An atlas of human endometrium from non-pregnant and pregnant women
To evaluate the heterogeneity and dynamic evolution characteristics of decidualized stromal cells, we analyzed the expression of marker molecules of decidualization in human endometrium during proliferative and secretory phases, and decidualized endometrium during early pregnancy. As shown, decidualization has begun at secretory endometrium during WOI time with a small number of IGFBP1+ and PRL+ cell population (Fig. 1A). Even in decidualized endometrium, some populations of stromal cell do not express IGFBP1 and PRL (Fig. 1A), suggesting that the heterogeneity of stromal cells is particularly significant in the process of decidualization. More interestingly, this hypothesis was verified in the pseudo-time analysis of decidual stromal cells in early pregnancy based on the previous report data (see “Materials and methods”) [28]. As shown, subset 1 of decidual stromal cells (dS1: IGF1high, IGFBP1−, PRL−) should be precursor cells of dS2 (IGF1low, IGFBP1+, PRL−) and dS3 (IGF1−, IGFBP1high, PRL+) (Fig. 1B).
To further determine the full repertoire of cell types and differentiation characteristics present in endometrium during decidualization, we isolated cells from endometrium from healthy controls at the WOI timing (endometrium, n = 3) and normal decidua from early pregnancy (decidua, n = 3) (see “Materials and methods”), and generated single-cell transcriptome libraries on the droplet-based 10X Genomics Chromium System (Additional file 1: Figure S1A). After computational quality control and integration of transcriptomes from both technologies, we obtained a total of 29, 855 cells (19 clusters) endometrial and decidual single-cell transcriptomes, and preformed graph-based clustering of uniform manifold approximation and projection (UMAP) (Fig. 1C, Additional file 1: Figure S1B) and used cluster-specific marker genes to annotate the clusters (Additional file 1: Figure S1C). Overall, all sequenced endometrial and decidual cells were assigned to six main classes of cells: fibroblast-like stromal cells (SC; expressing HOXA10, MME, COL1A1, and IGF1, 22,480 cells), epithelial cells (EPC; expressing KRT7, KRT8, KRT18, and EPCAM, 373 cells), NK cells (expressing PTPRC and NCAM1, 4152 cells), macrophages (expressing PTPRC and CD14, 1064 cells), T cells (expressing PTPRC and CD3D, 1132 cells), and endothelial cells (EC, expressing PECAM1 and ACKR1, 654 cells) (Fig. 1C, Additional file 1: Figure S1C).
Next, we displayed the characterization of cell contribution between endometrium and decidua from different donors (Fig. 1D, Additional file 1: Figure S1D and S1E). The subpopulation heterogeneity of six main cell clusters was obvious, especially SC (Fig. 1D). Accumulating evidence has indicated recruitment, enrichment, and redistribution of immune cells in decidua during early pregnancy [11,12,13]. As expected, more immune cell (IC) accumulation was observed in decidua compared to endometrium, especially NK cells and macrophages (Additional file 1: Figure S1D, S1E and Additional file 2: S2).
