Human and murine sickle cell disease (SCD) associated pulmonary hypertension (PH) is defined by hemolysis, nitric oxide depletion, inflammation, and thrombosis. Further, hemoglobin (Hb), heme, and iron accumulation are consistently observed in pulmonary adventitial macrophages at autopsy and in hypoxia driven rodent models of SCD, which show distribution of ferric and ferrous Hb as well as HO-1 and ferritin heavy chain. The anatomic localization of these macrophages is consistent with areas of significant vascular remodeling. However, their contributions toward progressive disease may include unique, but also common mechanisms, that overlap with idiopathic and other forms of pulmonary hypertension. These processes likely extend to the vasculature of other organs that are consistently impaired in advanced SCD.To date, limited information is available on the metabolism of macrophages or monocytes isolated from lung, spleen, and peripheral blood in humans or murine models of SCD.Here we hypothesize that metabolism of macrophages and monocytes isolated from this triad of tissue differs between Berkley SCD mice exposed for ten weeks to moderate hypobaric hypoxia  or normoxia  with normoxia exposed wild type mice evaluated as controls.This study represents an initial set of data that describes the metabolism in monocytes and macrophages isolated from moderately hypoxic SCD mice peripheral lung, spleen, and blood mononuclear cells. Sickle cell disease (SCD) is a genetic hemoglobinopathy that disproportionally affects ethnic groups worldwide. In the US, approximately 1 out of every 365 African Americans and 1 out of 16,300 Hispanic Americans are born with this disorder . Under hAdvancements in metabolite analysis have created a new, unbiased, sensitive, high-throughput tool for scientists to investigate the etiology of SCD-related comorbidities at a molecular level and potentially inform on novel therapies. Tissue and cell specific metabolomics provide a versatile tool to help define and validate biomarkers for measurement of disease progression . Further2)  .Macrophage metabolic reprogramming is associated with a pro- or anti-inflammatory state of tissue resident macrophages , with glycolysis, the Krebs cycle, and arginine/polyamine metabolism representing critical hubs regulating cell functional fates . Studies2) or sea level (21% O2). The hypothesis is formed based on our studies that demonstrate chronic moderate hypoxia induces PH in Berk SCD mice that associates with sickling, accelerated erythrophagocytosis of injured RBCs and macrophage accumulation of iron (In the current study macrophages were obtained from homozygous Berkeley (Berk) SCD mice following exposure to 10 weeks of moderate simulated hypobaric hypoxia to accelerate PH progression . Here, w of iron , 19, 20.ad libitum. Female heterozygous Berk mice were bred with male homozygous Berk mice to generate homozygous offspring. Specifically, Berk mice with genotype Tg(Hu-miniLCR α1 Gγ Aγ δ βs) 0/0 Hbb0/0Hba and the hemizygous with genotype Tg(Hu-miniLCR α1 Gγ Aγ δ βs) 0/0 Hbb0 Hbb+Hba were littermates. Genotyping of mice used for breeding and experiments was performed by TransnetYX . A total of 24 mice (n = 8 per group) were used to evaluate cardiovascular changes and a subset of 12 mice were utilized for metabolomics  were used in the present investigation. Levels of discomfort and distress were monitored daily by the in-house animal care staff, with a veterinarian available as needed. Mice presented with no pain or discomfort associated with moderate hypoxia and were alert as well as eating, drinking, and grooming normally while housed. Hypoxia exposure consisted of mice housed at 8,000 feet (15.4% O2) for 10 weeks as previously described  female C57Bl/6 WT and Berk mice were either obtained from Jackson Laboratories  or our in-house Berk mouse colony. Mice were housed and bred in an AAALAC accredited animal facility at the University of Colorado, Denver, Anschutz Medical campus and were maintained on a 12:12 light–dark cycle with food and water available escribed . All expysiology , the NatAfter 10 weeks in either normal or hypoxic environmental conditions, mice underwent terminal open chest right ventricular pressure measurements (RVSP) function measurements with a 1.2F, FTE-1212B-4,018 pressure volume catheter  inserted by direct cardiac puncture. Mice were induced inhaled isoflurane (4–5%), and tracheal incision (~1 cm) was performed. Next, a tracheal tube was inserted and connected to an Anesthesia Workstation or Hallowell EMC Microvent and an anesthetic plain was maintained at 1.0–2.5% isoflurane in 100% oxygen. After which, a thoracotomy was performed exposing the heart, the pericardium was resected, and a small hole made at the base of the right ventricle with a 30 g needle for insertion of the pressure-volume catheter. Steady state RVSPs, followed by contractility (Ees), and afterload (Ea) (during an occlusion by retracting the inferior vena cava), were captured and mice were humanely euthanized by exsanguination and cervical dislocation. Data was recorded continuously using LabScribe2 and analyzed offline.via cardiac puncture, using a syringe with a 26-gage needle, and placed in an EDTA treated tube. The blood was transferred to a 15 mL conical tube and diluted 2:1, sterile PBS: blood, and gently mixed. Lympholyte® Mammal Cell Separation media  was gently added to the bottom of the blood solution and spun at 1400 rpm for 30 min in a refrigerated centrifuge. After centrifugation, layers were visualized, the PBMC layer (midlayer) was extracted and resuspended in a new tube. The isolated PBMC’s were washed using ~14 mL sterile PBS, spun at 1800 rpm for 10 min, excess PBS was removed, cells were resuspended in 1-2 mL for counting, and the final pellet was frozen in liquid nitrogen and stored at -80C.Whole blood samples (1 mL) were obtained from animals The right lung and spleen organ tissues were harvested, finely minced, and each organ placed individually in 2 mL Eppendorf tubes containing 1 mL of Collagenase D  and DMEM media. The tissues were incubated at 37C with agitation for 30 min. After incubation, 100 μL of 0.1 M EDTA was added to the tissue-containing tubes and placed on ice. A single cell suspension was created by addition of Hanks buffered salt solution , passing through a 100 μm filter, and collected in a 15 mL conical tube. The tissue/cell suspension was spun at 500 g for 5 min and the supernatant discarded. The remaining tissue/cell solution was resuspended in 5 mL of RBC lysis buffer , incubated at room temperature for 15 min, and centrifuged at 500 g for 5 min–this step was repeated if RBC presence was sustained. Next, the cells are washed with Miltenyi buffer , resuspended in 180uL Miltenyi buffer, and incubated on ice with CD11b Microbeads  for 15 min. Positive cells were isolated using magnetic Ls columns , collected in 2 mL Eppendorf tubes, counted, and then final pellet aliquots were frozen in liquid nitrogen and stored at -80C.6/ml and 10 mg/mL ratios) prior to UHPLC–MS analyses , as previously described.Lung and spleen macrophages and PBMCs were extracted in methanol:acetonitrile:water . Other multivariate analyses, including hierarchical clustering analyses, heat maps, partial least square-discriminant analyses (PLS-DA), two-way ANOVA were performed with MetaboAnalyst 5.0 . Statistical significance was defined as p ≤ 0.05.Hemodynamics: statistical comparisons for tissue and hemodynamic data measurements were completed with the analysis of variance (ANOVA) and lyst 5.0 . Bar plolyst 5.0 , 24. Datp < 0.001; Berk hypoxia vs. wild type hypoxia; p < 0.005; p < 0.004; To demonstrate differences in cardiovascular function between healthy wild type and Berk mice housed at sea level and moderate hypoxia, right ventricular systolic pressures (RVSP), ventricular to vascular coupling (ratio of contractility to afterload), and the Fulton index (RV/LV + S) were also compared between the four groups. At 20 weeks of age there was no differences in RVSP pressures , ventricular vascular coupling ratio , and Fulton index (measurement of right ventricular hypertrophy) between the wild type (WT) and Berk Sea level cohorts . After 1Metabolomics analyses were performed on circulating PBMCs and resident splenic and lung macrophages . Two-wayFocusing on PBMCs, we noted a significant difference between WT and Berk mice, which was evident by clustering across principal component 1  in PLS-DA analyses . An overMetabolomics analyses of WT and Berk splenic macrophages following exposure to moderate hypoxia revealed a significant effect of reduced oxygen availability on metabolism . FollowiPLS-DA analysis of metabolomics data from lung macrophages showed distinct metabolic phenotypes between WT controls and Berk mouse lung macrophages, in normoxia and hypoxia  and moderate hypoxia . This hypothesis is formed on the basis that moderate hypoxia induces disease progression and accelerates erythrophagocytosis in Berk mice. To date we are unaware of any study that compares metabolomic profiles of circulating, lung, and spleen macrophages between healthy and SCD mice in normoxic and moderate hypoxic environments.To date our studies in human and murine sickle cell disease (SCD) associate pulmonary hypertension (PH) have focused on hemoglobin (Hb), heme, and iron accumulation in pulmonary macrophages and their potential contributions toward progressive disease , 25, 26.Confirming our previous work Berk mice housed in moderate hypoxia show increased RVSPs, ventricular to vascular coupling ratio, and RV hypertrophy that is consistent with the development PH associated with increased hemolysis , 19.ex-vivo. As such, the observation of elevated amino acid levels in Berk mice is consistent with phagocytosis of RBCs associated with hemoglobin polymerization, RBC sickling, and tissue damage from vaso-occlusion. Further, circulating PBMC are recruited to the lung and contribute to pulmonary vascular disease  and medium  chain acylcarnitines. The well-established biologic function of acylcarnitines is to transport acyl groups from the cytosol into the mitochondria matrix for B-oxidation and sustain cell activity , 39. HowIn conclusion, this study was designed to address an initial understanding of how macrophages may contribute to PH, splenomegaly, vascular, or other metabolic disorders associated with SCD. To better understand mechanistic underpinnings of macrophage function in the sequela of SCD, future studies will focus on interrogating macrophages, analyzing metabolomic signatures and associating these findings with end organ injury and disease endpoints. The work presented herein provides a platform to expand on these concepts.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care and Use Committee at Anschutz Medical.DI, AD’A, and PB: design of the work. CL, FC, DP, and DS: data collection. CL, DI, AD’A, and PB: Drafting the article. KH, RN, GG, and PD-C: critical revision of the article. All authors contributed to the article and approved the submitted version.The authors disclosed the receipt of the following financial support for the research, and publication of this article. This research was supported by NIH Grants RO1HL159862 (DI and PB), RO1HL152337 (DI and PB), RO1Hl161004 , RO1HL162120 (PB and DI), and K99HL156058 (PD-C).