extravillous trophoblast, and seven cases were positive in the chorionic villi  (E6/E7 staining was observed in 57% of cases with HPV DNA (n = 8/14). Minimal E6/E7 was present in each cell; however, multiple cells were positive  . In endopositive . In chorphoblast . E6/E7 expression. Cells with the most intense staining were identified as anchoring or extravillous trophoblasts based on the observation that areas with E6/E7-positive cells were also positive for Ki67 and cytokeratin, indicating proliferating cells and epithelial cells, respectively  showed positive ectively . In threE6/E7 in villous trophoblasts were separately pretreated with DNase I and RNase A before repeating the E6/E7 RNAscope assay. E6/E7 was present following pretreatment with DNase I, but not with RNase A, in all three cases  of those positive for high-risk E6/E7. E4 was present in the decidua basalis and no evidence of staining in the chorionic villus was found.The three cases exhibiting high-risk positive . To testThese results suggest that high-risk HPV genes associated with altered trophoblast function in vitro are expressed in the first-trimester placenta.E6/E7 RNA in PET-associated placentae is uncertain, but it is likely not due to the presence of different HPV types compared to placentae from uncomplicated pregnancies. The role of HPV as a pathogen likely extends beyond cancer promotion, as a growing number of studies have found associations between HPV and increased risks of pregnancy complications and cardiovascular diseases ,30,31. TE6/E7 was frequently expressed in HPV DNA-positive placentae from two cohorts of PET-complicated pregnancies, but it was rare in the placentae from uncomplicated pregnancies, including those with high-risk HPV DNA. This suggests that in PET cases, HPV oncoproteins affecting cell function likely occur in the placenta if high-risk HPV DNA is present [In the current study,  present ,32. In t present ,33,34,35 present . et al. (2014), cervical HPV during early pregnancy posed a two-fold increased risk of preeclampsia [E6/E7 expression in the anchoring trophoblast is consistent with E6/E7 being active early in placentation, when HPV would likely have the strongest effects on PET based on the in vitro effects of E6/E7 in trophoblasts.Few studies have investigated HPV DNA in the placenta and preeclampsia. Instead, most studies investigated the whole of cervical HPV in preeclampsia and reported varying associations that may relate to HPV prevalence in the study population ,36. In tclampsia . The finE6/E7 is expressed. In addition to decreased p53 and RB function, E6 and E7 degrade and interact with many other proteins, including those with roles in inflammation, and both proteins are required for the long-term episomal maintenance of HPV [Other consequences regarding placental function could occur when e of HPV ,38,39. E6/E7 had limited E6/E7 expression overall, which was much lower than that found with integrated HPV, as illustrated in HeLa cells in the current study, or than the amount likely achieved in cultured trophoblasts transfected with E6 and E7 constructs. Episomal HPV is capable of affecting cell function, as evidenced by the existence of invasive cervical cancers with only an episomal virus, but these cancers were associated with high E6 expression and a high viral load [E6/E7 is sufficient to affect overall placental function. If increased HPV activity resulted in increased placental cell apoptosis as found in vitro, cells with the highest E6/E7 expression may not be represented in the in situ analysis, therefore limiting the use of in situ analyses to predict the consequences of HPV gene expression [Establishing that high-risk E6 and E7 in the placenta are causative of PET requires more investigation. High-risk E6 and E7 are found in women with normal cervical cytology, suggesting that HPV gene expression is not always associated with pathology ,41. Mostral load . In thispression . HPV is pression . EstabliE6/E7 expression was not found in placentae using quantitative PCR in another study [E6/E7 is not always expressed in HPV-positive cases and may be more likely in PET compared to other pregnancy complications with HPV-positive placentae, which adds further complexities to deciphering how placental HPV E6/E7 expression is governed. High-risk E6/E7 was not significantly increased in placentae from FGR- and diabetes-complicated pregnancies; however, a considerable number of FGR placentae displayed E6/E7 expression, and an association between the two would be better established in a larger cohort of placentae.HPV 16 er study . The resE6/E7 was present in over 20% of PET placentae from two geographical locations. In the Japanese cohort, low-risk HPV E6/E7 was significantly increased compared to the control cohort. Whether low-risk HPV contributes to pregnancy complications in the absence of high-risk HPV is uncertain. Low-risk HPV types are not carcinogenic and instead cause benign proliferative lesions; thus, they are not considered a serious health concern. If high-risk HPV proves to be causative of PET, determining whether low-risk HPV is also of “low risk” for PET may be important. The function of low-risk E6 and E7 is less understood. Roles in maintaining viral episomes have been proposed [+/H+ exchanger regulatory factor 1 protein, resulting in activation of the Wnt signaling pathway [Low-risk proposed , and rec pathway .HPV 18 was the most frequent type in the PET, control, FGR and diabetic groups. Although there were no differences regarding individual HPV types in the PET cohort many PET and FGR cases contained multiple HPV types. The prevalence of multiple HPV types varies between populations. In cervical screening studies estimates of 4.6% and 43.2% of HPV positive women have multiple HPV types present ,47. WomeWhether HPV vaccination will reduce PET incidence is yet undetermined. Currently, the Gardasil 9 vaccine , the FDA-approved HPV vaccine effective against the most types, would cover the HPV types most frequently found in this study; however fourteen cases had an HPV type, including three cases with high-risk types, that would not be vaccinated against by Gardasil 9. Pregnancies complicated by PET are associated with a predisposition to cardiovascular disease for both the mother and child. Therefore, understanding the underlying mechanism and role of HPV may provide novel insights for preventing cardiovascular disease ,49.ubiquitin C , and FGR was defined as weight ≤5th centile for gestational age, as determined by the GROW-centile calculator with permission from the Perinatal Institute . For the cohort from Japan, growth estimates were calculated according to the standard gestational age-specific birth weight set by the Japan Pediatric Society, and preeclampsia was defined according to the guidelines of the Japanese Society of Hypertension [Placental samples were collected from New Zealand and Japan. For the New Zealand samples, placentae were selected from those collected during the Otago Placenta Study (OPuS) based at Dunedin Hospital, including previously studied cases and newer cases (collected since 2014) . The cohn C UBC; . For thertension .Chlamydia trachomatis or Neisseria gonorrhoeae DNA; vaginal swab culture for bacterial vaginosis, fungi, or Trichomonas vaginalis; and maternal serology . Cases with any doubt or suspicion of an organism other than HPV were excluded from the study. Clinical data were available concerning smoking status, medical history, maternal age, maternal BMI, and gestation. Ethnicity data was available for the New Zealand cohort . For New Zealand and Japanese participants: none of the women had been vaccinated against HPV. Cases without other infections were selected by clinical investigations during pregnancy; cervical swabs for E6/E7 was expressed in early pregnancy, a cohort of 40 first-semester placentae were obtained from elective terminations (<12 weeks’ gestation) collected from 2015 to 2017 in New Zealand on a convenience basis. Thirty-two cases contained decidua basalis and chorionic villus tissue and had sufficient RNA quality, as determined using RNAscope for UBC (>6 dots per cell). To determine whether HPV Ethical approval was obtained from the New Zealand Health and Disabilities Lower Regional South and Multiregional Ethics Committees, and all participants provided signed informed consent. In Japan, ethical consent was obtained from the ethics committee of Tokyo Metropolitan Tama Medical Center.The presence of high-risk HPV DNA was determined either in a previous study  using a To confirm that some placentae had HPV 18, an HPV 18-specific probe  was used on selected cases identified as likely positive for HPV 18 based on HPV array analysis. Cell clots containing HPV18-positive HeLa cells were used as positive tissues. For in situ hybridization, tissues were treated with Carezyme III Protease  for 10 min at room temperature (20–26 °C) and heated in Tris-EDTA (pH 9) buffer at 95 °C for 20 min. Then, the probe was applied and denaturation occurred at 95 °C, followed by hybridization for 20 h at 40 °C. After hybridization, slides were placed in SSC wash buffer  at 40 °C for 5–10 min, and the probe was detected after applying anti-digoxigenin antibody , EnVision Dual Link , DAB , and DAB enhancer . Slides were counter-stained, dehydrated, and mounted following a standard procedure. Positive staining examples are shown in Individual HPV types in placental tissues collected in New Zealand were identified using the EUROArray HPV Test . DNA was extracted from frozen tissue using the QIAamp DNA Mini Kit  according to the manufacturer’s protocol, which was modified by adding poly-A carrier RNA  to improve DNA extraction. HPV typing was conducted following the manufacturer’s protocol with minor modifications: the hybridization step was increased from 40 min to 90 min, and in some cases, more than the recommended DNA quantity was added (>100 ng). UBC, Negative Control Probe DapB towards the bacterial gene DapB, HPV-HR18 probe towards E6/E7 of 18 high-risk HPV types , and LR-HPV-HPVE6/E7 probe towards 10 low- or unclassified HPV types  were applied to placental tissue slides and FFPE HeLa cell slides as a control for high-risk E6/E7 expression . Slides were counter-stained, dehydrated, and mounted following a standard procedure.The RNAscope 2.5HD Assay-Brown manual assay protocol  was carried out on 5 µm FFPE sections using a protocol modified according to the manufacturer’s recommendations for placenta (the protease plus digestion step was decreased from 30 to 25 min). The Positive Control Probe Hs-UBC towards pression . The firE6/E7 assay were pretreated with RNase A  for 30 min at 40 °C and DNase I  separately for 30 min at 40 °C, and the E6/E7 RNAscope assay was repeated.Since the E6/E7 RNAscope assay could detect single-stranded DNA, three first-trimester placenta tissue sections that were positive for the Stained slides were scanned into the digital pathology scanners of the Aperio Scanscope CS or the Aperio VERSA System  at up to 630× magnification under oil. The entire tissue section was viewed to identify cells positive for each component. For the HPV DNA analysis, cases were classified as negative if <6 positive cells were present throughout the tissue section. For HPV RNAscope analyses, placentae were examined in different tissue compartments . Third-trimester placenta cohorts: E6/E7-positive staining was scored based on positive stains in the decidua basalis and/or chorionic villus, the number of positive high-power fields  in the chorionic villus, and the number of positive dots within chorionic villi cells. Stains were scored as 0 , 1 , 2 , or 3 .First-trimester placenta cohort: E6/E7 staining was scored as 0–3 in different cell types based on the positive staining intensity and positive cell number, which highlighted cell types with more E6/E7, as follows: 0, E6/E7 negative; 1, positive dots in at least six cells or intensely positive dots in <3% of cells overall; 2, intensely positive dots in >3% and ≤20% of cells; and 3, moderately positive dots in >20% of cells.DapB-stained slides to exclude non-specific staining.Equivalent areas with positive staining on HPV-stained sections were checked on the Immunohistochemistry for cytokeratin 7  and Ki67  was conducted according to an automated staining method using the Leica Bond Polymer Refine Detection System and the Leica Bond RX Research Autostainer . A double-staining method for p53  and HPV L1 1 HPV-viral capsid protein  was performed according to an automated staining method with the same Leica system. HPV L1 and p53 were detected using the BOND polymer refine red detection kit and the BOND polymer refine detection (DAB) platform  through a sequential staining method.Stained slides were scanned into the Aperio Scanscope CS digital pathology system  at up to 400× magnification. Slides were investigated manually to identify positive cells.2 tests and t-tests were used to compare differences between categorical measures and continuous measures, respectively. Statistical significance was indicated by a two-tailed p-value < 0.05. The Bonferroni correction used to correct for multiple comparisons. χE6/E7 is expressed in the PET placenta. This result, combined with the observed E6/E7 expression in the first-trimester trophoblast, suggests that HPV is active early in placental development when placental origins of preeclampsia are established. Further investigations are required to establish whether the E6/E7 quantity expressed is sufficient to affect placental function and if HPV is causative towards pregnancy complications. This study provides evidence that HPV


It can play a role in cervical and extra-cervical cancers. Penile cancer is rare, even if an increasing trend was recently reported. Aim of the present study was to assess the prevalence and distribution of HPV genotypes in cases of penile cancer diagnosed in Sardinia, Italy. Surrogate markers of HPV infection  expression.HPV prevalence was 28.1% (9/32); HPV-16 was the most prevalent genotype . p16INK4a positivity was found in 66.7% of the samples with a statistically significant difference between HPV-positive and -negative groups. E6-transcript was detected in 71% of the HPV-16 positive samples. The overall survival was not statistically different between HPV-positives and -negatives.The present study confirms the etiologic role of HPV in penile cancer and supports the adoption of vaccination strategies in men and women. Further studies should clarify the diagnostic and prognostic role of E6 and p16 proteins.HPV infection can favor the occurrence of penile cancer, whose diagnosis and prognosis could be improved with the implementation of validated molecular techniques. It has been estimated that > 90%, > 50%, 77%, and 25% of anal, penile, vaginal, and vulvar cancers are attributable to HPV, respectively [Human Papillomavirus (HPV) is one of the leading causes of cancer, with ~ 690,000 new annual cases worldwide. HPV vaccination and PAP-testing  specimens: they underwent histopathology and p16 immunohistochemistry. In total, 10 consecutive sections of 3 µm were cut from FFPE blocks. Hematoxylin and eosin stained sections were analyzed by two pathologists to confirm diagnosis and tumor staging, following the recommendations of the classification system of the American Joint Committee on Cancer (AJCCC) . ImmunohMolecular analyses were carried out as reported in another publication . After dHPV-16 positive samples were tested for E6-mRNA by reverse transcriptase polymerase chain reaction (RT-PCR), using a set of primers designed by Sotlar et al.  for the An ad hoc electronic form was prepared to collect demographic, clinical, and epidemiological variables. The following variables were retrieved from medical files: age, year of diagnosis, comorbidities, histopathological classification, HPV-DNA positivity, HPV genotype, p16INK4a and E6 proteins expression. Qualitative variables were summarized with absolute and relative (percentages) frequencies, whereas quantitative variables were summarized with means . Student t test was computed to detect statistical differences for quantitative variables following abnormal distribution. Chi-squared and Fisher exact test were computed to assess differences for qualitative variables. Kaplan–Meier survival analysis was performed to assess the mortality in different subgroups. A two-tailed p-value less than 0.05 was considered statistically significant. All statistical analyses were performed with the statistical software STATA version 16 .Thirty-two cases of penile cancer were diagnosed between 2002 and 2019. The mean (SD) age was 68 (11.6) years (Table HPV infection was diagnosed in 28.1% (9/32) samples. HPV-16 was the most frequent genotype , followed by HPV-56 , and HPV-35 . Only one patient had multiple infections, caused by the genotypes HPV-16 and -35. No statistically significant differences were found for HPV infection and genotype distribution according to age and year of diagnosis.p value: 0.01) ; 85.7% (6/7) of them were HPV-DNA positive (E6 transcript was found in 71.4% (5/7) of HPV-16 positive samples Table .Table 2C43.8% 14/32) of patients died, with only 21.4% (3/14) HPV-infected. However, no statistically significant differences were observed for the outcome mortality between HPV-positives and -negatives   cases . The most prevalent comorbidities were inguinal lymph node metastasis , colic adenoma , pulmonary metastasis , and squamous cell carcinoma . Three samples  were HPV-16 and E6 transcript positive in two patients  who showed HPV-positive penile samples.A total of fourteen patients showed comorbidities  [A recent systematic review carried out by Olesen et al. recruiting 4000 patients with penile cancer  reported(HGSILs) . These eThe p16 immunohistochemistryshowed a positivity of 22% with a statistically significant difference between HPV-positive and -negative cases: this finding could suggest its potential adoption for the diagnosis of HPV infection in patients with penile cancer [No statistically significant differences were found between HPV-positive and -negative groups when we compared epidemiological and clinical variables. The poor sample size could not potentially help assess differences which occur in the population. However, our data confirmed that the SCC is the most frequent histological subtype, mainly in persons aged ≥ 55 years .It is scientifically unclear a better prognosis has been found in HPV-positive cases . HoweverIt is confirmed a high risk of secondary lesions, mainly inguinal and pelvic lymphnode metastasis, whereas only in 1–10% of the cases other anatomic sites are affected . A strikAlthough our study has described for the first time the etiologic role of HPV in penile cancer in an Italian region, some limitations should be highlighted. First, the small sample size could be associated with the absence of statistically significant differences and associations between the collected epidemiological variables and HPV infection. Moreover, missing demographic and clinical data linked to the retrospective nature of the study did not provide a comprehensive picture. The lower prevalence of E6 transcript-positive cases, if compared with HPV-DNA positivity, could be partially explain by the RNA degradation often reported in FFPE samples; fresh samples can be used in future studies to assess the diagnostic and prognostic role of those molecular markers in HPV-related medical conditions. Furthermore, we could not assess the effectiveness of therapies prescribed the attending physicians.i.e., HPV vaccination) in men and women could rapidly decrease the national burden of HPV-related diseases. Moreover, further studies are needed to assess the role of HPV infection in secondary malignancies and the potential therapeutic role of HPV vaccines in the reduction of the risk of recurrence. Larger multicenter studies and standardization of molecular methods could be useful to validate the diagnostic and prognostic process of biomarkers in cases of penile cancers.Our study, the first of its kind in the Sardinian region, confirms the role played by HPV in the etiology of penile cancer. Moreover, the diagnostic and prognostic role of E6 and p16 proteins should be further investigated in larger studies. The implementation and scale-up of primary preventive strategies (