IGF1+ stromal cells initiate endometrial decidualization
Since SC is the most abundant cell type in the endometrium and decidua, we initially explored endometrial and decidual SC and identified seven subset clusters of SC: IGF1+MMP11+DIO2+MKI67−PRL−IGFBP1− Rem-SC (SC with high tissue remodeling property), IGF1lowADAMTS5highPRLlowIGFBP1+ dRem-SC (decidualized SC with high tissue remodeling property), IGF1+FABP5+IGFBP3+PRL−IGFBP1− PreSec-SC (SC with secretory ability), IGF1lowPLA2G2A+IGFBP1low Sec-SC (SC with high secretory ability), IGF1−PRLhighIGFBP1+ADAMTS5+ dSec-SC, TOP2A+MKI67+ Pro-SC (SC with high proliferation), and ACTA2+RGS5+ endometrial mesenchymal stem cells (eMSCs) (Fig. 2A,B, Additional file 3: Figure S3A). In particular, a large number of genes involved in tissue remodeling (e.g., extracellular matrix (ECM) organization, cell adhesion, embryo implantation and placenta development), response to cAMP and cellular metabolic process, and cell cycle and proliferation were highly enriched in dRem-SC and Rem-SC, dSec-SC, and Sec-SC and PreSec-SC, and Pro-SC, respectively (Additional file 3: Figure S3B). Together with a small number of eMSCs, FABP5+PreSec-SC, MMP11+Rem-SC, and MKI67+Pro-SC constituted the main population of SC in endometrium. Differentially, the stroma of decidua was mainly based on dRem-SC, dSec-SC, and PLA2G2A+Sec-SC (Fig. 2C, Additional file 4: Figure S4A and S4B). This finding indicates a dramatical subpopulation heterogeneity of SC in endometrium and decidua. With the implantation of embryo and the maturation of endometrial decidualization, ECM remodeling, angiogenesis, autophagy levels, immune regulation, cell metabolism, and responses to oxidative stress of SC were increasingly active. In contrast, the proliferative potential of SC gradually decreased, as well as IGF1 (Additional file 4: Figure S4C and S4D).
Surprisingly, common decidualization-related genes, including leukemia inhibitory factor (LIF), Indian hedgehog (IHH), and dipeptidyl Peptidase 4 (DPP4) [30,31,32], were rarely expressed in SC of both endometrium and decidua (Additional file 5: Figure S5A). The classical marker genes (i.e., PRL and IGFBP1) of decidualization, and the enzymes (i.e., adenylate cyclase 1, ADCY1) for catalyzing the formation of cAMP (a strong inducer of decidualization) were highly expressed in decidualized SC, but were barely expressed in SC (PreSec-SC, Rem-SC and Pro-SC) of endometrium at the WOI (Fig. 2B, Additional file 5: S5A and S5B). More importantly, this process was accompanied by the advantage exchange between IGF1 and its receptor (IGF1R) levels. The data together suggest that the IGF1+ stromal cells located earlier in the endometrium should be involved in the initiation of decidualization.
To explore the initiating mechanism of decidualization, we first focused on two subpopulations of SC (Rem-SC and dRem-SC) with tissue remodeling (Additional file 6: Figure S6A), and constructed a new trajectory about Rem-SC and dRem-SC by a standard pseudo-time analysis (Additional file 6: Figure S6B and S6C). Notably, we observed a notable discontinuity between Rem-SC and dRem-SC. Endometrial Rem-SC was the starting point, which went through Rem-SC coexisted in endometrium and decidua, and the final endpoint was decidual dRem-SC (Additional file 6: Figure S6C). Further analysis showed that the expression of cell proliferation and differentiation (e.g., IGF1, and SFRP1, a modulator of Wnt signaling), apoptosis regulation (e.g., BCL2 Interacting Protein 3 Like, BNIP3L), and ECM disassembly (e.g., MMP16)-related genes were markedly reduced from the Rem-SC to dRem-SC in the trajectory (Fig. 2D, Additional file 7: Figure S7A and S7B). Importantly, gene expression about translation, positive regulation of cAMP catabolic process, cellular response to growth factor, and insulin receptor signaling pathway reached the peak in the last stage of Rem-SC and rapidly decreased in dRem-SC. In contrast, dRem-SC was characterized by high expression of ECM organization, embryo implantation, cell adhesion, angiogenesis, positive regulation of macrophage, and cytokine production-related genes (Additional file 7: Figure S7A and S7B).
