Engineering human endometrial model systems in reproductive health and disease

Nowadays, the pathophysiology of several human endometrium disorders remains inadequately understood, posing challenges for the identification of effective treatments. Severe damage to the uterine endometrium, often results in scar formation and endometrial dysfunction, eventually leads to infertility or pregnancy-related complications. Studies over the last decade have demonstrated the feasibility of developing engineered endometrial models of intractable reproductive diseases, such as endometriosis and cancer, which may improve our understanding of the underlying pathophysiology and facilitate clinical and pharmaceutical interventions. In this section, we introduce representative examples of bioengineered in vitro models applied to reproductive diseases. Endometriosis, an estrogen-dependent disease, is associated with a myriad of complications such as pelvic pain and infertility, making it a prominent cause of female morbidity 4 , 106 . The development of in vitro endometrial models has provided valuable insights into the pathogenesis of this disease and serves as a foundation for novel treatments targeting disease-associated pain and infertility, given that most animals do not develop endometriosis spontaneously. Immortal cells (e.g., 12-Z cell line) and primary endometriotic cells, such as epithelial, stromal peritoneal or immune cells, have been used to simulate the endometriotic milieu and study the pathogenetic pathways 4 . However, carcinoma-derived cell lines are abnormal genome-wise and show aberrant hormone receptor expression and responses. Moreover, primary endometriotic cells are difficult to culture and have a limited lifespan. Simple endometrial explant culture systems have been employed to replicate crucial stages in the development of endometriosis. However, the utilization of explants is hindered by substantial heterogeneity, indeterminate cell composition, and low throughput. 3D cultures derived from patients with endometriosis (e.g., endometrial explants and spheroids) can help to reconstruct the pathological microenvironment and determine the interaction and growth state of cells under pathological conditions 107 , 108 , which provide better phenotype and gene expression of the lesion than 2D cultures. Endometrial organoids exhibit long-term expansion, genome and transcriptome stability, and replicate disease diversity. Recently, endometriosis organoids were established from patient endometriotic lesion biopsies to reproduce endometriotic lesions, showing disease-associated traits including invasive activity and expression of MMPs and specific WNT pathway components 109 . Of note, it will also be important to enhance organoid model by combining the epithelial cells with the stromal cells of the lesion, as well as with other cell types such as endothelial cells and immune cells, which may play major roles in endometriosis pathology. Organs-on-chips can also strongly help in modeling endometriosis to simulate the pathological environment of endometriosis, to explore the pathogenesis of endometriosis. Endometriosis-on-a-chip decipher epithelial-stromal crosstalk, angiogenesis and immune/inflammatory impact. By studying the interactions between endometrial cells and other cell types, such as immune cells or endometrial-derived stem cells, researchers have identified potential targets for therapeutic intervention. Chen et al. presented an in vitro model for the dynamic real-time monitoring of the microenvironmental interactions in endometriosis 110 . They developed a microfluidic system where endometrial stromal cells and human peritoneal mesothelial cells co-cultured in separate compartments. This platform enabled the study of pathophysiology of endometriosis and can be suitable for further investigation of other biological processes by modeling the interactions between different cell types. The unlimited expansion potential of organoids, while retaining properties, and their cryopreserving and biobanking ability also render this model ideal candidate for establishing drug screening platforms, even in a patient-personalized manner. Endometriosis organoids also have considerable potential for drug screening 109 . Endometriosis organoid drug screening provides a new tool, and patient-specific drug responses were demonstrated by testing four standard chemotherapy compounds and everolimus. Taken together, organoids can capture the diversity of endometrial diseases and will provide powerful research models and tools for drug screening and discovery. Asherman syndrome (AS), also known as uterine synechiae, is a leading cause of uterine infertility worldwide. It is characterized by obliteration of the uterine cavity with adhesions and the scar formation on the uterine surface lacking of endometrial epithelial cells, which prevents endometrial regeneration 111 – 113 . Most of the current research related to AS focused on the regeneration of the endometrium by restoring its regular thickness and rate of proliferation 114 . The transplantation of stem cells including MSCs, endometrial stromal cells and endometrial epithelial progenitor cells (EEPCs) is one of efficient strategies to regenerate the damaged endometrial tissue and have been used for the treatment of AS 113 , 115 – 118 . Recently, 3D spheroids or endometrial organoids have also been implanted to promote regeneration of endometrium in rat models of AS 113 , 119 , which significantly improved the rat fertility. To stabilize the stem cell niche during in vitro culture, human embryonic stem cells (ESCs) were induced into EEPCs that were accompanied by endometrial stromal cells to generate endometrial organoids in a 3D culture model 113 . Implantation of endometrial organoids into the damaged endometrium facilitates endometrial regeneration and angiogenesis, which may prove useful for the treatment of Asherman 113 . With the development of in vitro microphysiological systems, it is possible to establish Asherman models that can reproduce scarring, fibrosis and even adhesions between endometrial surfaces and study the pathology of Asherman syndrome 114 . In addition, the incorporation of perivascular cells, vascular smooth muscle cells and pericytes 120 , will allow researchers to probe the properties of these cells in the pathogenesis of disease. In the past decades, mouse models have been mostly used to simulate the pathological environment of endometrial cancer 121 , but mouse tumors have certain species differences compared to human tumors 122 , 123 . Compared with traditional 2D culture and animal models, 3D models such as organ chips or organoids can better reproduce the pathological characteristics of human endometrial cancer and have greater advantages in cancer prevention and treatment 124 . It has been shown that cells grown in 3D culture are more resistant to the common chemotherapeutic drugs adriamycin and cisplatin than cells grown in 2D culture, suggesting that the results of drug testing may be quite different in 3D models 124 . For instance, Maru, Y et al. established patient-derived organoids that retained key features of the original tumor, including histological features, mutational profiles, intratumor heterogeneity, and the feasibility of organoids for drug testing (e.g., paclitaxel and cisplatin) 125 . Recently, mouse endometrial cancer organoids were used to test 276 small molecules targeting epigenetic factors in vitro 126 . Human endometrial-cancer organoids were also used to test commonly used chemotherapeutic agents, which successfully predicted multidrug resistance in patients 127 . Endometrial cancer organoids reliably reconstruct tumor histology, genetics, and drug response and hold great potential for biobanking and precision medicine. Future treatment strategies for endometrial cancer may include creating endometrial cancer organoids from resistant tumors and screening for chemotherapy drugs to predict drug sensitivity 114 . However, organoid models have not fully captured tumor-matrix interactions that influence drug sensitivity and resistance, which are important for immune-oncology research 128 . The future advancement of organoid platforms holds the potential to overcome these limitations by the combination of tumor and stromal cells 129 . Inflammatory diseases account for a large proportion of gynecological diseases, especially in women of childbearing age 130 , 131 . The main cause of endometritis is considered to be intrauterine infection, and the occurrence and progression of endometritis may be related to the local ecological imbalance of female reproductive tract 132 – 134 . As such, constructing the interaction model between microbes and endometritis is crucial for understanding the pathogenesis of chronic endometritis. Inspired by the human gut-on-a-chip model 15 , the adoption of organs-on-chips for the reproductive tract could offer valuable insights into the interactions between pathogens and endometrial cells. Organoids have been used to study host responses to pathogens, such as bacteria and viruses. Recently, murine endometrial organoids have been developed to model Chlamydia trachomatis infection, revealing the formed inclusions within the cytosol of epithelial cells and a full developmental cycle of the bacteria 135 . This study of Chlamydia -host interactions using organoid models may contribute to elucidating the intracellular lifestyle of C. trachomatis. The maternal-fetal interface, consisting of the maternal decidua and fetal-derived placenta, plays a critical role in supporting fetal growth, maintaining maternal-fetal immune tolerance, and providing immune defenses to protect the fetus from infections 136 . Recently, matched trophoblast (TOs) and decidual organoids (DOs) derived from human placenta have been developed to examine the contribution of these cells to antiviral defense at the maternal-fetal interface. Moreover, the co-cultures of TOs and DOs demonstrated that trophoblast-derived factors protect decidual cells from human cytomegalovirus (HCMV) infection 137 . This model highlighted key differences in innate immune signaling between the fetal-derived trophoblast and the maternal decidual, providing a valuable platform for studying vertically transmitted infections 137 . Currently, there is no complete system capable of modeling all cell-to-cell interactions during human endometrial infection, particularly those involving blood vessels and immune cells. T cells, NK cells, macrophages and other immune cells may become dysfunctional during endometrial infection and promote immune escape. With the continuous progress of technology, more complete disease models incorporating immune cells are expected to be developed. Future