Although preoperative splenic artery embolism (SAE) has been widely used for splenomegaly, the efficiency and safety of preoperative SAE in patients with sinistral portal hypertension (SPH) is unknown.We designed a retrospective cohort of SPH patients who received preoperative SAE in our hospital (February 2018 to September 2020) and compared to those who received splenectomy only, in terms of intraoperative and postoperative outcomes.P = 0.041) and operation time . The incidence of complications including postoperative pancreatic fistula (POPF), bleeding, and thromboembolism was comparable. Multivariate analysis showed that SAE was a protective factor for intraoperative blood loss and operation time, while prior pancreatic pseudocyst/abscess was a risk factor.In all, 59 patients (18 patients received preoperative SAE) were analyzed. The median age was 44.7 years. Preoperative SAE reduced the intraoperative blood loss (637.0 vs. 420.3 ml, Preoperative SAE could reduce intraoperative blood loss and operation time in SPH patients without increasing the incidence of complications compared to splenectomy only. Sinistral portal hypertension (SPH), also named left-sided portal hypertension, is a rare clinical syndrome. Unlike common causes of portal hypertension, occlusion of the splenic vein results in hypertension in the drainage system for splenic blood, which further leads to the hypertension of gastric varices or esophageal varices . The patAlthough most patients are asymptomatic, upper gastrointestinal bleeding has been reported in 4–17% of cases , and theWe designed a retrospective cohort study to compare splenectomy only with SAE combined with splenectomy in patients with SPH to determine whether preoperative SAE could reduce intraoperative blood loss and the operation time. Secondary outcomes such as postoperative complications, expenses, and length of stay (LOS) were also analyzed.; (4) open splenectomy performed in the Department of Pancreatology. Patients with malignant pancreatic or splenic diseases and portal hypertension derived from liver diseases were excluded. Written informed consent was obtained from all patients. The study was performed according to the Declaration of Helsinki and approved by the local ethics committee .The study had a retrospective cohort design, and patients were grouped into two cohorts on the basis of the procedures they received: splenectomy with preoperative SAE and splenectomy only. Inpatients diagnosed with SPH in the Department of Pancreatology, West China Hospital, Sichuan University (February 2018 to September 2020), were identified, and the follow-up period ended in April 2021. In all, 59 patients were selected based on the following criteria: (1) age 18 to 75 years old; (2) diagnosis of SPH based on isolated gastric or gastric and esophageal varices confirmed by endoscopy, and results of CT/MRI/DSA which inferred the primary diseases; (3) a history of gastrointestinal bleeding and failure of conservative therapies or endoscopic therapiesSAE procedures were performed as previously described . A 2.5- This study focused on the effect of preoperative SAE on decreasing the amount of blood loss during surgery. Considering the difficulty in precise measurement of intraoperative blood loss, we chose the equation proposed by Mercuriali et al. , and theχ2 test or Fisher’s exact test were chosen to analyze categorical data. Two-tailed unpaired t-tests were employed to compare normally distributed data, whereas Mann–Whitney’s U tests were used for nonnormally distributed data or ranked data. Owing to the nonnormal distribution of the estimated blood loss data and operation time data, we transformed the continuous variables into binary variables based on cutoff values, defined as median blood loss and operation time in the splenectomy cohort, to perform the Firth logistic regression analysis [P < 0.1 for entry), and then a multivariate analysis was performed. A P < 0.05 denoted statistical significance. All statistical analyses were performed using RStudio 1.4 .Continuous data that followed the normal distribution are presented as means with standard deviations (SDs), while nonnormally distributed data are presented as medians with interquartile ranges (IQRs). Categorical data are presented as frequencies and percentages. Pearson’s analysis . A univaP = 0.009). One hundred percent (41/41) of patients in the splenectomy group and 94.4% (17/18) of patients in the preoperative SAE group had a history of pancreatitis (P = 0.305). Regarding pancreatitis attacks, no significant difference was noted (P = 0.258), and the median number of attacks was 2 in both groups. A total of 34.1% (14/41) of patients in the splenectomy group had previously undergone abdominal surgery, while the number is 44.4% (8/18) in SAE + splenectomy group (P = 0.451).A total of 59 patients were analyzed Table . The medP = 0.041) and a longer operation time (174 (145–212) min vs. 141.5 (120–166.25) min, P = 0.012) (Table P = 0.171). Notably, fewer patients in the SAE group (13/18) received distal pancreatectomies than patients in the splenectomy group (39/41) (P = 0.023). Regarding the postoperative outcomes, no deaths were reported during follow-up. The median follow-up was 18 months in the splenectomy group and 26 months in the preoperative group. POPF was the most common complication, with 22% (13/59) of patients experiencing it, yet the majority (12/13) had biochemical POPF, with only one patient in the SAE group suffering from grade B POPF (P = 0.146). Postoperative bleeding occurred in 5 of 59 patients. Only one patient in the preoperative SAE group reported melena during follow-up. For patients who received splenectomy, two patients presented with melena; one was diagnosed with bleeding from surgical sites, while the other one exhibited acute bleeding from postoperative stress ulcers and developed hemorrhagic shock. Thromboembolism in the portal vein developed in three patients, two patients received splenectomy only and 1 patient received preoperative SAE . Although one cohort received preoperative SAE, neither total LOS nor postoperative LOS showed any significant difference. In light of this finding, the cost of interventional therapy might account for the higher total cost in the SAE + splenectomy group  vs. 69,358.9 , P = 0.002), while the surgical costs were comparable.Compared to patients who received preoperative SAE, patients in the splenectomy-only group significantly exhibited more blood loss (637.0 (416.5–1109.9) ml vs. 420.3 (278.1–620.1) ml, We conducted univariate and multivariate analyses using demographic and clinical characteristics and intraoperative variables to identify risk factors for intraoperative blood loss Table . In the We also analyzed the risk factors for prolonged operation time Table . Prior aPortal hypertension is a well-discussed topic, and its causes can be groups as prehepatic, hepatic, and posthepatic while hepatic (mainly cirrhosis) is the most common one. However, for patients with SPH, the etiology evidently differed from those with hepatic portal hypertension. Considering that clinical manifestations of SPH mainly come from primary diseases and splenic vein occlusion, splenectomy is preferred for patients who present with gastrointestinal bleeding. Based on our experience, a history of pancreatitis could give rise to a greater risk of adhesions in the abdominal area, making splenectomy for SPH patients much more difficult than that for patients with other causes of splenomegaly. Thus, a safer, more effective procedure must be developed.With surgeons at the crossroads of treatment, interventional therapy has come to the center stage. Wang and colleagues conducted a study of 14 patients with SPH, and reported no severe complications, such as splenic abscess or rupture of the spleen . HoweverUnder these circumstances, the combination approach of surgical and interventional therapy finally drew the attention of surgeons. Preoperative SAE was first proposed by Poulin et al. in 1993 . This meP = 0.023), which might raise questions regarding whether this discrepancy could lead to a prolonged operation time and more blood loss. In our hospital, the diseased pancreas is divided with a stapler so that the tail of the pancreas and the spleen can be resected at almost the same time. In other words, distal pancreatectomy did not take a longer time, which was supported by the results of logistic regression analysis. Furthermore, we also conducted an analysis with patients who received distal pancreatectomy, and proved the effectiveness of preoperative SAE in reducing operation time (174 (145–212) vs. 148 (125–167.5) min, P = 0.046)) and blood loss (662.2 (421.4–1120.7) ml vs. 428.5 (326.8–600) ml, P = 0.048).There was one notably significant difference observed between the patients: more patients in the splenectomy-only group received distal pancreatectomy , but fewer developed pleural effusion (2/59) . We thouThere were some limitations to this study. First, it was retrospectively designed, and thus, selection bias may exist. However, there was no notable difference in the demographic and clinical characteristics, except for the ASA score, with more patients in preoperative SAE group seeming to have higher scores. Since preoperative SAE could not remain concealed, it was also challenging to implement blinding. Second, the size of the sample is small de facto. Only 18 patients received preoperative SAE, which might have caused relatively high heterogeneity, but we also have to point out that, to our knowledge, this study is the first to compare preoperative SAE to splenectomy only in SPH patients. Finally, we excluded patients with pancreatic or splenic malignancy because these diseases would require other surgical methods that might interfere with the analysis of outcomes such as intraoperative blood loss and operation time. Further studies with a larger sample size and stratified analysis might provide more supportive evidence.This study demonstrated that preoperative SAE could efficiently reduce intraoperative blood loss and the operation time for patients with sinistral portal hypertension when undergoing splenectomy. Preoperative SAE did not increase the incidence of complications  or mortality among these patients. Since the LOS and surgery costs were comparable between the two groups, patients who received preoperative SAE had higher overall healthcare expenses.


