Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids

Abstract

Decidual remodelling of midluteal endometrium leads to a short implantation window after which the uterine mucosa either breaks down or is transformed into a robust matrix that accommodates the placenta throughout pregnancy. To gain insights into the underlying mechanisms, we established and characterized endometrial assembloids, consisting of gland-like organoids and primary stromal cells. Single-cell transcriptomics revealed that decidualized assembloids closely resemble midluteal endometrium, harbouring differentiated and senescent subpopulations in both glands and stroma. We show that acute senescence in glandular epithelium drives secretion of multiple canonical implantation factors, whereas in the stroma it calibrates the emergence of anti-inflammatory decidual cells and pro-inflammatory senescent decidual cells. Pharmacological inhibition of stress responses in pre-decidual cells accelerated decidualization by eliminating the emergence of senescent decidual cells. In co-culture experiments, accelerated decidualization resulted in entrapment of collapsed human blastocysts in a robust, static decidual matrix. By contrast, the presence of senescent decidual cells created a dynamic implantation environment, enabling embryo expansion and attachment, although their persistence led to gradual disintegration of assembloids. Our findings suggest that decidual senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.

Keywords: assembloid; cell biology; decidualisation; embryo implantation; endometrium; human; organoid; senescence.

Figure 1.. Establishment of endometrial assembloids.

(A) Schematic for establishing endometrial assembloids. (B) Structural appearance of hematoxylin and eosin stained secretory endometrium, E-cadherin labelled gland-like organoids, and E-cadherin and vimentin stained endometrial assembloids. Scale bar = 50 µm. (C) Schematic summary of experimental design. (D) Secreted levels of PRL and CXCL14 were measured by ELISA in spent medium at the indicated timepoints. Data points are coloured to indicate secretion in assembloids established from different endometrial biopsies (n = 3). (E) Representative immunofluorescence labelling of laminin and vimentin, progesterone receptor (PR), glycodelin, and osteopontin (OPN) in undifferentiated (day 0, top panels) and decidualized (day 4; bottom panels) assembloids. Nuclei were counterstained with DAPI. Scale bar = 50 µm. ELISA data in (B) are available in Figure 1—source data 1.

Figure 2.. Characterization of a minimal differentiation medium for endometrial assembloids.

Parallel gland-like organoids (red) and assembloids (blue) were established from three endometrial biopsies and decidualized with 8-bromo-cAMP and MPA for 4 days in either expansion medium (ExM), base medium (BM), or BM with each exogeneous factor added back individually (+). Induction of PAEP and SPP1 was used to monitor the glandular differentiation. The grey bar indicates the composition of the minimal differentiation medium selected for further use (BM supplemented with NAC, E2, cAMP, and MPA). Data are presented as fold-change relative to expression levels in undifferentiated organoids or assembloids cultured in ExM+ E2. Bars present minimal, maximal, and median fold-change. * and ** indicate p<0.05 and p<0.01 obtained by Friedman’s test for matched samples. Relative expression values for biological replicates are available in Figure 2—source data 1.

Figure 3.. Decidualizing assembloids mimic midluteal endometrium.

(A) Schematic overview of experimental design. ExM: expansion medium; MDM: minimal differentiation medium. (B) Uniform Manifold Approximation and Projection (UMAP) visualizing epithelial and stromal subsets (EpS and SS, respectively) identified by single-cell transcriptomic analysis of undifferentiated and decidualized assembloids. A transitional population (TP) consisting of cells expressing epithelial and stromal markers is also shown. Dotted lines indicate the separation of EpS and SS in UMAP_1 and of undifferentiated and differentiated subpopulations in UMAP_2. Dotted circles indicate ciliated (EpS3) and TP, which did not fit these broad segregations. (C) Composite heatmaps showing relative expression (Z-scores) of epithelial marker genes across the menstrual cycle in vivo and in undifferentiated and decidualized assembloids. Highlighted in green are genes that mark the midluteal window of implantation (Díaz-Gimeno et al., 2011), whereas genes encoding secreted proteins are indicated by * (Uhlén et al., 2015). See also Figure 3—figure supplement 1. (D) Dot plots showing GO terms related to biological processes enriched in different epithelial populations in decidualizing assembloids. The dot size represents the number of genes in each GO term and the colour indicates FDR-corrected p-value. (E) Composite heatmaps showing relative expression (Z-scores) of stromal marker genes across the menstrual cycle in vivo and in undifferentiated and decidualized assembloids. Highlighted in green are genes that mark the midluteal window of implantation (Díaz-Gimeno et al., 2011), whereas genes encoding secreted proteins are indicated by * (Uhlén et al., 2015). (F) Dot plots showing GO terms related to biological processes enriched in different stromal subpopulations in decidualizing assembloids. See also Figure 3—figure supplements 1 and 2 and 3. Complete epithelial subpopulation marker lists can be found in Figure 3—source data 1. GO analysis outputs can be found in . Complete stromal subpopulation marker lists can be found in .

Figure 3—figure supplement 1.. Heatmap showing relative expression (Z-scores) of genes encoding the cyclin-dependent kinase inhibitors p16^INK4a and p21^CIP1 as well as SASP-related genes in epithelial and transitional subpopulations in decidualizing assembloids.

