The immunological paradox of tubal ectopic pregnancy: unraveling immune tolerance amidst inflammation via single-cell sequencing.

In this study, fallopian tube tissue samples were collected from six patients clinically diagnosed with TEP and treated surgically. For each patient, paired samples were obtained from the IS and a corresponding NIS (serving as the control). All participants were women aged 18 to 45 years. Inclusion criteria included the following: patients from whom both IS and NIS could be surgically removed, without apparent tissue degradation or contamination. Furthermore, patients were required to have a complete clinical history documented, including information on serum hCG levels, surgical history, and history of previous pregnancies. The exclusion criteria were ectopic pregnancies at non-tubal sites, concurrent reproductive system diseases (such as endometriosis, polycystic ovary syndrome), recent immunomodulatory treatments, poor sample quality, or lack of complete clinical records. IS samples were collected from the tubal segment containing the ectopic embryo, typically in the ampulla, confirmed intraoperatively. NIS samples were from adjacent unaffected segments, at least 2 cm distal to IS, ensuring no embryonic tissue contamination. The tissues collected during surgery were full-thickness fallopian tube tissues (including serosal layer, muscular layer, and mucosal layer). Table S1 provides the demographic and clinical characteristics of the 6 TEP patients included in this study, including age, body mass index (BMI), gestational age, gravidity, parity, serum hCG levels, and diagnosis, ensuring transparency and allowing assessment of potential confounding factors. During quality control of the single-cell transcriptome sequencing data, Souporcell v2 software was used to assess sample identity and clustering consistency [ 12 ]. Some paired samples showed discordant patterns in clustering analysis, possibly due to technical or biological factors. To ensure data reliability, these inconsistent samples were excluded, resulting in a final dataset of 4 IS samples and 5 NIS samples for subsequent analysis. The study was approved by the Ethics Committee of Shanxi Ervine Maternity Hospital (IRB-2022-KY-002), and all participants signed informed consent forms. All tissue samples were collected freshly during the surgical procedure. After collection, the samples were immediately washed with cold saline to remove excess blood and then placed in sterile centrifuge tubes containing 15 mL of tissue preservation solution (provided by LC-Bio Ltd). All tissue samples were transported to the LC-Bio laboratory within 24 h of collection using insulated foam boxes with ice packs, maintaining low temperature to preserve sample integrity. Tissue samples were transferred to a Petri dish containing ice-cold PBS (RNase-free, without RNase and Ca, Mg ions), washed thoroughly, and cut into small pieces of approximately 0.5 mm². The minced tissues were then incubated in a dissociation solution containing 0.35% Collagenase IV (Worthington Biochemical, LS004188 ) and 120 U/mL DNase I (Roche, 11284932001) at 37℃ in a water bath shaker (100 rpm) for ~ 20 min. The enzymatic reaction was terminated by adding PBS supplemented with 10% fetal bovine serum (FBS). The resulting cell suspension was filtered through a 70 μm cell strainer, followed by erythrocyte lysis (Miltenyi Biotec, 130-094-183) and dead cell removal (Miltenyi Biotec, 130-090-101), yielding high-quality single-cell suspensions for downstream analysis. Cell viability was assessed using Trypan Blue staining, and cell counting was performed with an automated cell counter, only samples with > 85% viable cells were used for downstream single-cell RNA sequencing. For each sample, the single-cell suspension was adjusted to a final concentration of 700–1200 cells/µL and loaded for subsequent single-cell capture and library preparation, with an expected capture of approximately 8,000 cells. In the single-cell RNA sequencing experiment, the 10x Genomics Chromium single-cell platform was used for library construction. First, the sorted single-cell suspensions were loaded onto the 10x Genomics Chromium chip in a low-throughput manner. The 10× Genomics Chromium Single-Cell 3’kit (V3) instructions were strictly followed to sequentially complete the steps of cell capture, reverse transcription to synthesize cDNA, cDNA generation, and library amplification to ultimately generate libraries for sequencing. Finally, high-throughput sequencing was performed using the Illumina NovaSeq 6000 platform, with an average sequencing depth of at least 20,000 reads/cell per sample to ensure sufficient depth and coverage for the sequencing data. At the initial stage of single-cell sequencing data processing, we applied Cell Ranger software (V6.0) to strictly filter and reduce noise on the raw sequencing data, and compared them with the human reference genome Homo sapiens GRCh38. During quality control, we screened and excluded low-quality cells based on the following criteria: median gene expression less than 500 or greater than 5000, mitochondrial gene proportion greater than 30%, and fewer than 500 unique molecular identifiers. Subsequently, we