Organoids as a model to study the human endometrium : Reproductive and Developmental Medicine

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Special Issue: Updates on Research in Endometrium

Organoids as a model to study the human endometrium

Li, Jianlin; Lin, Leqian; Zhong, Jiangming; Li, Xintong; Lee, Cheuklun; Chiu, Philip C. N.*,

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Reproductive and Developmental Medicine 6(4):p 215-224, December 2022. | DOI: 10.1097/RD9.0000000000000040
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Abstract

Introduction

The endometrium is the inner lining of the uterus, which can be divided into two layers: the stratum functionalis and stratum basalis[1–3]. Within the functionalis, luminal and glandular epithelia influence uterine receptivity, luminal fluid homeostasis, and blastocyst implantation[4]. Factors secreted by the endometrial glands play a role in maintaining embryo growth by regulating immune responses and trophoblast invasion throughout the initial period of pregnancy[5,6]. Unlike the stratum functionalis, the stratum basalis is responsible for regenerating the endometrium during each menstrual cycle[1,7]. During a woman’s lifetime, the endometrium undergoes cyclic shedding, restoration, and remodeling. Functional abnormalities of the endometrium are related to various diseases, including implantation failure, endometriosis, and endometrial cancers[8–14].

Physiological studies of the endometrium, especially the endometrial gland, are constrained by the limitations of easily accessible models. Development of a human endometrial organoid system has provided a novel way to study the endometrium. Organoids are three-dimensional (3D) self-renewing cellular structures that maintain the functions and morphology of their original tissues[15]. In this review, a basic introduction of the endometrium and endometrial models for the study of the human endometrium will be provided. In addition, the applications of organoids in endometrial research will also be highlighted, along with a discussion of their limitations and future perspectives.

Development, structure, and function of the human endometrial glands

Compared with other female reproductive tract organs that have already developed and differentiated at birth, the human uterus undergoes development after birth and has rudimentary or even undifferentiated glands[16,17]. In the neonatal endometrium, the luminal epithelium (LE) consists of low columnar or cuboidal cells, while the glandular epithelium (GE) is scattered and restricted to the adluminal stroma[18]. Some portions of the GE develop from invagination of the LE and gradually invade the mesenchyme, forming a network of epithelial glands that extends to the myometrium[19–22].

Three-dimensional models of the human endometrium show a plexus network of tubular structures that extends horizontally along the myometrium in the stratum basalis[23]. The plexus structure in the stratum basalis is preserved after menstruation. In the proliferative phase, the tubular glands radiate from the network vertically into the stratum functionalis[23]. Endometrial gland formation involves cell adhesion and apicobasal polarity. Histologically, endometrial gland cells have a highly polar phenotype in vivo with a basement membrane at their basal surface and microvilli on the apical surface where the cells adhere through tight junctions[24].

GE cells within and near the decidua have increased activity in the early secretory phase and during early pregnancy[5,25,26]. This activity can be correlated with uterine gland function. Secretions from the uterine gland contain growth factors, hormones, cytokines, and enzymes[26–32] important in regulating uterine receptivity, blastocyst implantation, and stromal cell decidualization[6,28,33,34]. The endometrial glands also provide histotrophic nutrition to the human conceptus during the initial 10–12 weeks of pregnancy before full establishment of the placenta[6,26,33,35]. It is well acknowledged that the uterine luminal fluid and histotroph are responsible for nutrition delivery to the peri-implantation embryos in mammalian species that experience an extended period of development before superficial implantation and placentation[17,36].

Models for the study of the human endometrium

In vivo and in vitro models have been used to investigate the molecular and biological characteristics of the endometrium[37,38] (Table 1). Nonhuman primates (eg, monkeys and baboons) enable researchers to explore cyclical changes unique to menstruating animals[39]. However, the considerable need for resources and infrastructure for nonhuman primate housing facilities hinders the large-scale use of these models[40]. In contrast, the availability of targeted gene-editing tools for rodents enhances their popularity for endometrial research[41–48], despite well-noted differences in endometrial development and function between rodents and humans. For instance, spontaneous decidualization of the human endometrium depends on progesterone changes during the menstrual cycle, whereas decidualization of the rodent endometrium only occurs when the embryo is implanted or when there is a mechanical stimulus[39,49].