To determine the prevalence of surgical site infections (SSIs) in neonatal congenital heart disease patients undergoing delayed sternal closure (DSC) and evaluate risk factors for SSI.Hospital records of 483 consecutive neonates who underwent surgical intervention between January 2013 and December 2017 were reviewed, and perioperative variables were recorded.p = 0.000).We found that the prevalence of SSI was 87.5% when the body weight was less than 1500 g. When the operative age was between seven and 14 days, the probability of no SSI is about 93.9%. When the duration of the aortic cross-clamp was more than 60 min, the prevalence of SSI was 91.2%. The prevalence without SSI was 96.6% when the duration of DSC was less than 24 h. However, when the duration of DSC was more than 120 h, the prevalence of SSI was 88.9% (With the prolongation of aortic clamping duration, the probability of occurrence of SSI increased in neonatal CHD with DSC. The age at operation and body weight are closely related to the occurrence of SSI in neonatal CHD patients with DSC. The field of pediatric and neonatal cardiac surgery has witnessed major advances in the past 30 years . At presThis work was conducted in line with the Strengthening the Reporting of Cohort Studies in Surgery (STROCSS) criteria ; it was A nested case-control study was conducted within the DSC cohort, comparing subjects with SSI to those without SSI. The SSI patients were diagnosed independently by two doctors, using published Centers for Disease Control and Prevention (CDC) criteria. SSI included incisional SSI and organ/space SSI. Control subjects were matched to case subjects in a 3:1 ratio based on year of surgery.Delayed sternal closure was generally as follows –20: 1) tPatients received prophylactic antibiotic therapy with cefazolin (60–80 mg/kg a day) , 21, whiAll data was retrospectively collected from the medical records of patients. This was a single-center, retrospective analysis. After all case and control subjects were identified, the primary author reviewed the medical record for all preoperative, intraoperative, and postoperative variables. Pertinent data collected included sex, weight, age at operation, genetic anomaly, prematurity (≤37 weeks [w]), time to find fetal congenital heart disease when prenatal detection was necessary, location of chest opening, type of surgical procedure, foreign material placed, duration of aortic cross-clamp, DSC, duration of DSC, duration of mediastinal chest tube, extracorporeal membrane oxygenation (ECMO), duration of mechanical ventilation, CICU length of stay, and hospital length of stay.2 test or Fisher’s exact test, as appropriate, for nominal variables. Variables found to be significantly associated with SSI in the univariate analyses (< 10) were investigated further by building multivariate logistic regression models to determine independent associations of risk factors with SSI. Results of the logistic regression are presented as odds ratio (OR) with 95% confidence intervals (CIs). A stepwise, multivariate model was constructed to evaluate risk factors. Variables that were not statistically significant (p > 0.05) were removed from the final multivariate model. All statistical analyses were performed with SPSS (version 23).Data is presented as frequencies, with percentage for categorical variables and median with interquartile range for continuous variables. Preoperative, intraoperative, postoperative, and clinical outcome variables were compared between cases and controls, using the χP < 0.0001 from χ2 test). A total of 188 patients (47 cases and 141 controls) were included in the analysis. Demographic and related data is shown in Tables p = 0.000, Table p = 0.919, Table p = 0.000, Table p = 0.000, Table p = 0.000, Table p = 0.000, Table p = 0.000, Table p = 0.000, Table p = 0.000, respectively) (Table p = 0.000) and thus excluded from the multivariate model to avoid multicollinearity. Multivariate logistic regression analysis showed that weight and age at operation were significantly associated with increased prevalence of SSI  , methicillin-susceptible Staphylococcus aureus (MSSA) (21.28%), Enterococcus sp. (12.76%), Enterobacter cloacae (8.51%), Pseudomonas sp. (6.38%), Klebsiella oxytoca (4.26%), Serratia marcescens (4.26%), and mycotic infection (6.38%).A total of 483 neonatal patients underwent DSC during the study period. Of these, 47 (9.7%) patients met CDC and National Healthcare Safety Network (NHSN) criteria for SSI . There were 28 cases of mediastinitis and two cases of endocarditis within the group of patients with organ/space SSI. There was a significantly higher prevalence of SSI in patients with DSC. The prevalence of SSI among neonatal CHD patients who did not undergo DSC at our center during the study period was 1.1% (21 out of 1832), significantly lower than among the DSC cohort  Placement of foreign bodies, including a drainage tube, increases the prevalence of SSI in neonatal patients with CHD who undergo DSC. b) The duration of aortic occlusion is less than 45 min, and the effect on SSI is negligible. However, with the prolongation of aortic clamping duration, the probability of developing SSI increased among neonatal CHD patients who underwent DSC. c) The study showed that the use of ECMO could also increase the prevalence of SSI among these patients. d) The age at surgery was closely related to the occurrence of SSI among these neonates as well. The probability of developing SSI was negatively correlated with the age at surgery. e) Compared with full-term infants, the prevalence of SSI is greater in premature infants. f) Weight has a great impact on the probability of SSI. Body weight was negatively correlated with the occurrence of SSI. g) Multivariate logistic regression analysis showed that weight and age at the time of surgery were significantly associated with increased prevalence of SSI. In this way, we can predict the prevalence of SSI in DSC neonates before surgery. h) In any case, the frequency of SSI is positively correlated with duration of DSC.