Subsequently, the early expression of IGF1 in Rem-SC, PreSec-SC, and Pro-SC of endometrium at the WOI, like ESR1 and PGR, had attracted our attention (Fig. 2B–D). Interestingly, IGF1R was mainly expressed in dRem-SC with high levels of decidualized genes (e.g., PRL, PRLR, and IGFBP1), which was present in small amounts in endometrium (Fig. 2D). Further bioinformatics analysis displays IGF1 should be involved in the PGR and ESR1-triggered decidualization (Additional file 8: Figure S8A). As expected, medroxyprogesterone (MPA) plus estradiol (E2) upregulated IGF1 expression of human ESCs dramatically in vitro (Additional file 8: Figure S8B). More importantly, the expression of decidualization-related genes, especially PRL and IGFBP1, was quickly increased in human ESCs or primary DSCs stimulated by recombinant human IGF1 protein rather than MPA plus E2 (Fig. 2E, Additional file 8: Figure S8C). This process should be dependent on the strong effect on ADCY1 and ADCY3 for cAMP production, and high levels of PRL for further accelerating the decidualization (Fig. 2E, Additional file 8: Figure S8C and S8D). The data above indicate that IGF1+ SC possibly initiates endometrial decidualization under regulation of progesterone and estrogen (Fig. 2F).
IL1B+ dSec-SC with active metabolism maintains decidual homeostasis
Decidual transformation is associated with the accumulation of glycogen and lipid droplets in the expanding cytoplasm, increased activities of endocytosis/exocytosis, and protein biosynthesis [1, 11, 33,34,35]. In other three subpopulations of SC, PreSec-SC and Sec-SC had high levels of protein translation-related genes (RPL12 and EIF3F), and dSec-SC, only found in the decidua, were rich in genes associated with cell growth, phagosome, and multiple metabolic pathways (e.g., oxidative phosphorylation, glutathione metabolism, glycolysis, gluconeogenesis, and lipid metabolism) (Fig. 3A–C, Additional file 9: S9A and S9B). Additionally, dSec-SC with a high level of PRL and IGFBP1 were involved in regulating macrophage chemotaxis and cytokine production, and NK cell-mediated cytotoxicity, and characterized by high levels of autophagy (e.g., MAP1LC3B, ATG5), cellular response to hypoxia (e.g., FOXO1, CAV1), oxidation–reduction process (e.g., P4HB, MAOA), response to reactive oxygen species (ROS) (e.g., GPX1, GPX4), responses to lipopolysaccharide (e.g., LITAF, CYP11A1, DADM9, and B2M), and glutathione derivative biosynthetic process (e.g., MGST1, MGST3)-related genes (Fig. 3C).
This process of blastocyst invasion of the maternal uterine endometrium has been reported to depend on an evolutionarily conserved inflammatory response including IL1B, INFG, TNF, and IL6 [36,37,38]. As shown, IL1B was mainly derived from dSec-SC in decidua, which also highly expressed IL1B and receptors (IL1R1 and IL1R2) (Fig. 3D and Additional file 9: S9C). The potential interaction of IL1B and receptors between these five clusters of SCs and other cells (EPC, NK cells, macrophages, T cells, and EC) in decidua were predicted (Fig. 3E). To confirm the role of IL1B in SC, primary decidual stromal cells were treated with recombinant IL1B protein in vitro. IL1B significantly upregulated the levels of ROS in DSC, along with the increases of various anti-oxidative stress genes (e.g., GPX1), but no influenced cell apoptosis (Fig. 3F–H). Collectively, these findings suggest that dSec-SC with activate metabolism, on the one hand, provides essential nutrients and immunotolerance environment for blastocyst implantation and placental development and, on the other hand, maintains decidual homeostasis by powerful phagocytosis, and strong protection against oxidative stress caused by various factors (e.g., inflammatory response, hypoxia, and lipid metabolism).
IL1B+ dSec-SC triggers the apoptosis of epithelial cells during decidualization
As the site of blastocyst adhesion, the luminal epithelium is perceived as the crucial site for uterine receptivity [39]. Markers that distinguish the different endometrial and decidual EPC populations identify 3 clusters: KRT18+FOXJ1+RPS2+ Ciliated epithelial cell (Cil-Epi), KRT18+FOXJ1−RPS2highDPP4− glandular epithelial cell (PreSec-Epi), and KRT18+FOXJ1−DPP4+RPS2+ glandular epithelial cell (Sec-Epi, with high decidualization and immune regulation abilities) (Fig. 4A, B, and Additional file 10: S10A-C). Of note, KRT18+HLA-G+ extravillous trophoblasts (EVT) are observed in two samples of decidua (Fig. 4B, C). PreSec-Epi predominate in endometrium at the WOI, but there is more Sec-Epi in decidua (Fig. 4C, D), which was characterized by high levels of decidualized genes (e.g., IGFBP1, LIF, DPP4, CXCL14) (Additional file 10: Figure S10B and S10C).