Table 1. - Pros and cons of different in vivo and in vitro models for studying the endometrium.
Features Primate Mouse Cell line Primary cell Tissue explant Organoids
Replicate physiology of human endometrium +++ + + ++ +++ +++
Cell-cell/cell-matrix interaction +++ +++ + ++
Resources/cost of labor/training +++ ++ + ++ + ++
Readiness for genetic manipulation +++ +++ ++ + +
Long-term experiment +++ +++ ++ + + +++
Sample variation +++ ++ + +++ +++ +++
Use of undefined culture supplement (eg, serum, matrix) + + ++ ++
Readiness for high-throughput screening +++ + + +++
Research ethical consideration +++ ++ + ++ ++ ++
–: not applicable; +: low; ++: medium; +++: high.

In vitro models of the endometrium can be classified into two-dimensional (2D) (primary cell and cell line) and 3D (3D cell aggregate, tissue explant, and organotypic culture) models[24,50–57]. The proliferative potential, phenotype, polarity, and functions of primary cells isolated from endometrial tissues may be gradually lost during subculture[58]. However, endometrial cell lines derived from carcinomas and immortalized primary cells may not fully recapitulate the physiological status of primary cells. Compared with cells cultured in 3D models, cells cultured as 2D monolayers lack stimulation from the surrounding matrix, which subsequently affects cellular functions such as cell adhesion, mechanotransduction, and response to stimulation[59]. Tissue explants (organotypic cultures) can mimic both complex structure and cellular heterogeneity of native organs. However, the lack of defined conditions to fully preserve their proliferative potential, cell phenotype, and function hinders their utility in research[56,60] (Table 1).

Organoids

The term “organoid” was first described as a cellular collection originating from cells with self-regeneration ability (such as adult stem/progenitor cells, embryonic stem cells, and induced pluripotent stem cells) to mimic the structural and biological role of the tissue of origin[61–72]. Compared with cell lines, self-renewing organoids recapitulate the morphology and physiological function of their origin, maintaining these features for a long time[68,73,74]. They offer scientists a simplified version of the organ with a detailed innate structure to investigate functional and pathological mechanisms.

Organoids can undergo extensive expansion while maintaining their genomic stability, making long-term storage and high-throughput screening possible. In addition, they recapitulate various biological and pathological characteristics of their organs of origin[65,67,75,76]. Organoids may also be manipulated and sequenced to develop disease models and to study drug responses[15,77]. These advantages make them a competitive candidate to replace the existing models for the study of the physiology and pathology of the female reproductive system (Table 1).

Development of endometrial organoids that resemble the endometrial gland

The initial organoid with glandular structures dates back to 1988, when Rinehart et al.[24] cultured glandular fragments. These organoids maintained their characteristics and ability to generate secretory vesicles over 6 months. Impeded by the technology at the time, Rinehart et al.[24] did not validate the relevant markers of their organoids or compare the transcriptomic profile of their organoids with that of the primary tissue. Endometrial organoids that resemble endometrial glands widely accepted by peer researchers were established by Turco et al.[67] and Boretto et al.[65] using normal and diseased endometrium. They cultured endometrial organoids in Matrigel, which imitated the mechanical support and exogenous environmental signals from the extracellular matrix (ECM) in vivo. To preserve the self-renewal competency of the organoids, a culture medium was used to activate Wnt signaling pathway and mitogen-activated protein kinase pathways and to constrain transforming growth factor (TGF)-β and bone morphogenetic protein signaling[65,67].

