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BACKGROUND: Residual peri-device leakage (PDL) is frequent after left atrial appendage occlusion (LAAO). Little is known about management strategies, procedural aspects and outcomes of interventional PDL closure. AIMS: The aim of this study was to assess the safety and feasibility of PDL closure after LAAO. METHODS: Fifteen centres contributed data on baseline characteristics, in-hospital and follow-up outcomes of patients who underwent PDL closure after LAAO. Outcomes of interest included acute success and complication rates and long-term efficacy of the procedure. RESULTS: A total of 95 patients were included and a cumulative number of 104 leaks were closed. The majority of PDLs were detected within 90 days (range 41-231). Detachable coils were the most frequent approach (42.3%), followed by the use of the AMPLATZER Vascular Plug II (29.8%) and the AMPLATZER Duct Occluder II (17.3%). Technical success was 100% with 94.2% of devices placed successfully within the first attempt. There were no major complications requiring surgical or transcatheter interventions. During follow-up (96 days [range 49-526]), persistent leaks were found in 18 patients (18.9%), yielding a functional success rate of 82.7%, although PDLs were significantly reduced in size (pre-leak size(max): 6.1±3.6 mm vs post-leak size(max): 2.5±1.3 mm, p5 mm. Major adverse events during follow-up occurred in 5 patients (2 ischaemic strokes, 2 intracranial haemorrhages, and 1 major gastrointestinal bleeding). CONCLUSIONS: Several interventional techniques have become available to achieve PDL closure. They are associated with high technical and functional success and low complication rates.

In women, establishment of pregnancy is dependent upon 'fine-tuning' of the endometrial microenvironment, which is mediated by terminal differentiation (decidualisation) of endometrial stromal fibroblasts (ESFs). We have demonstrated that intracrine steroid metabolism plays a key role in regulating decidualisation and is essential for time-dependent expression of key factors required for endometrial receptivity. The primary aim of the current study was to determine whether sulphated steroids can act as precursors to bioactive sex steroids during decidualisation. We used primary human ESF and a robust in vitro model of decidualisation to assess the expression of genes associated with sulphation, desulphation and transport of sulphated steroids in human ESF as well as the impact of the steroid sulphatase (STS) inhibitor STX64 (Irosustat). We found evidence for an increase in both expression and activity of STS in response to a decidualisation stimulus with abrogation of oestrone biosynthesis and decreased secretion of the decidualisation marker IGFBP1 in the presence of STX64. These results provide novel insight into the contribution of STS to the intracrine regulation of decidualisation.

Objective: To investigate the impact of the androgen precursor dehydroepiandrosterone on decidualization of human endometrial stromal cells isolated from women of advanced reproductive age. Design: In vitro study. Setting: University Research Institute. Patients: Proliferative phase primary human endometrial stromal fibroblasts (hESF) were isolated from women of advanced reproductive age (n=16, mean age 44.7 +/- 2.3). None of the women were receiving hormonal therapy or suffering from endometriosis. Interventions: Isolated hESF were decidualized in vitro by incubation with progesterone (1µM) and cAMP (0.1mg/ml) in the presence, or absence, of DHEA (10nM, 100nM). Main outcome measures: Secretion of androgens was assessed by ELISA. Expression of decidualization markers and endometrial receptivity markers was assessed by qPCR and ELISA. Results: Decidualization responses were retained in hESF isolated from women of advanced reproductive age. Supplementation with DHEA increased androgen biosynthesis and concentrations of testosterone (p

In women, endometrial breakdown, which is experienced as menstruation, is characterised by high concentrations of inflammatory mediators and immune cells which account for ~40% of the stromal compartment during tissue shedding. These inflammatory cells are known to play a pivotal role in tissue breakdown but their contribution to the rapid scarless repair of endometrium remains poorly 25 understood. In the current study we used a mouse model of menstruation to investigate dynamic changes in mononuclear phagocytes during endometrial repair and remodelling.Menstruation was simulated in MacGreen mice to allow visualisation of CSF1R+ mononuclear phagocytes. Immunohistochemistry revealed dynamic spatio-temporal changes in numbers and location of CSF1R-EGFP+ cells and Ly6G+ neutrophils. Flow cytometry confirmed a striking increase 30 in numbers of GFP+ cells during repair (24 h): influxed cells were 66% F4/80+Gr-1+ and 30% F4/80-Gr-1+. Immunostaining identified distinct populations of putative ‘classical’ monocytes (GFP+F4/80-), monocyte-derived macrophages (GFP+F4/80+) and a stable population of putative tissue-resident macrophages (GFP-F4/80+) localised to areas of breakdown, repair and remodelling respectively. Collectively, these data provide the first compelling evidence to support a role for different 35 populations of monocytes/macrophages in endometrial repair and provide the platform for future studies on the role of these cells in scarless healing.

