Xenograft model of heterotopic transplantation of human ovarian cortical tissue and its clinical relevance.

In brief: Xenografts of human ovarian cortical tissue provide a tractable model of heterotopic autotransplantation that is used for fertility preservation in patients undergoing ablative chemo/radiotherapy. This study describes the behavior of hundreds of xenografts to establish a framework for the clinical function of ovarian cortex following autotransplantation over short- and long-term intervals. Abstract: More than 200 live births have been achieved using autotransplantation of cryopreserved ovarian cortical fragments, yet challenges remain to be addressed. Ischemia of grafted tissue undermines viability and longevity, typically requiring transplantation of multiple cortical pieces; and the dynamics of recruitment within a graft and the influence of parameters like size and patient age at the time of cryopreservation are not well-defined. Here, we describe results from a series of experiments in which we xenografted frozen/thawed human ovarian tissue (n = 440) from 28 girls and women (age range 32 weeks gestational age to 46 years, median 24.3 ± 4.6). Xenografts were recovered across a broad range of intervals (1-52 weeks post-transplantation) and examined histologically to quantify follicle density and distribution. The number of antral follicles in xenografted cortical fragments correlated positively with the total follicle number and was significantly reduced with increased patient age. Within xenografts, follicles were distributed in focal clusters, similar to the native ovary, but the presence of a leading antral follicle coincided with increased proliferation of surrounding follicles. These results underscore the importance of transplanting ovarian tissue with a high density of follicles and elucidate a potential paracrine influence of leading antral follicles on neighboring follicles of earlier stages. This temporal framework for interpreting the kinetics of follicle growth/mobilization may be useful in setting expectations and guiding the parameters of clinical autotransplantation.

A guide to undertaking and understanding blood pressure measurement.

This article aims to provide background information on blood pressure. It outlines the anatomy and physiology associated with the skill of blood pressure measurement, and the varying techniques for taking blood pressure readings, both invasively and non-invasively. It further explains the steps for taking a manual blood pressure and provides top tips for carrying out this procedure in clinical practice.

Are Surgeons Working Smarter or Harder? A Systematic Review Comparing the Physical and Mental Demands of Robotic and Laparoscopic or Open Surgery.

BACKGROUND: Minimally invasive surgical techniques such as robotic surgical platforms have provided favourable outcomes for patients, but the impact on surgeons is not well described. This systematic review aims to synthesize and evaluate the physical and mental impact of robotic surgery on surgeons compared to standard laparoscopic or open surgery. METHODS: A search strategy was developed to identify peer-reviewed English articles published from inception to end of December 2019 on the following databases: MEDLINE, PubMed, PsycINFO and Embase. The articles were assessed using a modified Newcastle-Ottawa tool. RESULTS: Of the 6563 papers identified, 30 studies were included in the qualitative synthesis of this review. Most of the included studies presented a high risk of bias. A total of 13 and 21 different physical and mental tools, respectively, were used to examine the impact on surgeons. The most common tool used to measure physical and mental demand were surface electromyography (N = 9) and the NASA Task Load Index (NASA-TLX; N = 8), respectively. Majority of studies showed mixed results for physical (N = 10) and mental impact (N = 7). This was followed by eight and six studies favouring RS over other surgical modalities for physical and mental impact, respectively. CONCLUSION: Most studies showed mixed physical and mental outcomes between the three surgical modalities. There was a high risk of bias and methodological heterogeneity. Future studies need to correlate mental and physical stress with long-term impact on the surgeons.

Exogenous insulin-like growth factor 1 accelerates growth and maturation of follicles in human cortical xenografts and increases ovarian output in mice.