normalized the pre-processed data using the Seurat R package (version 4.1.0). The gene expression matrix was normalized using the log-normalization method. Based on this, we used the “NormalizeData” function in Seurat and applied its LogNormalize method to calculate the expression values of each gene. For cell clustering analysis, we used PCA and UMAP dimensionality reduction methods from the Seurat package to perform clustering on the cell population. Different cell populations were identified by analyzing the gene expression patterns of the cells. Subsequently, known cell-type specific gene markers were used to annotate the clustering results and determine the type of each cell population. To explore the gene expression differences between different cell types and groups, we used the FindAllMarkers function in Seurat for group comparisons. Differentially expressed genes (DEGs) were performed using the bimod test implemented in Seurat. The screening criteria included P value < 0.01, log2 fold change (log2FC) ≥ 0.26, and a minimum expression proportion of 0.1 in at least one group. To further investigate the biological functions and potential signaling pathways of DEGs, we performed functional enrichment analysis on the screened differentially expressed genes. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs using the ClusterProfiler R package to reveal the potential roles of these genes in cellular functions and metabolic pathways. In the analysis, we set a significance threshold of P value < 0.05. In addition, to further resolving the biological functions of the subpopulation of cells, we also utilized the Metascape online platform ( https://metascape.org/ ) to perform the functional enrichment analysis. The Metascape platform integrates multiple bioinformatics tools and databases, providing more comprehensive and in-depth gene function annotation and pathway analysis [ 13 ]. Through this integrated analytical approach, we were able to systematically understand the functional characteristics and potential regulatory mechanisms of subpopulation cells in different biological processes. To quantify site-specific enrichment, we applied the observed/expected ratio (Ro/e) following the STARTRAC-dist procedure [ 14 ]. Briefly, a contingency table of clusters by implantation status (IS and NIS) was constructed, and a chi-square test was performed to derive the expected number of cells for each cluster–location combination. Ro/e was then defined as the observed cell number divided by the expected cell number (Ro/e = observed/expected). Ro/e >1 indicates enrichment and Ro/e < 1 indicates depletion. Annotation symbols (“+++”, “++”, “+”, “+/−”, “−”) were assigned according to the STARTRAC legend. Data analysis and graphical generation were performed using R software (version 4.1.0) ( http://www.R-project.org ). A P value < 0.05 was considered statistically significant.

We performed single-cell sequencing analysis of tissues from the IS and NIS of patients with TEP (Fig.  1 ). A total of 28 different cell clusters were enriched by cluster analysis of the single-cell data. After cellular annotation, 13 cell types were identified, including trophoblast cells, mesenchymal cells, ciliated epithelial cells (CEs), non-ciliated secretory epithelial cells (NCSEs), fibroblasts, myofibroblasts, smooth muscle cells (SMCs), blood endothelial cells, lymphatic endothelial cells, B cells, T/NK cells, macrophages, and dendritic cells (DCs) (Fig.  1 B, C, and D). Further, we analyzed the relative proportions of these cell types. The results of stacked bar plot showed that the macrophages and T/NK cells populations had the highest proportions in both IS and NIS (Fig.  1 E). Fig. 1 Single-cell transcriptomic profiling of human tubal ectopic pregnancy. A Schematic overview of the experimental workflow. B UMAP plot depicting major cellular clusters classified by cell type. C UMAP plot illustrating major cellular clusters categorized by group. D Bubble plot displaying the expression patterns of canonical marker genes across distinct cell types. E Stacked bar plot representing the proportions of each cell type within the clusters Single-cell transcriptomic profiling of human tubal ectopic pregnancy. A Schematic overview of the experimental workflow. B UMAP plot depicting major cellular clusters classified by cell type. C UMAP plot illustrating major cellular clusters categorized by group. D Bubble plot displaying the expression patterns of canonical marker genes across distinct cell types. E Stacked bar plot representing the proportions of each cell type within the clusters To further analyze the possible molecular mechanisms of TEP, we performed differential gene analysis on the tissues of IS versus NIS (Fig.  2 A and B). We screened 905 DEGs between IS and NIS, with 512 up-regulated and 393 down-regulated in IS compared to NIS. Further, we performed function enrichment analysis on these DEGs (Fig.  2 C and D). GO analysis showed that these DEGs were mainly associated with immune and inflammatory response (GO:0006954, GO:0045087, and GO:0006955), extracellular matrix and function (GO:0062023, GO:0005576, GO:0005856, GO:0005886, GO:0016020, GO:0016021, GO:0070062, and