Endometrial Assembloids to Model Human Embryo Implantation In Vitro.

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.

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 < 0.005; CXCL14: P < 2.7e-6; DPP4: P < 3.7e-13). Pearson correlation between EndoTime estimates for the same sample set but based on RTq-PCR or RNA-sequencing data showed a high degree of congruency between the two (P = 8.6e-10, R2 = 0.687). Estimated timings did not differ significantly between control subjects and patients with recurrent pregnancy loss or recurrent implantation failure (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).

Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids.

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.

At the beginning of a human pregnancy, the embryo implants into the uterus lining, known as the endometrium. At this point, the endometrium transforms into a new tissue that helps the placenta to form. Problems in this transformation process are linked to pregnancy disorders, many of which can lead to implantation failure (the embryo fails to invade the endometrium altogether) or recurrent miscarriages (the embryo implants successfully, but the interface between the placenta and the endometrium subsequently breaks down). Studying the implantation of human embryos directly is difficult due to ethical and technical barriers, and animals do not perfectly mimic the human process, making it challenging to determine the causes of pregnancy disorders. However, it is likely that a form of cellular arrest called senescence, in which cells stop dividing but remain metabolically active, plays a role. Indeed, excessive senescence in the cells that make up the endometrium is associated with recurrent miscarriage, while a lack of senescence is associated with implantation failure. To study this process, Rawlings et al. developed a new laboratory model of the human endometrium by assembling two of the main cell types found in the tissue into a three-dimensional structure. When treated with hormones, these 'assembloids' successfully mimic the activity of genes in the cells of the endometrium during implantation. Rawlings et al. then exposed the assembloids to the drug dasatinib, which targets and eliminates senescent cells. This experiment showed that assembloids become very robust and static when devoid of senescent cells. Rawlings et al. then studied the interaction between embryos and assembloids using time-lapse imaging. In the absence of dasatinib treatment, cells in the assembloid migrated towards the embryo as it expanded, a process required for implantation. However, when senescent cells were eliminated using dasatinib, this movement of cells towards the embryo stopped, and the embryo failed to expand, in a situation that mimicks implantation failure. The assembloid model of the endometrium may help scientists to study endometrial defects in the lab and test potential treatments. Further work will include other endometrial cell types in the assembloids, and could help increase the reliability of the model. However, any drug treatments identified using this model will need further research into their safety and effectiveness before they can be offered to patients.

Organoids to model the endometrium: implantation and beyond.

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.