SUMMARY The inflammatory decidual reaction renders the cycling endometrium transiently permissive for embryo implantation before transforming it into the decidua, the maternal bed accommodating the fetal placenta during pregnancy. Disruptions in decidual tissue remodeling are linked to miscarriage and other pregnancy disorders. However, endometrial assessment is hampered by a lack of affordable technologies capable of mapping the spatiotemporal dysregulation of this dynamic and complex tissue. Employing a graph neural network on whole slide images of 493 CD56-immunostained endometrial samples, Endometronome was developed as a deep learning tool to spatially track the decidual reaction and provide accurate estimates of marker gene expression. When applied to 2,690 additional biopsies, this model consistently identified morphological correlates of prior miscarriage burden, a proxy for future risk. Further, a morphological signature indicative of metabolic glandular impairment discriminated between clinical miscarriage presentations. These findings illustrate how advanced imaging analysis of routine histology can transform miscarriage prevention strategies.

Does treatment with doxycycline in those with chronic endometritis (CE) and a history of recurrent miscarriage (RM) improve cumulative live-birth rates? Treatment with doxycycline did not increase live-birth rates in those with CE and a history of RM (Relative Risk (RR): 1.02; Credible Interval (CrI) 0.85-1.21). RM affects up to 5% of couples trying to conceive. A commonly purported cause of RM is CE. This asymptomatic inflammation of the endometrium has been differentially attributed to both pathogenic organisms and derangement of the endometrial microbiota. CE has been associated with RM in numerous uncontrolled studies. Similarly, CE resolution has been demonstrated in women following treatment with antibiotic therapy in uncontrolled studies. Based on this antibiotics treatment of CE remains a common intervention for RM prevention and features in several national guidelines. The CERM trial is an adaptive, multicentre, parallel-arm, double-blind, placebo-controlled, randomised trial. 2,178 women were screened for eligibility, with 728 women undergoing endometrial biopsy. Of these 505 screened positive for CE and 438 were randomised (219 doxycycline and 219 placebo). Randomised, participants were followed-up for a median of 525 (IQR: 329) days post-randomisation. 210 screen negative women were also followed-up. Women aged ≥18 to < 42 years with a history of ≥ 2 consecutive first trimester losses, attending 26 UK-wide clinics were screened for CE by endometrial biopsy and immunohistochemistry for CD138. Women who screened positive were randomised, 1:1, to pre-conceptual doxycycline 100mg or identical placebo commenced on day 1 of the menstrual cycle twice daily for 14 days. The primary outcome was cumulative live births and pregnancies ≥24+0 weeks gestation at cessation of the trial. No between group imbalances were detected in the baseline characteristics between screen positive and negative women and those randomised to doxycycline or placebo in terms of, age, number of previous miscarriages, BMI, previous live birth rate and ethnicity. 95% of randomised women met the criteria for treatment compliance. A small cohort of women (n = 17) received doxycycline external to the trial of these 6 women received doxycycline after the first pregnancy outcome. No significant difference in time to first conception was demonstrated. 113 live-births or ongoing pregnancies occurred in the doxycycline group with 109 in the placebo group (RR 1.02; CrI 0.85-1.21). Similarly, no between group difference in miscarriage rate was demonstrated (RR 0.90; CrI 0.67-1.22). There were 7 ectopic pregnancies in the placebo group and 1 in the doxycycline group. A per-protocol sensitivity analysis excluding known treatment non-compliance additionally showed no significance (RR 1.05; CrI 0.87-1.26). Pre-specified subgroup analyses also showed no significance for the impact of age (35 vs ≥ 35), miscarriage history (≤3 vs > 3) or CE severity. A comparison of first pregnancy outcome demonstrated more, (n = 89) live-births or ongoing pregnancies in the placebo group compared to the CE negative group (n = 62) (RR 0.73; CrI 0.56-0.95). The study was stopped prior to reaching target recruitment by the funder. This was for perceived futility based on the summation of the clinical and translational evidence available. Preconceptual doxycycline was not shown to be associated with improved live-birth rates in women with CE and RM. Screen negative women had worse pregnancy outcomes that screen positive women on placebo. The screening and treatment of CE should not form part of the routine management of RM. Yes

Understanding the process of human embryo implantation is impeded by the inability to study this phenomenon in vivo, thus limiting opportunities to gain knowledge to in vitro modeling. Previous models have relied on monolayer co-cultures, which do not replicate the complexity of endometrial tissue. Here, we detail the establishment of three-dimensional endometrial assembloids, comprising gland-like epithelial organoids in a stromal matrix. Endometrial assembloids mimic endometrial tissue structure more faithfully and can be used to study human embryo-endometrial interactions. Co-cultures of human embryos and endometrial assembloids will enhance our fundamental understanding of these processes as well as allowing us to study the mechanisms of persistent reproductive failure.