This protocol can generate endometrial organoids from diverse endometrial tissues, including tissues at different phases of the menstrual cycle, decidualized endometrium, and even atrophic endometrium. Self-regenerative endometrial organoids exhibit characteristics and functions similar to those of endometrial glands[65,67,75,76]. Endometrial organoids are usually spherical, with a central lumen lined by polar columnar cells with microvilli (Fig. 1). The expression of epithelial (eg, E-cadherin, CDH1) and glandular (eg, forkhead box A2 [FOXA2]) markers and steroid hormone receptors can recapitulate the dynamic changes in endometrial tissue throughout the menstrual cycle. Upon exposure to estrogen (E2), progesterone (P4), and cyclic adenosine monophosphate (cAMP), organoids have increased expression of IGFBP1 and leukemia inhibitory factor, consistent with the action of sex steroids on decidualization[50,65,67,78–81]. Organoids also demonstrate endometrial features by producing glandular products of the uterus, such as glycodelin-A (PAEP)[24,65,67,82]. The overall gene expression pattern of organoids is comparable to that of the glandular origin and stromal cells from the same patient[67]. Combining the gene ontology and hierarchical clustering analysis results, organoids are more similar to glands than to stroma[65,67,83]. Endometrial organoid models in recent years are shown in Table 2.

Table 2. - Endometrial organoid models in recent years.
Authors Year Summary of major findings
Rinehart et al.[24] 1988 The first researcher to establish organoids from human endometrial glandular epithelia in vitro
Serum-free medium with Matrigel
Cells changed from monolayer to glandular structures, with polar column facing a central lumen
Bläuer et al.[53] 2005 Glandular epithelial organoids in tissue culture inserts and stromal cells on plastic below the epithelial compartment
E2 induced cell proliferation and expression of hormonal receptor while along with MPA their expression decreased
Turco et al.[67] 2017 Organoids generated from normal, decidualized, and malignant endometrium
Genetically stable organoids for long-term expansion and response to treatment with sexual hormones
Human organoids shared genetic level similarity among different individuals
Boretto et al. [65] 2017 Organoids had similar physiological responses of the endometrial epithelium to hormones
Human endometrial organoids replicated the menstrual cycle under hormonal treatment at both the structural and genetic levels
Mouse and human endometrial organoids might respond to hormone treatment differently
Girda et al.[82] 2017 Patient-derived organoids were derived from endometrial cancer cells
Organoid growth assay was carried out to test the inhibitory effects of various drugs
Histological and immunohistochemical comparison had been made between organoids and the tumors of origin
Boretto et al.[84] 2019 Long-term expandable organoids were developed from a variety of endometrial pathologies (endometriosis, endometrial cancer, hyperplasia, and Lynch syndrome)
Organoids from endometrial disease reproduced the original lesion when transplanted to mice
Organoids might become tools for drug screening
Haider et al.[80] 2019 Endometrial organoids are established to study the E2-driven control of cell fate decisions in human endometrial epithelium
Induction of ciliated cells is under the control of the coordinated action of E2 and Notch signaling pathway signaling
Fitzgerald et al.[85] 2019 Endometrial epithelial organoids were treated with E2 or E2 and MPA
Bulk RNA sequencing were used to study the influence of hormone treatment on gene expression
Various epithelial cell types were identified in the epithelial cells generated organoids by single-cell RNA sequencing
Wiwatpanit et al. [86] 2020 Endometrial organoids consisted of both epithelial and stromal cells of the human endometrium
No exogenous scaffold materials
Luddi et al. [87] 2020 Organoids mimicking the early secretive phase showed reliable significant features of the implantation window
Organoids generated from eutopic endometrium of women with endometriosis and healthy endometrium show different glycodelin-A glycosylation pattern
Cochrane et al. [88] 2020 Single-cell sequencing was used to find new markers specific for endometrial ciliated or secretory cells
Secretory-like and ciliated-like tumor cells were found in endometrial and ovarian tumors
Bui et al. [89] 2020 Organoids were developed from infertile women and cryopreservation of the biopsy
Dolat and Valdivia[90] 2020 Infection process of Chlamydia in organoids was catalog from invasion to egress
Neutrophils were cocultured with infected organoids to observe the primary immune cell response
Simintiras et al. [91] 2021 Metabolomic profiles were compared between intra-organoid and extra-organoid fluid
Cheung et al. [92] 2021 Placenta-derived endometrial epithelial organoids were cocultured with pluripotent stem cell-derived endometrial stromal fibroblasts
Cindrova-Davies et al. [93] 2021 Organoids were derived from menstrual flow and shared the same transcriptome signature, derivation efficiency and proliferation rate with those from paired scratch biopsies
Organoids from two resources responded similarly to sex steroids and early pregnancy hormones
Maru et al. [94] 2021 Kras(G12D)-expressing endometrial organoids were transformed to immunodeficient mice
Epithelial-mesenchymal transition was observed during the transformation of endometrial cells and progression
MPA: medroxyprogesterone acetate.

