Human receptive endometrial assembloid for deciphering the implantation window

Yu Zhang; Rusong Zhao; Chaoyan Yang; Jinzhu Song; Peishu Liu; Yan Li; Boyang Liu; Tao Li; Changjian Yin; Minghui Lu; Zhenzhen Hou; Chuanxin Zhang; Zi-Jiang Chen; Keliang Wu; Han Zhao

doi:10.7554/eLife.90729.3

eLife Assessment

This important study reports an endometrial organoid culture system mimicking the window of implantation. The evidence supporting the conclusion drawn is convincing. The data will be of interest to embryologists and investigators working on reproductive biology and medicine.

https://doi.org/10.7554/eLife.90729.3.sa3

Significance of findings

important: Findings that have theoretical or practical implications beyond a single subfield

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important
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useful

Strength of evidence

convincing: Appropriate and validated methodology in line with current state-of-the-art

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Abstract

Human endometrial receptivity is a critical determinant of pregnancy success; however, in vivo studies of its features and regulation are particularly challenging due to ethical restriction. Recently, the development of human endometrial assembloids has provided a powerful model to examine this intricate biological process. In this study, we established a specialized human window-of-implantation (WOI) endometrial assembloid system that mimics the in vivo receptive endometrium. It recapitulates not only the structural attributes of pinopodes and cilia, but also molecular characteristics of mid secretory endometrium. Furthermore, the WOI endometrial assembloid exhibits hormone responsiveness, energy metabolism with larger and enhanced functional mitochondria, increased ciliary assembly and motility, and epithelial-mesenchymal transition (EMT), as well as promising potential for embryo implantation. As such, WOI assembloids hold great promise as a platform to unravel the intricate mechanisms governing endometrial receptivity regulation, maternal-fetal interactions, and associated diseases, ultimately driving impactful advancements in the field.

Introduction

The human endometrium, comprising various cell types, experiences shedding (menstrual phase), regeneration (proliferative phase), and differentiation (secretory phase) under the regulation of estrogen and progesterone during the menstrual cycle (1). A brief window, called the “window-of-implantation (WOI)” or mid-secretory phase, allows for embryo implantation into the endometrium (1). The endometrium at the embryo implantation site contains diverse cells, including ciliated epithelial cells, decidualized stromal cells and immune cells. Cyclic endometrial changes akin to those in humans are exclusive to apes, Old World monkeys, molossid bats, and spiny mice, but not mice (1), rendering typical mouse models insufficient for accurately simulating human endometrium. Existing endometrial cell lines, such as Ishikawa and HEEC cells, consist solely of a single cell type, thus failing to reproduce the intricate physiological structure and function of the endometrium.

Replicating and reconstructing human organs has become essential for exploring tissue physiology and function. O rganoid, a self-assembled 3D structure, closely resemble in vivo tissue or organ (2). They offer high expansibility, phenotypic, and functional properties, emerging as powerful tools for investigating tissue physiology and disease (3). In 2017, the first long-term and hormone-responsive human endometrial organoid was established using adult stem cells from endometrial biopsy(2, 3). Based on this, Margherita Y. Turco, et al developed noninvasive methods to construct endometrial organoids from menstrual flow(4). Takahiro Arima et al. developed polar reversal endometrial organoids to study embryo implantation(5). Apart from adult stem cells, pluripotent stem cells were also induced to endometrial stromal fibroblasts and epithelium, and then cocultured to form organoids(6, 7), which offered vigorous proliferative capacity but lacked immune cells and other components of the microenvironment. There are also studies that add immune cells to endometrial organoids for co-culture(8). In efforts to mimic human endometrial cell types in vitro, the endometrial organoids progressed from epithelial organoids(9), to assemblies of epithelial and stromal cells(10, 11) and then to stem cell-laden 3D artificial endometrium(12, 13), which were solely closer but not completely identical to the endometrium. Additionally, pathological endometrial organoid models have been established for conditions like endometriosis, endometrial hyperplasia, Lynch syndrome, and endometrial cancer (14). These organoids simulated endometrial morphology, hormone responsiveness, and physiological and pathological processes in vitro (3, 9, 15–17), facilitating the study of physiological phenomena (15, 18, 19), pathogenic mechanisms(16) and drug screening(14).

Although various regulators have been suggested to be involved in WOI, we are far from understanding how embryo implantation occurs during the WOI due to ethical limitations and fewer in vitro receptive endometrial model. Transforming from proliferative to receptive endometrium involves dynamic changes like decidualization, epithelial-mesenchymal transition (EMT) and ciliated epithelial development (1, 20), which have not been reconstructed in most in vitro model to date. Here, we successfully established a receptive endometrial assembloid system regulated by hormones and reflecting the in vivo WOI endometrial features, providing a platform for studying physiological and pathological endometrium and maternal-fetal interactions.

Results

Developing receptive endometrial assembloids in vitro

To establish endometrial assembloids in vitro, pre-receptive endometrium from reproductive-age women was dissociated into single cells or small cell masses. These cells then self-assembled into assembloids induced by various small molecules, such as Noggin, EGF, FGF2, WNT-3A and R-Spondin1 in expansion medium (ExM) (Fig.1A∼B, Fig.S1A). The assembloids derived from the first generation are used for experiments (Fig.S1B). The endometrial assembloids consist of vesicle-like glands and fibrous stromal cells (Fig. S1C, Video S1∼S4). The epithelium marker E-cadherin, stromal cell marker vimentin, and endometrial gland marker FOXA2 were expressed in the cultured endometrial assembloid, which resembled the endometrium morphologically (Fig. S1D). Moreover, the endometrial assembloids exhibited substantial expression of the proliferation marker Ki67, while the apoptosis marker cleaved caspase-3 was undetectable, signifying the assembloids’ robust proliferative capacity (Fig. S1E).

