Dynamic chromatin remodeling in cycling human endometrium at single-cell level

Estrogen-dependent proliferation followed by progesterone-dependent differentiation of the endometrium culminates in a short implantation window. We performed single-cell assay for transposase-accessible chromatin with sequencing on endometrial samples obtained across the menstrual cycle to investigate the regulation of temporal gene networks that control embryo implantation. We identify uniquely accessible chromatin regions in all major cellular constituents of the endometrium, delineate temporal patterns of coordinated chromatin remodeling in epithelial and stromal cells, and gain mechanistic insights into the emergence of a receptive state through integrated analysis of enriched transcription factor (TF) binding sites in dynamic chromatin regions, chromatin immunoprecipitation sequencing analyses, and gene expression data. We demonstrate that the implantation window coincides with pervasive cooption of transposable elements (TEs) into the regulatory chromatin landscape of decidualizing cells and expression of TE-derived transcripts in a spatially defined manner. Our data constitute a comprehensive map of the chromatin changes that control TF activities in a cycling endometrium at cellular resolution

Expression and function of the luteinizing hormone choriogonadotropin receptor in human endometrial stromal cells

The human luteinising hormone choriogonadotropin receptor (LHCGR) is a G-protein coupled receptor activated by both human chorionic gonadotropin (hCG) and luteinizing hormone (LH), two structurally related gonadotropins with essential roles in ovulation and maintenance of the corpus luteum. LHCGR expression predominates in ovarian tissues where it elicits functional responses through cyclic adenosine mononucleotide (cAMP), Ca2+ and extracellular signal-regulated kinase (ERK) signalling. LHCGR expression has also been localized to the human endometrium, with purported roles in decidualization and implantation. However, these observations are contentious. In this investigation, transcripts encoding LHCGR were undetectable in bulk RNA sequencing datasets from whole cycling endometrial tissue and cultured human endometrial stromal cells (EnSC). However, analysis of single-cell RNA sequencing data revealed cell-to-cell transcriptional heterogeneity, and we identified a small subpopulation of stromal cells with detectable LHCGR transcripts. In HEK-293 cells expressing recombinant LHCGR, both hCG and LH elicited robust cAMP, Ca2+ and ERK signals that were absent in wild-type HEK-293 cells. However, none of these responses were recapitulated in primary EnSC cultures. In addition, proliferation, viability and decidual transformation of EnSC were refractory to both hCG and LH, irrespective of treatment to induce differentiation. Although we challenge the assertion that LHCGR is expressed at a functionally active level in the human endometrium, the discovery of a discrete subpopulation of EnSC that express LHCGR transcripts may plausibly account for the conflicting evidence in the literature

EndoTime: non-categorical timing estimates for luteal endometrium

STUDY QUESTION Can the accuracy of timing of luteal phase endometrial biopsies based on urinary ovulation testing be improved by measuring the expression of a small number of genes and a continuous, non-categorical modelling approach? SUMMARY ANSWER Measuring the expression levels of six genes (IL2RB, IGFBP1, CXCL14, DPP4, GPX3 and SLC15A2) is sufficient to obtain substantially more accurate timing estimates and to assess the reliability of timing estimates for each sample. WHAT IS KNOWN ALREADY Commercially available endometrial timing approaches based on gene expression require large gene sets and use a categorical approach that classifies samples as pre-receptive, receptive or post-receptive. STUDY DESIGN, SIZE, DURATION Gene expression was measured by RTq-PCR in different sample sets, comprising a total of 664 endometrial biopsies obtained 4–12 days after a self-reported positive home ovulation test. A further 36 endometrial samples were profiled by RTq-PCR as well as RNA-sequencing. PARTICIPANTS/MATERIALS, SETTING, METHODS A computational procedure, named ‘EndoTime’, was established that models the temporal profile of each gene and estimates the timing of each sample. Iterating these steps, temporal profiles are gradually refined as sample timings are being updated, and confidence in timing estimates is increased. After convergence, the method reports updated timing estimates for each sample while preserving the overall distribution of time points. MAIN RESULTS AND THE ROLE OF CHANCE The Wilcoxon rank-sum test was used to confirm that ordering samples by EndoTime estimates yields sharper temporal expression profiles for held-out genes (not used when determining sample timings) than ordering the same expression values by patient-reported times (GPX3: P  0.05). LARGE SCALE DATA The RTq-PCR data files are available via the GitHub repository for the EndoTime software at https://github.com/AE-Mitchell/EndoTime, as is the code used for pre-processing of RTq-PCR data. The RNA-sequencing data are available on GEO (accession GSE180485). LIMITATIONS, REASONS FOR CAUTION Timing estimates are informed by glandular gene expression and will only represent the temporal state of other endometrial cell types if in synchrony with the epithelium. Methods that estimate the day of ovulation are still required as these data are essential inputs in our method. Our approach, in its current iteration, performs batch correction such that larger sample batches impart greater accuracy to timing estimations. In theory, our method requires endometrial samples obtained at different days in the luteal phase. In practice, however, this is not a concern as timings based on urinary ovulation testing are associated with a sufficient level of noise to ensure that a variety of time points will be sampled. WIDER IMPLICATIONS OF THE FINDINGS Our method is the first to assay the temporal state of luteal-phase endometrial samples on a continuous domain. It is freely available with fully shared data and open-source software. EndoTime enables accurate temporal profiling of any gene in luteal endometrial samples for a wide range of research applications and, potentially, clinical use

