BACKGROUND: The polymorphisms of the FOXP3 gene may mediate abnormalities in Tregs, leading to an imbalance in maternal-fetal immune tolerance and ultimately resulting in recurrent spontaneous abortion (RSA). This meta-analysis aims to assess the potential association between FOXP3 polymorphisms and susceptibility to RSA using five specific single nucleotide polymorphisms (SNPs).
METHODS: By conducting a comprehensive search across databases such as EMBASE, PubMed, Web of Science, Cochrane Library, CNKI, Wanfang, and CBM, we identified suitable studies for inclusion in the meta-analysis. The data extracted from these studies were subjected to analysis using Stata SE 15. To assess the degree of association, we utilized the odds ratio (OR) along with its corresponding 95% confidence intervals (CI). Five specific single nucleotide polymorphisms (SNPs) were employed in assessing the connection between FOXP3 gene polymorphisms and RSA.
RESULTS: The meta-analysis demonstrated a significant association between several polymorphisms (rs3761548, rs2232365, rs2232368, rs2280883, and rs2294021) and susceptibility to RSA. Conversely, the FOXP3 rs5902434 polymorphism was not associated with susceptibility to RSA.
CONCLUSIONS: Our meta-analysis suggests that these genetic variations within the FOXP3 gene might play a role in the progression of RSA disease. Meanwhile, large-scale studies that consider multiple factors are needed to validate this finding.

OBJECTIVE: We aimed to explore the risk factors in patients with unexplained recurrent spontaneous abortion (URSA) and to provide a basis for clinically targeted therapy.
METHODS: This case-control study comprised 202 patients with URSA treated at our hospital and 115 women in early pregnancy with a normal birth history during the same period. After procuring the data we conducted a multivariate logistic regression analysis of risk factors related to URSA.
RESULTS: Logistic regression analysis showed (i) that the number of spontaneous abortions (SAs; odds ratio [OR] = 492.123), the levels of autoantibodies (OR = 19.322) and tumor necrosis factor alpha (TNF-α; OR = 9.615), and the CT and TT genotypes of methylenetetrahydrofolate reductase (MTHFR) C677T (OR = 6.217 and 15.009, respectively) were risk factors for URSA and (ii) that 25-hydroxyvitamin D (25-(OH)D; OR = 0.919) was a protective factor. The most important risk factor was a history of one or more SAs, with the risk of pregnancy loss increasing 491.123-fold. Every unit increase in serum 25-(OH)D reduced the risk of SA by 8.1%.
CONCLUSIONS: The risk factors for URSA included the number of SAs, the levels of autoantibodies and TNF-α, and the MTHFR C677T T allele; 25-(OH)D was a protective factor. We recommend that women diagnosed with URSA receive intervention as soon as possible so as to actively reduce the incidence of recurrent SA.

Background and aims: Certain chromosomal structural variations (SVs) in biological parents can lead to recurrent spontaneous abortions (RSAs). Unequal crossing over during meiosis can result in the unbalanced rearrangement of gamete chromosomes such as duplication or deletion. Unfortunately, routine techniques such as karyotyping, fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA), and copy number variation sequencing (CNV-seq) cannot detect all types of SVs. In this study, we show that optical genome mapping (OGM) quickly and accurately detects SVs for RSA patients with a high resolution and provides more information about the breakpoint regions at gene level. Methods: Seven couples who had suffered RSA with unbalanced chromosomal rearrangements of aborted embryos were recruited, and ultra-high molecular weight (UHMW) DNA was isolated from their peripheral blood. The consensus genome map was created by de novo assembly on the Bionano Solve data analysis software. SVs and breakpoints were identified via alignments of the reference genome GRCh38/hg38. The exact breakpoint sequences were verified using either Oxford Nanopore sequencing or Sanger sequencing. Results: Various SVs in the recruited couples were successfully detected by OGM. Also, additional complex chromosomal rearrangement (CCRs) and four cryptic balanced reciprocal translocations (BRTs) were revealed, further refining the underlying genetic causes of RSA. Two of the disrupted genes identified in this study, FOXK2 [46,XY,t(7; 17)(q31.3; q25)] and PLXDC2 [46,XX,t(10; 16)(p12.31; q23.1)], had been previously shown to be associated with male fertility and embryo transit. Conclusion: OGM accurately detects chromosomal SVs, especially cryptic BRTs and CCRs. It is a useful complement to routine human genetic diagnostics, such as karyotyping, and detects cryptic BRTs and CCRs more accurately than routine genetic diagnostics.

