Tubal ectopic pregnancy (TEP) occurs when an embryo aberrantly implants in the fallopian tube, leading to abortive or ruptured tubal ectopic pregnancy (AEP or REP). Poor outcomes of REP include maternal infertility or mortality. Current studies on the prevention and treatment of ruptured tubal ectopic pregnancy (REP) are unfortunately hampered by a lack of the cell spectrum and cell–cell communications in the maternal–foetal interface. Here, we investigate the mechanisms of tubal rupture through single‐cell transcriptome profiling of the fallopian tube‐trophoblast interface in REP, AEP and intrauterine pregnancy patients. In REP, extravillous trophoblast (EVTs) cells form a dominant cell population, displaying aggressive invasion and proliferation, with robust differentiation into three subsets. Cell communication analysis identified colony‐stimulating factor 1 (CSF1), overexpressed by fallopian tube secretory epithelial cells in REP, with CSF1R on EVTs and macrophages, as a ligand/receptor pair that stimulates EVT invasion and macrophage accumulation. CSF1+ secretory epithelial cells stimulate EVTs migration and invasion, leading to a tubal rupture in REP. These results provide a mechanistic context and cellular milieu leading to tubal rupture, facilitating further study and development of therapeutics for REP in early pregnancy. Diagram of the main receptors and ligands expressed on the secretory epithelial cells, extravillous trophoblasts (EVTs) and macrophages that are involved in invasion and cellular recruitment. REP, ruptured tubal ectopic pregnancy. AEP, abortive tubal ectopic pregnancy. Three placenta‐specific cell types are responsible for its primary functions, including villous cytotrophoblasts , syncytiotrophoblasts (STBs), and extravillous trophoblasts (EVTs).The aberrant regulation of EVT invasion can lead to a series of obstetric syndromes. Incomplete invasion is associated with pre‐eclampsia, foetal growth restriction (FGR) and stillbirth,Under certain physiological conditions, the motion of tubal cilia, smooth muscle contraction and tubal secretory fluids play a central role in tubal transport. Altered cilia function, a perturbed chemotactic tubal microenvironment and dysregulation of migration by the fertilized egg can all result in stranding the embryo in the tube, and consequently, tubal pregnancy.Here, we conducted single‐cell sequencing to comprehensively determine the cell states and subtypes involved in the development of tubal rupture and maternal–foetal communication in the narrow space of fallopian tubal walls. Bioinformatic analyses were used to evaluate the functions of different cell types and subtypes, and generate detailed molecular and cellular maps of the human fallopian tubal–placental interface. These analyses provide an in‐depth understanding of tubal rupture at the single‐cell level and can help guide the development of novel therapeutic strategies to combat the aetiology of REP.22.1n = 14), abortive tubal EPs  or ruptured tubal EPs  at gestational weeks 6–8 were collected immediately after surgically resected chorionic villi and fallopian tube/uterine decidua. Then, transported tissues from the operation room to laboratory within 15 min. Voluntarily terminated intrauterine pregnancies that confirmed by ultrasonography combined with blood test or urine pregnancy test. For EP placental villi (including AEP and REP), the tubal implantation was first diagnosed by ultrasonography, followed by confirmation and removal by salpingectomy. All EP samples located in the ampulla of fallopian tube and were collected without receiving methotrexate treatment. AEPs are usually diagnosed by low β‐hCG levels and haemodynamic stability of unruptured fallopian tubes.All the maternal–foetal interface samples from normal intrauterine pregnancies IP, n = 4, abortiSamples collected by surgeries were processed immediately, including fixation in 10% formalin for histological and immunological analyses, immersed in Tissue Storage Solution  for following scRNA‐seq or flow cytometry staining and in RNAlater  for following real‐time PCR analyses, and cultured in appropriate media for explant experiments. Samples for histological and immunological analyses were obtained by the Department of Pathology, International Peace Maternity and Child Health Hospital (IPMCH), and processed by RecordBio company following standard protocols briefed later.2.2Decidual, fallopian tubal and placental tissue was isolated according to a previously published protocol with minor modifications.2.3This process was performed by Genergy Bio‐Technology Corporation and in accordance with the manufacturer's protocol. Cells were counted and loaded onto the Chromium Controller (10X Genomics) for a target recovery of 8000 single cells. Generation of gel beads in emulsion (GEMs), barcoding, GEM‐RT clean‐up, complementary DNA amplification and library construction were performed. Qubit was used for library quantification before pooling. The final library pool was sequenced on an Illumina Nova6000 platform using 150‐base‐pair paired‐end reads.2.4n = 2), 14,101 qualified cells of AEPs (n = 2) and 22,987 qualified cells of REPs (n = 3). Then, variation coefficient of genes was calculated with Seurat. Dimensionality reduction of data was performed by using principal component analysis based on the first 2000 highest variable genes. A k‐nearest neighbour graph was constructed from Euclidean distances in the space of the first 10 significant principal components. Louvain modularity optimization algorithm was utilized to cluster the cells in the graph and clustering results were visualized by using Uniform Manifold Approximation and Projection (UMAP) project.Mapping to GRCh38 human genome, quality control and read counting of Ensemble genes was performed by CellRanger software with default parameter (v6.0). The raw data and processed data were uploaded GEO datasets (GSE207630). Unsupervised clustering was performed with R (Seurat package version 4.0.5). Genes included in the analysis expressed in a minimum of five cells and the total number of expressed cells that expressed >100 and <6000 genes. The cells containing high percentages of mitochondrial genes to total genes, >20%, and haemoglobin genes, >3%, were flited out. A total of 61,119 qualified cells were used for analysis in scRNA‐seq, including 24,031 qualified cells of IPs . The list of DEGs  per cluster then were subjected to functional enrichment analyses using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) implemented in the clusterProfiler R Bioconductor package. Significantly enriched GO terms and KEGG pathways were selected by a threshold FDR  ≤ 0.05. Function Dotplot from R package ggplot2 (https://www.rdocumentation.org/packages/ggplot2/versions/3.3.5) and function FeaturePlot from R package Seurat (Version 4.0.5) have been used to generate the dot plot and violin plot.The Seurat FindAllMarkers function performed marker genes expression analysis for each cluster using the Wilcoxon rank sum test (adjust 2.6http://cole-trapnell-lab.github.io/monocle-release/monocle3/)http://velocyto.org/).The present study used two different algorithms to infer the development trajectories: one implemented in R package monocle3   for 48 h. The THP‐1 macrophages were subsequently cultured in THP‐1 medium for 24 h. Latterly, the THP‐1 macrophages the stimulated with human CSF1 (20 ng/mL) for 48 h to Macro2 phenotype.The human trophoblast cell line HTR8‐SVneo was obtained from Dr. PK Lala . The cells were cultured in DMEM/F12 containing 10%foetal bovine serum (FBS) with 1% penicillin–streptomycin (P/S) antibodies. HTR8‐SVneo were subsequently cultured in CSF1  medium (100 ng/mL) for 24 h, and then were extracted RNA. Human monocytic cell line (THP‐1) was obtained from the cell bank of Chinese Academy of Sciences . THP‐1 monocytes were cultured in THP‐1 medium  at 37°C and 5% CO2.9Explants were cultured according to a previously published protocol.2.10For the EVT were resuspended in cell staining buffer. For the macrophage, the single cells of the fallopian tube (location of embryo implantation) were resuspended in cell staining buffer . Cells were blocked with Human TruStain FcX Fc receptor blocking solution  and stained with Zombie Aqua Fixable Viability Kit  that eliminated the influence of dead cells. Then, the EVT was stained with PE. Anti‐human HLA‐G antibody . Cells were carried out using a FACS analyser (BD) and analysis after acquisition was conducted by FlowJo software (V10.4).2.11For assessing cell migration in vitro, the wound healing assay was performed. Cells were seeded into 6‐well plate. When the HTR8‐SVneo cells reached 90%–95% confluence, a 200 μL pipette tip was used to scrap the culture plate along the centre axis of the well. Next, the scraped cells were washed twice by DMEM/F12 medium, cultured in medium under differential conditions, and photographed at 0, and 18 h after scratching. Image J software was used to calculate wound healing percentage. The positive control: DMEM/F12 plus 10%FBS with P/S antibodies (positive medium). The negative control: DMEM/F12 plus 2%FBS with P/S antibodies (negative medium). The different concentrations of GW2580 were mixed in the positive medium. While the different concentrations of CSF1 were mixed in the negative medium. photographed at 0 and 18 h after scratching.2.125 cells per well were plated in the upper chamber precoated with Matrigel membrane. The lower chamber was filled with 500 μL DMEM containing 10% FBS, or 500 μL DMEM containing 10% FBS and 100 ng/mL CSF1. Cells in the lower chamber were stained for 30 min with crystal violet after 24 h of incubation. The number of migrated cells was counted using an inverted microscope (Nikon).Cell invasion was detected by the ability of HTR‐8/SVneo cell to cross the 8‐mm pores of polycarbonate membranes . 1 × 102.132O2 and 2% bovine serum albumin (BSA) in PBS. Subsequently, sections were incubated at 4 overnight with primary antibodies, Room temperature 1 h with secondary antibodies, followed by 10 15 mins with TYR‐488/TYR‐CY3 or TYR‐488/TYR ‐CY3/TYR‐CY5 solution. The primary antibodies including rabbit anti‐HLA‐G , rabbit anti‐CSF1 , rabbit anti‐CSF1R , rabbit anti‐MMP2 , mouse anti‐Oviductin , rabbit anti‐MKI67 , mouse anti‐ISYNA1, rabbit anti‐CD80 , and mouse anti‐CD206 .All the samples of paraffin blocks were collected by the Department of Pathology, IPMCH, followed by cut into 5‐μm slices using the microtome . The sections for immunological analyses were performed by the Double Homologous antibody Fluorescence labelling Kit  and in accordance with the manufacturer's protocol. Co‐staining of three antibodies was performed using a three‐colour Fluorescence kit  based on the tyramide signal amplification (TSA) technology according to the manufacture's instruction. In brief, paraffin sections were deparaffinized, rehydration, antigen retrieval, and followed by blocked with 3% H2.142O2 for 25 min. The sections were incubated with 0.2% Triton X‐100 and 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature and were then incubated overnight with primary antibodies at 4°C. Primary antibodies used for IHC include: rabbit anti‐HLA‐G , rabbit anti‐MMP2 , rabbit anti‐CSF1 , rabbit anti‐CSF1R  and rabbit anti‐PAX8 . After overnight, samples were washed in PBS and incubated with the secondary antibody. Slides were visualized using diaminobenzidine tetrahydrochloride (RecordBio), and nuclei were counterstained with haematoxylin.Paraffin blocks were cut into 5‐μm slices, then deparaffinized, rehydrated and processed for antigen retrieval by a standard microwave heating technique in antigen retrieval solution (pH = 8) . Endogenous peroxidase activity was quenched by incubating the sections in 