All tissues were collected from patients in The International Peace Maternity and Child Health Hospital of Shanghai Jiao Tong University from Feb 2025 to Aug 2025 with ethics committee approval (Approval No. GKLW-A-2025-020-01) and informed consent of the donors. Endometrial tissues of secretory phase (n=36) were collected from women of reproductive ages (29–38 years old) with normal pregnancy history, undergoing a hysteroscopic procedure with a pathological diagnosis of benign. These donors had not received any hormone-related treatment within the past six months. Decidual tissues were from women (n=63) with normal first-trimester pregnancy for selective termination (age, 21–35 years old; gestational age, 7–9 weeks) or patients (n=30) with unexplained recurrent miscarriage (age, 28–37 years old; gestational age, 6–9 weeks). The samples were collected in the operating room under strict aseptic conditions throughout the procedure and transported to the laboratory on ice within 30 minutes in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (HyClone, SH30023.01B) with 10% fetal bovine serum (FBS; Gibco, 26140-079) for further assays or cell isolation.

Primary endometrial stomal cells (ESCs) and endometrial immune cells (EICs) from endometrial tissues were isolated and collected for flow cytometry analysis according to our previous protocols (14). DSCs and DICs were also obtained according to the experimental methods previously reported by us (6). ESCs or DSCs were cultured in DMEM/F-12 (HyClone, SH30023.01) containing 10% FBS (Gibco, 26140-079). DMφ from DICs were obtained through positive selection by an CD14⁺ cell isolation kit according to the reagent instructions (Miltenyi Biotec, 130–050-201).

Single-cell RNA sequencing (scRNA-seq) data were obtained from the NCBI Gene Expression Omnibus (GEO) database, GSE183837 (endometrium) and GSE194219 (decidua). Data sequencing and analysis was conducted by NovelBio Bio-Pharm Technology Co., Ltd. Singlecell dissociation (15). QuSAGE analysis was used to describe the relative pathway activation of specific genes in stromal cells and macrophages. Gene Ontology analysis (http://www.geneontology.org) was performed to analyze the enriched pathways of differentially expressed genes in DSC and ESC (false discovery rate [FDR] < 0.05). Cell communication analysis was based on CellPhoneDB (16). Based on the interaction and normalized cell matrices obtained through Seurat normalization, significant mean values and cell communication significance (p-value < 0.05) were calculated. Pathways of ligands from DSC and receptors from dMφ were enriched by KEGG pathway in gene set enrichment analysis.

Human monocyte cell line U937 from American Type Culture Collection (ATCC, CRL-3253) was cultured with RPMI-1640 medium (HyClone, SH30027.01) containing 10% FBS. U937 cells (5 × 10⁵/well in 6-well plate) were treated with recombinant human (rH) macrophage colony stimulating factor (CSF1, 50 ng/mL; Pepro Tech, AF-300-25-100) to induce M0 macrophages and both rH-CSF1 (50 ng/mL) and rH-IL10 (50 ng/mL; Pepro Tech, AF-200-10-100) for 6 days to induce decidual-like M2 macrophages according to previous report (17).

M2φ were treated with exogenous COL4A1 (10 ug/mL; Proteintech, Ag34402), OPN (1 ug/mL; ABclonal, RP00989), and HA (High Molecular Weight; 50μM, 100μM; R&D system, GLR002) for 48 hours. Gene silencing plasmid targeting COL4A1 (siCOL4A1)/SPP1 (siSPP1) or control plasmid was from Shanghai Genechem Co., LTD. (China). DSCs were seeded in 6-well culture plates (5 × 10⁵ cells/well) and subsequently infected with siCOL4A1/siSPP1 or control plasmid. Twelve hours after infection, fresh complete culture medium was used to continue the culture. The infection efficiency was evaluated by RT-qPCR 96 hours after infection. DSCs were seeded in 6-well culture plates (5 × 10⁵ cells/well) and treated with 4-Methylumbelliferone (4-MU; 500 μM; MCE, HY-N0187), a hyaluronan synthase inhibitor or vehicle (1‰ DMSO) for 48 hours.

