Strategies for modelling endometrial diseases

Studies from the past 5 years have demonstrated that organoids derived from endometrial tumours recapitulate important histological, genetic and gene expression features of the original tumours 44 , 120 , 147 – 149 . A 2017 study from one of the first groups to develop endometrial organoids found that endometrial cancer organoids recreate characteristics of the original tumour, including a disorganized epithelial structure and breaching of the basement membrane 44 . Following up their initial study, Boretto et al. 120 then showed that organoids derived from endometrial tumours could be expanded long term, displayed long-term genomic stability and recapitulated tumour histology and morphology. Endometrial cancer organoids preserved the microsatellite instability, somatic copy number alterations and mutations of the original tumour and recreated the tumour when orthotopically transplanted in vivo. In addition, organoids established from other pathologies, such as endometrial hyperplasia and Lynch syndrome, maintained the mutations that occurred in the original tissue 120 . Patient-derived endometrial cancer organoids can also be subcutaneously injected or orthotopically engrafted into the uterus of immunocompromised mice, which provides a physiological tumour microenvironment 120 ( BOX 1 ). Xenografted organoids reproduced tumour histology and biomarkers, including loss of hormone receptor expression, and metastasis from the uterus to the peritoneum occurred in high-grade cancer organoids 120 . The accuracy with which endometrial cancer organoids reproduce patient tumours suggests that these organoids provide an excellent opportunity for testing drugs to treat endometrial cancer. In the first study to examine endometrial cancer cell lines in three dimensions, cells grown in 3D culture were more resistant to the common chemotherapeutic agents doxorubicin and cisplatin than those grown in 2D culture, indicating that the results of drug testing studies could be substantially different in 3D models 150 . Multiple studies have revealed that endometrial cancer organoids successfully recapitulate patient-specific responses to chemotherapeutic drugs 120 , 147 , 151 . In fact, a 2021 study tested common chemotherapeutic drugs in endometrial cancer organoids and successfully predicted that one patient’s tumour was resistant to several drugs 151 . Demonstrating the feasibility of using endometrial cancer organoids to screen drugs on a larger scale, a 2020 study used mouse endometrial cancer organoids to test 276 small molecules that target epigenetic factors in vitro. This study identified a menin-mixed-lineage leukaemia (menin-MLL) inhibitor and validated this inhibitor in an orthotopic xenograft mouse model of endometrial cancer 152 . Endometrial cancer organoids reliably recreate tumour histology, genetics and drug response, and have enormous potential for biobanking and use in precision medicine. Future endometrial cancer therapeutic strategies could involve creating endometrial cancer organoids from drug-resistant tumours and screening chemotherapeutic agents to predict which drugs a patient is likely to respond to. Although microfluidics systems are being used in endometriosis and adenomyosis research, they have not yet been used to study endometrial cancer pathogenesis. These studies are surely to come, as microfluidic models have been used to study breast cancer 153 and ovarian cancer 154 . Future microphysiological models of endometrial cancer could provide valuable information about how endometrial tumours metastasize to specific target organs by conditioning the pre-metastatic niche 155 . For example, endometrial cancer organoids could be grown in microfluidic co-culture with liver spheroids, allowing observations about how secreted factors from the cancer organoids alter the liver spheroids to make them more receptive to colonization. The inclusion of endothelial cells in these microphysiological models could determine how endometrial tumours might alter distant vasculature via secreted factors to promote the permeability of blood vessels to cancer cells. Finally, the inclusion of immune cells in microphysiological models of endometrial cancer would incorporate the role of inflammation as well as provide insights into responses to immunotherapy.