3% H2.15Total RNA of each sample was isolated by RNA Isolator Total RNA extraction Reagent . The cDNA was synthesized with the HiScript II Q RT SuperMix  according to manufacturer's instructions, and real‐time PCR reaction was done with QuantStudio 7 Flex Real‐Time PCR system (Life Technologies) and ChamQ Universal SYBR qPCR Master Mix . The amount of target mRNA  was determined using the ∆∆Ct method with GAPDH as the internal control with three replications. GAPDH‐F: GTCTCCTCTGACTTCAACAGCG. GAPDH‐R: CCACCCTGTTGCTGTAGCCAA. MMP2‐F: GATACCCCTTTGACGGTAAGGA. MMP2‐R: CCTTCTCCCAAGGTCCATAGC.2.16t‐test was used if the data were normally distributed and have equal variances, and Welch's t test was applied if the data were normally distributed and have unequal variances. One‐way ANOVA was applied to examine the differences of baseline characteristics among the three groups . Continuous variables are presented as mean ± SD/standard error of mean (SEM). The linear regressions of measured parameters were calculated with ages and gestational days as independent variables. Invasion depth of EVT in EP refers to the differentiated EVT close to cytotrophoblast cells column to the distance between the adjacent serous layer of EVT. Statistical analyses were performed using GraphPad Prism 8 . p‐Values < 0.05 were considered statistically significant.For continuous variables, Student's 33.1Since the placenta is implanted in the fallopian tube wall at different invasion depths in REP and AEP, we speculated that, in REP, EVT exhibit a high proliferative capacity and invade the serosal layer to ensure an adequate blood and nutrient supply to support embryo development, but which together result in tubal rupture Figure . Immunos3.2To systematically investigate differences in the respective REP, AEP and IP implantation microenvironments by identifying EVT subtypes and characterizing intercellular communications specific to REP, maternal and placental tissues were collected from the maternal–foetal interface of each pregnancy type  and isolated cells were mixed for single‐cell sequencing Figure . The cel3.3), which are associated with cell cycle and cell division , suggesting that this cell subtype could function in determining proliferative potential. GO analysis further showed enrichment in feature genes related to extracellular matrix (ECM) organization and positive regulation of EVT invasion in EVT3 cells , EVT2  and EVT3 (MMP2+) Figure . EVT1 cen Figure , suggess Figure .). Notably, invasive genes were expressed at a higher level in the EVT3 cells of REP patients than in the same subset of AEP patients  staining with anti‐ISYNA1, anti‐MKI67 and anti‐MMP2 antibodies in serial sections of human placental villi embedded in paraffin. Our results revealed that ISYNA1+ EVTs exhibited little or low expression of HLA‐G at the proximal site of the fallopian tube secretory epithelial cells (FTSECs), while MKI67+ EVTs were localized at the distal end of the column, with invasive EVTs (MMP2+) showing dispersed localization in the fallopian tube Figure . Notabls Figure .Trajectory analysis with Monocle3 and RNA velocity analysis of EVT subpopulations were consistent in REP samples, showing two distinct EVT lineages in which EVT2 cells could differentiate into either EVT1, with absorbing nutrients to support foetal growth (path 1), or into EVT3, with a high invasive capacity to break into the fallopian tubal wall (path 2) Figures and S2B. 3.4The implantation immune microenvironment is a complex, multilevel interaction network that provides major contributions to regulating EVT function. Identification of immune‐related cells using known marker genes revealed that macrophages/monocytes were the dominant cell type in both AEP and REP, while NK cells accounted for a relatively small population Figures  and S3A.Further clustering analysis and annotation with marker genes identified four macrophage subtypes and monocytes Figures , includi3.5To explore the association between maternal epithelia and EVT, we next identified epithelial cells based on their transcriptomic profile. Notably, epithelial cells (EPCAM+) could be partitioned into five cell types, including ciliated epithelia (FOXJ1+), unciliated epithelia (PAX8+), secretory epithelia , human placental epithelial cells  and undefined epithelia (EPPK1+) Figure . The latAside from epithelial cells, the fibroblast/stroma/endothelia cells were also identified in nine cell types Figure . The F3/CellChat analysis was conducted to explore this possibility, which identified FTSECs as the predominant intercellular communication “hub,” with secreted signals from these cells predicted to interact with both macrophages and EVTs Figure . To furt, transwell assay  and villous explant cultures . The results indicated that exogenous CSF1 could obviously stimulate the migration and invasion of HTR8‐SVneo, while exposure to GW2580 inhibited their migration and invasion  differentiation and function, which impacts pregnancy outcomes.Several studies have illustrated the different EVT subtypes that participate in IP. Based on their distinct spatial location, EVTs can be subdivided into two major subtypes: interstitial EVT cells , and endovascular EVT cells .CSF1 encodes a cytokine that positively regulates the production, differentiation and function of macrophages, and which is therefore likely involved in placental developmentTNF‐α and iNOS, which induces apoptosis in trophoblasts and inhibits EVT invasion, ultimately leading to abortion of the ectopic embryos.In the normal fallopian tube, in the nonpregnant state, the immune landscape is dominated by T cells ,While three EVT subsets are identified in EP, the mechanisms regulating the EVT subtype trajectories remain unexplored. In the present study, HTR8‐SVneo and villous explants were applied to verify the role of the CSF1/CSF1R ligand‐receptor pair in EVT proliferation and invasion. However, our results should be verified in a wider range of trophoblast cell lines or trophoblast organoids in future studies.In addition, the fallopian tube samples were collected after implantation and may not explain the aetiology responsible for differences in FTSECs between AEP and REP.In summary, our cell type‐specific data provide new insights into the aetiology of tubal rupture of REP patients by identifying key interactions between CSF1+ FTSECs and their surrounding immune macroenvironment. The present study thus emphasizes the contribution of FTSEC function in EP and further explores more complex interaction networks to promote the development of advanced interventions for tubal rupture.Xiaoya Zhao and Qian Zhu made the single cell suspension. Xiaoya Zhao performed scRNA‐seq computational analysis and experiments. Xiaoya Zhao wrote the manuscript. Qian Zhu cultured the villous explants. Li Yan, Sifan Ji and Yiqin Zhang collected clinical samples. Lisai Ha, Chuqing He, Yuan Tian, and Luting Chen performed pathological reviews. Jian Zhang conceived this study and provided funding. Mingqing Li, Jian Zhang and Qian Zhu designed the research, revised the manuscript.The authors have declared that no conflict of interest exists.All tissue samples used for this study were obtained through the International Peace Maternity and Child Health Hospital (IPMCH) in Shanghai. The study was approved by the institutional ethics committee of the IPMCH GKLW201909). Details about distribution of samples according to baseline characteristics are provided in the Table 909. DetaWritten consents for publication were obtained from all pregnancy patients .Figure S1: Major cell types identified by scRNA‐seq. (A) The harmony integrated data and present study visualized by UMAP. Colours indicate cluster or group. (B) Maternal–foetal interface from three groups visualized by UMAP. Colours indicate group. Violin plots showing log‐transformed, normalized expression levels to identify the 4 major clusters . Cells from Figure 2B are used for the violin plots.Figure S2: Spatially resolved developmental trajectories of EVT subsets.(A) Differential gene pseudotemporal expression trajectory map between AEP and REP group. EVT, extravillous trophoblast. (B) Pseudotemporal ordering trajectory map . REP, ruptured tubal ectopic pregnancy. AEP, abortive tubal ectopic pregnancy. IP, intrauterine pregnancy.Figure S3: Investigation of transcriptome characteristics of immune cell types and cell subtypes. (A) Dot plot presentation of scaled expression of each immune cell markers. Colours represent expression level and sizes of dot represent percentage of positive cells. (B) UMAP plots of NK subclusters. Each dot denotes one cell which is coloured by the identified subtypes. p, proliferative. (C) Violin plots of the expression of eight representative marker genes to identify the 5 major clusters. (D) Volcano plot showing DEGs  in REP compared to AEP. Blue and red represent down‐regulation and up‐regulation, respectively. (E) UMAP plots of macrophages. Each dot denotes one cell which is coloured by the groups. (F) Cell proportions of macrophages subpopulations in AEP and REP groups. Colours represent identified subtypes. M, macrophage. (G) Heat map showing relative expression of selected genes for macrophage subsets. Each column represents a single cell, and each row represents a selected gene. Colours indicate the expression levels as shown in the scale bar. (H) z‐scores of CD68, S100A9, S100A8, FCGR3A, GRP183, CCR7, CD206 and CD163 (mean expression levels) in the M1, M2 and M3. (I) Dot plot presentation of scaled expression of normal fallopian tube from Dinh et al. Colours represent expression level and sizes of dot represent percentage of positive cells. REP, ruptured tubal ectopic pregnancy. AEP, abortive tubal ectopic pregnancy. IP, intrauterine pregnancy.Figure S4: Identification of fibroblasts and endothelial types at the maternal–foetal interface. (A) UMAP plots of epithelial cells. Each dot denotes one cell which is coloured by the groups. (B) Subpopulation analysis UMAP plots of fibroblasts . ds, decidual stroma, endo, endothelia, F, fibroblasts. (C) Dot plot presentation of scaled expression of each subset markers. Colours represent expression level and sizes of dot represent percentage of positive cells. (D) GO analysis of enriched terms across feature genes of each subset. The terms with p < 0.05 are selectively shown. REP, ruptured tubal ectopic pregnancy. AEP, abortive tubal ectopic pregnancy. IP, intrauterine pregnancy.Figure S5: CSF1+ FTSECs promote EVT invasion and induce macrophage polarize to macro2. (A) Number of significant ligand‐receptor pairs between two cell subsets. The edge widths indicate the number of ligand‐receptor pairs. (B) z‐scores of genes between the FTSECs of AEP and REP (mean expression levels). Expression values were generated using scRNA‐seq data. (C) Double immunochemical staining for PAX8 and CSF1 in the FTSECs of AEP and REP . Scale bars, 100 μm. (D) Immunochemical staining for HLA‐G (EVTs marker) and CSF1R . Scale bars, 100 μm. (E) Representative images showed the migration of HTR8/SVneo cells in the differential treatments . CSF1 , GW2580  and 100 ng/mL CSF1 plus 20 μM GW2580. Negative control (DMEM/F12 + 2%FBS), positive control (DMEM/F12 + 10%FBS). (F) Immunofluorescence for CD206  and CD80  in macrophages. Scale bars, 50 μm. FTSECs, fallopian tube secretory epithelial cells. REP, ruptured tubal ectopic pregnancy. AEP, abortive tubal ectopic pregnancy. IP, intrauterine pregnancy.Click here for additional data file.Table S1. GO term analysis of the three EVT subsets.Click here for additional data file.Table S2. GO term analysis of the macrophage subsets.Click here for additional data file.Table S3. GO term analysis of the epithelial cell subsets.Click here for additional data file.