After 48 hours of culture of siCOL4A1/siSPP1/Ctrl DSCs or 4-MU/vehicle-treated DSCs in 24‐well plate (1 × 10⁵ cells/well), M2φ (1 × 10⁵ cells/well) was then placed in the upper compartment of the transwell chamber inserts (0.4 μm aperture, 12 mm diameter; Corning) in the non‐contact transwell co‐culture unit. After 48‐hour co‐culture, macrophages were collected and flow cytometry assays were carried out to analyze the levels of CD16 and other polarization markers in them.

The transcription levels of COL4A1 or SPP1 in DSCs were verified by RT-qPCR according to the standard protocols (RR036A and RR820A; Takara). All values obtained were normalized to the values obtained for β-actin (ACTB). The primer sequences were synthesized by Sangon Biotechnology Co., Ltd. COL4A1: forward 5′ -GGGATGCTGTTGAAAGGTGAA-3′, reverse 5′ -GGTGGTCCGGTAAATCCTGG-3′; SPP1: forward 5′ -CTCCATTGACTCGAACGACTC-3′, reverse 5′ -CAGGTCTGCGAAACTTCTTAGAT-3′; ACTB: forward 5′ - GCCGACAGGATGCAGAAGGAGATCA-3′, reverse 5′ - AAGCATTTGCGGTGGACGATGGA-3′. The fold change of the gene expression was calculated using the change in cycle threshold value method (ΔΔCt).

Paraffin sections were technically supported by Wuhan Servicebio Technology Co., Ltd. (China). Human endometrial or decidua samples were incubated with rabbit anti-human COL4A1 antibody (1:1000; Abcam, ab308360) or rabbit anti-human OPN antibody (1:1000; Abcam, ab214150) overnight at 4°C in a humid chamber. After washing three times with TBS (Beyotime, ST661), the sections were incubated and the signal was visualized with GTvision TMIII Immunohistochemical Detection Kit (Gene Tech, GK500710). The sections were counterstained with hematoxylin (Well Biotech, WH2013).

To detect the distribution of hyaluronic acid, de-waxed endometrial or decidua sample sections were incubated with Digestion Solution or Negative Solution at 37°C for 3 hours according to Hyaluronic Acid Stain Kit (Solarbio, G3710). After rinsed with running water for 5min, the sections were stained with Alican Stain Solution.

Primary ESCs and DSCs were cultured in 24-well plate with a density of 1 × 10⁵ cells for 24 hours and then the culture supernatant of ESCs or DSCs was harvested. The levels of COL4A1, OPN and HA of cell culture supernatant were detected by Human COL4A1 ELISA Kit (ELK Biotechnology, ELK2838), Human OPN ELISA Kit (ELK Biotechnology, ELK10182) and Human HA ELISA Kit (ELK Biotechnology, ELK10906) according to the standard directions.

EICs and dMφ were collected to detect the expression of CD45 (PE/CY5 anti-human CD45 antibody; BioLegend, 982324), CD14 (PE-CY7 anti-human CD14 antibody; BioLegend, 982510), CD16 (APC-CY7 anti-human CD16 antibody; BioLegend, 360710), polarization markers (PE anti-human CD86 antibody, BioLegend, 374206; PE anti-human CD209 antibody, BioLegend, 330108; APC anti-human HLA-DR antibody, BioLegend, 307610; FITC anti-IDO antibody, eBioscience, 11-9473-82) and adhesion markers (BV510 anti- CD49a antibody, BD Biosciences, 755208; FITC anti-human CD44 Antibody, BioLegend, 397518; FITC anti-LYVE1 antibody, eBioscience, 53-0443-82; APC anti-human Integrin αVβ3 antibody, BioLegend, 304416). DICs from normal pregnant women and women with RM were isolated to detect the expression of CD45 (BV 421 anti-human CD45 Antibody, 368522), CD14 (PE-CY7 anti-human CD14 antibody; BioLegend, 982510), CD16 (BV785 anti-human CD16 antibody; BioLegend, 360734), CD86 (FITC anti-human CD86 antibody; BioLegend, 374204), CD206 (APC anti-human CD206 antibody; BioLegend, 321110) and CD209 (PerCP/Cyanine5.5 anti-human CD209 antibody; BioLegend, 330110). CD16 (APC-CY7 anti-human CD16 antibody; BioLegend, 360710), CD86 (PE anti-human CD86 antibody, BioLegend, 374206), CD206 (APC anti-human CD206 antibody; BioLegend, 321110), CD209 (PerCP/Cyanine5.5 anti-human CD209 antibody; BioLegend, 330110) of M2φ treated with exogenous COL4A1, OPN or HA and collected from different co-culture system were also analyzed by flow cytometry assays. Flow cytometry was performed using a Beckman CytoFLEX S flow cytometer (Beckman) equipped with Becton CytExpert software. Data analysis was conducted using FlowJo V10 software.