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The purpose of this study was to investigate the value of a clinical radiomics model based on Positron emission tomography-computed tomography (PET-CT) radiomics features combined with clinical predictors of Lymphovascular invasion (LVI) in predicting preoperative LVI in patients with colorectal cancer (CRC).18F-fluorodeoxyglucose (FDG) PET-CT examination were retrospectively enrolled. Univariate and multivariate logistic regression analyses were used to analyse clinical factors and PET metabolic data in the LVI-positive and LVI-negative groups to identify independent predictors of LVI. We constructed four prediction models based on radiomics features and clinical data to predict LVI status. The predictive efficacy of different models was evaluated according to the receiver operating characteristic curve. Then, the nomogram of the best model was constructed, and its performance was evaluated using calibration and clinical decision curves.A total of 95 CRC patients who underwent preoperative P < 0.05). The clinical radiomics model obtained the best prediction performance, with an Area Under Curve (AUC) of 0.922 (95%CI 0.820–0.977) and 0.918 (95%CI 0.782–0.982) in the training and validation cohorts, respectively. A nomogram based on the clinical radiomics model was constructed, and the calibration curve fitted well (P > 0.05).Mean standardized uptake value (SUVmean), maximum tumour diameter and lymph node metastasis were independent predictors of LVI in CRC patients (The clinical radiomics prediction model constructed in this study has high value in the preoperative individualized prediction of LVI in CRC patients. At pCompared with CT images, high-resolution magnetic resonance imaging (MRI) can accurately evaluate extramural vascular invasion with a large vessel diameter (diameter ≥3 mm), but it has low sensitivity for the diagnosis of some early intramural invasion and extramural small vessel invasion , 8. The 2.2.1.Ethical approval for this retrospective study was obtained by the Medical Ethics Committee of Henan Provincial People's Hospital [approval number: (2019) No. 68], and the need for written informed consent was waived.This was a retrospective single-centre study. A total of 239 patients with CRC confirmed by postoperative pathology in Henan Provincial People's Hospital from January 2016 to May 2022 were retrospectively collected. Inclusion criteria were: (1) CRC was confirmed by postoperative pathology, (2) PET/CT was performed within 1 month before surgery, (3) complete preoperative PET-CT images were available. Exclusion criteria were: (1) preoperative neoadjuvant therapy , (2) vascular invasion was not clear in pathology, and (3) combined with other pelvic malignant tumours or poor image quality could not be evaluated.Clinical information of patients was obtained by searching medical records, including age, sex, serum levels of tumour markers: carcinoembryonic antigen (CEA ≤ 5 ng/ml), carbohydrate antigen 199 (CA199 ≤ 35U/ml), carbohydrate antigen125 (CA125 ≤ 35U/ml), and tumor differentiation degree. Maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), peak standardized uptake value (SUVpeak), metabolic tumor volume (MTV), total lesion glycolysis (TLG), maximum tumour diameter and lymph node metastasis were obtained by PET-CT information.2.2.18F-FDG PET-CT examination. All surgical specimens were reviewed by pathologists with more than 6 years of experience in abdominopelvic pathology, specifically to assess for the presence of LVI. LVI was diagnosed when tumour cells or tumour cell thrombi were observed within an endothelium-lined space or if tumour cells had destroyed a lymphovascular wall, as described in a previous study . The radiochemical purity of 18F-FDG was >95%. High-performance liquid chromatography was used for quality control. Fingertip blood glucose was collected before the examination, and the blood glucose level was controlled below 11.1 mmol/l. The patients fasted for 6–8 h before the examination, and the dose of 18F-FDG was 5.55 MBa/kg. After the injection, the patients rested for 40–50 min in a quiet and suitable temperature environment before imaging. The bladder was emptied, and the stomach was filled with 500 ml of water. PET and CT scanning were performed from the head to the feet. CT images were acquired with the following scanning parameters: tube voltage, 120 kV; tube current, 170 mA; field of view, 40 mm; slice thickness, 3.75 mm; tube speed, 0.6 ms/r; pitch, 0.984 mm; scanning time, 13.8 s; and matrix, 256 × 256. Three-dimensional PET scans were performed with 4–6 window levels according to height, and each window level took 3 min to scan. After CT attenuation correction and iterative reconstruction, the fused images of the coronal, sagittal and transverse planes were obtained by Integrated Registration software in the GE 4.6ADW postprocessing station.The imaging equipment used was a Discovery™ VCT PET-CT instrument from GE Company in the United States. 2.4.18F-FDG PET/CT diagnosing regional lymph nodes metastasis in CRC were lymph node short diameter ≥1.0 cm or SUVmax ≥2.5) were measured and recorded.The acquired PET-CT original data were imported into the uWS-MR, UIH workstation. The images were processed and analysed by two senior physicians. In cases of disagreement, the result was obtained after discussion. The region of interest (ROI) of CRC lesions was delineated , as shown in 2.5.http://www.itksnap.org/) to delineate the ROI layer by layer on rectal cancer lesions and obtain the tumour volume of interest (VOI), as shown in The original PET-CT images of all included patients were obtained from the MedEX System of Nuclear Medicine Workstation of Henan Provincial People's Hospital, and postprocessing was performed based on axial PET and CT sequences. Two radiologists with 5 years of experience applied the ITK-SNAP software (Version 3.8.0 Inter- and intra-class correlation coefficients (ICCs) were calculated for evaluation of the inter-reader reliability and intra-reader reproducibility of feature extraction. Radiologist 1 and radiologist 2 randomly selected 30 CT and PET images for radiomics ROI delineation and image feature extraction. Radiologist 1 repeated the segmentations 1 month later. An Intraclass correlation coefficient (ICC) greater than 0.75 indicated good agreement of feature extraction. The ROI segmentation for the remaining cases was performed by radiologist 1.http://github.com/Radiomics/pyradiomics) was used feature extraction and analysis. Feature extraction was performed using filters such as Original, BoxMean and AdditiveGaussianNoise during preprocessing. First Order Statistics (Firstorder), Grey Level Co-occurrence Matrix (GLCM), Shape-based (3D) (Shape), Grey Level Dependence Matrix (GLDM), Neighbouring Grey Tone Difference Matrix (NGTDM), Grey Level Run-Length Matrix (GLRLM), and Grey Level Size Zone Matrix (GLSZM) features were generated from the ROI of image. Finally, 2,264 radiomics features were extracted from each sequence, and the PET-CT features set was generated by means of features stitching.Radiomics features were calculated and combined for different imaging sequences . The open-source software PyRadiomics . The features with low correlation with the classification label were removed based on the K-best method . Finally, the irrelevant or redundant features were removed by the least absolute shrinkage and selection operator (LASSO), and the radiomics signature was constructed according to the weighted linear combination of the selected features and their corresponding regression coefficients. The radiomics score (Rad-score) of each patient was calculated accordingly. Finally, the training model is obtained based on logistic regression classifier.The dataset was randomly divided into training and validation cohorts at a ratio of 6:4, the training cohort was used for features selection and models construction, and the test cohort was used for models’ evaluation. First, Univariate logistic regression analysis was used to compare the differences in clinical information between the LVI-positive group and the LVI-negative group, and multivariate logistic regression analysis was used to identify the independent predictors significantly associated with LVI. Four datasets were constructed, and four prediction models were trained based on PET, CT radiomics features and clinical predictors. They were named PET_RS (PET radiomics features), CT_RS (CT radiomics features), PET-CT_RS (PET and CT radiomics features) and clinical radiomics model .2.7.The predictive ability of the PET_RS, CT_RS, PET-CT_RS and the clinical radiomics models was evaluated based on the Area Under Curve (AUC), sensitivity, and specificity in the training and test cohorts. A radiomics nomogram was constructed by combining the clinical predictors of LVI screened by multivariate logistic regression and the Rad-score of the best model. The calibration of the nomogram was evaluated by drawing the calibration curve between the actual probability of LVI and the predicted probability of LVI. Decision curve analysis (DCA) was used to evaluate the clinical utility of multimodal radiomics by calculating the net benefit at different threshold probabilities.2.8.x ± s, and comparisons between the two groups were analysed using the two independent sample t test. Measurement data not following a normal distribution are represented as M , and comparisons between the two groups were analysed using the Mann‒Whitney U test. Count data are expressed as cases (%), and the chi-square test was used for comparisons between groups. Univariate and multivariate logistic regression methods (Forward selection) were used to analyse the clinical indicators of patients with CRC. The MedCalc software was used for the ICC test, receiver operating characteristic (ROC) curve drawing and analysis. The nomogram, calibration plots, and DCA were performed using R statistical software . The “rms” package was used for nomogram and calibration curve drawing, and the “rmda” package was used for decision curve drawing. All statistical tests were two-sided, and P < 0.05 was considered statistically significant.All statistical analyses were performed using SPSS (version 26.0), R statistical software (version 4.0.2), and MedCalc (version 15.2.2). Measurement data following a normal distribution are represented as 3.3.1.P > 0.05). The above information indicates that the distribution of baseline clinical characteristics of patients in the training and validation cohorts was balanced. Among these patients, 44 (46.3%) were pathologically diagnosed to have LVI, and 51 (53.7%) were pathologically diagnosed to be free of LVI. A univariate logistic regression analysis of preoperative colorectal cancer LVI showed that maximum tumour diameter, lymph node metastasis, SUVmax and SUVmean metabolic values were independent risk factors for LVI (P < 0.05). Multivariate logistic regression analysis finally included maximum tumour diameter, lymph node metastasis and SUVmean metabolism as independent clinical predictors of LVI (P < 0.05).A total of 95 CRC patients were included in this study after screening, including 54 males and 41 females, with an average age of 62 years. None of the clinical factors significantly differed between the training cohort and the validation cohort (3.2.P < 0.001). The confusion matrix shown in Taking the clinical radiomics model as an example, firstly, to avoid subjective differences in segmentation of ROI, the radiomics features with both inter- and intra-reader ICCs > 0.75 were retained. Secondly, two PET features and four CT features were selected by variance threshold, K-best and LASSO regression models, and then combined with three clinical predictors, a total of nine features were obtained. The selected features were summed by weighting coefficients to calculate the Rad-score . Finally3.3.By comparing the area under the ROC curve (AUC), sensitivity, and specificity of each model , the cli4.In this study, PET-CT radiomics features and clinical predictors were used to construct a clinical radiomics model for preoperative prediction of pathological LVI in CRC. We found that the clinical radiomics nomogram integrating PET-CT radiomics score, SUVmean, maximum tumour diameter, and lymph node status had good predictive value for LVI in CRC and could assist clinical decision-making. Clinical factors add additional value to radiomics in predicting LVI, and it was expected to become a biomarker to assist clinical decision making in CRC patients.LVI refers to the invasion of cancer cells into the surrounding tissues or even the entire body through veins or small vessels . LVI is Consistent with the results of many previous studies, combining clinical risk factors can improve the performance of prediction models –26. The 18F-FDG PET-CT imaging metrics were associated with prognosis in patients with non-small cell lung cancer undergoing stereotactic ablative radiation therapy, and they