Functionally, endometrial assembloids effectively secreted glycogen into the lumen, mirroring the in vivo endometrial activity, thereby providing nourishment for embryo implantation (Fig. S1D). Moreover, after two days of estrogen (E2) treatment followed by fourteen days of medroxyprogesterone acetate (MPA) and cAMP administration, the assembloids exhibited a significant upregulation of progesterone receptor (PRA/B) expression, a modest increase in estrogen receptor α (ERα), and enhanced expression of estrogen-responsive genes EGR1 and OLFM4, along with progesterone-responsive genes PGR and PAEP at the transcriptional level (Fig. S1F∼S1G). It reflected a comparable hormonal responsiveness in the in vivo endometrium.

To identify hormonal regimens that induce implantation window, pregnancy-related hormones were supplemented into the culture system following the two-day E2. E2 and MPA promote the transition of endometrial assembloids into the secretory phase, while pregnancy hormones can promote the further differentiation. Prolactin (PRL) promotes immune regulation and angiogenesis during implantation(2, 21). Human chorionic gonadotropin (hCG) improves endometrial thickness and receptivity (22, 23). Human placental lactogen (hPL) promotes the development and function of endometrial glands(24). Hormone dosage was primarily based on peri-pregnant maternal body or localized endometrium levels (2). A comparison of multiple groups revealed similar counts, area, and average intensity of assembloids over time (Fig.1C-D, Fig.S1H). However, only the final recipe (i.e., a combination of E2, MPA, cAMP, PRL, hCG and hPL) exhibited an endometrial receptivity-related gene expression profile, which highly expressed genes positively correlated with endometrial receptivity, and lowly expressed genes negatively correlated with receptivity, compared to the other hormone formulations (Fig.1E). The assembloids induced with this scheme were defined as WOI assembloids (Fig. 1A). In contrast, endometrial assembloids maintained in ExM constituted the “control (CTRL)” group, whereas assembloids treated with E2 for two days, followed by E2, MPA, and cAMP for an additional six days, were induced to the secretory phase as previously reported(9), and designated as the “secretory (SEC)” group (Fig. 1A). There was no significant difference in the morphology of assembloids among the three groups (Fig.1B), but when comparing receptivity markers, WOI assembloids exhibited high expression of IGFBP1, MAOA, and DPP4 (Fig. 1F), with increased glycogen secretion (Fig.S1I). Theoretically, the WOI assembloids are initially in the secretory phase, thus sharing characteristics with the SEC assembloids. More importantly, they are expected to display traits typical of the mid-secretory phase.

Receptive endometrial assembloids mimicked the implantation - window endometrium

Single-cell transcriptomics analysis identified the presence of epithelium, stromal cells, and immune cells in WOI assembloids with reference to CellMarker, PanglaoDB, Human Cell Atlas, Human Cell Landscape, and scRNASeqDB, and previous endometrium related studies(1, 9, 11, 25) (Fig. 2A, Fig. S2A∼S2B). The WOI assembloids exhibited similarities to the mid-secretory endometrium in terms of glandular and luminal epithelium, secretory epithelium, LGR5 epithelium, ciliated epithelium, and EMT-derived cells, as evidenced by comparisons of scRNA-seq data from our assembloids and the mid-secretory endometrium as described by Stephen R. Quake in 2020 (1) (Fig.2A, Fig.S2C∼S2D). The morphology of immune and stromal cells was analyzed through 3D clearing staining and light sheet microscopy imaging, with vimentin labeling stromal cells (Vimentin⁺ or Vimentin⁺ F-actin⁺), CD45 and CD44 indicating immune cells, and FOXA2 identifying glands (Fig.2B∼2D). Furtherly, we confirmed the presence and types of immune cells using flow cytometry. White blood cells (WBC) were identified as CD45⁺ cells, with T cells, macrophages and NK cells characterized as CD45⁺CD3⁺ cells, CD45⁺CD68⁺CD11b⁺ cells and CD56⁺CD16^- cells, respectively (Fig. 2E).

The WOI assembloids displayed characteristic features of the receptive endometrium. On the one hand, the WOI assembloids secreted more glycogen into the lumen (Fig. S1I). On the other hand, the WOI assembloids possessed various characteristic microstructure of implantation window, including elongated microvilli and increased glycogen, pinopodes, and especially cilia (Fig.2F).

Further, we concentrated on the comparison of gene expression and transcriptional regulation during the mid-secretory phase, as this phase marks the implantation window and involves substantial transcriptional alterations (Fig.2G∼2H). We referred to the scRNA-seq data of the mid-secretory phase from Stephen R. Quake 2020 (1). Pathways related to mitochondrial energy metabolism and cell adhesion exhibited upregulation in both the WOI assembloid and mid-secretory endometrium in comparison to the CTRL and SEC assembloids (Fig. 2G). The crucial transcription factors (TFs) found in the secretory epithelium and EMT-derived cells, which are implicated in implantation, revealed similarities between WOI assembloids and mid-secretory endometrium. Specifically, the secretory epithelium exhibited comparable TFs related to hypoxia response (such as previously reported HIF1A(26)), embryo implantation (such as FBLN1(27)), lipid metabolism (such as VMP1(28)), cell migration, and cell junction (such as TJP1(29)) (Fig. 2H). Similarly, EMT-derived cells also expressed similar TFs involved in endometrial decidualization (such as S100A10(30)), EMT (such as FAT1(31) and FOXF2(32)), and receptivity (such as NEAT1(33), SERPINB9(34), SOX17(35) and SOX4(36)) (Fig. 2H).