A woman’s reproductive lifespan is marked by more than 400 menstrual cycles over the course of about 40 years ( FIG. 1 a ). Every month, growth and differentiation of the endometrium occurs in response to controlled fluctuations in levels of the ovarian steroid hormones oestrogen and progesterone, in preparation for the implantation of an embryo. If there is no embryo, the endometrium is no longer needed and is shed as ovarian hormone levels fall. There are two layers in the human endometrium. The functionalis layer is the upper two-thirds of the tissue, which is shed during menstruation and the basalis layer is the lower third of the endometrium closest to the myometrium, which remains during menses 1 – 3 , 17 . Multiple cell types make up the endometrium. The epithelial layer is composed of luminal epithelial cells, which line the endometrium, and glandular epithelial cells, which are invaginations of the epithelial cells extending from the lumen into the myometrium 18 , 19 . Stromal cells as well as the extracellular matrix (ECM) provide structural and endocrine support. Blood vessels are remodelled into spiral arteries and perivascular cells and immune cells play important roles in endometrial remodelling throughout the cycle 1 – 3 , 17 . Interestingly, the proportions of these cell populations change during the menstrual cycle 20 , 21 . In the past 2 years, 3D imaging of human endometrial tissue demonstrated that the endometrial glands form a unique network in the basalis layer that expands horizontally along the muscular layer. These structures are reminiscent of rhizomes, with glands vertically emanating from the rhizomes into the functionalis layer 22 , 23 .

Asherman syndrome is an acquired endometrial disorder characterized by intrauterine adhesions or adhesions of the endocervix. The prevalence of Asherman syndrome can vary substantially; depending on the subpopulation, it is reported in 0.8–45.5% of women with infertility 4 , 5 . The opposing uterine walls are partially or completely fused by fibrous adhesions, obliterating the uterine cavity 66 . The degree and localization of intrauterine adhesions lead to the complete or partial dysfunction of the endometrium, resulting in menstrual abnormalities, reduced fertility and recurrent pregnancy loss. Asherman syndrome is considered to be mainly caused by iatrogenic trauma to the endometrium due to postpartum or miscarriage curettage, but could also be induced by intrauterine infections 4 , 66 . Because the endometrium is not able to regenerate without scarring, as it normally does following menstruation or parturition, it is probable that the trauma reaches the basalis layer or further, resulting in a poorly vascularized and thin endometrium and a limited response to oestrogen and progesterone 67 – 71 . Much of the current research related to Asherman syndrome is focused on the regeneration of the endometrium, restoring its regular thickness and proliferation rate. The most common models for endometrial regeneration are mouse or rat animal models ( BOX 1 ) in which endometrial damage is induced mechanically via scraping 72 , 73 or excision 74 or by injecting ethanol or other cytotoxic solutions 75 , 76 . Notably, some studies of Asherman syndrome did use 3D organoids or spheroids, but only as a method to aid regeneration, and not as a tool to study the pathology itself 76 , 77 . The in vitro models that have been used to study Asherman syndrome directly were standard 2D wound healing assays to investigate the regenerative capabilities of certain compounds such as platelet-rich plasma 78 or media conditioned by mesenchymal stem cells 79 . Advances in technologies could potentially enable the development of in vitro models that can reproduce scarring, fibrosis or even adhesions between two endometrial surfaces, enabling the use of in vitro models to study the pathology of Asherman syndrome. In addition, incorporation of perivascular cells (vascular smooth muscle cells and pericytes) would enable researchers to examine the regenerative properties of these cells 41 – 43 in the context of Asherman syndrome.

Adenomyosis is characterized by the invasion of endometrial tissue into the myometrium of the uterus. The burden oflesions can range from several small lesions dispersed throughout the myometrium to larger nodules or cysts 6 , 7 . As a result, this condition can cause an enlarged uterus, heavy uterine bleeding and severe pain with one study reporting approximately 82% of patients with adenomyosis undergoing a hysterectomy and 37.6% reporting chronic use of pain medication 133 . The causes of adenomyosis are unknown although theories have been proposed, including invasion of endometrial cells through a damaged junctional zone or de novo generation of endometrium-like tissue owing to differentiation of misplaced embryonic Mullerian remnants. Similar to endometriosis, growth of the displaced tissue is dependent on oestrogen. A 2021 study used 3D visualization of the endometrial glands within adenomyotic lesions, revealing an ‘ant colony’-like organization with direct invasion of endometrial glands into the myometrium 23 . Such visualization of native tissues has provided novel insights into the structural pathology of adenomyosis.