In humans and mice, endometrial decidualization is characterized by apoptosis in the glands [40,41,42,43]. According to functional enrichment analysis, the genes involved in ECM organization, cell adhesion, embryo implantation, regulation of cell migration, aging, apoptotic process, macrophage activation, angiogenesis, and autophagy were highly enriched in Sec-Epi compared to PreSec-Epi (Fig. 4E). The results of TUNEL assay confirmed that there was a higher level of apoptosis of glandular epithelial cell in decidua than that in endometrium (Fig. 4F). Interestingly, we observed DPP4+ Sec-Epi in the lumen of decidua is discontinuous and interspersed with IL1B+ dSec-SC and PLA2G2A+ Sec-SC using the immunofluorescence staining (Fig. 4G and Additional file 11: S11A). To investigate the potential role of IL1B produced by dSec-SC in epithelial cells (Additional file 11: Figure S11B), human endometrial epithelial cell (EEC) line was treated with or without IL1B for 24 h. As shown, IL1B induced the apoptosis of EECs markedly in vitro (Fig. 4H). These data indicate that IL1B+ dSec-SC is involved in the apoptosis of epithelial cells during decidualization.
AREG+ NK cell accelerates decidualization by interacting with IGF1+ SC
Although the potential mechanisms are largely unknown, there is evidence for a large number of immune cell infiltration in the endometrium at the WOI, and enrichment and redistribution in decidua at the maternal–fetal interface, including NK, macrophage, T and dendritic cell (DC) [11,12,13, 44]. Here we observed a very rich population of immune cell, such as CD45+CD3−CD56+ NK cell, CD45+CD14+ macrophage, CD45+CD3+CD8−CD4+ T cell, and CD45+CD3+CD4−CD8+T cell in decidua compared with endometrium (Additional file 12: Figure S12 and Additional file 13: S13).
As an abundant leukocyte population in the non-pregnant endometrium and pregnant decidua, macrophages are considered to play a central role in the establishment and maintenance of normal pregnancy [45]. However, the change and characteristic of macrophages during decidualization remain unclear. Here, we identified seven main subsets from total macrophage/DC cluster (Additional file 14: Figure S14A and S14B). M1 cells highly expressed folate receptor beta (FOLR2), M2 cells highly expressed secreted phosphoprotein 1 (SPP1, also known as OPN) (Additional file 14: Figure S14B and S14C), M3 cells expressed CX3CR1 (an important chemokine receptor for macrophage migration and recruitment), pro-macrophages expressed MKI67, monocytes expressed S100 calcium-binding protein A9 (S100A9), cDC1 cells expressed c-type lectin domain containing 9A (CLEC9A) but not CD1C, and cDC2 cells expressed CD1C. Additionally, the closed crosstalk between four subpopulations (M1, M2, M3, and pro-macrophages) of macrophages and other cells was predicted in endometrium and decidua, including the regulation of focal adhesion, ECM receptor interaction, cytokine-cytokine interaction, and NK cell-mediated cytotoxicity (Additional file 15: Figure S15A). Further analysis showed that M1 cluster displays a M2-like phenotype (high levels of MRC1 and CD209) and high levels of IGF1 and PDGFB, suggesting that M1 cluster should be involved in tissue remodeling, decidualization, and immune tolerance (Additional file 15: Figure S15B). The chemokine genes (e.g., CCL2, CCL3, CCL4), SPP1, VEGFA, and VEGFB were enriched in M2 cluster, indicating that M2 cluster participates in the recruitment of immune cells and angiogenesis. M3 cluster had high levels of CD86, IFNGR1, HLA-DQA2, HLA-DPB1, and PLD4, contributing to the immune response and antigen presentation. Compared to endometrium, there are more macrophages in decidua, especially M1 cluster (Additional file 15: Figure S15C). FOLR2 has been reported to regulate folate uptake and absorption in tissue-resident M2-like macrophages, which should be dependent on the activin A (encoded by INHBA) [46]. Activins are important autocrine and paracrine regulators of endometrial decidualization and the priming of endometrium for implantation [42]. The expression of INHBA, INHBB, and INHA subunits are increased in decidualized stromal cells at the onset of decidualization [47]. More importantly, INHBA and INHA were expressed in IGF1+ Rem-SC, dRem-SC, and dSec-SC, respectively (Additional file 15: Figure S15D). These data above suggest that enrichment of FOLR2+IGF1+M1 macrophage cluster with a M2-like phenotype possibly induced by SC-derived activin A results in maternal–fetal immune tolerance, tissue remodeling, and decidualization during early pregnancy.