Supports Figure 3C.

Figure 3—figure supplement 2.. Heatmap showing relative expression (Z-scores) of epithelial-mesenchymal transition/mesenchymal-epithelial transition (EMT/MET), epithelial and mesenchymal marker genes in the transitional population (TP), epithelial (EpS4-5) and stromal (SS3-5) subpopulations in decidualizing assembloids.

Supports Figure 3C.

Figure 3—figure supplement 3.. Heatmap showing relative expression (Z-scores) of genes encoding the cyclin-dependent kinase inhibitors p16^INK4a and p21^CIP1 as well as secretory and SASP-related genes in stromal subpopulations (SS3-5) in decidualizing assembloids.

Supports Figure 3D.

Figure 4.. Putative receptor-ligand interactions in decidualizing assembloids.

(A) Heatmap showing the total number of cell-cell interactions predicted by CellPhoneDB between different subpopulations in decidualizing assembloids. (B) Dot plots of representative ligand-receptor interactions between stromal subsets (SS) and epithelial subsets (EpS) (upper panel) and EpS and SS (lower panel) in decidualizing assembloids. Circle size and colour indicate p-value and the means of the average expression value of the interacting molecules, respectively. Shaded boxes were used to group putative interactions by level of selectivity. (C) Dot plot of representative ligand-receptor and receptor-ligand interactions between stromal subpopulations in decidualizing assembloids. Direct and indirect tyrosine kinase interactions are indicated by red and blue labels, respectively. Complete tables of predicted ligand-receptor interactions can be found in Figure 4—source data 1.

Figure 5.. Tyrosine kinase-dependent stress responses determine the fate of decidual cells.

(A) Schematic overview of experimental design. ExM: expansion medium; MDM: minimal differentiation medium. (B) Uniform Manifold Approximation and Projection (UMAP) visualization (left panel) and relative proportions (right panel) of subpopulations in endometrial assembloid decidualized in the presence or absence of dasatinib. (C) Number of differentially expressed genes (DEGs) in each subpopulation in response to dasatinib pre-treatment. (D) Secreted levels of CXCL8 and decidual cell factors in spent medium from assembloids treated with or without dasatinib. Secreted levels in individual assembloids established from four different endometrial assembloids decidualized with or without dasatinib are shown by dotted and solid lines, respectively. Full lists of DEGs and associated GO analysis can be found in Figure 5—source data 1 and Figure 5—source data 2, respectively. Data used in (D) are available in Figure 5—source data 3.

Figure 6.. Impact of decidual senescence in assembloids on co-cultured human blastocysts.

(A) Diagram showing experimental design. ExM: expansion medium; MDM: minimal differentiation medium; EM: embryo medium. (B) Schematic drawing of co-culture method. (C) Representative time-lapse images of blastocysts embedded in assembloids following decidualization for 96 hr in the absence (upper panels) or presence (lower panels) of dasatinib. Scale bar = 100 µm. See also Figure 6—figure supplement 1. (D) Embryo diameters (µm) measured over 72 hr when embedded in decidualizing assembloids pre-treated with or without dasatinib. (E) OCT4 and GATA6 immunofluorescence marking the epiblast and hypoblast, respectively, in a blastocyst attached by proliferating polar trophectoderm (arrowhead) to decidual assembloids. Scale bar = 50 µM. (F) Secreted levels of human chorionic gonadotropin (hCG) in blastocyst-endometrial assembloid co-cultures. Individual embryo diameter measurements for biological replicates in (D) are available in Figure 6—source data 1. Individual ELISA data used in (F) are available in Figure 6—source data 2.

Figure 6—figure supplement 1.. Stromal migration towards the polar trophectoderm of expanding embryos in differentiated endometrial assembloids.

Images were captured by time-lapse microscopy and annotated to indicate the frontier of the stromal egress (white dotted line).

Figure 6—figure supplement 2.. Dasatinib prevents disintegration of decidualizing assembloids.

Time-course images of endometrial assembloids in minimal differentiation medium (MDM) supplemented or not with dasatinib. Scale bar = 50 µm. Supports Figure 6C.

Author response image 1.. (A) Expression of FOXA2, a glandular epithelial marker (green font), and putative luminal epithelial marker genes (black font) in assembloid subpopulations.

Dot size indicates the proportion of cells expressing the marker, while colour indicates the level of expression. Note that none of the cell populations express a compelling luminal epithelial marker gene signature. (B) Expression of the same markers in laser-capture micro-dissected endometrial glands in vivo, obtained during the early- and mid-luteal phase (LH⁺5 and LH⁺8, respectively).

Author response image 2.. Comparison of secreted levels of IL-8, CXCL14 and TIMP3 in two independent primary endometrial stromal cells maintained in standard 2D cultures in response to standard growth medium (10% DCC-DMEM+E2) or assembloid expansion medium (ExM+E2) and standard differentiation medium (2% DCC-DMEM+cAMP+MPA) or assembloid differentiation medium (MDM+E2+cAMP+MPA), as indicated.

Author response image 3.