endometrial models may include multiple immune cells from healthy or pathologic endometrium to reveal the complex interactions between endometrial, immune and vascular cells 114 . Endometrial full-thickness morphology is widely accepted as a key prognostic indicator of endometrial receptivity and is associated with pregnancy and live birth rates 138 . Damaged endometrium caused by AS or thin endometrium adversely affects reproductive success in fertility treatments. While conventional treatments for thin endometrium remain common, regenerative medicine including the use of platelet-rich plasma and stem cells (e.g., menstruation-derived stem cells and MSCs) therapies has emerged as a promising therapy strategy for restoring uterine function and promoting endometrial repair 118 , 139 . Several studies have demonstrated that stem cell injections can enhance endometrial thickness and vascularity. Additionally, autologous transplantation of exogenously prepared endometrial tissue or epithelial cells shows potential as a therapeutic option for thin endometrium. However, the limited expansion potential of endometrial epithelial cells remains a challenge. Recent studies have performed long-term culture of endometrial epithelial cells in vitro as a potential therapeutic option for refractory thin endometrium. Yokomizo et al. demonstrated that co-culturing endometrial epithelium with feeder cells (endometrial stroma) can support their long-term propagation 140 . They also established an endometrial 3D model using frozen-thaw endometrial epithelial cells and stromal cells, which could provide a new platform for investigating the mechanisms underlying implantation. Additionally, biomaterials served as scaffolds have been developed to reconstruct the injured uterus by incorporating endometrial cells or growth factors. For instance, Zhu et al. designed a GelMA microneedle patch loaded with antioxidant nanozyme and stem cells, which enhanced endometrial repair and improved pregnancy rates in an AS rat model 141 . Furthermore, methacrylated hyaluronic acid (HAMA) microspheres carried VEGF factor were found to promote angiogenesis at the injured sites, yielding excellent therapeutic effects in the mouse model 142 . Collagen is prominent in the endometrium and important for the cell-ECM interactions. A collagen scaffold loaded with MSCs was developed to facilitate endometrial regeneration 143 . Upon transplantation, the collagen/MSCs construct preserved the normal luminal structure, promoted the proliferation of intrinsic endometrial cells, and supported endometrium functional recovery. Moreover, Wei et al. developed a recombinant human type III collagen (RHC)-HA hydrogel as an intervention for endometrial damage 144 . The RHC-HA hydrogel promoted endometrial regeneration and restored fertility, which offers a therapeutic strategy for endometrial injuries. Bioprinting technology have also been employed to support endometrial cell survival and facilitate the 3D construction of tissues aimed at restoring the full-thickness morphology and fertility of injured uterine endometrium. An HA-based bioprinted endometrial construct was developed to restore the morphology and fertility of the injured uterine endometrium 104 . Similarly, a bioprinted hydrogel scaffold loaded with G-CSF sustained-release microspheres were demonstrated to support endometrial regeneration 103 . In the future, hydrogel modification can be considered to construct a more complex system by co-culture of endometrial cells with other cell types, such as immune or vascular cells. It could better mimic human endometrial physiology, thus facilitating the restoration of tissue morphology and function.

In vivo, the uterus provides a suitable intrauterine environment for embryo implantation and fetal development. Human endometrium is the inner lining the uterus, which is divided into functionalis and basalis layers 20 . The endometrium is highly dynamic, and its shedding, regeneration and differentiation are finely controlled by periodic changes in ovarian hormones (Fig. 2 ). The morphology, ultrastructure and biochemical characteristics of endometrium vary greatly in response to hormones during the menstrual cycle. The endometrium consists of multiply cell types, including epithelial cells, endometrial stroma, stem/progenitor cells, immune cells and basal/spiral arteries, which undergo different changes during the menstrual cycle 21 . The epithelium consists of monomeric lamellar lumen epithelium and branched columnar glandular epithelium. The luminal epithelium contains ciliated cells and secreting cells. The glandular epithelium is mainly composed of columnar secretory cells with a small number of ciliated cells at the gland opening. The stroma decidualization leads to the recruitment and angiogenesis of populations of immune cells that are essential for a successful pregnancy 22 , 23 . The basal gland expresses markers found in endometrial stem/progenitor cells, but the pinpointing of endometrial stem cells at present still not conclusively defined 24 . Fig. 2 Architecture and functions of the human endometrium during the menstrual cycle. The healthy endometrium comprises epithelial, stromal, vascular, and immune cells, organized into two main layers: the stratum basalis and stratum functionalis. The functional layer grows out from the basal layer, sloughs off and is regenerated every 28 days. This dynamic process is controlled by female sex hormones, estrogen and progesterone, produced by the ovary during the proliferative and secretory phases of the menstrual cycle. Adapted from ‘Uterine Cycle’, by BioRender. com (2022). The healthy endometrium comprises epithelial, stromal, vascular, and immune cells, organized into two main layers: the stratum basalis and stratum functionalis. The functional layer grows out from the basal layer, sloughs off and is regenerated every 28 days. This dynamic process is controlled by female sex hormones, estrogen and progesterone, produced by the ovary during the proliferative and secretory phases of the menstrual cycle. Adapted from ‘Uterine Cycle’, by BioRender. com (2022). During the menstrual phase, the functional layer of the endometrium is shed due to the withdrawal of ovarian steroid hormones. It then enters the proliferative stage, where estrogen in the ovarian follicles affects the regeneration and growth of the functional layer. Following ovulation and progesterone secretion by the luteum, decidualization occurs. The glands become increasingly tortuous and secretory (secretory phase), and stromal cells surrounding spiral arterioles differentiate into large rounded decidual cells with enhanced secretory function. Epithelial cells also undergo morphological changes, increasing ciliogenesis and secretory activity, thereby relaying multiple molecular signals to prime the endometrium toward the receptive state in preparation for a potential pregnancy. If pregnancy does not occur, the functional layer is again shed due to the drop of progesterone levels during menstruation and the cycle repeats 8 , 25 . The dynamic and regenerative features of endometrium is essential for human reproduction. In early pregnancy, the blastocyst contacts with the uterine wall between the 5th and the 6th day post fertilization, attaches to the epithelium, and invades the receptive decidua to implant. After implantation, cytotrophoblasts fuse to form the syncytiotrophoblast, which promotes early placentation and develops into the intervillous space and villous tree. The cells localized at the tips of villi differentiate into extravillous trophoblasts (EVTs) that migrate across the endometrium, initiating the process of EVT invasion 26 . A batch of EVTs is responsible to remodel the maternal spiral arteries (SA) to ensure an adequate maternal blood supply for normal fetal growth and development 27 , 28 . Shallow invasion of the EVTs is associated with the pregnancy complications, such as pre-eclampsia, fetal growth restriction, and stillbirth 29 . EVTs further invade the decidua and reach as far as the inner third of the myometrium. The uterine microenvironment must be suitable for an effective implantation during early pregnancy, which is coordinately regulated by a combination of EVTs, decidual cells, stromal components, vascular features, and soluble factors. Immune cells play a key role in endometrial function, pregnancy, and endometrial tissue shedding and regeneration 23 . Many immune cells, such as dendritic and uterine natural killer (uNK) cells, largely increase during decidualization and early pregnancy. uNK cells, the main type of immune cells in the decidualized endometrium, can target and clear the senescent decidual cells to maintain proper endometrial receptivity 30 . Moreover, they contribute to decidual angiogenesis regulation and SA remodeling and control EVT invasion 31 . In addition to uNK cells, regulatory T (Treg) cells are recruited to the decidualized endometrium where they play a key role in immune tolerance of the fetus 32 . Insufficiency in Treg cells in the decidualized endometrium is correlated with recurrent pregnancy loss, preterm births and preeclampsia 33 . Antigen presenting cells (APC) and NK cells are involved in vascular remodeling of decidual spiral arteries, and the absence of APC leads to pregnancy failure 34 . The ECM is also an important environmental factor. The decidual ECM is invaded by the trophoblast lineages. This matrix is composed of a variety of proteins including collagen, fibronectin, laminin, vitronectin 35 . Matrigel contained laminin, collagen IV, and entactin, is often used to mimic the basal membrane for generating endometrial organoids. ECM modulate EVT functions and EVTs degrade and induce ECM remodeling to enable migration at the same time 36 , 37 . Decidual cells tightly regulate the secretion of MMPs and their inhibitors (TIMP-1 and TIMP-2) to control EVT migration and prevent an exacerbate invasion 38 . In addition, mechanical forces are thought to regulate cell fate and behaviors during organogenesis as progenitor cells differentiate into diverse specialized functions in fetal organs 39 . Abbas Y et al., examined the stiffness of human endometrium and placenta samples, and probed the influence of mechanics on blastocyst implantation at the maternal-fetal interface 40 . They found the stiffness of decidua basalis is 10 3  Pa while that in nonpregnant endometrium is about 10 2  Pa, indicating the mechanics change during implantation. Thus, correct placentation requires coordinated uterine microarchitecture remodeling operated by multiple players. Both the compatibility of ECM protein with multicellular tissue models and the mechanical cue as regulator of cellular behaviors should be considered for the endometrial models.