It is apparent that the new advancements in culture models and platforms hold incredible potential to unravel unknown mechanisms that drive endometrial disease development and growth. As previously mentioned, the challenge lies in how best to implement these technologies to obtain the most meaningful data. It is tempting to use the newest tool, as it grows in popularity, and try to fit it into our research. However, specific biological questions should be answered using the correct approach that is customized to reflect the disease and the niche of interest. For now, a ‘one-size-fits-all’ approach does not exist, and biology should serve as the blueprint for technological innovation. In the past few years, many researchers have used endometrial organoids and organotypic cultures derived from healthy endometrium and have shown these systems to reflect in vivo characteristics of the tissue. Now, efforts should pivot towards generating reliable disease models using the appropriate cells that interact in a physiological manner using organoids and microfluidics, among others.

More than half a million new cases of endometrial cancer occur per year worldwide 140 . The USA has the highest incidence of endometrial cancer in the world, and disease rates continue to increase globally 141 . Endometrial cancer is typically divided into two major subtypes, with type 1 being the most prevalent and associated with risk factors such as marked obesity, diabetes mellitus and hypertension 9 - 142 , whereas type 2 endometrial cancers are more aggressive and less common, carrying a different mutation profile. The Cancer Genome Atlas study of endometrial carcinoma revealed four molecular subtypes of endometrial cancer: polymerase epsilon ultramutated; microsatellite instability hypermutated; copy number low; and copy number high 143 . Polymerase epsilon ultramutated tumours are characterized by somatic mutations in polymerase epsilon accompanied by a very high mutation rate (232 × 10 −6 mutations per Mb), whereas hypermutated tumours are characterized by mutations in the DNA mismatch repair pathway, resulting in microsatellite instability and a high mutation rate (18 × 10 −6 mutations per Mb). Copy number high and copy number low tumours are distinguished on the basis of the number of copy number alterations they contain. These genomic features permit a reclassification that could better inform post-surgical adjuvant treatment for women with aggressive tumours. Most cases of endometrial cancer are strongly associated with continuous sex steroid imbalance due to excessive oestrogen and insufficient progesterone action in endometrial tissue 9 . Endometrial cancer develops when benign cells of the endometrium grow in an uncontrolled manner, accumulating mutations and transforming into malignant cells. Oestrogen promotes genotoxicity and proliferation of the endometrium, whereas progesterone antagonizes oestrogen-driven growth by promoting differentiation 9 . Depending on the grade and stage of the cancer, hysterectomy is the first line of treatment, which can be accompanied by radiation or chemotherapy. Preclinical studies for drug testing have been limited to cell lines 144 , 145 and animal models 146 ( BOX 1 ), and many targeted drugs have had a modest impact in clinical trials. Given the rising incidence of endometrial cancer, the risk factors associated with lifestyle and behaviour, and the lack of therapeutic solutions, additional tools for mechanistic investigation as well as drug testing are needed at the level of both prevention and treatment ofthis disease.