OBJECTIVE: To measure the influence of exogenous insulin-like growth factor 1 (IGF1) on follicle growth and maturation in human ovarian cortical xenografts. DESIGN: Xenotransplantation model. SETTING: University-based research laboratory. PATIENTS/ANIMALS: Ovarian tissue was donated with consent and institutional review board approval by brain-dead organ donors or patients undergoing ovarian tissue cryopreservation for fertility preservation. Cortical fragments were transplanted into immunocompromised mice. INTERVENTIONS: Cryopreserved ovarian cortical fragments from four women (aged 19, 25, 33, and 46 years) were transplanted into the gluteus muscle of immunocompromised mice in a fibrin matrix containing endothelial cells that were transduced with lentiviral particles encoding secreted IGF1. Xenografts were recovered after 3, 8, and 14 weeks. In addition, C57/Bl6 mice underwent intraovarian injection of saline or recombinant IGF1 (60 µg), followed by superovulation, analysis of ethynyl-deoxyuridine incorporation, and ribonucleic acid sequencing of the whole ovaries. MAIN OUTCOME MEASURES: For xenografts: follicle count and distribution; antral follicle count; and corpora lutea/albicans count. For mice: follicle count and distribution; oocyte yield, ethynyl-deoxyuridine incorporation (granulosa cell proliferation); and ovarian transcriptomic signature. RESULTS: At 3 weeks, xenografts in the IGF1 condition revealed a decreased percentage of primary follicles and increased percentage of secondary follicles that were concentrated in the preantral subtype; at 8 weeks, an increase in secondary follicles was concentrated in the simple subtype; after 14 weeks, primordial follicles were reduced, and while the number of advanced follicles did not power the experiment to demonstrate significance, antral follicles reduced and corpora lutea increased. Supporting experiments in mice revealed an increase in normal oocytes following intraovarian injection of recombinant IGF1 (60 µg) as well as increased proliferative index among follicles of secondary and preantral stages. Ribonucleic acid sequencing analysis of the whole ovaries following injection of recombinant IGF1 (25 µg) revealed an acute (24 hours) upregulation of transcripts related to steroidogenesis and luteinization. CONCLUSIONS: Exogenous IGF1 advances the pace of growth among primordial, primary, and secondary stage follicles but results in near absence of antral stage follicles in long-term (14 weeks) xenografts. In mice, acute administration of IGF1 promotes follicle advance and increased oocyte yield. The results suggest that while superphysiological IGF1 alone advances the pace of growth among early/preantral follicles, a sustained and/or later-stage influence undermines antral follicle growth/survival or promotes premature luteinization. These findings provide a temporal framework for interpreting follicle growth/mobilization and may be useful in understanding the clinical application of human growth hormone in the context of assisted reproduction.

Serotonin system genes and hoarding with and without other obsessive-compulsive traits in a population-based, pediatric sample: A genetic association study.

BACKGROUND: Hoarding, originally only considered a symptom of obsessive-compulsive disorder (OCD), is now categorized as a separate disorder in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). We studied candidate serotonergic genes and the distinctness of hoarding in children and adolescents and hypothesized that unique gene variants would be associated with hoarding alone. METHODS: We examined obsessive-compulsive (OC) traits, including hoarding, in a total of 5,213 pediatric participants in the community. We genotyped candidate serotonin genes (5-HTTLPR polymorphism in SLC6A4 for 2,018 individuals and single nucleotide polymorphisms [SNPs] across genes SLC6A4, HTR2A, and HTR1B for 4,711 individuals). In a previous study conducted by our group in the same sample, we identified a significant association between 5-HTTLPR and hoarding in males. In this study, we examined hoarding more closely by testing the association between serotonin gene variants and hoarding traits with and without other accompanying OC traits. RESULTS: The [LG +S] variant in 5-HTTLPR was significantly associated with hoarding alone in males (p-value of 0.009). There were no significant findings for 5-HTTLPR in females. There were no significant findings after correction for multiple comparisons using SNP array data, but top SNP findings suggested that variation downstream of HTR1B may be implicated in hoarding alone in females. CONCLUSIONS: Our results suggest specific serotonin gene variants are associated with hoarding traits alone, differing between sexes. Top findings are in line with our former study, suggesting that individuals with hoarding alone were driving previous results. Our paper supports hoarding disorder's new designation.

Intensive LDL cholesterol-lowering treatment beyond current recommendations for the prevention of major vascular events: a systematic review and meta-analysis of randomised trials including 327 037 participants.