reported that metabolic tumour volumes with pretreatment MTV values higher than SUV 7 or 10 could be used to predict overall survival, whereas metabolic tumour volumes with low thresholds SUV 2 and SUV 4) were not associated with overall survival. However, our study used a lower SUV threshold of 4.0 (median SUV). Although TLG can reflect both tumour metabolism and tumour volume, TLG and LVI did not significantly correlate in this study, which may be due to different thresholds for calculating MTV.SUVmax is calculated based on the maximum voxel grey value and is the most commonly used metabolic parameter in PET-CT. It is not affected by the size of the ROI and can be used to initially differentiate benign and malignant tumours. However, it only represents the maximum metabolic value of the tumour and reflects the highest activity of the lesion but cannot reflect the overall characteristics of the tumour. Multiple studies –31 have In addition, we concluded the presence or absence of lymph nodes with abnormal metabolism is also one of the predictors of LVI. Although the lymph node status determined by PET-CT is not as accurate as that determined by surgical pathology, it reflects the overall situation of lymph node metastasis to a certain extent. Colorectal cancer lesions with positive lymph node metastasis are likely to have microscopically invaded the lymphovascular tissue around the intestine, which is consistent with the findings of Aktekin et al. . In addi5.Our study has some limitations. First, due to the strict inclusion and exclusion criteria, the sample size was small, and we will continue to expand the sample size for further research in the future. Second, this study was a single-centre study and lacked independent external validation. We plan to include multicentre samples in future studies to address these issues.6.In summary, based on PET-CT imaging omics combined with clinical risk prediction factors, a clinical radiomics prediction model for the prediction of LVI was developed. The model has good diagnostic performance and can be used as an individualized decision support tool to evaluate the preoperative LVI status of colorectal cancer patients, which can better assist clinicians to make preoperative decision-making, postoperative evaluation, and formulate individualized clinical treatment plans for patients.


Dietary effects on GI symptoms were reported more frequently by children with HD than controls , as were dietary adjustments to improve GI symptoms , and social limitations due to dietary adjustments . Of 90 food items, children with HD reported that more of the items induced GI symptoms compared to controls (7 (0–66) vs. 2 (0–34), p = 0.001). Diet-induced GI symptoms and dietary adjustments’ impact on daily life are reported more frequently by children with HD than controls. Moreover, the number and types of food items causing GI symptoms differ. The results indicate the need for disease-specific dietary advice to improve support for families of children with HD.Hirschspung’s disease (HD) is a congenital gastrointestinal (GI) disorder frequently accompanied by GI complaints. Despite the lack of evidence regarding whether diet affects GI symptoms, advice on dietary changes is common. The aim was to investigate self-reported dietary effects on GI symptoms, comparing children with HD with healthy children. This was an observational, cross-sectional, self-reported case-control study using the validated Diet and Bowel Function questionnaire. All children with HD aged 1–18 years were surgically treated during 2003–2021 at a national HD center, and their parents were invited to participate. Healthy children served as controls. The data were presented as median (range) and  Hirschsprung’s disease (HD) is a congenital disorder characterized by the absence of intestinal ganglion cells (aganglionosis) and streching of various lengths cranially from the anus, causing chronic bowel obstruction ,2,3. DesThis was an observational, cross-sectional, case-control, and patient-reported questionnaire study. Children with HD and healthy children, serving as controls, were studied. This study was conducted at the Department of Pediatric Surgery in Lund, in the south of Sweden. In 2018, the department was appointed a national referral center for HD, covering a catchment area of 5 million residents. Data collection was performed from March 2020 to March 2021.All children aged 1–18 years treated for HD were identified in the local HD register and invited to participate in this study together with their parents. Invitations and questionnaires were sent by postal mail, and a study announcement was published online by the National Hirschsprung Disease Patient Organization of Sweden . HealthyThe questionnaire included 32 questions in total and Likert scale graded answering options  ,20 and sThe third section was an assessment of the dietary effects on GI symptoms and daily life by using the Diet and Bowel Function questionnaire . This inn (%). Data on continuous variables did not fulfill assumptions for a normal distribution. They were therefore reported as the median (range). The non-parametrical Mann-Whitney U-test was used for continuous variables and for graded data on an ordinal scale. Binary categorical variables were analyzed by means of the Chi-square test. Statistical significance was set at p < 0.05. The program IBM SPSS Statistics Version 27 for MacOS was used.The statistical design was planned by a statistician. Only questionnaires with a minimum of 90% (29/32) questions answered were included in the study. Descriptive data were reported as HD was classified as either rectosigmoid aganglionosis, long-segment aganglionosis (extending past the rectosigmoid), or total colonic aganglionosis (TCA). Children with long-segment aganglionosis and TCA were grouped since the numbers of patients were few and no one had a longer extension of aganglionosis than in the colon. Answers to questions about constipation were dichotomized into No versus Yes . GI complaints were defined as constipation (Yes), abdominal pain (Often/Always) and/or bothersome gases (Often/Always).All methods were carried out in accordance with national guidelines and regulations. This study had received ethical approval from the Swedish Ethical Review Authority (registration number 2018/720). Participants received age-adapted oral and written information. Informed consents were obtained from both legal guardians for children younger than 15 years old, while adolescents aged 15 years or older gave their own consent.In total, 71/85 (84%) children with HD and 265/300 (88%) controls fulfilled the inclusion criteria. This corresponded to a case-control ratio of 1:3.7. Among children with HD, 58 (82%) had rectosigmoid aganglionosis, eight (11%) had long-segment colonic aganglionosis, and five (7%) had total colon aganglionosis (TCA), including a maximum resection of 10 cm of small bowel.In univariate subgroup analyzes, neither aganglionic extension, age, or sex constituted risk factors for dietary GI effects or GI complaints (p < 0.001) A ‘Good bowel function’ (BFS 18–20) was reported by 15/50 (30%) children with HD, compared to 182/201 (90%) controls (p ≤ 0.001). Every single GI symptom listed in the BFS was reported inferiorly by children with HD (p ≤ 0.001 per item) compared to controls  vs. 20 (16–20), controls .n = 71) and for controls (n = 265) (p ≤ 0.001). For specific symptoms, abdominal pain (Often/Always) was reported by 20% of children with HD and 8% of controls (p = 0.004), while bothersome gases (Often/Always) were reported by 53% and 10% of the children, respectively (p ≤ 0.001).The GI symptoms of constipation, abdominal pain, and/or bothersome gases were analyzed for children aged 1–18 years old with HD (n = 265) . Overallp = 0.013) (p ≤ 0.001).Constipation was reported by 33% of children with HD vs. 19% of controls (= 0.013) . Among cp ≤ 0.001). Children with HD also reported more frequently that they used dietary adjustments to improve bowel function , both with regard to choosing and especially avoiding foods in order to improve GI symptoms. ‘Choosing’ was reported by 38% of children with HD vs. 16% of controls (p ≤ 0.001), and ‘avoiding’ by 62% vs. 18%, respectively (p ≤ 0.001). In sub-analysis including only children with GI complaints, these differences remained . Moreover, concerns about dietary effects on GI symptoms differed between children and their parents: 26/71 children with HD (38%) vs. 60/265 controls (23%) (p = 0.003 for children’s views), and 62/71 parents of children with HD (89%) vs. 161/265 parents of control children (63%) (p ≤ 0.001 for parents’ views). Emotional effects as a result of dietary issues were reported to affect children with HD more frequently than controls  and also their parents . Children with HD more frequently answered that they were interested in improving their knowledge about the effect of diet on GI symptoms compared to controls . In sub-analyzes including only children reporting GI complaints, these differences remained  vs. 53/265 (20%), respectively, p ≤ 0.001. GI effects as a result of consuming any of the 18 fruits inquired about did not differ significantly between children with HD and controls: 30/71 (42%) vs. 92/265 (35%), respectively, p = 0.056.More children with HD than controls reported the development of GI effects as a result of consuming any of the 18 vegetables enquired about in the questionnaire: 36/71 (51%) vs. 84/265 (32%), respectively, p = 0.001), while GI effects of dairy products and fruits did not differ between the groups .In children with GI complaints, children with HD more frequently than healthy controls reported GI effects of vegetables: 31/51 (61%) vs. 24/77 (31%), respectively (p ≤ 0.001) and for children with GI complaints (9 (0–59) for children with HD vs. 3 (0–34) for controls, p = 0.012). Specific GI effects as a result of consuming selected types of food items are displayed in Among all the 90 food items enquired about, the median number of items reported to induce GI symptoms was significantly higher for all children with HD than healthy controls (7 (0–66) vs. 2 (0–34), p ≤ 0.001, then banana 24/71 (34%) vs. 27/265 (10%) controls, p ≤ 0.001, and corn 24/71 (34%) vs. 13/265 (5%) controls, p ≤ 0.001. Exactly the same ranking  was reported by children with GI complaints (p = 0.013). For controls, the next most frequently reported food items were plums , and cream, reported by 38/265 (14%) controls, which was still less than in children with HD  (The types of food items reported to induce GI symptoms and the frequency of children reporting on each type differed between children with HD and controls (mplaints . For con≤ 0.001) . The sam≤ 0.001) .Children with HD reported more frequently than controls that diet induced GI symptoms and made dietary adjustments accordingly, which influenced their daily lives. Specific food items inducing GI symptoms were also cited more frequently by children with HD than by controls, and these differed between the two groups. The differences persisted when comparing only children reporting GI complaints. Similar explicit dietary differences comparing children with HD and controls have never been presented before. The results reveal that diet is a stronger extrinsic factor for GI symptoms in children with HD than is the case for healthy children . These qIn this study, 77% of children with HD reported that diet induced GI symptoms. This proportion was higher than that reported previously, where 58–64% of patients with HD reported dietary-induced GI symptoms ,10. AddiA well-known GI symptom in children with HD is let-out obstruction ,2,3,4. TMost parents of children with HD (89%) reported concerns about dietary effects on their child’s GI complaints, and nearly 40% of children with HD reported an influence of dietary adjustments on daily life. The results confirm our previous findings, revealing frequent parental worries about worsening their child’s GI symptoms by giving them the wrong foods . SimilarThe main strength of our study was to address diet’s role in GI complaints in HD, which has been shown to be a highly ranked research topic by patient organizations ,25,26. TOne limitation of the study was the cross-sectional design, implying that neither changes over time nor causalities were investigated. Moreover, the large number of food items specified in the questionnaire could be criticized as a result of the risk of answer exhaustion. However, on the other hand, very few participants added other food items in the free text section, suggesting that most of the food items relevant to them had been covered in the questionnaire. Regarding food allergies, it cannot be excluded that the few IgE-mediated food allergies reported affected the results, but the probability of bias was considered to be small. This is since the frequencies were similar in the groups and also since effects on GI symptoms caused by milk allergy are reported to occur only in up to 30% of cases . A selecThe study’s results support the inclusion of dietary changes in bowel management programs within HD guidelines , and theChildren with HD report more frequently than controls that diet induces GI symptoms and make dietary adjustments that influence their daily lives. Children with HD also report that more food items induce GI symptoms and that their GI effects are different and more frequent compared to controls. These dietary differences are also evident in children with GI complaints. The results strongly suggest a need for disease-specific dietary advice for children with HD, and for further studies.