We then conducted the endometrial receptivity test (ERT) (37) to further assess the receptive state of WOI assembloids. ERT is a kind of gene analysis-based method for detecting endometrial receptivity, which combines high-throughput sequencing and machine learning to analyze the expression of endometrial receptivity-related genes (37). It is currently used in clinical practice to determine endometrial receptivity and guide personalized embryo transfer. The WOI assembloids derived from pre-receptive endometrium were observed to transit into the receptivity phase (Fig. S1J).

Collectively, these results demonstrated that the WOI assembloids closely resemble the in vivo endometrium during the implantation window in terms of structures and molecular characteristics.

Receptive endometrial assembloids recapitulate WOI-associated hormone response

We analyzed the transcriptome and proteome profiles of WOI assembloids compared to those of CTRL or SEC assembloids to assess WOI associated biological characteristics (Fig. 3A, Fig. S3A∼S3E).

WOI assembloids exhibited a robust hormone response, as demonstrated by the upregulation of PGR at the transcriptome level (Fig. 3B). Integrated analysis of the transcriptome and proteome data identified 179 upregulated genes/proteins in WOI assembloids compared to the CTRL group, with most of these genes/proteins implicated in the estrogen response (Fig. S3F∼S3G). Furthermore, the analysis of progesterone response levels using PRA/B immunostaining indicated the highest levels in the WOI group, accompanied by an upregulation of the estrogen responsive protein OLFM4 (Fig. 3C). FOXO1, a crucial marker of endometrial receptivity reliant on PGR signaling, exhibited significantly elevated expression in WOI assembloids compared to CTRL assembloids (Fig.3D, Video S5), suggesting the involvement of progesterone signaling in the establishment of WOI assembloids. These findings collectively demonstrated a strong response of WOI assembloids to estrogen and progesterone.

Hormone response increased secretory epithelium and decreased proliferative epithelium in the SEC and WOI assembloids, suggesting the transformation from proliferation to secretory phase (Fig. 3E). The secretory epithelium, critical for the implantation window, contributed to cellular metabolic processes and HIF-1 signaling pathway response to hypoxia at the single-cell level (Fig. S2E∼S2F). The secretory epithelium of WOI assembloids exhibited enhanced peptide metabolism and mitochondrial energy metabolism compared to the SEC group, supporting endometrial decidualization and embryo implantation (Fig. 3F). Proliferative epithelium differentiates into secretory epithelium under the regulation of branch nodal genes between state 5 and state 6, such as the KRT19, MALAT1, MT2A, RPL and RPS families, as revealed by single-cell trajectory (Fig. 3G, Fig. S2H∼S2J). The WOI assembloids showed more thoroughly differentiation from proliferative to secretory epithelium in relation to the SEC assembloids (Fig. 3G).

Overall, the WOI assembloids possessed the hormone response characteristic of implantation window, closely resembling the gene traits of embryo implantation.

Receptive endometrial assembloids possess enhanced energy metabolism

The WOI assembloids exhibited upregulation of monocarboxylic acid and lipid metabolism (represented by SLC25A1(38)), and hypoxia response (represented by HIF1α(26)) (Fig.3B, Fig.S3G∼S3I). Likewise, the secretory epithelium, critical for the implantation window, contributed to cellular metabolic processes and HIF-1 signaling pathway response to hypoxia at the single-cell level (Fig. S2E∼S2F).

To further investigate this trend, the Mfuzz algorithm was utilized to analyze gene expression across these three groups, focusing on gene clusters that were progressively upregulated or downregulated. It was observed that mitochondrial genes exhibited the highest expression levels in WOI endometrial assembloids (Fig. 4A). Additionally, at the protein level, WOI endometrial assembloids showed sustained high expression of mitochondrial proteins compared to SEC assembloids (Fig. 4B). TEM analysis revealed that WOI endometrial assembloids had the largest average mitochondrial area (Fig. 4C). The expression of mitochondrial-related genes increased from CTRL to SEC to WOI assembloids, with COA1 ensuring proper nuclear-mitochondrial connection(39), OXA1L promoting mitochondrial translation(40), and TIMMDC1 being crucial for mitochondrial complex I assembly(41) (Fig. 4D). The WOI assembloids notably expressed higher levels of OXA1L and TIMMDC1 than the SEC assembloids (Fig. 4D). Furthermore, WOI assembloids produced more ATP and IL8(42) (Fig. 4E).