Advanced approaches such as organoids, organs-on-chips and biomaterials are emerging as promising complements or alternatives to conventional in vitro cell cultures and animal models. These innovations are driven by their ability to replicate key aspects of human organ physiology and disease, providing more accurate and relevant models for studying human reproduction. Recent advances in bioengineered in vitro endometrial models have shown great potential for investigating various aspects of human reproduction, including pregnancy and related disorders. These models offer a more physiologically relevant platform by mimicking the tissue microenvironment and facilitating endocrine crosstalk between distinct cell types and organs. They have significantly improved our understanding of reproductive physiology and the mechanisms underlying reproductive diseases, potentially facilitating the development of novel and personalized therapeutics in reproductive biology and medicine. However, achieving high-fidelity endometrial models that closely resemble native tissues remains a major challenge. The human endometrium is particularly difficult to replicate ex vivo due to the intricate cyclic changes it undergoes, each involving distinct morphologies and functions. Current in vitro models still struggle to fully replicate the intact architecture of the endometrium, including the lumen, functionalis and basalis layers, and myometrium. Furthermore, many of these models lack essential cell types, such as endothelial, perivascular, and immune cells, which are crucial for normal endometrial function and play key roles in disease pathogenesis. This limitation poses a significant challenge for further advancing our understanding of reproductive biology. Additionally, the translation of current endometrial models into clinical practice is still confronted with a multitude of challenges. For instance: (1) the heterogeneity of endometrial organoids can compromise the reproducibility of drug screening results and reduce their clinical predictability; (2) Hormone absorption by PDMS or other polymeric materials used in organ-on-a-chip platforms may hinder drug response assessment and endocrine pathway studies; (3) Bioprinted endometrial constructs often exhibited limited cell viability and functionality primarily due to insufficient vascularization or inadequate nutrient exchange. With the advancement of new biotechnologies, these challenges may be addressed by the integration of multiple cell types and tailored matrix components into endometrial models. For instance, the incorporation of endothelial cells and uterine immune cells within hydrogels or the use of pre-vascularized scaffolds, may help establish a vascular and immune microenvironment critical for endometrial function. Furthermore, employing chemically defined hydrogels as substitutes for Matrigel may improve the reliability of endometrial organoids and organ-on-a-chip systems by enabling more precise control over the extracellular microenvironment. The development of alternative hydrogel materials to replace PDMS, along with surface engineering strategies that prevent steroid hormone absorption, would further facilitate the broader applications of endometrial models in basic research and translational medicine. In addition, integrating a readout system within the organs-on-chips devices will be crucial for real-time analysis of biological data in these biomimetic endometrial models. The inclusion of biosensors that enable continuous monitoring of cellular behaviors, important signaling molecules (e.g., estradiol and progesterone), and microenvironmental parameters will be essential. Organs-on-chips also allow for the recreation of mechanical forces, such as peristaltic movements in native tissues, including the myometrium. This is particularly important for studying conditions like adenomyosis, where mechanical stress plays a key role in disease progression. Furthermore, the use of multiple organs-on-chips models will allow for the study of inter-organ communications within the reproductive system, which could aid in mimicking endometrial diseases and identifying the primary drivers of such conditions. The convergence of organoids and organs-on-chips (namely organoids-on-chips) offers a promising avenue for building 3D models with higher physiological relevance. The integration of defined hydrogels with organoids and organs-on-chips advanced the 3D models with more precise spatiotemporal control of niche factors 145 . Recent studies have demonstrated the feasibility of engineering perfused intestinal organoids by combining a 3D matrix, mechanical fluid flow and stem cell self-organization 146 , 147 . Similar strategies could be applied to construct biomimetic endometrial tissue with gland-like architecture and multiply cell types. Looking ahead, the development of advanced and clinically relevant bioengineered endometrial models demands an interdisciplinary approach. Endometrial models derived from diseased tissue (e.g., endometriosis, cancer, Asherman’s) or patient-derived stem cells could exhibit human relevant pathological features, which may provide powerful tools for testing therapeutic agents. For example, endometriosis organoids have been utilized to elucidate stromal-epithelial crosstalk and disease phenotypes in response to inflammatory stimuli 100 . Adenomyosis-derived organoids were also established to recapitulate native endometrial features and disease-specific traits 148 , providing a promising preclinical model to study infertility. By integrating with genetic manipulation, these models would deepen our understanding of endometrial gland function during pregnancy and in female reproductive disorders. The integration of living imaging and multi-omics approaches (e.g., genomics, epigenomics, transcriptomics, proteomics and metabolomics) will facilitate the access to study human organogenesis, disease mechanisms, and personalized medicine (Fig. 6 ). Importantly, the correlation between data generated from these in vitro advanced models and clinical trial outcomes are expected to provide new diagnostic strategies and therapeutic targets for endometrial disorders and infertility, thereby advancing the translational medicine applications of bioengineered endometrial models. Fig. 6 Applications of advanced models of the human endometrium. Emerging technologies, such as organs-on-chips, organoid, hydrogels and bioprinting, provide physiologically relevant platforms to reproduce many characteristics of the native endometrium and disease states. Further innovations in integrating genome editing, biosensors and multi-omics approaches will facilitate the development of advanced endometrial models, enabling more accurate applications in studying endometrial biology, disease modeling, drug testing, tissue repair and therapy and reproductive health. Image created with Biorender.com with permission. Emerging technologies, such as organs-on-chips, organoid, hydrogels and bioprinting, provide physiologically relevant platforms to reproduce many characteristics of the native endometrium and disease states. Further innovations in integrating genome editing, biosensors and multi-omics approaches will facilitate the development of advanced endometrial models, enabling more accurate applications in studying endometrial biology, disease modeling, drug testing, tissue repair and therapy and reproductive health. Image created with Biorender.com with permission.

