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Compared to most mammals, human pregnancy is unusual in that it involves chromosomally diverse embryos, cyclical breakdown and regeneration of the uterine mucosa, and intimate integration of fetal and maternal cells at the uteroplacental interface. Not surprisingly, pregnancy often falters in early gestation. Whether these losses result in clinical miscarriages depends on the origins and impacts of chromosomal errors on fetal development and the ability of the decidualizing endometrium to engage in embryo biosensing and selection. Aneuploidy originating in oocytes during meiosis drives the age-related risk of miscarriage. By contrast, the frequency of endometrial cycles with an impaired decidual response may account for the stepwise increase in miscarriage rates with each pregnancy loss independently of maternal age. Additional physiological mechanisms operate in early gestation to ensure that most failing pregnancies are lost before vascular maternal-fetal connections are established by the end of the first trimester. Here, we summarise how investigations into the mechanisms that cause miscarriage led to new insights into the processes that govern maternal selection of human embryos in early gestation.

Embryo implantation in humans is interstitial, meaning the entire conceptus embeds in the endometrium before the placental trophoblast invades beyond the uterine mucosa into the underlying inner myometrium. Once implanted, embryo survival pivots on the transformation of the endometrium into an anti-inflammatory placental bed, termed decidua, under homeostatic control of uterine natural killer cells. Here, we examine the evolutionary context of embryo implantation and elaborate on uterine remodelling before and after conception in humans. We also discuss the interactions between the embryo and the decidualising endometrium that regulate interstitial implantation and determine embryo fitness. Together, this Review highlights the precarious but adaptable nature of the implantation process.

Supplementary Figure 1 from Definition of microRNAs That Repress Expression of the Tumor Suppressor Gene FOXO1 in Endometrial Cancer

Supplementary Figure 2 from Definition of microRNAs That Repress Expression of the Tumor Suppressor Gene FOXO1 in Endometrial Cancer

Endometrial cancer is the most common malignancy of the lower female reproductive tract. The tumor suppressor FOXO1 is downregulated in endometrial cancer compared with normal endometrium but the underlying mechanisms are not well understood. Using microRNA (miR) target prediction algorithms, we identified several miRs that potentially bind the 3′-untranslated region of FOXO1 transcripts. Expression profiling of normal and malignant endometrial samples by quantitative real-time PCR and Northern blot analysis revealed an inverse correlation between the levels of FOXO1 protein and the abundance of several of the in silico–predicted miRs, suggesting that loss of FOXO1 expression in endometrial cancer may be mediated by miRs. To determine the role of candidate miRs, we used the endometrial cancer cell lines HEC-1B and Ishikawa, which express FOXO1 at high and low levels, respectively. Expression of miR-9, miR-27, miR-96, miR-153, miR-182, miR-183, or miR-186, but not miR-29a, miR-128, miR-152, or miR-486 mimetics in HEC-1B cells was sufficient to significantly reduce the abundance of FOXO1. Conversely, FOXO1 expression was efficiently restored in the Ishikawa cell line upon simultaneous inhibition of miR-9, miR-27, miR-96, miR-153, miR-183, and miR-186. Moreover, induction of FOXO1 in Ishikawa cells by miR inhibitors was accompanied by G1 cell cycle arrest and cell death, and was attenuated by the small interfering RNA–mediated downregulation of FOXO1 expression. Our findings identify several miRs overexpressed in endometrial cancer that function in concert to repress FOXO1 expression. Further, aberrant miR expression results in deregulated cell cycle control and impaired apoptotic responses, and thus, may be central to endometrial tumorigenesis. Cancer Res; 70(1); 367–77

Supplementary Figure Legends 1-2 from Definition of microRNAs That Repress Expression of the Tumor Suppressor Gene FOXO1 in Endometrial Cancer

Women who have had repeated miscarriages often have uncertainties about the cause, the likelihood of recurrence, the investigations they need, and the treatments that might help. Health-care policy makers and providers have uncertainties about the optimal ways to organise and provide care. For this Series paper, we have developed recommendations for practice from literature reviews, appraisal of guidelines, and a UK-wide consensus conference that was held in December, 2019. Caregivers should individualise care according to the clinical needs and preferences of women and their partners. We define a minimum set of investigations and treatments to be offered to couples who have had recurrent miscarriages, and urge health-care policy makers and providers to make them universally available. The essential investigations include measurements of lupus anticoagulant, anticardiolipin antibodies, thyroid function, and a transvaginal pelvic ultrasound scan. The key treatments to consider are first trimester progesterone administration, levothyroxine in women with subclinical hypothyroidism, and the combination of aspirin and heparin in women with antiphospholipid antibodies. Appropriate screening and care for mental health issues and future obstetric risks, particularly preterm birth, fetal growth restriction, and stillbirth, will need to be incorporated into the care pathway for couples with a history of recurrent miscarriage. We suggest health-care services structure care using a graded model in which women are offered online health-care advice and support, care in a nurse or midwifery-led clinic, and care in a medical consultant-led clinic, according to clinical needs.

We tested the hypothesis that a panel of placental mammal-specific miRNAs and their targets play important to establish receptivity to implantation and their dysregulated expression may be a feature in women with early pregnancy loss. Relative expression levels of miR-340-5p, −542-3p, and −671-5p all increased following treatment of Ishikawa cells with progesterone (10 μg/ml) for 24 hrs (p < 0.05). RNA sequencing of these P4-treated cells identified co-ordinate changes to 6,367 transcripts of which 1713 were predicted targets of miR-340-5p, 670 of miR-542-3p, and 618 of miR-671-5p. Quantitative proteomic analysis of Ishikawa cells transfected with mimic or inhibitor (48 hrs: n=3 biological replicates) for each of the P4-regulated miRNAs was carried out to identify targets of these miRNAs. Excluding off target effects, mir-340-5p mimic altered 1,369 proteins while inhibition changed expression of 376 proteins (p < 0.05) of which, 72 were common to both treatments. A total of 280 proteins were identified between predicted (mirDB) and confirmed (in vitro) targets. In total, 171 proteins predicted to be targets by mirDB were altered in vitro by treatment with miR-340-5p mimic or inhibitor and were also altered by treatment of endometrial epithelial cells with P4. In vitro targets of miR-542-3p identified 1,378 proteins altered by mimic while inhibition altered 975 a core of 200 proteins were changed by both. 100 protein targets were predicted and only 46 proteins were P4 regulated. miR-671-mimic altered 1,252 proteins with inhibition changing 492 proteins of which 97 were common to both, 95 were miDB predicted targets and 46 were also P4-regulated. All miRNAs were detected in endometrial biopsies taken from patients during the luteal phase of their cycle, irrespective of prior or future pregnancy outcomes Expression of mir-340-5p showed an overall increase in patients who had previously suffered a miscarriage and had a subsequent miscarriage, as compared to those who had infertility or previous miscarriage and subsequently went on to have a life birth outcome. The regulation of these miRNAs and their protein targets regulate the function of transport and secretion, and adhesion of the endometrial epithelia required for successful implantation in humans. Dysfunction of these miRNAs (and therefore the targets they regulate) may contribute to endometrial-derived recurrent pregnancy loss in women.