F1
Fig. 1.:
Endometrial organoids in different forms. (A) Bright-field microscopy (40×); (B) Schematic; (C) Hematoxylin and eosin-stained endometrial organoids (200×); (D) Hematoxylin and eosin-stained endometrial tissue (100×).

Comparing the culture conditions of human and mouse endometrial organoids, Boretto et al.[65] found that factors such as Wnt family member 3A were not necessary for effective growth and expansion of human endometrial organoids. They also found that mouse and human endometrial organoids responded differently to hormone treatment. For example, while the expression of leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) decreased in mouse endometrial organoids with E2 and P4 treatment, the expression of LGR4 and LGR5 was not influenced by the same treatment in human endometrial organoids.

Turco et al.[67] derived organoids from matched normal and malignant endometria in postmenopausal women. Organoids from adenocarcinomas exhibited the morphology of the primary tumor (grade I endometrioid cancer), including nuclear pleomorphism and a disorganized epithelium with an irregular basement membrane. The organoids not only resembled the histological structure and phenotype of endometrial cancer, such as the expression of mucin 1 (MUC1) and SRY-box transcription factor 17 (SOX17), but also confirmed their glandular origin. This model was beneficial for the comparison between cancer tissue and its adjacent healthy tissue, offering an isogenic control without the variability of genetic background among individuals.

Besides transcervical procedures to obtain endometrial biopsies from donors, assessment of the endometrium can also be performed noninvasively. Cindrova-Davies et al.[93] proposed a method to derive organoids from menstrual flow, with these organoids faithfully presenting the in vivo characteristics of the endometrium. Menstrual flow was obtained from patients undergoing in vitro fertilization (IVF) treatment. After downregulation of the hypothalamic-pituitary-gonadal axis, menstrual flow was collected and ovarian stimulation was subsequently performed. Similar to organoids originating from paired scratch biopsies, those derived from menstrual flow showed similar morphology, derivation efficiency, and proliferation rate. The transcriptomes of the endometrial-derived and menstrual flow-derived organoids from each patient were also paired, indicating the conservation of transcripts between the two sources. Upon treatment with sex steroids and early pregnancy hormones, the organoids from the two sources showed increased expression of glycodelin-A and MUC1. In addition to menstrual blood, organoids can also be derived from cryopreserved biopsy catheter-derived endometrial tissue[89].

With the identification of different epithelial populations, their possible functions have raised the curiosity of scientists. Using bulk RNA sequencing and single-cell RNA sequencing simultaneously, Fitzgerald et al.[85] disclosed the components of epithelial cell types in organoids affected by E2 and P4. Consistent with previous research, the organoids had a transcriptional signature similar to that of uterine glands. They contained several epithelial cell types that are also found in the human endometrium. In a chemical-specific culture medium, endometrial organoids continued to proliferate until the addition of E2, P4, and cAMP, which decreased the proportion of the candidate stem/progenitor cell population within the organoids[85].

To gain better understanding of the heterogeneity of endometrial cancer and the physiology of endometrial epithelium, single-cell RNA sequencing was performed to compare the cell types in organoids originating from healthy endometrial epithelial tissue and those from endometrial tumors[88]. A secretory cell-specific marker, MPST, and several ciliated cell markers, including DYDC2, were identified and validated by immunohistochemistry in both organoids and endometrial tissue sections. Additionally, the above markers were found in endometrial tumors and were associated with improved overall survival in patients with endometrial cancer.

The ability of organoids to undergo extensive expansion while maintaining their genomic stability makes them possible tools for investigating the genomic features of potential stem cells. Using single-cell sequencing and spatial transcriptomics, Garcia-Alonso et al.[95] revealed the periodic change in the expression of SRY-box transcription factor 9 (SOX9) and LGR5 in the human uterus throughout the menstrual cycle. SOX9-positive cells with LGR5 expression in the LE would not enter the menstrual cycle, while those without LGR5 expression were promising stem cell candidates.