Scurvy is uncommon in the developed world, and clinical presentation may mimic other pathologic states. A thorough social and dietary history is essential to identifying patients at risk of vitamin C deficiency, which can then be easily treated.Scurvy is a disease of defective collagen synthesis that is characterized by easy bruising, gingival hemorrhages, poor wound healing, fatigue, and arthralgias. It is caused by dietary deficiency of vitamin C. This is the case of a patient with multiple risk factors for malnutrition who was diagnosed with scurvy. Scurvy may present with petechiae, purpura, abnormal hair growth, and poorly healing wounds, as seen in this image of the patient's left arm. Following his circumnavigation of South America, the crew spent nearly 4 months crossing the Pacific Ocean, during which time food and water supplies dwindled. Nineteen crew members died of scurvy, and dozens more were made invalid by the time they reached the Philippines.Scurvy is a disease that primarily affects the connective tissue and is caused by vitamin C deficiency. Vitamin C serves as a reducing agent for the hydroxylation of the amino acids proline and lysine, both essential reactions in the synthesis of collagen. Defective collagen synthesis reduces vascular integrity and results in the classic presentation of easy bruising, petechiae, gingival hemorrhages, and poor wound healing. Additional non‐specific symptoms of scurvy include anemia, fatigue, musculoskeletal pain, joint pain and swelling, and hypotension. Given the differential diagnosis for these signs and symptoms is broad, a dietary history is key to considering and making a diagnosis of scurvy.Manifestations of vitamin C deficiency have been recorded in ancient Egyptian hieroglyphics,Here we present the case of a 55‐year‐old male who was hospitalized for non‐specific symptoms and found to have poor dietary variety. Scurvy was diagnosed by history and physical exam, and confirmed by serum ascorbic acid concentration. Treatment of scurvy is by supplemental vitamin C, which can bring about full recovery.2A 55‐year‐old male came to the Emergency Department complaining of progressive weakness over the past 2–3 weeks and several pre‐syncopal falls at home. He had no known medical history or consistent primary care. He lived alone and smoked one pack of cigarettes daily and consumed 6–8 cans of beer daily. He used marijuana but no other recreational drugs. His diet had consisted almost exclusively of packaged deli meat, hot dogs, and white bread for the past 3 months. Collateral history from a relative indicated that he declined offers for more balanced food because large meals attenuated the intoxicating effects of his alcohol and tobacco use.On arrival to the Emergency Department, he was hypotensive with a blood pressure of 84/53 mmHg; all other vital signs were within normal range. He was alert and oriented with normal mental status. He had lost all teeth several years prior; there was no gingival inflammation. There were telangiectasias across the chest and purpura and petechiae on the bilateral upper extremities. There was a large eschar, several centimeters in diameter, on the left wrist which the patient reported had been present for several weeks Figure . The rig3, Hgb 6.1 g/dL, platelets 121 thousand/mm3) with macrocytosis (MCV 107.5 fL). Serum folate and cobalamin levels were within the reference range. His coagulation profile was notable for prothrombin time 14.8 s (reference 10.1–12.9 s), INR 1.3, PTT 32 s (reference 28–35.7 s). His metabolic profile was significant for a serum sodium level of 130 mEq/L (reference 132–146 mEq/L) and serum BUN 8 mg/dL (reference 9–23 mg/dL). Serum osmolality was 271 mOsm/kg (reference 275–295 mOsm/kg). Renal function was preserved. Liver function tests showed decreased serum albumin of 2.5 g/dL (reference 3.2–4.8 g/dL), mildly elevated AST of 42 U/L (reference 11–35 U/L), normal ALT and alkaline phosphatase , and elevated total bilirubin of 3.6 mg/dL (reference 0.3–1.2 mg/dL). Inflammatory markers included a normal ESR (18 mm/h) and elevated C‐reactive protein of 4.62 mg/dL (reference 0.0–0.50 mg/dL).His hematologic workup revealed pancytopenia . He was started on ascorbic acid 250 mg once daily by mouth. He was discharged home with prescriptions for vitamin C, a daily multivitamin, dietary protein supplements, and home physical therapy.He was seen for follow‐up in the outpatient clinic after 3 months and was noted to be recovering well. He reported abstinence from alcohol and demonstrated improvement in skin lesions and resolution of pancytopenia.3While the morbidity of scurvy in the general population is unknown, the prevalence of vitamin C deficiency in the United States has been estimated to be 5.9%.Dietary history is not heavily emphasized in clinical training, nor is it a required component of clinical documentation. Nevertheless, the clinician should be aware of risk factors for poor nutritional intake in adults; these include institutionalization, old age, mental illness, loss of teeth, social isolation, and drug and alcohol abuse.Rapid improvement in scurvy symptoms can be seen within days to weeks of initiating therapy with vitamin C supplementation.Scurvy is a debilitating but preventable condition that is uncommon in the developed world. The clinical presentation may mimic other pathologic states, obscuring the proper diagnosis. The incidence of scurvy is highly influenced by social factors. By paying careful attention to social and dietary risk factors, clinicians should be able to promptly identify and treat patients with scurvy, leading to full recovery.Michael Pope: Conceptualization; formal analysis; investigation; writing – original draft. Joshua Elder: Supervision; writing – review and editing.The authors received no funding for writing this article.The authors have no conflict of interest to disclose.Written informed consent to share de‐identified personal information including photographs was obtained from the subject by the authors.


Compared to the first quartile, it was observed that HBI in the last quartile was significantly related to lower odds of MetS in the multivariate analysis . Also, we observed a significant association between the last quartile of HBI with lower odds of high waist circumference (WC) . Our findings showed that the higher HBI score reduced MetS odds and WC. Therefore, to reduce the odds of MetS, a healthy pattern of beverage consumption, including drinking water, low‐fat milk, unsweetened tea, and coffee, and reducing the consumption of SSB are recommended. More studies are needed to confirm the findings.Some studies indicated that sugar‐sweetened beverages (SSBs) were related to MetS risk, and others found no relationship between MetS and SSBs. Therefore, the present study aimed to investigate the relationship between healthy beverage index (HBI) and MetS in Iranian adults. This cross‐sectional study was performed on baseline data FASA cohort. Out of 10,127 participants in the FASA cohort study, 8838 participants were included in this study. The National Cholesterol Education Program's Adult Treatment Panel (ATP) III was used for MetS definition. The HBI was calculated by a 125‐item food frequency questionnaire with standard criteria. The association between HBI and MetS and its components was evaluated by univariate regression. Multivariate regression with the backward method was used for adjusting confounders.  Our findings showed that the higher HBI score reduced MetS risk and WC. Therefore, to prevent MetS, it is recommended to have a good pattern of beverage consumption, such as drinking more water, low‐fat milk, unsweetened tea, and coffee, and reducing the consumption of SSB. More studies are needed to confirm the findings. This syDietary habits are related to some factors included in the definition of MetS and can play an important role in preventing obesity and MetS  risk factors in the rural population of FASA (Fars province of Iran) who participated in the Prospective Epidemiological Research Studies in Iran (PERSIAN) FASA cohort. At first, 11,079 adults were invited from Sheshdeh and 24 nearby city villages. General, medical and nutritional information, physical assessments, and biological sampling for laboratory examination were collected by educated experts at the beginning of the study (the details of this study have been published previously). A semi‐125 food frequency questionnaire (FFQ) was used for food intake evaluation  was used to measure height and weight. Also, the nonstretchable tape was used to evaluate hip circumference (HC) and waist circumference (WC). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were checked in both arms after 5 min in a sitting position and repeated after 15 min. Also, based on the study protocol, after 12‐ to 14‐h fasting, a 25‐mL sample was taken from all subjects, and samples were stored at −80°C for further analysis. Lipid profiles and fasting blood sugar (FBS) were assessed by frozen plasma samples  III, if each participant has three bottom criteria were considered as the MetS: SBP > 130 mmHg and/or DBP > 85 mmHg, fasting blood glucose (FPS) > 100 mg/dL, WC > 102 cm for men and > 88 cm for women, triglyceride (TG) ≥ 150 mg/dL, and high‐density lipoprotein‐cholesterol (HDL‐C) < 50 mg/dL for females and < 40 mg/dL for males . The significance level of the tests was considered less than .05. At first, HBI was converted into quartiles, and then the analysis was performed accordingly. Mean and standard deviation (SD) or percentage were used to describe the basic differences between the HBI quartiles. The one‐way analysis of variance (ANOVA) or Kruskal–Wallis for quantitative and Chi‐square test for qualitative variables analysis were used. Univariate analysis was used to determine the relationship between HBI and MetS and its related factors. Multivariate analysis with the backward method was used to adjust potential confounders , physical activity, education, energy intake, smoking, alcohol, medication use, and disease history).3p ˂ .001 for all). Also, we observed a significant difference in BMI (p ˂ .001), WC (p ˂ .001), HC (p = .004), FBS (p = .006), total cholesterol (TC) (p = .005), and HBI score (p ˂ .001) among HBI quartiles. Moreover, individuals in the last quartile of HBI had a lower intake of energy and fat, but protein, carbohydrate, and fiber intakes were higher than the first quartile of HBI score . But we observed no significant association among education, SBP, DBP, TG, and HDL‐C mean in HBI quartiles , also lower intake of SSBs (p < .001), but there were no differences in 100% fruit juice intakes among the HBI quartile (p ˃ .05).As shown in Table p < .001). Also, compared to the first quartile, it was observed that HBI in the last quartile was significantly related to lower odds of MetS in the multivariate analysis .According to Table 3) was significantly related to higher odds of high FBS , but in Q4 was significantly associated with lower odds of high WC , high TG , and low HDL‐C . A multivariate analysis of MetS components is reported in Table p < .002).The association between MetS components in univariate analysis is shown in Table 4The present study demonstrated that Iranian adults with the highest HBI had significantly lower odds of MetS. Also, the findings indicated that those with the highest HBI were likely to have lower WC than those with the lowest HBI after adjusting for confounders. Regarding other components of MetS, no significant relationship was found with HBI scores in the adjusted model. Further, the results showed that in a higher HBI, intake of water, unsweetened coffee and tea, low‐fat milk, and full‐fat milk was significantly higher than in a lower HBI. In contrast, SSB intake was significantly lower in the last quartile of HBI compared to the first quartile.The HBI was applied to assess the overall quality of beverage intake and determine whether changes in beverage consumption patterns were related to improvements in a health condition . Moreover, another study on Mexican adults showed that sweetened beverage intake increases MetS risk ; investigation ; writing – original draft . Zainab Shateri: Writing – original draft . Mahshid Rezaei: Writing – original draft . Ali Zangene: Writing – original draft . Reza Homayounfar: Data curation ; methodology ; writing – review and editing . Parisa Keshani: Investigation ; methodology ; writing – review and editing .The authors declare that they have no conflict of interest.