Women living with human immunodeficiency virus (WLWH), especially those living in low- and middle-income countries (LMIC), are at increased risk of cervical cancer. The optimal cervical-cancer screening strategy for WLWH has not been determined. We therefore conducted a pilot study of screening methods in WLWH living in Limbe, Cameroon.Five-hundred sixty-six WLWH, aged 25–59 years, were enrolled. After self-collecting a cervicovaginal specimen, they underwent a pelvic exam, during which a provider also collected a cervical specimen and visual inspection after acetic acid (VIA) was performed. Both self- and provider-collected specimens were tested for high-risk HPV by the Xpert HPV Test , with the residual of the latter used for liquid-based cytology. Women testing HPV positive on either specimen and/or VIA positive were referred to colposcopy and biopsies. However, because of poor attendence for follow-up colposcopy for the screen positives due to civil strife and technical issues with biopsies, high-grade cytology and/or clinical diagnosis of cancer was used as the primary high-grade cervical abnormality endpoint. Clinical performances for high-grade cervical abnormality of HPV testing and VIA for screening WLWH, and the most carcinogenic HPV genotypes and/or VIA to triage high-risk HPV-positive WLWH, were evaluated.p < 0.01 for both comparisons) for identifying women with high-grade cervical abnormalities. HPV testing on provider- and self-collected specimens was less specific than VIA  for identifying women with high-grade cervical abnormalities; HPV testing on provider-collected specimens was more specific than on self-collected specimens (p < 0.01). Among HPV-positive women, HPV16/18/45 detection or VIA positivity had a sensitivity and positive predictive value of 73.5 and 29.0%, respectively, for provider-collected specimens and 68.8 and 22.9%, respectively, for self-collected specimens for high-grade cervical abnormalities.Four-hundred eighty-seven (86.0%) WLWH had results for HPV testing on both specimen, VIA, and cytology and were included in the analysis. Forty-nine (10.1%) had a high-grade cervical abnormality. HPV testing on provider- and self-collected specimens was more sensitive than VIA ; retrospectively registered on May 26, 2020 Women living with human immunodeficiency virus (HIV) (WLWH) are at a significantly higher risk of cervical cancer than women who are not infected by HIV –4. ProphCameroon is a high cervical-cancer burden country in Central sub-Saharan Africa (SSA). Cameroon has age-standardized cervical cancer incidence rate of 31.3 per 100,000 (28th in the world) and mortality rate of 21.9 per 100,000 (26th in the world) . High raRecent studies –12 compaWe enrolled a convenience, consecutive sample of 878 consenting women, aged 25–59 years, receiving health services at the Limbe Regional Hospital in Limbe, Cameroon from May 01, 2017 to April 26, 2018. Inclusion criteria were aged 25–59 years, confirmed to be either a WLWH or HIV [−] woman, never undergone cervical cancer screening, no history of invasive cervical cancer, and willing, and able to competently understand and provide written, informed paper-based consent to participate. Exclusion criteria were those who did not meet the inclusion criteria or were pregnant, had signs of abnormalities or non-menstrual bleeding suggestive of invasive cervical cancer, undergone hysterectomy, and/or, based on the judgment of the clinicians, were not sufficiently healthy to participate in a research study. Women were recruited according to HIV status. Of the 878 women enrolled in the study, 566 were WLWH attending HIV Treatment Center. Inclusion and exclusion criteria were previously described . InstituEnrolled women were escorted to a private room and provided a device  for self-collection and instructions on how to self-collect their own sample, which was then placed into ThinPrep® medium . Women then underwent a pelvic exam, at which time they had a specimen collected using Cervex Combi Brush (Rovers) into PreservCyt for ThinPrep liquid-based cytology (LBC) and molecular testing. Finally, VIA was performed by a physician trained for this purpose.The Xpert HPV Test  on the GeneXpert platform was used to test 1-mL aliquots of the self-collected and provider-collected specimens for high-risk HPV per the manufacturer’s instructions . Xpert HWomen who tested positive for HPV on either specimen or were VIA positive were referred to colposcopy that included a 4-quadrant microbiopsy protocol that included endocervical curettage (ECC) for diagnosis , 17. Scrn = 201), in part due to civil unrest at the time of the study, or inadequate quality of histology (n = 24). We therefore defined our endpoint of high-grade cervical abnormality based cytologic interpretation of high-grade cytology, as was previously done in Senegal [The original study design proposed to use histologic endpoints for analyses. However, of the 316 WLWH who screened positive for HPV on either specimen or by VIA, only 91 had histologic results due to failure to return for colposcopy n = 1, in par Senegal , and/or Fisher’s exact test for categorical variables and Kruskal-Wallis test for continuous variables were used to test for differences between the sub-groups included in and excluded from these analyses. We calculated the percent positive of each test for normal, low-grade, and high-grade cervical cytology. Ordinal logistic regression was used to assess whether of HIV status and median age influenced the trend of a positive screening test with increasing severity of cervical abnormalities.p < 0.05) using an exact version of the McNemar chi-square test.Sensitivity, specificity, negative (NPV) and positive (PPV) predictive values, and odds ratio (OR) with 95% confidence intervals (95%CI) of each screening test for high-grade cervical abnormalities were calculated. Differences in sensitivity and specificity were tested for statistical significance , tested positive for HPV on the provider-collected specimen (p = 0.13) and self-collected specimen (p = 0.01), but not VIA (p = 0.84), compared to those excluded from the analysis (missing cytology results). There was no difference in age (p = 0.27) or CD4 counts (p = 0.71) between those with and without cytology results.Five hundred forty-seven of the 566 (96.6%) recruited WLWH had results for all three screening tests; 6 were missing HPV testing results for the provider-collected specimens, 6 were missing HPV testing results for the self-collected specimens, and 7 VIA results were missing. Of the 547 WLWH with all three screening results, 487  of 316 screen positives) also had cytology results and were included in the analysis. Those included in the analysis (with cytology results) were more  likely to screen positive overall  were currently on anti-retroviral therapy at the time of enrollment. More demographic data are shown in Table WLWH included in this analysis had a mean, median, and age range of 42, 42, and 25–59 years, respectively, and a mean, median, and CD4 count range of 569, 544, and 13–2406 per mmn = 49; 10.1% of 487 enrolled women) had evidence of high-grade cervical abnormalities: 47 had high-grade cytology, 1 had clinical diagnosis of cancer and high-grade cytology, and 1 had a clinical diagnosis of cancer. All three screening results were more likely to be positive with increasing severity of the cervical abnormality  . CD4 counts were independently and inversely associated with increasing severity of the cytologic interpretation after controlling for screen test positivity (p = 0.03 for both HPV test results and p < 0.01 for VIA). All three screening tests were positive for the two women with a clinical diagnosis of cancer.Forty-nine women (l) Table . This trp < 0.01). This was due to the greater likelihood of self-collected specimens testing HPV positive than provider-collected specimens among those with negative cytology  whereas there was no significant difference in the two specimens testing HPV positive among those who had low-grade  or high-grade  cervical abnormalities.Self-collected specimens were more likely to test HPV positive than provider-collected specimens overall (p = 0.01), HPV18 and 45 (p = 0.01), HPV31, 33, 35, 52, and 58 (p < 0.01), HPV51 and 59 (p = 0.06), and HPV39, 56, 66, and 68 (p < 0.01).Among WLWH with negative cytology, the ct (cycle threshold) values of HPV-positive self-collected specimens for those whose provider-collected specimen also tested positive was lower  (Self+/Provider+) than for those provider-collected specimen tested negative (Self+/Provider-) for all Xpert Channels : HPV16 (p = 0.50) but both were more sensitive than VIA . HPV testing of the provider-collected specimen was more specific than of the self-collected specimen , and both were less specific than VIA .Table p = 0.04) and self-collected (p = 0.01) specimens for women with high-grade cervical abnormalities was greater for WLWH with CD4 counts of ≥350 cells/ mm3 compared to those with CD4 counts of < 350 cells/mm3. There was no appreciable difference in sensitivity stratified on CD4 counts (data not shown). Similar results were observed if the endpoint was restricting to HSIL cytology  (data not shown).The specificity of HPV testing for provider-collected  were generally lower than that of concordant HPV-positive results (Self+/Provider+). We suggest that there might have been small, focal, vaginal HPV infections that were sampled only by self-collection and tested HPV positive by Xpert. Using lower ct positive cutpoints might eliminate some of these extraneous HPV-positive results .We acknowledge that a major limitation of the study was the absence of histologically confirmed endpoints. The well-known challenges of pathology diagnosis still remain in many countries , 25, andAnother limitation was the study population consisted of only women who sought care at the Limbe clinic and therefore may not be representative of the general population. This convenience sample excludes WLWH who do not present at clinics for a variety of reason. Therefore, this study population may have different, and quite possibly lower, carriage of HPV infection than those not included in the study, resulting in better specificity.Finally, low-cost strategies for the management of HPV-positive WLWH remains an important research goal. VIA combined with detection of the most carcinogenic and predictive HPV genotypes offers modest performance and the decision on how to use them may depend on resources available for follow-up care and cultural sensitivities related to overtreatment vs. cancer prevention. Promising new technologies such as automated visual evaluation  and in vAdditional file 1: Table S1. A comparison of ct (cycle threshold) values of HPV-positive self-collected specimens for those whose provider-collected specimen also tested positive was lower  (Self+/Provider+) to those whose provider-collected specimen tested negative (Self+/Provider-) for each Xpert human papillomavirus (HPV) test channel (group) among women living with HIV who had negative cytology. Abbreviation: IQR, interquartile range, *Kruskal-Wallis.