Satisfaction has become a key measure of quality and an important tool for improvement. Laboratories are increasingly required to regularly assess satisfaction of their customers. This study aimed to assess clinicians’ satisfaction with laboratory services and associated factors at public health facilities.p-value < 0.05 was considered significant.A facility-based cross-sectional study was conducted in Northeast Ethiopia from May to June 2019. Eight hospitals and 24 health centres were first selected using a stratified sampling method, and a total of 224 randomly selected clinicians were included. Satisfaction with multiple aspects of laboratory services was assessed using a self-administered questionnaire, on a rating scale of 1 (very dissatisfied) to 5 points (very satisfied). Laboratory quality assessment was performed using WHO-AFRO’s stepwise accreditation checklist. Multivariable logistic regression model was fitted to determine the association between independent variables and clinicians’ overall satisfaction level using STATA ver14.1. A Overall, 72.8% of the clinicians were satisfied. Lowest mean ratings were obtained for the helpfulness of the laboratory handbook (3.3), provision of STAT/urgent services (3.7), and adequacy of tests provided (3.8). The clinicians’ timely receipt of results , notification of panic results , perceived quality/reliability of test results , and the laboratories’ rate of concordant malaria microscopy results , were significantly associated with satisfaction.Nearly one-third of clinicians were not satisfied with the laboratory services. Laboratory managers should emphasize the timely communication of STAT/urgent and panic results, and the reliability of test results, to improve users’ satisfaction and overall quality of care. Medical laboratories are an essential component of effective healthcare system. Laboratory results must be accurate to ensure that subsequent medical decisions will lead to the best possible patient outcomes . ResultsQuality is providing excellent care as per published norms, thereby meeting customers’ needs and expectations , 5. CustIn Ethiopia, the laboratory structure is integrated with the healthcare tier, which includes health centres, and primary, general and specialized hospitals . The couMany aspects of laboratory services could be investigated from the clinicians’ perspective, such as the availability of ordered tests, courtesy and respect, report format, turnaround time (TAT), critical result notification, and reliability , 6. PrevAssessment from multiple perspectives could provide a better basis to inform opportunities for balanced and thus sustainable improvement , 6, 20. A facility-based cross-sectional study was conducted from May to June 2019 in East Amhara, Northeast Ethiopia. This area covers six of the 15 zones of Amhara region. There were 402 governmental health facilities, of which only 252 facilities  were diagnostic. The laboratories provide basic tests such as serology, urinalysis, parasitology, malaria, tuberculosis (TB) microscopy and Gram staining / [(d2/Z21−α/2)*(N−1)+ p*(1−p)]. Considering a 95% confidence level (Z1−α/2 = 1.96), p = 80.0% from a study conducted in Ethiopia [n = 272 clinicians.The sample size of clinicians was determined using OpenEpi ver3.03 with the common formula for a single population proportion and applying a finite population correction: Ethiopia , and a mThe required sample size was then pre-allocated to each facility proportional to facility size but kept as fixed average quotas by facility type  – 10 clinicians from a hospital and eight from a health centre.Facilities were selected using a stratified random sampling method with probability proportionate to facility size (client load) . List frThe dependent variable was the overall satisfaction level of a clinician. It was measured based on satisfaction ratings towards multiple aspects of laboratory service . Independent variables include demographics  and clinician-reported experiences . Facility-level variables include objective key performance indicators .A pre-tested, structured self-administered questionnaire was used to collect data from clinicians. It was customized for local use from a nationally validated tool for clinician customers of general laboratories in Ethiopia . The queAt the facility level, WHO-AFRO’s stepwise accreditation checklist was used to audit quality system practices and readiness of required tests , 9. BlinData collectors and supervisors were senior laboratory experts recruited from external facilities. They were then trained on the data collection tools and procedures. All tools were pre-tested in nearby facilities before use for the actual data collection.Satisfaction ratings given for the nine satisfaction measuring items were averaged to create an overall mean score for each respondent. Respondents with a score of ≥ 4 out of five points  were classified as satisfied, while the rest were classified as not satisfied. For each laboratory, the availability of required tests was measured as a percentage of the standard test menu expected for the respective facility type . The steTraining was given to data collectors and supervisors on the recruitment of participants and data collection. Pre-tested, structured, and standardized tools were used to ensure consistent data collection. Certified external laboratory assessors conducted the laboratory assessments. Supervisors oversaw the data collection and checked the consistency and completeness of the completed questionnaires.Satisfaction level is the degree of perception or feeling to which service quality attributes have fulfilled the customer’s needs and expectations [ctations . It is ap-value < 0.20 in the bi-variable analysis were considered to be included in the final multivariable model. A p-value < 0.05 was considered statistically significant. Adjusted odds ratios (AORs) with 95% confidence intervals (CIs) were reported.Data were entered using EpiData ver3.1 and analysed using STATA ver14.1. Descriptive statistics  were computed and compared by facility type. Stepwise logistic regression analysis was used to identify individual- and organizational-level factors affecting clinicians’ overall satisfaction level. Those variables with a Ethical clearance was obtained from the regional Ethical Review Board of Amhara Public Health Institute (APHI) Head office, Bahir dar, Amhara region, Ethiopia. The respective zone health departments and facility administrators were informed about the general aim and significance of the study through an official permission letter. The purpose of the study was described to each eligible clinician and all voluntary participants gave informed consent before enrolment. Data were collected anonymously without personal identifiers to ensure confidentiality. The study complied with the Helsinki Declaration.A total of 224 clinicians participated in this study accounting for a response rate of 82.7%. The mean age (± SD) of the participants was 30.9 (± 8.6) years, and nearly two-thirds of them (66.5%) had work experiences of four and more years. Table Three-fourths of clinicians reported the absence of a laboratory handbook (75.1%). Nearly one-third claimed to receive results out of the expected TAT (34.4%), poor quality/reliability of test results (35.3%), and lack of backup/specimen referral system 42.5%). Compared to health centres, more percentages of hospital clinicians reported poorer experiences on the timely receipt of results, notification of panic results and quality/reliability of test results  and 24 health centres (75.0%). The total number of tests provided increased with an increase from the health centre to hospital levels. The overall mean scores observed were generally poor, such as for test availability 53.6% ±16.3) and quality systems (accreditation) practice (33.0% ±16.2) . In Likert scale, the overall mean (± SD) score was 3.8 (± 0.62), with mean ratings for specific aspects ranging from 3.3 to 3.9. The lowest mean ratings were obtained for the handbook’s helpfulness (3.3), provision of STAT/urgent services (3.7), and adequacy of tests provided (3.8) Table .The overall percentage of satisfied clinicians was relatively lower at hospitals (56.0%) compared to health centres (76.8%). Specific aspects with more dissatisfaction at the hospitals include STAT/urgent service and result completeness Fig. .all, p ≤ 0.20). However, other variables, such as socio-demographic characteristics, and the stepwise accreditation score or star grade of the laboratories, did not show associations with clinicians’ satisfaction   Table .all, p ≤ 0.036). Respondents who received timely results  or were notified of panic results  were about two or three times more likely to be satisfied than their respective counterparts. Respondents who were comfortable with the quality/reliability of results  had higher odds of being satisfied compared to those who were not comfortable   and soutLaboratory handbook can play an important role in communicating relevant information to users , 8. In tTimeliness of results is one of the most noticeable signs of performance that is often stressed by users , 6. In tThe technical quality of laboratory results is often a point of emphasis in quality assurance frameworks. Many clinicians considered test results unreliable 35.3%), in line with findings of similar studies (39.8-87.0%) 5.3%, in , 23, 27.The first limitation is the small sample size with a 17.3% nonresponse rate, as some clinicians could not return the completed questionnaire due to workload. Second, a survey of clinician customers may not reflect the views of other laboratory customer groups. Third, there might be important variables that we did not consider, such as workload. Finally, satisfaction level can often be highly subjective and may not be fully reliable to measure actual or technical quality. However, we have tried to measure satisfaction based on multiple items and examine additional objective measures of laboratory practice.The study revealed that nearly one-third (27.2%) of the clinicians were not satisfied with the laboratory services provided. Specific areas related to both clinicians’ experiences and technical quality aspects were driving dissatisfaction. These include the absence of a helpful handbook, STAT/urgent services, communication of panic results, and reliability of test results. Therefore, laboratory managers should devise and take appropriate corrective actions to solve the root causes of the identified gaps, thereby addressing users’ needs. Particularly, emphasis should be given to improving the timely communication and quality of test results that will improve clinicians’ trust and thus utilization.