GO:0007155). KEGG analysis showed that these DEGs were mainly associated with immune and inflammatory responses (hsa04612, hsa05323, hsa05152, and hsa04145), cell adhesion and tissue remodeling (hsa04514, hsa04530, and hsa04151), infection and pathogen-related pathways (hsa05169, hsa05132, hsa05130, and hsa05140). These results further suggest that the occurrence of tubal ectopic pregnancy is associated with inflammation, immune response, cell adhesion, and tissue remodeling. Fig. 2 Integrated analysis of DEGs across cell subpopulations in human tubal ectopic pregnancy. A Scatter plot showing gene expression in patients with human tubal ectopic pregnancy. B Volcano plot depicting DEGs in human tubal ectopic pregnancy. C GO enrichment analysis of DEGs. D KEGG enrichment analysis of DEGs. Note: DEGs, differentially expressed genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes Integrated analysis of DEGs across cell subpopulations in human tubal ectopic pregnancy. A Scatter plot showing gene expression in patients with human tubal ectopic pregnancy. B Volcano plot depicting DEGs in human tubal ectopic pregnancy. C GO enrichment analysis of DEGs. D KEGG enrichment analysis of DEGs. Note: DEGs, differentially expressed genes; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes To further explore the potential pathogenetic mechanisms involving T/NK cells in TEP, we conducted a T/NK subpopulation analysis (Fig.  3 A, B and C). The results showed that T/NK cells were categorized into 8 subpopulations, including CD4 + memory T cells (CD4 + Tm), CD8 + GZMB + T cells (CD8 + GZMB + Teff), CD8 + KLRC2 + effector T cells (CD8 + KLRC2 + Teff), CD8 + memory T cells (CD8 + Tm), CD16 − CD56 + natural killer cells (CD16 − CD56 + NK), CD16 + CD56 − natural killer cells (CD16 + CD56 − NK), interferon-stimulated gene - expressing T cells (ISG-T), and regulatory T cells (Treg). The stacked bar plot showedthat both IS and NIS had the most CD4 + Tm, CD8 + KLRC2 + Teff and CD8 + Tm cells (Fig.  3 D). In addition, CD4 + Tm were higher in the IS group than in the NIS group, whereas CD8 + KLRC2 + Teff and CD8 + Tm cells were decreased in IS. In addition, Ro/e analysis showed that CD4 + Tm cells were strongly enriched at the implantation site (IS, “+++”) compared with the non-implantation site (NIS, “+”), whereas CD8 + KLRC2 + Teff and CD8 + Tm cells were strongly enriched at NIS (NIS, “+++”) relative to IS (IS, “+”) (Fig.  3 E). Fig. 3 Subtypes of T/NK cells in human tubal ectopic pregnancy. A UMAP plot showing the T/NK cell subsets by cell type. B UMAP plot illustrating the T/NK cell subsets by group. C Dot plot displaying the gene expression characteristics of each T/NK cellsubset. D Stacked bar plot representing the proportions of each T/NK cell subset. E Ro/e plot showing the enrichment or depletion of each T/NK cell subset. Subtypes of T/NK cells in human tubal ectopic pregnancy. A UMAP plot showing the T/NK cell subsets by cell type. B UMAP plot illustrating the T/NK cell subsets by group. C Dot plot displaying the gene expression characteristics of each T/NK cellsubset. D Stacked bar plot representing the proportions of each T/NK cell subset. E Ro/e plot showing the enrichment or depletion of each T/NK cell subset. Further, we performed DEGs and functional enrichment analyses on CD4 + Tm, CD8 + KLRC2 + Teff, CD8 + Tm. For CD4 + Tm cells, we screened a total of 468 DEGs, of which 183 were up-regulated and 285 were down-regulated (Fig.  4 A). The functional enrichment analysis of CD4 + Tm showed that these DEGs were mainly associated with immune cell proliferation and activation (CORUM:306, CORUM:308, and GO:0006364), immune regulation and inflammatory response (CORUM: 5266, R-HSA-1280215, and GO:0071345), angiogenesis and tissue repair (WP3888), regulation of immune tolerance (GO:0030099, GO:0030097, and R-HSA-909733) and stress adaptation and tissue remodeling (GO:1901798 and hsa04933) (Fig.  4 B). These results suggest that CD4 + Tm cells may play a dual role of pro-inflammatory and immune tolerance in the local immune microenvironment at the implantation site of tubal ectopic pregnancy. Fig. 4 Integrated analysis of DEGs for T/NK cell subsets in human tubal ectopic pregnancy. A Volcano plot showing DEGs in CD4 + Tm cell subsets in human tubal ectopic pregnancy. B Bar chart depicting the functional enrichment of DEGs in the CD4 + Tm cell subsets. C Volcano plot illustrating DEGs in the CD8 + KLRC2 + Teff cell subsets in human tubal ectopic pregnancy. D Bar chart showing the functional enrichment of DEGs in CD8 + KLRC2 + Teff cell subsets. E Volcano plot of DEGs for CD8 + Tm cell subsets in human tubal ectopic pregnancy. F Bar chart illustrating the functional enrichment of DEGs in CD8 + Tm cell subsets. Note: DEGs: differentially expressed genes. Integrated analysis of DEGs for T/NK cell subsets in human tubal ectopic pregnancy. A Volcano plot showing DEGs in CD4 + Tm cell subsets in human tubal ectopic pregnancy. B Bar chart depicting the functional enrichment of DEGs in the CD4 + Tm cell subsets. C Volcano plot illustrating DEGs in the CD8 + KLRC2 + Teff cell subsets in human tubal ectopic pregnancy. D Bar chart showing the functional enrichment of DEGs in CD8 + KLRC2 + Teff