Receptive endometrial assembloids possess enhanced energy metabolism
(A) The Mfuzz trend analysis displayed the transcriptional variation trends of five clusters from CTRL, SEC, and WOI groups (with a focus on the differences between SEC and WOI assembloids). The heatmap illustrated corresponding gene expression profile (where color represents Z-score). The bubble plot showed the associated GO functions (with bubble size representing the number of genes and bubble color indicating the P value).
(B) The circular heatmap illustrated the functional differences of SEC and WOI assembloids at the proteomic level. The color represents protein expression levels, and the innermost circle color represents GO functions.
(C) Transmission electron microscopy images displayed the mitochondrial morphology of CTRL, SEC, and WOI assembloids, along with a quantitative comparison of mitochondrial area. Scale bar = 1 μm.
(D) RT-qPCR assessed the expression levels of mitochondrial function-related genes in the three assembloid groups.
(E) Quantitative comparison of the concentration of ATP (left) and IL8 (right) released by CTRL, SEC and WOI assembloids. *P<0.05<**P<0.005<***P<0.0005<****P<0.0001.

Thus, compared to SEC assembloids, WOI assembloids exhibited increased energy metabolism with larger and enhanced functional mitochondria.

Receptive endometrial assembloids increased the ciliary assembly and motility

The growth and development, assembly and movement of cilia, a characteristic endometrial structure, are essential for the establishment of endometrial implantation window and embryo implantation. Under the electron microscopy, the cilia were most observed in the WOI assembloids (Fig. 2E). Cilia-related genes and proteins exhibited the highest expression levels in WOI endometrial assembloids (Fig. 5A∼5B). Compared to SEC assembloids, WOI endometrial assembloids showed upregulation in ciliary assembly, ciliary basal body, and motile cilia-related gene expressions, while genes related to non-motile cilia were downregulated (Fig. 5A).

Transcriptomic analysis further confirmed the upregulation of genes involved in ciliary assembly (NEK2), ciliary basal body (CFAP36), and motile cilia (DNAH9 and TPPP) in WOI endometrial assembloids, with NEK2 showing the most significant difference (Fig.5B). Meanwhile, expressions of proteins related to ciliary assembly, ciliary basal body, and motile cilia were also increased in WOI assembloids, with the most notable differences observed in TBC1D1, IFT22, and IFT57 (ciliary assembly), and PJA2 (ciliary basal body) (Fig. 5C). Acetyl-α-tubulin (cilia marker (15)) were highly expressed in the WOI assembloids (Fig. 5D).

Single-cell transcriptome analysis revealed hormone treatment increased ciliated epithelium and decreased unciliated epithelium in SEC and WOI groups (Fig. 3E). Ciliated epithelium functioned in protein binding, cilium organization and assembly, while unciliated epithelium acted on actin cytoskeleton and translation (Fig. S2E∼F). The WOI assembloids’ ciliated epithelium regulated vasculature development and displayed higher transcriptional activity than the SEC group (Fig. 5E). Ciliated-unciliated epithelium transition occurred during the menstrual cycle, which was regulated by key genes, such as GAS5, JUN, RPL and RPS families (Fig. 5F, Fig.S2K∼S2M). The implementation of aforementioned functions depended on its interaction with other cells. CellPhoneDB is a useful tool to investigate ligand-receptor interactions between the cells(43). Ciliated epithelium of WOI assembloids interacted with immune cells and secretory epithelium, showing enhanced invasion ability via CD74-COPA(44), and ROR2-WNT5A(45), which was validated by proximity ligation assay (PLA) (Fig. 5G∼5H).

In summary, the WOI assembloids revealed accumulated ciliated epithelium’s role in preparing the implantation window, with increased ciliary assembly and motility compared to SEC assembloids.

Receptive endometrial assembloids experienced epithelial-mesenchymal transition (EMT)

The WOI assembloids displayed upregulated cell differentiation not only at assembloid level (Fig. 3B) but also at cellular level, which is represented by EMT. EMT is a common and crucial biological event in the endometrium during the implantation window(46). During the EMT process, epithelial cells lose their epithelial characteristics while gaining migratory and invasive properties of fibroblasts. Synthetic analysis of the transcriptome and proteome revealed increased EMT in WOI assembloids (Fig. S3G).

EMT-derived cells, exhibiting gene expression patterns typical of epithelial and stromal cells, as well as EMT, are more abundant in the SEC and WOI groups, and act on protein binding, cell cycle, organelle organization, and reproduction (Fig. S2E). They performed enhanced lamellipodium-mediated cell migration, cell junction and cytoskeleton regulation in the WOI assembloids compared to the SEC assembloids (Fig. S4A). The WOI assembloids exhibited more thoroughly differentiation from proliferative epithelium to EMT-derived cells than the SEC assembloids, which was regulated by key genes such as DOC2B, FXYD3, and LPCAT3 (Fig. S4B∼S4D). EMT-derived cells and epithelium cooperated during the implantation window (Fig. S4E). We found that NRP1 and SLC7A1 were highly expressed by EMT-derived cells, and their receptors (SEMA3A and CSF1) were more upregulated in the epithelium of WOI group than SEC group (Fig. S4E, S4G). NRP1-SEMA3A has been reported to promote vascularization and responds to hypoxia(47). SLC7A1(48) and CSF1(49) both support receptivity establishment, embryo implantation and development. Compared with common stromal cells, EMT-derived cells communicate slightly differently with epithelial or immune cells. CD44 and CD46, known for their roles in cell adhesion (51) and immunoregulation(52), are highly expressed in stromal cells and EMT-derived cells, respectively, and bind separately with SPP1 and JAG1 in epithelial and stromal cells (Fig. S4E∼S4G).

In general, WOI assembloids demonstrated EMT’s role in mediating endometrial transformation towards the implantation window.