Malignant peripheral nerve sheath tumors (MPNSTs) are malignancies that arise or differentiate from or infiltrate peripheral nerves and account for approximately 5% of soft-tissue malignancies. Approximately half of MPNSTs develop in patients with neurofibromatosis type 1 (NF1), a hereditary disease. MPNSTs occur mainly in the trunk, proximal extremities, and neck, but can on rare occasion arise in or near the gastrointestinal tract, and intestinal complications have been reported. We describe herein a case with resection of an MPNST arising in the pelvic region.A 51-year-old woman had undergone repeated resections for systemic neurofibrosis associated with NF1. This time, a pelvic tumor was noted on follow-up positron emission tomography computed tomography (CT). She presented with slowly progressive radiating pain in the lower extremities and was referred to our hospital for tumor resection. Contrast-enhanced CT showed a 75 × 58-mm mass in the right greater sciatic foramen directly below a 24 × 28-mm mass. Open pelvic tumor resection was performed for pelvic neurofibroma. The obturator nerve was identified lateral to the main tumor and the sciatic nerve was identified dorsally, then dissection was performed. The closed nerve was spared, while the sciatic nerve was partially dissected and the two tumors were removed. Both tumors were elastic and hard. Pathologic findings were MPNST for the large specimen and neurofibroma with atypia for the small specimen. The patient developed temporary postoperative ileus, but is generally doing well and is currently free of recurrence or radiating pain. The patient is at high risk of recurrence and close monitoring should be continued.We encountered a rare case of MPNST. Due to the high risk of recurrence, surgery with adequate margins was performed, with a requirement for appropriate follow-up. Malignant peripheral nerve sheath tumors (MPNSTs) account for approximately 5% of all soft-tissue sarcomas, and about half occur in patients with neurofibromatosis-1 (NF1) , 3. In aThe patient was a 51-year-old woman on previous medical follow-up with NF1. A pelvic mass was noted on follow-up PET–CT. She was referred to our hospital for close examination and treatment due to slowly progressive radiating pain in the right lower extremity. The patient had no contributory family history, but had undergone several previous tumor resections associated with NF1. Contrast-enhanced CT showed a heterogeneous mass 75 mm in diameter in the right greater sciatic foramen Fig. , and PETMPNSTs are soft-tissue malignancies that arise or differentiate from or infiltrate peripheral nerves and account for approximately 6% of all soft-tissue sarcomas . The peaKim et al. reviewed 389 cases of peripheral schwannoma . Of thesAbout 70% of cases were detected as painless swellings, and clinical symptoms are said to become apparent as the tumor grows [–3 mm2/s for benign nerve sheath tumors and 1.12 ± 0.37 × 10–3 mm2/s for MPNST. Bredella et al. also reported on the utility of PET–CT [–3 mm2/s, and PET–CT showed an SUV of 12.7, suggestive of malignancy when examined retrospectively.Yun et al. reported on the usefulness of MRI for diagnosing MPNST , 9. Theyf PET–CT . In the Definitive diagnosis was reached on histopathological examination, which showed dense bundles of atypical spindle-shaped cells . The chrVarious treatment options are available for MPNST, including doxorubicin plus ifosfamide and peripheral blood stem cell transplantation. However, no clear benefits from these have been reported, and resistance to radiation has also been described , 14, 15.We encountered a rare case of MPNST. Due to the high risk of recurrence, surgery with adequate margins was performed, with a requirement for appropriate follow-up.


Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multifaceted condition that affects most body systems. There is currently no known diagnostic biomarker; instead, diagnosis is dependent on application of symptom-based case criteria following exclusion of any other potential medical conditions. While there are some studies that report potential biomarkers for ME/CFS, their efficacy has not been validated. The aim of this systematic review is to collate and appraise literature pertaining to a potential biomarker(s) which may effectively differentiate ME/CFS patients from healthy controls.This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and Cochrane review guidelines. PubMed, Embase and Scopus were systematically searched for articles containing “biomarker” and “ME/CFS” keywords in the abstract or title and if they included the following criteria: (1) were observational studies published between December 1994 and April 2022; (2) involved adult human participants; (3) full text is available in English (4) original research; (5) diagnosis of ME/CFS patients made according to the Fukuda criteria (1994), Canadian Consensus Criteria (2003), International Consensus Criteria (2011) or Institute of Medicine Criteria (2015); (6) study investigated potential biomarkers of ME/CFS compared to healthy controls. Quality and Bias were assessed using the Joanna Briggs Institute Critical Appraisal Checklist for Case Control Studies.A total of 101 publications were included in this systematic review. Potential biomarkers ranged from genetic/epigenetic (19.8%), immunological (29.7%), metabolomics/mitochondrial/microbiome (14.85%), endovascular/circulatory (17.82%), neurological (7.92%), ion channel (8.91%) and physical dysfunction biomarkers (8.91%). Most of the potential biomarkers reported were blood-based (79.2%). Use of lymphocytes as a model to investigate ME/CFS pathology was prominent among immune-based biomarkers. Most biomarkers had secondary (43.56%) or tertiary (54.47%) selectivity, which is the ability for the biomarker to identify a disease-causing agent, and a moderate (59.40%) to complex (39.60%) ease-of-detection, including the requirement of specialised equipment.All potential ME/CFS biomarkers differed in efficiency, quality, and translatability as a diagnostic marker. Reproducibility of findings between the included publications were limited, however, several studies validated the involvement of immune dysfunction in the pathology of ME/CFS and the use of lymphocytes as a model to investigate the pathomechanism of illness. The heterogeneity shown across many of the included studies highlights the need for multidisciplinary research and uniform protocols in ME/CFS biomarker research.The online version contains supplementary material available at 10.1186/s12916-023-02893-9. Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition that may affect between 17 and 24 million people worldwide [There is currently no definitive diagnostic test; however, there is a significant body of biological evidence for this condition. Currently, ME/CFS is diagnosed using self-report case criteria following exclusion of any other potential medical explanation . A systeA biomarker is defined as biological entity that can effectively and objectively differentiate one condition from healthy controls (HC) or from another condition . A biomaDespite no definitive consensus on a molecular biomarker multiple research studies have investigated the prognostic potential of some markers for ME/CFS. This systematic review aims to collate evidence on suggested biomarkers for ME/CFS, critically examine their methodology, and evaluate their efficacy as well as their  suitability as a biomarker for ME/CFS.This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) and Cochrane review guidelines , 12. PriStudies were selected if they complied with the following inclusion criteria: (1) observational study published after 1994; (2) study involved human participants aged 18 years or older; (3) full text available in English; (4) original research; (5) diagnosis of ME/CFS patients were made according to the FC (1994), CCC (2003), ICC (2011), or IOMC (2015); (6) study investigated potential biomarker/s of ME/CFS compared to HC.Studies were excluded if they met any of the following exclusion criteria: (1) was an interventional study; (2) written prior to the establishment of the  FC in December 15th 1994; (3) not available as full text or written in English; (4) was not an original study type including reviews, duplicate studies, or case reports; (5) use of other diagnostic criteria instead of FC, CCC, ICC, IOMC; (6) studies that are not within the scope of this review or did not compare to HC.Duplicates were removed using Endnote version 20  through the automated duplicate removal tool. The references were scanned for any remaining duplicates which were manually removed. The reference management tool Rayyan was used to sort and store the remaining publications . AbstracFollowing the selection of papers, relevant data were manually extracted including (1) study type; (2) criteria; (3) sample size (4) age; (5) sex; (6) body mass index (BMI); (7) illness duration; (8) biomarker; (9) classification 10) selectivity; (11) ease of detection; and (12) receiver operator characteristic (ROC) area under the curve (AUC)/sensitivity/specificity/accuracy; (13) findings. Studies were sorted according to type of biomarker from the following: genetic, immunological, metabolomics/mitochondrial/microbiome, endovascular/circulatory, neurological, ion channel and physical biomarkers. Due to lack of homogeneity between studies, a meta-analysis could not be conducted. Selectivity and ease of detection were included based on Byrnes and Weigl’s analytical biomarker for diagnostic application framework [ selectivAll publications were assessed for quality and bias using the Joanna Briggs Institute Critical Appraisal Checklist for Case Control Studies (JBI CACCCS). JBI CACCCS was selected for quality analysis as the checklist is validated and internationally recognised . This chPubMed (303), Embase (340) and Scopus (259) retrieved a total of 902 articles. Following removal of duplicates and application of inclusion and exclusion criteria, the total number of articles were refined to 101. The systematic process as conducted by PRISMA guidelines is outlined in Fig. The study and participant characteristics as well as findings of each article is summarised in Additional File n = 43.83 and n = 38.34, respectively. The average age of ME/CFS patients was 45.40 ± 8.76 years old and the average age of HC was 43.43 ± 9.15. Females were more prominent whereby on average 76% of ME/CFS patients and 72% of HC were female. Mean illness duration for ME/CFS patients ranged between 4.5 and 22 years with the average duration being 10.30 years.The average sample size of ME/CFS patients and HC included across all the studies was In total, 56 of the studies recruited patients that met the FC , 111–115n = 35, 34.65%). Due to the limited number of studies that included these values, an average was not calculated.In total, 76 of the biomarkers