Sexually transmitted infections (STIs) can be associated with infertility. Human papillomavirus (HPV) has been identified as a potential agent in male infertility. Also, anti-sperm antibodies (ASA) have been detected in men with infertility. The aim of this study was to investigate the prevalence and association of HPV and ASA in infected semen of infertile men.This cross-sectional study was performed on 96 infertile men referring to infertility treatment center of Kashan University of Medical Sciences during March 2017 till September 2017 in Iran. Semen analysis and diagnostic PCR test were performed for detection of HPV DNA. The semen parameters in HPV infected and ASA positive samples were compared with HPV non-infected and ASA negative samples. Chi square test was used to determine the correlation between variables and p<0.05 was considered statistically significant.HPV DNA and ASA were detected in 17.4% and 15.2% of 96 semen samples, respectively. Semen volume, sperm count, sperm motility and the normal morphology rate were significantly decreased in HPV-positive subjects . Also, sperm motility was significantly decreased in ASA-positive subjects (p=0.002), also patients with HPV infection had a higher rate of ASA than the non-HPV group. In contrast to ASA, HPV infection had a significant correlation with education level (p=0.039).The findings suggest that asymptomatic seminal infection of HPV and ASA by adversely affecting sperm quality, in particular sperm motility and count, may play an important role in male infertility. Infertility refers to the inability to have a child after at least one year of unprotected sex . Infertihr of sexual abstinence prior to sampling and the subjects didn’t take antibiotic during the last one week. None of the patients had clinical symptoms of genital herpes and genital warts. After liquefaction at room temperature, semen volume, pH, sperm count, viability, motility, and normal morphology were determined according to World Health Organization guidelines for semen analysis  according to the protocol.Two-hundred microliters of the sample was centrifuged at 2500 μl and each reaction contained 5.5 μl of 2x master mix , 3 μl of DNA, 0.5 μl of Taq DNA polymerase , 0.5 μl of each primer and 20 μl of DEPC water. Amplification cycles were set as follows: for HPV16, 95°C for 30 s, 57°C for 45 s, 72°C for 60 s and 72°C for 5 min; for HPV18, 95°C for 30 s, 55°C for 45 s, 72°C for 60 s and 72°C for 5 min.After DNA extraction, using general screening primers and then with the specific primers of HPV18 E6 and HPV16 E7, HPV detection and genotyping were performed  accordinThe detection of IgG anti-sperm antibody in each semen sample was performed using ELISA kit . All processes were performed according to the manufacturer’s protocol.After determining the frequencies, Chi square test was used to determine the correlation between variables. The p<0.05 was considered significant. In this study, SPSS soft-ware version 17 was employed for statistical analysis.ml, the mean total number of sperm (SD)×106 was 64.11±32.39, the mean pH=7.80±0.5 and the average duration of infertility (SD) was 3.92±4.33 years. The findings of this study showed that viral infection rates of HPV and distribution of ASA in infertile men was 16(17.4%) and 14(15.2%), respectively. Of 16 positive cases for HPV, eight persons were infected with HPV 16, four cases with HPV 18 and HPV 16/18 co-infection was detected in four patients  age was 32.15±6.66 years. The mean semen volume (SD) was 3.56±1.47 patients .In addition, HPV infection and ASA infection were not associated with age (p=0.608), duration of infertility (p=0.865) and pH (p=0.843); the means are shown in The frequency of semen volume (p=0.004), total sperm count (p=0.016), morphology (p=0.017), and sperm motility (p<0.001) were significantly associated with HPV . AdditioThe results of this study showed that HPV infection was positive in 17.4% of infertile men and there is a significant relationship between HPV and sperm quality. Until now, widespread studies have surveyed HPV prevalence in infertile men that ranged from 10 to 30% in the world as well as Iran , 16–19. The relationship between HPV and its negative effect on sperm morphology remains weakly understood, but it could be due to binding of HPV to the spermatozoa head . Like prIn this study, high-risk HPV infection did significantly affect total sperm count in infertile men. The studies reported a significant relationship between the lower total sperm count and HPV infection in semen . In thisThe results of this study showed that HPV infection and ASA were not associated with age, pH and duration of infertility. In contrast to ASA, HPV infection had a significant correlation with the education level (p<0.039). This result is in accordance with previous studies , 23. PerHPV infection and antisperm antibody via the effect on total sperm count, morphology and sperm motility, decrease sperm quality and result in male infertility. Also, these results suggest that young couples can be tested for HPV and ASA along with other causes of infertility.


Background and Purpose: The role of the cartilage oligomeric matrix protein (COMP) in epithelial-mesenchymal transition (EMT) in tumor progression has been studied, but its exact regulatory mechanism remains unknown.Methods: The interaction between COMP and the actin-binding protein transgelin (TAGLN) was identified by interaction protein prediction and co-immunoprecipitation and verified through the stochastic optical reconstruction microscopy (STORM) and duolink experiments. Western blot and immunofluorescence analyses were conducted to detect the changes in EMT-related markers after COMP overexpression and knockdown. Molecular docking and Biacore of the interaction interface of COMP/TAGLN revealed that Chrysin directly targeted COMP. The promotion of COMP and the Chrysin inhibition of EMT were detected through the cell migration, invasion, apoptosis, and xenotransplantation of nude mice.Results: COMP interacts with TAGLN in EMT in colorectal cancer to regulate cytoskeletal remodeling and promote malignant progression. COMP is highly expressed in highly malignant colorectal cancer and positively correlated with TAGLN expression. COMP knockdown can inhibit colorectal cancer metastasis and invasion, whereas COMP overexpression promotes EMT in colorectal cancer. Through virtual screening of the protein interaction interface, Chrysin, a flavonoid compound extracted from Oroxylum indicum, was found to have the highest docking score to the COMP/TAGLN complex. Chrysin inhibited COMP, thereby preventing EMT and the malignant progression of colorectal cancer.Conclusions: This study illustrated the role of COMP in EMT and suggested that COMP/TAGLN may be a potential tumor therapeutic target. Chrysin exhibits obvious antitumor effects. This work provides a preliminary antitumor therapy to target COMP or its interaction protein to inhibit EMT. Colorectal cancer (CRC), which has the highest incidence in the age group of 40-50 years, is one of the most common malignant tumors worldwide. Approximately 8 million new cases occur globally every year, accounting for 10%-15% of all malignant tumors The cartilage oligomeric matrix protein (COMP) contains five subunits of homologous polymeric glycoprotein, each of which have a molecular weight of 87 kD Transgelin (TAGLN) is an actin-binding protein that regulates the dynamics of the actin cytoskeleton and promotes the aggregation of G-actin to F-actin In vivo and in vitro experiments showed that Chrysin could significantly inhibit the proliferation and metastasis of colorectal cancer. The role of COMP and TAGLN in colorectal cancer was fully described by combining bioinformatics analysis and functional and mechanistic studies. This study may help improve the understanding of colorectal cancer metastasis and provide new targets for follow-up treatment.In this study, 471 patients with colorectal cancer and 41 normal colon tissues were analyzed to describe the differentially expressed genes (DEGs) of colorectal cancer and the functions of these DEGs. COMP was highly expressed in the colorectal cancer tissues, as observed through weighted gene co-expression network analysis (WGCNA) and Venn analysis. The high expression of COMP substantially affected the advanced clinicopathological features and poor prognosis of 597 patients. Moreover, COMP was found for the first time to interact with TAGLN to promote the migration and invasion of colorectal cancer cells. On the basis of the protein structure of the COMP/TAGLN complex, the specific inhibition of the COMP/TAGLN protein interaction by Chrysin from the in-house traditional Chinese medicine (TCM) database was determine through high-throughput screening technology The expression of COMP and TAGLN in normal adenoma-adenocarcinoma sequences was evaluated by IHC staining from 45 human colon tissue specimens . There were no significant differences in age or gender between the groups. These patients were excluded if they were suffering from other clinical diseases. All patients signed the informed consent, and ethical approval was obtained from the Ethics Committee of General Hospital, Tianjin Medical University, China 2, digested by trypsin, and subcultured every 2 days. Full-length COMP or mock (empty vector) or shCOMP was stably expressed in both cell lines by transfection with Lipofectamine 2000  and selection with hygromycin or puromycin . COMP expression was verified by WB. Single clones with good COMP expression were chosen for further experiments.HCT116, HCT-8, and SW620 cells were purchased from KeyGEN BioTECH. Cells were cultured in RPMI 1640 or DMEM containing 10% FBS  and 1% penicillin and streptomycin . The cells were cultured in a cell incubator at 37 ℃ with 5% COHuman subjects were not involved in this study. The colorectal cancer data used were downloaded from the TCGA dataset, which contained 471 colorectal cancer and 41 normal colon tissue control samples. The DEGs were analyzed using the edgeR packages of Bioconductor. The cutoff values were set at FDR < 0.05 and |logFC| ≥ 2. A total of 969 DEGs, including 420 upregulated and 549 downregulated genes, were calculated.GSEA software was used to input the gene expression matrix of colorectal cancer and normal control samples. All genes were sequenced to show the trend of gene expression between the two groups. The top and bottom of the sorted list of genes were viewed as the upregulated and downregulated DEGs, respectively. In GSEA results, ES indicated enrichment score, and the FDR q-value indicated q-value, which was the p value corrected by multiple hypothesis tests and represented the credibility