Resveratrol, a natural polyphenolic compound, is widely studied for its anti-inflammatory and antisenescent properties. Recently, two studies reported seemingly conflicting findings on the actions of resveratrol on decidualization of human endometrial stromal cells (HESCs). One study by Ochiai et al. demonstrated that resveratrol inhibits decidual transformation of primary cultured HESCs. The other study by Mestre Citrinovitz et al., showed that resveratrol enhances decidualization of HESCs in culture. At a glance, the reason for these opposing observations seems puzzling. However, recent studies demonstrated that decidualization is a multistep process, which starts with an acute proinflammatory stress response that lasts for several days and is followed by the emergence of stress-resistant decidual cells as well as senescent decidual cells. The balance between these decidual subpopulations may determine if the cycling endometrium can successfully transition into the decidua of pregnancy upon embryo implantation. Here, we explore the importance of timing of drugs aimed at modulating the decidual response. We posit that resveratrol treatment during the initial proinflammatory decidual phase, i.e., coinciding with the implantation window in vivo, inhibits decidual transformation of the endometrium. However, when given after the initial phase, resveratrol may promote decidualization by inhibiting decidual senescence. Further, if restricted to the proliferative phase, resveratrol may promote ovarian function without adversely impacting on embryo implantation or decidualization. Thus, failure to align drug interventions with the correct phase of the menstrual cycle may negate beneficial clinical effects and results in adverse reproductive outcomes.Summary SentenceResveratrol treatment during the initial proinflammatory decidual phase inhibits decidualization of human endometrial stromal cells but enhances decidualization when given after the initial phase.Graphical Abstract

Historically, a lack of methodologically strong and generalizable studies has limited policymakers fromrecommending the use of progesterone supplementation to improve outcomes in women at high risk of miscarriage. The PROMISE and PRISM trials were carried out to rectify this and generate robust evidence on the role of progesterone supplementation to prevent miscarriage. This review aims to evaluate the PROMISE and PRISM trials and their impact on collective academic understanding of the use of progesterone supplementation for miscarriage prevention, as well as to provide recommendations for clinical practice. The PROMISE trial was a randomized double-blind, placebo-controlled study that included 836 women from 45 hospitals in the United Kingdom and the Netherlands and found a 3% increase in live birth rate with progesterone supplementation; however, the P value associated with this finding was 0.45. A post hoc subgroup analysis was performed with cohorts stratified according to number of previous miscarriages that revealed a trend for greater benefit with increasing number of previous miscarriages. Small sample sizes in this subgroup analysis resulted in a P value of 0.41; however, these results were hypothesis-generating that a biological gradient existed with respect to improved benefit of progesterone treatment among women with an increasing number of prior miscarriages. The PRISMtrial was a randomized, double-blind, placebo-controlled study that involved 4153 women from48 hospitals in the United Kingdom and noted a 3% increase in live birth rate with vaginal micronized progesterone; however, the P value associated with this finding was 0.08. To followup on the gradient effect observed in the PROMISE trial, this study included a prespecified subgroup analysis analyzing the number of previous miscarriages with population split into 3 subgroups: women with zero prior miscarriages, women with 1 to 2 prior miscarriages, and women with 3 or more prior miscarriages. Among women with 1 or more priormiscarriages and bleeding in the current pregnancy, the live birth rate was 75%with progesterone versus 70% with placebo (risk ratio, 1.09; 95% confidence interval, 1.03 1.15; P = 0.003). Among women with 3 or more prior miscarriages and bleeding in the current pregnancy, live birth rate was 72% with progesterone versus 57% with placebo (risk ratio, 1.28; 95% confidence interval, 1.08 1.51; P = 0.004). Guidelines for interpretation of subgroup analyses translate into 11 criteria to assess credibility and therefore clinical applicability; 5 on design, 2 on analysis, and 4 on context. The PRISM trials subgroup analysis meets all 11 criteria in women with dual risk factors of previous miscarriage and current pregnancy bleeding, suggesting the subgroup effect of a gradient existing in the context of improving live birth rate with progesterone treatment is highly plausible. Given biologic plausibility, lack of short-term safety concerns, and observed positive treatment effect, providers should consider offering women with vaginal bleeding and a history of 1 or more previous miscarriages a course of treatment with vaginal micronized progesterone 400 mg twice daily.

Background Emerging evidence supports an association between vaginal microbiota composition and risk of miscarriage; however, the underlying mechanisms are poorly understood. We aim to investigate the vaginal microbial composition and the local immune response in chromosomally normal and abnormal miscarriages and compare this to uncomplicated pregnancies delivering at term. Methods We used 16S rRNA gene based metataxonomics to interrogate the vaginal microbiota in a cohort of 167 women, 93 miscarriages (54 euploid and 39 aneuploid using molecular cytogenetics) and 74 women who delivered at term and correlate this with the aneuploidy status of the miscarriages. We also measured the concentrations of IL-2, IL-4, IL-6, IL-8, TNF-α, IFN-γ, IL-1β, IL-18 and IL-10 in cervical vaginal fluid. Results We show that euploid miscarriage is associated with a significantly higher prevalence of Lactobacillus spp. deplete vaginal microbial communities compared to aneuploid miscarriage (P = 0.01). Integration of matched cervicovaginal fluid immune-profiles showed that Lactobacillus spp. depleted vaginal microbiota associated with pro-inflammatory cytokine levels most strongly in euploid miscarriage compared to viable term pregnancy (IL-1β; P < 0.001, IL-8; P = 0.01, IL-6; P < 0.001). Conclusions Our data suggest the vaginal microbiota plays an important aetiological role in euploid miscarriage and may represent a target to modify risk of pregnancy loss.