The endometrium is a female specific reproductive tissue, while many details of the female growth, development and endometrial disease remain unclear 41 – 43 . It is critical to create in vitro endometrial models by considering the complexity of the endometrial lining, the abundance of cell types, and many morphological and pathological changes of cell types that occur during the menstrual cycle. Advanced engineered strategies including organ chips, organoids, biomaterials and bioprinting have been integrated to mimic the in vivo-like endometrial tissue microenvironment and explore its physiopathology in normal and disease (Fig. 3 ), providing valuable tools for reproductive medicine. Table 1 outlines various bioengineering strategies for modeling the endometrium in vitro. Additionally, Table 2 summarized the key limitations and advantages of currently available in vitro endometrial models, including 2D cell cultures (e.g., cell lines, primary cells, and tissue explants), organoids, organs-on-chips, and hydrogel-based bioprinted constructs under physiological and pathological conditions. Fig. 3 Bioengineering strategies for constructing the human endometrium. A The derivation of endometrial organoids typically relies on 3D culture systems such as Matrigel or air-liquid interface cultures. Endometrial epithelial organoids consist of epithelial cells that form hollow, gland-like structures within a 3D matrix, often using Matrigel. These epithelial cells preserve their polarity, with their apical side oriented towards the lumen of the organoid. Endometrial assembloids are composed of multiply cell types, including both endometrial epithelial cells and stromal cells. When cultured at the air-liquid interface, endometrial assembloids can form a luminal epithelium, closely mimicking the functional architecture of native endometrial tissue. B Organ-on-chip technology has been used to model the endometrial niche and functions by integrating with multicellular co-cultures, vascularized structures, the maternal-fetal interface and organ-organ interactions. It enables the dynamic simulation of endometrial responses in a highly controlled manner, providing insights into the complex interactions that occur during the menstrual cycle, implantation, and tissue remodeling. C Biomaterials with tunable properties are promising tools for regulating the ECM microenvironment, which in turn guides the growth and self-organization of endometrial cells and organoids. These biomaterials can include natural hydrogels, decellularized matrices, synthetic polymers, and hybrid materials. Hydrogel-based modules, such as scaffolds, defined blocks, and patterned substrates, can be used to generate endometrial tissue with a well-defined architecture, more closely resembling native endometrial tissue. D Bioprinting is an advanced manufacturing technique that involves cell layers, spheroid or hydrogel scaffolds by layer-by-layer deposition of living cells, biomaterials and growth factors to create 3D functional tissues, holding great promise for tissue repair and regeneration of the endometrium. Image created with Biorender.com with permission. Table 1 Summary of recent progress in engineered endometrium models Engineered models Types of cells or materials Key features of device Main findings and applications References Organ-on-a-chip •Primary human cells • Co-culture of endometrial stroma and endothelial cells • A dual chamber microfluidic chip with a porous membrane •Simulating the temporal hormone changes during menstrual cycle •Shear stress promoted cytoskeleton alignment and tight junction formation in the endothelial layer •Enhanced stromal decidualization under perfusion via endothelial- derived PGE2 and prostacyclin 68 , 69 •Primary human cells isolated from clinical specimens including uterine ECs, EVTs, decidualized stromal cells (DSCs) and uterine natural killer (uNK) cells •Implantation-on-a-chip consisting of three parallel lanes • Co-culture of uterine ECs and EVTs to study EVT invasion and spiral artery remodeling • Reconstructing the 3D structural organization of the maternal-fetal interface • Modeling the invasion of specialized fetal EVT s into the maternal uterus •Demonstrating in vivo-like directional EVT migration towards maternal vessel •Revealing the role of DSCs as a regulator of EVT migration and effects of uNK on EVT invasion 81 •Primary human cells •Cell lines •3D microengineered vascularized endometrium on chip •Consisting of three distinct layers including epithelial cells, stromal fibroblasts and endothelial cells in a 3D ECM •Recapitulating the endometrial microenvironment including endometrial vasculo-angiogenesis and hormonal responses •Evaluating the effect of the emergency contraception drug levonorgestrel •Modeling embryo implantation 70 •Primary human cells •A dual reproductive organ on a chip •Complex multicellular architectures including endometrial and ovarian follicular cells in biodegradable natural polymers •Recapitulating bidirectional endocrine crosstalk between endometrium and ovary •Predicting the reproductive toxicity of various hazardous materials using a SERPINB2 luciferase reporter system 85 •Animal and human reproductive tissues •A microfluidic platform with single, dual and multiple unit •Integrating human female reproductive tract and peripheral tissue dynamics •Supporting murine ovarian follicles to produce the human 28-day menstrual cycle hormone profile •Simulating the female reproductive tract and the endocrine loops between organ modules for the ovary, fallopian tube, uterus, cervix and liver under circulating flow 86 Organoids •Matrigel-based 3D cultures •Primary human cells •Human adult stem-cell-derived organoid cultures of normal and decidualized endometrium •Expanded long-term, be genetically stable •Differentiated following treatment with reproductive hormones 46 •Matrigel-based 3D cultures Primary endometrium •Multiply patient-derived endometrial organoids from distinct diseases, including endometriosis, endometrial cancer, endometrial hyperplasia and lynch syndrome •Recapitulated endometrial disease diversity and displayed patient-specific drug responses. 109 •Human PSC-derived endometrial stromal fibroblasts (ESFs) •Primary human endometrial epithelial cells •Coculture of PSC-ESFs with endometrial epithelial organoids •The coculture model cyclically respond to hormone treatment and withdrawal •PSC-ESFs respond to epithelial cell signaling in cocultures 53 •Matrigel-based 3D cultures mouse and human primary endometrial biopsies •Organoid model from both mouse and human endometrium •Human endometrial epithelial organoid •Showed the endometrial epithelium physiology and long-term expansion capacity, and replicated the menstrual cycle under hormonal treatment •High WNT yielded cystic organoids displaying a more differentiated phenotype than low WNT 47 , 48 •Mice endometrial biopsies •Modeling Chlamydia trachomatis infection in murine endometrial organoid system •Revealing the formed inclusions within the cytosol of epithelial cells and a full developmental cycle of the bacteria 135 •Human endometrial biopsies •Established human endometrial assembloids consisting of gland- like organoids and primary stromal cells •Coculture decidualized assembloids with human blastocysts •Resembled midluteal endometrium and contained differentiated and senescent subpopulations in both glands and stroma •Modeling the impact of decidual senescence on embryo implantation 54 Matrigel-based 3D cultures human placentas and decidua tissue •Developed matched trophoblast and decidua organoids to study vertically transmitted infections •Showed differential susceptibility and innate immune signaling to human cytomegalovirus (HCMV) in TO and EO •Trophoblast-derived factors protected decidual cells from HCMV infection in cocultures of TO and DO 137 Hydrogels •Nature hydrogels •GelMA or collagen as scaffolds for engineering endometrial tissues •Collagen/MSCs construct for endometrial regeneration •Recombinant human type III collagen (RHC)-HA hydrogel for endometrial regeneration • Modeling the layers of endometrial epithelial cells • Modeling human endometrium by coculture of epithelial and stromal cells • Preserving the normal luminal structure and supporting endometrium functional recovery • Enhancing endometrial regeneration and restoring fertility 90 , 91 , 143 , 144 •Endometrial Decellularized ECM (EndoECM) •EndoECM from the endometrium-specific layer or the entire uterus •EndoECM as a soluble additive •Inducing endometrial regeneration through the regulation of IGF1and IGFBP3 •Improving human endometrial organoid culture systems •Supporting the growth of normal and cancerous endometrial organoids 96 – 98 •Synthetic hydrogels • PEG-based hydrogels cross linked with integrin-binding or MMP peptides •Endometrial organoids showed cell-specific and apicobasal polarity markers upon differentiation •Enabling co-culture of endometrial stroma and epithelial organoids 99 , 100 Bioprinting •Human iPSC-derived MSCs (hiMSC) •Gelatin-alginate hydrogel •3D-printed hydrogel scaffold loaded with hiMSCs for endometrial repair •Promoting endometrial morphology recovery and the regeneration of endometrial and endothelial cells in an endometrial injury rat model 102 •Rat primary cells •Alginate-hyaluronic acid (HA) hydrogel •Bioprinted bilayer endometrial construct consisting of upper endometrial epithelial layer and lower stromal layer •Restoring the morphology and structure of the endometrial wall •Improving the reproductive outcome in the surgical area after implantation in a partial uterine excision rat model 104 •Alginate hydrogel •Human endometrial epithelial and stromal cell lines •Bioprinted bilayer endometrial model consisting of epithelial and stromal cells •Exhibiting hormone responsiveness upon simulation with estradiol and progesterone •Simulating early embryo implantation events 105 •G-CSF-PLGA •Gelatin-alginate •3D-printed G-CSF sustained-release microsphere system •Suppressing endometrium tissue fibrosis, and improving endometrial epithelial, stromal and vascular cells regeneration in an IUA rat model 103 Table 