The association between SOX14 and cancer has been reported. The aim of this study was to identify and validate the potential value of SOX14 methylation in the early detection of cervical cancer.AUC.First, we extracted the data for SOX14 methylation and expression within cervical cancer from The Cancer Genome Atlas (TCGA) database and analysed them via UALCAN, Wanderer, MEXPRESS and LinkedOmics. Subsequently, according to the bioinformatics findings, primers and probes were designed for the most significantly differentiated methylation CpG site and synthesized for methylation-specific PCR (MSP) and quantitative methylation-specific PCR (QMSP) to verify SOX14 methylation in both cervical tissuses and liquid-based cell samples. Eventually, the clinical diagnostic efficacy of SOX14 methylation in the normal, cervical intraepithelial neoplasia, and cancer groups was analysed by ROCP < 0.001). Both the verification and validation cohorts indicated that the methylation level and the positive rate of SOX14 gradually increased with increasing severity from normal to cancer samples (P < 0.01). When the cut-off value was set as 128.45, the sensitivity and specificity of SOX14 hypermethylation in the diagnosis of cervical cancer were 94.12 and 86.46%, respectively. When taken as a screening biomarker (>CINII), the sensitivity was 74.42% and the specificity was 81.48%, with a cut-off value of 10.37.Pooled analysis demonstrated that SOX14 methylation levels were significantly increased in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) compared to normal tissues (SOX14 hypermethylation is associated with cervical cancer and has the potential to be a molecular biomarker for the screening and early diagnosis of cervical cancer.The online version contains supplementary material available at 10.1186/s12885-021-08406-2. Cervical cancer ranks as the fourth most frequently diagnosed cancer and the fourth leading cause of cancer death among women, with an estimated 604,000 cases and 342,000 deaths worldwide in 2020 . It is tCurrently, the Pap smear, the ThinPrep cytologic test (TCT), and human papillomavirus (HPV) testing are the most commonly used methods for cervical cancer screening . HoweverSOX14 is a member of the SOX gene family, which mediates the binding of high-mobility group (HMG) domains to DNA and has regulatory functions in development, the cell cycle and differentiation . PreviouSOX14 is also involved in the development of tumours. Deb S et al. found that SOX14 can induce apoptosis of cervical cancer cell lines by activating the p53 pathway . Li F etTherefore, the aims of this study were to 1) further explore the relationship of SOX14 methylation in cervical cancer using integrated datasets and web tools and 2) validate the potential value of SOX14 methylation in the screening and early diagnosis of cervical cancer.http://ualcan.path.uab.edu/index.html) [http://maplab.imppc.org/wanderer/) [http://gepia.cancer-pku.cn/) [https://mexpress.be/) [http://www.linkedomics.org/login.php) [Datasets of DNA methylation was extracted from the TCGA database and included clinical information from cervical tissues Table . The metex.html) . The meanderer/) . The rawpku.cn/) . The assess.be/) . The assgin.php) .Cervical frozen tissues and liquid-based cell specimens were available from the Department of Gynecology and Obstetrics, Tianjin Medical University General Hospital, and Tianjin First Central Hospital from January 2016 to June 2017. No patients included had a history of hysterectomy, radiotherapy or chemotherapy and a history of taking immunosuppressive agents or other tumours. Moreover, none of the patients involved were pregnant. Normal tissue samples and normal TCT samples from benign disease patients with fibroids, uterine prolapse, hypermenorrhoea, etc.All the samples were collected before treatment and a diagnosis was made by experienced gynaecological pathologists, with histological classification as the reference; the samples were classified as normal, cervical intraepithelial neoplasia II (CINII), CINIII or cancer. This study was approved by the Medical Ethics Committee of Tianjin Medical University, and all the patients gave informed consent.A total of 36 tissue specimens and 113 TCT specimens were collected for the tests. The frozen tissue samples consisted of 19 normal samples, and 17 cancer samples stored at − 80 °C. The TCT specimens included 27 normal cervical samples (median age: 43 years), 36 CINII stage specimens (median patient age: 42 years), 33 CINIII stage specimens (median patient age: 40 years), and 17 cervical cancer specimens (median patient age: 54 years). The FIGO staging of cervical cancer results were as follows: 6 cases of IA1, 3 cases of IB1, 1 case of IB2, 4 cases of IIA, 2 cases of IIB, and 1 case of IIIB. The specimens were obtained by clinicians using a disposable cervical specimen collection brush, stored in TCT preservation solution , and stored at 4 °C.Genomic DNA from frozen tissues was extracted using a TIANamp Genomic DNA Kit  according to the manufacturer’s instructions. DNA from cervical liquid-based cell specimens was isolated by phenol/chloroform extraction . DNA conOne microgram of genomic DNA per sample was modified using the EZ DNA methylation kit  according to the manufacturer’s instructions. Leukocyte DNA from healthy women was used as a negative control for methylation, while in vitro methylated leukocyte DNA produced using M. SssI methyltransferase  was used as a positive control.Methylated primers were designed using Methyl Primer Express v1.0 and synthesized by Sangon Biotech  with sufficient methylated DNA (200 pg DNA). The relative level of SOX14 methylation was expressed as (the average quantity of methylated SOX14 /the average quantity of ACTB) × 10,000 [The methylated primers for QMSP were the same as those used for MSP. The probes were designed using Cone Manager 9.0 software and synthesized by Sangon Biotech. The ACTB gene was used as a methylation reference gene. Each reaction was performed in a total 10 μl reaction volume containing 5 μl 2 × Master Mix, 2.5 μl BS-DNA (10 ng/μl), forward and reverse primer (10 μmol/L) at 0.3 μl each, probe (5 μmol/L) at 0.4 μl, and ddH× 10,000 .P-value of less than 0.05 was considered statistically significant.Statistical analysis were performed using IBM SPSS 25.0  and GraphPad Prism 8 . The association between methylation in cg4945331 and expression from TCGA was analysis by Spearman. Kruskal-Wallis test was used to compare methylation levels and clinical stages. The threshold of each two groups was defined with the maximum value of the Youden index (YI), and the sensitivity, specificity were calculated accordingly. GraphPad Prism version 8.0 was used for statistically significant differences between two stages by the Mann–Whitney test. A P < 0.001). Furthermore, the methylation level was increased with tumour stage, and that of stage IV was the highest (P < 0.001) . The most differential methylated CpG site was cg4945331   Fig. . Fig. 2bP < 0.01, n = 292).  or T stage  . Fig. a It was 44) Fig. b,c.Fig.We performed MSP with primer sets targeting the sequence including the cg4945331 site with the top differential value Table  to detecP < 0.01) (Table P < 0.05)  Table . Paired 5) Table . SimilarAUC was 0.99 in the normal and cancer tissues, and the sensitivity and specificity in the diagnosis of cancer were 94.12 and 96.3%, respectively, with the optimal cut-off value (87.21)  into a disease group. Table AUC was 0.81, the sensitivity (74.42%) was decreased, but the specificity (81.48%) were comparable.The sensitivity and specificity between the groups were analysed according to ROC curves Fig. . The higAs a common malignancy of the female reproductive system, cervical carcinogenesis is a complicated process with multiple stages and steps. There is a gradual development process from precancerous lesions to cervical cancer, and early detection of cervical lesions is particularly important in clinical practice. Aberrant DNA methylation is an epigenetic hallmark of cancer and is known to play an important role in tumorigenesis and progression. Meanwhile, with the advancement of bioinformatics technology, it is feasible to identify novel methylation biomarkers via integrative multi-web tools –33. DefiHence, to further reveal