In each menstrual cycle, the endometrium becomes receptive to embryo implantation while preparing for tissue breakdown and repair. Both pregnancy and menstruation are dependent on spontaneous decidualization of endometrial stromal cells, a progesterone-dependent process that follows rapid, estrogen-dependent proliferation. During the implantation window, stromal cells mount an acute stress response, which leads to the emergence of functionally distinct decidual subsets, reflecting the level of replication stress incurred during the preceding proliferative phase. Progesterone-dependent, anti-inflammatory decidual cells (DeC) form a robust matrix and recruit bone marrow-derived decidual progenitors to accommodate the conceptus whereas pro-inflammatory, progesterone-resistant stressed and senescent decidual cells (senDeC) control tissue remodelling and breakdown. To execute these functions, each decidual subset engage innate immune cells: DeC partner with uterine natural killer (uNK) cells to eliminate senDeC, while senDeC co-opt neutrophils and macrophages to assist with tissue breakdown and repair. Thus, successful transformation of cycling endometrium into the decidua of pregnancy not only requires continues progesterone signalling but dominance of DeC over senDeC, aided by recruitment and differentiation of circulating NK cells and bone marrow-derived progenitors. We discuss how the frequency of cycles resulting in imbalanced decidual subpopulations determines the recurrence risk of miscarriage and discuss emerging therapeutic strategies.\ud \ud

Evolutionary changes in the anatomy and physiology of the female reproductive system underlie the origins and diversification of pregnancy in Eutherian (‘placental’) mammals. This developmental and evolutionary history constrains normal physiological functions and biases the ways in which dysfunction contributes to reproductive trait diseases and adverse pregnancy outcomes. Here, we show that gene expression changes in the human endometrium during pregnancy are associated with the evolution of human-specific traits and pathologies of pregnancy. We found that hundreds of genes gained or lost endometrial expression in the human lineage. Among these are genes that may contribute to human-specific maternal–fetal communication (HTR2B) and maternal–fetal immunotolerance (PDCD1LG2) systems, as well as vascular remodeling and deep placental invasion (CORIN). These data suggest that explicit evolutionary studies of anatomical systems complement traditional methods for characterizing the genetic architecture of disease. We also anticipate our results will advance the emerging synthesis of evolution and medicine (‘evolutionary medicine’) and be a starting point for more sophisticated studies of the maternal–fetal interface. Furthermore, the gene expression changes we identified may contribute to the development of diagnostics and interventions for adverse pregnancy outcomes., eLife digest Pregnancy is a complicated process. It has three phases: the body recognizes the embryo, it maintains the pregnancy, and finally, it induces labor. These stages happen in all mammals, but the details are different in humans. Human pregnancy and labor last longer. We menstruate. Our placentas invade deeper into the uterus, and the cues that signal pregnancy is done and induce labor are different than in most other mammals. We are also more likely to have pregnancy complications, including infertility, a dangerous rise in blood pressure called preeclampsia, and premature birth. The reasons for these differences are unknown. Human pregnancy relies on close communication between the placenta and the uterus. The immune system must allow the placenta to grow large enough to support the developing embryo, and blood vessels need to adapt to supply gases and nutrients and to remove waste. Understanding how the genes used by the human uterus are different to those used in other species could help explain why human pregnancies are so unusual. Mika, Marinić et al. compared the genes used by the pregnant human uterus to those used in 32 other species, including monkeys, marsupials and other mammals, birds, and reptiles. The analysis revealed that the humans use almost a thousand genes that other animals do not. These genes have roles in the invasion of the placenta, the growth of blood vessels, and control of the immune system. Several have links to the hormone serotonin, which had not been connected with the uterus before. Mika, Marinić et al. suggest that it might control the length of pregnancy, the timing of labor, and communication between parent and baby. The genes identified here provide a starting point for further investigation of human pregnancy. In the future, this may help to prevent or treat infertility, preeclampsia, or premature birth. A possible next step is to examine our closest living relatives, the great apes. Performing similar experiments using tissues or cells from chimpanzees, gorillas, and orangutans could reveal more about the genes unique to human pregnancy.

Decidual remodelling of midluteal endometrium leads to a short implantation window after which the uterine mucosa either breaks down or is transformed into a robust matrix that accommodates the placenta throughout pregnancy. To gain insights into the underlying mechanisms, we established and characterised endometrial assembloids, consisting of gland organoids and primary stromal cells. Single-cell transcriptomics revealed that decidualized assembloids closely resemble midluteal endometrium, harbouring differentiated and senescent subpopulations in both glands and stroma. We show that acute senescence in glandular epithelium drives secretion of multiple canonical implantation factors, whereas in the stroma it calibrates the emergence of anti-inflammatory decidual cells and pro-inflammatory senescent decidual cells. Pharmacological inhibition of stress responses in pre-decidual cells accelerated decidualization by inhibiting senescence and mesenchymal-epithelial transition, processes involved in endometrial breakdown and regeneration, respectively. Accelerated decidualization resulted in entrapment of co-cultured human blastocysts in a largely static decidual matrix. By contrast, the presence of senescent decidual cells created a dynamic implantation environment, enabling embryo expansion and attachment, although their persistence led to gradual disintegration of assembloids. Our findings demonstrate that senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.

Embryo implantation is a complex and tightly regulated process. In humans, uterine luminal epithelium functions as a biosensor gauging the embryo quality and transmitting this information to the underlying endometrial stromal cells. This quality control ensures that only high quality embryos are implanted, while aberrant ones are rejected. The mechanisms of the embryo-uterine mucosa crosstalk remain incompletely understood. Trypsin, a serine protease secreted by the blastocyst, has been implicated in the cross-signaling. Here we address the mechanisms by which trypsin triggers the intracellular calcium signaling in uterine epithelium. We found that protease-activated G-protein coupled receptors are the main mechanism mediating the effects of trypsin in human uterine epithelium. In addition, trypsin activates the epithelial sodium channels thus increasing the intracellular Na+concentration and promoting Ca2+entry on the reverse mode of the sodium/calcium exchanger.