2 The comparisons of different in vitro endometrium models in normal and disease In vitro Models Advantages Limitations 2D cell cultures (e.g., cell lines, primary cells, tissue explants) • Convenient for the isolation and cultivation • Studying mechanism of endometrial diseases • Large-scale drug screening • Lacking cell-cell/matrix interactions and complex 3D tissue organization • Different from native endometrial tissue in terms of gene profiles, epigenetics and functions • Limited sources and rapidly losing phenotype and hormone responsiveness function of primary cells/tissues Organoids • Recapitulating key features and function of the endometrium • Amenable to extended expansion and cryopreserved • Long-term preservation of cell phenotype and genotype in vitro • Available for studying embryo implantation, endometrial receptivity and reproductive diseases • Patient-derived organoids allowing for biobanking and precision medicine • High-throughput drug screening • Uncontrolled biochemical and biophysical environmental cues • Lack of relevant mechanical signals, such as blood perfusion and stretch force • Often use of ill-defined animal-derived matrices (e.g., Matrigel) • Lack of maternal-fetal interface • Lack of vascular network or immune cells Organs-on-chips • Mimicking in vivo-like tissue microenvironment • Recapitulating the human-relevant endometrial physiology and pathology • Precise control of mechanical cues • Mimicking maternal-fetal interface • Studying multicellular or multi-organ interactions • Enable apical surface accessibility of embryo or pathogens • The PDMS material of chip device may influence drug testing • Low-throughput for drug screening Hydrogel-based bioprinting models • Assisted in repairing the damaged endometrium and restoring its ability to support embryos • Facilitating the delivery of growth factors that induced endometrial regeneration and fertility enhancement • Tuning mechanical properties by control over their compositions and stiffness • Development of suitable tissue-specific bio-inks for improving tissue fidelity • Limited resolution of bioprinting process • Exhibiting shear stress-induced cell damage due to high cell densities A The derivation of endometrial organoids typically relies on 3D culture systems such as Matrigel or air-liquid interface cultures. Endometrial epithelial organoids consist of epithelial cells that form hollow, gland-like structures within a 3D matrix, often using Matrigel. These epithelial cells preserve their polarity, with their apical side oriented towards the lumen of the organoid. Endometrial assembloids are composed of multiply cell types, including both endometrial epithelial cells and stromal cells. When cultured at the air-liquid interface, endometrial assembloids can form a luminal epithelium, closely mimicking the functional architecture of native endometrial tissue. B Organ-on-chip technology has been used to model the endometrial niche and functions by integrating with multicellular co-cultures, vascularized structures, the maternal-fetal interface and organ-organ interactions. It enables the dynamic simulation of endometrial responses in a highly controlled manner, providing insights into the complex interactions that occur during the menstrual cycle, implantation, and tissue remodeling. C Biomaterials with tunable properties are promising tools for regulating the ECM microenvironment, which in turn guides the growth and self-organization of endometrial cells and organoids. These biomaterials can include natural hydrogels, decellularized matrices, synthetic polymers, and hybrid materials. Hydrogel-based modules, such as scaffolds, defined blocks, and patterned substrates, can be used to generate endometrial tissue with a well-defined architecture, more closely resembling native endometrial tissue. D Bioprinting is an advanced manufacturing technique that involves cell layers, spheroid or hydrogel scaffolds by layer-by-layer deposition of living cells, biomaterials and growth factors to create 3D functional tissues, holding great promise for tissue repair and regeneration of the endometrium. Image created with Biorender.com with permission. Summary of recent progress in engineered endometrium models • Co-culture of endometrial stroma and endothelial cells • A dual chamber microfluidic chip with a porous membrane •Simulating the temporal hormone changes during menstrual cycle •Shear stress promoted cytoskeleton alignment and tight junction formation in the endothelial layer •Enhanced stromal decidualization under perfusion via endothelial- derived PGE2 and prostacyclin •Implantation-on-a-chip consisting of three parallel lanes • Co-culture of uterine ECs and EVTs to study EVT invasion and spiral artery remodeling • Reconstructing the 3D structural organization of the maternal-fetal interface • Modeling the invasion of specialized fetal EVT s into the maternal uterus •Demonstrating in vivo-like directional EVT migration towards maternal vessel •Revealing the role of DSCs as a regulator of EVT migration and effects of uNK on EVT invasion •Primary human cells •Cell lines •3D microengineered vascularized endometrium on chip •Consisting of three distinct layers including epithelial cells, stromal fibroblasts and endothelial cells in a 3D ECM •Recapitulating the endometrial microenvironment including endometrial vasculo-angiogenesis and hormonal responses •Evaluating the effect of the emergency contraception drug levonorgestrel •Modeling embryo implantation •A dual reproductive organ on a chip •Complex multicellular architectures including endometrial and ovarian follicular cells in biodegradable natural polymers •Recapitulating bidirectional endocrine crosstalk between endometrium and ovary •Predicting the reproductive toxicity of various hazardous materials using a SERPINB2 luciferase reporter system •Supporting murine ovarian follicles to produce the human 28-day menstrual cycle hormone profile •Simulating the female reproductive tract and the endocrine loops between organ modules for the ovary, fallopian tube, uterus, cervix and liver under circulating flow •Matrigel-based 3D cultures •Primary human cells •Expanded long-term, be genetically stable •Differentiated following treatment with reproductive hormones •Matrigel-based 3D cultures Primary endometrium •Human PSC-derived endometrial stromal fibroblasts (ESFs) •Primary human endometrial epithelial cells •The coculture model cyclically respond to hormone treatment and withdrawal •PSC-ESFs respond to epithelial cell signaling in cocultures •Organoid model from both mouse and human endometrium •Human endometrial epithelial organoid •Showed the endometrial epithelium physiology and long-term expansion capacity, and replicated the menstrual cycle under hormonal treatment •High WNT yielded cystic organoids displaying a more differentiated phenotype than low WNT •Established human endometrial assembloids consisting of gland- like organoids and primary stromal cells •Coculture decidualized assembloids with human blastocysts •Resembled midluteal endometrium and contained differentiated and senescent subpopulations in both glands and stroma •Modeling the impact of decidual senescence on embryo implantation •Showed differential susceptibility and innate immune signaling to human cytomegalovirus (HCMV) in TO and EO •Trophoblast-derived factors protected decidual cells from HCMV infection in cocultures of TO and DO •GelMA or collagen as scaffolds for engineering endometrial tissues •Collagen/MSCs construct for endometrial regeneration •Recombinant human type III collagen (RHC)-HA hydrogel for endometrial regeneration • Modeling the layers of endometrial epithelial cells • Modeling human endometrium by coculture of epithelial and stromal cells • Preserving the normal luminal structure and supporting endometrium functional recovery • Enhancing endometrial regeneration and restoring fertility •Endometrial Decellularized ECM (EndoECM) •EndoECM from the endometrium-specific layer or the entire uterus •EndoECM as a soluble additive •Inducing endometrial regeneration through the regulation of IGF1and IGFBP3 •Improving human endometrial organoid culture systems •Supporting the growth of normal and cancerous endometrial organoids •Endometrial organoids showed cell-specific and apicobasal polarity markers upon differentiation •Enabling co-culture of endometrial stroma and epithelial organoids •Human iPSC-derived MSCs (hiMSC) •Gelatin-alginate hydrogel •Rat primary cells •Alginate-hyaluronic acid (HA) hydrogel •Restoring the morphology and structure of the endometrial wall •Improving the reproductive outcome in the surgical area after implantation in a partial uterine excision rat model •Alginate hydrogel •Human endometrial epithelial and stromal cell lines •Exhibiting hormone responsiveness upon simulation with estradiol and progesterone •Simulating early embryo implantation events •G-CSF-PLGA •Gelatin-alginate The comparisons of different in vitro endometrium models in normal and disease • Convenient for the isolation and cultivation • Studying mechanism of endometrial diseases • Large-scale drug screening • Lacking cell-cell/matrix interactions and complex 3D tissue organization • Different from native endometrial tissue in terms of gene profiles, epigenetics and functions • Limited sources and rapidly losing phenotype and hormone responsiveness function of primary cells/tissues • Recapitulating key features and function of the endometrium • Amenable to extended expansion and cryopreserved • Long-term preservation of cell phenotype and genotype in vitro • Available for studying embryo implantation, endometrial receptivity and reproductive diseases • Patient-derived organoids allowing for biobanking and precision medicine • High-throughput drug screening • Uncontrolled biochemical and biophysical environmental cues • Lack of relevant mechanical signals, such as blood perfusion and stretch force • Often use of ill-defined animal-derived matrices (e.g., Matrigel) • Lack of maternal-fetal interface • Lack of vascular network or immune cells • Mimicking in vivo-like tissue microenvironment • Recapitulating the human-relevant endometrial physiology and pathology • Precise control of mechanical cues • Mimicking maternal-fetal interface • Studying multicellular or multi-organ interactions • Enable apical surface accessibility of embryo or pathogens • The PDMS material of chip device may influence drug testing • Low-throughput for drug screening • Assisted in repairing the damaged endometrium and restoring its ability to support embryos • Facilitating the delivery of growth factors that induced endometrial regeneration and fertility enhancement • Tuning mechanical properties by control over their compositions and stiffness • Development of suitable tissue-specific bio-inks for improving tissue fidelity • Limited resolution of bioprinting process • Exhibiting shear stress-induced cell damage due to high cell densities Cell-cell communications are essential for normal uterine functions and their dysregulation. 