cell subsets. E Volcano plot of DEGs for CD8 + Tm cell subsets in human tubal ectopic pregnancy. F Bar chart illustrating the functional enrichment of DEGs in CD8 + Tm cell subsets. Note: DEGs: differentially expressed genes. For CD8 + KLRC2 + Teff cells, we screened a total of 242 DEGs, of which 147 were up-regulated and 95 were down-regulated (Fig.  4 C). The functional enrichment analysis showed that these DEGs were mainly associated with diminished immune effector function (M54, hsa04612, hsa04010, and M167), immunosuppressive microenvironment formation (WP5090) and decreased cytokine responsiveness (GO:0071345) were associated (Fig.  4 D). These results suggest that functional inhibition of CD8 + KLRC2 + Teff cells at the IS may be directly related to enhanced local immune tolerance and defective immune surveillance. For CD8 + Tm cells, we screened a total of 263 DEGs, of which 130 were up-regulated and 133 were down-regulated (Fig.  4 E). The functional enrichment analysis showed that these DEGs were mainly associated with abnormal regulation of hormone signaling (GO:0048545 and R-HSA-8939211), defective antigen presentation (hsa04612), oxidative stress and proliferation inhibition (GO:0006979 and hsa04010) were associated (Fig.  4 F). These findings suggest that CD8 + Tm cells may suffer from multiple effects of hormonal imbalance, dysfunction of the immune microenvironment, and accumulation of oxidative stress at the IS, resulting in a reduced ability to immune surveillance and clearance ability due to multiple factors, which in turn exacerbates fallopian tube structural damage. To explore the potential role of macrophages in TEP, we performed macrophage subpopulation analysis (Fig.  5 A, B and C). The results showed that macrophage cells were divided into 3 subpopulations, including M1 macrophage, M2 macrophage, and monocytes. Stacked bar plot showed that M2 macrophages were higher in the IS group than in the NIS group, whereas M1 was lower in the IS group (Fig.  5 D). Ro/e analysis showed that M2 and monocytes cells were strongly enriched at the implantation site (IS, “+++”) compared with the non-implantation site (NIS, “+” and “++”), whereas M1 cells were strongly enriched at NIS (NIS, “+++”) relative to IS (IS, “+”) (Fig.  5 E). Fig. 5 Subtypes of macrophage cells in human tubal ectopic pregnancy. A UMAP plot showing macrophage subsets by cell type. B UMAP plot illustrating macrophage subsets by group. C Dot plot depicting the gene expression characteristics of each macrophage cell subset. D Stacked bar plot representing the proportions of each macrophage cell subset. E Ro/e plot showing the enrichment or depletion of each macrophage cell subset. Further, we performed differential gene and functional enrichment analysis on monocytes, M1 and M2 macrophages. For moncytes, we screened a total of 1337 differential genes, of which 785 were up-regulated and 552 were down-regulated (Fig.  6 A). The functional enrichment analysis showed that these DEGs were mainly associated with the formation and maintenance of inflammatory microenvironment (GO:0006954, GO:0071345, and GO:0001819), imbalance of immune regulation and cell migration (GO:0045087, GO:0002683, GO:0050900, and GO:2000147) and angiogenesis and metabolic adaptation (WP3888, GO:1901342, GO:0051248, and GO:0070482) (Fig.  6 B). These features suggest that monocytes may influence local immune responses and participate in tissue repair and remodeling processes by participating in the formation and maintenance of the inflammatory microenvironment, regulating immune cell migration, and promoting angiogenesis and metabolic adaptation. Fig. 6 Integrated analysis of DEGs in macrophage subsets in human tubal ectopic pregnancy. A Volcano plot showing DEGs for monocyte subsets in human tubal ectopic pregnancy. B Bar chart illustrating the functional enrichment of DEGs in the monocyte subsets. C Volcano plot showing DEGs for M1 macrophage subsets in human tubal ectopic pregnancy. D Bar chart illustrating the functional enrichment of DEGs in the M1 macrophage subsets. E Volcano plot showing DEGs for M2 macrophage subsets in human tubal ectopic pregnancy. F Bar chart illustrating the functional enrichment of DEGs in the M2 macrophage subsets. Note: DEGs, differentially expressed genes. Subtypes of macrophage cells in human tubal ectopic pregnancy. A UMAP plot showing macrophage subsets by cell type. B UMAP plot illustrating macrophage subsets by group. C Dot plot depicting the gene expression characteristics of each macrophage cell subset. D Stacked bar plot representing the proportions of each macrophage cell subset. E Ro/e plot showing the enrichment or depletion of each macrophage cell subset. Integrated analysis of DEGs in macrophage subsets in human tubal ectopic pregnancy. A Volcano plot showing DEGs for monocyte subsets in human tubal ectopic pregnancy. B Bar chart illustrating the functional enrichment of DEGs in the monocyte subsets. C Volcano plot showing DEGs for M1 macrophage subsets in human tubal ectopic pregnancy. D Bar chart illustrating the functional enrichment of DEGs in the M1 macrophage subsets. E Volcano plot showing DEGs for M2 macrophage subsets in human tubal ectopic pregnancy. F Bar