the role of SOX14 methylation in cervical cancer and validate the potential clinical value of SOX14 methylation in the diagnosis of cervical cancer, we combined information from databases and clinical samples. Based on an integrative multi-web tool approach, we found that SOX14 was hypermethylated in CESC and correlated with its expression. Clearly, these findings indicate that SOX14 methylation participates in carcinogenesis by perturbing SOX14 gene regulation. More exciting, it was feasible for us to select a CpG site with the most differentiated methylation level between normal and cancer samples to design primers and probes for subsequent verification and validation via the Wander web tool. As expected, the MSP results demonstrated that the methylation positive rate of SOX14 in cancer tissues (70.59%) was obviously higher than that in normal tissues (5.26%). Thus, the biomarker of SOX14 hypermethylation in cervical cancer moved forward to the validation step. The QMSP results showed that both the SOX14 methylation positive rate and methylation level were higher than those in normal cervical samples.In a recent review that about the translational road of DNA methylation biomarkers to the clinic, Warwick J. Locke et al. mentioned that there are several diagnostic areas including primary diagnosis,triage,choice of therapy etc. for adopting the DNA methylation biomarkers . Hence,wRecently, the World Health Organization (WHO) called for action towards achieving the global elimination of cervical cancer by 2030. To achieve this goal, one of the challenges is to build effective and scaled screening strategies . AlthougFurthermore, the detection of methylation biomarker is an objective test and can be performed on the same material used for hrHPV testing, which makes it promising for self-sampled tests. This will benefit more Chinese women who live in remote rural places . As one What’s more, consistent with studies that have revealed that DNA methylation occurs in a tissue-specific, cell type-specific and stage-specific manner , 40,SOX1Our results demonstrated that it was feasible and convenient to identify DNA methylation biomarkers through coupling with bioinformatics analysis and clinical samples. SOX14 presented hypermethylation in cervical cancers; furthermore, with the optimal cut-off value, the sensitivity and specificity in the differentiation of precancer and cancer were 94.12 and 86.46%, respectively. Additionally, the sampling method can be combined with HPV tests. Thus, scale-up screening can be performed via high-throughput detection. Nevertheless, more sustainable and large cohort clinical trials are needed to confirm the identified biomarkers worldwide to push forward the rapid development of translation medicine in the near future.Additional file 1: Table S1.1. TCGA cervical squamous cell carcinoma and endocervical adenocarcinoma patient characteristics (cancer tissues). Table S1.2. TCGA cervical squamous cell carcinoma and endocervical adenocarcinoma patient characteristics . Table S2. Each CpG sites of SOX14 methylation in normal vs. CSEC. The difference value indicated that the beta value of cancer minus the normal. The adj.pval represents adjusted P-value.Additional file 2: Figure S1. A full-length gel figure was presented for DNA Ladder PCR. Among the 38 samples of frozen tissues whose DNA fragments can be determined, 2 samples  have DNA fragments with severe fragmentation or low quality and are unavailable, while the remaining 36 samples are of high quality and can be used in the following experiment.Additional file 3: Figure S2. The level of SOX14 expression in different normal tissues,the samples were ordered by their expression value.Additional file 4: Figure S3. Full length gel pictures for the verification results of SOX14 methylation in total 36 cervical tissues using MSP.Additional file 5: Figure S4. We further repeated samples  with multiple bands from Fig S2. The PCR product for methylation was 85 bp,unmethylation was 94 bp.Additional file 6: FigureS5. ROC curve for SOX14 methylation in each of the differentiated groups.


Assessment of patients at emergency departments (EDs) is a potential opportunity to achieve early TB diagnosis, and interrupt transmission. An earlier study in London found that 39% of patients diagnosed with TB had attended an ED in the preceding 6 months [4]. Of these, 76% had a chest radiograph performed, of which 86% and 40% were abnormal in cases of pulmonary and extrapulmonary TB, respectively. Attendance at EDs provides an opportunity to identify individuals with LTBI, who may be at risk for progression to active disease and unlikely to engage with healthcare services  LTBI screening among high-risk groups at EDs could be implemented to identify those at risk of progression to TB disease. Large-scale studies are required to investigate effective TB disease screening strategies in EDs.https://bit.ly/3bTkoOn To the Editor:via other routes.Most tuberculosis (TB) cases in low-incidence settings are thought to be due to reactivation of latent TB infection (LTBI) in high-risk populations –3. AssesBetween July 2013 and May 2017, we recruited individuals over the age of 16 years, who were recent entrants from, or prolonged travellers to, high TB incidence countries, or people with a history of homelessness, imprisonment or problem drug use attending EDs at seven London hospitals. We investigated the yield of interferon-γ release assay (IGRA) and TB disease screening among eligible ED attendees, regardless of their reason for ED attendance. Participants were tested with either the QuantiFERON Gold-in-Tube , or QuantiFERON-TB Plus. A subset of participants was screened for TB disease; symptomatic individuals  underwent chest radiograph, sputum Xpert MTB/RIF testing and subsequent referral for further evaluation and management . Those sa priori variables , and variables found to be significant in univariable analyses (p<0.2).Descriptive analyses were performed to assess the yield of screening and incidence rates of TB disease among those tested by IGRA. Logistic regression models were used to examine factors associated with IGRA positivity. The final multivariable model included A total of 1407 participants were recruited to the IGRA screening study, of whom 241 (17.1%) had a history of substance use disorders, homelessness or imprisonment, while the remainder were migrants from high TB burden countries. Of the 1407 participants, 642 (45.6%) were female, and the majority  were over 35 years of age (median 45 years). Among those recruited due to recent migration from, or travel to, a high TB incidence country, most participants  were South Asian, while the largest ethnic group among those with social risk factors was white . Almost one fifth  of participants reported previous contact with a TB case. Diabetes was common, affecting 239/1407 (17.0%). Of the 1407 participants, 109 (7.7%) were not registered with a general practitioner; this was more common among those with social risk factors  when compared with the migrant group .IGRA results were available for 1232 participants, of which 34 (2.4%) were indeterminate. A total of 256/1198 (21.4%) participants with valid available results were IGRA positive. The prevalence of IGRA positivity was 24% among migrants and 19% among those with social risk factors . In a muvia linkage to national TB surveillance records until 31 December 2017. Of the 256 with a positive IGRA, five were notified with TB disease during follow-up, giving a TB incidence rate among those with a positive IGRA of 1476.4/100 000 person-years (95% CI 529.4–3173.2). All five TB cases had extrapulmonary disease (lymph node (notified 5 days after recruitment), spinal (35 days), disseminated (52 days), genito-urinary (162 days) and intra-abdominal (412 days)), and none reported recent TB contact at study recruitment. Median quantitative interferon-γ responses to Mycobacterium tuberculosis antigens were 6.02 IU·mL−1 (range 0.62 to >10 IU·mL−1) among the five progressors to TB disease, compared to 2.57 IU·mL−1 (0.35 to >10 IU·mL−1) among non-progressors. No TB cases were notified among participants who had negative or indeterminate IGRAs.Participants screened by IGRA were followed up for a median of 381 days (interquartile range 303–605 days), Of the 513 