New technologies facilitate new discoveries. There have been great advancements in modelling the endometrium as well as improving culture systems to provide dynamic flow. As these technologies are adapted to understand the pathogenesis of endometrial diseases by the research community, the challenge now lies in knowing how to apply these new technologies in a meaningful way. The overarching goal is to mimic endometrial tissue physiology in vitro. The human endometrium is one of the most challenging tissues to model ex vivo given the intricate cyclic changes of the tissue, each with its morphologies and functions. The ideal endometrial model, as depicted in FiG. 4 , would represent the architecture of the endometrium, including the lumen, functionalis and basalis layers and the myometrium. The model would contain all the important cell types of the endometrium, including epithelial, stromal, endothelial, perivascular and immune cells, and the appropriate matrix, all within an MPS that promotes dynamic flow of medium. The medium could include circulating immune cells, and biosensors could be integrated within the cultures to measure activities in real time. Additionally, multiple MPS units can be connected to study inter-organ communication. Components of this model can be changed to mimic the endometrial disease of interest or to determine the major drivers of disease. A complete in vitro model of endometrial disease must include immune cells. Immune cells such as T cells, NK cells and macrophages can be dysfunctional during the establishment of endometriosis, adenomyosis and endometrial cancer and can promote immune evasion. Future models could include various immune cells from healthy or pathological endometrium to reveal the complex interplay between endometrial cells and immune cells. Vascular remodelling and angiogenesis have a key role in normal endometrial function and promote growth of diseases when unregulated, especially in endometriosis 156 and endometrial cancer 157 . Incorporation of a vascular network would involve endometrial endothelial cells, as well as an appropriate niche for vessel formation and branching. Studies from the past year have shown that pericytes have mesenchymal stem cell properties and display a limited ability to differentiate into endometrial stromal-like cells 41 , 43 . In addition to their role in regulating angiogenesis, pericytes could be included in pathological models to examine their potential for regenerating the endometrium in diseases such as Asherman syndrome 41 . The matrix that organoids or 2D cultured cells are in contact with is an important aspect of the pathological microenvironment and could come from the specific niche of the diseased tissues, such as ECM derived from myometrium for adenomyosis studies, or ECM from peritoneal tissues for endometriosis. The ability to 3D print matrix materials and cells could enable easy creation of customized scaffolds and structures. Human endometrial ECM can be obtained through decellularization of endometrial tissue 158 or synthetic hydrogels can be used to generate matrices with similar properties to the human endometrial ECM 159 . The molecular and biophysical properties of the matrices can be modulated to recreate specific healthy or pathological niches to examine how diseased cells interact with these microenvironments. It will be important to improve our understanding of how the endometrium regenerates after menses, especially for pathologies such as Asherman syndrome, in which regeneration does not occur. 3D models have not yet reproduced the degeneration of the functionalis during menses. Future models could seek to incorporate organoids derived from both functionalis and basalis endometrium. Notably, menstrual flow-derived organoids would by definition consist of the functionalis tissue. Finally, there is an undeniable dysregulation of hormones in all of the diseases mentioned in this Review. Hormones are essential for endometrial function and must be included in cultures. Fine tuning hormonal conditions to mimic physiology is needed, as current concentrations to induce decidualization in vitro are very high 30 – 32 , probably because other cell types that contribute to the decidualization process are absent. It was interesting to note that many studies included in this Review on in vitro models of endometrial diseases did not include or describe hormonal stimulation. Inclusion of hormones will be essential as we strive towards representing the endometrial diseases in vitro. Microfluidic platforms offer advancements from the standard static cultures in terms of mechanical flow dynamics, constant replenishment of new medium and elimination of toxic metabolites. Improvements in microfluidic technology that would facilitate wider use of these systems would include fabrication materials that do not absorb sex steroid hormones, improved affordability and a user-friendly platform. Depending on the needs of the study, integration of biosensors for important signalling molecules or hormones such as oestradiol and progesterone and bioanalytical tools within the microfluidic platform would allow researchers to collect data in real time ( FIG. 4 ). Microfluidic platforms that allow extended culture time of endometrial models, for example, through multiple menstrual cycles with cyclic levels of menstrual hormones, would enable researchers to observe alterations in the endometrium that might accumulate over time in response to risk factors such as unopposed oestrogen action. Microfluidic platforms also allow the recreation of mechanical influences from peristaltic movements in the myometrium, which can continue to improve our understanding of how mechanical stresses affect neighbouring endometrial cells or endometrial tissue within the myometrium, as in adenomyosis. With regard to effective drug testing platforms, a robot-assisted high-throughput microfluidic system would be essential. MPSs could include multiple different tissue types relevant to the disease in question. As we consider the use of multiple organs in one platform, the notion of deriving all organ models from the same patient is most attractive and is becoming possible with induced pluripotent stem cell technologies. This approach would permit organs with the same genetics to communicate with each other and enables researchers to test drugs in a culture system that is personalized.