BACKGROUND: The benefits of LDL cholesterol-lowering treatment for the prevention of atherosclerotic cardiovascular disease are well established. However, the extent to which these effects differ by baseline LDL cholesterol, atherosclerotic cardiovascular disease risk, and the presence of comorbidities remains uncertain. METHODS: We did a systematic literature search (MEDLINE, Embase, and the Cochrane Central Register of Controlled Trials, from inception up to June 15, 2019) for randomised controlled trials of statins, ezetimibe, and proprotein convertase subtilisin/kexin type 9 inhibitors with at least 1000 patient-years of follow-up. Random-effects meta-analysis and meta-regressions were done to assess for risk of major vascular events (a composite of cardiovascular mortality, non-fatal myocardial infarction, non-fatal ischaemic stroke, or coronary revascularisation) per 1 mmol/L (38·7 mg/dL) reduction in LDL cholesterol concentrations. FINDINGS: 327 037 patients from 52 studies were included in the meta-analysis. Each 1 mmol/L reduction in LDL cholesterol was associated with a 19% relative risk (RR) reduction for major vascular events (RR 0·81 [95% CI 0·78-0·84]; p<0·0001). Similar reductions (per 1 mmol/L reduction in LDL cholesterol) were found in trials with participants with LDL cholesterol 2·60 mmol/L or lower, 2·61-3·40 mmol/L, 3·41-4·10 mmol/L, and more than 4·1 mmol/L (p=0·232 for interaction); and in a subgroup of patients who all had a baseline LDL cholesterol less than 2·07 mmol/L (80 mg/dL; RR 0·83 [95% CI 0·75-0·92]; p=0·001). We found greater RR reductions in patients at lower 10-year atherosclerotic cardiovascular disease risk (change in RR per 10% lower 10-year atherosclerotic cardiovascular disease 0·97 [95% CI 0·95-0·98]; p<0·0001) and in patients at younger age across a mean age of 50-75 years (change in RR per 10 years younger age 0·92 [0·83-0·97]; p=0·015). We found no difference in RR reduction for participants with or without diabetes (p=0·878 for interaction) and chronic kidney disease (p=0·934 for interaction). INTERPRETATION: For each 1 mmol/L LDL cholesterol lowering, the risk reduction of major vascular events is independent of the starting LDL cholesterol or the presence of diabetes or chronic kidney disease. Patients at lower cardiovascular risk and younger age might have a similar relative reduction in risk with LDL-cholesterol lowering therapies and future studies should investigate the potential benefits of earlier intervention. FUNDING: None.

Serotonin system genes and obsessive-compulsive trait dimensions in a population-based, pediatric sample: a genetic association study.

BACKGROUND: Serotonin system genes are commonly studied in obsessive-compulsive disorder (OCD), but genetic studies to date have produced inconsistent results, possibly because phenotypic heterogeneity has not been adequately accounted for. In this paper, we studied candidate serotonergic genes and homogenous phenotypic subgroups as presented through obsessive-compulsive (OC) trait dimensions in a general population of children and adolescents. We hypothesized that different serotonergic gene variants are associated with different OC trait dimensions and, furthermore, that they vary by sex. METHODS: Obsessive-compulsive trait dimensions (Cleaning/Contamination, Counting/Checking, Symmetry/Ordering, Superstition, Rumination, and Hoarding) were examined in a total of 5,213 pediatric participants in the community using the Toronto Obsessive-Compulsive Scale (TOCS). We genotyped candidate serotonin genes (directly genotyping the 5-HTTLPR polymorphism in SLC6A4 for 2018 individuals and using single nucleotide polymorphism (SNP) array data for genes SLC6A4, HTR2A, and HTR1B for 4711 individuals). We assessed the association between variants across these genes and each of the OC trait dimensions, within males and females separately. We analyzed OC traits as both (a) dichotomized based on a threshold value and (b) quantitative scores. RESULTS: The [LG + S] variant in 5-HTTLPR was significantly associated with hoarding in males (p-value of 0.003 and 0.004 for categorical and continuous analyses, respectively). There were no significant findings for 5-HTTLPR in females. Using SNP array data, there were significant findings for rumination in males for HTR2A SNPs (p-value of 1.04e-6 to 5.20e-6). CONCLUSIONS: This represents the first genetic association study of OC trait dimensions in a community-based pediatric sample. Our strongest results indicate that hoarding and rumination may be distinct in their association with serotonin gene variants and that serotonin gene variation may be specific to sex. Future genetic association studies in OCD should properly account for heterogeneity, using homogenous subgroups stratified by symptom dimension, sex, and age group.