3D culture systems incorporating different cell types (e.g., epithelial cell and stromal cell) of the uterus have been developed for deciphering the cellular crosstalk in uterine normal and disease states 44 , 45 . Recently, 3D endometrial organoids were successfully established from human and mouse endometrium, recapitulating the key structure and function of endometrium 46 – 48 . Generally, endometrial epithelial cells isolated from endometrial biopsy or decidua were embedded in Matrigel and cultured in a chemically defined medium with various factors (e.g., R-spondin1, EGF, FGF10, Noggin, and A83-01). The generated endometrial epithelial organoids (EEOs) can be extended expansion for a long time and cryopreserved, which overcomes the limitation of primary endometrial epithelial cells in 2D cultures. Moreover, the establishment and preservation of EEOs from multiple donors allows for biobanking and diseases studies, providing a powerful platform to study endometrial development and explore the mechanisms of infertility caused by implantation failure 49 . At present, the generated endometrial organoids still lack key cellular components, such as mesenchymal cells, immune cells and blood vessel cells, that are important for endometrial functions 50 . Co-culture of various cell types including endometrial epithelial and stromal cells is required for providing an appropriate microenvironment and accurately understanding the crosstalk between distinct cellular components of endometrium 51 . Wiwatpanit et al. established a scaffold-free multicellular endometrial organoids by co-culturing primary endometrial epithelial and stromal cells in a micromolded agarose gel 52 . These organoids showed organization with stromal cells in the center surrounded by polarized epithelial cells lining the outer surface, recapitulating the complex stromal-epithelial interactions in healthy and disease states across a simulated menstrual cycle. Recent study further demonstrated that human PSC-derived endometrial stromal fibroblasts (ESFs) were capable of self-assembling with primary EEOs to form endometrial model cyclically respond to hormones 53 . In addition, a study constructed an assembloid consisting of gland-like organoids and primary stromal cells, which responded to hormones and kinase inhibitors (Fig. 4A ) 54 . The assembloid-embryo interactions showed that senescent decidual cells enabled embryo attachment by providing a dynamic implantation environment. Fig. 4 Engineered organoid models of the human endometrium that recapitulate the endometrial niche and pathologies. A Human endometrial assembloids consisting of gland-like organoids and primary stromal cells were established to study the impact of decidual senescence on embryo implantation. Reproduced with permission 54 . B Human endometrial assembloids with a luminal epithelium were generated by combining endometrial epithelial and stromal cells using air-liquid interface cultures, recapitulating human endometrium anatomy, menstrual cycle changes and dynamic ciliogenesis. Reproduced with permission 56 . C Apical-out endometrial organoids comprised of an apical epithelium surface, stromal cells and endothelial network were developed to recapitulate human embryo implantation and feto-maternal interactions. Reproduced with permission 58 . D A human endometrial organoid co-culture model in a fully synthetic extracellular matrix enables the study of epithelial-stromal crosstalk. Reproduced with permission 100 . A Human endometrial assembloids consisting of gland-like organoids and primary stromal cells were established to study the impact of decidual senescence on embryo implantation. Reproduced with permission 54 . B Human endometrial assembloids with a luminal epithelium were generated by combining endometrial epithelial and stromal cells using air-liquid interface cultures, recapitulating human endometrium anatomy, menstrual cycle changes and dynamic ciliogenesis. Reproduced with permission 56 . C Apical-out endometrial organoids comprised of an apical epithelium surface, stromal cells and endothelial network were developed to recapitulate human embryo implantation and feto-maternal interactions. Reproduced with permission 58 . D A human endometrial organoid co-culture model in a fully synthetic extracellular matrix enables the study of epithelial-stromal crosstalk. Reproduced with permission 100 . Generally, endometrial organoids are self-organized 3D gland-like structures, primarily modeling the epithelial gland component of the endometrium. Compared to endometrial organoids, assembloids are more complex systems by combining gland organoids with other cell types (like stroma) to model interactions between different cell types. Additionally, an air-liquid interface culture can be used to help these models develop more mature by submerging the basal cell surface in liquid and the apical portion exposed to air, which may better replicate polarized epithelial layer and in vivo-like endometrial functional morphology. Recent studies have demonstrated that human endometrial epithelial cells cultured at the air-liquid interface displayed discontinuous multilayer phenotypes 55 . Bioinspired the physiological microenvironment of the endometrium, Tian et al. constructed multicellular endometrial assembloids with a luminal epithelium in a 3D matrix and at the air-liquid interface cultures (Fig. 4B ) 56 . The endometrial assembloids generated by combining endometrial epithelial cells and stromal cells contained intact glandular and luminal epithelium, recapitulating the anatomy of human endometrium and key features of menstrual cycle changes and dynamic ciliogenesis. This model may provide a potential platform to facilitate the studies of embryo implantation, endometrial receptivity and reproductive diseases. Although endometrial organoids can recapitulate cell composition and hormone responses of uterine epithelium, they have apical-in polarity that is different from in vivo gland structure. It hinders access to the apical surface for studying epithelial interactions with the embryo or pathogens. Recently, apical-out EEOs were established based on suspension cultures to preserve a distinct apical-basolateral orientation, which remained physiological hormone responsiveness and showed susceptibility to bacterial infections 57 . Furthermore, a novel apical-out endometrial organoids were eatablished by coculture of an apical epithelium surface, stromal cells and endothelial network (Fig. 4C ) 58 . When coculturing with human blastoids, endometrial organoids recapitulated the initial stages of embryo implantation, including apposition, adhesion, and invasion. The organoid model provides a valuable paltform for studying feto-maternal interactions. Despite the significant progress of endometrial organoid study, they often represent partial properties of native endometrium with some major limitations, such as the lack of vascular network or immune cells. Thus, advanced bioengineering strategies, such as organs-on-chips technology and biomaterials, are expected to construct vascularized or immunized endometrial organoids to promote the study of endometrium intercellular interactions in normal and diseased states and translational applications. Human endometrium decidualization occurs from the mid-secretory phase after ovulation, regulated by periodic changes in ovarian steroid hormone levels and other factors. Endometrial decidualization is critical process for successful establishment and maintenance of pregnancy to term 59 , 60 . During decidualization, stromal cells and vascular endothelial cells undergo morphological and biochemical changes to support embryo implantation and vascular development 61 – 63 . Impaired decidualization is a vital reason for early pregnancy miscarriages and pregnancy-related complications, such as preeclampsia (PE) 64 – 66 . Therefore, in vitro recreate of human endometrial decidualization process is very important for understanding related diseases and treatment 67 . Organs-on-chips have the advantages of reconstructing the microstructure, mechanical properties and biochemical functionalities of organs as well as mimicking dynamic tissue microenvironment in vitro. Researchers have demonstrated the potential of organs-on-chips for modeling human endometrium structure, functions, multicellular interactions and decidualization process, which are essential for the successful establishment of pregnancy. Specifically, a compartmentalized organ chip device was designed to enable co-culture of primary human endometrial fibroblasts and human umbilical vein endothelial cells (HUVECs) on the opposite sides of porous membrane to mimic the interface between the stroma and vasculature in the maternal decidua (Fig. 5A ) 68 . The design allows for the controlled delivery of ovarian hormones to the stromal cells through the vascular compartment, enabling simulation of the temporal changes in the levels of estrogen and progesterone during menstrual cycle. Moreover, this model revealed that hemodynamic forces promoted decidualization through a paracrine mechanism mediated by prostaglandin E2 and prostacyclin secreted from endothelial cells 69 . Another study established a vascularized