D) and increased nematode diversity by 31% (Shannon‐Weaver H'). However, burning increased plant parasites  and shifted community to more bacterial‐feeding genera . Generally, burning increased soil bio‐available nitrogen (NH4+–N and NO3−–N) content, which would be the main drivers causing nematode community to flourish via a “bottom‐up” effect. These results suggest that prescribed fire increases nematode diversity and alters community composition toward more plant parasites and bacterial feeders. Our findings highlight the importance of prescribed fire management in shaping short‐term nematode community structure and function, but the long‐term effects and impacts of these changes on soil nutrient and carbon cycling remain unknown.Fire is a common disturbance in many biomes, with both beneficial and detrimental effects on soil biology, which largely depend on fire intensity. However, little is known about the impact of fire on soil nematode communities in terrestrial ecosystem. In the present study, we investigated the effects of short‐term prescribed fire on soil nematode communities and soil properties in an old‐field grassland in Northern China. The results showed that burning significantly increased soil nematode abundance by 77% and genus richness by 49% compared to the control. Burning also decreased taxon dominance by 45% (Simpson's  Prescribed burning increased soil nematode abundance and richness. Prescribed fire significantly altered the nematode community structure. N‐rich food sources would cause the nematode community to flourish via a “bottom‐up” effect. The experimental site was originally under wheat‐maize crop rotation until 2000, when the land was left uncultivated and natural successional developed into an “old‐field” grassland. The dominant plant species in the old‐field grassland mainly include Cynodon dactylon, Gaura parviflora, Conyza canadensis, Melilotoides ruthenica, Humulus scandens, and Setaria viridis.The study was conducted at the Research and Education Farm, Henan University , Kaifeng, Henan. Mean annual precipitation and temperature are 625 mm and 14.4°C, respectively. Soil parent material and texture are Yellow River sediment and sandy loam according to FAO classification, respectively, with 65.7% sand, 14.1% silt, and 20.3% clay  and microbial biomass nitrogen (MBN) concentration. The third subsample was used to extract and identify soil nematodes.Ten 3 m × 3 m plots were established in the present study. Each plot had 2 m wide buffer zones with removed vegetation and litter to prevent fire spread. Five plots were randomly selected for prescribed burning. For the burning treatment, we fired the withered grass with a blowtorch and burned itself out to mimic the nongrowing season natural fire on December 3, 2018. Soil samples were collected on March 8, 2019. Four cores were randomly taken in each plot with a soil auger  and mixed carefully to obtain a total of 10 composite samples. Stones, large roots, and macro‐arthropods were excluded by hand. Each soil sample was divided into three subsamples. The first subsample was used to analyze soil physicochemical properties including soil gravimetric water content, SOC, soil total N (TN), soil pH, NO2.34+‐N) and nitrate (NO3−‐N)—was extracted from 10 g fresh soil with 50 mL 2 M KCl and measured by Discrete Auto Analyzer .A total carbon analyzer  was used to analyze soil organic C and total N contents. Soil organic C was determined by the Walkley‐Black's wet digestion method with a Total Organic Carbon Analyzer . The pH values were determined with a combination glass electrode (soil/water W/V ratio 1:2.5). Microbial biomass was measured by fumigation‐extraction method  according to Bongers  ]    represent c‐p values and feeding groups following Bongers and Bongers  and the percentage data  were ASIN‐transformed to meet the conditions of normality. All data were analyzed using ANOVAs. Simple linear regressions were used to develop the relationships of nematode population density and genus richness with NO3−‐N, NH4+‐N, MBN, MBC, and soil pH. All data analyses were conducted using SAS 9.3 statistical software package (SAS Institute Inc.).The nematode population density and genus richness were logarithmically transformed  with nematode community. Finally, a multiple ANOVA, or MANOVA, was conducted using manova function with top three principal components from the PCA. Treatment differences at the level of p < .05 were considered as significant, but correlates in PCA were mapped with less stringent value of p < .1.We used principal components analysis (PCA) to determine the prescribed burn effect on the overall nematode community in R (version 3.4.3). Using e Dixon, , we anal33.13−‐N and NH4+‐N concentration by 60% and 44%, respectively. Additionally, burning also increased soil MBN and MBC by 44% and 61%, respectively. However, burned sites significantly lowered soil pH (8.76 vs. 8.89).Three months after the prescribed burn, there was no detectable effect on soil water content, SOC and TN concentration Table . Burning3.2Across both treatments, we found 29 taxa from 21 nominal families with frequencies of detection ranging from <99% to >1% of the samples Table . Their fD) and increased Shannon‐Weaver Index (H′) by 45% and 31%, respectively.The prescribed burn had strong effects on overall abundance and measures of diversity Figure . Burning3.3Prescribed burning had striking effects mostly on each trophic group. Burning significantly increased relative abundance of PP by 98% compared to the control Figure .r‐selected), at least compared to fungi.Prescribed burning had mixed effects on ecological indices Table . Burning3.4Acrobeloide, from 49% to 26% relative abundance. Burning also decreased the most abundant Fu, Aphelenchoides, from 10% to 4% relative abundance. However, burning increased the dominant PP genus, Cephalenchus, from 10% to 22% relative abundance.Prescribed burning had notable effects on nematode genera Figure . Burningp = .036). Soil MBC, nitrate, and ammonium were positively correlated with prescribed burn soil nematode community, but pH negatively correlated , we can both determine overall community differences and elucidate factors driving community shifts. The PCA showed rather low dominance of top principal components  but suppressed the dominance . In terms of ecological indices, burning positively affected the EI and the CI, but had no effects on the SI and MI  or even long‐lasting impacts like increasing parasitation of the recovering plant community. Our findings highlight the importance of prescribed fire management in shaping short‐term nematode community structure and function. However, the long‐term effects remain unclear and warrant further exploration, in particular how they might be related to these short‐term changes in nematode community dynamics.While controlled burning increased nematode abundance and diversity, it also increased the relative abundance of plant pathogens. In particular, it increased the abundance of Min Song: Conceptualization ; data curation (lead); methodology ; writing – original draft ; writing – review and editing . Marshall D. McDaniel: Data curation ; methodology ; writing – review and editing . Chen Zhu: Data curation ; investigation . Feng Lin: Writing – review and editing . Yaojun Zhang: Investigation ; supervision ; visualization ; writing – review and editing .The authors declare that they have neither personal relationships nor competing financial interests that could affect the work.Data S1Click here for additional data file.