The receptive endometrial assembloids possess the potential for embryo implantation

We further investigated the key biological function of the implantation window, specifically embryo implantation, using the assembloids. Given the rarity and ethical constraints associated with human embryos, we employed blastoids (corresponding to the human embryo at 6 days post-fertilization, referred to as “Day 6”) for implantation into the endometrial assembloids (Fig.6A). By Day 9, we observed that the blastoids could grow within the endometrial assembloids and interact with them (Fig.6B). The co-cultured blastoids displayed normal tri-lineage differentiation, characterized by markers for the epiblast (OCT4), hypoblast (GATA6), and trophoblast (KRT18) (Fig.6C). Additionally, we conducted a comparative analysis of the survival rates of blastoids and their interaction rates with endometrial assembloids across the CTRL, SEC and WOI assembloids. Remarkably, the survival and interaction rates of blastoids in the WOI endometrial assembloids were significantly higher compared to the CTRL and SEC groups, with survival rates of 66%, 19%, and 28%, and interaction rates of 90%, 47%, and 53% respectively, indicating the promising potential for embryo implantation (Fig.6D∼E). This demonstrates that we have provided an innovative and supportive model suitable for embryo implantation.

In summary, WOI endometrial assembloids not only exhibited the typical structural and molecular features of the implantation window but also demonstrated significant potential for embryo implantation.

Discussion

In our study, we constructed the WOI endometrial assembloids, and observed the remarkable resemblance in structure and function to the in vivo endometrium. The assembloids consist of three primary types of endometrial cells, specifically epithelial, stromal, and immune cells, with epithelial cells assembling into glands and surrounded by immune cells and stromal cells. This similarity in cellular composition and tissue architecture to the endometrium lays the foundation for simulating receptive endometrium.

Although previous studies treated endometrial assembloid with E2, P4, and cAMP to induce a transition to the secretory phase, the methodology to accurately simulate the implantation window or mid-secretory endometrium remains elusive and warrants further investigation. Previous research suggested that placental signals could further promote the differentiation of endometrial assembloids (2), and PRL, hCG and HPL have been implicated in processes such as decidualization, implantation, immunoregulation, and angiogenesis (21, 22, 24). Detailly, PRL, synthesized by the adenohypophysis, endometrium, and myometrium, plays a vital role in implantation, immunoregulation, and angiogenesis, with the secretory endometrium producing PRL in response to MPA and E2, leading to ciliated cell formation and stromal cell decidualization (2, 21). HCG, secreted by trophoblasts in early pregnancy, influences decidual cells (22) and improves endometrial thickness and receptivity(23). The introduction of hCG has been shown to elevate the expression of critical factors associated with endometrial receptivity, such as endocytosis proteins, hypoxia-inducible factor 1 (HIF1), chemokines, and glycodelin (53). HPL contributes to the development and function of uterine glands(24). Consequently, the additional supplementation of PRL, hCG and HPL in our system augmented hormone responsiveness and receptivity, while promoting cell differentiation, ultimately yielding a model more representative of the receptive endometrium.

Here, the WOI assembloid exhibited the characteristic ultra-structures, such as cilia. During the human menstrual cycle, motile cilia are present in the epithelium of the endometrium(1). These hair-like organelles extend from the cell surface and beat rhythmically, facilitating cell and tissue movement while driving fluid transport across the epithelium (54). During the decidualization of the endometrium, the number and length of cilia increase, a process driven by the two primary regulatory factors for embryo implantation: estrogen and progesterone (15, 55, 56). However, with aging, the expression of cilia-related genes in the endometrium is downregulated(57). In patients with recurrent implantation failure (RIF), ciliary defects in the endometrium are observed, leading to abnormal decidualization and repeated implantation failure(56). Thus, cilia play a crucial role in endometrial decidualization and embryo implantation. Consistently, the WOI assembloid exhibited increased ciliated epithelium, along with enhanced assembly and motility of cilia.

Furthermore, the WOI assembloid demonstrated energy and lipid metabolism patterns resembling those of the in vivo receptive endometrium. Enhanced energy metabolism involves monocarboxylic acid metabolism and mitochondrial oxidative phosphorylation. Monocarboxylic acids, exemplified by lactate, pyruvate, and ketone bodies, are vital metabolites in most mammalian cells. During the implantation window, elevated lactate levels mobilize endometrial monocarboxylic acid metabolism and function as a pregnancy-related signal, stimulating secretion in the epithelium. Subsequently, ATP, the primary product of energy metabolism, induces neighboring epithelial cells to release IL8, promoting decidualization of stromal cells (42). It was also observed that WOI assembloids indeed possessed larger and functionally active mitochondria, producing much more ATP and IL8 than CTRL and SEC assembloids. Lipid metabolism, responsible for energy storage, signal transduction, cell proliferation, apoptosis, and membrane trafficking, plays a crucial role in endometrial receptivity and implantation, although the precise mechanisms are not fully understood (58–60). Thus, the WOI assembloids possessed metabolic characteristics of in vivo implantation window.

However, our WOI endometrial assembloids also exhibit some limitations. It is undeniable that the assembloids cannot perfectly replicate the in vivo endometrium, which comprises functional and basal layers with a greater abundance of cell subtypes, under superior regulation by systemic hormones and nutrients. Stromal cells and immune cells are difficult to pass down stably and their proportion is lower than that in the in vivo endometrium. We are looking forward to combining stem cell induction, 3D printing technology, and microfluidic systems to alter the culture environment in order to optimize the existing model.

