In every menstrual cycle, progesterone acting on estrogen-primed endometrium elicits an inflammatory decidual reaction, rendering it poised for embryo implantation and transformation into the decidua of pregnancy. Here, we show that the sequential functions of the decidual reaction-implantation and decidualization-pivot on the time-sensitive loss of progesterone-resistant DIO2+ stromal cells that form a specialized implantation niche and reciprocal expansion of progesterone-dependent PLA2G2A+ predecidual cells. Simultaneously, uterine natural killer (uNK) cell proliferation results in the accumulation of immunotolerant subsets. Examination of endometrial biopsies from 924 women revealed that the recurrence risk of miscarriage closely aligns with the incidence of a weakened or stalled decidual reaction, more so than poor uNK cell expansion. Analysis of paired biopsies obtained in different cycles and modeling in assembloids intimated that prior miscarriages disrupt intercycle endometrial homeostasis and calibration of the decidual reaction. Our findings show that erosion of the decidual reaction following a miscarriage drives the recurrence risk irrespective of maternal age.

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.

Decidualization denotes the process of inflammatory reprogramming of endometrial stromal cells (EnSC) into specialized decidual cells (DC). During this process, EnSC are subjected to endoplasmic reticulum (ER) stress as well as acute cellular senescence. Both processes contribute to the proinflammatory mid-luteal implantation window and their dysregulation has been implicated in reproductive failure. Here, we evaluated the link between ER stress, decidual differentiation and senescence. In-silico analysis identified HSPA5 gene, codifying the ER chaperone BiP, as a potentially critical regulator of cell fate divergence of decidualizing EnSC into anti-inflammatory DC and proinflammatory senescent decidual cells (snDC). Knockdown of HSPA5 in primary EnSC resulted both in decreased expression of DC marker genes and attenuated induction of senescence associated βgalactosidase activity, a marker of snDC. Stalling of the decidual reaction upon HSPA5 knockdown was apparent at 8 days of differentiation and was preceded by the upregulation of ER stress associated proteins IRE1α and PERK. Further, HSPA5 knockdown impaired colony-forming unit activity of primary EnSC, indicative of loss of cellular plasticity. Together, our results point to a key role for HSPA5/BiP in decidual transformation of EnSCs and highlight the importance of constraining ER stress levels during this process.

Abstract Study question Is de-differentiation of committed endometrial stromal and epithelial cells in response to decidual senescence involved in regulating stemness of cycling human endometrium? Summary answer Transient decidual senescence promotes endometrial tissue rejuvenation by reprogramming stromal and epithelial cells into progenitor stem-like cells whereas chronic senescence causes stem cell depletion. What is known already In wound healing, acute but not prolonged senescence, a cellular state characterised by permanent cell cycle arrest and production of a complex secretome rich in inflammatory mediators, ECM proteins and proteinases, growth factors and angiogenic modulators, has been shown to promote de-differentiation of committed cells into stem-like progenitors, thereby enhancing tissue regeneration upon immune clearance of senescent cells. Menstrual repair depends on endometrial progenitor cells but whether they represent stromal and epithelial cells that have de-differentiated in response to acute premenstrual decidual senescence is not known. Study design, size, duration Endometrial 'instant' assembloids, consisting of gland organoids and primary stromal cells in collagen hydrogels, were established from freshly isolated cells from mid-luteal endometrial biopsies. The assembloids were then subjected over 36 days to 4 cycles of decidualization, a process associated with acute inflammatory senescence, followed by hormonal withdrawal. To induce chronic senescence, assembloids were decidualized continuously for 14 days. Stemness of assembloids was assessed in undifferentiated and decidualized cultures at the end of each ‘cycle’. Participants/materials, setting, methods 'Instant' assembloids, which closely recapitulate native endometrium, were established in collagen hydrogels from 10 midluteal biopsies and subjected to cyclical or prolonged decidualization. Decidualization was monitored by RT-qPCR analysis, using epithelial and stromal cells isolated at regular timepoints. The level of stemness of stromal and epithelial cells was measured by colony-forming unit (CFU) and organoid formation efficacy (OFE) assays, respectively. Main results and the role of chance Repeated cycles of hormonal stimulation and withdrawal resulted in cyclical decidualization of instant assembloids, as characterised by the induction of decidual stromal (PRL, SCARA5 and DIO2) and epithelial (PAEP and SPP1) marker genes when compared to parallel undifferentiated assembloids. Cyclicity enabled cells to maintain a healthy state and preserved the structural integrity of the assembloids. Further, cyclical decidualization of assembloids enhanced CFU activity and OFE activity of stromal and epithelial cells, respectively, indicative of active de-differentiation of committed cells. By contrast, chronic senescence, as induced by a prolonged decidualization, resulted in stem cell depletion in both glandular and stromal compartments and progressive loss of structural integrity of assembloids. We observed a reduced expression of the decidual marker gene PRL and an enhanced expression of IGFBP1 representing the increased stress state of cells. Taken together, induction of acute decidual senescence resulted in a robust de-differentiation response and increased abundance of stromal and epithelial progenitor cells, whereas prolonged senescence caused stem cell exhaustion in both cellular compartments. Limitations, reasons for caution Although the cellular responses observed in our ‘instant’ assembloid model were robust, caution is warranted when extrapolating from in vitro observations. Further, although the ‘instant’ assembloid model enables co-culturing of endometrial endothelial and immune cells, current hydrogels greatly limit their migratory capacity. The mechanisms of cellular de-differentiation are incompletely understood. Wider implications of the findings Our findings indicate that the level of premenstrual decidual senescence in the superficial layer regulates stemness in the basal layer, thus ensuring inter-cycle endometrial homeostasis. Conversely, prolonged decidual senescence associated with clinical miscarriages may plausibly increase the risk of further pregnancy loss by depleting stemness of the regenerative basal layer. Trial registration number N/A