Cenococcum geophilum (C. geophilum) is a widely distributed ectomycorrhizal fungus that plays a crucial role in forest ecosystems worldwide. However, the specific ecological factors influencing its global distribution and how climate change will affect its range are still relatively unknown. In this study, we used the MaxEnt model optimized with the kuenm package to simulate changes in the distribution pattern of C. geophilum from the Last Glacial Maximum to the future based on 164 global distribution records and 17 environmental variables and investigated the key environmental factors influencing its distribution. We employed the optimal parameter combination of RM = 4 and FC = QPH, resulting in a highly accurate predictive model. Our study clearly shows that the mean temperature of the coldest quarter and annual precipitation are the key environmental factors influencing the suitable habitats of C. geophilum. Currently, appropriate habitats of C. geophilum are mainly distributed in eastern Asia, west‐central Europe, the western seaboard and eastern regions of North America, and southeastern Australia, covering a total area of approximately 36,578,300 km2 globally. During the Last Glacial Maximum and the mid‐Holocene, C. geophilum had a much smaller distribution area, being mainly concentrated in the Qinling‐Huaihe Line region of China and eastern Peninsular Malaysia. As global warming continues, the future suitable habitat for C. geophilum is projected to shift northward, leading to an expected expansion of the suitable area from 9.21% to 21.02%. This study provides a theoretical foundation for global conservation efforts and biogeographic understanding of C. geophilum, offering new insights into its distribution patterns and evolutionary trends. Cenococcum geophilum. C. geophilum mainly occurs in East Asia, Europe, and North America. Temperature and precipitation were critical to the distribution of C. geophilum. The habitat of C. geophilum is projected to shift northward under future climate scenarios. New insights have been provided into C. geophilum distribution pattern formation and evolution.MaxEnt Modeling was used to predict the distribution of  When geographic coordinates were unavailable occurrence data, we used Google Earth to determine latitude and longitude. To address issues of spatial autocorrelation and pseudo‐replication of species occurrence data impacting model outputs, we used ENMTools software to ensure that each 5 km2 grid cell contained only one distribution record , Middle Holocene (MH), current (1970–2000), and future , were obtained from the Worldclim database (http://worldclim.org). The topographic variable data were downloaded from the WorldClim database, while the soil variable data were gathered from the Harmonized World Soil Database . For future climate scenarios, we used BCC CSM2‐MR climate change modeling data to predict the distribution of C. geophilum under two Shared Socioeconomic Pathways (SSPs): SSP 1–2.6 (2.6 Wm−2) and SSP 5–8.5 (5.8 Wm−2) at two time points  of the environmental variables  and Regularization Multiplier (RM) using the “kuenm” package in the R software  curve ws employed , as described by Pearson et al. (C. geophilum was divided into four categories: unsuitable habitat (0.00–0.2217), poorly suitable habitat (0.2217–0.40), moderately suitable habitat (0.40–0.60), and highly suitable habitat (0.60–1.00). Furthermore, we analyzed the changes in spatial patterns of C. geophilum under future climate scenarios relative to its current range, identifying areas of range expansion, contraction, and no change.We used ArcGIS v10.6 to divide and visualize the layers representing potentially suitable habitats for n et al. . The Max33.1C. geophilum and 17 environmental variables, we simulated the potential global distribution of C. geophilum at different periods using the MaxEnt model. The default parameters of the model were RM = 1 and FC = LQPH. Through optimization, the parameters of the model were changed to RM = 4, FC = QPH, and delta AICc = 0  and annual precipitation  were the two environmental variables with the highest percentage contribution  as the threshold value, the range of key environmental factors influencing the distribution of C. geophilum was evaluated. The suitable range for Bio11 was approximately −17.92 to 17.42°C, with an optimal value of around 0.05°C. The suitable range for Bio4 was approximately 188.54%–1548.8%, with an optimal value of about 510.95%. Bio12 had a suitable range greater than 280.73 mm, and an optimal value of 840.40 mm. Bio17 had a suitable range greater than 4.87 mm, with an optimum value of 178.18 mm. T_ECE had a suitable range greater than 0.034 dS/m, and an optimal value of 0.14 dS/m.To further clarify the climatic characteristics of potentially suitable areas for s Figure . We foun3.3C. geophilum in the Northern Hemisphere are concentrated in East Asia, west‐central Europe, the western seaboard, and eastern regions of North America , appropriate habitats for r Figure . The ove2 Figure . From th2 Figure .3.5C. geophilum habitats, we utilized four scenarios  to model habitat suitability , southern and eastern Canada, northwestern and central Alaska, and the United States , annual precipitation (≥280.73 mm), temperature seasonality (188.54%–1548.8%), precipitation of the driest quarter (≥4.87 mm), and topsoil electrical conductivity (≥0.034 dS/m) are the dominant factors restricting the current distribution of C. geophilum survival are closely associated with climatic factors, particularly temperature and precipitation. Temperature has a greater relative importance compared with precipitation. A recent global meta‐analysis on mycorrhizal fungi also suggests that temperature and precipitation, especially warming, are strong predictors of climate change impacts on ECM fungi ; methodology ; software ; writing – original draft ; writing – review and editing . Chao Yuan: Investigation ; resources ; writing – review and editing . Norihisa Matsushita: Investigation ; resources ; writing – review and editing . Chunlan Lian: Investigation ; resources ; writing – review and editing . Qifang Geng: Data curation ; investigation ; resources ; writing – original draft ; writing – review and editing .The authors declare there are no conflicts of interest.Appendix S1.Click here for additional data file.


Epstein–Barr virus (EBV)‐associated gastric lymphoepithelioma‐like carcinoma (EBVaGLELC) represents a small number of gastric cancer (GC), and research on tumor microenvironment (TME) and treatment strategy are still lacking.Here, we aim to elucidate the immune features of this rare disease and further help to develop more effective treatment options.A retrospective analysis was conducted between 2019 to 2022 in West China Hospital to reveal the immunological characteristics of EBV‐positive GLELC. The difference of immune cell subset and tumor vascular structure between gastric denocarcinoma (GAC) and EBVaGLELC will be pointed out.+ T cells, CD8+ T cells, and Treg cells in the EBV‐associated GLELC group. Such a distinct TME may provide therapeutic advantages, and patients with this rare subtype of GC could be good candidates for immune checkpoint inhibitors (ICIs). Angiogenesis in EBV‐positive GLELC may be less intense than that in gastric adenocarcinoma (GAC), a feature that might decrease their susceptibility to antiangiogenic therapy. Furthermore, we reported a 52‐year‐old male with advanced EBV‐positive GLELC who showed a favorable response to the combined therapy with . A repeat evaluation showed sustained partial response (PR), and the progression‐free survival (PFS) was more than 34 months until now.13 patients with GELEC and 8 patients with GAC were retrospectively studied. The heterogeneity of the immune cell profile was then confirmed through multiplexed immunofluorescence staining (mIF), which revealed a higher proportion of CD3Compared with GAC, EBVaGLELC revealed higher T cell infiltration and less intense of angiogenesis. It displays relatively “hot” TME that may provide the rationality to treat with immunotherapy in EBV‐related GLELC. Compared with gastric adenocarcinoma, EBV‐positive GLELC has distinct TME characteristics. A “hot” TME may provide therapeutic advantages for those patients. Furthermore, our case provides an underlying therapeutic option for patients with EBV‐associated GLELC. This study supplements the limited literature, which could also contribute to the clinic's treatment decision‐making, especially for advanced patients. For advanced‐stage GC, chemotherapy‐based comprehensive treatment is the main treatment method.Of note, the reported rate of EBVaGLELC in patients of Chinese descent has increased in recent years. Thus, a systematic study is required to elucidate the immune features of this rare disease and further help to develop more effective treatment options. In this study, we aimed to comprehensively characterize the clinical characteristics, immune cells, tumor vascular structure, and expression of PD‐L1 in the TME of EBVaGLELC. Furthermore, we report on an EBVaGLELC patient with a PFS of more than 34 months to date who exhibited a marked response to combined therapy with immune checkpoint blockade.22.1N = 8) were eligible if they were aged ≥18 years, histologically confirmed diagnosis and blocks were sufficient to perform mIF analysis. All samples were gastrectomy or gastric biopsy samples and were obtained prior to treatment at the Department of Pathology, West China Hospital between 2019 and 2022. The clinical features of these individuals, including baseline characteristics and tumor‐related information, were retrieved from our hospital's electronic medical record system.Patients with EBVaGLELC were retrospectively studied in West China Hospital between January 2019 and 2022. The inclusion criteria were as follows: (1) a histologically confirmed diagnosis of EBV‐positive GLELC in West China Hospital; (2) age ≥18 years; (3) EBV status was identified using the polymerase chain reaction (PCR) technique to detect EBERs in tissue samples; (4) no prior radiotherapy, chemotherapy, or any other targeted therapies for the current gastric cancer; and (5) patient blocks were sufficient to perform mIF analysis. Patients with incomplete data were eliminated from this study, and finally, a total of 13 cases of gastric primary LELC were included. To define the distinction of the TME between GELEC and GAC more thoroughly, patients with GAC were also included in our study. Randomly selected GAC patients .2.3+ T cells, helper T cells , CD8+ T cells, regulatory T (Treg) cells , PD‐1+ effector T lymphocytes , M1‐like tumor‐associated macrophages (TAMs) , M2 TAMs , PD‐L1+ macrophages , NK cells (CD56 bright and CD56 dim), and B lymphocytes (CD20). Cancer‐associated fibroblasts (CAFs) are associated with tumor growth, invasion, and metastasis and are characterized by high α‐SMA expression.The blocks of the above 21 patients were analyzed through mIF. All steps were performed on a Leica Bond Rx autostainer (Leica Microsystems), and then, images were acquired on an Akoya Vectra Polaris. The HALO image analysis platform (Indica Labs) was used for image analysis. This study includes antibodies against: CD163(ab182422), CD20(L26 IR604), CD3(A0452 IR503), CD4(ab133616), FoxP3(ab20034), CD56(ab75813), CD68(ab213363), CD8(ab178089), PD‐1(D4W2J), and PD‐L1(E1L3N). Immune cells were analyzed, including CD32.4https://www.r‐project.org) and GraphPad Prism 8 software. Unless otherwise indicated, data are expressed as the means ± SDs or means ± SEMs. A p < 0.05 was considered statistically significant.The counts and percentages of immune cells were obtained, and statistical analyses were performed using R v.4.0.3 (33.1n = 13) who developed EBV‐associated GLELC was 59.69 ± 7.70 years, with 84.6% males (Table p = 1.00), a high percentage of advanced T stage (p = 0.699), less positive node metastasis (p = 0.377), and well cell differentiation (p = 0.25). EBV infection was not found in patients with GAC, and most were MMR‐proficient patients. The HER‐2 status in most patients was negative. Differences between the two types of GC are summarized in Table The mean age of patients n = 3 who dev3.2p = 0.04).PD‐L1 serves as a ligand of programmed cell death protein 1 (PD‐1), which is expressed on tumor cells