Endometriosis is a disease in which endometrial tissue grows outside the uterus in the peritoneal cavity and ovaries. Symptoms include severe pain and infertility in approximately 10% of women who are of reproductive age globally 8 . The treatment of endometriosis is estimated to cost approximately $22 billion each year in the USA alone 80 and there is currently no cure. Theories for the development of endometriosis include Sampson’s retrograde menstruation theory, as well as metaplasia of cells that abnormally transdifferentiate into endometrial cells at ectopic sites 8 , 81 . Hormone responses in endometriosis in both the ectopic lesions and the eutopic endometrium are aberrant, with a dependence on oestrogen and a suboptimal response to progesterone 82 – 85 . It remains unclear whether the presence of ectopic lesions influences the eutopic endometrium or whether there are other inherent differences in the eutopic endometrium that promote endometrial tissue survival at ectopic sites 86 . With the exception of some non-human primates 87 , 88 ( BOX 1 ), animals do not develop endometriosis spontaneously and thus in vitro models using human tissues have been used to study the pathogenesis of this disease. Primary endometriotic cells are difficult to culture and have a limited lifespan, leading to the development of other in vitro culture systems 89 . Simple endometrial explant culture systems have been used to model important steps in endometriosis development. To study the interactions between ectopic endometrial fragments and fibrin, a protein that is deposited on damaged mesothelium and which is implicated in the formation of peritoneal adhesions 90 , a 2003 study grew endometrial explants in a 3D fibrin matrix 91 . Endometrial glands and stroma invaded the matrix and formed tubular structures by 2 weeks of culture, and after 5 weeks, CD31 + capillaries had sprouted. Follow-up studies identified glycodelin and COX2 as molecules that can promote neovascularization in endometriosis 92 and showed that lovostatin, a VEGF inhibitor, inhibited cell growth and angiogenesis in a dose-dependent manner 93 . The original endometriotic explants used in the 2003 study became necrotic at 5 weeks of culture; however, by that time, the outgrowth from the explant exceeded the size of the original tissue 91 . Another study of endometrial explants found that treatment with the progestin dienogest decreased outgrowth of the explants in a dose-dependent manner, suggesting that dienogest might reduce early endometriosis invasion 94 . Thus, although explants have limited viability, they can be useful to study the outgrowth and invasion of endometriosis explants through a matrix, as the growing tissue continues to be viable. Co-culture of endometriotic cells with other cell types of interest in endometriosis has revealed much about the important interactions between cells and their microenvironment. Co-cultures have provided an important tool to observe early steps in endometriosis establishment, including the beginning of adhesion, invasion and angiogenesis, which are difficult to observe in vivo 94 , 95 . Early assays to study angiogenesis in the endometrium included co-culturing endometrial explants directly on chick embryo chorioallantoic membrane (CAM) 96 . CAM-endometrial co-cultures revealed that angiostatic agents significantly decreased angiogenesis in endometriosis-like lesions in CAM, showing that this assay could be used for testing anti-angiogenesis drugs for endometriosis 97 . A 2008 study also showed that transplanting endometrium from women taking combination oral contraceptives onto CAM resulted in fewer endometriosis-like lesions compared with transplanting endometrium from women not taking oral contraceptives 98 . These studies demonstrated the utility of the CAM-endometrial co-culture model in studying the early stages of endometriosis, including lesion establishment and angiogenesis, as well as its use in drug testing. Co-culture of endometrial explants in direct contact with peritoneal explants were carried out to demonstrate that the endometrium mostly adhered in regions where mesothelial cells were missing, suggesting that a damaged mesothelium would allow endometriosis lesions to become established 99 – 101 . Similarly, endometrial explants adhered to stripped amniotic membranes, showing that this model could be used to study interactions between endometrial cells and the ECM 100 , 102 . Co-culture of endometrium with amniotic or peritoneal membranes thus offers the ability to observe the interactions between ectopic endometrium and the mesothelium or matrix at the implantation site. Bone