Abstract Study question Is de-differentiation of committed endometrial stromal and epithelial cells in response to decidual senescence involved in regulating stemness of cycling human endometrium? Summary answer Transient decidual senescence promotes endometrial tissue rejuvenation by reprogramming stromal and epithelial cells into progenitor stem-like cells whereas chronic senescence causes stem cell depletion. What is known already In wound healing, acute but not prolonged senescence, a cellular state characterised by permanent cell cycle arrest and production of a complex secretome rich in inflammatory mediators, ECM proteins and proteinases, growth factors and angiogenic modulators, has been shown to promote de-differentiation of committed cells into stem-like progenitors, thereby enhancing tissue regeneration upon immune clearance of senescent cells. Menstrual repair depends on endometrial progenitor cells but whether they represent stromal and epithelial cells that have de-differentiated in response to acute premenstrual decidual senescence is not known. Study design, size, duration Endometrial 'instant' assembloids, consisting of gland organoids and primary stromal cells in collagen hydrogels, were established from freshly isolated cells from mid-luteal endometrial biopsies. The assembloids were then subjected over 36 days to 4 cycles of decidualization, a process associated with acute inflammatory senescence, followed by hormonal withdrawal. To induce chronic senescence, assembloids were decidualized continuously for 14 days. Stemness of assembloids was assessed in undifferentiated and decidualized cultures at the end of each ‘cycle’. Participants/materials, setting, methods 'Instant' assembloids, which closely recapitulate native endometrium, were established in collagen hydrogels from 10 midluteal biopsies and subjected to cyclical or prolonged decidualization. Decidualization was monitored by RT-qPCR analysis, using epithelial and stromal cells isolated at regular timepoints. The level of stemness of stromal and epithelial cells was measured by colony-forming unit (CFU) and organoid formation efficacy (OFE) assays, respectively. Main results and the role of chance Repeated cycles of hormonal stimulation and withdrawal resulted in cyclical decidualization of instant assembloids, as characterised by the induction of decidual stromal (PRL, SCARA5 and DIO2) and epithelial (PAEP and SPP1) marker genes when compared to parallel undifferentiated assembloids. Cyclicity enabled cells to maintain a healthy state and preserved the structural integrity of the assembloids. Further, cyclical decidualization of assembloids enhanced CFU activity and OFE activity of stromal and epithelial cells, respectively, indicative of active de-differentiation of committed cells. By contrast, chronic senescence, as induced by a prolonged decidualization, resulted in stem cell depletion in both glandular and stromal compartments and progressive loss of structural integrity of assembloids. We observed a reduced expression of the decidual marker gene PRL and an enhanced expression of IGFBP1 representing the increased stress state of cells. Taken together, induction of acute decidual senescence resulted in a robust de-differentiation response and increased abundance of stromal and epithelial progenitor cells, whereas prolonged senescence caused stem cell exhaustion in both cellular compartments. Limitations, reasons for caution Although the cellular responses observed in our ‘instant’ assembloid model were robust, caution is warranted when extrapolating from in vitro observations. Further, although the ‘instant’ assembloid model enables co-culturing of endometrial endothelial and immune cells, current hydrogels greatly limit their migratory capacity. The mechanisms of cellular de-differentiation are incompletely understood. Wider implications of the findings Our findings indicate that the level of premenstrual decidual senescence in the superficial layer regulates stemness in the basal layer, thus ensuring inter-cycle endometrial homeostasis. Conversely, prolonged decidual senescence associated with clinical miscarriages may plausibly increase the risk of further pregnancy loss by depleting stemness of the regenerative basal layer. Trial registration number N/A