chart illustrating the functional enrichment of DEGs in the M2 macrophage subsets. Note: DEGs, differentially expressed genes. For M1 macrophage, we screened a total of 744 differential genes, of which 477 were up-regulated and 267 were down-regulated (Fig.  6 C). The functional enrichment analysis showed that these DEGs were mainly associated with the suppression of immune and inflammatory responses (GO:0001775, GO:0006959, GO:0071345), regulation of cell death and survival (GO:0043068, GO:0051248), negative regulation of hematopoiesis and differentiation (GO:0030097, GO:1903706, GO:0045596), and impaired angiogenesis and signaling (WP3888, R-HSA-9006934) (Fig.  6 D). These findings suggest that M1 macrophages at the ectopic implantation site of the fallopian tube exhibit broad functional suppression, including reduced immune activation, weakened proapoptotic signaling, limited differentiation potential, and impaired angiogenesis. For M2 macrophage, we screened a total of 816 differential genes, of which 450 were up-regulated and 366 were down-regulated (Fig.  6 E). The functional enrichment analysis showed that these DEGs were mainly associated with immune regulation and inflammatory response (R-HSA-1280215, GO:0009617), interferon signaling (RHSA-913531, WP619), cell migration and angiogenesis (GO:0030335, GO:0001944), metabolic reprogramming (GO:0032787), and extracellular matrix organization (M5885, M53) (Fig.  6 F). These results indicate that M2 macrophages at the implantation site exhibit typical polarization characteristics and shape a microenvironment supporting the survival of the ectopic embryo through multifunctional activation.

Tubal ectopic pregnancy (TEP) is a serious pregnancy complication in the field of obstetrics and gynecology, accounting for more than 95% of ectopic pregnancies. Despite recent advances in diagnostic techniques, TEP remains an important cause of early maternal mortality, with an incidence rate of approximately 1% to 2% of all pregnancies [ 1 ]. The occurrence of TEP not only has a serious impact on the reproductive health of patients, but may also lead to long-term complications such as tubal injury, chronic pelvic pain, and secondary infertility [ 2 ]. Therefore, in-depth exploration of the pathogenesis of TEP is important for early diagnosis, intervention and prevention. The occurrence of TEP is closely related to abnormal changes in immune cells. Immune cells such as T cells, macrophages and dendritic cells play a key role in the pathological process of TEP. Wang et al. compared the distribution differences of maternal-fetal interface immune cells in normal pregnancy and TEP, suggesting that changes in immune cell composition and function may be an important factor in the occurrence of TEP [ 3 ]. Rigby et al., through a systematic review, found that T lymphocytes were the most abundant immune cell population in healthy fallopian tubes, whereas macrophages were significantly increased in pathological conditions such as TEP [ 4 ]. Shaw et al. further verified the distribution characteristics of lymphoid and myeloid cell populations in non-pregnant fallopian tubes by flow cytometry and immunohistochemistry, and found that the number of CD11c + cells was significantly increased in the fallopian tubes of patients with TEP [ 5 ]. Dereli et al. found that low neutrophil-to-lymphocyte ratio (NLR) and systemic immune-inflammation index (SII) were significantly associated with treatment success [ 6 ]. These studies suggest that immune cells play a crucial role in the function of the fallopian tubes and the pathophysiological mechanisms of TEP. Although previous studies have revealed the distribution and functional changes of immune cells in TEP, the understanding of their specific mechanisms remains limited. In recent years, the rapid development of single-cell sequencing technology has provided powerful tools for studying cell heterogeneity and function in complex tissues. It enables comprehensive analysis of the gene expression profiles of individual cells, thereby revealing functional differences and interactions between different cell types. It has been widely used in multiple biomedical fields, especially in oncology, developmental biology and immunology. In tumor research, it has helped identify specific cell types and gene expression patterns associated with tumor progression and metastasis, providing a basis for new therapeutic developments [ 7 , 8 ]. In developmental biology, it is used to study for cell differentiation and fate determination during embryonic development [ 9 ]. In the field of immunology, it has been used to analyze the diversity and functional status of immune cells, revealing dynamic changes in immune responses [ 10 , 11 ]. However, the application of single-cell sequencing in TEP is still limited. In this study, we collected tissues from implantation site ( IS) and non-implantation site (NIS) in the patients with TEP, and proposed to explore the possible mechanisms of TEP by single-cell sequencing. Through in-depth analysis of cell heterogeneity and the microenvironment, it may provide new insights into the pathogenesis of TEP and help refine our understanding of its underlying immune mechanisms.

