Journal: Frontiers in Immunology

Article Title: Integrative genetic and multi-omics analysis reveals the interleukin-6 receptor’s role in recurrent spontaneous abortion

doi: 10.3389/fimmu.2025.1659251

Figure Lengend Snippet: Schematic representation of the research design. MR analysis of plasma proteomics data from the UK Biobank Protein Plasma Project (UKB-PPP) and the deCODE Health Study identified 47 and 34 causal plasma proteins associated with RSA risk, respectively. The genetic data for RSA were obtained from the FinnGen R12_N14_HABITABORT dataset. Four candidate proteins were identified by overlapping the two datasets, with IL6R being the only candidate protein that overlapped with DEGs from mid-secretory phase endometrial RNA-seq data of RSA patients and controls ( GSE165004 ). Reanalysis of single-cell RNA-seq data from the decidual tissues identified elevated IL6R expression in RSA-derived decidua and stromal cells. The IL6R expression was significantly upregulated at both the plasma protein and decidua mRNA levels in RSA patients. Finally, MR-PheWAS was performed using the ukb-ppp-IL6R- OID20385 dataset as the exposure variable and 2467 phenotypic traits from the finngen_R12 GWAS dataset as the outcome to evaluate the potential side effects and additional indications for IL6R as a drug target for RSA. DEGs, Differentially expressed genes; SNP, Single nucleotide polymorphism; IL6R, interleukin 6 receptor; UKB-PPP, UK Biobank Pharma Proteomics Project; RSA, Recurrent spontaneous abortion; RNA-seq, RNA sequencing; MR, Mendelian randomization; MR-PheWAS, MR-Phenome-wide association study; GWAS, genome-wide association study.

Article Snippet: Antigen retrieval was performed using citrate buffer (pH 6.0) before incubation with rabbit polyclonal anti-IL6R antibody (Proteintech; 1:200 dilution) overnight at 4 °C.

Techniques: Clinical Proteomics, RNA Sequencing, Expressing, Derivative Assay, GWAS

Journal: Frontiers in Immunology

Article Title: Integrative genetic and multi-omics analysis reveals the interleukin-6 receptor’s role in recurrent spontaneous abortion

doi: 10.3389/fimmu.2025.1659251

Figure Lengend Snippet: Analysis of the association between plasma proteins and the risk of RSA using the IVW method. The forest plot depicts the causal effects of plasma proteins on RSA risk based on a two-sample MR analysis using data from UKB-PPP and the Icelandic cohort. The four overlapping proteins identified were IL6R, CNTNAP2, DLL1 and NPTX1. OR and 95% CI were estimated using the IVW method. UKB-PPP, UK Biobank Prospective Proteomic Study; deCODE, Icelandic cohort; IVW, Inverse variance weighted; OR, Odds ratios; CI, confidence intervals.

Article Snippet: Antigen retrieval was performed using citrate buffer (pH 6.0) before incubation with rabbit polyclonal anti-IL6R antibody (Proteintech; 1:200 dilution) overnight at 4 °C.

Techniques: Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Integrative genetic and multi-omics analysis reveals the interleukin-6 receptor’s role in recurrent spontaneous abortion

doi: 10.3389/fimmu.2025.1659251

Figure Lengend Snippet: Comparative analysis of IL6R expression and pathway interactions in endometrial and decidual tissues of control group and RSA group. (A) Heatmap of IL6R expression in the mid-secretory endometria from RSA cases (n=24) compared to controls (n=24). Each row represents a sample, and color intensity indicates expression levels, with scale provided. (B) RNA-seq analysis of IL6R expression in mid-secretory endometrium derived from RSA group (n=24) compared to control group (n=24). * P < 0.05. (C) Bar chart displaying GO and KEGG pathway enrichment of DEGs in mid-secretory endometria derived from RSA cases compared to controls. (D) Chord Diagram of pathway-gene associations visualizes associations between DEGs and enriched biological processes and pathways. Ribbon widths indicate the strength of gene-pathway interactions, with significant links between IL6R, JAK-STAT signaling, and α-β T cell differentiation. (E) IL6R mRNA levels in deciduas from women with RSA and those with normal early pregnancies were analyzed using quantitative real-time polymerase chain reaction. The results were normalized to RNA18SN1 expression and are presented as the mean ± SD.* P < 0.05. n=18. (F, G) immunohistochemistry (IHC) staining and analysis of IL6R expression in early pregnancies deciduas from control group and RSA group. Scale bars,100 μ m. n=13. IL6R, interleukin 6 receptor; CON, Control; RSA, recurrent spontaneous abortion. GO,0002181, cytoplasmic translation; GO,2000644, regulation of receptor catabolic process; GO,0006611, protein export from nucleus; GO,0046632, alpha-beta T cell differentiation; GO,0050684, regulation of mRNA processing; hsa05171, Coronavirus disease- COVID-19; hsa03010, Ribosome; hsa04630, JAK-STAT signaling pathway.