were blood-based (75.25%). Most of the biomarkers had either secondary (43.56%) or tertiary 53.47%) selectivity % selecti, 98–115 The studies have been grouped according to type of biomarker in Additional File There were 20 studies that discussed genetic/epigenetic biomarkers in ME/CFS patients compared with HC  have significantly higher Ca2+ influx in ME/CFS compared with HC [2+ influx through TRPM3 was also significantly lower in ME/CFS as described by Eaton-Fitch et al. [Three studies reported on TRPM3 function in NK cells , 29, 83.the cell . In cont with HC . Cabanas with HC . Ca2+ inIn total, nine studies used physical based biomarkers (Additional File Nacul et al. assessed hand-grip strength differences between ME/CFS patients and HC . Mild/moQuality of each included publication was assessed using the JBI CACCCS. Full results and justification can be found in Additional File Although there is no consensus on a biomarker for ME/CFS, several markers have been suggested as potential candidates. The aim of this systematic review was to collate and appraise available literature on suggested biomarkers for ME/CFS.This systematic review used “biomarker” keywords in the abstract and title to determine the search. Therefore, the only articles that were retrieved were limited to those that specifically contained the keywords as according to Cochrane’s guidelines . StudiesFemales were more prominent across the studies where 76% of the ME/CFS patients and 72% of the HC were female. In literature, ME/CFS is more commonly reported in females . There iMajority of the participants in this review are diagnosed according to FC , 111–115ME/CFS is a multifactorial condition that is also often associated with comorbid conditions including, but not limited to, IBS and fibromyalgia. Presence of multiple comorbidities complicates determining a biomarker specific for ME/CFS. Where possible, stratification of patients with and without comorbidities may assist with further understanding ME/CFS-specific pathomechanisms. Furthermore, there are other illnesses that are currently only diagnosed with case criteria and have significant symptom and pathological overlap with ME/CFS such as Gulf War illness . InclusiAppropriate selection criteria of HC are a necessary experimental consideration. There were two studies that investigated pathology in ME/CFS patients and blood-related relatives compared with HC , 109. ImThe majority of the biomarkers were blood-based. Blood-based biomarkers are recognised as accessible, direct and non-invasive, especially in at-risk populations. Importantly, condition of the blood or blood product is important to consider. Recovery of frozen lymphocyte samples resulted in reduced viability . This reThere were 20 studies that investigated genetic and epigenetic changes in ME/CFS patients compared with HC , 111. MaImmune dysfunction was the most prevalent biomarker type investigated in the included publications. Among these studies supporting immune dysfunction, there were five that investigated NKCC , 114. NK2+ influx in NK cells from ME/CFS patients compared to HC [2+ in regulating NK cell function and maintenance of cellular homeostasis. In contrast, Nguyen et al. did not show any significant differences in Ca2+ influx through TRPM3 in ME/CFS patients compared to HC; however, this was potentially due to interfering small inward currents activated by PregS through the flow cytometry technique which was not apparent in the confocal microscopy approach [2+ influx. While patch clamp is the gold standard for investigating ion channel physiology, it can be conducted complimentary with confocal live imaging. TRP channels are also stress-activated including infection. ME/CFS development post-infection was described in three of the included studies [Cabanas et al. and Eaton-Fitch et al. assessed potential physiological processes underlying NK cell dysfunction including measurement of TRPM3 ionic currents and Caed to HC , 29. Theapproach . Patch c studies –49. An e studies .Three studies described changes in mitochondria , 96, 113Neurological changes in ME/CFS were investigated in eight  of the included studies , 112. Th2+ influx [Many of these biomarkers were studied in isolation but may be part of a complex multidisciplinary process as displayed by some of the overlap between observations made and extensive crosstalk between each system. There is evidence of widespread genetic, immune, neurological, mitochondrial and endocrine differences in ME/CFS compared with HC. Genetic abnormalities lead physiological disruption at the cellular and tissue level. Potential linking pathways have been described in Fig. + influx , 62, 77.Use of exercise to promote PEM was commonly used in some studies. There are significant limitations to use of exercise to stimulate and measure biological changes in ME/CFS patients. The studies also do not cater for potential delays in onset of PEM where some patients reported symptoms presenting post 24 h. In many cases, inducing PEM through exercise requires specialised equipment and transport of patients to specific locations in order to collect data and many patients are unable to participate due to the severity of their illness. There is also a trade-off for specificity in all studies, for example Frith et al. had a sensitivity of 77% but a specificity of 53% . Many MEMost of these biomarkers fall under “moderate to difficult-to-detect” and requires extensive multi-step laboratory experiments by trained scientists in order to make these observations. Considerations for translation of the biomarker from laboratory to more automated high-throughput industry technology are critical. A rigorous approach is required for biomarkers to enter into the clinical stages of development . Byrnes Integration of machine learning algorithms with the development of biomarkers will allow to assess a model for classification and feature selection. With machine learning, you can select multiple strong candidates for ME/CFS and train the software to recognise these patterns and appropriately classify patients and HC with great sensitivity, specificity and accuracy as described by Missailidis et al. . In thisStability is an important component when considering biomarkers. In some of the biomarkers investigated, changes in certain parameters were influenced by illness severity, illness duration, and onset patterns such as infectious onset and time , 75, 100Many of the studies investigated were standalone studies with insufficient or proof-of-concept sample sizes. It is difficult to compare studies of the same marker as their methodology varied significantly including cell or tissue type. Some studies implemented two phases, discovery and validation phase and this process was effective in demonstrating test-test reproducibility. In some cases, the sample sizes are relatively small; however, reproducibility is also demonstrated through assessing many individual cells across multiple samples and shows robustness between and within samples , 29, 83.Levels of quality varied across the included papers, with six studies appropriately addressing all quality criteria , 115. AlThere is currently no consensus on a biomarker for ME/CFS; however, there is significant body of biological evidence demonstrating that ME/CFS results in widespread immunological disruption. Effective biomarkers are accessible and have strong sensitivity, specificity and selectivity to detect disease targets. Additionally, having multiple layers of evidence such as genetic and physiological high test-test reproducibility of results is paramount. The included studies ranged in efficacy, quality, and potential to be developed into a diagnostic biomarker. This review also corroborates the use of NK cells as a suitable model to investigate the pathomechanism of this illness due to the consistency observed over time. This systematic review identifies potential associations between the findings and highlights that these systems do not work independently and rather they could be part of a complex integrative network. The heterogeneity shown across many of the included studies highlights the need for multidisciplinary research and uniform protocols in ME/CFS biomarker research.Additional file 1. Database search terms.Additional file 2. Study and participant results. Table S1. Genetic biomarkers; Table S2. Immunological biomarkers; Table S3. Metabolomics/ mitochondrial/ microbiome biomarkers; Table S4. Endovascular/circulatory biomarkers; Table S5. Neurological biomarkers; Table S6. Ion channel biomarkers; Table S7. Physical biomarkers. Additional file 3. JBI quality assessment table and descriptions


We aimed to explore the value of gut microbiota and tricarboxylic acid (TCA) metabolites in early diagnosis of necrotizing enterocolitis (NEC) among infants with abdominal manifestations.n = 16) and NEC (n = 16) groups. Faecal samples were collected when the infants were enrolled. The gut microbiota was analysed with high-throughput sequencing, and TCA metabolites were measured with multiple reaction monitoring (MRM) targeted metabolomics. Receiver operating characteristic (ROC) curves were generated to explore the predictive value of the obtained data.Thirty-two preterm infants with abdominal manifestations at gestational age ≤ 34 weeks were included in the study and were divided into non-NEC (p > 0.05). At the phylum level, Proteobacteria increased, and Actinomycetota decreased in the NEC group (p < 0.05). At the genus level, Bifidobacterium and Lactobacillaceae decreased significantly, and at the species level, unclassified Staphylococcus, Lactobacillaceae and Bifidobacterium animalis subsp. lactis decreased in the NEC group (p < 0.05). Further Linear discriminant analysis effect sizes (LEfSe) analysis showed that the change in Proteobacteria at the phylum level and Lactobacillaceae and Bifidobacterium at the genus level scored higher than 4. The concentrations of succinate, L-malic acid and oxaloacetate in the NEC group significantly increased (p < 0.05), and the areas under the ROC curve for these metabolites were 0.6641, 0.7617, and 0.7344, respectively.There was no significant difference in alpha diversity or beta diversity between the two groups (unclassified Staphylococcus, Lactobacillaceae and Bifidobacterium animalis subsp. lactis at the species level as well as the increase in the contents of some TCA metabolites, including succinate, L-malic acid and oxaloacetate, have potential value for the early diagnosis of NEC.Decreased  AccordiAt present, the diagnosis of NEC is mainly based on clinical manifestations such as bloody stools, vomiting, diarrhea, abdominal distension, and imaging presentation of pneumatosis intestinalis, portal venous gas or pneumoperitoneum . HoweverProteobacteria, and corresponding underrepresentation of Firmicutes and Bacteroidota , branched-chain amino acids, trimethylamine oxide, tryptophan and