This study systematically characterized the immune microenvironment of implantation versus non-implantation sites in the fallopian tubes of TEP using single-cell transcriptomic analysis, revealing the composition and functional reprogramming of key immune cells. Out Results showed that CD4 + Tm cells at implantation sites in TEP exhibited the dual function of pro-inflammatory and immune tolerance, participating in local inflammation while maintaining partial immune tolerance. CD8 + KLRC2 + Teff cells and CD8 + Tm cells displayed impaired function and reduced immune surveillance, creating conditions for embryonic immune evasion. Monocytes maintained chronic inflammation and promoted tissue remodeling by secreting pro-inflammatory and angiogenic factors. The proportion of M1 macrophages decreased, with restricted pro-inflammatory and apoptotic signaling, weakening early immune defense, whereas M2 macrophages increased in number and activation, contributing to inflammation suppression, immune tolerance maintenance, angiogenesis, and ECM remodeling. Collectively, the local immune microenvironment in TEP exhibits a complex state characterized by coexisting pro-inflammatory and immune tolerance, alongside parallel tissue injury and repair. This immune imbalance may permit transient embryo survival while simultaneously increasing the risk of tubal rupture and hemorrhage, revealing the cellular and molecular mechanisms underlying TEP development. These findings not only deepen our understanding of TEP pathophysiology but also provide potential strategies and targets for future interventions through modulation of the local immune microenvironment.

TEP is a serious threat to women’s reproductive health, with its pathogenesis involving multiple cellular and molecular abnormalities. This study employed single-cell sequencing to systematically analyze the immune cell infiltration and gene expression profiles at both IS and NIS in TEP fallopian tube tissues, revealing changes in various immune cells and potential mechanisms. Results showed that immune cell distribution and functional disorders at the implantation sites of TEP collectively promote a shift from immune rejection to a coexistence of immune tolerance and inflammation, providing conditions for abnormal embryo implantation. The occurrence of TEP is closely related to significant changes in the fallopian tube microenvironment, and single-cell sequencing technology provides a powerful tool for in-depth understanding of this process. In this study, we performed single-cell sequencing on IS and NIS tissues from TEP patients and found significant changes in the proportion and functional state of immune cells. Through cell clustering analysis, we identified 28 distinct cell clusters and annotated 13 cell types. Furthermore, the DEGs analysis and functional enrichment analysis revealed significant gene expression differences between IS and NIS in TEP patients, and mainly involved in immune and inflammatory responses, extracellular matrix remodeling, and infection-related pathways. These findings suggest that abnormal activation and functional disorders of immune cells may play a key role in the development of TEP. Notably, the stacked bar plot showed that T/NK cell and macrophage populations had the highest proportions in both IS and NIS, with significant differences, indicating that T/NK cells and macrophages may be closely related to the occurrence and development of TEP. Therefore, we further investigated the composition and functional states of T/NK cell and macrophage subpopulations to elucidate their specific roles in TEP and their association with disease progression. Among T/NK cells, CD4 + Tm cells constitute a core component of adaptive immunity, playing a pivotal role in maintaining tissue immune memory, regulating local inflammation, and mediating immune tolerance. Previous studies have demonstrated that CD4⁺ Tm cells can rapidly activate and proliferate upon recognition of previously encountered antigens, participating in infection defense, autoimmune regulation, and tissue repair [ 15 ]. During normal pregnancy, CD4⁺ Tm cells at the maternal-fetal interface balance pro-inflammatory and anti-inflammatory signals to eliminate maternal infections while maintaining immune tolerance toward the embryo, thereby ensuring successful gestation [ 16 ]. This study reveals that in TEP, the proportion of CD4⁺Tm cells at the IS significantly exceeds that at the NIS, suggesting this subset is actively recruited to the site of abnormal implantation and participates in the local immune response. Functional enrichment analysis of DEGs revealed a dual regulatory role for CD4⁺ Tm cells in the local microenvironment. On one hand, their gene expression profiles enriched for immune cell proliferation and activation, as well as immune regulation and inflammatory responses, indicating an activated state with the potential to drive local inflammation. On the other hand, DEGs were also enriched in angiogenesis, tissue repair, immune tolerance, and stress adaptation and tissue remodeling. These findings suggest that within the TEP microenvironment, CD4⁺ Tm cells are not a singular pro-inflammatory or immunosuppressive population, but