participants screened in the active TB study, only 14 (2.7%) were symptomatic. Of these, 13 had a chest radiograph, with eight providing an adequate sputum sample for Xpert MTB/RIF testing. None of these patients were diagnosed with TB during the study.Previous studies have retrospectively reviewed ED presentations among notified TB cases in low-incidence settings , 8–11, aMajor study limitations were that patients with positive IGRA results were not routinely linked to TB services for evaluation for TB disease and consideration of LTBI treatment, since LTBI screening among risk groups attending EDs was not part of national policy at the time. Thus, we are unable to estimate the proportions of IGRA-positive participants who would have started and completed preventative therapy if referred to TB services following detection. This remains a key knowledge gap, since effectiveness (and cost-effectiveness) of screening is dependent upon completion of LTBI treatment for these individuals, to reduce risk of TB disease. In addition, although the vast majority of participants in the study were registered with general practitioners, it is unclear how engaged they were with these services, limiting our ability to assess precisely how well ED screening could complement the primary care screening programme. Data were not available on individuals who attended EDs and were screened for eligibility but not recruited to the study.In the TB disease screening study arm, we proactively screened for pulmonary TB disease using a symptom screen followed by chest radiograph and sputum testing. Consequently, pulmonary TB cases without typical symptoms and extrapulmonary cases may have been missed. Moreover, our sample size of 513 participants screened for TB symptoms may have been too small to detect active cases; we therefore could not evaluate risk factors for active TB or cost-effectiveness of TB disease screening.via other screening approaches. Such screening must be supported by a dedicated protocol that detects individuals at higher risk of TB for screening, and resources to facilitate onward referral of those with a positive IGRA to local TB services. In contrast, this study suggests that resource-intensive, symptom-based active TB screening in EDs is unlikely to be worthwhile as the yield is likely to be too low to justify the resource. However, other high-throughput screening models such as automated, routine chest radiograph review in EDs may be evaluated in future research.In summary, our study suggests that ED IGRA screening among TB risk groups could be implemented to identify individuals at risk of TB who may be difficult to engage 10.1183/13993003.03831-2020.Shareable1This one-page PDF can be shared freely online.ERJ-03831-2020.ShareableShareable PDF


In children with myocardial ischemia, myocardial fibrosis was observed with greater frequency. Myocardial perfusion defects may reflect an ischemic process which (1) affects the clinical manifestations and (2) is an important predictor of adverse clinical events and risk of death in children with HCM. Myocardial ischemia in HCM patients frequently correlates with myocardial fibrosis.Myocardial ischemia caused by microvascular dysfunction is an important pathophysiologic component of hypertrophic cardiomyopathy (HCM), promoting myocardial fibrosis, adverse left ventricular remodeling, and impacting on clinical course and outcome in HCM patients. The aim of study was to assess the prevalence and clinical significance of myocardial ischemia in children with HCM using 99mTc-MIBI single-photon emission computed tomography (SPECT). Ninety-one children with HCM, median age 13.6 years, underwent SPECT evaluation from 2006 to 2017. Imaging was performed at rest and after maximal exercise. Myocardial perfusion defects were identified in 70 children , median age 13.8 years. Fixed perfusion defects were evident in 22 of them, while reversible at rest in 48. In 21 children , median age 11 years, myocardial perfusion defects were not detected. Patient demographics, echocardiography, resting electrocardiogram (ECG), 24-h Holter ECG, myocardial fibrosis in cardiovascular magnetic resonance imaging, and cardiovascular events were analyzed and compared between the groups. During follow-up at a median of 8.3 years in children with myocardial ischemia, clinical endpoints occurred more often (47 vs. 5;  Hypertrophic cardiomyopathy (HCM) is the second most common cardiomyopathy in children, with an estimated annual incidence of 0.24–0.47 per 100,000 , 4. The From January 2006 to November 2017 we prospectively enrolled pediatric patients with diagnosed HCM hospitalized in the cardiology department. All our HCM patients underwent 99mTc-MIBI SPECT evaluation and then were followed prospectively for a median of 8.3 years to assess the occurrence of cardiovascular events. Criteria for inclusion in the study were age < 18 years at time of diagnosis and echocardiographic evidence of LV hypertrophy defined as a maximal left ventricular wall thickness greater than two standard deviations above the body surface area-corrected mean (z-score ≥ 2) and not explained by abnormal loading conditions , 16. ThePatients demographics, clinical symptoms, as well as the results of echocardiography, resting and ambulatory 12 lead electrocardiograms (ECG), CMR, and 99mTc-MIBI SPECT at rest and after exercise test were collected. Cardiac examinations were repeatedly performed during the follow-up period.In all children, routinely planned cardiac examinations  and clinical evaluation every 6 months were performed. In the case of impairment of the general condition, onset of clinical symptoms and disease progression, cardiac tests, including repeated CMR, were performed more frequently, according to individual indications.For analysis, the most recent or latest results before the endpoint were chosen. The period from diagnosis of HCM to the most recent study was median 10.8 (6.5–13.2) years. The median period between SPECT and recent results was 8.3 (4.3–10.8) years.Sudden cardiac death, unexplained syncope and non-sustained ventricular tachycardia (NSVT) were defined as recommended in the literature . EchocarCardiovascular magnetic resonance imaging (CMR) was performed using a 1.5-T scanner . Cine images were acquired by the breath-hold, electrocardiographic-gated, segmented k-space steady-state free-precession technique using 25 phases per cardiac cycle. LGE images were obtained in the long-axis and short-axis imaging planes using a breath-hold-segmented inversion recovery sequence implemented 10–15 min after intravenous administration of 0.1 mmol/kg of gadobutrol ; gadodiamide  was used instead gadobutrol if the patient was under 2 years of age. The presence of left ventricular LGE and myocardial fibrosis was determined using visual assessment by two independent observers and if positive, quantified. Quantification was performed both as a signal intensity threshold of > 6 SD above remote myocardium. The extent of LGE is presented in grams and as the percentage of total LV mass .The 99mTc-MIBI SPECT was performed for evaluation of MP and prevalence of perfusion defects indicative of myocardial ischemia. The tracer used was sestamibi (MIBI) labeled with technetium 99 m. The 99mTc‑MIBI chelate was administered intravenously at a dose of 111 to 740 MBq per kilogram of body weight . MP scanPatient events were recorded by communication with children‘s parents. Medical records were reviewed after attendance in outpatient clinics or hospital stay. No patient was lost to follow-up. Clinical endpoints included these cardiovascular events: SCD, HF-related death, resuscitated cardiac arrest (rCA), appropriate ICD discharges (ICDdx), heart transplant (HTx), NSVT, syncope, and progression of heart failure from New York Heart Association (NYHA) class I or II to class III. Moreover, arrhythmic endpoints was predefined and included: SCD, rCA, adequate ICDdx, NSVT.All children were divided into group I (gI) with and group II without myocardial ischemia in SPECT (gII). The presence and extent of the perfusion defects were analyzed by two independent experts and relative myocardial perfusion was assessed. In a descriptive way, the presence of perfusion defects was determined in the following projections: short axis , vertical long axis  