The results were representatives of multiple experiments. All analyses were conducted by SPSS 25 (IBM). Normally distributed data with uniform variance were analyzed by a t test between two groups or one-way ANOVA test among multiple groups. Data were presented as mean ± standard error (SEM). P < 0.05 was considered to indicate a statistically significant difference.

Based on our single-cell sequencing data (15), stromal cells constituted the largest cellular component in both endometrial and decidual tissues (Figure 1A). Compared to other cell populations, genes associated with ECM-receptor interaction were highly enriched in stromal cells (Figure 1B). We performed data analysis on differentially genes in endometrial stromal cells (ESCs) and DSCs (Figure 1C). As showed in Figure 1D, GO enrichment analysis was performed using the R package to obtain information about biological process (BP), cellular component (CC), and molecular function (MF). Compared with ESCs, ECM-related pathways were significantly enriched in DSCs, such as ECM organization, collagen-containing ECM and ECM constituent conferring. In DSCs, genes associated with ECM organization and remodeling were generally significantly overexpressed (Figure 1E), suggesting that the matrix undergoes profound changes during decidualization. Since collagen, hyaluronic acid (HA), and osteopontin (OPN) have been reported in previous studies to be important ECM components regulating the immune state at the maternal-fetal interface (3, 13, 18), we focused on detecting and validating their expression in the decidua and endometrium. Combined with scRNA sequencing results, including alterations in ECM-related genes during decidualization and insights from cell communication signal analysis we observed subsequently (Figure 2A), we confirmed through Immunohistochemical or Alcian blue staining analysis that the levels of type IV collagen (COL4A1), osteopontin (OPN), and hyaluronic acid (HA) in normal early pregnancy decidua were significantly higher than those in secretory phase endometrium (Figures 3A-C). To further confirm that they can be synthesized from DSCs and transferred to the matrix to exert regulatory functions, we supplemented the study with ELISA assays (Figure 3D). Compared to ESCs, higher levels of COL4A1, OPN, and HA were detected in the culture supernatants of DSCs, which may play a crucial regulatory role in cell-to-cell communication within the matrix in decidualization.

In single-cell sequencing analysis of cell-cell communication pathways, over 100 interaction pathways were identified between as ligands on DSCs and receptors on dMφ (Figure 2A). Then, we performed enrichment analysis on genes associated with ligands and receptors (Figure 2B). Genes about ECM receptor interaction were significantly enriched in ligands from DSC, while cell adhesion molecule-related genes were significantly enriched in receptors from dMφ (Figure 2B). This result suggested that cross-talk between decidua and macrophages might occur via ECM-adhesion molecules. Furthermore, we identified a high expression of cell adhesion molecules in macrophages through gene set activity clustering analysis, consistent with their characteristics as tissue-resident macrophages (Figure 2C). Compared to endometrial macrophages (eMφ; Macrophge_Ctrl), dMφ (Macrophage_NP) exhibited higher expression levels of adhesion molecules, which may participate in interactions between DSCs (Figure 2D).