marrow-derived stem cells (BMSCs) have been shown to migrate into the eutopic endometrium and are speculated to contribute to endometrial regeneration 103 . BMSCs are also known to associate with endometriotic lesions; however, their impact remains unclear 104 . Co-culture of commercially purchased BMSCs with endometriotic cells using transwell inserts revealed that BMSCs promote the proliferation of endometriotic stromal cells but not healthy stromal cells, possibly owing to stem cell-derived factors 104 . Reciprocally, co-culture of primary endometrial stromal cells from mice with BMSCs isolated from mouse bone marrow in a transwell system promoted the migration of BMSCs 105 . However, much controversy remains surrounding the role of BMSCs in the endometrium. It is still unclear whether BMSCs have a key role in endometrial physiology or whether they simply acquire endometrial characteristics after migrating into the uterus 106 . As bone marrow contains a heterogeneous mixture of cells 107 , and because there are no established markers for BMSCs in the endometrium 106 , future studies could be improved by ensuring that the BMSC types used display the same markers as the BMSCs that associate with endometriotic explants, or by examining BMSCs isolated from explants themselves. It has been suggested that immune dysfunction in women with endometriosis could allow ectopic endometrial cells to escape destruction by the immune system 108 . Co-culture of endometriotic cells and immune cells has revealed important paracrine interactions between these two cell types. Studies have shown that co-culturing macrophages with endometriotic cells altered the characteristics of the macrophages towards the more tolerant M2 phenotype 109 , 110 . Additionally, macrophages co-cultured with endometriotic stromal cells either directly or within a tissue culture insert were less phagocytic than those cultured with normal stromal cells 109 , 110 . Treatment with oestradiol and the dioxin TCDD synergistically promoted M2 activation in macrophages that were cultured in direct contact with endometriotic stromal cells 111 . It is clear that the presence of endometriotic cells shifts macrophages towards a more permissive phenotype, which could make the establishment of endometriotic lesions easier. Reciprocally, immune cells can alter the characteristics of the endometrial cells, resulting in a more endometriosis-like phenotype. Studies have shown that co-culture with macrophages resulted in increased proliferation, clonogenicity and invasion of endometrial stromal cells as well as secretion of the pro-inflammatory cytokine IL-6 (REFS. 112 – 115 ). These co-culture studies demonstrate that immune cells can influence endometrial cell behaviour. Natural killer (NK) cells have been shown to affect macrophages and endometriotic cells as well. Conditioned medium from co-cultures of endometriotic stromal cells and NK cells promoted increased macrophage migration compared with conditioned medium from endometriotic stromal cells alone 116 . Co-culture of endometriotic stromal cells with NK cells added directly to the culture wells caused a decrease in CD16 expression on NK cells compared with NK cells cultured alone, possibly decreasing NK cell activity. IL-15 secreted by endometriotic stromal cells decreased the expression of NK cell functional molecules such as granzyme B and interferon-y (IFNy) 117 . Another study showed that NK cell viability, cytotoxicity and functional marker expression were decreased by co-culturing endometriotic stromal cells and macrophages in direct contact with NK cells in a transwell insert 118 . These studies support both possibilities, first, that endometriotic cells in ectopic lesions dysregulate immune cell function, promoting immune escape, and second, that immune cells in endometriosis are pro-inflammatory and promote ectopic endometrial cells to establish themselves and become pathological. Although co-cultures are valuable models to study endometriosis-immune cell interactions, the in vivo situation involves more than two cell types. Future co-culture studies could include a diverse immune cell mixture of macrophages, NK cells and T cells, along with both epithelial and stromal endometriotic cells. Various combinations of these cells from healthy and diseased tissues could reveal whether immune cells are actively promoting lesion development or are compromised and unable to clear ectopic lesions. The first in vitro spheroid endometriosis model was developed in 2014 by culturing both new and established endometriosis cell lines in low-adhesion plates 119 . Cells in 3D cultures more closely