Lack of evidence for a causal role of CALR3 in monogenic cardiomyopathy

The pathogenicity of previously published disease-associated genes and variants is sometimes questionable. Large-scale, population-based sequencing studies have uncovered numerous false assignments of pathogenicity. Misinterpretation of sequence variants may have serious implications for the patients and families involved, as genetic test results are increasingly being used in medical decision making. In this study, we assessed the role of the calreticulin-3 gene (CALR3) in cardiomyopathy. CALR3 has been included in several cardiomyopathy gene panels worldwide. Its inclusion is based on a single publication describing two missense variants in patients with hypertrophic cardiomyopathy. In our national cardiomyopathy cohort (n = 6154), we identified 17 unique, rare heterozygous CALR3 variants in 48 probands. Overall, our patient cohort contained a significantly higher number of rare CALR3 variants compared to the ExAC population (p = 0.0036). However, after removing a potential Dutch founder variant, no statistically significant difference was found (p = 0.89). In nine probands, the CALR3 variant was accompanied by a disease-causing variant in another, well-known cardiomyopathy gene. In three families, the CALR3 variant did not segregate with the disease. Furthermore, we could not demonstrate calreticulin-3 protein expression in myocardial tissues at various ages. On the basis of these findings, it seems highly questionable that variants in CALR3 are a monogenic cause of cardiomyopathy

The human endometrium as a sensor of embryo quality

Human reproduction is characterized by a high degree of embryo wastage, which is largely ascribed to a high prevalence of embryo aneuploidy. It is proposed that maternal strategies have evolved that prevent inappropriate investment in invasive, but poorly viable embryos. Key to this is the emerging concept of the endometrium as biosensor, first identified in human in vitro embryo/decidualized stromal cell coculture systems and recently confirmed in an in vivo mouse model. In this review, the growing supporting experimental evidence for the biosensor component of decidualized endometrium is outlined, and recent insights into the nature of the embryo-derived signal detected by the endometrium and the biological processes by which this signal is thought to be converted into a go or no-go endometrial response are described. Finally, the clinical implications of this new paradigm of the choosy uterus are addressed

The motile and invasive capacity of human endometrial stromal cells: implications for normal and impaired reproductive function

Background: mechanisms underlying early reproductive loss in the human are beginning to be elucidated. The migratory and invasive capacity of human endometrial stromal cells (ESCs) is increasingly recognized to contribute to the intense tissue remodelling associated with embryo implantation, trophoblast invasion and endometrial regeneration. In this review, we examine the signals and mechanisms that control ESC migration and invasion and assess how deregulation of these cell functions contributes to common reproductive disorders.Methods: the PubMed database was searched for publications on motility and invasiveness of human ESCs in normal endometrial function and in reproductive disorders including implantation failure, recurrent pregnancy loss (RPL), endometriosis and adenomyosis, covering the period 2000–2012.Results: increasing evidence suggests that implantation failure and RPL involve abnormal migratory responses of decidualizing ESCs to embryo and trophoblast signals. Numerous reports indicate that endometriosis, as well as adenomyosis, is associated with increased basal and stimulated invasiveness of ESCs and their progenitor cells, suggesting a link between a heightened menstrual repair response and the formation of ectopic implants. Migration and invasiveness of ESCs are controlled by a complex array of hormones, growth factors, chemokines and inflammatory mediators, and involve signalling through Rho GTPases, phosphatidylinositol-3-kinase and mitogen-activated protein kinase pathways.Conclusions: novel concepts are extending our understanding of the key functions of ESCs in effecting tissue repair imposed by cyclic menstruation and parturition. Migration of decidualizing ESCs also serves to support blastocyst implantation and embryo selection through discriminate motile responses directed by embryo quality. Targeting regulatory molecules holds promise for developing new strategies for the treatment of reproductive disorders such as endometriosis and recurrent miscarriage; and harnessing the migratory capacity of progenitor mesenchymal stem cells in the endometrium may offer new opportunities in regenerative medicin

Diagnostic accuracy of contrast-enhanced diffusion-weighted MRI for liver metastases of pancreatic cancer: towards adequate staging and follow-up of pancreatic cancer - DIA-PANC study: study protocol for an international, multicenter, diagnostic trial

Contains fulltext : 225255.pdf (publisher's version ) (Open Access)BACKGROUND: At the time of surgery, approximately 10-20% of the patients with pancreatic cancer are considered unresectable because of unexpected liver metastasis, peritoneal carcinomatosis or locally advanced disease. This leads to futile surgical treatment with all the associated morbidity, mortality and costs. More than 50% of all liver metastases develop in the first six months postoperatively. These (subcentimeter) liver metastases are most likely already present at the time of diagnosis and have not been identified pre-operatively, due to the poor sensitivity of routine preoperative contrast-enhanced CT (CECT). METHODS: The DIA-PANC study is a prospective, international, multicenter, diagnostic cohort study investigating diffusion-weighted, contrast-enhanced MRI for the detection of liver metastases in patients with all stages of pancreatic cancer. Indeterminate or malignant liver lesions on MRI will be further investigated histopathologically. For patients with suspected liver lesions without histopathological proof, follow up imaging with paired CT and MRI at 3-, 6- and 12-months will serve as an alternative reference standard. DISCUSSION: The DIA-PANC trial is expected to report high-level evidence of the diagnostic accuracy of MRI for the detection of liver metastases, resulting in significant value for clinical decision making, guideline development and improved stratification for treatment strategies and future trials. Furthermore, DIA-PANC will contribute to our knowledge of liver metastases regarding incidence, imaging characteristics, their number and extent, and their change in time with or without treatment. It will enhance the worldwide implementation of MRI and consequently improve personalized treatment of patients with suspected pancreatic ductal adenocarcinoma. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03469726 . Registered on March 19th 2018 - Retrospectively registered

Medullary Pancreatic Carcinoma Due to Somatic POLE Mutation A Distinctive Pancreatic Carcinoma With Marked Long-Term Survival

Contains fulltext : 221545pub.pdf (publisher's version ) (Open Access