Hand hygiene and handwashing: key to preventing the transfer of pathogens.

Claire Ford, Lecturer, Adult Nursing ( claire.ford@northumbria.ac.uk ), and Laura J Park, Graduate Tutor, of Northumbria University, describe how to maintain good skin health to reduce the risk of infection.

Heritability of obsessive-compulsive trait dimensions in youth from the general population.

Obsessive-compulsive disorder (OCD) is a heritable childhood-onset psychiatric disorder that may represent the extreme of obsessive-compulsive (OC) traits that are widespread in the general population. We report the heritability of the Toronto Obsessive-Compulsive Scale (TOCS), a new measure designed to assess the complete range of OC traits in youth. We also examined the dimensional nature of the TOCS and the degree to which genetic effects are unique or shared between dimensions. OC traits were measured using the TOCS in 16,718 youth (6-18 years) at a science museum. We conducted a factor analysis to identify OC trait dimensions. We used univariate and multivariate twin models to estimate the heritability of OC trait dimensions in a subset of twins (220 pairs). Six OC dimensions were identified: Cleaning/Contamination, Symmetry/Ordering, Rumination, Superstition, Counting/Checking, and Hoarding. The TOCS total score (74%) and each OC dimension was heritable (30-77%). Hoarding was not highly correlated with other OC dimensions, but did share genetic effects. Shared genetics accounted for most of the shared variance among dimensions, whereas unique environment accounted for the majority of dimension-specific variance. One exception was Hoarding, which had considerable unique genetic factors. A latent trait did not account for the shared variance between dimensions. In conclusion, OC traits and individual OC dimensions were heritable, although the degree of shared and dimension-specific etiological factors varied by dimension. The TOCS may be informative for genetic research of OC traits in youth. Genetic research of OC traits should consider both OC dimension and total trait scores.

Expression of N471D strumpellin leads to defects in the endolysosomal system.

Hereditary spastic paraplegias (HSPs) are genetically diverse and clinically characterised by lower limb weakness and spasticity. The N471D and several other point mutations of human strumpellin (Str; also known as WASHC5), a member of the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex, have been shown to cause a form of HSP known as spastic paraplegia 8 (SPG8). To investigate the molecular functions of wild-type (WT) and N417D Str, we generated Dictyostelium Str- cells and ectopically expressed StrWT-GFP or StrN471D-GFP in Str- and WT cells. Overexpression of both proteins apparently caused a defect in cell division, as we observed a clear increase in multinucleate cells. Real-time PCR analyses revealed no transcriptional changes in WASH complex subunits in Str- cells, but western blots showed a twofold decrease in the SWIP subunit. GFP-trap experiments in conjunction with mass-spectrometric analysis revealed many previously known, as well as new, Str-interacting proteins, and also proteins that no longer bind to StrN471D At the cellular level, Str- cells displayed defects in cell growth, phagocytosis, macropinocytosis, exocytosis and lysosomal function. Expression of StrWT-GFP in Str- cells rescued all observed defects. In contrast, expression of StrN471D-GFP could not rescue lysosome morphology and exocytosis of indigestible material. Our results underscore a key role for the WASH complex and its core subunit, Str, in the endolysosomal system, and highlight the fundamental importance of the Str N471 residue for maintaining lysosome morphology and dynamics. Our data indicate that the SPG8-causing N471D mutation leads to a partial loss of Str function in the endolysosomal system. This article has an associated First Person interview with the first author of the paper.

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery.

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

Frailty in advanced liver disease.

Prognostication of patients with cirrhosis is complex, depending on more than just the severity of liver disease. Scores such as the model for end-stage liver disease (MELD) and Child Pugh can assist with prognostication, yet by focusing on physiological parameters they fail to completely capture the elements contributing to a patient's clinical status. Evidence is increasing to support an important role for physical functioning in patient outcomes. Frailty has been increasingly recognised in medical literature over recent years, including in hepatology where it is identified in nearly half of cirrhosis patients. It is a complex construct consisting of multisystemic physiological decline and increased vulnerability to stressors. Diagnosis is complicated by lack of a consensus definition and measurement tool for frailty in cirrhosis. Frailty heralds a poor prognosis, predicting increased morbidity and mortality both pre- and postliver transplant, independent of MELD score. It is thought to be reversible, with promising data supporting prehabilitation and lifestyle intervention programs. In the future, assessment of patients with cirrhosis is likely to incorporate a measure of frailty, however, further research is required.

hsa-mir183/EGR1-mediated regulation of E2F1 is required for CML stem/progenitor cell survival.