Abstract Study question Can embryo implantation be recapitulated in vitro using human blastocysts and a new 3D model of the human endometrium representing the architecture of the tissue? Summary answer We present an innovative implantation model consisting of blastocysts attaching on endometrial assembloids containing stromal cells, gland-like epithelial organoids, and overlaid with an epithelial monolayer. What is known already A successful pregnancy denotes the implantation of an embryo to the receptive, luminal lining of the uterus, the endometrium. The tissue transforms into the semi-permanent decidua which provides an optimal microenvironment and nourishes a healthy embryo. The transformation of the tissue is driven by a differentiation process, known as decidualisation, which accounts for the change of tissue-resident stromal cells to specialised decidual cells or senescent decidual cells. Simultaneously, endometrial glands of epithelial origin become secretory and provide histotrophic nutrition to the implanting embryo. Perturbations in this process of decidualisation may also result in an implantation failure or a pregnancy loss. Study design, size, duration Endometrial 'instant' assembloids, consisting of gland organoids and primary stromal cells in collagen hydrogels, were established from freshly isolated cells from endometrial biopsies. Additional cell types were incorporated including immune and endothelial cells creating a layer of epithelial cells recapitulating the luminal epithelium. Hormonal stimulated assembloids were further co-cultured with human blastocysts at 37 °C, 5% O2 and 6% CO2. Attachment of the blastocysts to the luminal epithelium was assessed upon overnight co-culture. Participants/materials, setting, methods Endometrial biopsies (n = 3) were obtained from consenting hormonally stimulated, healthy oocyte donors. After informed consent, human embryos (n = 14) were donated to research after 5 years of cryopreservation. Decidualisation was monitored by RT-qPCR analysis using epithelial and stromal cells from undifferentiated or decidualised assembloids. Attachment of the embryos (n = 11) to the model was assessed by disturbance of the liquid and this was further confirmed by immunofluorescence antibody labelling. Main results and the role of chance 'Instant' assembloids mimic the endometrium morphologically and functionally since hormonal stimulation results in the induction of decidual stromal (PRL, SCARA5 and DIO2) and epithelial (PAEP and SPP1) marker genes. The presence of immune and endothelial cells was confirmed with the induction of IL2RB and VWF respectively. Immunofluorescent antibody labelling confirmed the similarity in morphology of the model to the human endometrium. Aiming to mimic implantation, assembloids underwent a 3-day long differentiation in a chemically defined medium, 8-bromo-cAMP, estradiol, and a progestin, that induced in vitro decidualisation. Following the 3-day treatment, instant assembloids were overlaid with single epithelial cells, isolated from instant assembloids which had undergone prolonged decidualisation treatment to imitate the physiology of the endometrium. Following a 24-hour long incubation period, human hatched day 6 blastocysts were positioned on the top epithelial layer of the instant assembloid. Immunofluorescent antibody labelling using makers for the epiblast (NANOG), primitive endoderm (GATA4) and epithelial cells (E-cadherin) were used to visualise the co-culture. Our findings signify the newly established model as the most advanced method to study embryo implantation in vitro. The uniqueness of the model is owned by the preservation of endometrial cell types and the potential to model disease through patient specificity. Limitations, reasons for caution The proposed system is an innovative method to recapitulate apposition and adhesion during implantation. Our results are preliminary and further experiments will be performed to increase our repeats and therefore reliability. Moreover, due to the complex nature of the co-culture system, imaging remains a challenge that requires further optimisation. Wider implications of the findings The embryo-assembloid co-culture system may provide a useful tool to aid our understanding of the mechanisms of implantation. Future work will include the labelling of the embryos with a nuclear dye to enable tracking using time-lapse microscopy. We further aim to expand our protocol to study the process of invasion. Trial registration number N/A

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 chro-matin with sequencing on endometrial samples obtained across the menstrual cycle to investigate the regu-lation of temporal gene networks that control embryo implantation. We identify uniquely accessible chro-matin 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 ele-ments (TEs) into the regulatory chromatin landscape of decidualizing cells and expression of TE-derived tran-scripts 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.

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.