NK cells are the most distinguishable lymphocytes during the first trimester of pregnancy, constituting 50 ~ 70% of all leukocytes in human decidua [27, 28]. Here, we identified 5 clusters of NK cells in endometrium and decidua (Fig. 5A, and Additional file 16: Figure S16), including ITGA1+NCAM1highITGAE−TNFRSF4− NK1, NCAM1+CD160+CXCR4+ NK2, ITGA1+NCAM1highTNFRSF4+AREG+ NK3, ITGA1+NCAM1highTNFRSF4+AREG+CSF1+ NK4 cells and MKI67+TOP2A+ pro-NK cells (Fig. 5B, C). Among these, pro-NK cells with high proliferation potential are metabolically active (e.g., pyruvate metabolism, carbon metabolism, glycolysis / gluconeogenesis, and oxidative phosphorylation) (Additional file 17: Figure S17). NK2 cells highly expressed various chemokines (e.g., CCL5, CCL3L3, CCL4L2, and CCL3), CXCR4 and CD160, but had a low cytotoxicity, which were mainly located in endometrium at the WOI (Fig. 5C, Additional file 18: Figure S18A and S18B). Therefore, NK2 cells should contribute to immune recruitment and infiltration. NK1 cells with high levels of ITGAX, NCAM1, and COL1A1 are involved in ECM receptor interaction with other cells, such as SC and EVT (Fig. 5C, Additional file 18: Figure S18C and S18D). NK3 cells with high levels of GZMB and GZMH, KLRC1, and AREG, and NK4 cells with high levels of KIR genes (KIR3DL1 and KIR3DL2), granule protein-coded genes (GNLY and GZMB), AREG and CSF1, were mainly enriched in decidua (Fig. 5C and Additional file 18: S18A). Among these, NK4 and pro-NK, NK3, and NK2 are consistent with the characteristics of dNK1 and dNKp, dNK2, and dNK3 reported by Vento-Tormo and colleagues using scRNA-seq [28], respectively. AREG, also known as amphiregulin, has been reported to regulate the production of GM-CSF, co-operate with IGF1 for regulation of cell growth, and directly stimulate trophoblast invasion [48,49,50]. CSF1 contributes to trophoblast invasion (Additional file 18: Figure S18E and S18F), the recruitment, and M2 differentiation of macrophage [51, 52]. Additionally, AREG is predicted to regulate the IGF1 and CSF1 by the protein–protein interaction (PPI) network analysis (Fig. 5D). The data above and the results in vitro (Fig. 5E,F) suggest that AREG+ NK3 cells should promote the differentiation of CSF1+ NK4 cells and IGF1+ SC, and further contributing to the decidualization and embryo implantation. Importantly, cAMP-dependent regulation of ovulatory response genes (e.g., AREG) has been reported to be amplified by IGF1 [53], suggesting that there is a close dialog between AREG+ NK cells and IGF1+ SC during decidualization (Fig. 5G).