indole derivatives  and someEubacterium coprostanoligenes group, Faecalibacterium, and Oscillibacter (Bacteroidota and Prevotella (Some of intermediates of the tricarboxylic acid (TCA) cycle such as succinate, malic acid and oxaloacetate are not only the metabolites of cellular metabolism , but alslibacter  and succevotella . Thus, tevotella . Some inevotella . HoweverThe aim of this study was to clarify whether the gut microbiota and its metabolites contribute to the early diagnosis of NEC among those infants with abdominal distention or vomiting, diarrhoea or bloody stools.2.A prospective cohort study was performed in Children’s Hospital of Chongqing Medical University from April to November in 2021. This study was approved by the Ethics Committee of the Children’s Hospital of Chongqing Medical University (No. 2021.23) and registered in the China Clinical Trial Canter (ChiCTR2100044842). Parents of the enrolled infants all signed an informed consent form, and all infants were treated according to conventional treatment without any extra interventions.2.1.Infants who met the following criteria were enrolled: (1) infants with gestational age ≤ 34 weeks and (2) infants with one of the following manifestations including abdominal distention, vomiting, diarrhoea or bloody stools.2.2.The exclusion criteria were as follows: (1) infants who died during hospitalization for causes other than NEC, (2) infants with congenital gastrointestinal malformations , (3) infants who were discharged against medical service, (4) infants who did not complete the determination of gut microbiota and metabolites, and (5) infants whose parents refused to participate in this study.2.3.Neonates were divided into NEC and non-NEC groups according to the final diagnosis. Infants in the NEC group were required to meet Bell’s diagnostic criteria (stage II or above) . The non2.4.Naturally excreted faecal samples from the enrolled infants were collected with disposable sterile swabs on the day of enrolment and stored in the refrigerator at −80 degrees for further examination. Clinical data such as gender, gestational age, birth weight, feeding strategy, maternal condition and treatments in hospital duration were routinely collected. All infants enrolled were followed up until they were diagnosed with NEC or discharged from the hospital.2.5.Half of each sample were used for the determination of microbiota and sequencing was performed with the standard kit by Applied Protein Technology Co., Ltd.  according to the standard protocol. Total genome DNA from samples was extracted using CTAB method and the concentration and purity was monitored on 1% agarose gels. According to the concentration, DNA was diluted to 1 ng/μl using sterile water. 16S rRNA genes were amplified used the specific primer 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT) to amplify the V3-V4 region . All PCR2.6.g, 4°C). The supernatant was dried in a vacuum centrifuge and in the liquid chromatograph mass spectrometer (LC–MS) analysis, the dried supernatant was re-dissolved in 100 μL acetonitrile/water , adequately vortexed, and then centrifuged   at 4°C to remove the protein. The mixture was centrifuged for 20 min  . The sam 15 min) .2.7.correlation t test. Quantitative data from nonnormal distributions were expressed as interquartile ranges (IQRs) and analysed with the Wilcoxon signed rank sum test. Qualitative data were analysed with Fisher’s exact test. Reads were clustered into operational taxonomic units (OTUs) at 97% similarity  obtained by the Kruskal–Wallis rank sum test were used to further characterize the variation among microbiomes (Spearman rank correlation in R language. Receiver operating characteristic (ROC) curves and all data were generated with GraphPad Prism .All data of clinical recorders and metabolites were analysed with SPSS statistical software . Quantitative data with a normal distribution are expressed as the mean ± standard deviation (SD) and were analysed with a milarity . A core milarity . The betrobiomes . A corre3.3.1.n = 14), failure to collect faecal samples (n = 35). Other 4 infants were also excluded from further study because of failure to complete determination of the microbiota and metabolites (n = 4) in NEC infants. Therefore, 16 infants were enrolled in the NEC group and the other 16 infants without NEC were matched as the control group. The flow chart of this study was shown in p > 0.05). Infants in the NEC group had a longer hospital duration and a higher incidence of sepsis than those in the non-NEC group . Firmicutes and Bacteroidota showed no difference (p > 0.05) . Enterococcus, Escherchia-Shigella, Staphylococcus, Klebsiella, Enterobacter and Acinetobacter et al. showed no significant difference (p > 0.05)  . At the  > 0.05) . At the 3.5.p < 0.05, p > 0.05, To determine the difference of metabolites between the two groups, the concentrations of TCA intermediates were analyzed, and we found that some of them were significantly different between the two groups. Succinate [956.42  vs. 166.19 ], L-malic acid [193.05  vs. 80.33 ] and oxaloacetate [2960.10  vs. 705.67 ] concentrations were significantly increased in the NEC group compared to the non-NEC group , 0.7617 (95%CI: 0.5946 ~ 0.9289) and 0.7344 (95%CI: 0.5538 ~ 0.9149), respectively, which showed low-to-medium early diagnosis values –C.3.6.Proteobacteria abundance and negatively correlated with Firmicutes and Actinomycetota abundances , and other data can be obtained from the corresponding author.The microbiota databases for this study can be found in the NCBI , Joint Medical Research Project of Chongqing Science and Technology Commission (2022MSXM039), Science and Health Project of Chongqing Health Commission (2020FYYX217), National Natural Science Foundation of China (82001602), China Postdoctoral Science Foundation (No. 2022MD713716), and the General Basic Research Project from the Ministry of Education Key Laboratory of Child Development and Disorders (No. GBRP-202107).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Square dance is gaining increasing popularity among middle-aged and older Chinese women who are also at high risk of sleep disturbance. Although previous studies have shown exercise could improve sleep quality, the association between square dance and sleep quality remains to be discussed, and even less is known about the potential mechanism underlying this association.This study aims to investigate the relationship between square dance and sleep quality and test if social support and depressive symptoms together play a serial mediating role in the influence of square dance on sleep quality.A cross-sectional study was conducted among 549 middle-aged and older Chinese females from September to December 2020 in Shao Yang City, Hunan Province of China, with ethics approval granted (SYU [2020]002). Square dance involvement was assessed by three questions about the time participants spent in square dance. Social support, depressive symptoms, and sleep quality were measured using the Pittsburgh Sleep Quality Index (PSQI), Social Support Self-Rating Scale (SSRS), and 9-item Patient Health Questionnaire (PHQ-9), respectively. The serial mediation model was analyzed by the bootstrapping method to assess whether social support and depressive symptoms mediate the relationship between square dance and sleep quality.Two-thirds of the participants had high involvement in square dance and most reported a moderate and high level of social support (98.54%). The prevalence of depressive symptoms and sleep disturbance was 19.49 and 26.78%, respectively. The serial mediation model showed a significant association between square dance and sleep quality, which was fully mediated by social support and depressive symptoms in a serial model .Our study extends the understanding of how square dance is associated with sleep quality through the serial mediating roles of social support and depressive symptoms. It provides crucial implications for developing square dance interventions to improve sleep quality among middle-aged and older Chinese females. The The beneficial roles of square dance in promoting human health have been well-documented, with abundant evidence showing its health-promoting effects in terms of physical, mental, and social wellbeing . As a moSleep disturbance, characterized by persistent inability to fall asleep or maintain sleep, is a significant public health problem affecting over 10% of the worldwide population , 17. TheThe current study aims to fill those gaps by studying the mechanisms by which square dance impacts sleep quality in middle-aged and older Chinese females. Based on previous research regarding the significant associations among square dance, social support, depressive symptoms, and sleep disturbance, this study hypothesized that square dance indirectly reduces the risk of sleep disturbance through social support and depressive symptoms in two separate serial mediation models. Serially, we hypothesized that higher involvement in square dance would be associated with higher levels of social support and, in turn, fewer depressive symptoms, thus decreasing the risk of sleep disturbance. Therefore, we proposed the following hypotheses.Hypothesis 1. There is an association between square dance and sleep disturbance ;Hypothesis 2. There is a specific indirect effect of square dance on sleep disturbance through social support (indirect effect a1b1);Hypothesis 3. There is a specific indirect effect of square dance on sleep disturbance through depressive symptoms (indirect effect a2b2);Hypothesis 4. There is a serial multiple mediation effect of square dance on sleep disturbance through social support and then through depressive symptoms .22.1n = z  was estimated at 40% based on past studies . Data were collected by a research team composed of 10 college students from the Music and Dance Academy of Shaoyang College. The research team received unified and standardized training combining lecture and role-play practice on questionnaire interviews prior to the formal investigation. The research team approached the participants in each square during the night when they were performing square dance and explained the purpose and procedure of the study in detail to each interested participant. After providing written informed consent, the eligible participants were invited to complete a battery of questionnaires. Considering most participants were middle-aged and older people who may have difficulty reading and filling in the questionnaires by themselves, the research team conducted face-to-face interviews with the participants and then filled in the questionnaires for the participants. The answers were then checked by a quality control person on the spot to ensure integrity, accuracy, and consistency.2.32.3.1Square dance involvement was assessed by three questions asking about the time participants spent in square dance. The first