rather exhibit dual functions of coexisting pro-inflammatory–immune tolerance. This dual function may temporarily support ectopic embryo survival, but persistent inflammation could damage the fallopian tube wall, thereby increasing the risk of rupture and bleeding. CD8⁺ KLRC2⁺ Teff cells express the activating receptor NKG2C (encoded by KLRC2) and exhibit potent cytotoxicity in antiviral and antitumor immunity [ 17 , 18 ]. In normal pregnancy, placental-derived HLA-E maintains the balance between immune defense and tolerance at the maternal-fetal interface through interaction with NKG2C/NKG2A receptors [ 19 , 20 ]. However, this study reveals a significant reduction in CD8⁺ KLRC2⁺ Teff cells proportion within the IS. The DEGs are enriched in pathways associated with weakened immune responses, reduced cytokine responsiveness, and immune suppression. These findings indicate a functional defect that may weaken local immune surveillance and promote enhanced immune tolerance. Previous studies indicate that the ordered assembly of HLA-I molecules on the cell membrane is crucial for the efficient cytotoxic function of CD8⁺ KLRC2⁺ Teff cells [ 21 ]. Disruption of this process markedly impairs their cytotoxic capacity. In this study, we observed functional impairment of CD8 + KLRC2 + Teff cells, consistent with this mechanism. Moreover, pathogens can achieve immune evasion by suppressing effector T cell cytokine secretion and cytotoxic activity [ 22 ]. Similarly, the observed functional suppression of CD8⁺ KLRC2⁺ Teff cells in this study may reflect the immune evasion process of abnormal embryos through specific mechanisms within the local microenvironment. Thus, the dysfunction of this cell population provides an immunological basis for TEP occurrence, promoting chronic tubal inflammation through disrupted immune regulation and increasing rupture risk. CD8⁺ Tm cells, serving as an immune memory reservoir, rapidly secrete cytokines such as IFN-γ and TNF-α upon reinfection. They participate in fallopian tube mucosal repair while mitigating inflammatory damage under estrogen receptor β (ERβ)-mediated hormonal signaling regulation [ 23 , 24 ]. In this study, we revealed a significantly reduced proportion of CD8⁺ Tm cells at IS in TEP. Their DEGs enriched in pathways involving abnormal hormone signaling, impaired antigen presentation, oxidative stress, and proliferation inhibition, indicating multifactorial functional impairment. Previous research has confirmed that downregulation of ERβ expression suppresses CD8⁺ Tm cell proliferation and cytokine secretion capacity [ 25 ], while oxidative stress disrupts T cell mitochondrial structure and weakens their effector functions [ 22 ]. Furthermore, this immune cell dysfunction often exacerbates local inflammatory responses and tissue damage [ 26 ]. Taken together, CD8 + Tm dysfunction may promote chronic inflammation and impair mucosal repair in the fallopian tube, thereby creating a permissive microenvironment for abnormal embryo adhesion and increasing the risk of TEP-related complications. Monocytes, as circulating immune cells, serve as the primary source of tissue macrophages. In response to inflammatory or injury signals, they are rapidly recruited to affected sites and differentiate into macrophages with distinct functional phenotypes depending on local cues. Previous studies have reported a marked increase in monocyte-derived cells in the fallopian tubes of patients with ectopic pregnancy [ 5 ], yet their specific phenotypes and functional pathways within the TEP microenvironment remain poorly understood. This study reveals that monocyte clusters constitute the predominant immune cell population, with a significantly higher proportion in the IS compared to the NIS. DEGs enrichment analysis indicates these monocytes are primarily involved in forming and maintaining the inflammatory microenvironment, immune dysregulation and migration, as well as angiogenesis and metabolic adaptation, suggesting their multifaceted roles in local immune imbalance and tissue remodeling. Studies have shown that monocytes sustain chronic local inflammation by secreting proinflammatory cytokines such as TNF-a and IL-6, and by differentiating into macrophages, they further regulate the intensity and persistence of the inflammatory response [ 27 ]. In addition, monocytes and their derivatives can secrete angiogenic factors such as VEGF and PDGF, promoting aberrant neovascularization and tissue remodeling-a feature commonly observed in tumor microenvironments and chronic diseases [ 28 ]. Therefore, the findings of this study suggest that in the local fallopian tube environment of TEP, monocytes may contribute to the formation and maintenance of the ectopic pregnancy microenvironment by secreting pro-inflammatory factors to sustain chronic inflammation and by promoting abnormal angiogenesis and tissue remodeling through factors such as VEGF and PDGF. M1 macrophages are typically activated by classical signals such as IFN-γand LPS, characterized by the production of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, IL-6) and iNOS, playing a crucial role in pathogen clearance and antitumor immunity [ 29 , 30 ]. Subpopulation analysis in this study