EVT promotes decidualization by multiply pathways
The embryonic EVT invade the decidua and uterine myometrium, infiltrating the uterine vessels and glands to direct nutrients to the developing fetus. Therefore, HLA-G+ EVT direct contact with maternal-derived decidual cells, including SC, EPC, IC, and EC (Fig. 6A, B, and Additional file 19: Figure S19A). With embryo implantation, the expression of some classic decidualization-related genes in SC (e.g., PRL, IGFBP1, CXCL14, MAP3K5) and EPC (e.g., LIF, DPP4, IGFBP1, CXCL14) in decidua were upregulated rapidly (Fig. 6C and Additional file 19: Figure S19B). Further analysis confirmed co-culture with human trophoblast HTR8/SVneo cell line led to the elevation of these decidualization-related genes in human ESC and EEC cells in vitro (Fig. 6D and Additional file 19: S19C), indicating that EVT promote the decidualization. The top 100 genes (e.g., CSH1, FN1, NOTUM, SERPINE2, QSOX1, ISM2, FLT1, HSPG2, FSTL3, HLA-G, PAPPA2, PAPPA, COL4A1, CDKN1C, HTRA4, FSTL1, COL4A2, CTSL, HPGD, PTPRF) in EVT were mainly associated with ECM remodeling, cell adhesion, angiogenesis and blood vessel development, contributing to embryo implantation, embryonic and placental development, and immune regulation (Fig. 6E and Additional file 2: Figure S20). As a member of the somatotropin/prolactin family of hormones, chorionic somatomammotropin hormone 1 (CSH1) promotes fetal growth and metabolism by activating PRLR. More importantly, CSH1 and PRL are enriched in EVT, and predicted to be associated with the IGF1, CSF1, and AREG (Additional file 21: Figure S21A and S21B). Additionally, pregnancy-associated plasma protein A (PAPPA) and PAPPA2 encode secreted metalloproteinases which cleave IGFBPs, and are thought to be local regulators of IGF bioavailability [54]. Therefore, the stimulatory effect of EVT on decidualization should be dependent on the regulation of CSH1, PRL, and PAPPAs (Fig. 6F).
The aberrant ratio of IGF1+SC to IGF1R+SC is observed in repeated implantation failure patients
To systematically study the interactions of endometrial and decidual cells, we developed a repository of ligand-receptor interacting pairs, representing a complex regulatory network by intercellular communication analysis (Fig. 7A and Additional file 22: Figure S22). Of note, there was the most intense crosstalk between SC (Rem-SC and dRem-SC) and EVT. EVT with powerful cell adhesion and epithelial cells should stimulate decidualization and embryo implantation by PRL/PRLR, pleiotrophin (PTN)/PLXNB2, LIF/LIFR, and or INHBB/ACVR, respectively (Additional file 22: Figure S22). In addition to decidualization development, NK cells and macrophage contribute to stromal cell homeostasis by FASLG/FAS, GAS6/AXL, GAS6/MERTK, PROS1/AXL, AREG/EGFR, TGFB/EGFR, and PDGFs/PDGFRs (Additional file 22: Figure S22). In turn, SC facilitates embryo adhesion, implantation and development, lymphocyte recruitment, immune tolerance, and angiogenesis by FN/integrins, CCL8/CCR1, CXCL12/CXCR4, IL15/IL15R, TGFB1/TGFBR, and VEGFs/VEGFRs, especially dSec_SC and dRem_SC (Additional file 23: Figure S23).
As noted above, IGF1+ SC was considered to start decidualization by interacting with IGF1R+ SC. More importantly, the conversion of IGF1+SC to IGF1R+SC in stromal cells was hampered in some cases of unexplained RIF patients (Fig. 7B, C), which was also confirmed by immunohistochemical staining (Fig. 7D). These data together suggest the subset imbalance between IGF1+SC and IGF1R+SC is involved in the pathogenesis of RIF possibly by decidualization deficiency.