Single-cell and spatial transcriptomics reveal alterations in trophoblasts at invasion sites and disturbed myometrial immune microenvironment in placenta accreta spectrum disorders.

BACKGROUND: Placenta accreta spectrum disorders (PAS) are a severe complication characterized by abnormal trophoblast invasion into the myometrium. The underlying mechanisms of PAS involve a complex interplay of various cell types and molecular pathways. Despite its significance, both the characteristics and intricate mechanisms of this condition remain poorly understood. METHODS: Spatial transcriptomics (ST) and single-cell RNA sequencing (scRNA-seq), were performed on the tissue samples from four PAS patients, including invasive tissues (ST, n = 3; scRNA-seq, n = 4), non-invasive normal placenta samples (ST, n = 1; scRNA-seq, n = 2). Three healthy term pregnant women provided normal myometrium samples (ST, n = 1; scRNA-seq, n = 2). ST analysis characterized the spatial expression landscape, and scRNA-seq was used to identify specific cellular components in PAS. Immunofluorescence staining was conducted to validate the findings. RESULTS: ST slices distinctly showed the myometrium in PAS was invaded by three subpopulations of trophoblast cells, extravillous trophoblast cells, cytotrophoblasts, and syncytiotrophoblasts, especially extravillous trophoblast cells. The pathways enriched by genes in trophoblasts, smooth muscle cells (SMC), and immune cells of PAS were mainly associated with immune and inflammation. We identified elevated expression of the angiogenesis-stimulating gene PTK2, alongside the cell proliferation-enhancing gene EGFR, within the trophoblasts of PAS group. Trophoblasts mainly contributed the enhancement of HLA-G and EBI3 signaling, which is crucial in establishing immune escape. Meanwhile, SMC regions in PAS exhibited upregulation of immunomodulatory markers such as CD274, HAVCR2, and IDO1, with CD274 expression experimentally verified to be increased in the invasive SMC areas of the PAS group. CONCLUSIONS: This study provided information of cellular composition and spatial organization in PAS at single-cell and spatial level. The dysregulated expression of genes in PAS revealed a complex interplay between enhanced immune escape in trophoblasts and immune tolerance in SMCs during invasion in PAS. These findings will enhance our understanding of PAS pathogenesis for developing potential therapeutic strategies.

Exploring myometrial microenvironment changes at the single-cell level from nonpregnant to term pregnant states.

The microenvironment and cell populations within the myometrium play crucial roles in maintaining uterine structural integrity and protecting the fetus during pregnancy. However, the specific changes occurring at the single-cell level in the human myometrium between nonpregnant (NP) and term pregnant (TP) states remain unexplored. In this study, we used single-cell RNA sequencing (scRNA-Seq) and spatial transcriptomics (ST) to construct a transcriptomic atlas of individual cells in the myometrium of NP and TP women. Integrated analysis of scRNA-Seq and ST data revealed spatially distinct transcriptional characteristics and examined cell-to-cell communication patterns based on ligand-receptor interactions. We identified and categorized 87,845 high-quality individual cells into 12 populations from scRNA-Seq data of 12 human myometrium tissues. Our findings demonstrated alterations in the proportions of five subpopulations of smooth muscle cells in TP. Moreover, an increase in monocytic cells, particularly M2 macrophages, was observed in TP myometrium samples, suggesting their involvement in the anti-inflammatory response. This study provides unprecedented single-cell resolution of the NP and TP myometrium, offering new insights into myometrial remodeling during pregnancy.NEW & NOTEWORTHY Using single-cell RNA sequencing and spatial transcriptomics, the myometrium was examined at the single-cell level during pregnancy. We identified spatially distinct cell populations and observed alterations in smooth muscle cells and increased M2 macrophages in term pregnant women. These findings offer unprecedented insights into myometrial remodeling and the anti-inflammatory response during pregnancy. The study advances our understanding of pregnancy-related myometrial changes.