recapitulated the histological and molecular characteristics of endometriotic lesions than did those in 2D cultures. A 2019 study generated organoids from endometriotic lesions, healthy endometrium and matched eutopic endometrium of patients with endometriosis 120 . Ectopic organoids were morphologically different from eutopic and disease-free organoids, with a thicker, stratified epithelial layer, and they could produce endometriosis-like lesions when engrafted into mice. Bulk RNA sequencing of these three organoid types revealed differential gene expression in ectopic organoids compared with healthy organoids, while eutopic organoids and healthy organoids displayed similar gene expression. Ectopic organoids showed altered expression of ECM-receptor interaction genes, adhesion and invasion genes and PI3K-AKT pathway genes, among others. Other studies have confirmed the ability of the endometrial epithelial organoid model to faithfully reproduce the characteristics of ectopic endometriotic lesions, eutopic endometrium of women with endometriosis and healthy endometrium, enabling the comparison of these tissue types 121 – 123 . Organoids derived from ectopic lesions and eutopic endometrium preserve the methylation patterns found in ectopic and eutopic biopsy samples, respectively, and ectopic and eutopic organoids have methylation patterns that differ from each other 121 . Organoids from the eutopic endometrium of women with endometriosis display a different expression of glycodelin A compared with healthy organoids 123 . Additionally, an epithelial organoid model revealed that progesterone receptor B is downregulated in both ectopic and eutopic endometrium compared with healthy endometrium, but through different mechanisms 122 . Endometriotic stromal cell spheroids are also useful to study the early stages of endometriosis in the context of endometriotic lesion invasion of the ECM. In a 2021 study, endometriotic stromal spheroids, with or without endometriotic epithelial cells, were grown in either collagen I matrix or Matrigel 124 . Endometriotic stromal spheroids, but not single stromal cells or spheroids co-cultured with endometriotic epithelial cells, directionally migrated through the matrix while remodelling it. Thus, spheroid and organoid models of endometriosis are useful in reproducing and identifying differences in ectopic lesions compared with normal endometrium, as well as differences between the eutopic endometrium of women with and without endometriosis. Spheroids and organoids can be used to study gene expression, hormone response, DNA methylation and invasion of endometriosis. Microfluidic technology has been used to understand the interactions between endometriotic cells and peritoneal mesothelial cells. A 2012 study used microfluidic channels made from PDMS to create a 2D micropatterned static co-culture of endometrial stromal cells and human peritoneal mesothelial cells (HPMCs) taken from patients with endometriosis and from healthy control individuals 125 . Here, the microenvironment found in peritoneal endometriosis was recreated by seeding one channel with endometrial stromal cells and the other with HPMCs ( FIG. 3e ). Then, the channels were removed and cell interaction and migration speed were monitored dynamically. Interestingly, when healthy HPMCs were co-cultured with both healthy and endometriotic stromal cells they were able to withstand the invasion of the stromal cells. Diseased HPMCs lost cell-cell contact and died when co-cultured with both types of endometrial stromal cell, suggesting that peritoneal health could be an important factor in the establishment of endometriosis. Different microfluidic platforms were also used to create sensitive methods that characterized the matrix metalloproteinase activity and mechanical properties of endometrial cells from patients with endometriosis and were able to distinguish them from endometrial cells from healthy individuals 126 , 127 . These findings demonstrate the versatility of this technology to provide detailed insights into the pathophysiology of endometriosis. Studies have attempted to recapitulate the peristaltic movement of the smooth uterine muscle layer and its effects on the endometrium 128 – 131 ( FIG. 3f ). Developing such systems would be especially useful for modelling endometriosis, in which patients experience a higher frequency of uterine contractions compared with women without endometriosis 132 . Microphysiological systems could potentially be used to support long-term co-cultures of endometrial cells with other disease-relevant cell types including immune cells, mesothelial cells and perivascular cells.