Key Gene and Functional Pathways Identified in Unexplained Recurrent Spontaneous Abortion Using Targeted RNA Sequencing and Clinical Analysis

Abstract

Identifying the mechanisms underlying unexplained recurrent spontaneous abortion (URSA) can help develop effective treatments. This study provides novel insights into the biological characteristics and related pathways of differentially expressed genes (DEGs) in URSA. Nineteen patients with URSA and three healthy fertile women with regular menstruation (control group) were recruited. RNA was extracted from the two groups to determine the differential expression of immunoregulatory gene sequences. Gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses were used to identify the biological functions and pathways of the identified DEGs. A protein-protein interaction (PPI) network was constructed using the STRING database. Furthermore, qRT-PCR and ELISA were performed to validate the differential expression of the hub genes. We also explored the regulatory mechanism of Th1/Th2 imbalance. A total of 99 DEGs were identified, comprising 94 upregulated and five downregulated genes. Through GO analysis, nine immune cell function-related clusters were selected, and genes with significant differential expression were primarily enriched in eight immune regulatory functions related to the KEGG signalling pathway. Subsequently, five hub genes (TLR2, CXCL8, IFNG, IL2RA, and ITGAX) were identified using Cytoscape software; qRT-PCR confirmed the differential expression among the hub genes, whereas ELISA revealed a significant difference in extracellular IFN-γ and IL-8 levels. The levels of Th1 (IFN-γ) and the Th1/Th2 ratio were higher in the peripheral blood of URSA patients than in control group patients. These findings suggest that the occurrence of URSA may be associated with the abnormal expression of some specific immunoregulatory genes involved in T-cell activation and differentiation. Among the identified DEGs, IFNG may play a key role in regulating maternal immune response. Although further validation is required, our data provide an important theoretical basis for elucidating the pathogenesis of recurrent spontaneous abortion.

METTL3-Mediated m6A RNA Methylation of ZBTB4 Interferes With Trophoblast Invasion and Maybe Involved in RSA

“…Studies have shown that TLR was expressed in placental cells and participated in the initiation of signal mediators that were essential for the immune response in choriocarcinoma cell lines, which may be related to placental inflammation ( Klaffenbach et al, 2005 ). Similarly, cytotoxicity mediated by natural killer cells (NK) can lead to endometritis, form a uterine immune state, and then lead to infertility, recurrent abortion, and so on ( Chen et al, 2020 ; Li et al, 2020 ; Gu et al, 2021 ). Other studies have shown that apoptosis can affect the differentiation and invasion of trophoblast cells, block the process of vascular recasting, change the function of the placenta, and finally lead to embryo implantation, growth restriction, and abortion ( Sharp et al, 2010 ).…”

“…In this study, we found that m 6 A modification in RSA trophoblast cells was significantly different from that in the normal control group through MeRIP-seq, and the pathways of the differential m 6 A modified genes enriched have been reported to be related to the pathogenesis of RSA such as toll-like receptor signaling pathway, natural killer cell–mediated cytotoxicity, and necrotizing apoptosis. ( Klaffenbach et al, 2005 ; Sharp et al, 2010 ; Chen et al, 2020 ; Li et al, 2020 ; Gu et al, 2021 ). The authors pointed out that URSA patients, compared with normal pregnant women, were enriched in several signaling pathways associated with immune regulatory functions, including natural killer cell–mediated cytotoxicity, cytokine receptor interaction, and other signaling pathways which were consistent with our findings ( Klaffenbach et al, 2005 ; Sharp et al, 2010 ; Chen et al, 2020 ; Li et al, 2020 ; Gu et al, 2021 ).…”

“…( Klaffenbach et al, 2005 ; Sharp et al, 2010 ; Chen et al, 2020 ; Li et al, 2020 ; Gu et al, 2021 ). The authors pointed out that URSA patients, compared with normal pregnant women, were enriched in several signaling pathways associated with immune regulatory functions, including natural killer cell–mediated cytotoxicity, cytokine receptor interaction, and other signaling pathways which were consistent with our findings ( Klaffenbach et al, 2005 ; Sharp et al, 2010 ; Chen et al, 2020 ; Li et al, 2020 ; Gu et al, 2021 ).…”

Transcriptome sequencing identified the ceRNA network associated with recurrent spontaneous abortion

“…At present, some researchers have obtained some results about recurrent abortion through transcriptome sequencing [ 27 , 28 ]. In this study, we firstly performed transcriptome sequencing analysis on the tissues of 3 patients with RSA and 3 patients with normal abortion, and found key molecules by constructing lncRNA-related ceRNA network, which is helpful to explore the pathogenic mechanism of RSA.…”

Research on the Mechanism of Asperosaponin VI for Treating Recurrent Spontaneous Abortion by Bioinformatics Analysis and Experimental Validation

“…According to statistics, over 5% of women at reproductive age su er from RSA [3]. Multiple pathological factors lead to RSA, including genetic factors, anatomical abnormalities, endocrine disorders, infectious diseases, and thyroid dysfunction [4,5]. Embryonic chromosomal abnormality is the main cause of spontaneous abortion in early pregnancy, which results in over 50% of rst trimester miscarriages [6][7][8].…”