Three-dimensional observation of endometrial lumen morphology before embryo attachment.

a A schematic diagram of embryo implantation. b Schematic of the longitudinal and cross-sectional views of the uterine horns. c Two-dimensional (2D) and Tridimensional (3D) views of the uterine epithelium stained for E-cadherin. The luminal epithelium was segmented and magenta colored using Imaris. Scale bar: 1 mm (left) and 200 µm (middle and right). Schematic diagrams of luminal shapes on each pregnancy day are shown in the right panel. d The area of the luminal epithelium per sectional view was quantified. n = 3 for each sample and n = 3 for each day of pregnancy; in total, n = 9 sections were quantified. Data are presented as means ± SEM, ***P < 0.001, ****P < 0.0001 by one-way ANOVA followed by Bonferroni’s post-hoc test.

Dynamic morphological changes in the endometrial luminal epithelium occur on day 4 night, just before embryo attachment.

a Representative photographs of pregnant uteri from day 4 morning to day 5 morning, which were injected with blue dye to depict embryo attachment sites. Scale bar: 1 cm. b Percentage of implantation-positive females in (a). The number of replicates and percentage of implantation-positive females are shown above each bar. c The number of implantation sites in (a) and (b). Data are represented as means ± SEM. d 2D and 3D longitudinal views of uterine epithelia stained for E-cadherin. In the right panels, the luminal epithelium is segmented and colored in magenta using Imaris. Scale bar: 1 mm (left) and 200 µm (right). Asterisks indicate the locations of embryos.

Stress-related signals are activated in the uteri during the peri-attachment phase.

a UMAP of scRNA-seq cell types on days 4 and 5 of pregnancy. Dots indicate individual cells, and colors indicate different clusters. b Enrichment analyses for upstream transcription factors (left) and gene ontology (GO) (right) of highly expressed genes in the LE_activated vs. LE cells. c Network of GO terms related to the upregulated genes in the LE_activated cells compared with those in the LE cells. Each node represents an enriched term and is colored according to its cluster ID. d UMAP of different stromal cell types on days 4 and 5 of pregnancy. Dots indicate individual cells, and colors represent different clusters. e Enrichment analyses for upstream transcription factors (left) and gene ontology (GO) (right) of highly expressed genes in the Attached cluster compared with those in the other stromal clusters. f Network of GO terms related to the upregulated genes in the Activated cells compared with those in the other stromal clusters. Each node represents an enriched term and is colored according to its cluster ID. g Representative images of p38α (top) and phosphorylated p38α (pp38α; bottom) immunohistochemistry during days 1, 4, 6, and 8 of pregnancy. Scale bar: 100 µm. LE: luminal epithelia, GE: glandular epithelia, Str: Stroma, Em; embryo, Le; Luminal epithelium, St; Stroma. Arrowheads indicate embryos. At least three independent samples were evaluated for each day of pregnancy.

Morphological changes in the pre-attachment endometrial luminal epithelium are impaired in uterus-specific p38α KO mice.

a Efficient p38α deletion was confirmed by immunostaining for p38α and phosphorylated p38α (pp38α) in the uteri on day 4 of pregnancy. Scale bar = 100 µm. b Average litter size for each genotype. The numbers of replicates are shown in the graphs. Data represent the mean ± SEM, and ****P < 0.0001 by Student’s t-test. c Comparable numbers of flushed blastocysts (left) and their morphology (right). The graph shows the number of dams tested. Data represent the mean ± SEM, n.s.: not significant according to Student’s t-test. d Representative photographs of the uteri from each genotype (left) and the average number of implantation sites on day 5 of pregnancy. Flushed embryos from p38α uKO are shown next to the uterine photographs. Scale bar = 1 mm (uteri) and 100 µm (embryos). The number of replicates is shown on the graph. Data represent the mean ± SEM, and ****P < 0.0001 by Student’s t-test. e Representative photographs of the uteri from each genotype (left) and the average number of implantation sites on day 6 of pregnancy. Scale bar = 1 mm. The number of replicates is shown on the graph. Data represent the mean ± SEM, and ****P < 0.0001 by Student’s t-test. f Representative images of day 5 pregnant uteri stained for E-Cadherin in 3D. The luminal and glandular epithelia were segmented and colored magenta and cyan, respectively. Scale bar = 100 µm. g Representative 3D views of luminal epithelia during days 1–4 of pregnancy, segmented based on epithelial staining for E-cadherin. Scale bar = 100 µm.

P₄ supplementation to p38α uKO mice partially rescues structural changes and proliferation-differentiation switching (PDS) in the endometrial luminal epithelium before embryo attachment.

a, b Representative images of Ki67 immunofluorescence in the luminal epithelium of the uteri on day 4 morning. The percentages of Ki67-positive cells per total luminal cells are shown in (b). The number of replicates is shown on the graph. Data represent the mean ± SEM, **P < 0.01 by Student’s t-test. c The schedule of P4 treatment to p38α uKO females during days 1 to 5 of pregnancy. d Representative 3D longitudinal views of luminal epithelia from the control and p38α uKO uteri with or without P4 treatment, segmented from epithelial staining for E-cadherin. Scale bar = 100 µm. e Cross-sectional view of the luminal epithelia in (d). Scale bar = 100 µm. f, g Representative images of Ki67 immunofluorescence in the luminal epithelium on day 4 morning in the uteri from control and p38α uKO mice with or without P4 treatment. The percentages of Ki67-positive cells per total luminal cells are shown in (g). The number of replicates is shown on the graph. Data represent the mean ± SEM, **P < 0.01 and ***P < 0.001 by one-way ANOVA followed by Bonferroni’s post-hoc test.