revealed a significantly reduced proportion of M1 macrophages at IS in TEP. DEGs were primarily enriched in downregulated immune and inflammatory responses, weakened apoptosis signaling, restricted hematopoiesis and differentiation, and diminished angiogenic potential. This phenomenon indicates that within the local TEP microenvironment, M1 macrophages undergo a shift from a typical pro-inflammatory state to a functionally suppressed tolerant phenotype. In normal uterine pregnancy, M1 macrophages initially limit excessive trophoblast invasion through moderate inflammation, subsequently being gradually replaced by M2 macrophages to maintain immune tolerance [ 31 ]. The observed functional suppression of M1 macrophages in TEP may represent an adaptive mechanism that mitigates embryonic and tubal tissue damage by reducing local immune attacks and cell death signals. This conversion pattern resembles the immune evasion mechanism of M1-to-M2-like phenotype shifts in tumor microenvironments and may be driven by elevated progesterone levels, trophoblast-derived immunomodulatory factors (e.g., HLA-G, IL-10), and local hypoxia [ 32 – 34 ]. Although this regulation may favor transient embryonic survival, it simultaneously compromises tubal wall repair capacity and stromal remodeling functions, thereby increasing the risk of rupture [ 35 ]. Consequently, targeting M1 macrophage polarization or restoring partial immune function may offer potential strategies for immune intervention in TEP. M2 macrophages represent a polarized subtype of macrophages, regulated by multiple cytokines such as IL-4, IL-13, and IL-10. They exhibit anti-inflammatory, tissue repair, and immunoregulatory functions [ 36 ]. Our study demonstrates that the proportion of M2 macrophages at IS in TEP is significantly higher than at the NIS. Their DEGs are enriched in processes including immune regulation and inflammatory response, interferon signaling pathways, cell migration and angiogenesis, metabolic reprogramming, and ECM remodeling. This indicates that M2 macrophages are functionally activated across multiple dimensions in the TEP microenvironment, acting as central drivers for maintaining immune tolerance and a supportive tissue environment. In normal pregnancy, M2 macrophages suppress local immune responses by secreting anti-inflammatory factors such as IL-10 and TGF-β, promote angiogenesis via VEGF, and regulate ECM remodeling through matrix metalloproteinases (MMPs), thereby supporting embryo implantation and placental formation [ 37 ]. We hypothesize that in TEP, M2 macrophages are misguidedly activated, engaging pathways similar to uterine decidualization, creating a locally permissive environment that supports embryonic survival but may also induce structural remodeling of the tubal wall and increase rupture risk. This environment supports local embryo survival while potentially inducing tubal wall structural remodeling and rupture risk [ 38 , 39 ]. This aberrant activation resembles the excessive M2 activity observed in wound healing or fibrotic diseases, where tissue repair is promoted but patgical structural changes can occur simultaneously [ 40 ]. Additionally, trophoblast-derived signaling molecules (e.g., HLA-G, IL-10, Galectin-1) and progesterone may sustain the abnormal hyperactivation of M2 macrophages [ 41 ]. Therefore, targeting the regulation of M2 polarization or its downstream VEGF/MMP pathways holds promise as a novel immunological intervention strategy for the prevention and treatment of TEP. The present study revealed changes in the distribution and dysfunction of immune cells at IS in TEP by single-cell analysis and suggested a shift from immune rejection to immune tolerance and inflammation coexistence. However, this study has several limitations. First, the limited sample size may impact the generalizability and statistical power of the findings, which need to be validated in larger samples in the future. Second, this study mainly focused on immune cells, but the pathomechanism of TEP may also involve other factors such as genetic background and hormone influences, which may interact with immune cell changes, and this study failed to adequately consider these confounding factors. Third, the present study only revealed an association between immune cell distribution and embryo implantation abnormalities, but did not demonstrate a causal relationship. Future studies should employ functional experiments, such as cell co-culture and animal models, to further investigate the causal links and underlying mechanisms. Finally, this study lacks direct comparison with the endometrial implantation sites of normal intrauterine pregnancies, limiting a comprehensive understanding of the TEP-specific immunological characteristics. Future research will involve multicenter collaboration to incorporate endometrial samples from normal intrauterine pregnancies, in compliance with ethical guidelines, to further validate the features of the TEP-specific immune microenvironment and elucidate the mechanisms underlying differences between abnormal immune imbalance and normal pregnancy immune tolerance.

Supplementary Material 1. Supplementary Material 1. Supplementary Material 2. Supplementary Material 2.