Serpin family E member 1 enhances myometrium contractility by increasing ATP production during labor.

The uterine contraction during labor, a process with repetitive hypoxia and high energy consumption, is essential for successful delivery. However, the molecular mechanism of myometrial contraction regulation is unknown. Serpin family E member 1 (SERPINE1), one of the most upregulated genes in laboring myometrium in both transcriptome and proteome, was highlighted in our previous study. Here, we confirmed SERPINE1 is upregulated in myometrium during labor. Blockade of SERPINE1 using small interfering RNA (siRNA) or inhibitor (Tiplaxtinin) under hypoxic conditions in myocytes or myometrium in vitro showed a decrease contractility, which was achieved by regulating ATP production. Chromatin immunoprecipitation (ChIP-seq), Co-immunoprecipitation (Co-IP), and glutathione-S-transferase (GST) pull down explored that the promoter of SERPINE1 is directly activated by hypoxia-inducible factor-1α (HIF-1α) and SERPINE1 interacts with ATP Synthase Peripheral Stalk Subunit F6 (ATP5PF). Together they enhance hypoxia driven myometrial contraction by maintaining ATP production in the key oxidative phosphorylation pathway. The results provide new insight for uterine contraction regulation, and potential novel therapeutic targets for labor management.

Lipid reprogramming induced by the NNMT-ABCA1 axis enhanced membrane fluidity to promote endometrial cancer progression.

Elucidating the mechanism for the high metastasis capacity of Endometrial cancer (EC) is crucial to improve treatment outcomes of EC. We have recently reported that nicotinamide N-methyltransferase (NNMT) is overexpressed in EC, especially in EC, and predicts poor survival of chemotherapy patients. Here, we aimed to determine the function and mechanism of NNMT on metastasis of EC. Additionally, analysis of public datasets indicated that NNMT is involved in cholesterol metabolism. In vitro, NNMT overexpression promoted migration and invasion of EC by reducing cholesterol levels in the cytoplasm and cell membrane. Mechanistically, NNMT activated ABCA1 expression, leading to cholesterol efflux and membrane fluidity enhancement, thereby promoting EC's epithelial-mesenchymal transition (EMT). In vivo, the metastasis capacity of EC was weakened by targeting NNMT. Our findings suggest a new molecular mechanism involving NNMT in metastasis, poor survival of EC mediated by PP2A and affecting cholesterol metabolism.

Upregulated TIMP1 facilitates and coordinates myometrial contraction by decreasing collagens and cell adhesive capacity during human labor.

Myometrial contraction is one of the key events involved in parturition. Increasing evidence suggests the importance of the extracellular matrix (ECM) in this process, in addition to the functional role of myometrial smooth muscle cells, and our previous study identified an upregulated tissue inhibitor of metalloproteinase 1 (TIMP1) in human laboring myometrium compared to nonlabor samples. This study aimed to further explore the potential role of TIMP1 in myometrial contraction. First, we confirmed increased myometrial TIMP1 levels in labor and during labor with cervical dilation using transcriptomic and proteomic analyses, followed by real-time PCR, western blotting, and immunohistochemistry. Then, a cell contraction assay was performed to verify the decreased contractility after TIMP1 knockdown in vitro. To further understand the underlying mechanism, we used RNA-sequencing analysis to reveal the upregulated genes after TIMP1 knockdown; these genes were enriched in collagen fibril organization, cell adhesion, and ECM organization. Subsequently, a human matrix metalloproteinase (MMP) array and collagen staining were performed to determine the TIMPs, MMPs and collagens in laboring and nonlabor myometrium. A real-time cell adhesion assay was used to detect cell adhesive capacity. The results showed upregulated MMP8 and MMP9, downregulated collagens, and attenuated cell adhesive capacity in laboring myometrium, while lower MMP levels and higher collagen levels and cell adhesive capacity were observed in nonlabor. Moreover, TIMP1 knockdown led to restoration of cell adhesive capacity. Together, these results indicate that upregulated TIMP1 during labor facilitates and coordinates myometrial contraction by decreasing collagen and cell adhesive capacity, which may provide effective strategies for the regulation of myometrial contraction.