In summary, we developed a receptive endometrial assembloid that mimics the features of in vivo implantation-window endometrium (Fig. 7). With typical ultra-structures of the implantation window, including pinopodes and cilia, it not only demonstrated hormonal responsiveness and characteristic glycogen secretion functions, but also recapitulated the processes such as decidualization, metabolic changes and EMT, while retaining the potential for embryo implantation. This receptive endometrial assembloid serves as a platform to investigate peri-implantation endometrial physiology and pathology, maternal-fetal interactions, with potential practical applications and clinical translation.

Schematic diagram displaying the establishment and validation of receptive endometrial assembloids, and summarizing the characteristic biological events of implantation-window endometrium.

Materials and Methods

Establishment of endometrial assembloids

All experiments involving human subjects followed medical ethical principles and the Declaration of Helsinki, and were approved by the Ethics Committee of Qilu Hospital of Shandong University. Informed consents were obtained from patients. Females of reproductive age who received hysterectomy for benign diseases were selected for this study. Clinical information of patients providing endometrial tissue was listed in Dataset S1. Experimental procedures are presented in detail in the Supporting Information.

Hormone treatment of endometrial assembloids

The hormone regimen for inducing endometrial secretory phase, as described by Thomas E. Spencer et al (9), consisted of estradiol for 2 days followed by a combination of estradiol (Sigma E2758), medroxyprogesterone acetate (Selleck S2567) and N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (cAMP) (Sigma D0627) for 6 days.

To simulate the endometrium of implantation window, we developed a model by incorporating various pregnancy-related hormones. The hormone regimen included estradiol for the first two days, followed by a combination of estradiol, medroxyprogesterone acetate, cAMP, Human Chorionic Gonadotropin (HCG) (Livzon Pharmaceutical Group Inc), Human Placental Lactogen (HPL) (R&D Systems 5757-PL), and prolactin (Peprotech 100-07) for 6 days. The CTRL group was cultured in ExM without the addition of hormones. (Fig. 1A) (Table S2)

Developing receptive endometrial assembloids in vitro
(A) Brightfield of endometrial assembloids on day2, day4 and day11. Scale bar = 200 μm.
(B) Brightfield of endometrial assembloids in the primary generation (P0) and first generation (P1). Scale bar = 200 μm.
(C) Screenshot of video S1 showing endometrial glands gradually developing into a vesicular shape, and the surrounding stromal cells arranging in a fibrous pattern in the CTRL endometrial assembloids (100x, up-left) (The yellow arrows indicate stromal cells and the white arrows indicate endometrial glands). Screenshot of video S2 displaying the stromal cells growing in fibrous pattern and forming an extensive network in the CTRL endometrial assembloids (200x, up-right) (The white arrows indicate stromal cells). The epithelial cells arrange like paving stones (middle and down, left). Stromal cells formed an extensive network (middle and down, right) (The arrowhead indicates stromal cells). Scale bar = 100 μm (middle), Scale bar = 50 μm (down).
(D) Validation of epithelial, stromal cell and endometrial gland markers (E-cadherin, vimentin and FOXA2, respectively) with immunofluorescence (IF) in the endometrium in vivo and CTRL endometrial assembloids in vitro. Nuclei were counterstained with DAPI. Scale bar = 40 μm. Periodic acid-schiff staining (PAS) of endometrium in vivo and endometrial assembloid in vitro. Scale bar = 20 μm.
(E) IF analysis of proliferation and apoptosis indicated by Ki67 and cleaved caspase-3 in the CTRL assembloids, respectively. Nuclei were counterstained with DAPI. Scale bar = 40 μm.
(F) Verification of hormone responsiveness by the expression levels of ERα and PRA/B (E-cadherin indicated the marker of epithelial cell). Scale bar = 40 μm. ***P ≤ 0.0005.
(G) Relative expression of PGR, PAEP, EGR1, and OLFM4 in the CTRL and hormone-treated assembloids by RT-qPCR. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005.
(H) The dynamic changes of the average intensity of assembloids over time in each hormone regimen.
(I) PAS of CTRL, SEC and WOI assembloids. Scale bar = 20 μm. Quantitative comparison of glycogen staining area in the CTRL, SEC and WOI assembloids. *P ≤ 0.05, ****P ≤ 0.0001.
(J) Endometrial receptivity evaluation of endometrium and their derived WOI assembloids through ERT. Asterisks indicate individual samples.