question asked how many years participants had been involved with square dance, with optional answers ranging from 1 (less than 1 year) to 5 (more than 10 years). The second question asked how often participants performed square dance, with optional answers ranging from 1 (less than once a month) to 5 . The third question asked about the average length of each square dance session, with optional answers ranging from 1 (less than 10 min) to 5 (more than 60 min). A total involvement score was calculated by summing up the three question scores and ranged from 3 to 15, with a higher score indicating higher involvement in square dance.2.3.2Sleep quality was measured using the Pittsburgh Sleep Quality Index (PSQI), which is the most commonly used standardized self-administered scale to assess sleep quality and sleep disturbance over the previous month . Ever si2.3.3Social support was measured using the Social Support Self-Rating Scale (SSRS) developed by Xiao  to asses2.3.4Depressive symptoms were measured using the 9-item Patient Health Questionnaire (PHQ-9)  to asses2.3.5To account for observable potential confounding effects of sociodemographic variables, we collected the following variables as covariates in the fully adjusted models: age, race (ref: Han), education (ref: primary and below), employment (ref: not employed), income , marital status (ref: not married), illness (ref: no), hospitalization (ref: no), and medical insurance (ref: no).2.4Descriptive and correlation analyses were conducted using Stata v.15  for all The serial mediation analysis was carried out using SPSS PROCESS v.4.3 macro (Model 6) . A seria33.1n = 107) with a mean PHQ-9 score of 5.40 ± 1.93. Prevalence of depression at a mild level , moderate level , and severe level (PHQ-9 ≥ 15) was 30.05%, 15.85%, and 3.65%, respectively. The prevalence of sleep disturbance was 26.78% (n = 147), with a mean global PSQI score of 5.94 ± 2.78.The mean total score of square dance involvement was 9.19 ± 2.14. Most participants had been involved in square dance for less than 3 years (65.94%). Most went to square dance at least three times per week (67.94%) for 20–60 min each time (66.67%). The mean score of SSRS was 41.78 ± 7.54. Most participants reported having a moderate (61.38%) or high (37.16%) level of social support. The prevalence of depressive symptoms was 19.49% (3.2r > 0.80)  and negatively correlated with depressive symptoms  and sleep disturbance . Sleep disturbance was positively correlated with depressive symptoms  and negatively correlated with social support . Both mediators, social support and depressive symptoms, were negatively associated with each other . > 0.80) . Specifi3.3Confirming hypothesis 1, the association between square dance involvement and sleep disturbance after controlling for covariates was statistically significant , indicating higher square dance involvement was associated with less sleep disturbance. The combined contribution of square dance involvement and covariates explained 14.2% of the total variance. However, the association between square dance involvement and sleep disturbance was not statistically significant when mediators  were added , indicating a significant full mediation effect by the mediators. Square dance involvement was unrelated to sleep disturbance, independent of the effect of social support and depressive symptoms. The total indirect effect of square dance involvement on sleep disturbance was significant .p < 0.001), which in turn was associated with lower sleep disturbance . The indirect effect of square dance involvement on sleep disturbance through depression was significant , indicating that those who had higher square dance involvement had lower depressive symptoms , which in turn was associated with lower sleep disturbance , confirming hypothesis 3. Finally, the indirect effect of square dance involvement on sleep disturbance through social support and depressive symptoms serially was significant . Thus, hypothesis 4 was also confirmed. Those with higher square dance involvement had a higher level of social support , which was associated with fewer depressive symptoms , and which, in turn, was associated with less sleep disturbance . Among the three mediation paths, depressive symptoms account for the largest proportion of the mediation effect (38%), followed by social support and depressive symptoms serially (34%) and social support (28%).Confirming hypothesis 2, The indirect effect of square dance involvement on sleep disturbance through social support was significant , indicating that those who had higher square dance involvement had higher social support . The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.JW: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Visualization, Writing – original draft. YY: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing. KQ: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing. ZO: Conceptualization, Data curation, Investigation, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing.


To date, few studies on the factors related to vaginal intraepithelial neoplasia (VaIN) have been published. In this study, we aimed to analyze the features of VaIN and identify underlying risk factors.Patients with VaIN or vaginitis histologically confirmed at the Industrial Street Branch of Chengdu Women’s and Children’s Central Hospital from July 2020 to December 2021 were included. We statistically analyzed their baseline clinical characteristics, human papillomavirus (HPV) infection status, cytology results, and pathology results. Categorical indicators were analyzed using the chi-square test or Fisher’s exact test, as appropriate. Differences were considered to be statistically different with p < 0.05.A total of 62 patients with VaIN (mean age: 39.06 ± 11.66 years) and 32 with vaginitis (mean age: 41.13 ± 13.43 years) were included. Synchronous cervical intraepithelial neoplasia (CIN) was histologically identified in 46 (74.2%) patients with VaIN and 7 (21.9%) with vaginitis (p < 0.001). Low-grade squamous intraepithelial lesions (LSILs) and atypical squamous cells of undetermined significance (ASC-US) were the most frequent cytological abnormalities in both groups. Patients with VaIN only (62.5%) were more likely to be negative for intraepithelial lesion or malignancy than patients with synchronous CIN . No statistically significant difference in HPV infection was noted between patients with VaIN and those with vaginitis (p = 0.439). The most prevalent HPV genotype in patients with VaIN or vaginitis was HPV16, whereas both HPV58 and HPV16 were the most common in patients with concurrent CIN.Attention should be paid to HPV16- and HPV58-positive patients with cytological abnormalities such as ASC-US and LSILs  to avoid misdiagnosis or underdiagnosis and to facilitate early interventions for VaIN. The incidence of vaginal intraepithelial neoplasia (VaIN) is difficult to accurately determine, given that VaIN is an asymptomatic disease. VaIN is rare, with an incidence of approximately 0.2–0.4 cases per 100,000 women, accounting for approximately 1% of all lesions in the epithelial layer of the lower genital tract . Early sVaIN is classified similarly to cervical intraepithelial neoplasia (CIN), with HPV considered the major initiator. In particular, VaIN I is a benign form of HPV infection that typically resolves on its own, whereas VaIN II and III are considered precancerous lesions . VaIN isVarious factors, such as age, HPV infection, cytology results, gravidity, parity, menopausal status, and number of male sexual partners, can increase an individual’s risk of VaIN; therefore, regular screening must be prioritized to avoid underdiagnosis. Owing to the rarity of VaIN, published data on the associated risk factors are limited. We have been surprised by the difficulty in reduce the underdiagnosis of VaIN. Given the limited data on risk factors, we conducted this retrospective analysis of VaIN in 62 patients with VaIN and 32 with vaginitis as a control group, with an emphasis on the importance of early detection of potential VaIN-related issues via vaginal examination and screening.A retrospective review was performed on women with histologically confirmed VaIN who were referred to a gynecological outpatient clinic at the Industrial Street Branch of Women’s and Children’s Central Hospital  from July 2020 to December 2021. All women diagnosed with VaIN or vaginitis via colposcopy-directed biopsy were included. VaIN and vaginitis were histologically diagnosed by two independent gynecologic pathologists. Women with synchronous lower genital malignancies, acute inflammation of the lower genital tract, or incomplete documentation were excluded from analysis. Those with vaginitis were included as a control group.This study was conducted in accordance with the ethical principles embodied in the Declaration of Helsinki and was approved by the Ethics Committee of Chengdu Women’s and Children’s Central Hospital, reference number 2023(41). The requirement for the acquisition of informed consent from participants was waived by the Ethics Committee of Chengdu Women’s and Children’s Central Hospital owing to the retrospective nature of the data.General clinical data, including age, menopausal status, gravidity, parity, number of sexual partners, HPV infection status, cytology results, and cervical epithelium condition were collected from hospital medical records.All statistical analyses were performed using GraphPad Prism software version 8.0 , with statistical significance set at p < 0.05. Categorical indicators were analyzed using the chi-square test or Fisher’s exact test, as appropriate.Table Except for one patient with vulvar itching, almost all patients were asymptomatic but had abnormal cytology and/or HPV test results during regular screening for cervical cancer. Overall, 37 (59.7%) of patients with VaIN and 18 (56.3%) with vaginitis exhibited cytological abnormalities (p = 0.798). Among those, low-grade squamous intraepithelial lesions (LSILs) and atypical squamous cells of undetermined significance (ASC-US) were the most common in both groups, with no cases of atypical glandular cells on Pap smears Table . Analysin = 25] in patients with VaIN and 37.9% [n = 11] in patients with vaginitis). No cases of a single, low-risk HPV infection were observed among patients with vaginitis. Patients with vaginitis seemed more susceptible to multiple, high-risk HPV infections than patients with VaIN (p < 0.001)  developed an HPV infection. The HPV positivity rate was marginally higher in patients with VaIN (96.8%) than that in patients with vaginitis (90.6%); however, the difference did not reach statistical significance p = 0.439). In patients diagnosed with HPV infection, the most prevalent type of HPV infection was a single, high-risk HPV infection  to avoid misdiagnosis or underdiagnosis and to facilitate early interventions for VaIN.