Through single-cell sequencing analysis (15), we have discovered that FCGR3A (gene encoding CD16) was primarily expressed in macrophages in both endometrium (Macrophge_Ctrl) and decidua (Macrophage_NP) in the UMAP visualization (Figure 4A). Compared with eMφ (Macrophge_Ctrl), there was an upward trend in FCGR3A expression in dMφ (Macrophage_NP) in the violin analysis diagram (Figure 4B). Confirmed by flow cytometry, CD16 expression on CD45⁺CD14⁺ dMφ was significantly higher than on CD45⁺CD14⁺ eMφ (Figure 4C). We then detected the polarized molecules in CD45⁺CD14⁺CD16^- dMφ and CD45⁺CD14⁺CD16⁺ dMφ by flow cytometry assays. CD16⁺ dMφ exhibited an immunoregulatory phenotype, characterized by higher expression of CD209 and IDO, along with lower levels of CD86 and HLA-DR compared to CD16^- dMφ (Figure 4D).

Specifically, we found in flow cytometry that CD45⁺ CD14⁺CD16⁺ Mφ in both endometrium (Figure 5A) and decidua (Figure 5B) exhibited higher expression of adhesion molecules compared with CD45⁺ CD14⁺CD16^- Mφ, suggesting a tendency toward tissue residency. Here, we focused on detecting CD49a (collagen IV receptor), CD44 and LVYE1 (HA receptor), and integrin αVβ3 (OPN receptor), whose expression levels in CD16⁺ dMφ were significantly higher than in CD16^- dMφ (Figure 5B). This suggests that ECM derived from the decidua may exert regulatory effects of CD16⁺ Mφ via these adhesion molecules.

U937 cells were induced into dMφ-like macrophages using the previously mentioned method (17). Exogenous COL4A1 (10 ug/mL) and OPN (1 ug/mL) for 48 hours promoted CD16 expression on dMφ-like macrophages (Figure 6A) in flow cytometry assays. As shown in Figure 5B, the higher levels of receptors of COL4A1 and OPN were detected on CD16⁺ Mφ. The regulatory effects of COL4A1 and OPN on the polarized molecules in CD16⁺ Mφ were more pronounced in Figure 6B. Specifically, COL4A1 (10 ug/mL) and OPN (1 ug/mL) for 48 hours significantly downregulated CD86 expression on CD16⁺ Mφ while upregulating CD206 or CD209 expression on them. However, this effect was not significant on CD16^- Mφ. We then treated dMφ-like macrophages with high molecular weight HA at different concentrations (0, 50, 100μM) for 48 hours. In the high-concentration HA treatment group (100μM), the proportion of CD16⁺ macrophages highly expressing CD206 and CD209 significantly increased by flow cytometry (Figures 6C, D).

Subsequently, we further validated this result by a co-culture system. Following co-culture with siCOL4A1 or siSPP1 DSCs (gene knockout efficiency was validated by RT-qPCR in Figure 7A) for 48 hours, the proportion of CD16⁺ macrophages from the co-culture system significantly decreased (Figures 7B, C), accompanied by upregulation of CD86 and downregulation of CD206 or CD209 (Figure 7D) assessed by flow cytometry. Similarly, when HA synthesis was inhibited by 4-MU (500 μM for 48 hours) in DSCs, CD16 expression on macrophages in the co-culture system was suppressed (Figure 7E), leading to pro-inflammatory differentiation characterized by higher expression of CD86 and lower expression of CD206 and CD209 (Figure 7F).

Dysfunction of the decidualized DSCs and abnormal expression of related genes correlate with adverse pregnancy outcomes, particularly early pregnancy failure (19). Specifically, abnormal interactions between DSCs and dMφ also hinder the establishment and maintenance of normal pregnancy, leading to recurrent miscarriage (RM) and other complications (10). We then assessed the ECM synthesis of decidua and CD16 expression of dMφ from RM patients to determine whether these alterations are associated with the disease.

In Immunohistochemical or Alcian blue staining experiments, we detected significantly reduced levels of target ECM components in the decidua of RM patients compared to the normal pregnancy group (NP), including COL4A1 (Figure 8A), OPN (Figure 8B), and HA (Figure 8C). Through flow cytometry, we found that RM patients exhibited reduced CD16 expression in CD45⁺CD14⁺ dMφ, representing an immune-activated phenotype characterized by higher levels of CD86 and lower levels of CD206 and CD209 (Figure 8D). The above findings suggest that insufficient interaction between ECM-deficient DSCs and CD16⁺ dMφ may be associated with the occurrence of RM, which requires further exploration.