or stromal immune cells. PD‐L1 can inhibit the activation of cytotoxic T cells by combining with PD‐1 and contribute to evading immune surveillance. Here, we assessed PD‐L1 expression in tumor cells and immune cells through immunohistochemistry (IHC) analysis for all samples. No significant difference was observed between the PD‐L1 expression levels in the tumor cells. A substantial proportion of patients did not express high levels of PD‐L1 in both groups either group. PD‐1 is mainly expressed in T cells. The expression of PD‐1 was higher in the GAC group, but there was a significant difference between those two groups (p = 0.513). The percentages in the tumor region were 0.2% and 0.04%, respectively. The levels of expression (low or high) of PD1 and PD‐L1 are shown in Figure Immune cell PD‐L1 colocalization with macrophages (PD‐L1 + CD68+) was also measured in our study. Consequently, no statistically significant difference was found  staining was conducted for untreated tumor tissues. + T cells and CD8+ T cells were significantly elevated in EBVaGLELC compared with GAC . The infiltration of NK cells was poor in EBV‐positive GLELC. Although Treg cells correlated with the formation of a cold TME in the tumor region and less NK cells infiltration in both the tumor region and stroma region, the proportion was far less than tumor‐infiltrating lymphocytes. Other markers did not show a significant difference.Tumor classifications through characteristics of the tumor microenvironment have been proposed.p < 0.001, Figure p < 0.001, Figure In addition, there were significant differences between tumor vascular structure assessed by CD31 and α‐SMA IF double staining Figure . The exp3.4). After excluding the diagnosis of nasopharyngeal carcinoma, the patient was diagnosed with EBV‐associated GLELC and staged as IVA .A 52‐year‐old male was referred to the West China Hospital Sichuan (China) with a swallowing disorder in July 2019. Endoscopy analysis revealed an ulcerated lesion in the stomach. The biopsy was performed, indicating poorly differentiated carcinoma, and the subsequent immunohistochemistry results showed PCK (+), EMA (±), CDX2 (−), CgA (−), Syn (−), CD20 (−), CD3 (−), Ki67 , CK8/18 (+), P63 (+), CK5/6 (+), CK7 (−), and EBER1/2‐ISH (+) Figure . GeneticThe patient was not considered for surgery. After obtaining consent for cancer treatment , the patient commenced systemic treatment in September 2020. Sustained partial response (PR) was achieved based on CT or PET/CT evaluation after eight treatment cycles from September 2020 to May 2021. In June 2021, a biopsy through gastroscopy confirmed that the primary tumor had achieved a complete pathological response. Since the lymph node beside the left renal artery area did not decrease, radiotherapy was given in July 2021 (45 Gy/25 f). Then, the patient was changed to maintenance immunotherapy monotherapy. After seven cycles of maintenance therapy, this advanced patient still benefited. We performed mIF analysis to investigate the immune context before treatment Figure . Higher 4EBV is an oncogenic virus that infects approximately 90% of the world's population.Helicobacter pylori (HP) infections, higher PD‐L1 expression, more PI3K/AKT pathway mutations, and better outcomes were reported in EBVaGC.EBVaGCs represent approximately 10% of all gastric cancers, and EBVaGC has distinct characteristics that distinguish it from EBV‐negative GC.Cancer immunotherapy has revolutionized cancer treatment both as monotherapy or in combination strategies, especially ICIs that block cytotoxic T lymphocyte‐associated protein‐4 (CTLA‐4) and the PD‐1/PD‐L1 axis.+ T cells and CD8+ T cells not only helps form a hot TME but also contributes to predicting ICB therapeutic efficacy.+ T cells might reprogram the TME to a hotter TME by facilitating the normalization of tumor vessels.In addition, based on mIF, we thoroughly clarified the characterization of the TME in EBVaGLELC. The TME plays a key role in tumor formation and progression, providing a beneficial environment for tumor cell proliferation. With the notion of TME, a new tumor classification was also proposed, “hot” or “cold” tumors, which contribute to stratifying patients who may benefit from immunotherapy.In our study, patients with EBVaGLELC exhibited an increased number of CD3+ T cells and effector T‐cell infiltration, suggesting that these patients may benefit from immunotherapy. Moreover, the fraction of Treg cells was enriched in EBVaGLELC tumors compared to GAC, which may be associated with an immunosuppressive TME and mediate resistance to ICIs. However, the proportion was far less than tumor‐infiltrating lymphocytes. Additionally, NK cells have the potential to magnify immune responses, and interest in harnessing NK cells for cancer immunotherapy is rapidly growing.Additionally, vasculature networks in the TME are essential for tumor development and metastasis to other tissues.Understanding the crosstalk between the TME and tumor cells is necessary in tumor immunotherapy to identify ICB responders, help predict prognosis, and guide immunotherapy. However, the complex connection in the TME has not been sufficiently illustrated until now. Here, we comprehensively studied the immune cells and the tumor vascular structure in EBVaGLELC, which may facilitate the understanding of the complex interaction between cancer cells and the TME and thus further encourage future research and guide treatment. Above all, EBV‐associated GLELC showed different patterns in immune cell infiltration and tumor vascular structure compared to patients with GAC. An immunostimulatory TME was shown in patients with EBV‐associated GLELC, which may raise the potential of checkpoint immunotherapy for this rare disease.4.1There are some limitations in the present study. First, the number of patients in our study is limited due to a relatively low incidence of EBVaGLELC, and expanding the sample size is still needed in future research to verify our results. Second, we only assessed the TME before treatment due to the limited accessibility of tumor tissue during and after treatment. Dynamic changes in the tumor microenvironment could not be detected. Moreover, the results of analysis from biopsy and resection specimens may be discordant. Finally, single‐cell RNA sequencing or bulk RNA sequencing could provide more detailed information than the multiplexed immunofluorescence technique.5Compared with gastric adenocarcinoma, EBV‐positive GLELC has distinct TME characteristics. A “hot” TME may provide therapeutic advantages for those patients. Furthermore, our case provides an underlying therapeutic option for patients with EBV‐associated GLELC. Relevant clinical experiments are in preparation in our hospital to verify its clinical application value. Our study supplements the limited literature, which could also contribute to the clinic's treatment decision‐making, especially for advanced patients. Future studies with a larger sample size and a better design are still needed to verify our findings and enhance our understanding of EBV‐positive GLELC.Yanna Lei: Data curation (lead); investigation ; methodology ; validation ; writing – original draft ; writing – review and editing . Peng Cao: Investigation ; visualization ; writing – original draft . Xiufeng Zheng: Methodology ; supervision . Jing Wei: Validation . Mo Cheng: Validation . Ming Liu: Conceptualization ; data curation ; supervision ; validation .All authors declare no conflicts of interest concerning this work.Written informed consent was obtained from the patient for publication of this case report.FIGURE S1. EBER staining (200X) of tumor specimen which shows a number of EBER‐positive cells.Click here for additional data file.


Pre‐exposure prophylaxis (PrEP) delivery based on user needs can enhance PrEP access and impact. We examined whether telehealth for daily oral PrEP delivery could change the indicators of care related to prophylactic use in five Brazilian public HIV clinics .Between July 2019 and December 2020, clients on PrEP for at least 6 months could transition to telehealth or stay with in‐person follow‐up. Clients were clinically monitored until June 2021. A desktop or mobile application was developed, comprising three asynchronous consultations and one annual in‐person consultation visit. Predictors influencing telehealth preference and care outcomes were examined. The analysis encompassed intent‐to‐treat (first choice) and adjustments for sexual practices, schooling, age, duration of PrEP use and PrEP status during the choice period.p = 0.486) or at pre‐ and post‐telehealth follow‐ups (p = 0.245). Sexually transmitted infections increased between the pre‐follow‐up and post‐follow‐up choices and were not associated with in‐person or telehealth (p = 0.528). No HIV infections were observed.Of 470 users, 52% chose telehealth, with the adjusted odds ratio (aOR) increasing over time for PrEP use , having discontinued PrEP at the time of the choice  and having health insurance  and decreasing for those who reported higher‐risk behaviour . After an average follow‐up period of 1.6 years (95% CI: 1.5–1.7), the risk of discontinuing PrEP (not having the medication for more than 90 days) was 34% lower with telehealth . When adjusted by mixed linear regression, no differences in adherence (measured by mean medication possession rate) were found between in‐person and telehealth (Our findings indicate that telehealth for PrEP delivery can enhance service rationalization and reinforce the prevention cascade. This approach reduces prophylaxis interruptions and is mainly preferred by individuals with lower demands for healthcare services. We evaluated the acceptability and predictive factors of choice for telehealth and the effect of telehealth follow‐up on discontinuation of PrEP, adherence and frequency of occurrence of sexually transmitted infections (STIs) and HIV infection.22.1The evaluation occurred as part of a demonstration study  of dailyThe study has been registered in the “Brazilian Registry of Clinical Trials” with “The Universal Trial Number”: U1111‐1165‐7889s.2.2The telehealth protocol included three quarterly remote assessments and annual in‐person evaluations. A web platform, accessible via cell phone or computer, was developed in consultation with professionals and clients for the clinical evaluations see . A totalAll the participants consented to participate in the study. This study was approved by the Ethics Committees of the Clinical Hospital of the School of Medicine of the University of São Paulo (3438329/2019) and the Pan American Health Organization (PAHO‐2019‐06‐0048).2.3Telehealth was offered between July 2019 and December 2020 to 740 individuals who had started PrEP at least 6 months earlier . For those with regular PrEP use, telehealth was offered preferentially at consultation, and for those who discontinued use, contact was made by telephone, social networks or e‐mail. Clients can change the type of follow‐up at any time.n = 5) or clinical conditions requiring in‐person evaluation (n = 7), defined at the physician's discretion, were excluded. Therefore, 728 PrEP users were eligible for telehealth.After the first evaluation, individuals without internet access . At intermediate levels, the influence would occur due to HIV risk behaviour  and vulnerability . Demographic characteristics  were recorded at the most distal level.Data refer to the 6 months preceding enrolment in the Combine! Study, which was obtained by the applied questionnaire. Sex work was defined as sexual relations for money, favours, presents or self‐identification as a sex worker. The question used to define risk self‐assessment was: “How do you evaluate your risk of becoming infected with HIV with your casual partners, assigning a score from 1 to 10?”