STUDY QUESTIONCan 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 ANSWERMeasuring the expression levels of six genes (IL2RB, IGFBP1, CXCL14, DPP4, GPX3, and SLC15A2) is sufficient to obtain substantially more accurate timing estimates and assess the reliability of timing estimates for each sample.WHAT IS KNOWN ALREADYCommercially available endometrial timing approaches based on gene expression require much larger gene sets and use a categorical approach that classifies samples as pre-receptive, receptive, or post-receptive.STUDY DESIGN, SIZE, DURATIONGene expression was measured by RT-qPCR in 260 endometrial biopsies obtained 4 to 12 days after a self-reported positive home ovulation test. A further 36 endometrial samples were profiled by RT-qPCR as well as RNA-sequencing.PARTICIPANTS/MATERIALS, SETTING, METHODSA 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 CHANCEThe 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 < 0.005; CXCL14: p < 2.7e-6; DPP4: p < 3.7e-13). Pearson correlation between EndoTime estimates for the same sample set but based on RT-qPCR or RNA-sequencing data showed high degree of congruency between the two (p = 8.6e-10, R2 = 0.687).LIMITATIONS, REASONS FOR CAUTIONTiming estimates are predominantly 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 FINDINGSOur 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.STUDY FUNDING/COMPETING INTEREST(S)This study was supported by a Wellcome Trust Investigator Award (Grant/Award Number: 212233/Z/18/Z) and the Tommy’s National Miscarriage Research Centre. None of the authors have any competing interests. JL was funded by the Biotechnology and Biological Sciences Research Council (UK) through the Midlands Integrative Biology Training Partnership (MIBTP).TRIAL REGISTRATION NUMBERN/A.

Pregnancy depends on the wholesale transformation of the endometrium, a process driven by differentiation of endometrial stromal cells (EnSC) into specialist decidual cells. Upon embryo implantation, decidual cells impart the tissue plasticity needed to accommodate a rapidly growing conceptus and invading placenta, although the underlying mechanisms are unclear. Here we characterize a discrete population of highly proliferative mesenchymal cells (hPMC) in midluteal human endometrium, coinciding with the window of embryo implantation. Single-cell transcriptomics demonstrated that hPMC express genes involved in chemotaxis and vascular transmigration. Although distinct from resident EnSC, hPMC also express genes encoding pivotal decidual transcription factors and markers, most prominently prolactin. We further show that hPMC are enriched around spiral arterioles, scattered throughout the stroma, and occasionally present in glandular and luminal epithelium. The abundance of hPMC correlated with thein vitrocolony-forming unit activity of midluteal endometrium and, conversely, clonogenic cells in culture express a gene signature partially conserved in hPMC. Cross-referencing of single-cell RNA-sequencing data sets indicated that hPMC differentiate into a recently discovered decidual subpopulation in early pregnancy. Finally, we demonstrate that recurrent pregnancy loss is associated with hPMC depletion. Collectively, our findings characterize midluteal hPMC as novel decidual precursors that are likely derived from circulating bone marrow-derived mesenchymal stem/stromal cells and integral to decidual plasticity in pregnancy.Significance statementTransformation of cycling endometrium into the decidua of pregnancy requires extensive tissue remodeling. Perturbations in this process lead to breakdown of the maternal-fetal interface and miscarriage. Here we report the characterization of decidual precursor cells during the window of implantation. We demonstrate that decidual precursors are clonogenic and primed for exponential growth. They likely originate from bone marrow-derived MSC and give rise to a distinct decidual subpopulation in pregnancy. Recurrent pregnancy loss is associated with loss of decidual precursor cells prior to conception, raising the possibility that they can be harnessed for the prevention of pregnancy disorders, including miscarriages and preterm labor.

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. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by a Wellcome Trust Investigator Award (Grant/Award Number: 212233/Z/18/Z) and the Tommy's National Miscarriage Research Centre. None of the authors have any competing interests. J.L. was funded by the Biotechnology and Biological Sciences Research Council (UK) through the Midlands Integrative Biology Training Partnership (MIBTP, BB/M01116X/1).

Study question To investigate the vaginal microbial composition and the local immune response in chromosomally normal and abnormal miscarriages and compare this to uncomplicated pregnancies delivering at term. Summary answer We show that euploid miscarriage is associated with a significantly higher prevalence of Lactobacillus spp. deplete vaginal microbial communities compared to aneuploid miscarriage. What is known already Emerging evidence supports the role of the vaginal microbiota in adverse pregnancy outcome, but the underlying mechanisms are poorly understood. A dominance of Lactobacillus spp. in pregnancy provides protection against pathogenic bacteria by producing lactic acid and antimicrobial compounds. A depletion in Lactobacillus spp. is often linked to adverse pregnancy outcomes.Current work also implicates the reproductive tract microbiota as a key modulator of local inflammatory and immune pathways. We have previously shown that miscarriage is associated with vaginal dysbiosis but without knowledge of the cytogenetic status of those miscarriages or the local immune profile. Study design, size, duration This study was a prospective observational cohort study based at Queen Charlotte’s & Chelsea Hospital, Early Pregnancy Unit, London between March 2014-February 2019. Vaginal swabs were collected from the posterior vaginal fornix of 167 patients. Participants/materials, setting, methods We used 16S rRNA gene based metataxonomics to interrogate the vaginal microbiota in a cohort of 167 women, 93 miscarriage patients (54 euploid and 39 aneuploid using molecular cytogenetics) and 74 women who delivered at term and correlate this with the aneuploidy status of the miscarriages. We also measured the concentrations of IL-2, IL-4, IL-6, IL-8, TNF-α, IFN-γ, IL-1β, IL-18 and IL-10 in cervical vaginal fluid using Human Magnetic Luminex Screening Assay (8-plex). Main results and the role of chance We show that euploid miscarriage is associated with a significantly higher prevalence of Lactobacillus spp. deplete vaginal microbial communities compared to aneuploid miscarriage (P=0.008). In women having Lactobacillus spp. deplete vaginal microbial communities, euploid miscarriage associates with higher concentrations of pro-inflammatory cytokines IL-1β, IL-8, IL-6 (P Limitations, reasons for caution There were no longitudinal samples in this cohort and our results are based on the assumption that the vaginal microbial composition is stable throughout the first trimester.Future longitudinal studies with larger sample sizes are needed to corroborate these findings and provide insights to the mechanisms that trigger the inflammatory response. Wider implications of the findings These findings support the hypothesis that the vaginal microbiota plays an important aetiological role in euploid miscarriage and may represent a target to modify the risk of pregnancy loss. Trial registration number n/a

Preeclampsia is an important cause of maternal and perinatal morbidity, especially in first-time pregnant adolescent women. Although prevention of preeclampsia has been attempted for many decades, effective intervention can only be achieved upon the full elucidation of the risk factors and mechanisms of disease. As the pathogenesis of preeclampsia during adolescence may differ from that in older women, preventive interventions should be tailored accordingly. During adolescence, 4 putative drivers of preeclampsia can be identified. First, uterine immaturity in very young teenagers is likely a major cause of defective deep placentation and adverse reproductive outcome, underscoring the importance of educational programs and public health initiatives focused on teen pregnancy prevention. Second, the association between adolescent obesity and preeclampsia merits further studies on the benefits of weight loss and dietary interventions to improve pregnancy outcome. Third, there is a need for greater awareness of the link between cardiovascular risk factors in young women and early-onset preeclampsia associated with atherosclerosis of the uteroplacental arteries. Finally, infrequent menstruations may prolong uterine immaturity because of lack of "menstrual preconditioning." This risk factor may be amenable to pharmacological/hormonal preconditioning prior to conception.