he human endometrium undergoes regular cycles of synchronous tissue shedding (wounding) and repair that occur during menstruation before estrogen‐dependent regeneration. Endometrial repair is normally both rapid and scarless. Androgens regulate cutaneous wound healing, but their role in endometrial repair is unknown. We used a murine model of simulated menses; mice were treated with a single dose of the nonaromatizable androgen dihydrotestosterone (DHT; 200 μg/mouse) to coincide with initiation of tissue breakdown. DHT altered the duration of vaginal bleeding and delayed restoration of the luminal epithelium. Analysis of uterine mRNAs 24 h after administration of DHT identified significant changes in metalloproteinases (Mmp3 and ‐9; P < 0.01), a snail family member (Snai3; P < 0.001), and osteopontin (Sppl; P < 0.001). Chromatin immunoprecipitation analysis identified putative androgen receptor (AR) binding sites in the proximal promoters of Mmp9, Snai3, and Spp1. Striking spatial and temporal changes in immunoexpression of matrix metalloproteinase (MMP) 3/9 and caspase 3 were detected after DHT treatment. These data represent a paradigm shift in our understanding of the role of androgens in endometrial repair and suggest that androgens may have direct impacts on endometrial tissue integrity. These studies provide evidence that the AR is a potential target for drug therapy to treat conditions associated with aberrant endometrial repair processes.

BackgroundTranscatheter left atrial appendage (LAA) ligation may represent an alternative to oral anticoagulation for stroke prevention in atrial fibrillation.ObjectivesThis study sought to assess the early safety and efficacy of transcatheter ligation of the LAA for stroke prevention in atrial fibrillation.MethodsThis was a retrospective, multicenter study of consecutive patients undergoing LAA ligation with the Lariat device at 8 U.S. sites. The primary endpoint was procedural success, defined as device success (suture deployment and

The human endometrium is a complex multicellular tissue, located within the cavity of the uterus. Its luminal surface is defined by a layer of epithelial cells supported on a multicellular stroma containing fibroblasts, glands (lined by a secretory epithelium), blood vessels (lined with endothelial cells) and several populations of immune cells; the latter includes a unique population of natural killer (uNK) cells. The endometrium undergoes dynamic remodelling across the menstrual cycle in response to fluctuating levels of sex steroids secreted by ovarian cells. The phases of the endometrial cycle include an oestrogendominated proliferative phase, a progesterone-dominated secretory phase and menses (endometrial shedding precipitated by falling levels of progesterone). A key feature of the secretory phase is differentiation (decidualisation) of endometrial stromal fibroblasts (ESC) an event characterised by transformation of cell shape, secretion of growth factors/cytokines, angiogenesis/vascular remodelling and an increase in the numbers of resident immune cells. Decidualisation ensures an appropriate nutritional and hormonal environment exists during the establishment of pregnancy. Studies in mice suggest that de novo biosynthesis of oestrogen within the uterus may play an essential role in regulation of decidualisation but no data exist for human. Endometrial endothelial and uNK cells both contain oestrogen receptors but the impact of oestrogens on their function has not been explored. In the current studies three questions have been addressed: 1. Is oestrogen biosynthesis a feature of human endometrial stromal cell decidualisation? 2. What is the impact of oestrogen on uNK cell function? 3. What role (if any) does oestrogen play in the interplay between decidual, immune and vascular cells within the human endometrial stroma? Results obtained provide the first evidence that de novo biosynthesis of oestrogens occurs during decidualisation of human ESC. This was attributed to changes in expression patterns of mRNAs encoding proteins that play a critical role in regulation of oestrogen biosynthesis (STAR, CYP11A1, CYP19A1 [aromatase], HSD17B2 [17βHSD2] and STS [steroid sulphatase]). Changes in the pattern of metabolism were confirmed using thin layer chromatography and analysis of concentrations of oestrone (E1) and oestradiol (E2) in culture media. Secretion of E1 and E2 was reduced by addition of an aromatase inhibitor. Data derived from studies described within this thesis also show for the first time that incubation of uNK cells with E2 not only enhanced cell migration but also stimulated secretion of factors that had a significant impact on endothelial cell angiogenesis. These findings were supported by novel evidence that E2 had a significant impact on expression of genes associated with cell motility and angiogenesis. In addition, factors, including E1/E2, secreted by decidualised stromal cells, stimulated chemotaxis of uNK cells. Future experiments will focus on determining the identity of the angiogenic factors secreted by uNK cells in response to E2 and the mechanisms responsible for uNK cell movement. In summary, new data presented in this thesis provide evidence that local biosynthesis of oestrogens within the endometrial stroma may play a previously unrecognised role in regulating the function of uNK cells and endometrial endothelial cells in women. These results have implications for treatment of disorders such as infertility, heavy menstrual bleeding and endometriosis.