Recurrent spontaneous abortion (RSA) is defined as two or more pregnancy loss, affecting the happiness index of fertility couples. The mechanisms involved in the occurrence of RSA are not clear to date. The primary problem for the maternal immune system is how to establish and maintain the immune tolerance to the semi-allogeneic fetuses. During the pregnancy, decidual macrophages mainly play an important role in the immunologic dialogue. The purpose of this study is to explore decidual macrophages, and to understand whether there is a connection between these cells and RSA by analyzing their phenotypes and functions. Pubmed, Web of Science and Embase were searched. The eligibility criterion for this review was evaluating the literature about the pregnancy and macrophages. Any disagreement between the authors was resolved upon discussion and if required by the judgment of the corresponding author. We summarized the latest views on the phenotype, function and dysfunction of decidual macrophages to illuminate its relationship with RSA.

Recurrent spontaneous abortion (RSA) is a disturbing pregnancy disorder experienced by ~2.5% of women attempting to conceive. The pathogenesis of RSA is still unclear. Previous findings revealed that transcription factor YIN-YANG 1(YY1) was related to the pathogenesis of RSA by influence trophoblastic cell invasion ability. Present study aimed to investigate more specific molecular mechanism of YY1 playing in trophoblastic cells. In our research, RNA-seq and Chip-seq were used to find significant changed genes between si-YY1(Knock down of YY1) HTR-8/SVneo cells(n = 3) and HTR-8/SVneo cells(n = 3). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis results suggested that Integrins related pathway maybe necessary to biological functions of trophoblastic cells. Chip-seq dataset analysis results predict YY1 can regulate ITGA3/7 expression by binding to the promoter region of ITGA3/7. Furthermore, results from chip experiment, RT-PCR, Dual-luciferase reporter gene assay showed that YY1 was able to bind to the promoter region of ITGA3 and regulate ITGA3 mRNA and protein expression. However, ITGA7 could not be significant influenced by YY1. Besides, gene silencing experiment, Western blot and Immunofluorescence assay confirmed that both YY1 and ITGA3 can accelerate phosphorylation focal adhesion kinase and affect cytoskeleton formation in HTR-8/SVneo cells. In conclusion, YY1/ITGA3 play a critical role in trophoblast invasion ability by regulating cytoskeleton formation.

Impaired spermatozoa immunogenicity can result in pregnancy complications such as recurrent spontaneous abortion (RSA). Given that spermatozoa contact with microbiota, it is possible that inappropriate microbiota composition in the reproductive tract could result in the alteration of spermatozoa antigenicity. Probiotics, as a representative of microbiota, may therefore have a beneficial effect on this altered immunogenicity. The objective of this study was to determine the effect of probiotics on spermatozoa immunogenicity.
Twenty-five fertile couples and twenty-five RSA couples were included in this study. Spermatozoa were purified and treated with probiotics. Untreated and probiotic treated spermatozoa were evaluated for human leukocyte antigen (HLA) class I & II expression by flow cytometry. Untreated and probiotic treated spermatozoa were also cocultured with the wife\'s peripheral blood mononuclear cells (PBMC) for 12 days. Then, the supernatant was assessed for IgG and APCA by enzyme-linked immunosorbent assay (ELISA) and complement-dependent cytotoxicity (CDC) assay respectively.
Probiotic treatment of spermatozoa leads to an increase of HLA class I & II expression in both the fertile and RSA groups. The probiotic treatment resulted in a decrease in both IgG and APCA in the fertile group, but an increase in both IgG and APCA in the RSA group.
The results of this study suggest that a supplementary probiotic treatment may be useful in couples suffering from RSA with an immunologic cause, because it improves disturbed HLA expression on spermatozoa and improves disturbed APCA and IgG production in the presence of spermatozoa.

Human uterine stromal cell undergoes decidualization for pregnancy establishment and maintenance, which involved extensive proliferation and differentiation. Increasing studies have suggested that recurrent spontaneous abortion (RSA) may result from defective endometrial stromal decidualization. However, the critical molecular mechanisms underlying impaired decidualization during RSA are still elusive. By using our recently published single-cell RNA sequencing (scRNA-seq) atlas, we found that MYC-associated factor X (MAX) was significantly downregulated in the stromal cells derived from decidual tissues of women with RSA, followed by verification with immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). MAX knockdown significantly impairs human endometrial stromal cells (HESCs) proliferation as determined by MTS assay and Ki67 immunostaining, and decidualization determined by F-actin, and decidualization markers. RNA-seq together with chromatin immunoprecipitation sequencing (ChIP-seq) and cleavage under targets and release using nuclease sequencing (CUT&RUN-seq) analysis were applied to explore the molecular mechanisms of MAX in regulation of decidualization, followed by dual-luciferase reporter assay to verify that MAX targets to (odd-skipped related transcription factor 2) OSR2 directly. Reduced expression of OSR2 was also confirmed in decidual tissues in women with RSA by IHC and qRT-PCR. OSR2 knockdown also significantly impairs HESCs decidualization. OSR2-overexpression could at least partly rescue the downregulated insulin-like growth factor binding protein 1 (IGFBP1) expression level in response to MAX knockdown. Collectively, MAX deficiency observed in RSA stromal cells not only attenuates HESCs proliferation but also impairs HESCs decidualization by downregulating OSR2 expression at transcriptional level directly.