In vitro three-dimensional (3D) models are better able to replicate the complexity of real organs and tissues than 2D monolayer models. The human endometrium, the inner lining of the uterus, undergoes complex changes during the menstrual cycle and pregnancy. These changes occur in response to steroid hormone fluctuations and elicit crosstalk between the epithelial and stromal cell compartments, and dysregulations are associated with a variety of pregnancy disorders. Despite the importance of the endometrium in embryo implantation and pregnancy establishment, there is a lack of in vitro models that recapitulate tissue structure and function and as such a growing demand for extracellular matrix hydrogels that can support 3D cell culture. To be physiologically relevant, an in vitro model requires mechanical and biochemical cues that mimic those of the ECM found in the native tissue. We report a semisynthetic gelatin methacryloyl (GelMA) hydrogel that combines the bioactive properties of natural hydrogels with the tunability and reproducibility of synthetic materials. We then describe a simple protocol whereby cells can quickly be encapsulated in GelMA hydrogels. We investigate the suitability of GelMA hydrogel to support the development of an endometrial model by culturing the main endometrial cell types: stromal cells and epithelial cells. We also demonstrate how the mechanical and biochemical properties of GelMA hydrogels can be tailored to support the growth and maintenance of epithelial gland organoids that emerge upon 3D culturing of primary endometrial epithelial progenitor cells in a defined chemical medium. We furthermore demonstrate the ability of GelMA hydrogels to support the viability of stromal cells and their function measured by monitoring decidualization in response to steroid hormones. This study describes the first steps toward the development of a hydrogel matrix-based model that recapitulates the structure and function of the native endometrium and could support applications in understanding reproductive failure.

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 assess the reliability of timing estimates for each sample. WHAT IS KNOWN ALREADY Commercially 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, DURATION Gene 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, 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 < 0.005; 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 CAUTION Timing 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 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.

Despite advances in assisted reproductive techniques in the 4 decades since the first human birth after in vitro fertilisation, 1–2% of couples experience recurrent implantation failure, and some will never achieve a successful pregnancy even in the absence of a confirmed dysfunction. Furthermore, 1–2% of couples who do conceive, either naturally or with assistance, will experience recurrent early loss of karyotypically normal pregnancies. In both cases, embryo-endometrial interaction is a clear candidate for exploration. The impossibility of studying implantation processes within the human body has necessitated the use of animal models and cell culture approaches. Recent advances in 3-dimensional modelling techniques, namely the advent of organoids, present an exciting opportunity to elucidate the unanswerable within human reproduction. In this review, we will explore the ontogeny of implantation modelling and propose a roadmap to application and discovery. Lay summary A significant number of couples experience either recurrent implantation failure or recurrent pregnancy loss. Often, no underlying disorder can be identified. In both cases, the interaction of the embryo and maternal tissues is key. The lining of the womb, the endometrium, becomes receptive to embryo implantation during each menstrual cycle and provides a nourishing and supportive environment to support ongoing pregnancy. It is not possible to study early pregnancy directly, therefore, modelling embryo-endometrium interactions in the laboratory is essential if we wish to understand where this goes wrong. Advances in the lab have resulted in the development of organoids in culture: 3D cellular structures that represent the characteristics of a particular tissue or organ. We describe past and present models of the endometrium and propose a roadmap for future work with organoid models, from fundamental understanding of the endometrial function and implantation processes to the development of therapeutics to improve pregnancy outcomes and gynaecological health.

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 characterized endometrial assembloids, consisting of gland-like 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 eliminating the emergence of senescent decidual cells. In co-culture experiments, accelerated decidualization resulted in entrapment of collapsed human blastocysts in a robust, 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 suggest that decidual senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.