ObjectivesThe purpose of this study was to compare the relative risk of major bleeding with left atrial appendage (LAA) closure compared with long-term warfarin therapy.BackgroundLAA closure is an alternative approach to chronic oral anticoagulation for the prevention of thromboembolism in patients with atrial fibrillation (AF).MethodsWe conducted a pooled, patient-level analysis of the 2 randomized clinical trials that compared WATCHMAN (Boston Scientific, Natick, Massachusetts) LAA closure with long-term warfarin therapy in AF.ResultsA total of 1,114 patients were included, with a median follow-up of 3.1 years. The overall rate of major bleeding from randomization to the end of follow-up was similar between treatment groups (3.5 events vs. 3.6 events per 100 patient-years; rate ratio [RR]: 0.96; 95% confidence interval [CI]: 0.66 to 1.40; p = 0.84). LAA closure significantly reduced bleeding >7 days post-randomization (1.8 events vs. 3.6 events per 100 patient-years; RR: 0.49; 95% CI: 0.32 to 0.75; p = 0.001), with the difference emerging 6 months after randomization (1.0 events vs. 3.5 events per 100 patient-years; RR: 0.28; 95% CI: 0.16 to 0.49; p < 0.001), when patients assigned to LAA closure were able to discontinue adjunctive oral anticoagulation and antiplatelet therapy. The reduction in bleeding with LAA closure was directionally consistent across all patient subgroups.ConclusionsThere was no difference in the overall rate of major bleeding in patients assigned to LAA closure compared with extended warfarin therapy over 3 years of follow-up. However, LAA closure significantly reduced bleeding beyond the procedural period, particularly once adjunctive pharmacotherapy was discontinued. The favorable effect of LAA closure on long-term bleeding should be considered when selecting a stroke prevention strategy for patients with nonvalvular AF. (WATCHMAN Left Atrial Appendage System for Embolic PROTECTion in Patients With Atrial Fibrillation; NCT00129545; and Evaluation of the WATCHMAN LAA Closure Device in Patients With Atrial Fibrillation Versus Long Term Warfarin Therapy [PREVAIL]; NCT01182441)

http://archive.org/details/solutionofproble1094513055 Lieutenant, United States Navy