endometrium on chip that consists of three different layers including epithelial cells, stromal cells, and endothelial cells (Fig. 5B ) 70 . This model recapitulates the interactions between different cells in the human menstrual cycle and simulated the endometrial microenvironment, including endometrial vasculo-angiogenesis and hormonal responses. In addition, the model has been used to model embryo implantation and evaluate the effects of the emergency contraceptive drug levonorgestrel, which provides a new tool for drug screening and discovery. Fig. 5 Representative in vitro engineered models of the human endometrium using organs-on-chips technology. A A microfluidic model of the human endometrium was established by co-culturing perivascular endometrial stromal and endothelial cells in a compartmentalized device, which simulated the temporal hormone changes during the menstrual cycle. Reproduced with permission 68 . B A 3D vascularized endometrium-on-a-chip was developed by co-culturing endometrial stromal fibroblasts, endometrial epithelial cells and endothelial cells, recapitulating in vivo endometrial vasculo-angiogenesis and hormonal responses. Reproduced with permission 70 . C A dual reproductive organ-on-a-chip was proposed by integrating with interconnected organs, which reflected the bidirectional endocrine crosstalk between the endometrium and the ovary. Reproduced with permission 85 . D A microfluidic culture model of the human reproductive tract was established to produce the human 28-day menstrual cycle hormone profile and simulate the endocrine loops between various reproductive tract organ modules. Reproduced with permission 86 . A A microfluidic model of the human endometrium was established by co-culturing perivascular endometrial stromal and endothelial cells in a compartmentalized device, which simulated the temporal hormone changes during the menstrual cycle. Reproduced with permission 68 . B A 3D vascularized endometrium-on-a-chip was developed by co-culturing endometrial stromal fibroblasts, endometrial epithelial cells and endothelial cells, recapitulating in vivo endometrial vasculo-angiogenesis and hormonal responses. Reproduced with permission 70 . C A dual reproductive organ-on-a-chip was proposed by integrating with interconnected organs, which reflected the bidirectional endocrine crosstalk between the endometrium and the ovary. Reproduced with permission 85 . D A microfluidic culture model of the human reproductive tract was established to produce the human 28-day menstrual cycle hormone profile and simulate the endocrine loops between various reproductive tract organ modules. Reproduced with permission 86 . Six or seven days after fertilization, the embryo starts implantation on the uterine wall. The blastocyst adheres tightly and penetrates the uterine epithelium, while trophoblasts invade the endometrium and uterine vasculature 5 , 71 , 72 . The key implantation process is regulated by intercellular paracrine and autocrine processes, mobilizing complex and synchronized molecular and cellular events between the uterus and the implanted embryo 73 – 76 . In order to better understand the embryo implantation process, the combination of blastocysts or blastocyst substitutes with in vitro 3D endometrial models has been extensively studied 77 . A 3D in vitro model was established to evaluate trophoblast migration and invasion by co-culturing endometrial cells and trophoblasts. This model simulated the process of attachment, migration and early invasion of trophoblasts 78 . In the decidua, the migration and invasion of EVTs play important roles for successful placentation 77 , 79 . There is growing evidence that various types of maternal uterine cells including endothelial cells, immune cells, and deciduated stromal cells, coordinate to regulate EVT invasion 80 . Huh et al. proposed a microengineered system to reconstruct the 3D structure of the mother-to-fetus interface, simulating the invasion of specialized fetal ectrophoblastic cells into the mother’s uterus 81 . This system recapitulates the complex sequence of multicellular events in early pregnancy. The study demonstrated the directed migration of trophoblast cells to microengineered maternal blood vessels and their interactions necessary for vascular remodeling, and that deindividualized stromal cells are important regulators of extracellular trophoblast migration. In addition, the study revealed the influence of maternal immune cells on extracellular trophoblast invasion before implantation. This will greatly help in understanding these diseases and in developing and testing potential treatments. Recently, Hiraoka et al. developed an ex vivo uterine system by co-culturing mouse embryos and uterine tissue at the air-liquid interface, which recapitulated bona fide implantation, embryogenesis and trophoblast invasion 82 . This model also demonstrated robust induction of COX-2, suggesting a possible signaling mediated by uterine COX-2 and embryonic AKT1 that accelerates implantation. These implantation-on-chip models hold potential for identifying biomarkers or therapeutic targets for implantation failure or recurrent pregnancy loss. Additionally, they can also be used to screen the effects of drugs or environmental factors on implantation and assess their potential impact on fertility and pregnancy outcomes. Multi-organ-on-a-chip is a promising technology that can provide a species-specific platform for studying the crosstalk between various tissues by reconstructing organ-level function 83 . The female reproductive system consists of a variety of interrelated organs, including placenta, uterus and ovary, etc. The endometrium is subject to ovarian steroid hormones, and there is bidirectional regulation of cytokines between the ovary and endometrium 84 . This reproductive organ crosstalk is essential for maintaining the various physiological characteristics and functions of the tissues. Park et al. developed an endometrium-ovarian organ on a chip that reflects the bidirectional endocrine crosstalk and complex multicellular structure by combining various cell types of endometrium and ovaries with natural polymers (Fig. 5C ) 85 . This system significantly improve the viability of loaded cells in each chamber, which provides a new platform to study reproductive biology. In addition, Xiao et al. created a multi-organ chip system that functionally re-creates the entire 28-days menstrual cycle in vitro, where human and mouse cells from several reproductive organs were grown in a network of interconnected microunits (Fig. 5D ) 86 . This innovative system allowed the modeling of multi-tissues interaction and the reproduction of characteristic reproductive functions, providing a new avenue for the study of diseases such as cervical cancer or infertility. Moreover, this system could be adapted for rapid screening several drugs or new compounds to evaluate potential reproductive toxicity. Further advancements in this platform may involve the integration of other organs, such as the immune system and the placenta, to produce a more complex model of the in vivo system. The endometrial ECM mainly contains collagen IV, fibrillin, laminin and fibronectin 87 . The ECM provides scaffold and support for constructing 3D endometrial tissue models. Matrigel, a commonly used ECM, supports the self-organization and formation of 3D endometrial organoids with structure and molecular signatures similar to in vivo tissue. Many studies have demonstrated that human endometrial epithelial cells can maintain long-term hormonal responses using 3D culture patterns 88 , 89 . Other materials, such as gelatin methacryloyl (GelMA) 90 or collagen 91 have also been used as scaffolds for engineering endometrial tissues. For instance, Abbas et al. developed a porous collagen I scaffold to construct human endometrium model by coculture of epithelial and stromal cells 91 . Stromal cells proliferated and organoid fragments formed a luminal-like epithelial layer on the scaffold, which were functionally responsive to hormones. In future, the construction of endometrial model at air-liquid interface by organ on chips or biomaterials may be beneficial for exploring the cellular characteristics of endometrial and potential molecular mechanisms. Endometrial decellularized ECM (EndoECM) recapitulates the complexity of natural ECMs with its intrinsic characteristics. The different forms of EndoECM as soluble additive, acellular ECM scaffolds or injectable carriers have been developed to support in vitro culture of endometrial cells and enhance uterine repair by facilitating the delivery of growth factors 92 – 95 . For instance, Ahn et al. developed EndoECM hydrogels from the endometrium-specific layer or the entire uterus 96 , which induced endometrial regeneration and fertility enhancement through the regulation of IGF1and IGFBP3. Recently, EndoECM hydrogels as a soluble additive were used to improve human endometrial organoid culture systems by allowing epithelial cells to interact with soluble molecules in ECM, which recapitulated the in vivo phenotype of endometrial glands and increased proliferation rates of endometrial organoids 97 . Furthermore, Jamaluddin et al. developed endometrium-derived ECM hydrogel scaffolds to support the growth of normal and cancerous endometrial organoids 98 , which more closely recapitulates the native tissue compared to culture in Matrigel. Endometrium-specific ECM hydrogels provide a physiologically relevant microenvironment that enables tissue repair and faithful organoid growth, thereby enhancing their applicability in reproductive medicine research. Synthetic polyethylene glycol (PEG)-based hydrogels have also been used to engineer endometrial organoids. Hernandez-Gordillo et al. designed fully synthetic ECMs consist of PEG-macromers cross linked with integrin-binding or MMP peptides for in culturing primary endometrial organoids in vitro 99 . These organoids showed cell-specific and apicobasal polarity markers upon differentiation. Similarly, Gnecco et al. developed an endometrial organoid co-culture model in a fully synthetic PEG-based hydrogel 100 , which enabled stable co-culture of endometrial stroma and epithelial organoids (Fig. 4D ). The model recapitulates the epithelial-stromal crosstalk during the human menstrual cycle. The endometrial ECM composition changes throughout menstrual cycle and pregnancy, which have a direct impact on mechanical properties such as tissue stiffness 40 , 101 . PEG-based hydrogels have tunable physicochemical properties by control over their compositions and stiffness. The organoids in defined hydrogels may recapitulate tissue ECM remodeling and facilitate to study ECM correlated endometrial diseases. In future, tailored matrix scaffolds for the culture of endometrial organoids may contribute to advance organoids toward defined and standardized tools for basic research and translational applications. 