of enrichment results. GSEA adopted the p- and q-values < 5% and < 25%, respectively, to filter the results.The ClueGo plug-in in Cytoscape software was used to conduct pathway enrichment analysis of DEGs in colorectal cancer, and p < 0.05 was used as the threshold to set the standard for visual analysis of enrichment pathway results.A scale-free network topological analysis of mRNA expression data from colorectal cancer samples was performed via WGCNA. During the analysis, the data were collated and computed using the default standard parameters. Excel was used to sort the gene expression amount of patients with colorectal cancer and the age, lymphatic invasion, pathologic M, pathologic N, pathologic T, venous invasion, gender, tumor stage, status, and other information of each sample for follow-up analysis. In accordance with the expression of genes, the correlation of genes was determined; the genes with high expression correlation were clustered into a module. The correlation between the collected modules and the clinical data of patients was analyzed. The purpose of module clustering analysis was to integrate the acquired genes and further narrow the scope of key genes.http://biocc.hrbmu.edu.cn/CancerSEA/) was used for the single-cell level comprehensive exploration database for cancer cell function. The CancerSEA database was used to analyze the COMP gene. The t-SEN distribution of COMP in all individual cells in colorectal cancer single-cell sequencing data was analyzed by functional correlation among different cell populations. The different colors represented the expression level of the input gene. Correlation analysis was conducted between COMP and the functions, angiogenesis, metastasis, and invasion of the EMT to further understand the COMP functions.CancerSEA  is the TCGA database data mining platform that provides the gene expression level and survival, correlation, and DNA promoter region methylation data analyses. This study directly investigated COMP expression in colorectal cancer and its relationship with prognosis.The UALCAN website  was incubated with control or specific antibodies (1-2 μg) at 4 °C for 8 h. The HCT116 cell lysates were prepared on ice with 0.3% Nonidet P-40 lysis buffer under the action of the protease inhibitor mixture. The lysate was centrifuged at 12,000 rpm for 10 min at 4 °C. The precipitates were discarded, and the beads coupled with antibodies were incubated at 4 °C. After incubation for 12 h, the precipitates were washed with cold 0.1% Nonidet p-40 buffer five times. The precipitated protein was resuspended in 2×SDS-PAGE loading buffer, boiled at 99 °C for 10 min, and eluted from the beads. The boiled immune complex was subjected to SDS-PAGE and WB and then treated with COMP (1:1000) and TAGLN (1:1000).Intracellular proteins were isolated using 10% or 15% SDS-PAGE gel and then transferred to polyvinylidene difluoride membranes. After 5% skim milk powder was sealed at room temperature for 1 h, the specific primary antibodies, including COMP , Vimentin , E-cadherin , N-cadherin , Occludin , Twist1 , Snail1 , MMP2 , and β-actin , were incubated overnight at 4 °C. The corresponding secondary antibodies were re-incubated, and the membranes were tested. Results were standardized using β-actin.The Duolink experiment followed the steps described previously. In brief, a blocking solution was added to the samples for 30 min. After the blocking solution was removed, the samples were added with diluted primary antibodies (1:100). The primary antibodies were rinsed off for 3×5 min. The samples were added with PLA® PROBES, and two PLA probes were diluted and incubated for 1 h at 37 °C. The ligase was washed twice with Wash Dilute solution (1:40), added to the samples, and incubated for 30 min at 37 °C. The mixture was then washed in 1× Wash Buffer A for 2×2 min, and the amplification stock in water (1:5) was diluted. Polymerase was also diluted in the solution (1:80), added to the samples, incubated for 100 min at 37 °C, and washed with 1×Wash Buffer B for 2×10 min and 0.01×Wash Buffer B for 1 min. The samples were prepared for DAPI staining and confocal microscopy imaging.The Lipofectamine RNAi MAX  was used to transfect all siRNAs as recommended by the manufacturer. The final concentration of siRNA was 10 nM, and the cells were transfected for 72 or 96 h and collected for experiments.4, and fixed for 60 min. The OsO4 solution was removed, and the cells were gently rinsed twice for 5 min. The cells were gradually dehydrated with gradient ethanol, and ethanol was replaced by gradient tert-butanol. Finally, the samples were subjected to vacuum drying, sprayed with gold, and imaged by SEM .Cell slides were prepared in 24-well plates. After sticking to the wall, the cells were treated and fixed with 4% glutaraldehyde in PBS for 60 min. The slides were washed with PBS three times for 10 min, washed with 1% OsOThe cell slides were prepared in 24-well plates and treated as follows after cell adherence. The cells were washed three times with PBS for 5 min each time and fixed for 10 min by using precooled 3.7% formaldehyde. The sealed serum was diluted to 5% with 1×PBS  at room temperature and incubated at room temperature for 30 min. The major antibodies used were TAGLN , COMP , E-cadherin , and Vimentin . After the incubation of cells with the primary antibody, the antibody was gently rinsed using PBS for three times and Alexa Fluor®488 . Alexa Fluor®568  was used to mark the secondary antibody. The cells were observed and photographed by confocal microscopy .In the invasion assay, the Transwell chamber was first coated with Matrigel (BD Biosciences), and cells of different treatments were added to the upper chamber of the Transwell. The lower chamber was treated with a medium containing 10% FBS. The cells were cultured at 37 °C for 24 h, washed three times with 1×PBS, fixed with 4% paraformaldehyde (precooled at 4 °C), and stained with crystal violet (KeyGEN BioTECH). The cells that passed were counted under a microscope .In the migration experiment, HCT116, HCT8, and SW620 cells with different treatments were uniformly scratched in the center of the cell culture plate. The cells after the scratch were photographed using a microscope every 24 h, and the gap size of the scratch was counted.COMP- or mock-transfected cells (1:1 ratio) were cultured until 80% confluent in a six-well plate. The cells were treated with 15 μg/mL brefeldin A  or an equal volume of PBS for 48 h to induce apoptosis in vitro with the COMP shRNA- or overexpressed plasmid. Subsequently, 2×106 cells were injected into BALB/c nude mice  bearing subcutaneous tumors. A total of 2×106 HCT116 and HCT-8 cells or COMP knockdown cells were subcultured in BALB/c nude mice (4-6 weeks old) to study the inhibition of colorectal cancer due to Chrysin. After formation, the tumors were evenly divided by size. Experimental mice were treated with 20 mg/kg Chrysin every day. The control mice received the same amount of saline. The long and short diameters of the tumor were measured every 4 days by using a Vernier caliper. The tumor volume was calculated as follows: V = ab2/2 . After reaching the endpoint of the experiment, the mice were sacrificed, and the tumors were removed. The tumors were fixed using 4% paraformaldehyde, embedded in paraffin, and sectioned for subsequent detection.HCT116, HCT-8, and SW620 cells were infected 6 SW620 cells with stable overexpression and low COMP expression were suspended in PBS and injected into the spleen with a needle. The spleen was gently returned to the abdominal cavity, and the wound was sutured and disinfected. After splenic injection modeling, the experimental mice were treated with 20 mg/kg Chrysin every day to evaluate the Chrysin inhibitory effect on metastasis. After 5 weeks, the mice were sacrificed, and the liver was isolated to observe metastasis A mouse model of liver metastasis was constructed as follows. Mice were anesthetized by intraperitoneal injection of 0.5% pentobarbital sodium . An incision of about 1 cm was made on the left side of the abdominal cavity. The spleen was gently isolated, and 2×10The tumor sections were removed of paraffin by using gradient xylene and dehydrated with gradient ethanol. Hydrogen peroxide (3%) was used to block the endogenous levels of peroxidase. The protein was denatured in a microwave oven with citrate buffer brine (pH 6.0) to expose the antigen. At room temperature, normal goat serum was incubated for 30 min to seal the nonspecific binding. Anti-COMP (1:100), anti-E-cadherin (1:100), anti-Vimentin (1:100), anti-Snail1 (1:100), and anti-Twist1 (1:500) were incubated overnight at 4 °C. Diaminobenzidine and hematoxylin were used as color developing and redyeing agents, respectively. The expression levels of COMP, E-cadherin, Vimentin, Snail1, and Twist1 were independently evaluated by two researchers.http://www.rcsb.org) protein database. Hydrogenation, steric hindrance optimization, and hydrogen bond optimization were carried out on the initial structure to eliminate stereoscopic conflict. Protein docking was performed using HEX software. COMP was selected as receptor, and the TAGLN structure was selected as the ligand. The docking parameters were selected based on the protein surface structure and surface potential. On the basis of the COMP/TAGLN complex structure obtained by docking, the protein interaction interface was selected as the active pocket for high-throughput screening of the TCM database. The XYZ size of the docking grid box was set to 60 × 60 × 60. Ligprep was used to generate the three-dimensional structure of the TCM monomer, and the OPLS-2005 force field was used to minimize the energy of the structure.The protein crystal structure of COMP and TAGLN was downloaded from the PDB  and MEDC (200 mM) solutions were mixed in equal proportions. The mixture was then injected at a rate of 10 μL/min, followed by COMP at 10 μL/min. The activation of the conjugated protein steps was repeated until the desired signal strength was reached. The remaining active groups were injected into the chip channel at a rate of 10 µM/min to seal it. The buffer used by the system was 1×PBS. Small molecules of Chrysin  were injected at a rate of 30 μL/min for 180 s. In the dissociation phase, the running buffer was injected at a rate of 30 μL/min for 300 s. All signals were corrected by channel 1 as the control channel. The data were analyzed and processed using BIAevaluation Software .Statistical analyses were performed using GraphPad Prism . Data from biological triplicate experiments were presented with an error bar as the mean ± standard deviation. Two-tailed unpaired Student's t-test was used to compare two groups of