. Individuals rated ≥ 8 were classified as having a “higher risk perception.” Intimate partner violence was defined as coercion, humiliation, physical aggression with or without weapons, and sex and drug use without consent.Participants completed an online questionnaire regarding their reasons for choosing telehealth, considering various factors related to the individual, the service and clinical follow‐up.2.5Discontinuation was defined as a delay of ≥ 90 days since the last withdrawal was supposed to have occurred. This was measured at the end of the study, assuming that the date of the previous medication withdrawal was the date of discontinuation.1,2… = NP1../((DtW2…‐DtW1…)+). An MPR <1 was considered non‐adherence.Adherence was measured using medication possession rates (MPR). For each medication withdrawal period, the number of pills delivered (NP) was divided by the number of days between withdrawal dates (DtW). Leftover medication (LO) or days without medication (DN) at each interval were added to the subsequent interval. Thus, MPR2.6The incidence of STIs was identified as new reports of indicative signs and symptoms in the genitals, anus and mouth in medical records, irrespective of treatment, clinical conduct or laboratory test results.HIV infection was screened at each visit or upon medication withdrawal using a third‐generation rapid test.2.7Descriptive analysis was used to study the reasons for choosing telehealth. Associations with individual characteristics were verified using the chi‐square test. When differences in the distributions were observed, the standardized adjusted residual (Zres) was used to identify excess or absence | 1.96 | of the frequency.The predictors of opting for telehealth were analysed using logistic regression. The model included the variables from distal to proximal levels, as defined in the predictive model. Variables with a significance level of ≤ 0.10 remained in the model for the analysis of subsequent levels and, in the final model, only variables with a significance of ≤ 0.05.Assuming telehealth does not impact PrEP outcomes , this study compared these outcomes before and after telehealth implementation and between telehealth and in‐person follow‐up. The intention‐to‐treat method was used for the analyses. A complementary analysis was conducted, reclassifying individuals by the last type of clinical follow‐up performed at the end of the follow‐up period to consider the change in the type of follow‐up after the first choice.Survival analysis for discontinuation was performed using the Kaplan−Meier method. Survival curves for each follow‐up type were compared using the log‐rank and Tarone−Wares tests. Cox regression was used to adjust for the effect of the follow‐up type on PrEP discontinuation, and Schoenfeld residuals were used to verify the model's assumptions.Mixed linear models were employed to calculate the mean MPR for both telehealth and in‐person consultations in the pre‐ and post‐implementation periods of telehealth. This calculation was adjusted for variables reflecting risk, vulnerability, PrEP status and demographic characteristics. The study participants were considered random, and the other variables were considered fixed effects. The rates for each type of follow‐up were compared, and the interaction between time and type of follow‐up was analysed.We also investigated whether non‐adherence was associated with either telehealth or in‐person follow‐ups. We employed logistic regression with MPR <1 tablet/day as the outcome variable to examine this. The model was adjusted for variables reflecting risk, vulnerability, PrEP status and demographic characteristics.A generalized estimating equation model was used to analyse the evolution of STI occurrence in the pre‐ and post‐telehealth onset periods. The study subjects were considered clustering factors, and a negative binomial marginal distribution was employed, considering the exposure time. Analysis was adjusted for risk variables, vulnerability, PrEP status and demographic characteristics.The analyses were conducted using a confidence interval of 95% (95% CI) in the software packages SPSS 20.0 and STATA 17.33.1p <0.05) by not using PrEP at the time of telehealth delivery (66.7%), were sex workers (31.5%), aged 16–25 years old (28.1%), were transgender women (14.1%) and had incomplete primary education (14.1%).A total of 470 individuals  chose to undergo clinical follow‐up. Non‐choice occurred in 79.3% (214) of the cases because of the inability to locate the participant or because the participant did not select the invitation . Participants who did not choose (270) differed  chose telehealth, which varied between 92.9% and 15.8%, depending on client characteristics  left the study because they moved to another city  or switched to an event‐driven regimen , with a similar distribution between the follow‐up types. In total, 110 participants (23.4%) discontinued PrEP: 53 telehealth and 57 in‐person. Of these, 30  were not followed up after choosing. Additionally, 20 participants (4.2%) changed the type of follow‐up, and most  returned to in‐person follow‐up. The decision to return was mainly driven by personal and organizational challenges in conducting remote assessments and the physical requirement to visit the service for tests or medication pickups.PrEP discontinuation curves showed a homogeneous pattern by follow‐up type , with a mean time of PrEP use for post‐telehealth choice of 1.6 years  and 1.5 years for in‐person . The risk of discontinuing PrEP at 6, 12 and 18 months were similar, with 11.0% (95% CI: 7.7–14.3), 17.2% (95% CI: 13.8–20.6) and 26.0% (95% CI: 22.6–29.4) for telehealth and 14.0% (95% CI: 11.4–16.6), 21.4% (95% CI: 18.7–24.1) and 33.8% (95% CI: 29.1–38.5) for in‐person, respectively.When adjusted using the Cox model Figure , the ris3.3p = 0.486) and between the pre‐ and post‐onset periods (p = 0.054), with mean MPR indices higher than 1 medication/day. A slight decline in MPR  occurred in person between the pre‐ and post‐choice and was unrelated to the type of follow‐up or time .Medication was withdrawn twice or more from 401 participants. The mean MPR values Figure  were simp = 0.259) were found between the groups with MPR <1/day and MPR ≥ 1/day. The proportion of people with MPR <1/day was 19.5% (95% CI: 14.2–24.9) in telehealth and 24.2% (95% CI: 18.0–30.4) in in‐person. This remained unchanged (Table p = 0.921). The only variable associated with an MPR <1/day was the duration of PrEP use, and an increase of 1 day of use reduced the chance of insufficient adherence by 2% .No significant differences p = 0.29 were fo3.4p <0.001) in the mean frequency of occurrence between the pre‐ and post‐choice periods , showing that the pattern of average occurrence of STIs in telehealth and in‐person was similar in the pre‐ (p = 0.772) and post‐onset periods (p = 0.438).A total of 300 syndromes indicative of STIs were recorded in telehealth and 420 in‐person, with an increase .Alan Clausen da Silveira, Bruno Silva Kauss, Dennis Soares Leite, Jonatan da Rosa Pereira Da Silva, Maria Eugênia Paiva do Amaral, Romário Miranda Alexandre, Thiago Souza Reis, Vanessa Souza Brito and Wilver Cunha Portella.Table S1. Adjust hazard ratio (aHR) and 95% confidence intervals (95% CI) for discontinuation of PrEP, according to the first type of follow‐up choice in the studyTable S2. Adjust hazard ratio (aHR) and 95% confidence intervals (95% CI) for discontinuation of PrEP, according to the last type of follow‐up performed in the studyTable S3. Adjust hazard ratio (aHR) and 95% confidence intervals (95% CI) for discontinuation of PrEP, according to the last type of follow‐up performed and excluding people without follow‐up after the first choice in the studyClick here for additional data file.Supplementary File: TelePrEP Platform Development History.Screenshot 01: Login screen.Screenshot 02: Professional profile initial menu.Screenshot 03: Screen for scheduling face‐to‐face appointments.Screenshot 04: Access screen to the remote PrEP clinical evaluation form.Screenshot 05: Tab for filling in the results of routine clinical exams.Screenshot 06: Tab for final conduct and PrEP prescription.Screenshot 07: Chat screen between professional and user.Click here for additional data file.


H3K27M‐mutant diffuse midline glioma (DMG) is a rare and aggressive central nervous system tumor. The biological behavior, clinicopathological characteristics, and prognostic factors of DMG have not yet been completely uncovered, especially in adult patients. This study aims to investigate the clinicopathological characteristics and identify prognostic factors of H3K27M‐mutant DMG in pediatric and adult patients, respectively.A total of 171 patients with H3K27M‐mutant DMG were included in the study. The clinicopathological characteristics of the patients were analyzed and stratified based on age. The Cox proportional hazard model was used to determine the independent prognostic factors in pediatric and adult subgroups.p < 0.001). In the overall population, the multivariate analysis identified adult patients, single lesion, concurrent chemoradiotherapy/radiotherapy, and intact ATRX expression as independent favorable prognostic factors. In the age‐stratified subgroups, the prognostic factors varied between children and adults, with intact ATRX expression and single lesion being independent favorable prognostic factors in adults, while infratentorial localization was significantly associated with worse prognosis in children.The median overall survival (OS) for the entire cohort was 9.0 months. Significant differences were found in some clinicopathological characteristics between children and adults. The median OS was also significantly different between the pediatric and adult subgroups, with 7.1 months for children and 12.3 months for adults  is a heterogeneous type of gliomas with distinct clinicopathological features and showed a significant difference in overall survival between children and adults. Notably, there were differences in prognostic factors between them. Our results suggested DMG should be clinically and molecularly segregated according to different age. On the other hand, mutations in P53 and amplification of platelet‐derived growth factor receptor A (PDGFRA) often co‐occur with H3K27M mutation and impact the prognosis of DMG.ACVR1), fibroblast growth factor receptor 1 (FGFR1), BRAFV600E, and alpha‐thalassemia mental retardation X‐linked (ATRX) mutations, have been associated with the survival of DMG.The distinctive molecular feature of DMG is the presence of H3K27M mutations in H3.3 or H3.1 histones. Consensus genetic findings indicate that mutations in isocitrate dehydrogenase  for pediatric patients with H3K27M‐mutant DMG ranges from 9 to 12 months, with a 2‐year OS of less than 10% and a 5‐year OS of less than 1%.In this single‐center large cohort study of 171 patients with H3K27M‐mutant DMG, one of the largest sample sizes to date, we presented the clinical and pathological characteristics of the entire population and subgroups stratified by age. We conducted univariable and multivariable analyses on the entire cohort and age‐stratified subgroups to identify factors impacting survival, respectively.22.1In this retrospective single‐center study, H3K27M‐mutant DMG patients who were diagnosed and treated at Xiangya Hospital of Central South University in China between October 2016 and January 2022 were reviewed. Two experienced neuropathologists reviewed the histopathologic findings according to the 2016 WHO classification of CNS tumors.The inclusion criteria for this study were as follows: (1) pathologic diagnosis of H3K27M‐mutant DMG based on the 2016 WHO classification of CNS tumors, including tumors located in midline structures of CNS with histological characteristics of glioma, diffuse and infiltrative growth pattern, and confirmed H3K27M mutation through immunohistochemistry (IHC) and Sanger sequencing; (2) newly diagnosed DMG with the first operation or biopsy; and (3) availability of complete follow‐up materials (see “Data Collection” section for details). The exclusion criteria were as follows: (1) patients with ganglioglioma or ependymoma with H3K27M mutation; (2) patients who had relapsed DMG or received chemotherapy, radiotherapy, or targeted therapy before the pathologically confirmed diagnosis of H3K27M‐mutant DMG.Based on the inclusion and exclusion criteria, 24 patients were excluded, and the final analysis included 171 patients. There was one patient having a previous diffuse astrocytoma (WHO grade II) diagnosis in 2014 before the recognition of DMG. This patient underwent a second operation in 2016 because of local recurrence and was subsequently diagnosed with H3K27M‐mutant DMG. We re‐examined the tissue sample from the first operation and confirmed the presence of H3K27M mutation. Therefore, this patient