Strategies for modelling endometrial diseases

Abstract

Each month during a woman's reproductive years, the endometrium undergoes vast changes to prepare for a potential pregnancy. Diseases of the endometrium arise for numerous reasons, many of which remain unknown. These endometrial diseases, including endometriosis, adenomyosis, endometrial cancer and Asherman syndrome, affect many women, with an overall lack of efficient or permanent treatment solutions. The challenge lies in understanding the complexity of the endometrium and the extensive changes, orchestrated by ovarian hormones, that occur in multiple cell types over the period of the menstrual cycle. Appropriate model systems that closely mimic the architecture and function of the endometrium and its diseases are needed. The emergence of organoid technology using human cells is enabling a revolution in modelling the endometrium in vitro. The goal of this Review is to provide a focused reference for new models to study the diseases of the endometrium. We provide perspectives on the power of new and emerging models, from organoids to microfluidics, which have opened up a new frontier for studying endometrial diseases.

Fig. 1 |. The human endometrium and its most common pathologies.

a | The healthy endometrium consists of epithelial, stromal, vascular and immune cells that are distributed in the two main layers of the endometrium, the stratum basalis and stratum functional. Every 28 days, on average, the functional layer grows out from the basal layer, sloughs off and is regenerated. This process is controlled by female sex steroids produced by the ovary during the proliferative and secretory phases of the menstrual cycle. Adapted from ‘Uterine Cycle’, by BioRender. com (2022). Retrieved from https://app.biorender.com/biorender-templates. b | Asherman syndrome is an acquired pathology presenting with intrauterine adhesions in the uterine cavity, which has little to no vascularization or glands. These adhesions replace the functional luminal epithelium. c | Endometriosis is a disease in which endometrium-like tissue is found outside the uterus, growing within the peritoneal cavity. d | In adenomyosis, endometrial glands and stroma invade the underlying myometrial layer, which can result in an enlarged uterus. e | Endometrial hyperplasia and cancer. Endometrial hyperplasia is commonly described as the pathological proliferation of endometrial glands and is considered to be a precursor of endometrial carcinoma.

Fig. 2 |. In vitro models of endometrial diseases.

a | 2D, adherent, monolayer cell culture is the traditional method of growing cells in vitro. The method is simple and cost-effective, but endometrial epithelial cells lose their polarity and hormone response during culture. b | Endometrial explants are short-lived in culture, but they preserve native cell-cell interactions, diverse cell populations and tissue organization. c | 2D and 3D endometrial co-cultures can include one cell type in a monolayer on the bottom of a culture plate, with another cell type or organoids in a transwell tissue culture insert (left). The cells communicate through paracrine factors released into the culture medium. Other common 3D endometrial co-cultures consist of endometrial epithelial organoids or whole glands plated in Matrigel in the bottom of a culture well, with stromal cells cultured in two dimensions on the surface of the Matrigel (right). d | Endometrial epithelial organoids consist of epithelial cells that form a hollow, gland-like structure in a 3D matrix, typically made out of Matrigel. Epithelial cells preserve their polarity, with their apical side oriented towards the lumen of the organoid. e | Multicellular endometrial organoids are made up of both endometrial epithelial cells and stromal cells. The stromal cells, located in the interior of the organoid, serve as a scaffold for the epithelial cells, which are polarized with their apical side facing the exterior.

Fig. 3 |. Current approaches to model the endometrial niche and pathologies using microfluidic technologies.

a | Microfluidic device recreating the endometrial perivascular niche using a perfused endothelial chamber (composed of human umbilical vein endothelial cells (HUVECs)) and a static stromal chamber to study the role of the endometrial vasculature during decidualization. b | The design of a multichannel microfluidic device able to recapitulate the vascularized endometrium. Two channels with stromal fibroblasts generate a pro-angiogenic gradient that influences the angiogenesis and morphological changes of blood vessels residing in a third channel. The blood vessels in this system are composed of HUVECs. c | The multi-organ microfluidic platform (MFP) called EVATAR can be used to recreate the female reproductive tract in vitro. The Quintet MFP has been used for five different tissues. d | A co-culture device created by 3D printing a mould for polydimethylsiloxane (PDMS) enables the culture of an endometrium construct and ovarian cells (granulosa and theca cells). The endometrial well consists of endometrial stem cells, stromal fibroblasts and HUVECs supported in a hyaluronic acid, collagen and agarose hydrogel. e | Microfluidic channels are used to selectively pattern healthy or diseased endometrial stromal cells and human peritoneal mesothelial cells to study the peritoneal niche found in endometriosis. Here, cells were loaded into microfluidic channels (1), which were then removed (2), leaving the micropatterned cells (3). f | Microfluidic device that recreates healthy and diseased peristaltic flow patterns, introducing selective wall shear stresses on endometrial tissue-engineered constructs. PTFE, polytetrafluoroethylene. Part a reprinted from REF, CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). Part b reprinted with permission from REF, Oxford University Press. Part c reprinted from REF, CC BY 4.0 (https://creativecommons.org/1icenses/by/4.0/). Part d reprinted with permission from REF, IOP Publishing. Part e reprinted with permission from REF, Oxford University Press. Part f adapted from REF, CC BY 4.0 (https://creativecommons.org/1icenses/by/4.0/).

Fig. 4 |. Idealized design of MPS of the healthy endometrium and its diseases.

An idealized microfluidic device incorporates various cell types and niches of the natural endometrium, including a myometrial layer and both basalis and functional layers of the endometrium. The myometrium ideally contains inner myometrial smooth muscle cells of the junctional zone and outer smooth muscle cells that form bundles and can contract with certain frequencies. This contractile frequency is important to recapitulate certain aspects of endometriosis and particularly adenomyosis, in which lesion invasion can also be assessed. Cell health and behaviour are monitored continuously by on-chip imaging and incorporated biosensors. The basalis region contains rhizome-like epithelial structures that are vascularized and perfused with physiological capillary structures that contain endothelial cells and pericytes, while the functional layer contains vascularized organoids generated from menstrual flow. The addition of new cell types to endometrial organoid models will allow researchers to interrogate new aspects of endometrial diseases in vitro. In particular, immune cells, including macrophages, natural killer (NK) cells and T cells, play an important part in the pathogenesis of diseases such as endometriosis. Endothelial cells are important in the growth and remodelling of the endometrium, and angiogenesis is essential to the establishment and progression of endometrial pathologies, including endometriosis and cancer. Furthermore, fibrosis and scarring, modelled with stromal fibroblasts, would help in investigation of intrauterine adhesions. This idealized microphysiological system (MPS) can be integrated with other organs-on-a-chip for toxicity screening, drug metabolism, pharmacokinetics and studying diseases within a system. Finally, new technologies that allow 3D printing of both matrix proteins and live cells can offer improved precision and adaptability in generating complex bioengineered structures.