Upon embryo implantation, the uterine mucosa - the endometrium - transforms into a robust decidual matrix that accommodates the fetal placenta throughout pregnancy. This transition is driven by the differentiation of endometrial fibroblasts into specialised decidual cells. A synchronised influx of circulating natural killer (NK) cells and bone marrow-derived mesenchymal stem/progenitor cells (BM-MSC) is pivotal for decidual homeostasis and expansion in early pregnancy. We hypothesise that pathological signals interfering with the recruitment or activity of extrauterine cells at the maternal-fetal interface link miscarriage to subsequent adverse pregnancy outcomes, including further pregnancy losses and preterm labour. NK cells and BM-MSC are key homeostatic regulators in multiple tissues, pointing towards a shared aetiology between recurrent miscarriage and age-related disorders, including cardiometabolic disease. We propose the term ‘miscarriage syndrome’ to capture the health risks associated with miscarriage and discuss how this paradigm can inform clinical practice and accelerate the development of preventative strategies.\ud \ud

Embryo implantation is a complex and tightly regulated process. In humans, uterine luminal epithelium functions as a biosensor gauging the embryo quality and transmitting this information to the underlying endometrial stromal cells. This quality control ensures that only high quality embryos are implanted, while aberrant ones are rejected. The mechanisms of the embryo-uterine mucosa crosstalk remain incompletely understood. Trypsin, a serine protease secreted by the blastocyst, has been implicated in the cross-signaling. Here we address the mechanisms by which trypsin triggers the intracellular calcium signaling in uterine epithelium. We found that protease-activated G-protein coupled receptors are the main mechanism mediating the effects of trypsin in human uterine epithelium. In addition, trypsin activates the epithelial sodium channels thus increasing the intracellular Na+ concentration and promoting Ca2+ entry on the reverse mode of the sodium/calcium exchanger.

The physical and psychological effect of miscarriage is commonly underappreciated. The journey from diagnosis of miscarriage, through clinical management, to supportive aftercare can be challenging for women, their partners, and caregivers. Diagnostic challenges can lead to delayed or ineffective care and increased anxiety. Inaccurate diagnosis of a miscarriage can result in the unintended termination of a wanted pregnancy. Uncertainty about the therapeutic effects of interventions can lead to suboptimal care, with variations across facilities and countries. For this Series paper, we have developed recommendations for practice from a literature review, appraisal of guidelines, and expert group discussions. The recommendations are grouped into three categories: (1) diagnosis of miscarriage, (2) prevention of miscarriage in women with early pregnancy bleeding, and (3) management of miscarriage. We recommend that every country reports annual aggregate miscarriage data, similarly to the reporting of stillbirth. Early pregnancy services need to focus on providing an effective ultrasound service, as it is central to the diagnosis of miscarriage, and be able to provide expectant management of miscarriage, medical management with mifepristone and misoprostol, and surgical management with manual vacuum aspiration. Women with the dual risk factors of early pregnancy bleeding and a history of previous miscarriage can be recommended vaginal micronised progesterone to improve the prospects of livebirth. We urge health-care funders and providers to invest in early pregnancy care, with specific focus on training for clinical nurse specialists and doctors to provide comprehensive miscarriage care within the setting of dedicated early pregnancy units.

Recurrent Pregnancy Loss (RPL) affects 2-4% of couples, and with increasing numbers of pregnancy losses the risk of miscarrying a euploid pregnancy is increased, suggesting RPL is a pathology distinct from sporadic miscarriage that is due largely to lethal embryonic aneuploidy. There are a number of conditions associated with RPL including unspecified ‘immune’ pathologies, one of the strongest candidates for dysregulation remains T regulatory cells as depletion in the very early stages of pregnancy in mice leads to pregnancy loss.Human endometrial Treg and conventional CD4T cells were isolated during the peri-implantation period of the menstrual cycle in normal women. We identified an endometrial Treg transcriptomic signature and validated an enhanced regulatory phenotype compared to peripheral blood Treg. Parous women had an altered endometrial Treg transcriptome compared to nulliparity, indicating acquired immunity memory of pregnancy within the Treg population, by comparison endometrial conventional CD4T cells were not altered. We compared primary and secondary RPL to nulliparous or parous controls respectively. Both RPL subgroups displayed differentially expressed Treg gene transcriptomes compared to controls. We found increased cell surface S1PR1 and decreased TIGIT protein expression by Treg in primary RPL, confirming the presence of altered Treg in the peri-implantation RPL endometrium.

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27me3) to maintain repression of genes specific for other cell types and is essential for cell differentiation. In endometrial stromal sarcoma, the PRC2 subunit SUZ12 is often fused with the NuA4/TIP60 subunit JAZF1. Here, we show that JAZF1-SUZ12 dysregulates PRC2 composition, recruitment, histone modification, gene expression and cell differentiation. The loss of the SUZ12 N-terminus in the fusion protein disrupted interaction with the PRC2 accessory factors JARID2, EPOP and PALI1 and prevented recruitment of PRC2 from RNA to chromatin. In undifferentiated cells, JAZF1-SUZ12 occupied PRC2 target genes but gained a JAZF1-like binding profile during cell differentiation. JAZF1-SUZ12 reduced H3K27me3 and increased H4Kac at PRC2 target genes, and this was associated with disruption in gene expression and cell differentiation programs. These results reveal the defects in chromatin regulation caused by JAZF1-SUZ12, which may underlie its role in oncogenesis.