was also included in our study. The study protocol was approved by the Ethics Committee of Xiangya Hospital, Central South University. Informed consent was obtained from each patient or their legal guardian, and all patient data were treated with confidentiality.2.2In this study, we collected the following data for each patient: general data including gender, age, and Karnofsky Performance Score (KPS); radiographic data including preoperative, postoperative, and follow‐up magnetic resonance (MR) imaging; extent of surgical resection; and postoperative treatments such as radiotherapy, chemotherapy, and palliative treatment. The evaluation criteria of extent of surgical resection were as follows: Based on postoperative imaging, gross total resection (GTR) was defined as a removal of >95% of the tumor mass; non‐GTR included subtotal resection and partial resection of craniotomy.TP53 mutation and interpreted as P53 positive. For ATRX, no nuclear staining was considered as evidence of loss. For Olig2 and GFAP, >50% of nuclear and cytoplasmic staining were interpreted as positive, respectively. For IDH1, cytoplasmic staining was interpreted as positive. For MGMT, nuclear staining was determined as MGMT promoter unmethylation. Molecular detections were also conducted to investigate MGMT promoter methylation, H3.3K27M mutation, and IDH mutation. Sanger sequencing was used for IDH1/2 and H3.3K27M mutation analysis, and methylation‐specific quantitative PCR was employed to determine the status of MGMT promoter methylation.Primary antibody used for histopathology and IHC in this study are listed as follows: H3K27M , Ki‐67 , P53 , ATRX , GFAP , Olig2 , MGMT , and IDH1 . For H3K27M, tumor cells nuclear staining was determined as positive and no nuclear staining was determined as negative. For Ki‐67, the index was calculated as a percentage of positively stained nuclear by 1000 tumor cells counted under 400× magnification in the areas with highest density of positive nuclear. For P53, >30% of nuclear staining was the evidence of 2.3t‐test or Chi‐square (Fisher exact) test as appropriate. The starting point for OS analysis in DMG patients was the time of the first operation, and the endpoint was either death or the last follow‐up date. The study's follow‐up period ended in June 2022. OS was assessed using Kaplan–Meier survival curves and compared using the log‐rank test. Multivariate survival analysis was performed using the Cox proportional hazard model. p‐Value < 0.05 was considered statistically significant.The patient baseline data were analyzed by direct counting, and non‐normally distributed measurement data were reported as medians. Data processing was conducted using GraphPad Prism 8 (GraphPad Software) and SPSS 23.0. Statistical analyses included the Student 33.1n = 74), spinal cord (n = 19) and supratentorial region including the thalamus (n = 53), the basal ganglia (n = 11), the ventricles (n = 4), corpus callosum (n = 5), pineal gland (n = 3), and suprasellar region (n = 2). Majority of the DMG located in the infratentorial region in children (68.1%), while it more frequently occurred in the supratentorial region in adults (57.8%) (p < 0.001). In our series, 146 patients (85.4%) underwent surgical resection, including 15.8% with GTR and 69.6% with non‐GTR, while 25 patients (14.6%) underwent stereotactic biopsy. After surgery, the majority of patients  received either concurrent chemoradiotherapy (CCRT) or radiotherapy alone (RT), while 82 patients (48.0%) chose palliation therapy or chemotherapy alone. The median follow‐up duration was 14.6 months . At the final follow‐up, only 43 patients (25.1%) were still alive, including 11 in the pediatric subgroup and 32 in the adult subgroup. All alive pediatric patients underwent postoperative CCRT; two patients achieved GTR and nine received non‐GTR. Among the alive adult patients, 27 patients received CCRT, one underwent chemotherapy alone, and four chose palliative treatment; as for extent of surgical resection, six with GTR, 22 with non‐GTR, and four with stereotactic biopsy.A total of 171 patients with H3K27M‐mutant DMG were included in the study. Among them, 69 (40.4%) cases were children (age below 18 years) and 102 (59.6%) cases were adults. Tumors were anatomically located in the infratentorial region . Furthermore, positive P53 status was more frequently observed in children than in adults . The loss of ATRX expression was uncommon in DMG (26.9%), and there was no significant difference between children and adults. The clinical, histologic, and molecular characteristics are summarized in Table H3.3K27M mutation was confirmed in tumor tissue from all 171 patients using IHC or Sanger sequencing. Based on Ki‐67 staining, pediatric patients showed a higher proliferative index compared to adults . Additionally, it also showed adult patients (p < 0.001), supratentorial/spinal cord localization (p < 0.001), single lesion (p = 0.015), GTR (p = 0.002), CCRT/RT (p < 0.001), lower Ki‐67 index (p = 0.003), and negative P53 status (p = 0.042) were associated with longer OS. However, tumor size (p = 0.448), and other molecular markers such as MGMT promoter methylation (p = 0.106) and loss of ATRX expression (p = 0.095) did not show a significant effect on prognosis in the entire cohort  and loss of ATRX expression  were independent factors associated with poor survival. Conversely, single lesion  and CCRT/RT  were independent favorable prognosticators for longer overall survival in the entire cohort  and intact ATRX expression (p = 0.020) were significantly associated with a better prognosis. In the pediatric patients, GTR (p = 0.034) was associated with better survival. Specifically, children aged 8 years or younger had even worse survival (p = 0.027)  and adult  subgroups. Additionally, infratentorial tumors  associated with poor survival in pediatric DMG patients. In contrast, in adult subgroup, single lesion  was an independent favorable factor, while loss of ATRX expression  was an independent adverse factor in adult patients , and ATRX mutations were more common in adults.When stratified by age, the median OS for adults was 12.3 months, longer than the 7.1 months observed in children. Cox regression analysis revealed that pediatric patient was an unfavorable independent factor in the whole cohort. Most retrospective studies have demonstrated that the prognosis for adult patients was superior to that of historical pediatric survival.Furthermore, in the pediatric subgroup, we found that children aged ≤8 years had a shorter OS. Several previous studies have also investigated the prognosis of pediatric DIPG in different age subgroups. An earlier study with 316 pediatric patients showed that age ≤3 years positively predicted the prognosis of DIPG.Recently, some retrospective studies have revealed that DMG in different locations may exhibit distinct outcomes, and H3K27M‐mutant DMG tumors in the infratentorial region indicated poorer survival than those in the supratentorial region.Single lesion was another favorable prognosticator in the entire cohort and the adult subgroup when compared with multiple lesions or LMD. The prognostic value of dissemination characteristics was rarely reported. LMD was related to shorter progression‐free survival in DIPG.Surgical resection is one of the standard therapeutic approaches in H3K27M‐mutant DMG. However, due to the deep location and diffused and infiltrative behavior of DMG, total resection is usually impractical, especially in the brainstem. Moreover, unlike glioblastoma, the benefit from surgery in DMG is unconfirmed. Most experts advocate maximal resection or even extended resection for glioblastoma because the extent of resection is unquestionably associated with prognosis.According to guidelines and consensus, radiotherapy is recommended as a standard and efficient strategy for DMG, as it improves the patient's quality of life and prognosis.Moreover, CCRT/RT was identified as a favorable prognostic factor both in pediatric and adult patients. Similarly, several retrospective studies focusing on either adults or children have shown that postoperative adjuvant treatments are associated with improved prognosis. A study with 96 adult DIPG patients found that postoperative treatment was an important predictor for H3K27M‐mutant tumors, especially in patients with a symptom duration of ≤4 months.P53 mutation is frequently observed in H3K27M‐mutant DMG,P53 overexpression or mutation has been identified as an independent risk factor for shorter OS in H3K27M‐mutant DMG.ACVR1 and FGFR1 mutations and PDGFRA amplification are related to the prognosis of H3K27M‐mutant DMG.TERT) mutation, cyclin‐dependent kinase inhibitor 2A (CDKN2A) deletion, and EGFR amplification were correlated with worse prognosis in adult midline gliomas. Our study identified loss of ATRX expression as an unfavorable prognosticator in the entire cohort and adult subgroup. ATRX mutation is observed in various gliomas, particularly in low‐grade gliomas with IDH mutation.ATRX deficiency impairs non‐homologous end joining (NHEJ) by hindering the recruitment of pDNA‐PKcs, potentially through conformational changes in heterochromatin, leading to genetically unstable tumors and aggressive tumor behavior.ATRX loss in H3K27M‐mutant DMG remains unknown, but studies have suggested that ATRX loss played an essential role in DMG development.ATRX induces an immunosuppressive microenvironment in gliomas by promoting the secretion of immunosuppressive cytokines and upregulating the expression of immune checkpoint molecules, suppressing anti‐tumor immunity.ATRX loss was associated with shorter overall survival,ATRX loss have significantly longer survival compared to those with ATRX intact expression in diffuse gliomas.ATRX loss in H3K27M‐mutant DMG and its implications in different age populations.Recently, molecular characteristics have been recognized as predictive factors in gliomas. There are limitations to this study. Firstly, since most of the patients (n = 153) were enrolled before June 2021, we did not include those who harbored EZHIP overexpression, which also results in a global reduction of H3K27me3 according to the 5th edition of the WHO Classification of CNS Tumors. Thus, instead of the new term “diffuse midline glioma, H3 K27‐altered,” we still use the term “diffuse midline glioma, H3 K27‐mutated.” In future studies, we plan to adopt the newest diagnostic criteria to examine EZHIP and H3K27me3 expression. Secondly, we did not acquire comprehensive genetic information on the patients. Therefore, the next steps in our study are to collect tumor tissue samples and explore the transcriptome and genetic alterations of H3K27M‐mutant DMG using RNA‐sequencing and next‐generation sequencing. Moreover, this retrospective study has inherent selection bias, and the sample size is not large enough for robust subgroup analysis.5This study provided a comprehensive analysis of the clinicopathological and genetic features of the entire population and stratified them by age in H3K27M‐mutant DMG. We have identified postoperative concurrent chemoradiotherapy/radiotherapy as a significant independent prognostic factor for both the entire cohort and pediatric/adult subgroups. Additionally, we have made innovative findings, including the identification of single lesion and ATRX intact expression as independent favorable prognostic factors in adults. We have also observed that tumor location significantly impacts survival of pediatric patients, and children below the age of 8 years tended to have even poorer outcomes. These results suggest that the clinical outcomes of H3K27M‐mutant DMG should be stratified based on molecular characteristics and age. This finding can aid neuro‐oncologists in risk stratification and selecting individualized treatment options.Conception and design of the study: CL. Statistics analysis: XG, SK, and CL. Manuscript drafting: SK, XG, and CL. Acquisition of data: DD, JW, and LZ. All authors have read and agreed to the published version of the manuscript.This study was supported by the National Natural Science Foundation of China (Grant No. 81701285), the Natural Science Foundation of Hunan Province (Grant No. 2018JJ3824), and the Nature Science Youth Foundation of Hunan Province (Grant No. 2018JJ3856).The authors declare no conflict of interest.