data, whereas one-way ANOVA was used to compare multiple groups of data. According to the statistical correlation requirements, Pearson correlation coefficient was used to calculate the co-expression correlation of the two proteins. A survival curve was generated by the Kaplan-Meier method and assessed by a log-rank test. Differences were considered significant when *p < 0.05, **p < 0.01, or ***p < 0.001.The DEGs in colorectal cancer were identified, and enrichment analysis was conducted. Colorectal cancer and normal control colon tissues were pretreated using the limma package. With p < 0.05 and |logFC| > 1 as the threshold, 969 DEGs were identified. Among these DEGs, 420 upregulated  and 549 WGCNA was conducted to describe the correlation between the gene expression levels and clinical information of patients with colorectal cancer and further screen key genes in colorectal cancer. WGCNA is a systematic biological approach that describes the pattern of genetic associations among patients with different colorectal cancers. It can identify candidate biomarker genes or therapeutic targets based on the association of gene sets and the association between gene sets and phenotypes. WGCNA uses information from thousands of the most variable genes in colorectal cancer to identify the genes of interest that are significantly correlated with the clinical phenotype of patients with colorectal cancer. Finally, the key genes of colorectal cancer can be enriched and selected.A highly synergistic set of genes was integrated to divide colorectal cancer expression genes into 11 modules through WGCNA Figure A. The coThe correlation between the gene set and the clinical phenotype of patients with colorectal cancer was determined through WGCNA, but the module itself still contained a large number of genes. The most important genes should be further searched. To extract valuable clues from these data, key genes were discovered from the poor prognosis of colorectal cancer gene sets , WGCNA gThe effect of COMP in the EMT of colorectal cancer cells based on the biological function analysis of COMP was studied to understand the biological significance of COMP. Scanning electron microscopy (SEM) results showed that COMP knockdown promoted epithelial changes in colorectal cancer cells, whereas COMP overexpression promoted mesenchymal phenotypic changes in colorectal cancer cells Figure A.Western blot (WB) results further confirmed that the expression levels of epithelial markers (E-cadherin and occludin) were upregulated after the knockdown of COMP by colorectal cancer cells. In addition, the expression levels of mesenchymal markers (Vimentin and N-cadherin) and those of Twist1, Snail1, and MMP2 decreased in the siCOMP group. The overexpression of related markers of COMP and EMT led to opposite findings Figure B. The inin vivo. Compared with the control group, the COMP-deficient nude mice had slow tumor growth and small tumor volume, and the COMP-overexpressed nude mice exhibited fast tumor growth and large tumor volume  staining Figure I. The rehttp://dcv.uhnres.utoronto.ca/FPCLASS/ppis/). TAGLN, an actin-binding protein, was also found in the COMP-containing protein complex. The interaction between COMP and TAGLN was further verified by co-immunoprecipitation. COMP efficiently co-immunoprecipitated with TAGLN, and TAGLN was efficiently co-immunoprecipitated with COMP  showed that TAGLN expression was positively correlated with clinical stage  is associated with many biological effects. Thus, small molecules that specifically interfere with protein-protein recognition are becoming increasingly important in future antitumor drug development. However, some characteristics and properties of the protein complex interaction interface, such as large interaction interface and flat binding interface, introduce difficulties to PPI and inhibition research. In this study, COMP and TAGLN were not kinases and did not have traditional drug binding pockets. However, their PPI interface could be used as a good active site. Some properties and characteristics of the interaction interface between COMP and TAGLN were analyzed and summarized, and Chrysin from the TCM monomer database was screened to obtain the highest docking scores. Finally, the combination of COMP and Chrysin and the inhibitory effect of Chrysin on colorectal cancer were demonstrated through Oroxylum indicumTCM has attracted increasing attention worldwide. A broad space for the development of TCM, including its therapeutic effect on malignant tumors, is expected in the future. Chrysin, also known as aspen flavin, is a flavonoid compound extracted from In this study, we found that COMP regulated tumor cytoskeletal remodeling and promoted mesenchymal transition by interacting directly with TAGLN. Chrysin inhibited EMT by inhibiting the COMP/TAGLN interaction interface and showed excellent antitumor activity. Research on the mechanism of the EMT and the structural modification of lead compounds is of great significance for the development of clinical antitumor drugs targeted at COMP/TAGLN.Supplementary figures and tables.Click here for additional data file.


These results indicate that the proposed AFE can provide a feasible balance between sensitivity and stability in CBMs, and it could be a versatile replacement for the conventional voltage follower used in CBMs.This paper proposes a novel analogue front end (AFE) that has three features: voltage-dependent input impedance, bandpass amplification, and stray capacitance reduction. With a view to applying the AFE to capacitive biopotential measurements (CBMs), the three features were investigated separately in a schematic and mathematical manner. Capacitive electrocardiogram (cECG) or capacitive electromyogram (cEMG) measurements using the AFE were performed in low-humidity conditions (below 35% relative humidity) for a total of seven human subjects. Performance evaluation of the AFE revealed the following: (1) the proposed AFE in cECG measurement with 1.70-mm thick clothing reduced the baseline recovery time and root mean square voltage of respiratory interference in subjects with healthy-weight body mass index (BMI), and increased R-wave amplitude for overweight-BMI subjects; and (2) the proposed AFE in cEMG measurement of biceps brachii muscle yielded stable electromyographic waveforms without the marked DC component for all subjects and a significant ( As population aging is accelerating globally and high medical costs are becoming increasingly problematic, the concept of smart healthcare is gaining attention as an effective solution ,2. SmartThere is growing interest in various types of wearable devices that detect physiological information such as the electrocardiogram (ECG) and electromyogram (EMG) ,7. PreviCBM is a measurement method that exploits capacitive coupling, which is comprised of skin, insulator (or cloth), and electrodes. Some researchers have already successfully used CBM in detecting ECG ,19, EMG Another issue related to the ever-higher input impedance of AFE is an increased susceptibility to stray interference such as power-line noise and its harmonics. In many cases, the housing and lead wires around AFE adopt a driven shield for interference suppression. A double shield, which is a combination of the driven shield and ground shield, was applied to multi-layered fabric electrodes and was found to mitigate motion-induced artifacts as well as the stray interference . Furthercf), which is shown in cf was first proposed by Thakor et al. in 1980 for conventional ECG measurements [aR1, aR2 are resistance, and aC3 is the capacitance for feedback in f is frequency. According to Equation (1), the magnitude of A bootstrapped voltage follower with capacitance feedback Z˙inaR1 + aR2) and contributes to shortening the BR time as reported in [aC3. However, positive proportionality to frequency in cf in CBM, combined with capacitance of the skin-electrode coupling can cause resonant ringing in the signal and lead to signal instability [For an input frequency much lower than orted in . With a tability .rf), which has three features: (F-1) voltage-dependent input impedance, (F-2) bandpass amplification, and (F-3) stray capacitance reduction. The first feature (F-1) is attained by a bootstrapping part composed of a resistor bR1, a varistor with voltage-dependent resistance bvR and a feedback resistor bR3 as shown in vvrst relative to threshold voltages ±thrV as follows:The proposed novel AFE shown in rf, iv . By applying star-delta transformation to the bootstrapping part in Then the input impedance of BNAiv is within a range between bR3, a medium (or high) resistance for bR1, and a high resistance for h.R Since normal iv  is in the order of millivolts at most, iv falls within the range between thrV in the order of volts. On the other hand, for iv outside the range, bR1  and contributes to shortening the BR time . So, that means that the voltage-dependent input impedance of the proposed BNArf in Equation (4) can achieve a feasible balance between sensitivity and stability in the CBM. Furthermore, when the range between CCV, the bootstrapping part that includes the varistor also works as pre-over-voltage protection for the subsequent OP Amp, leading to increased durability of the proposed AFE.According to Equation (4), when rf  is attained, as shown in fR and fC, and a series connection of sR and sC. With the use of complex impedances of these connections, The second feature (F-2) of the proposed BNAFor Equation (5), we can define corner frequencies of f between rf is a constant rf as a non-inverting amplifier for the frequency band of concern. Meanwhile, for the lower or higher frequency region of the relevant band, the amplitude of BNArf gradually changes from f  of BNArf contributes to improvement in the signal-to-noise ratio (SNR) in CBM signals.For a frequency rf,  is only attained by inserting a capacitor nC as indicated in stC in nC yields compensating current f  of the proposed BNAnC to be negative capacitance stC. Since nC has to be set to satisfy Equation (8):According to Equation (7), we can consider the inserted All experimental procedures were approved by the Human Life Ethics Committee of Tokyo Denki University. All subjects provided informed consent prior to participation in our experiments.rf), cECG and cEMG were measured using BNArf and using a previous AFE (BVFcf) as a reference. Both AFEs were prototyped using off-the-shelf components. The same type of OP Amp with high input impedance  was employed to prevent degrading the input impedance synthesized in parallel with the bootstrapping part. The values of the elements used for circuit implementation and their specifications are shown in