The endometrium is the inner lining of the uterine cavity, composed of distinct epithelial and stromal cell compartments with the latter containing fibroblasts, a vascular compartment and fluctuating populations of immune cells. The endometrium is a highly dynamic tissue that undergoes cycles of proliferation and stromal cell differentiation (decidualisation) followed by tissue shedding (menstruation) and rapid repair/remodelling, all under control of fluctuating concentrations of steroid hormones secreted from the ovaries. This is known as the menstrual cycle. In response to the ‘injury’ inflicted as a consequence of decidual breakdown and shedding, the endometrium exhibits a unique capacity to restore tissue architecture by rapid tissue repair. This repair process is tightly regulated to ensure that the endometrium heals consistently every month throughout a woman’s reproductive lifespan, without the accumulation of fibrotic scar tissue which could have a negative impact on fertility. The initiation of menstruation is triggered by the withdrawal of progesterone as a consequence of the demise of the corpus luteum within the ovaries which precipitates an increase in production of inflammatory mediators, focal hypoxia and activation of matrix metalloproteinase enzymes culminating in endometrial shedding. In contrast the cellular and molecular mechanisms responsible for the rapid and scar-free tissue repair of endometrium remain poorly understood. Parallels can be drawn between the repair process of the endometrium and that of the foetal skin and oral mucosa which also exhibit ‘scarless’ healing, including rapid reepithelialisation, widespread cellular proliferation and migration and a short time to wound closure. However endometrial repair also shares key features of the wound healing experienced by adult tissues that exhibit scarring including extensive angiogenesis and a substantial inflammatory response. The endometrium appears to be unique, fitting in a gap between tissues that typically undergo ‘scarless’ or ‘scarring’ tissue repair. In women, endometrial shedding is considered an inflammatory event and the culmination of a cascade of inflammatory signals result in the accumulation of a diverse population of immune cells within the tissue. Whilst we believe immune cells play a key role in regulating spatial and temporal tissue breakdown and shedding their role in repair and restoration of tissue homeostasis remains poorly understood. One process essential for endometrial repair is restoration of the luminal epithelial cell layer (re-epithelialisation) and imaging studies have demonstrated that this appears not only to be rapid but also to occur synchronously with tissue degeneration and shedding. Re-epithelialisation was previously thought to be governed by proliferation and migration of glandular epithelial cells in the basal (unshed) tissue compartment, however new data suggest a role for trans-differentiation of stromal cells into epithelial cells which merits further investigation. In addition to role(s) for immune and stromal cells in regulation of endometrial tissue function, a role for somatic stem/progenitor cells capable of differentiating into mature endometrial cells to regenerate the tissue has also been claimed. In summary, whilst progress has been made in understanding the processes governing endometrial decidualisation, breakdown and shedding the regulation and roles of the different cell types that participate in scar-free repair of the tissue remain poorly defined. The studies in this thesis set out to address this gap by addressing three key aims: Aim 1. To investigate the phenotype and location of immune cell populations during scarless tissue repair. Aim 2. To identify and characterise a putative population of mesenchymal stem/progenitor cells in endometrium. Aim 3. To investigate the contribution of putative mesenchymal stem/progenitor cells to endometrial tissue repair. The aims were addressed using a recently refined and extensively characterised mouse model in which endometrial shedding (‘menstruation’) and repair occurs over a 48 hour period following removal of a progesterone stimulus. Importantly the Saunders’ group have already demonstrated that this model recapitulates the key features of human menses including overt vaginal bleeding, immune cell influx, tissue necrosis, transient hypoxia, re-epithelialisation and most importantly simultaneous breakdown and repair. Uterine tissues recovered 12, 24, and 48hrs after removal of progesterone and were investigated using immunohistochemistry (spatial organisation), flow cytometry (quantitation of cell subpopulations), FACS sorting (isolation of subpopulations) and molecular profiling (qPCR, RNAseq and single cell sequencing) with additional insights from bioinformatic analysis. To address Aim 1 endometrial shedding and repair was studied in Macgreen® mice: in this transgenic line all the cells of the mononuclear phagocyte lineage (monocytes, monocyte-derived macrophages) express green fluorescent protein. Immunohistochemistry revealed striking spatio-temporal changes in both numbers and location of GFP+ cells during endometrial breakdown and repair, the most prominent changes occurring 24hrs after removal of progesterone. Flow Cytometry quantified several immune cell populations with a significant increase in GFP+ cells during repair, the majority of which were GR1+F4/80- (inflammatory monocytes). These novel data provided compelling evidence to support a role for inflammatory monocytes in endometrial repair and provide the platform for future studies on the role of these cells in scarless healing. To address Aims 2 and 3 Pdgfrβ-BAC-eGFP® transgenic mice in which GFP was expressed under control of promoter elements of the Pdgfrβ gene was used to identify putative mesenchymal progenitor cells and investigate their role in endometrial repair. GFP+ cells were located exclusively within the endometrial stromal compartment and examination of tissue sections revealed that two subpopulations could be distinguished based on the both the intensity of GFP and expression of CD146 (Mcam). Characterisation by immunohistochemistry, flow cytometry and qPCR identified a GFPbright subpopulation located adjacent to CD31+ endothelial cells that were classified as pericytes based on location and phenotype (NG2+, CD146+, CD31-). When menstruation was stimulated in Pdgfrβ-BAC-eGFP® mice detailed analysis using flow cytometry revealed an increase in the perivascular pericyte subpopulation during active healing (24hrs) and also identified a new previously unidentified subpopulation of GFP+ cells which had a unique phenotype during repair. Evidence that GFP+ cells contribute to restoration of epithelial repair was obtained with an increase in expression of the epithelial cell marker EpCAM and GFP+ cells in the renewed epithelial cell layer. RNAseq and single cell sequencing combined with bioinformatics complemented these findings by identifying novel changes in gene expression in both endometrial fibroblasts and pericyte populations consistent with induction of novel pathways and trans-differentiation of stromal cells by a mesenchymal-to-epithelial transition (MET). In conclusion, using a mouse model of endometrial breakdown and repair a heterogeneous population of myeloid cells and a putative population of endometrial progenitors (pericytes) have been characterised, quantified and novel changes in gene expression identified. Adaptation of these cell types to the insult of endometrial shedding appears to play a key role in temporal and spatial regulation of rapid, scar-free endometrial tissue repair. These novel findings may inform the development of new approaches to treating gynaecological disorders associated with aberrant endometrial repair such as heavy menstrual bleeding, Asherman’s syndrome and endometriosis as well as other disorders associated with excessive fibrosis and scar formation.