Various functions performed by all kinds of cells identified with scRNA-seq.
(A) Box plot of the gene numbers detected, UMI numbers, and ratios of mitochondrial gene expression in single cells of the CTRL, SEC and WOI groups.
(B) Bubble diagram showing the distribution of marker gene expression in each cluster.
(C∼D) Comparison of cell composition between CTRL, SEC, WOI endometrial assembloids and mid-secretory endometrium demonstrating samples (C) and cell types (D).
(E) Bubble diagram and heatmap showing the corresponding upregulated genes and GO function of each cluster of endometrial cells.
(F) Bubble diagram and heatmap showing corresponding upregulated genes and KEGG functions of SOX9⁺ proliferative epithelium, stem-derived epithelium, secretory epithelium, proliferative epithelium, stromal cells, ciliated epithelium and unciliated epithelium. Color is proportional to log-transformed fold change of gene expression.
(G) GSEA between the SEC and WOI groups for proliferative epithelium.
(H) The single-cell pseudotime trajectory of SOX9⁺ proliferative epithelium, secretory epithelium and proliferative epithelium. Cells start at proliferative epithelium and progress to SOX9⁺ proliferative epithelium and secretory epithelium (left). There are seven major states over pseudotime (right). The black spot indicates the differentiation node between state 5 and state 6, indicating the direction from proliferative epithelium to SOX9⁺ proliferative epithelium and secretory epithelium, respectively.
(I)(L) The horizontal axis is the pseudotime point, and the vertical axis is the gene expression level. The solid line represents states 1, 2, 4, and 5 corresponding to Fig. S2H (I) or Fig. S2K (L). Different colors represent samples in the CTRL, SEC and WOI groups.
(J)(M) Heatmap of genes at the branch node regulating differentiation into SOX9⁺ proliferative epithelium or secretory epithelium (J), and ciliated epithelium or unciliated epithelium (M). The horizontal axis is the pseudo-time point (the pseudo-time point gradually increases from the middle to both sides). The vertical axis is the gene expression level, representing two differential directions on the left and right sides. Clusters represent the gene sets with a similar branch gene expression trend. Different colors represent the level of gene expression.
(K) Pseudotime trajectory of ciliated and unciliated epithelium. Cells start at ciliated epithelium and progress to unciliated epithelium (left). There are seven major states over pseudotime (right). The black spot indicates the differentiation node between state 5 and state 6, indicating the direction of ciliated and unciliated epithelium, respectively. Arrows indicate the direction of the pseudotime trajectory.

Comparisons between CTRL, SEC and WOI assembloids at the level of transcriptome and proteome.
(A) Venn diagram showing differential genes in pairs among the three groups. Log₂ FC (Fold Change)>1.2 or<-1.2, q value<0.05.
(B) Histogram showing pairwise comparison of differential gene expression (DEG) from bulk RNA-seq among the CTRL, SEC and WOI groups. qvalue<0.05, |logFC|>1.
(C) PCA plot computed with differentially expressed proteins in the micro proteomics of endometrial assembloids belonging to the CTRL, SEC and WOI groups.
(D) Venn diagram showing differential proteins in pairs among the three groups. Log₂ FC>1.2 or <-1.2, q value<0.05.
(E) Histogram showing pairwise comparison of differential proteins in micro proteomics among the CTRL, SEC and WOI groups.
(F) Scatterplot depicting the correlation between the transcriptome and proteome as for the WOI group and CTRL group. For transcriptome, FC>2 and p value<0.05 were defined as significantly upregulated. For proteome, FC>1.2 and p value<0.05 were defined as significantly upregulated.
(G) Circle diagram showing the functions of genes upregulated in both the transcriptome and proteome as for the WOI group compared to CTRL group.
(H) Verification of hypoxia response by the expression levels of HIF1α. Scale bar = 50 μm. *P ≤ 0.05, **P ≤ 0.005.
(I) Verification of lipid metabolism by the expression levels of SLC25A1. Scale bar = 50 μm. ****P ≤ 0.0001.

Receptive endometrial assembloids experienced epithelial-mesenchymal transition (EMT)
(A) GSEA between the SEC and WOI groups for proliferative epithelium.
(B) The single-cell pseudotime trajectory of proliferative epithelium, stromal cell, EMT derived cell and stem derived epithelium. Cells start at proliferative epithelium and progress to EMT derived cell. There are seven major states over pseudotime. The black spot indicates the differentiation node between state 4, 5 and state 6, indicating the direction from proliferative epithelium to EMT derived cell. Arrows indicate the direction of the pseudotime trajectory.
(C) The horizontal axis is the pseudotime point, and the vertical axis is the gene expression level. The solid line represents states 1, 2, 4, and 5 corresponding to Fig. S4B. Different colors represent samples in the CTRL, SEC and WOI groups.
(D) Heatmap of genes at the branch node regulating differentiation into EMT derived cell and stem-derived epithelium. The horizontal axis is the pseudo-time point (the pseudo-time point gradually increases from the middle to both sides). The vertical axis is the gene expression level, representing two differential directions on the left and right sides. Clusters represent the gene sets with a similar branch gene expression trend. Different colors represent the level of gene expression.
(E-F) Dot plots demonstrating the Cellphone DB analysis of relevant receptors and ligands of EMT derived cell (E) or stromal cell (F) with other cell types. The size of the dot represents the level of significance. The color of the dot indicates the mean of the average expression level of interacting molecule 1 in EMT derived cells (E) or stromal cells (F) and molecule 2 in other cell types.
(H) Proximity ligation assay (PLA) validating the interactions of SEMA3A-NRP1 and CD46-JAG1 in the CTRL, SEC and WOI assembloids. Red signals the interaction of two proteins. Nuclei were counterstained with DAPI. Scale bar = 20 μm.

Acknowledgements

This work was supported by National Natural Science Foundation of China (82192874 to H.Z), the National Key Research and Development Program of China (2021YFC2700301 to K.W), the Basic Science Center Program (31988101 to Z-J.C.), Fundamental Research Funds for the Central Universities(2022JC006 to K.W), the Fundamental Research Funds of Shandong University Taishan Scholars Program of Shandong Province (ts20190988 to H.Z.; tsqn201909194 to K.W), Innovative research team of high-level local universities in Shanghai (SHSMU-ZLCX20210201 to K.W).

We sincerely appreciate Prof. Tianqing Li (Kunming University of Science and Technology) and Prof. Shaorong Gao (Tongji University) for insightful comments and discussions.