Endometrial organoid systems including epithelial and stromal cells

A functional endometrium is composed of a superficial layer of epithelial cells with glands and an underlying stroma. Endometrial epithelial organoids recapitulate the in vivo characteristics of GE. Studies have been conducted to determine the communication between epithelial and stromal cells.

An organoid system containing endometrial epithelial and stromal cells without an exogenous basement membrane matrix was published in 2020[86]. In this model, human endometrial epithelial cells and stromal cells were resuspended in the commercially available medium MammoCult (STEMCELL Technologies Inc., Vancouver, Canada) at similar densities and then seeded together in a micromolded agarose gel. The organoids obtained using this method showed a structure with central stromal cells surrounded by polarized epithelial cells. The epithelial cell markers E-cadherin and pan-cytokeratin and the glandular marker FOXA2 were expressed at the margin of the organoids, while the stromal cell marker vimentin was located at the center.

Cheung et al.[92] used a monolayer protocol to differentiate human pluripotent stem cells into endometrial stromal fibroblasts (PSC-ESFs). They further established a 3D hormone-responsive human decidua model of PSC-ESFs cocultured with placenta-derived endometrial epithelial cells to generate organoids to study the interaction between the stroma and epithelium. Moreover, gene expression by PSC-ESFs and endometrial epithelial-stromal cell signaling relationships were also identified in this coculture model. It is not known whether these results could be applied to primary ESFs. Despite this limitation, this 3D model may be a potential scaffold for the addition of other decidual cell types to study maternal-fetal signaling[92].

Application of the endometrial organoid model—the construction of a 3D endometrium

In 2020, Abbas et al.[96] built a collagen scaffold-based multicellular system comprising endometrial stromal cells and epithelial organoids. Supported by porous collagen scaffolds, organoids developed into a superficial luminal-like epithelial layer on the scaffold. The apical surface of polarized endometrial epithelial cells contained microvilli and cilia facing the pore cavities, while the basal part was appended onto the scaffold with the production of ECM proteins. The addition of hormones to this model led to epithelial differentiation and decidualization of stromal cells. In addition, different cell types could be detached from the scaffold using 0.2% trypsin for downstream analysis, which made this model suitable for investigating the interactions between endometrial epithelial cells, stromal cells, and other cell types such as immune cells and endothelial cells.

Endometrial organoids as a study model of endometriosis

The dysfunction of endometrial precursors and/or their niche may lead to the pathogenesis of endometrial diseases. Abnormal propagation of endometrial cells occurs in endometriosis, endometrial hyperplasia, endometrial carcinoma, and abnormally thin endometrium[97]. Organoids originating from pathological endometria, such as endometriosis and endometrial cancer, provide an advantageous tool for studying the molecular mechanisms underlying endometrial diseases[84].

Approximately 12% of women of reproductive age have endometriosis, whereas 70% of women have chronic pelvic pain[98]. Moreover, 20%–50% of infertile women also have endometriosis[99]. It is generally believed that the endometrial tissue or cells of patients with endometriosis are translocated to the peritoneal cavity through retrograde menstruation[100–102]. To the best of our knowledge of the etiology and pathogenesis of endometriosis, recent therapies for the disease aim to relieve the symptoms with risk of recurrence.

It is challenging to mimic the variability and phenotypic heterogeneity in different phases (I to IV) as well as categories (eg, surface and profound peritoneal lesions) of endometriosis[103,104]. An expandable endometrial organoid model, developed from several clinical stages of ectopic lesions and eutopic endometrium from matched individuals, was established by Boretto et al[65,84]. Ectopic organoids exhibited a denser stratified epithelial cell layer, which was absent in the normal control group. The expression of estrogen receptor 1 (ESR1) and progesterone receptor (PGR), secretion of mucus into the lumen, and the structure of the microvilli and cilia were comparable between the two groups. Regarding genomic and transcriptomic features, ectopic organoids from patients with advanced phases of endometriosis expressed endometrial cancer-linked driver genes, including Kirsten rat sarcoma virus, accompanied by reported mutations related to cancer. The levels of phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT), WNT, and Hippo signaling pathway-related genes previously associated with endometriosis were different between ectopic and normal organoids. Most importantly, the characteristics of endometriosis could be reproduced by subrenal transplantation with intraperitoneal injection of ectopic organoids in immunodeficient mice[84]. Ectopic organoids derived from high-grade patients induced an invasive cell mass with high proliferative ability, while those from low-grade patients remained in their position with lower growth.

