Endometrial organoids possess endometrial morphology and function

(A) Human endometrial organoids constructed from adult stem cells were treated with expansion medium (ExM) (CTRL) or subjected to hormonal stimulation. Timeline of endometrial organoid cultured by ExM (CTRL), ovarian steroid hormones simulating secretory phase (SEC), ovarian steroid hormones combining PRL and placental hormones to mimic the window of implantation (WOI).

(B) Endometrial organoids in the CTRL, SEC and WOI groups displayed similar growth patterns during the culture period. 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 endometrial organoids (100x, up-left). Screenshot of video S2 displaying the stromal cells growing in fibrous pattern and forming an extensive network in the endometrial organoids (200x, up-right). (The yellow arrows indicate stromal cells and the white arrows indicate endometrial glands). 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 endometrial organoids in vitro. Nuclei were counterstained with DAPI. Scale bar = 40 μm. of endometrium in vivo and endometrial organoid in vitro. Scale bar = 20 μm.

(E) IF analysis of proliferation and apoptosis indicated by Ki67 and cleaved caspase-3, respectively. Nuclei were counterstained with DAPI. Scale bar = 40 μm.

Developing receptive endometrial organoids in vitro mimicking the implantation-window endometrium

(A) T-SNE plot of scRNA-seq data from three individual endometrial organoids of the CTRL, SEC and WOI groups (left). T-SNE plot of combined scRNA-seq data from the three kinds of organoids and mid-secretory endometrium (right).

(B) Exhibition of stromal cell marked by vimentin through combination of organoid clearing, IF and light sheet microscopy. Nuclei were counterstained with DAPI. The arrowhead indicates stromal cells. Scale bar = 40 μm (left), Scale bar = 30 μm (right).

(C) Exhibition of immune cell marked by CD44 and endometrial gland marked by FOXA2 through combination of organoid clearing, IF and light sheet microscopy. Nuclei were counterstained with DAPI. The arrowhead indicates immune cells. Scale bar = 30 μm (left), Scale bar = 10 μm (right).

(D) Flow cytometric analysis of T cells and macrophages in the endometrial organoid. Gating strategy used for determining white blood cells (CD45+ cells), T cells (CD45+CD3+ cells) and macrophages (CD45+CD68+CD11b+ cells).

(E) Heatmap and bubble diagram illustrating highly expressed genes as well as GO functions enriched in both organoids during the WOI and mid secretory endometrial tissue in terms of SOX9+LGR5+ epithelium, stem-derived epithelium, secretory epithelium, proliferative epithelium, unciliated epithelium, stromal cells and EMT-derived stromal cells. The color of heatmap represents log-transformed fold change of gene expression.

(F) Heatmaps showing differentially expressed TFs of endometrial organoids and endometrium in the secretory epithelium (left) and EMT-derived stromal cells (right). The color represents log-transformed fold change of gene expression.

(G) Endometrial receptivity evaluation of endometrium and their derived WOI organoids through ERT. Asterisks indicate individual samples.

Receptive endometrial organoid exhibits the implantation window at the transcription and protein levels

(A) Principal component analysis (PCA) plot computed with differentially expressed genes in the bulk transcriptome of endometrial organoids belonging to the CTRL, SEC and WOI groups.

(B) Heatmap showing that enrichment of differentially expressed genes for the terms of extracellular matrix remodeling, hormone response, negative regulation of cell proliferation, monocarboxylic acid metabolism, lipid metabolism, cell adhesion, negative regulation of cell differentiation and RHO GTPase signaling. The color represents log-transformed fold change of gene expression.

(C) Responsiveness to progesterone and estrogen was evaluated by IF to PRA/B and OLFM4 with IF respectively. Scale bar = 40 μm, **P ≤ 0.005.

(D) Exhibition of implantation marker (FOXO1) and endometrial gland marker (FOXA2) through combination of organoid clearing, IF and light sheet microscopy. Nuclei were counterstained with DAPI. **P ≤ 0.005.

(E) PCA plot computed with differentially expressed proteins in the microproteomics of endometrial organoids belonging to the CTRL, SEC and WOI groups.

(F) Dot bar diagram exhibiting protein expression levels related to cilia in the CTRL and WOI groups (left). Dot bar diagram displaying tubulin, integrin and PI3K-AKT pathway-related protein expression levels in the SEC and WOI groups (right).

(G) IF analysis of cilia assembly marked by acetyl-α-tubulin. Nuclei were counterstained with DAPI. The arrowhead indicates cilia. Scale bar = 50 μm. *P ≤ 0.05.

(H) Electron micrograph of the CTRL (top) and WOI (bottom) endometrial organoid showing pinopodes (P), glycogen granule (asterisk), microvilli (white arrows) and cilia (orange arrows). Scale bar = 1 μm.

Receptive endometrial organoid exhibits the implantation window at single cell level

(A) Bar graph exhibiting various percentages of each cell type in the three groups.

(B) Gene set enrichment analysis (GSEA) between the SEC and WOI groups for the secretory epithelium.

(C-E) Pseudotime trajectory showing the transformation between proliferative and secretory epithelium (C), ciliated and unciliated epithelium (D), proliferative epithelium and EMT-derived stromal cells (E) in the CTRL, SEC and WOI groups. Arrows indicate the direction of the pseudotime trajectory.

(F-G) Dot plots demonstrating the Cellphone DB analysis of relevant receptors and ligands of ciliated epithelium (F) and EMT-derived stromal cells (G) 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 ciliated epithelium or EMT-derived stromal cells and molecule 2 in other cell types.

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