and horizontal long axis . The presence of perfusion defects indicated a significantly lower concentration of 99mTc-MIBI in the myocardium. In the case of fixed myocardial perfusion defects SPECT images showed significantly lower concentration of 99mTc‑MIBI in stress and in rest images indicating severe myocardial ischemia. In the case of reversible perfusion defects, SPECT scans showed significant radiotracer accumulation improvement in rest images.The demographic, clinical, echocardiographic, CMR parameters, and occurrence of endpoints were analyzed and compared between the two groups.U Mann–Whitney test. Binary variables were compared using Fisher test or chi-square test. The Kaplan–Meier survival curves were constructed to compare the groups with and without myocardial ischemia and with fixed or reversible perfusion defects in terms of reaching endpoints. Values of p < 0.05 were considered statistically significant.Statistical analysis was performed using Statistica software. Continuous variables are described as median with interquartile range (25th to 75th centile). Nominal variables are presented as a numerical value and the percentage of the study group. Continuous variables were compared by A total of 91 patients with HCM, median age 13.6 (10.3–15.8) years, 64% male, in NYHA class I or II, underwent SPECT evaluation from January 2006 to November 2017. Patients were followed prospectively for 8.3 (4.3–10.8) years. MP defects were identified in 70 of the 91 children , median age 13.8 years (10.7–15.9). Fixed perfusion defects were evident in 22 (31.4%) patients . A comparable number of children in both groups experienced LVOTO. The median LA diameter z-score did not differ significantly between the two groups studied (3.4 [gI] and 2.6 [gII]). Fibrosis in CMR was found at a significantly greater frequency in children with perfusion defects compared to children without myocardial ischemia , , while the gII median was 6.6 (p = 0.02) and significantly more patients reached a clinical endpoint .During follow-up, clinical endpoints occurred significantly more often in children with recognized perfusion defects and myocardial ischemia , rCA (n = 1), ICDdx (n = 6), NSVT (n = 15), syncope (n = 10), progression of heart failure from NYHA class I or II to class III (n = 9), HF-related death (n = 1), and HTx (n = 2). In children with myocardial ischemia, significantly more frequent myocardial fibrosis was observed . The data are presented in Table In children with perfusion defects, the following cardiovascular events occurred: SCD ( rCA n = , ICDdx (SVT n = 1, syncopeMyocardial ischemia can occur in patients with HCM in the absence of coronary artery disease  and is aThe LGE CMR sequences identify the persistence of gadolinium-based contrast agents within areas of fibrosis due to their accumulation and delayed washout from an expanded extracellular space . A growiIn agreement with other investigations , 39, 40,During follow-up lasting longer than eight years, 40% of patients with myocardial ischemia had cardiovascular events. Moreover, an association was found between the presence of MP abnormalities and the prevalence of cardiovascular endpoints. It should also be emphasized that the number of endpoints was significantly higher in children with MP defects, which confirms current literature reporting that, in patients with HCM, the degree of microvascular dysfunction is a strong, independent predictor of clinical deterioration and death .The results of our study showed that myocardial ischemia was strongly associated with an unfavorable outcome, which was confirmed by the analysis of the survival curves. This finding is consistent with the results of similar studies by others , 41. It As in the studies of other authors, the maximum LV wall thickness correlated with the presence of perfusion disorders . In chilIn children with hypertrophic cardiomyopathy and diagnosed myocardial perfusion defects in SPECT, we consider extending the cardiac tests to include computed tomography angiography to assess coronary arteries, modify pharmacological treatment by increasing the dose of beta blocker and recommend low or moderate intensity physical activity. We advise against competitive sports in these children. We determine the indications for ICD implantation on an individual basis.In conclusion, we demonstrated that MP defects may reflect an ischemic process which affects clinical manifestations and is an important predictor of adverse clinical events and risk of death in children with HCM. Today's routine diagnostic protocols for HCM do not include testing for myocardial ischemia. It is hoped this and other studies emphasize the value of including this type testing as a key element for developing future risk strategies.


Ovarian tissue transplantation methods using cooled and cryopreserved samples have been attractive options for fertility preservation in animal models and humans. The aim of this study was to evaluate the impact of previous exposure to cooling, cryopreservation, and VEGF on the overall efficiency of equine ovarian tissue after heterotopic xenotransplantation in mice. The end points evaluated were follicular morphology and development, follicular and stromal cell densities, angiogenesis (i.e. the density of new and mature blood vessels), collagen types I and III fiber densities, and total fibrosis. Ovaries of adult mares were harvested after ovariectomy, and ovarian fragments were xenografted in the i.p. wall of BALB nude mice. Ten types of treatments involving different combinations of cooling, cryopreservation, xenografting procedures, and VEGF exposure were compared. The novel aspect of this study was the use of equine ovarian tissue xenotransplantation in mice, challenging the fragments with different combinations of treatments. The main findings were (i) cooling but not cryopreservation was effective in preserving the follicular morphology, (ii) a greater percentage of developing follicles but lower follicular and stromal cell densities were observed after ovarian tissue engraftment, (iii) exposure to VEGF increased new and mature vessels in cryopreserved-transplanted tissue, and (iv) an appropriate balance in the collagen types I and III fiber ratio in cooling-transplanted tissue was observed after exposure to VEGF. This study contributes to advancing knowledge in the preservation of ovarian tissue after cooling-cryopreservation and transplantation aiming to be applied to genetically superior/valuable horses, livestock, endangered animals, and, possibly, humans.n = 3), exposed or not to vascular endothelial growth factor (VEGF) before transplantation, better survives for 7 days after transplantation into mouse hosts (n = 12). Tissues submitted to different combinations of cooling, freezing, and transplanting treatments, along with control groups, were evaluated using the parameters morphology, development, the density of immature eggs (follicles), the density of supportive  cells, collagen protein proportions, and density of blood vessels. Frozen-thawed treatments had lower percentages of normal follicles. Exposure to VEGF increased blood vessel densities in frozen tissue and favored adequate collagen levels in cooled-transplanted treatments. In conclusion, VEGF exposure seems to be beneficial for mare ovarian tissue transplantation and warrants further investigation.Due to ethical limitations involving humans, the female horse (mare) has recently emerged as an alternative model for reproductive comparisons with women to optimize fertility restoration using ovarian tissue transplantation techniques. This study determined if ovarian tissue from donor mares ( Cryopreservation and ovarian tissue transplantation (OTT) have been successfully used to restore fertility in animal models . In addiXenotransplantation is an appealing OTT technique that uses either knock-out-immunodeficient or drug-immunosuppressed animals  has been used as a strategy to promote neoangiogenesis and revascularization, consequently reducing hypoxia, preserving follicles in the grafted tissue  (ethylene glycol (EG) and DMSO) were obtained from Dinâmica .n = 3) from three adult reproductively sound non-pregnant cycling mixed-breed mares were collected using right flank surgical ovariectomy during different procedures  from the three