Endometrial organoids as supporters for uterine carcinosarcoma establishment

Maru et al.[94] rebuilt organoids from genetically engineered mice using organoid- and allograft-based genetic methods. This approach has been applied to many organs including the pancreas[105] and lungs[106]. In a study on the endometrium, they found that cyclin-dependent kinase inhibitor (CDKN2) knockdown or tumor protein P53 (TRP53) deletion with KRASG12D-expressing endometrial organoids developed carcinosarcoma, indicating the oncogenic potential of KrasG12D. An epithelial-mesenchymal transition process was observed during endometrial cell transformation and progression, as spindle-shaped organoids could only form monophasic sarcomas. This study offered a novel way of understanding the mechanisms underlying tissue-specific KRAS-driven tumorigenesis.

Endometrial organoids as a model of studying endometrial biology associated with implantation

Embryo implantation is a process in which human embryos adhere to and invade the endometrium, which depends on the establishment of maternal-embryo crosstalk[107–109]. However, investigation into this process is hindered by ethical issues and the scarcity of reliable and reproducible in vitro endometrial models. Endometrial organoids provide an opportunity to study the endometrial interface during embryo implantation[87]. Under transmission electron microscopy, the organoids in the E2-induced proliferative phase showed a pseudostratified columnar phenotype, apicobasal polarity with nuclei near the basement membrane, and apical microvilli directed toward the lumen. In addition, in the upper region of these cells, ultrastructural features showed electron-dense particles forming apical tight junctions that signified the cellular epithelial barrier, suggesting cell-cell communication between epithelial cells. In the mid-secretory phase induced by E2 + P4 + cAMP, pinopodes were observed on the luminal surface using scanning electron microscopy. The presence of pinopodes have been reported as a good marker for the implantation window[110], and this was the first time that such cellular morphology was recapitulated in in vitro endometrial models. When seeded into a 2D layer, the endometrial organoid specifically interacted with stem cell-derived blastoids under the stimulation of E2 and P4, recapitulating the implantation process in vivo[111]. To investigate the implantation process, Rawlings et al.[112] constructed an assembloid model consisting of endometrial glandular organoids and stromal cells. Upon treatment with hormones, the assembloids showed gene expression patterns similar to those of the endometrium during implantation. Cocultured with embryos, cells in the assembloids moved toward the embryo as it grew, which mimicked the process required for implantation. Additionally, when senescent cells were killed by dasatinib, the migration of cells was discontinued and the embryo stopped developing, imitating the condition of implantation failure.

Glycodelin-A is produced by the endometrium and is involved in several physiological events related to human reproduction, fetal-maternal immunity, and endometrial receptivity[113–116]. Glycodelin-A interacts with its target receptors via its unique carbohydrate side chains, and its specific glycosylation is a prerequisite for biological activities[117–119]. Endometrial organoids derived from healthy donors faithfully recapitulated the quantitative and qualitative characteristics of glycodelin-A detected in the endometrium in vivo, while the glycosylation pattern of glycodelin-A was altered in organoids originating from eutopic endometrium from a patient with endometriosis[87].

Limitation and future research of endometrial organoid systems

Polarity

With a basal out/apical in phenotype, endometrial organoids are not completely similar to the endometrium in vivo, which hampers the exploration of their surface markers in binding experiments. Moreover, the presence of tight junctions in the organoid epithelium makes it difficult to transfect nucleic acids into cells. Simintiras et al.[91] found that some metabolites were unique to extra-organoid fluid, a product of asymmetrical apical and basolateral secretion by the organoid system. In this situation, microinjection could be used to investigate apical proteins in organoids[90]. To reverse polarity, a matrix gel-independent suspension culture system could be used, and an organoid-specific transfection protocol should be developed to overcome these obstacles[120].