Article Snippet: Antigen retrieval was performed using citrate buffer (pH 6.0) before incubation with rabbit polyclonal anti-IL6R antibody (Proteintech; 1:200 dilution) overnight at 4 °C.

Techniques: Expressing, Control, RNA Sequencing, Derivative Assay, Cell Differentiation, Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Integrative genetic and multi-omics analysis reveals the interleukin-6 receptor’s role in recurrent spontaneous abortion

doi: 10.3389/fimmu.2025.1659251

Figure Lengend Snippet: Abnormal upregulation of decidual IL6R mRNA in RSA patients was associated with IL6-signaling pathway dysregulation and compromised intercellular communication at the maternal-fetal interface. (A) Comparative single-cell RNA-seq (scRNA-seq) profiling of the IL6R transcript levels in decidual tissues from RSA patients (n = 6) versus gestational age-matched healthy controls (n = 5). The IL6R mRNA expression demonstrated a significant elevation in the RSA cohort (**** P < 0.0001). (B) DSCs-specific analysis of scRNA-seq data demonstrated significantly elevated IL6R mRNA levels within DSCs populations from RSA patients (n = 6) when compared to gestational age-matched healthy controls (n = 5). (C) Analysis of scRNA-seq data revealed how different cell subtypes serve as senders, receivers, mediators, or influencers in the IL6-signaling pathway in the control group. The importance score (bar height) represents the communication probability (range 0–1), quantifying the relative strength of IL-6 ligand–receptor interactions between "sender" and "receiver" cell clusters. Higher values indicate stronger inferred communication. (D) Analysis of scRNA-seq data revealed how different cell subtypes serve as senders, receivers, mediators, or influencers in the IL6-signaling pathway in the RSA group. (E) Analysis of scRNA-seq data indicated the crosstalk between DSCs and the other subtypes in the control group, with a focus on the IL6-signaling pathway. Solid circles on the left represent different DS cell subpopulations, hollow circles in the center represent target cell subpopulations, and solid circles on the right represent other distinct cell subpopulations. The size of each circle is proportional to the number of cells within the corresponding subpopulation. Lines connecting nodes represent inferred significant ligand–receptor interactions, indicating potential cellular communication. The width of each line denotes the relative strength of the interaction. (F) Analysis of scRNA-seq data indicated the crosstalk between DSCs and other cellular subtypes in the RSA group, focusing on the IL6-signaling pathway. DS1-DS5, Decidual stromal cells; dNK1-dNK2, Decidual natural killer cells; Endo1-Endo4, Endometrial cells; Epi1-Epi2, Epithelial cells; FB1-FB2, Fibroblast cells; Macro1-Macro3, Macrophages; peri_NK, Peripheral blood natural killer cells; Plasma, Plasma cells; pMacro, placental macrophages; pNK, Placental natural killer cells; TPV, Trophoblast blood vessels; T, T cells.

Article Snippet: Antigen retrieval was performed using citrate buffer (pH 6.0) before incubation with rabbit polyclonal anti-IL6R antibody (Proteintech; 1:200 dilution) overnight at 4 °C.

Techniques: RNA Sequencing, Expressing, Control, Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: Integrative genetic and multi-omics analysis reveals the interleukin-6 receptor’s role in recurrent spontaneous abortion

doi: 10.3389/fimmu.2025.1659251

Figure Lengend Snippet: Phenome-wide causal estimates for IL6R perturbation are displayed, as derived from MR analyses of Finnish database clinical traits. The -log10(P-values) are plotted along the ordinate, with 20 labels applied to associations surpassing predefined statistical significance criteria (FDR < 0.05).

Article Snippet: Antigen retrieval was performed using citrate buffer (pH 6.0) before incubation with rabbit polyclonal anti-IL6R antibody (Proteintech; 1:200 dilution) overnight at 4 °C.

Techniques: Derivative Assay