We are grateful to Guangzhou Genedenovo Biotechnology Co., Ltd for assisting in sequencing and bioinformatics analysis. We also thank to YiKon Medical from China for technological assistance of ERT, Jingjie PTM Biolab (HangZhou) Co., Inc. for proteomics.

Additional information

Author contributions

H. Zhao, K. Wu and Z.-J.C conceived the project and supervised research. Y. Zhang, R. Zhao and H. Zhao designed the experiments. Y. Zhang and R. Zhao performed the experiments, analyzed and interpreted data. Y. Zhang wrote the manuscript. R. Zhao, Y. Li, B. Liu, and H. Zhao revised the manuscript. P. Liu provided human endometrium samples. C.Yang, J. Song, T. Li, C. Yin, M. Lu, Z. Hou and C. Zhang provided technical support. All authors revised and approved the manuscript.

Additional files

supporting information

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Article and author information

Author information

Yu Zhang
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China
- These authors contributed equally
Rusong Zhao
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China, Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China, Research Unit of Gametogenesis and Health of ART-Offspring, Chinese Academy of Medical Sciences (No.2021RU001), Jinan, China
- These authors contributed equally
Chaoyan Yang
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China
Jinzhu Song
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China
Peishu Liu
Department of Obstetrics and Gynecology, Qilu Hospital of Shandong University, Jinan, China
Yan Li
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China, Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China, Research Unit of Gametogenesis and Health of ART-Offspring, Chinese Academy of Medical Sciences (No.2021RU001), Jinan, China
Boyang Liu
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China, Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China, Research Unit of Gametogenesis and Health of ART-Offspring, Chinese Academy of Medical Sciences (No.2021RU001), Jinan, China
ORCID iD: 0000-0002-1285-4842
Tao Li
Department of Gynecology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China, Department of Gynecology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
Changjian Yin
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China
Minghui Lu
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China
Zhenzhen Hou
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China
Chuanxin Zhang
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China
Zi-Jiang Chen
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China, Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China, Research Unit of Gametogenesis and Health of ART-Offspring, Chinese Academy of Medical Sciences (No.2021RU001), Jinan, China, Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, China, Department of Reproductive Medicine, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
- For correspondence: chenzijiang@hotmail.com
Keliang Wu
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China, Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China, Research Unit of Gametogenesis and Health of ART-Offspring, Chinese Academy of Medical Sciences (No.2021RU001), Jinan, China
- For correspondence: wukeliang_527@163.com
Han Zhao
State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproductive Medicine, Institute of Women, Children and Reproductive Health, Cheeloo College of Medicine, Shandong University, Nanjing, China, National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China, Key Laboratory of Reproductive Endocrinology (Shandong University), Ministry of Education, Jinan, China, Shandong Technology Innovation Center for Reproductive Health, Jinan, China, Shandong Provincial Clinical Research Center for Reproductive Health, Jinan, China, Shandong Key Laboratory of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China, Research Unit of Gametogenesis and Health of ART-Offspring, Chinese Academy of Medical Sciences (No.2021RU001), Jinan, China
- For correspondence: hanzh80@sdu.edu.cn

Author Notes

Competing interests: No competing interests declared

Version history

Sent for peer review: July 19, 2023
Preprint posted: July 28, 2023
Reviewed Preprint version 1: October 30, 2023
Reviewed Preprint version 2: July 19, 2024
Reviewed Preprint version 3: September 19, 2025

Cite all versions

You can cite all versions using the DOI https://doi.org/10.7554/eLife.90729. This DOI represents all versions, and will always resolve to the latest one.

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This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Significance of findings

Strength of evidence

Abstract

Introduction

Results

Developing receptive endometrial assembloids in vitro

Developing receptive endometrial assembloids in vitro

Receptive endometrial assembloids mimicked the implantation - window endometrium

Receptive endometrial assembloids mimicked the implantation - window endometrium

Receptive endometrial assembloids recapitulate WOI-associated hormone response

Receptive endometrial assembloids recapitulate WOI-associated hormone response

Receptive endometrial assembloids possess enhanced energy metabolism

Receptive endometrial assembloids possess enhanced energy metabolism

Receptive endometrial assembloids increased the ciliary assembly and motility

Receptive endometrial assembloids increased the ciliary assembly and motility

Receptive endometrial assembloids experienced epithelial-mesenchymal transition (EMT)

The receptive endometrial assembloids possess the potential for embryo implantation

The receptive endometrial assembloids possess the potential for embryo implantation.

Discussion

Schematic diagram displaying the establishment and validation of receptive endometrial assembloids, and summarizing the characteristic biological events of implantation-window endometrium.

Materials and Methods

Establishment of endometrial assembloids

Hormone treatment of endometrial assembloids

Developing receptive endometrial assembloids in vitro

Various functions performed by all kinds of cells identified with scRNA-seq.

Comparisons between CTRL, SEC and WOI assembloids at the level of transcriptome and proteome.

Receptive endometrial assembloids experienced epithelial-mesenchymal transition (EMT)

Acknowledgements

Additional information

Author contributions

Additional files

References

Article and author information

Author information

Yu Zhang*

Rusong Zhao*

Chaoyan Yang

Jinzhu Song

Peishu Liu

Yan Li

Boyang Liu

Tao Li

Changjian Yin

Minghui Lu

Zhenzhen Hou

Chuanxin Zhang

Zi-Jiang Chen

Keliang Wu

Han Zhao

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Yu Zhang

Rusong Zhao