Decidualizing endometrial stromal cells (EnSC) critically determine the maternal response to an implanting conceptus, triggering either menstruation‐like disposal of low‐fitness embryos or creating an environment that promotes further development. However, the mechanism that couples maternal recognition of low‐quality embryos to tissue breakdown remains poorly understood. Recently, we demonstrated that successful transition of the cycling endometrium to a pregnancy state requires selective elimination of pro‐inflammatory senescent decidual cells by activated uterine natural killer (uNK) cells. Here we report that uNK cells express CD44, the canonical hyaluronan (HA) receptor, and demonstrate that high molecular weight HA (HMWHA) inhibits uNK cell‐mediated killing of senescent decidual cells. In contrast, low molecular weight HA (LMWHA) did not attenuate uNK cell activity in co‐culture experiments. Killing of senescent decidual cells by uNK cells was also inhibited upon exposure to medium conditioned by IVF embryos that failed to implant, but not successful embryos. Embryo‐mediated inhibition of uNK cell activity was reversed by recombinant hyaluronidase 2 (HYAL2), which hydrolyses HMWHA. We further report a correlation between the levels of HYAL2 secretion by human blastocysts, morphological scores, and implantation potential. Taken together, the data suggest a pivotal role for uNK cells in embryo biosensing and endometrial fate decisions at implantation.

Miscarriage is generally defined as the loss of a pregnancy before viability. An estimated 23 million miscarriages occur every year worldwide, translating to 44 pregnancy losses each minute. The pooled risk of miscarriage is 15·3% (95% CI 12·5–18·7%) of all recognised pregnancies. The population prevalence of women who have had one miscarriage is 10·8% (10·3–11·4%), two miscarriages is 1·9% (1·8–2·1%), and three or more miscarriages is 0·7% (0·5–0·8%). Risk factors for miscarriage include very young or older female age (younger than 20 years and older than 35 years), older male age (older than 40 years), very low or very high body-mass index, Black ethnicity, previous miscarriages, smoking, alcohol, stress, working night shifts, air pollution, and exposure to pesticides. The consequences of miscarriage are both physical, such as bleeding or infection, and psychological. Psychological consequences include increases in the risk of anxiety, depression, post-traumatic stress disorder, and suicide. Miscarriage, and especially recurrent miscarriage, is also a sentinel risk marker for obstetric complications, including preterm birth, fetal growth restriction, placental abruption, and stillbirth in future pregnancies, and a predictor of longer-term health problems, such as cardiovascular disease and venous thromboembolism. The costs of miscarriage affect individuals, health-care systems, and society. The short-term national economic cost of miscarriage is estimated to be £471 million per year in the UK. As recurrent miscarriage is a sentinel marker for various obstetric risks in future pregnancies, women should receive care in preconception and obstetric clinics specialising in patients at high risk. As psychological morbidity is common after pregnancy loss, effective screening instruments and treatment options for mental health consequences of miscarriage need to be available. We recommend that miscarriage data are gathered and reported to facilitate comparison of rates among countries, to accelerate research, and to improve patient care and policy development.

Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible adhesion molecule and a primary amine oxidase involved in immune cell trafficking. Leukocyte extravasation into tissues is mediated by adhesion molecules expressed on endothelial cells and pericytes. Pericytes play a major role in the angiogenesis and vascularization of cycling endometrium. However, the functional properties of pericytes in the human endometrium are not known. Here we show that pericytes surrounding the spiral arterioles in midluteal human endometrium constitutively express VAP-1. We first characterize these pericytes and demonstrate that knockdown of VAP-1 perturbed their biophysical properties and compromised their contractile, migratory, adhesive and clonogenic capacities. Furthermore, we show that loss of VAP-1 disrupts pericyte-uterine natural killer cell interactions in vitro. Taken together, the data not only reveal that endometrial pericytes represent a cell population with distinct biophysical and functional properties but also suggest a pivotal role for VAP-1 in regulating the recruitment of innate immune cells in human endometrium. We posit that VAP-1 could serve as a potential biomarker for pregnancy pathologies caused by a compromised perivascular environment prior to conception.

Differentiation of endometrial fibroblasts into specialized decidual cells controls embryo implantation and transforms the cycling endometrium into a semi-permanent, immune-protective matrix that accommodates the placenta throughout pregnancy. This process starts during the midluteal phase of the menstrual cycle with decidual transformation of perivascular cells (PVC) surrounding the terminal spiral arterioles and endometrial stromal cells (EnSC) underlying the luminal epithelium. Decidualization involves extensive cellular reprogramming and acquisition of a secretory phenotype, essential for coordinated placental trophoblast invasion. Secreted metabolites are an emerging class of signaling molecules, collectively known as the exometabolome. Here, we used liquid chromatography-mass spectrometry to characterize and analyze time-resolved changes in metabolite secretion (exometabolome) of primary PVC and EnSC decidualized over 8 days. PVC were isolated using positive selection of the cell surface marker SUSD2. We identified 79 annotated metabolites differentially secreted upon decidualization, including prostaglandin, sphingolipid, and hyaluronic acid metabolites. Secreted metabolites encompassed 21 metabolic pathways, most prominently glycerolipid and pyrimidine metabolism. Although temporal exometabolome changes were comparable between decidualizing PVC and EnSC, 32 metabolites were differentially secreted across the decidualization time-course. Further, targeted metabolomics demonstrated significant differences in secretion of purine pathway metabolites between decidualized PVC and EnSC. Taken together, our findings indicate that the metabolic footprints generated by different decidual subpopulations encode spatiotemporal information that may be important for optimal embryo implantation.

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 (H3K27me3) to maintain gene repression and is essential for cell differentiation. In low-grade endometrial stromal sarcoma (LG-ESS), the PRC2 subunit SUZ12 is often fused with the NuA4/TIP60 subunit JAZF1. We show that JAZF1-SUZ12 dysregulates PRC2 composition, genome occupancy, histone modification, gene expression, and cell differentiation. Loss of the SUZ12 N terminus in the fusion protein abrogates interaction with specific PRC2 accessory factors, reduces occupancy at PRC2 target genes, and diminishes H3K27me3. Fusion to JAZF1 increases H4Kac at PRC2 target genes and triggers recruitment to JAZF1 binding sites during cell differentiation. In human endometrial stromal cells, JAZF1-SUZ12 upregulated PRC2 target genes normally activated during decidualization while repressing genes associated with immune clearance, and JAZF1-SUZ12-induced genes were also overexpressed in LG-ESS. These results reveal defects in chromatin regulation, gene expression, and cell differentiation caused by JAZF1-SUZ12 that may underlie its role in oncogenesis.