Recurrent spontaneous abortion (RSA) is a common complication during early gestation, which is associated with aberrant DNA methylation. Zinc Finger and BTB Domain Containing 24 (ZBTB24) plays a critical role in facilitating DNA methylation and cell proliferation. However, the regulatory role of ZBTB24 on trophoblast development in RSA remains unclear. In this study, ZBTB24 expression was compared between decidua tissues of RSA patients and induced abortion controls from a published dataset, which was further validated in placental villi tissues by RT-qPCR and Western blot. The roles of ZBTB24 in trophoblast proliferation, differentiation, and migration were investigated by functional assays after ZBTB24 knockdown or overexpression in HTR-8/SVneo cells. Our results showed that ZBTB24 expression was significantly decreased in RSA patients, and ZBTB24 expression level positively regulated cell viability, differentiation, and migration in HTR-8/SVneo cells. We further demonstrated that ZBTB24 modulated the expression of E-cadherin by altering the DNA methylation at the promoter region. Overall, the downregulation of ZBTB24 is implicated in RSA by inhibiting trophoblast proliferation, differentiation, and migration. Therefore, ZBTB24 may serve as a promising therapeutic target and diagnostic marker for RSA.

UNASSIGNED: One of the major genetic causes of recurrent spontaneous abortions is parental chromosomal abnormalities. The objectives of the study were to determine, compare and analyze the incidence and distribution of chromosomal abnormalities in couples with recurrent miscarriages from Northeastern Iran.
UNASSIGNED: This study was conducted at Ghaem Hospital, Mashhad, Iran. We evaluated karyotype results of 608 couples with history of recurrent spontaneous abortion. The standard method was used for culturing peripheral venous blood lymphocytes.
UNASSIGNED: Chromosome aberrations were detected in 43 patients (3.54%), including 25 females and 18 males. Structural chromosomal abnormality was detected in 40 cases, including balanced translocations (25 cases), robertsonian translocations (4 cases), inversions (10 cases) and numerical chromosome aberrations (3 cases). Polymorphic variants were observed in 22 individuals.
UNASSIGNED: The frequency of chromosomal abnormalities in couples with Recurrent Spontaneous Abortion (RSA) in our study is 3.54%. Reciprocal translocation, pericentric inversions, robertsonian translocations, and numerical abnormality observed among couples who had experienced recurrent spontaneous abortions and that these couples might benefit from cytogenetic analysis.

Previous studies have reported the involvement of γδ T cells in recurrent spontaneous abortion (RSA); however, both pathogenic and protective effects were suggested. To interrogate the role of γδ T cells in RSA, peripheral blood from RSA patients and healthy women with or without pregnancy were analyzed for γδ T cells by flow cytometry (n = 9-11 for each group). Moreover, the decidua from pregnant RSA patients and healthy controls (RSA-P and HC-P group, respectively) was simultaneously stained for γδ T cells by immunohistochemistry (IHC) and bulk sequenced for gene expression. Our results demonstrated that the frequencies of peripheral γδ T cells and their subpopulations in RSA patients were comparable to that in healthy subjects, but the PD1 expression on Vδ2+ cells was increased in pregnant patients. Furthermore, peripheral Vδ2+ cells in RSA-P patients demonstrated significantly increased expression of CD107a, as compared to that in pregnant healthy controls. In addition, RSA-P patients had higher proportion of IL-17A-secreting but not IL-4-secreting Vδ2+ cells compared to the control groups. In decidua, an inflammatory microenvironment was also evident in RSA-P patients, in which CCL8 expression and the infiltration of certain immune cells were higher than that in the HC-P group, as revealed by transcriptional analysis. Finally, although the presence of γδ T cells in decidua could be detected during pregnancy in both RSA patients and healthy subjects by multicolor IHC analysis, the expression of CD107a on γδ T cells was markedly higher in the RSA-P group. Collectively, our results indicated that the increased activation, cytotoxicity, and inflammatory potential of peripheral and/or local γδ T cells might be responsible for the pathogenesis of RSA. These findings could provide a better understanding of the role of γδ T cells in RSA and shed light on novel treatment strategies by targeting γδ T cells for RSA patients.