Gene manipulation

Earlier research has demonstrated the possibility of gene editing in various organoid systems using CRISPR (clustered regularly interspaced short palindromic repeats) - Cas9 (CRISPR-associated protein 9) technology[121–123]. Similar methods have been used to edit cell lines of the endometrium[124,125]. However, with experimental constraints such as the conveyance of targeting reagents and selection of positive organoids after manipulation, the efficacy of this technology remains unsatisfactory. Meanwhile, side effects, such as off-target effects and functional alteration of oncogenes or tumor suppressor genes due to fragment insertion, may occur during gene editing[126]. After tackling these issues, endometrial organoids with CRISPR-Cas9 technology will become excellent models to explore the endometrium. Fujii et al.[127] have successfully edited the genome of intestinal organoids using electroporation. piggyBac plasmids expressing a puromycin-resistant gene could be transferred simultaneously with a single-guide RNA (sgRNA) to select organoids. Additionally, Ringel et al.[128] have captured all sgRNAs in human intestinal organoids to construct high-quality genome-scale pooled-library CRISPR screens. This screening approach could be used in endometrial organoids for better understanding of both genetic basis and pathology of the diseased endometrium.

Patient variation

Similar to other organoid systems originating from different patients, it is inevitable for endometrial organoids to have heterogeneity in cells, as well as inconsistency in expansion and biological performance. The reproducibility of organoid culture should be improved for it to be useful as a drug screening tool[15]. A recognized standard protocol for organoid culture would enable the establishment of a biobank for comparative studies among different individuals.

More robust and defined culture conditions

The culture conditions for organoids are complicated and require costly growth factors to maintain their expansion and differentiation. Organoid culture conditions could be improved by better studying the signaling pathway and using small molecules to replace cytokine/growth factors. The replacement of chemically undefined materials like animal-derived Matrigel with synthetic substances that simulate the structure of the in vivo ECM would also be helpful in improving the system[129].

Interaction with other cells/tissues/organoids

The interaction of organoids with other tissues is not fully understood, mainly due to the polarity issue and the problem of whether endometrial organoid medium or matrix gel would have an impact on cocultured cells/tissues/organoids. Corresponding coculture systems should be established to mimic actual in vivo environments. Furthermore, microfluidic technologies could be applied to address the complicated alignment between different biological systems in vitro. For example, a microfluidic system with different tissues from the human female reproductive tract and sex hormones from ovarian follicles was established to recapitulate the features of the human menstrual cycle in 28 days[130]. An organ-on-a-chip system was also developed to study the mutual communication between the human endometrium and ovary[131]. The ability to replace 2D cell cultures and tissue explants with chip models and organoids will provide better biological relevance to the tissue of origin.

Summary

With the ability to recapitulate the nature of the endometrial gland, endometrial organoids have shed light on our knowledge of endometrial development. Combined with systemic drug screening methods, organoids derived from patients with endometrial diseases enable researchers to gain more comprehensive understanding of their pathological features. Currently, models of endometriosis[65,84] and endometrial carcinosarcoma[94] have been successfully developed. Although several challenges remain, combined with emerging technologies, this system offers a promising future to understand the physiology of the endometrium by providing novel screening and treatment strategies for endometrial diseases.

Acknowledgments

None.

Author contributions

J.L.L. was responsible for the article preparation and editing. L.Q.L., J.M.Z., and X.T.L. edited the article. C.L. L. and P.C.N.C. reviewed the article.

Funding(s)

This work was supported in part by the National Natural Science Foundation of China (81971396), Hong Kong Research Grant Council Grant (17115619 & 17115320), the High Level-Hospital Program, Health Commission of Guangdong Province, China (HKUSZH201902015), and HKU-SZH Fund for Shenzhen Key Medical Discipline (SZXK2020089).

Conflicts of interest

All authors declare no conflict of interest.

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Keywords:

Endometrial gland; Endometrium; Epithelial cell; Estrous cycle; Organoid; Three-dimensional culture model

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