Endometrial mesenchymal stem cells (eMSC) drive the extraordinary regenerative capacity of the human endometrium. Clinical application of eMSC for therapeutic purposes is hampered by spontaneous differentiation and cellular senescence upon large-scale expansion in vitro. A83-01, a selective transforming growth factor-β receptor (TGFβ-R) inhibitor, promotes expansion of eMSC in culture by blocking differentiation and senescence, but the underlying mechanisms are incompletely understood. In this study, we combined RNA-seq and ATAC-seq to study the impact of sustained TGFβ-R inhibition on gene expression and chromatin architecture of eMSC. Treatment of primary eMSC with A83-01 for 5 weeks resulted in differential expression of 1,463 genes. Gene ontology analysis showed enrichment of genes implicated in cell growth whereas extracellular matrix genes and genes involved in cell fate commitment were downregulated. ATAC-seq analysis demonstrated that sustained TGFβ-R inhibition results in opening and closure of 3,555 and 2,412 chromatin loci, respectively. Motif analysis revealed marked enrichment of retinoic acid receptor (RAR) binding sites, which was paralleled by the induction of RARB, encoding retinoic acid receptor beta (RARβ). Selective RARβ inhibition attenuated proliferation and clonogenicity of A83-01 treated eMSC. Taken together, our study provides new insights into the gene networks and genome-wide chromatin changes that underpin maintenance of an undifferentiated phenotype of eMSC in prolonged culture.

Decidualizing endometrial stromal cells (EnSC) critically determine the maternal response to an implanting conceptus, triggering either menstruation-like disposal of low-fitness embryos or creating an environment that promotes further development. However, the mechanism that couples maternal recognition of low-quality embryos to tissue breakdown remains poorly understood. Recently, we demonstrated that successful transition of the cycling endometrium to a pregnancy state requires selective elimination of pro-inflammatory senescent decidual cells by activated uterine natural killer (uNK) cells. Here we report that uNK cells express CD44, the canonical hyaluronan (HA) receptor, and demonstrate that high-molecular weight HA (HMWHA) inhibits uNK cell-mediated killing of senescent decidual cells. By contrast, low-molecular weight HA (LMWHA) did not attenuate uNK cell activity in co-culture experiments. Killing of senescent decidual cells by uNK cells was also inhibited upon exposure to medium conditioned by IVF embryos that failed to implant, but not successful embryos. Embryo-mediated inhibition of uNK cell activity was reversed by recombinant hyaluronidase 2 (HYAL2), which hydrolyses HMWHA. We further report a correlation between the levels of HYAL2 secretion by human blastocysts, morphological scores, and implantation potential. Taken together, the data suggest a pivotal role for uNK cells in embryo biosensing and endometrial fate decisions at implantation.

A limited window of receptivity is a prerequisite of reproductive success. Indispensable receptivity genes include cyclooxygenase 2 (COX2), an enzyme accomplishing formation of prostaglandin E2 (PGE2). A powerful regulator of PGE2 formation is Annexin A7 (ANXA7). The present study thus explored whether ANXA7 impacts on implantation and fertility. Here we show that ANXA7 is expressed in endometrial tissue and increases upon decidual transformation of human endometrial stromal cells (HESCs) in a time-dependent manner. Silencing ANXA7 significantly decreased the expression of PRL and IGFBP1, canonical decidual marker genes, but enhances COX2 and PGE2 levels. Genetic knockout of AnxA7 in mice significantly increases the number of implantation sites and litter sizes. Further, analysis of human endometrial biopsies showed that ANXA7 transcript and protein levels are decreased during the midluteal window of implantation in women suffering from recurrent pregnancy loss (RPL) when compared to subfertile patients. Taken together, the data indicate that ANXA7 has a conserved role in regulating endometrial receptivity and implantation.

Dear Editor,We would like to comment on the systematic review by Li et al.(1)The use of steroid hormones in the first trimester is a serious issue as organogenesis takes place at this time and therefore there is the possibility of harm from not only congenital anomalies, but also long-term, and even inter-generational effects. Anyone investigating the use of steroid hormones in the first trimester should remember the diethylstilbestrol legacy of devastating harm. Oestrogen (C18H24O2) and diethylstilbestrol (C18H20O2) have similar molecular composition, but their effects are poles apart. In this review, the authors have combined progesterone with progestogens; however they are not the same, in the same way that oestrogen and diethylstilbestrol are not the same. Vaginal micronized progesterone, which we used in our large and high-quality trials (the PROMISE (2) and PRISM (3) trials), has identical molecular structure to natural progesterone, but the other drugs included in this review do not (Table 1). We chose to study vaginal micronized progesterone, as it is identical in structure to natural progesterone, and the available evidence and expert opinion suggested that this is least likely to cause harm. It is important to note that there is evidence of potential harm from dydrogesterone, particularly congenital heart disease.(4)The authors make a bold statement in the abstract about the effects of dydrogesterone on live birth rate. However, they don’t fully address the weaknesses in the evidence. Therefore, we wish to highlight the significant deficiencies in the two trials that contributed live birth data that led to the assertion of beneficial effects from dydrogesterone. Both studies were single centre, open-label studies without placebo control. El-Zibdeh et al did not randomise participants, but instead allocated patients to dydrogesterone on Saturdays, Mondays and Wednesdays, and to no treatment on Sundays, Tuesdays and Thursdays. The trial by Pandian RU was not just a single-centre, but also a single-author study, with insufficient details of the methods to assess its quality. Thus, the effectiveness evidence from these trials cannot be considered reliable.Approximately 80% (4038 of 5056) of the data used in this systematic review come from our PRISM trial.(3) The PRISM trial is a prospectively-registered, randomised, placebo-controlled, multi-centre trial conducted to the highest standards in the UK. The trial found a 3% increase in live birth rate, but with borderline statistical significance (RR, 1.03; 95% CI, 1.00 to 1.07; P=0.08). A pre-specified subgroup analysis in women with the dual risk factors of current pregnancy bleeding and one or more previous miscarriages found a 5% increase in live birth rate (RR, 1.09; 95% CI, 1.03-1.15; P=0.003). In those with three or more previous miscarriages, a 15% increase in live birth rate was observed (RR, 1.28; 95% CI, 1.08 to 1.51; P=0.004).(3, 5) No short-term safety concerns were identified. Based on these data, our recommendation is to consider vaginal micronized progesterone for women with early pregnancy bleeding and one or more previous miscarriages. As for the role of dydrogesterone, we need not only high-quality, randomised trial evidence of its effects but also credible evidence of its safety. As dydrogesterone is a synthetic progesterone-like drug, i.e. a progestogen but not progesterone, the burden of proof to demonstrate short- and long-term safety rests on those promoting this drug.