rf , falling within the range of healthy-weight (18.5–25) or overweight (25–30) BMI, participated in this experiment. Subject information and the measurement conditions are presented in 2, the BMI of the subject under the force of gravity correlates roughly with the pressure of the capacitive coupling between the subject’s dorsal surface in a supine position and the electrode placed under the subject via the clothing. We selected these subjects to examine the effect of the coupling pressure on the quality of the obtained cECG signal. In the experiment, the subjects were requested to sit on a bed for 10 s, change their posture to a supine position on cue, and remain lying supine on the bed for 12 min with a constant breathing rate at 0.25 Hz in tune with the sound from a metronome.In order to verify compatibility between the sensitivity and stability of the proposed BNAAs shown in rf, but to 60 dB in the measurement using BVFcf, so that the total gain of the measurement system including AFE became 60 dB in both measurements.The cECG signal was measured using the system shown in cf and BNArf. As the first step to determine BR time for each cECG measurement, the baseline component was extracted from each cECG signal by applying a 0.15 Hz low-pass filter (LPF). Next, the steady-state baseline level  was computed as the mean of the baseline component from 60 s to 80 s after the onset of lying down on the bed. Finally, BR time was determined as the time required for the baseline component to return to a range of ±0.5 mV of the steady-state baseline level and to stay within the range for more than 10 s. A shorter BR time reflects the higher stability of the AFE used in the measurement against out-of-range input.Measured voltage of cECG signals was converted to input-referred voltage as a preprocessing common to BVFresp) was calculated by the minute from the onset of lying down on the bed. A smaller RMSVresp means the used AFE has higher insusceptibility to RM disturbance.In order to evaluate the insusceptibility of the used AFE to RM disturbance in cECG measurement, the RM component around 0.25 Hz was extracted from each cECG signal by applying a 0.15–0.5 Hz bandpass filter. For each time-series RM component, the root mean square voltage  signal was simultaneously measured with a commercially available EMG amplifier , telemetry system , and disposable electrodes . The disposable electrodes were directly attached on the skin near the right biceps brachii muscle so that they did not interfere with the pad electrode for cEMG measurement.During the experiment, cEMG signal was measured using the system shown in ref signals was converted to input-referred voltage as a preprocessing similar to that described in 3.3. For cEMG signals, the 10 s period of “elbow bended” and the subsequent 10 s period of “elbow straightened” were considered as a pair, and two segments of 4000 samples, from 4 s to 8 s (The measured voltage of cEMG and EMGS+N), while “elbow straightened” segments include noise only (N).We considered that the “elbow bended” segments contain both myoelectric signal and noise , there was little difference in the baseline stability of the systems, as shown in resp extracted from cECG signals measured with the previous or proposed AFE for all four subjects. Comparing the transitions between the subjects irrespective of AFE type, transitions in the subjects within the overweight BMI range  dropped rapidly after 1 min as in resp obtained with the proposed AFE in every one minute was smaller in most times. Having grouped minute-by-minute twelve RMSVresp data for each subject together to compare the two AFEs, the RMSVresp with the proposed AFE was significantly smaller (p < 0.01) than that with the previous AFE for both subjects #A and #D (data not shown). These results were consistent with the results in ref and cEMG recordings measured with the previous and the proposed AFE under low-humidity conditions for subject #E. For both of the AFEs, electromyographic firings during the “elbow bended” period in the cEMG signals were synchronized with those in the simultaneously measured EMGref signals. However, at times around elbow bending (t = 0 s) and elbow straightening (t = 10 s), there was a specific difference between the two AFEs in terms of baseline stability. When the previous AFE was used, the baseline of cEMG fluctuated widely around these timings, whereas the baseline settled with the use of the proposed AEF. This superiority in the baseline stability of the proposed AFE in the cEMG signal was observed in all subjects. In addition, with the use of the proposed AFE, the undesirable DC component in the signal was close to 0 in all subjects.p < 0.01) as shown in The results depicted in A comparison of the transient response of the cECG baseline shown in According to Equation (12), a small When considering the fluctuation of resp, which reflects the amplitude of disturbance caused by respiratory motion in cECG signals. In resp were obtained during 2–12 min periods. Presumably, these were because the lower coupling pressure of the healthy-weight-BMI subjects, #A and #D, resulted in a longer discharge process and in larger residue of resp values in subject #A and #D for the two AFEs shows the superior capability of the proposed AFE for quick discharge at low coupling pressure.While the first member in Equation (13) represents the discharging process of cf  can cause resonant ringing at a frequency The oscillatory fluctuations observed in the cEMG signal shown in a at the Given ystem in  against f in Equation (6). On the other hand, the gain for the components with the frequencies of interest is 20 dB in the proposed AFE but it is 0 dB in the previous AFE. Consequently, even when both measurement systems have the same total gain  for the components with the frequency of concern, the relative magnitude of the DC component decreases in the system with the proposed AFE. Similar logic can be applied to the components with non-DC frequencies of no concern. Smaller amplitudes in lower frequency (< 20 Hz) in the spectrum shown in Comparison of the baseline in the cEMG signal in nC being introduced only in the proposed AFE. Similarly, larger amplitudes of cEMG in the spectrum for the elbow-bended segment in nC in the proposed AFE. Note that the introduction of nC in the previous AFE does not reduce stray capacitance because compensating current is not produced due to no potential difference between the output and input nodes of the voltage follower. Hence, a change in AFE type from a voltage follower to a non-inverting amplifier is also necessary for the introduction of nC.Comparison of the R-wave amplitudes in resp for healthy-weight-BMI subjects, and increased the R-wave amplitude for overweight-BMI subjects. (2) The proposed AFE in cEMG measurements of biceps brachii muscle yielded stable electromyographic waveforms without a visibly biased baseline for all subjects and a significant (p < 0.01) increase in SNR. These results indicate that the proposed AFE can provide a feasible balance between sensitivity and stability in the CBM, and could be a versatile replacement for conventional voltage followers used in CBMs.This study proposed a novel AFE that has three features, which include voltage-dependent input impedance, bandpass amplification, and stray capacitance reduction. With a view to applying the AFE to CBMs, the three features were separately investigated in a schematic and mathematical manner. Measurements of cECG or cEMG using the AFE were performed under low-humidity conditions (below 35% relative humidity) for a total of seven human subjects. Performance evaluation of the AFE revealed the following: (1) The proposed AFE in cECG measurements with 1.70 mm thick clothing reduced the BR time and RMSV


This study focuses on factors that may disproportionately affect female veterans’ mental health, compared to men, and is part of a larger study assessing the prevalence of mental health disorders and treatment seeking among formerly deployed US military service members.We surveyed a random sample of 1,730 veterans who were patients in a large non-VA hospital system in the US. Based on previous research, women were hypothesized to be at higher risk for psychological problems. We adjusted our results for confounding factors, including history of trauma, childhood abuse, combat exposure, deployments, stressful life events, alcohol misuse, psychological resources, and social support.Among the veterans studied, 5% were female (n = 85), 96% were White , 22.9% were Iraq/Afghanistan veterans (n = 398), and the mean age was 59 years old (SD = 12). Compared to males, female veterans were younger, unmarried, college graduates, had less combat exposure, but were more likely to have lifetime PTSD (29% vs. 12%.), depression (46% vs. 21%), suicidal ideation (27% vs. 11%), and lifetime mental health service use (67% vs. 47%). Females were also more likely to have low psychological resilience and to have used psychotropic medications in the past year. Using multivariate logistic regression analyses that controlled for risk and protective factors, female veterans had greater risk for lifetime PTSD, depression, suicidal thoughts, and for lifetime use of psychological services, compared to males. Since 95% of the population in this study were male and these results may have been statistically biased, we reran our analyses using propensity score matching. Results were consistent across these analyses.Using a sample of post-deployment veterans receiving healthcare services from a large non-VA health system, we find that female veterans are at greater risk for lifetime psychological problems, compared to male veterans. We discuss these findings and their implications for service providers. Currently, women are one the fastest growing demographic groups in the military, and the proportion of female military service members and veterans is at its highest level ever in the United States (US) and other industrialized countries , 2. AlthBased on previous research, we hypothesize that female veterans receiving healthcare will have higher rates of PTSD and other mental health problems, compared to male veterans. This hypothesis is tentative, however, as research on sex differences on veterans’ mental health is inconsistent. Some research reports similar combat experiences and stress exposures for men and women among active duty military personnel, as well as commensurate rates of mental health problems , 6. ConvMany of the inconsistent results related to sex differences in US veterans are also seen in studies from other industrialized countries. In their study of Canadian veterans, Brunet et al. report that females were less