3D bioprinting enables the reconstruction of complex tissue architectures by precisely arranging cells, biomaterials, and growth factors within customized scaffolds. It has been applied to engineer reproductive tissues, including the regeneration of damaged endometrium often associated with intrauterine adhesions (IUA). Cell therapy has emerged as an alternative for endometrium regeneration since the limited effects of therapeutic drugs. Ji et al. utilized a 3D bioprinted hydrogel scaffold loaded with human iPSC-derived MSC (hiMSC) to facilitate endometrial repair and regeneration in a rat model 102 . Moreover, they developed a gelatin and alginate-based 3D-printed granulate colony-stimulating factor (G-CSF) sustained-release microsphere system 103 . The system suppressed endometrium tissue fibrosis, and improved the regeneration of endometrial epithelial, stromal and vascular cells in an IUA rat model. It provided a precise and individualized treatment method for repair of damaged endometrium. Although some cell sheet patches and organoid-based 3D model systems have replicated key architectural and functional aspects of the endometrium, they often exhibit limitations in spatial organization between cells and the matrix. It may compromise the precision and controllability of engineered tissue models. In contrast, 3D bioprinting enables precise spatial arrangement of cells within bioinks or cell-laden scaffolds, supporting the controlled reconstruction of tissue ultrastructure. Moreover, ECM components secreted by cells could contribute to tissue remodeling and help maintain histological integrity in the hydrogel environment. For example, Nie et al. leveraged 3D bioprinting to construct a bilayer endometrial tissue for endometrial repair 104 . The bio-printed endometrial construct was consisted of an upper endometrial epithelial layer and a lower stromal layer in alginate-hyaluronic acid (HA)-based hydrogel. The tissue significantly restored the endometrium morphology and improved the reproductive outcome in the surgical area in a partial uterine excision rat model. In addition, Catane et al. developed an alginate-based 3D-printed endometrial model consisting of epithelial and stromal cells arranged in a bilayer structure 105 . This model replicated the layered architecture of the native endometrium and exhibited hormone responsiveness upon simulation with estradiol and progesterone. Furthermore, human trophoblast-like spheroids adhered to and infiltrated the hormone-treated bilayer construct, simulating early embryo implantation events. This system provides a promising platform for studying endometrial receptivity and implantation mechanisms in vitro, as well as personalized therapeutics in recurrent implantation failure. In short, these examples highlight the abilities of these bioengineering approaches including organoids, organ chips, biomaterials and bioprinting for constructing in vitro endometrial models by mimicking tissue microenvironment. These models hold great potential for studying physiology and pathology of the human endometrium as well as expediting the development of new therapeutics for endometrial diseases. Various models possess unique advantages in biomedical research, yet numerous challenges persist. Although 2D cell cultures are widely used for endometrium research and convenient for drug screening, organoid and organ chip models exhibit more physiological relevance by recapitulating multicellular types, 3D structure and functions of the endometrium. Specially, organoids are superior for personalized disease modeling, and organ chips can simulate the dynamic tissue microenvironment and enable multi-organ interactions. In addition, bioprinting integrated with hydrogels offers significant advantages for endometrial repair in clinical settings.

The endometrium is a dynamic and complex tissue that plays a critical role in woman’s health. It is responsible for stabilizing the menstrual cycle, facilitating tissue regeneration and cell proliferation, and providing the site for fertilization, embryo implantation and fetal development 1 . These processes are primarily governed by the cyclical fluctuations of ovarian steroids, particularly estrogen and progesterone 2 – 4 . However, understanding the intricate processes of cell regeneration and differentiation in the human endometrial lining during the menstrual cycle, as well as the intercellular communication that supports pregnancy, remains a major challenge. Additionally, endometrium-related diseases, such as endometriosis, adenomyosis, endometrial cancer and Asherman syndrome, pose serious threats to female health. Yet, the pathogenesis of these diseases is still poorly understood, largely due to the absence of robust human endometrial models, thereby hindering the development of effective therapies. Furthermore, limited access to human reproductive tissues and the lack of reliable experimental models, especially for studying maternal-embryo interactions, continue to impede progress in this field. Current research on endometrial physiology and pathology relies heavily on animal models and two-dimensional (2D) cell cultures. However, animal models often fail to faithfully recapitulate the anatomy and physiology of the human endometrium and reproductive system due to significant species differences 5 – 7 . A key distinction lies in the menstrual cycle: unlike humans and some primates, rodents do not menstruate and are physiologically distinct. While 2D cultures of immortalized cell lines (e.g., Ishikawa cells) have yielded numerous insights into endometrial biology, they often exhibit abnormal genomes and karyotypes, and cannot replicate the complex three-dimensional (3D) in vivo microenvironment, including cell-cell interactions 8 , 9 . Moreover, isolated primary endometrial cells in 2D culture rapidly lose their phenotypic features and hormone responsiveness. These limitations have driven the development of new bioengineered in vitro models to advance the study of reproductive biology and medical research. Significant advances in bioengineered technologies, such as organs-on-chips, stem cell organoids, biomaterials and bioprinting, have shown great promise in accelerating research on female endometrial biology and related diseases (Fig. 1 ). Organs-on-chips are evolving from microfabrication technologies and bioengineering strategies, which contains microchannels inhabited by living cells in a microfluidic cell culture device 10 – 12 . It can mimic the complexity of cellular microenvironment by precise control of biochemical and biophysical cues. Considerable progress has been made in developing organs-on-chips to recreate miniaturized, functional units of various organs, such as the reproductive tract 13 , lung 14 , intestine 15 and liver 16 . In contrast, organoids are 3D multicellular tissues via stem cell self-organization relying on developmental biology principles 17 – 19 . Organoids possess a remarkable ability to mimic endometrial pathophysiology and other complex biological processes more accurately than traditional 2D cultures. However, their utility is constrained by some limitations, including high batch-to-batch variability and the lack of vascularization and immune components. Hydrogels are water-absorbent and polymeric materials with tunable features that could provide biomimetic extracellular matrix (ECM) environment for cell/tissue cultures. Additionally, bioprinting technology that uses living cells and biomaterials (such as hydrogels) to create complex, multi-cellular tissue architecture with highly organized and precision, which is essential for replicating physiology of tissues/organs. Recently, the convergence of organs-on-chips, organoids, biomaterials or bioprinting has led to produce organotypic models with more physiological relevance, which may provide new opportunities for engineering human endometrium and advancing biomedical research. Fig. 1 Illustration of bioengineered human endometrial models for biomedical research. The human endometrium is a dynamic tissue composed of multiply cell types and complex tissue microenvironment. These models can recapitulate key features of tissue architecture and functions by simulating the endometrial microenvironment, including the maternal-fetal interface and multicellular interactions, thereby advancing their applications in studying fundamental biology, disease modeling and regenerative medicine. Image created with Biorender.com with permission. The human endometrium is a dynamic tissue composed of multiply cell types and complex tissue microenvironment. These models can recapitulate key features of tissue architecture and functions by simulating the endometrial microenvironment, including the maternal-fetal interface and multicellular interactions, thereby advancing their applications in studying fundamental biology, disease modeling and regenerative medicine. Image created with Biorender.com with permission. In this review, we provide an overview of recent progresses in bioengineered in vitro models that enable to study the human endometrium, and emphasize their biomedical applications. We also discuss the prospects and challenges of building advanced in vitro models by integrative engineering strategies, aiming to enhance our understanding of reproductive biology and biomedicine.