animals were equally and randomly assigned to ten treatments : cooled (4°C) fragments in the absence of VEGF. All cooling periods were performed for 24 h at the same temperature, aiming to maintain a high quality of ovarian tissue and survivability of preantral follicles, as previously reported (VEGF+): cooled fragments in the presence of VEGF; (v) cooling and transplantation without VEGF (CTVEGF–): cooled fragments in the absence of VEGF, followed by xenotransplant; (vi) cooling and transplantation with VEGF (CTVEGF+): cooled fragments in the presence of VEGF, followed by xenotransplantation; (vii) cryopreservation and cooling without VEGF (CryoCVEGF–): vitrified/warmed fragments, followed by cooling in the absence of VEGF; (viii) cryopreservation and cooling with VEGF (CryoCVEGF+): vitrified/warmed fragments, followed by cooling in the presence of VEGF; (ix) cryopreservation + cooling + transplantation without VEGF (CryoCTVEGF–): vitrified/warmed fragments, followed by cooling in the absence of VEGF and submission to xenotransplantation; and (x) cryopreservation + cooling + transplantation with VEGF (CryoCTVEGF+): vitrified/warmed fragments cooled in the presence of VEGF and submitted to xenotransplantation. The fragments were vitrified and warmed after 7 days of storage (see below). The purpose of the cooling step in the CryoC and CryoCT groups was to ensure an adaptation period for the ovarian tissue immediately after warming but before xenotransplantation. This novel approach was based on the rationale that previous reports have shown the necessity of an adaptative period (hours or days) for the cryopreserved/thawed ovarian cells to recover their metabolism in in vitro culture conditions . The criterion used to select these treatments was based on the potential stimulus of neoangiogenesis (i.e. increase in blood vessel staining) after the transplantation procedure.The ovarian fragments . Briefly, ovarian fragments were exposed to two vitrification solutions (VS). The VS1 composed of MEM-HEPES supplemented with 10 mg/mL BSA, 0.25 M sucrose, 10% EG, and 10% DMSO was used at room temperature (~25°C). The composition of the VS2 and the temperature applied were similar to the VS1 but with a higher concentration of CPAs (20% EG and 20% DMSO). The fragments were exposed to VS1 for 4 min, followed by exposure to VS2 for 1 min. After exposure to CPAs, both VSs were removed, and the OTC containing the ovarian tissue was closed and immediately plunged into liquid nitrogen at –196°C. After the storage period (7 days), the OTC device containing the vitrified fragments was taken off from the liquid nitrogen container and kept at room temperature for 1 min and then immersed in a water bath at 37°C for 30 s. After that, the VS removal was performed in three steps using three different washing solutions (WS) composed of MEM-HEPES supplemented with 3 mg/mL BSA and decreasing sucrose concentrations . The ovarian fragments were kept for 5 min in each WS.Ovarian tissue vitrification was performed using a solid-surface technique with the ovarian tissue cryosystem (OTC) as previously described  were housed under a 12 h light: 12 h darkness cycle at 22°C and fed ad libitum. Non-castrated females were used since no difference in the quality of xenotransplanted ovarian tissue has been reported when compared with castrated hosts  and counterstained with hematoxylin  was calculated by considering the number of normal developing follicles divided by the total number of normal preantral follicles multiplied by 100. All histological sections were examined by the same blind operator, who was unaware of mare identity and treatment, thereby ensuring that each follicle was counted only once.2) was verified after scale calibration. Thereafter, the follicular density was determined by dividing the number of normal preantral follicles by the area of the ovarian section (cm2). The ovarian stromal cell density was manually evaluated by counting the cell nucleus in a total of 10% of all histological sections (2) per selected section were recorded to calculate the mean stromal cell density per ovarian fragment using the DS Cooled Camera Head DS-Ri1 coupled with a Nikon Eclipse 80i microscope at 400× magnification. Afterward, pictures were obtained and evaluated using the ImageJ software. All evaluations and measurements were made by the same operator.The follicular density was determined as reported previously  or anti-α-SMA . Afterward, the slides were incubated with goat anti-rabbit IgG secondary antibody for 30 min . Then, an additional incubation for 30 min was performed with the avidin–biotin enzyme complex  for reaction with 3,3’-diaminobenzidine in chromogenic solution . Finally, the slides were counterstained with hematoxylin and Scott’s solution. Negative controls were performed by incubating the tissue sections without the primary antibodies, and for the positive control, mouse spleen tissue was used. The number of stained new and mature vessels was quantified by evaluating the mean number of each type of vessel in four fields (12100 µm2 each) from each section of all slides  using a microscope at 400× magnification  and mature (α-SMA) blood vessels was determined with slight modifications fromn = 30; three slides/animals per treatment) were examined using polarized microscopy (Nikon E200) at 400× magnification coupled to an image capture system . The total collagen in the connective tissue and the differences in the polarizing colors were analyzed for both types I (stained yellow/orange birefringence) and III (stained green birefringence) collagen fibers  destined for tissue fibrotic evaluation was similarly histologically processed using 7 µm section intervals and stained using Masson’s trichrome kit . Four ovarian fragment sections were examined by light microscopy at 400× magnification . Data are presented as mean (± s.e.m.), and the statistical significance was defined as P ≤ 0.05 (two-sided). Probability values > 0.05 and < 0.1 indicate that a difference approached significance.Statistical calculations were carried out using Sigma Plot 11.0 (Systat Software Inc.). Normality (Shapiro–Wilk test) and homogeneity of variance (Levene’s tests) were evaluated. One-way ANOVA was used to compare either the Fresh Control group or the cryopreservation control group against each treatment evaluated. Two-way ANOVA (for VEGF exposure and procedure effects) followed by Fisher LSD A total of 595 preantral follicles were analyzed in 6186 histological sections, with an overall mean number of 213.3 ± 11.5  sections evaluated per fragment. The number of follicles per fragment was only available from the fragments destined for histological processing. In the histological analysis, a total of 29/58 fragments (50%) were evaluated; two fragments were not harvested due to the death of one mouse host. The remaining 29 fragments were destined to IHC and Picrosirius/Masson’s trichrome analyses, in which only tissue features (and not follicular number) were evaluated. Therefore, the mean number of follicles evaluated per fragment considering histology only was 20.5 ± 9.5 . In this regard, only four (13.8%) histological fragments from different treatments did not contain any follicles not affecting, therefore, the number of follicles in each treatment. All grafted ovarian fragments were successfully recovered after transplantation  and K. IP < 0.05) percentages of normal follicles were found in the CryoCVEGF–, CryoCVEGF+, CryoCTVEGF–, and CryoCTVEGF+ treatments compared with the Fresh Control group; however, a higher (P < 0.05) percentage was observed in the CVEGF+ treatment than in the Fresh Control group. When comparisons were performed within each treatment (i.e. in the absence or presence of VEGF), a lower (P < 0.05) percentage of normal follicles was observed under the cooling condition alone in the absence of VEGF (CVEGF– vs CVEGF+). Comparing the treatments in the absence of VEGF, a higher (P < 0.05) percentage of normal follicles was observed in the CTVEGF– compared with the CryoCVEGF– treatment only. However, in the presence of VEGF, a higher (P < 0.05) percentage of normal follicles was found in the CVEGF+ treatment compared with the CryoCVEGF+ and CryoCTVEGF+ treatments. When considering the overall procedure effect (P < 0.05) percentages of