Golgi Reassembly Stacking Protein 2 Modulates Myometrial Contractility during Labor by Affecting ATP Production.

The mechanism of maintaining myometrial contractions during labor remains unclear. Autophagy has been reported to be activated in laboring myometrium, along with the high expression of Golgi reassembly stacking protein 2 (GORASP2), a protein capable of regulating autophagy activation. This study aimed to investigate the role and mechanism of GORASP2 in uterine contractions during labor. Western blot confirmed the increased expression of GORASP2 in laboring myometrium. Furthermore, the knockdown of GORASP2 in primary human myometrial smooth muscle cells (hMSMCs) using siRNA resulted in reduced cell contractility. This phenomenon was independent of the contraction-associated protein and autophagy. Differential mRNAs were analyzed using RNA sequencing. Subsequently, KEGG pathway analysis identified that GORASP2 knockdown suppressed several energy metabolism pathways. Furthermore, reduced ATP levels and aerobic respiration impairment were observed in measuring the oxygen consumption rate (OCR). These findings suggest that GORASP2 is up-regulated in the myometrium during labor and modulates myometrial contractility mainly by maintaining ATP production.

Integrating single-cell RNA sequencing with spatial transcriptomics reveals an immune landscape of human myometrium during labour.

BACKGROUND: The transition of the myometrium from a quiescent to a contractile state during labour is known to involve inflammation, which is characterized by the infiltration of immune cells and the secretion of cytokines. However, the specific cellular mechanisms underlying inflammation in the myometrium during human parturition are not yet fully understood. METHODS: Through the analysis of transcriptomics, proteomics, and cytokine arrays, the inflammation in the human myometrium during labour was revealed. By performing single-cell RNA sequencing (scRNA-seq) and spatiotemporal transcriptomic (ST) analyses on human myometrium in term in labour (TIL) and term in non-labour (TNL), we established a comprehensive landscape of immune cells, their transcriptional characteristics, distribution, function and intercellular communications during labour. Histological staining, flow cytometry, and western blotting were applied to validate some results from scRNA-seq and ST. RESULTS: Our analysis identified immune cell types, including monocytes, neutrophils, T cells, natural killer (NK) cells and B cells, present in the myometrium. TIL myometrium had a higher proportion of monocytes and neutrophils than TNL myometrium. Furthermore, the scRNA-seq analysis showed an increase in M1 macrophages in TIL myometrium. CXCL8 expression was mainly observed in neutrophils and increased in TIL myometrium. CCL3 and CCL4 were principally expressed in M2 macrophages and neutrophils-6, and decreased during labour; XCL1 and XCL2 were specifically expressed in NK cells, and decreased during labour. Analysis of cytokine receptor expression revealed an increase in IL1R2, which primarily expressed in neutrophils. Finally, we visualized the spatial proximity of representative cytokines, contraction-associated genes, and corresponding receptors in ST to demonstrate their location within the myometrium. CONCLUSIONS: Our analysis comprehensively revealed changes in immune cells, cytokines, and cytokine receptors during labour. It provided a valuable resource to detect and characterize inflammatory changes, yielding insights into the immune mechanisms underlying labour.

Retinoic acid receptor gamma is required for proliferation of pancreatic cancer cells.