Endometrial mesenchymal stem cells (eMSC) drive the extraordinary regenerative capacity of the human endometrium. Clinical application of eMSC for therapeutic purposes is hampered by spontaneous differentiation and cellular senescence upon large-scale expansionin vitro. A83-01, a selective transforming growth factor-β receptor (TGFβ-R) inhibitor, promotes pharmacological expansion of eMSC in culture by blocking differentiation and senescence, but the underlying mechanisms are incompletely understood. In this study, we combined RNA-seq and ATAC-seq to study the impact of sustained TGFβ-R inhibition on gene expression and chromatin architecture of eMSC. Treatment of primary eMSC with A83-01 for 5 weeks resulted in differential expression of 1,463 genes. Gene ontology analysis showed enrichment of genes implicated in cell growth whereas extracellular matrix genes and genes involved in cell fate commitment were downregulated. ATAC-seq analysis demonstrated that sustained TGFβ-R inhibition results in opening and closure of 3,555 and 2,412 chromatin loci, respectively. Motif analysis revealed marked enrichment of retinoic acid receptor (RAR) binding sites, which was paralleled by the induction ofRARB, encoding retinoic acid receptor beta (RARβ). Selective RARβ inhibition attenuated proliferation and clonogenicity of A83-01 treated eMSC. Taken together, our study provides new insights into the gene networks and genome-wide chromatin changes that underpin maintenance of an undifferentiated phenotype of eMSC in prolonged culture.Significance statementCycling human endometrium is a rich source of adult stem/progenitor cells that could be exploited for clinical purposes. Small molecules, such as A83-01, that modulate cell identity may open new avenues to maintain the functional properties of eMSC upon expansion in culture. By integrating complementary genome-wide profiling techniques, we mapped the dynamic changes in chromatin landscape and gene expression in response to prolonged A83-01 treatment of eMSC. Our findings provide new insights into the mechanisms of action of TGFβ-R inhibition that may lead to the development of more targeted pharmacological approaches for MSC expansion.

Study question Does an early proliferative phase endometrial biopsy harvested during ovarian stimulation harbour information predictive of the outcome following fresh embryo transfer (ET) in that same cycle? Summary answer Transcriptome analysis of the whole-tissue endometrium did not reveal significant differential gene expression (DGE) in relation to the outcome; however, the secretome profile of isolated, cultured and in vitro decidualized endometrial stromal cells (EnSCs) varied significantly between patients who had a live birth compared to those with an implantation failure following fresh ET in the same cycle as the biopsy. What is known already In the majority of endometrial receptivity research protocols, biopsies are harvested during the window of implantation (WOI). This, however, precludes ET in that same cycle, which is preferable as the endometrium has been shown to adapt over time. Endometrial biopsies taken during ovarian stimulation have been reported not to harm the chances of implantation, and in such biopsies DGE has been observed between women who achieve pregnancy versus those who do not. The impact of the endometrial proliferative phase on human embryo implantation remains unclear, but deserves further attention, especially since in luteal phase endometrial biopsies, a transcriptional signature predictive for repeated implantation failure has been associated with reduced cell proliferation, possibly indicating proliferative phase involvement. Isolation, culture and in vitro decidualization (IVD) of EnSCs is a frequently applied basic research technique to assess endometrial functioning, and a disordered EnSC secretome has previously been linked with failed implantation. Study design, size, duration This study was nested in a randomized controlled trial (RCT) investigating the effect of endometrial scratching during the early follicular phase of ovarian stimulation on clinical pregnancy rates after IVF/ICSI. Of the 96 endometrial biopsies available, after eliminating those without fresh ET and after extensive matching in order to minimize the risk of potential confounding, 18 samples were retained to study two clinical groups: nine biopsies of patients with a live birth versus nine biopsies of patients with an implantation failure, both following fresh ET performed in the same cycle as the biopsy. We studied the proliferative endometrium by analysing its transcriptome and by isolating, culturing and decidualizing EnSCs in vitro. We applied this latter technique for the first time on proliferative endometrial biopsies obtained during ovarian stimulation for in-cycle outcome prediction, in an attempt to overcome inter-cycle variability. Participants/materials, setting, methods RNA-sequencing was performed for 18 individual whole-tissue endometrial biopsies on an Illumina HiSeq1500 machine. DGE was analysed three times using different approaches (DESeq2, EdgeR and the Wilcoxon rank-sum test, all in R). EnSC isolation and IVD was performed (for 2 and 4 days) for a subset of nine samples, after which media from undifferentiated and decidualized cultures were harvested, stored at −80°C and later assayed for 45 cytokines using a multiplex suspension bead immunoassay. The analysis was performed by partial least squares regression modelling. Main results and the role of chance After correction for multiple hypothesis testing, DGE analysis revealed no significant differences between endometrial samples from patients who had a live birth and those with an implantation failure following fresh ET. However secretome analysis after EnSC isolation and culture, showed two distinct clusters that clearly corresponded to the two clinical groups. Upon IVD, the secretome profiles shifted from that of undifferentiated cells but the difference between the two clinical groups remained yet were muted, suggesting convergence of cytokine profiles after decidualization. Limitations, reasons for caution Caution is warranted due to the limited sample size of the study and the in vitro nature of the EnSC experiment. Validation on a larger scale is necessary, however, hard to fulfil given the very limited availability of in-cycle proliferative endometrial biopsies outside a RCT setting. Wider implications of the findings These data support the hypothesis that the endometrium should be assessed not only during the WOI and that certain endometrial dysfunctionalities can probably be detected early in a cycle by making use of the proliferative phase. This insight opens new horizons for the development of endometrial tests, whether diagnostic or predictive of IVF/ICSI treatment outcome. Study funding/competing interest(s) This study was supported by Fonds Wetenschappelijk Onderzoek (FWO, Flanders, Belgium, 11M9415N, 1 524 417N), Wetenschappelijk Fonds Willy Gepts (WFWG G160, Universitair Ziekenhuis Brussel, Belgium) and the National Medicine Research Council (NMRC/CG/M003/2017, Singapore). There are no conflicts of interests. Trial registration number NCT02061228.