normal follicles than the cryopreservation-cooling-transplantation technique.With regard to follicle survival , lower  values were observed in the CT and CryoCT treatments regardless of the presence of VEGF and also in the CryoCVEGF– treatment. Within treatments, more (P < 0.05) developing follicles were found in the CryoC in the absence of VEGF (CryoCVEGF–). When examining the overall procedure effect (P < 0.05) developing follicles compared with the cooling technique alone. Representative images of preantral follicle morphology and categories in grafted ovarian tissue are shown  follicular density was observed in all treatments compared with the Fresh Control group (P < 0.05) follicular density was observed in the CVEGF+ treatment when compared with the cryopreservation control (Cryo Control) group and the cooling treatment without exposure to VEGF (CVEGF–). In the absence of VEGF, the cryopreserved fragments submitted to the cooling technique (i.e. CryoCVEGF–) had a higher (P < 0.05) follicular density compared with the other treatments  under the same conditions. In contrast, in the presence of VEGF, a higher (P < 0.05) follicular density was observed in the cooling treatment (CVEGF+) when compared with the other treatments . Regarding the overall procedure effect (P < 0.05) follicular densities were observed in the transplanted groups.The follicular and stromal cell densities in equine ovarian tissue with or without VEGF treatments and the effects of VEGF exposure and procedures in both end points are shown  value was observed in the cooling treatment exposed to VEGF (CVEGF+) compared with the Fresh Control and cryopreservation control (Cryo Control) groups. On the other hand, lower (P < 0.05) stromal cell density was observed in both CT and CryoCT treatments regardless of VEGF exposure. Within treatments, a higher (P < 0.05) stromal cell density was observed only in the cooling treatment in the presence of VEGF (CVEGF+ vs CVEGF–). Besides this, within the same VEGF condition (i.e. absence or presence of VEGF), higher (P < 0.05) stromal cell densities were observed in both cooling treatments when compared with the other treatments. Regarding the overall procedure effect (P < 0.05) stromal cell densities compared with their non-transplantation treatment counterparts (P < 0.05) stromal cell density was observed in the cooling procedure alone.Concerning the stromal cell density , a highee effect , fragmenterparts , while t2) of tissue was determined using the markers CD31 and α-SMA, respectively (P < 0.05) only in the CTVEGF– and CryoCTVEGF+ treatments (P < 0.05) CD31 density in the CTVEGF+ treatment and increased (P < 0.05) in the CryoCTVEGF+ treatment. Concerning the treatments without exposure to VEGF, a higher (P < 0.05) vessel density was observed in the CTVEGF– when compared with the CryoCTVEGF– treatment. Concerning the overall procedure effect (P > 0.05) was observed for CD31 labeling density between the procedures cooling-transplantation and cryopreservation-cooling and transplantation.The density of new and mature blood vessels per microscopic field  density was found in the CryoCTVEGF– treatment. Within treatments, a higher (P < 0.05) density of α-SMA labeling was observed in the CryoCTVEGF+ treatment. Concerning the treatments in the absence of VEGF, a higher (P < 0.05) staining density of α-SMA was found in the CTVEGF– than in the CryoCTVEGF– treatment. Regarding the overall procedure effect (P < 0.05) α-SMA density than those in the cryopreservation-cooling-transplantation technique. Representative images of CD31  in the cryopreservation control group and CTVEGF– and CryoCTVEGF– treatments  percentages of collagen type I fibers. Within treatments, a higher (P < 0.05) intensity of collagen type I was observed only in the CT treatment in the presence of VEGF (CTVEGF+). In the absence of exposure to VEGF, greater (P < 0.05) percentages of collagen type I were found in both CVEGF– and CryoCVEGF– treatments than in the CTVEGF– treatment. However, after VEGF exposure, fragments in the CTVEGF+ treatment had a higher (P < 0.05) percentage of collagen type I than those in the CVEGF+ treatment.The percentages of pixels for collagen types I and III fibers per histological section for the different treatments are shown , F and Geatments . AdditioP < 0.05) values were observed only in the cryopreservation control group and CTVEGF– treatments. Compared with the cryopreservation control group, lower (P < 0.05) percentages of collagen type III were observed in the CVEGF–, CTVEGF+, both CryoC, and CryoCTVEGF+ treatments. Within treatments, higher (P < 0.05) percentages of collagen type III were observed in the CVEGF+ and CTVEGF– treatments. In the absence of VEGF, a higher (P < 0.05) percentage of collagen type III fibers was found only in the CTVEGF– treatment compared with the CVEGF– treatment. Furthermore, after exposure to VEGF, a greater (P < 0.05) percentage of collagen type III fibers was observed in the CVEGF+ treatment than in the CTVEGF+ treatment. Representative histological merged images of ovarian tissue stained with Picrosirius red stain for collagen type I (red) and III (green) fibers in all treatments are shown  percentages of tissue fibrosis than the cryopreservation control treatment (VEGF+ and CryoCVEGF+) had greater (P < 0.05) percentages of tissue fibrosis. Concerning the treatments exposed to VEGF, higher (P < 0.05) percentages of tissue fibrosis were found in the CTVEGF+ and CryoCVEGF+ than in the CryoCTVEGF+ treatment. When considering the overall effect of VEGF (P < 0.05) percentages of tissue fibrosis. Representative histological images of total collagen in ovarian tissue for all treatments are shown  and cryopreserved-cooling (CryoCVEGF– and CryoCVEGF+) treatments, compared with the Fresh Control. However, the cryopreservation procedure alone (Cryo Control group) was efficient for preserving follicle morphology since the percentage of morphologically normal follicles was maintained similarly to the Fresh Control group. Interestingly, when fragments were submitted to cooling alone or followed by a transplantation procedure, the percentage of morphologically normal follicles was not significantly affected, demonstrating that the cooling and transplantation procedures herein used were appropriate for preserving the morphology of follicles enclosed in ovarian tissue. However, as expected in the most challenged treatments, when ovarian fragments were cryopreserved before cooling (regardless of VEGF exposure) and followed or not by transplantation, the percentages of normal follicles were reduced compared with the cooling treatment alone. Previous reports using the same cryodevice , the grafting process stimulated primordial follicle activation as expected  the cooling procedure had greater preservation of follicular morphology when compared to the cryopreservation followed by cooling procedures; (ii) a greater percentage of developing follicles but lower follicular and stromal cell densities were observed after ovarian tissue engraftment; (iii) exposure to VEGF increased new and mature blood vessels in cryopreserved-transplanted tissue; and (iv) an appropriate balance in collagen types I and III fiber ratio in cooling-transplanted tissue was observed after exposure to VEGF. Finally, this study contributes to advancing knowledge in this appealing field of ovarian tissue preservation using cooling-cryopreservation and transplantation techniques aiming to be applied to genetically superior/valuable horses, livestock, endangered animals, and, possibly, humans.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.Research supported by Coordination for the Improvement of Higher Education Personnel . Samara Silva de Souza was the recipient of a doctorate scholarship from the National Council for Scientific and Technological Development (CNPq), Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Conceived and designed the experiments: S S S, B G A, K A A, D I A T, and E L G. Performed the experiments: S S S, B G A, K A A, F A S B, D C C B, and E L G. Analyzed the data and prepared figures: S S S, F L N A, B G A, K A A, M O G, D I A T, and E L G. Contributed reagents/materials/analysis tools: D I A T, R S R, A P R R, J R F, and E L G. Wrote the paper: S S S, F L N A, B G A, K A A, J R F, and E L G.