Despite the expectation that retinoic acid receptor could be the potential therapeutic targets for pancreatic cancers, there has been the lack of information about the role and the impact of retinoic acid receptor gamma (RARγ, RARG) on pancreatic cancer, unlike other two RARs. Herein, we applied TCGA and GEO database to show that the expression and prognosis of RARG is closely related to pancreatic cancer, which demonstrates that RARG is commonly overexpressed in human pancreatic cancer and is an independent diagnostic marker predicting the poor prognosis of pancreatic cancer patients. In addition, we demonstrated that the reduction in the expression of RARG in human pancreatic cancer cells dramatically suppress their proliferation and tumor growth in vivo, partially attributable to the downregulation of tumor-supporting biological processes such as cell proliferation, antiapoptosis and metabolism and the decreased expression of various oncogenes like MYC and STAT3. Mechanistically, RARG binds on the promoters of MYC, STAT3, and SLC2A1 which is distinguished from well-known conventional Retinotic acid response elements (RAREs) and that the binding is likely to be responsible for the epigenetic activation in the level of chromatin, assessed by the measurement of deposition of the gene activation marker histone H3 K27 acetylation (H3K27ac) using ChIP-qPCR. In this study, we reveal that RARG plays important role in the tumorigenesis of pancreatic cancer and represents new therapeutic targets for human pancreatic cancer.

Proteomic and miRNA Profiles of Exosomes Derived from Myometrial Tissue in Laboring Women.

Myometrial contraction is essential for successful delivery. Recent studies have highlighted the vital roles of tissue-derived exosomes in disease diagnostic, prognostic, and therapeutic applications; however, the characteristics of uterine myometrium-derived exosomes are unclear. Here, we successfully isolated exosomes from myometrial tissues, human myometrial smooth muscle cells (HMSMCs), and human umbilical vein endothelial cells (HUVECs), then performed quantitative liquid chromatography-tandem mass spectrometry and miRNA sequencing to investigate the cargo of the exosomes. Fifty-two proteins and five miRNAs were differentially expressed (DE) in term non-labor and term labor myometrium-derived exosomes. Among them, seven proteins (SERPINE1, THBS1, MGAT1, VIM, FGB, FGG, and VWF) were differentially expressed both in the myometrial exosomes and tissues, three miRNAs (miR-363-3p, miR-203a-3p, and miR-205-5p) target 13 DE genes. The top three miRNA derived from HMSMCs (miR-125b-1-3p, miR-337-5p, and miR-503-5p) and HUVECs (miR-663a, miR-4463, and miR-3622a-5p) were identified. Two proteins, GJA1 and SLC39A14, exist in female blood exosomes and are highly expressed in HMSMCs exosomes, are also upregulated in the laboring myometrium, which verified increased in laboring blood samples, might be novel potential biomarkers for myometrial activation. The proteomic and miRNA profile of exosomes derived from laboring myometrium revealed some molecules in the exosomes that affect the intercellular communication and the function of the myometrium.

HIF-1α is essential for the augmentation of myometrial contractility during labor†.

Uterine contraction is crucial for a successful labor and the prevention of postpartum hemorrhage. It is enhanced by hypoxia; however, its underlying mechanisms are yet to be elucidated. In this study, transcriptomes revealed that hypoxia-inducible factor-1alpha was upregulated in laboring myometrial biopsies, while blockade of hypoxia-inducible factor-1alpha decreased the contractility of the myometrium and myocytes in vitro via small interfering RNA and the inhibitor, 2-methoxyestradiol. Chromatin immunoprecipitation sequencing revealed that hypoxia-inducible factor-1alpha directly binds to the genome of contraction-associated proteins: the promoter of Gja1 and Ptgs2, and the intron of Oxtr. Silencing the hypoxia-inducible factor-1alpha reduced the expression of Ptgs2, Gja1, and Oxtr. Furthermore, blockade of Gja1 or Ptgs2 led to a significant decrease in myometrial contractions in the hypoxic tissue model, whereas atosiban did not remarkably influence contractility. Our study demonstrates that hypoxia-inducible factor-1alpha is essential for promoting myometrial contractility under hypoxia by directly targeting Gja1 and Ptgs2, but not Oxtr. These findings help us to better understand the regulation of myometrial contractions under hypoxia and provide a promising strategy for labor management and postpartum hemorrhage treatment.