Chronic myeloid leukemia (CML) stem/progenitor cells (SPCs) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPCs maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase-dependent pathway mediated by the upregulation of hsa-mir183, the downregulation of its direct target early growth response 1 (EGR1), and, as a consequence, upregulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPCs. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPCs, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183/EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication.

Wnt inhibition promotes vascular specification of embryonic cardiac progenitors.

Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogenesis and developed protocols that enrich for cardiac derivatives during in vitro differentiation of human pluripotent stem cells (hPSCs). However, few studies have investigated the role of Wnt signaling in the specification of cardiac progenitor cells (CPCs) toward downstream fates. Using transgenic mice and hPSCs, we tracked endothelial cells (ECs) that originated from CPCs expressing NKX2.5. Analysis of EC-fated CPCs at discrete phenotypic milestones during hPSC differentiation identified reduced Wnt activity as a hallmark of EC specification, and the enforced activation or inhibition of Wnt reduced or increased, respectively, the degree of vascular commitment within the CPC population during both hPSC differentiation and mouse embryogenesis. Wnt5a, which has been shown to exert an inhibitory influence on Wnt signaling during cardiac development, was dynamically expressed during vascular commitment of hPSC-derived CPCs, and ectopic Wnt5a promoted vascular specification of hPSC-derived and mouse embryonic CPCs.

Engineered endothelium provides angiogenic and paracrine stimulus to grafted human ovarian tissue.

Despite major advances in tissue cryopreservation and auto-transplantation, reperfusion ischemia and hypoxia have been reported as major obstacles to successful recovery of the follicular pool within grafted ovarian tissue. We demonstrate a benefit to follicular survival and function in human ovarian tissue that is co-transplanted with exogenous endothelial cells (ExEC). ExECs were capable of forming functionally perfused vessels at the host/graft interface and increased both viability and follicular volume in ExEC-assisted grafts with resumption of antral follicle development in long-term grafts. ExECs that were engineered to constitutively express anti-mullerian hormone (AMH) induced a greater proportion of quiescent primordial follicles than control ExECs, indicating suppression of premature mobilization that has been noted in the context of ovarian tissue transplantation. These findings present a cell-based strategy that combines accelerated perfusion with direct paracrine delivery of a bioactive payload to transplanted ovarian tissue.

Notch hyper-activation drives trans-differentiation of hESC-derived endothelium.

During development, endothelial cells (EC) display tissue-specific attributes that are unique to each vascular bed, as well as generic signaling mechanisms that are broadly applied to create a patent circulatory system. We have previously utilized human embryonic stem cells (hESC) to generate tissue-specific EC sub-types (Rafii et al., 2013) and identify pathways that govern growth and trans-differentiation potential of hESC-derived ECs (James et al., 2010). Here, we elucidate a novel Notch-dependent mechanism that induces endothelial to mesenchymal transition (EndMT) in confluent monolayer cultures of hESC-derived ECs. We demonstrate density-dependent induction of EndMT that can be rescued by the Notch signaling inhibitor DAPT and identify a positive feedback signaling mechanism in hESC-ECs whereby trans-activation of Notch by DLL4 ligand induces elevated expression and surface presentation of DLL4. Increased Notch activation in confluent hESC-EC monolayer cultures induces areas of EndMT containing transitional cells that are marked by increased Jagged1 expression and reduced Notch signal integration. Jagged1 loss of function in monolayer hESC-ECs induces accelerated feedback stimulation of Notch signaling, increased expression of cell-autonomous, cis-inhibitory DLL4, and EndMT. These data elucidate a novel interplay of Notch ligands in modulating pathway activation during both expansion and EndMT of hESC-derived ECs.