Impaired Lif-Stat3 pathway affects embryo attachment in p38α uKO mice.

a Representative images of Lif in situ hybridization (top and middle) and phosphorylated Stat3 (pStat3) immunostaining (bottom) in day 4 uteri from control and p38α uKO mice with or without P4 treatment. Epithelial cells immunostained for CK-8 are shown in the top and middle panels. The area indicated by a dashed line in the top panel is shown in the middle panel. Scale bar = 50 µm. b The schedule of P4 and rLif treatment in p38α uKO female mice during days 1 to 5 of pregnancy. c Representative photographs of day 5 pregnant uteri from control and p38α uKO mice with or without P4 and rLif treatment. Arrowheads indicate sites of embryo attachment. Scale bar = 5 mm. d The number of implantation sites in (c) was calculated. The number of replicates is shown on the graph. Data represent the mean ± SEM, ***P < 0.001, ****P < 0.0001 and n.s.: not significant by one-way ANOVA followed by Bonferroni’s post-hoc test. f e Representative images of COX2 immunostaining on day 5 implantation sites from control and p38α uKO uteri treated with P4 and rLif. Scale bar = 100 µm. M: mesometrial pole; AM: anti-mesometrial pole. Arrowheads indicate embryos. f A representative image of day 20 pregnant uteri from p38α uKO mice treated with P4 and rLif. The percentage of deliveries per total number of pregnant females is shown on the right graph. The number of replicates is shown on the graph. Data represent the mean ± SEM, ***P < 0.001 by Student’s t-test.

Embryo attachment in p38α uKO mice is rescued by supplementation with P₄ and Lif, but luminal positioning remains inappropriate.

a Representative 3D views of the luminal epithelia collected from each genotype on days 5 and 6 of pregnancy. Scale bar = 100 µm, asterisks indicate embryos. b Representative 3D views of luminal epithelia on day 4 midnight, immediately before embryo attachment, collected from each genotype. Scale bar = 100 µm, asterisks indicate embryos. The graphs in the right panels show the luminal shapes for each genotype and condition. c Average area per sectional view calculated from the images shown in (b). Data represent the mean ± SEM, and P-values were determined by one-way ANOVA followed by Bonferroni’s post hoc test. d Average number of luminal branches per sectional view calculated from the images shown in (b). Data represent the mean ± SEM, and *P < 0.05, ***P < 0.001, ****P < 0.0001, n.s.: not significant by one-way ANOVA followed by Bonferroni’s post-hoc test.

p38α plays an important role in stromal differentiation to ensure appropriate epithelial-stromal interactions before embryo attachment.

a UMAP of scRNA-seq for various cell types from control uteri on day 4 evening and midnight, and from p38α uKO with or without P4 and rLif treatment on day 4 midnight. Dots indicate individual cells, and colors indicate different clusters. b UMAP of scRNA-seq of stromal cell types from control uteri on day 4 evening and midnight, and from p38α uKO with or without P4 and rLif treatment on day 4 midnight. Dots indicate individual cells, and colors indicate different clusters. c UMAP plots show the diffusion pseudotime of stromal cells. The colors of the spots indicate the pseudotime from early (blue) to late (dark red). d Boxplot showing the distribution of pseudo-time within different stromal cell clusters. Colors and labels indicate the cell types corresponding to those shown in (b). e Heatmap showing the outgoing (left) or incoming (right) communication strengths of key pathways between the major uterine endometrial cell types in each genotype and condition.

p38α uKO mice supplemented with P₄ and Lif show embryo invasion failure owing to sustained epithelial polarity.

a, b Luminal epithelial (a) and stromal (b) expression of Igf1 and Wnt5a determined using scRNA-seq in each genotype and condition. ***P < 0.001, ****P < 0.0001, ns: not significant by one-way ANOVA followed by Bonferroni’s post-hoc test. c Representative images of immunostaining for β-actin (red) and E-cadherin (green) in day 4 uteri from each genotype. M: mesometrial pole, AM: anti-mesometrial pole. Scale bar = 50 µm. d Representative images of immunostaining for CK-8 (green) on day 6 implantation sites from the control and p38α uKO mice treated with P4 and rLif. Areas demarcated by dashed lines in the top panels are shown in the bottom panels. M: mesometrial pole, AM: anti-mesometrial pole. Asterisks indicate embryos. Scale bar = 200 µm (top) and 100 µm (bottom). e H&E staining (upper) and visualization of the spatial transcriptome (lower) in day 5 implantation sites from the control and p38α uKO mice treated with P4 and rLif. M: mesometrial pole; AM: anti-mesometrial pole, LE: luminal epithelia, GE: glandular epithelia, Str: stroma. Arrowheads indicate embryos. f UMAP analysis of the spatial transcriptome dataset colored according to cell type for day 5 implantation sites in control (left) and p38α uKO uteri treated with P4 and rLif (right). Each dot color is mapped according to the spatial transcriptome visualized in the uterine sections, as shown in (e). g, h Network of GO terms related to the upregulated (g) or downregulated genes (h) in Str_uKO_specific compared to other stromal clusters. Each node represents an enriched term and is colored according to its cluster ID.

The role of uterine p38α in embryo attachment.

p38α contributes to flattening of the endometrial luminal surface and induction of luminal narrowing prior to embryo attachment, through pathways activated by P₄. It is also suggested that uterine p38α induces glandular Lif, thus inducing embryo attachment. In addition to these pathways, p38α contributes to a decrease in epithelial polarity by activating epithelial-stromal interactions, thus resulting in successful embryo attachment and invasion.