Photo-Cross-linked Gelatin Methacryloyl Hydrogels Enable the Growth of Primary Human Endometrial Stromal Cells and Epithelial Gland Organoids.

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.

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.

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.

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.