likely to experience combat related traumas, but more likely to suffer from sexual assaults compared to male Canadian veterans . Like soMuch of the previous research on deployment and veteran well-being analyze data from veterans seeking services from the US Department of Veterans Affairs (VA) –13, or ohttps://womenvetsusa.org/about.php). These VSOs report connecting women with active duty, Reserves, and National Guard military service members and their families, and with caregivers, advocates, and with local, state, and federal resources. In the discussion section below, we briefly discuss the potential impact of these VSOs on mental health outcomes for woman. While we do not directly address the impact of these activities have on women veterans, we would expect that they would likely reduce sex differences in well-being and treatment seeking over time.Finally, in recent years, the growth of women’s veterans service organizations (VSOs) in the US has been extensive. To date, over 150 active women’s VSOs and related auxiliary groups have been identified (www.geisinger.org), serving more than 3 million residents. Starting in 2007, Geisinger Clinic collected electronic records on veteran status and patients were asked to complete a military registry form , which keeps track of the survey responses and administers the survey electronically, which is a common research practice [https://www.ptsd.va.gov/professional/assessment/list_measures.asp). Those interested in the depression scale used should go to: https://www.apa.org/depression-guideline/patient-health-questionnaire.pdf to get permission to use and to download this scale from the APA. Those interested in the “Audit-c” scale should go to: https://www.hepatitis.va.gov/alcohol/treatment/audit-c.asp to get permission and to download this scale from the VA.Data were collected via a telephone survey from a sample of community-based US military veterans recruited to assess the health effects of military service . All parpractice . Those iDiagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5) [The current study focused on four main outcome variables: PTSD, depression, suicidal ideation, and use of mental health services. To assess lifetime and past year PTSD, we used a diagnostic instrument—the PTSD Checklist based on the Association’s  (DSM-5) . To rece (DSM-5) . Nearly  (DSM-5) . Lifetim (DSM-5) , 16. Con (DSM-5) , 20. We  (DSM-5) . Specifi (DSM-5) –16 and aOur survey included demographic, military experiences, recent stressors, social connections, and psychological variables known to affect mental health –16. DemoThe analysis also included three measures of stress based on previous work , 16. StrLastly, we included several psychological, social, and physical health factors that could help explain sex differences in well-being. Psychological resilience was assessed by the 5-item version of the Connor-Davidson Resilience Scale , with reFinally, we assessed several variables related to health services use. VA service use was assessed using single item questions inquiring about current and lifetime use of VA healthcare service. We also used a single item to ask about current VA disability status (yes vs. no). In addition, the survey asked about the use of psychotropic medications in the past year: anti-depressants, tranquilizers, sleeping pills or other medicines for problems with emotions, nerves, concentrating, sleeping or coping with stress over the past 12 months. Any reported use of these medications was coded yes or no, based on whether the participant used any of these medications in the past year. Thus, this medication measure represented the percent of those that used these drugs in the past year. All these measures were used in previous studies , 16 and We present descriptive statistics and bivariate differences between male and female veterans Table . Given tFor propensity score matching we first included the confounding covariates listed in Table The basic characteristics of the sample Table  show thaFemale veterans in our study were more likely to meet criteria for lifetime and past year PTSD, meet criteria for lifetime and past year depression, and lifetime suicidal thoughts, but not recent ones. Health service results show no sex differences in the current use of VA services, but women were less likely to report a current VA disability, and more likely to have used any (VA or non-VA) psychological services and psychotropic medications in the past year.Multivariate logistic regression results Table  revealedIn addition to sex, age was statistically related to lifetime depression (OR = 0.51), as were childhood adversities (OR = 2.13), stressful events (OR = 2.27), lifetime trauma (OR = 1.64), combat exposure (OR = 1.86), concussion history (OR = 1.50), low self-esteem (OR = 3.40), low resilience (OR = 2.32), and low social support (OR = 1.62). The model for lifetime suicidal thoughts showed that childhood adversities (OR = 2.25), low self-esteem (OR = 4.25), and low resilience (OR = 1.95), along with sex, were statistically related to this psychological problem. Finally, besides statistically significant differences by sex, the model for lifetime psychological service use showed that child abuse (OR = 1.87), high stress in the past year (OR = 2.06), high lifetime trauma (OR = 1.54), high combat exposure (OR = 1.69), service related concussion (OR = 2.05), low self-esteem (OR = 2.37), and low resilience (OR = 2.02) were associated with a higher likelihood of service use, while being older than 65 (OR = 0.53) or married (OR = 0.66) was related to a lower likelihood.As an additional check on sex differences in well-being among our sample of veterans, we replaced lifetime outcomes with current measures of each variable. That is, we replaced lifetime PTSD, depression, suicidal thoughts, and use of psychological services, with PTSD past year, current depression, recent suicidal thoughts, and use of services in the past year. As shown in Table In Table Using data collected from a sample of post-deployment veterans receiving their care from a non-VA healthcare system, we assess sex differences in health outcomes. Regarding our three research questions, we find significant sex differences in military and non-military experiences, with female veterans less likely to experience high combat and in-service concussion, but more likely to perceive low unit support. Women veterans were also more likely to meet criteria for lifetime and past year PTSD, lifetime depression, and lifetime suicide ideation. They are more likely to report using mental health services in their lifetime and in the past year. These sex differences in health outcomes persisted even after controlling for other variables. Our study is consistent with earlier research which finds female veterans at greater risk for lifetime PTSD and other mental health problems, and higher users of psychological services , 11, 18.Past research by Lehavot et al. analyzed data from the 2012–2013 National Epidemiologic Survey on Alcohol and Related Conditions-III and, like our findings, report that women veterans have the highest rates of lifetime and past year PTSD and treatment utilization, compared to male veterans . Using VThere are two points about our results that should be noted, one related to the propensity score and sensitivity analyses and one on the sex differences observed in the lifetime and past year psychological service use. The logistic regression models for our four outcomes included demographic, military experiences, and non-military experiences variables. Nevertheless, all four of the models continued to show sex differences. The propensity score analysis confirmed these results, but the sensitivity analysis indicated the possibility of omitted factors in the models for PTSD, depression, suicidal thoughts, and use of psychological services. Research shows that female veterans not only have different backgrounds , but also have different life experiences  than male veterans , 9, 41. The statistical significance for sex in the models for lifetime and past year use of psychological services also suggests the need for an examination of the differences in male and female veteran experiences. It is noted that in the lifetime model, the odds ratio for the logistic regression reached statistical significance, and the propensity score results showed statistically significant sex differences for the 1:3 and 1:5 models. Studies suggest sex differences can influence both the experience of stressful events and how persons respond to such events, including treatment seeking . FurtherIn terms of study limitations, our data were cross-sectional, which precludes assessment of causality. Second, we only were able to successfully recruit 85 female veterans. As noted, this may have biased our results and may not represent the larger population of female veterans receiving care. Although our propensity score analysis confirmed the multivariate analyses, it is possible that unmeasured variables, such as a more detailed history of sexual assault, might change our results. It is also possible that only certain types of female veterans seek healthcare in non-VA facilities, and we have not included this confounding factor in our models. Future research should sample veterans receiving care from both VA and non-VA facilities to broaden generalizations for this population. Third, the current study only included previously deployed U.S. veterans seen at a large non-VA multihospital system in Pennsylvania and our results may not generalize to all veterans. Research which includes veterans from both VA and non-VA systems may better clarify gaps in care. Fourth, the findings may not generalize to non-White US veterans because over 90% of our sample was White. Finally, we did not ask about gender identity, sexual assault/harassment in the military, or sexual orientation. Future studies should explore these issues in more detail, as these have been related to poor mental and physical outcomes among veterans . Many ofIn recent years, the growth of Veteran Service Organizations (VSOs) for women has been extensive. While outcome data related to VSOs are limited, preliminary studies are encouraging –44. For Despite these limitations, our findings support the case for more gender-informed planning, given the projected increase in female veterans in both the US and other industrial countries. These changes might include hiring more female healthcare providers, more explicit training in the health needs of female veterans, and training on greater sensitivity to a female’s unique life experiences, such as sexual harassment , 40. FurAdditional file 1. Veterans registry forms.Additional file 2. Survey script.