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Background: Mothers experiencing recurrent spontaneous abortion (RSA) along with repeated implantation failures (RIF) could potentially have abnormalities in their immune systems. Vitamin D is known as a crucial immunomodulatory agent. This study aimed to assess the ratio of Natural Killer T-cells (NKTs) and the correlation between this ratio with serum vitamin D levels among women with RSA and RIF. Methods: In this research, blood samples were collected from both patients and a group of healthy individuals. The flow cytometry technique was used to determine the proportion of NKT and activated NKT cells. Additionally, Vitamin D and IFN-γ levels were measured using the ELISA technique. Results: The mean ratio of NKT cells and IFN-γ levels increased significantly in those women with RSA relative to our healthy control group [(P < 0.018) and (p < 0.031), respectively]. Nevertheless, women in the RIF and control groups did not show any significant differences. Serum vitamin D levels significantly decreased in RIF (p < 0.04) and RSA (p < 0.01) groups relative to the control group. Conclusions: It was found that increasing ratio as well as inflammatory activity of NKT cells correlated with repeated miscarriage. Reduced vitamin D levels could cause immune system disorder along with pregnancy complications. [ABSTRACT FROM AUTHOR]

Aims: We aimed to elucidate the mechanism leading to polycystic ovarian syndrome (PCOS) and recurrent spontaneous abortion (RSA). Background: PCOS is an endocrine disorder. Patients with RSA also have a high incidence rate of PCOS, implying that PCOS and RSA may share the same pathological mechanism. Objective: The single-cell RNA-seq datasets of PCOS (GSE168404 and GSE193123) and RSA GSE113790 and GSE178535) were downloaded from the Gene Expression Omnibus (GEO) database. Methods: Datasets of PSCO and RSA patients were retrieved from the Gene Expression Omnibus (GEO) database. The "WGCNA" package was used to determine the module eigengenes associated with the PCOS and RSA phenotypes and the gene functions were analyzed using the "DAVID" database. The GSEA analysis was performed in "clusterProfiler" package, and key genes in the activated pathways were identified using the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Real-time quantitative PCR (RT-qPCR) was conducted to determine the mRNA level. Cell viability and apoptosis were measured by cell counting kit-8 (CCK-8) and flow cytometry, respectively. Results: The modules related to PCOS and RSA were sectioned by weighted gene co-expression network analysis (WGCNA) and positive correlation modules of PCOS and RSA were all enriched in angiogenesis and Wnt pathways. The GSEA further revealed that these biological processes of angiogenesis, Wnt and regulation of cell cycle were significantly positively correlated with the PCOS and RSA phenotypes. The intersection of the positive correlation modules of PCOS and RSA contained 80 key genes, which were mainly enriched in kinase-related signal pathways and were significant high-expressed in the disease samples. Subsequently, visualization of these genes including PDGFC, GHR, PRLR and ITGA3 showed that these genes were associated with the PI3K-AKT signal pathway. Moreover, the experimental results showed that PRLR had a higher expression in KGN cells, and that knocking PRLR down suppressed cell viability and promoted apoptosis of KGN cells. Conclusion: This study revealed the common pathological mechanisms between PCOS and RSA and explored the role of the PI3K-AKT signaling pathway in the two diseases, providing a new direction for the clinical treatment of PCOS and RSA. [ABSTRACT FROM AUTHOR]

Aims We aimed to elucidate the mechanism leading to polycystic ovarian syndrome (PCOS) and recurrent spontaneous abortion (RSA). Background PCOS is an endocrine disorder. Patients with RSA also have a high incidence rate of PCOS, implying that PCOS and RSA may share the same pathological mechanism. Objective The single-cell RNA-seq datasets of PCOS (GSE168404 and GSE193123) and RSA GSE113790 and GSE178535) were downloaded from the Gene Expression Omnibus (GEO) database. Methods Datasets of PSCO and RSA patients were retrieved from the Gene Expression Omnibus (GEO) database. The “WGCNA” package was used to determine the module eigengenes associated with the PCOS and RSA phenotypes and the gene functions were analyzed using the “DAVID” database. The GSEA analysis was performed in “clusterProfiler” package, and key genes in the activated pathways were identified using the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Real-time quantitative PCR (RT-qPCR) was conducted to determine the mRNA level. Cell viability and apoptosis were measured by cell counting kit-8 (CCK-8) and flow cytometry, respectively. Results The modules related to PCOS and RSA were sectioned by weighted gene co-expression network analysis (WGCNA) and positive correlation modules of PCOS and RSA were all enriched in angiogenesis and Wnt pathways. The GSEA further revealed that these biological processes of angiogenesis, Wnt and regulation of cell cycle were significantly positively correlated with the PCOS and RSA phenotypes. The intersection of the positive correlation modules of PCOS and RSA contained 80 key genes, which were mainly enriched in kinase-related signal pathways and were significant high-expressed in the disease samples. Subsequently, visualization of these genes including PDGFC, GHR, PRLR and ITGA3 showed that these genes were associated with the PI3K-AKT signal pathway. Moreover, the experimental results showed that PRLR had a higher expression in KGN cells, and that knocking PRLR down suppressed cell viability and promoted apoptosis of KGN cells. Conclusion This study revealed the common pathological mechanisms between PCOS and RSA and explored the role of the PI3K-AKT signaling pathway in the two diseases, providing a new direction for the clinical treatment of PCOS and RSA.

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. [ABSTRACT FROM AUTHOR]

Abstract Background 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. Methods 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. Results 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. Conclusions 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.

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.

Background & Objective: 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.Methods: 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.Results: 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.Conclusion: 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.

Background: 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. Methods: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Background & Objective: 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. Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

Purpose: Vitamin D (VD) deficiency seems to be associated with the risk of recurrent spontaneous abortion (RSA). Vitamin D receptor (VDR) and cytochrome P450 family 2 subfamily R member 1 (CYP2R1) are two genes which are vital for VD metabolism and actions. However, whether single-nucleotide polymorphisms (SNPs) in these genes are correlated with the risk of RSA are poorly understood. Therefore, we aimed to characterize the relationships among VDR SNPs, CYP2R1 SNPs and RSA.Methods: This case-control study enrolled 75 RSA patients and 83 controls. Serum VD and some cytokines were detected with LC-MS/MS and flow cytometry, respectively. Genotyping for three SNPs of CYP2R1 (rs10741657, rs10766197 and rs12794714) and five SNPs of VDR (rs7975232, rs1544410, rs2189480, rs2228570 and rs2239179) was done with polymerase chain reaction (PCR) and high-throughput sequencing. All the data were analyzed with appropriate methods and in different models.Results: The results revealed a significant correlation between the AG genotype of CYP2R1 rs12794714 and VD levels (OR 0.686; 95% CI 0.49-0.96; p = 0.028). Besides, the AG and GG genotypes of CYP2R1 rs12794714 were markedly related to the risk of RSA (OR 52.394, 59.497; 95% CI 2.683-1023.265, 3.110-1138.367; p = 0.009, 0.007, respectively).Conclusion: Our results indicate that CYP2R1 rs12794714 might be a risk factor for RSA. Hence, early screening of pregnant women for CYP2R1 rs12794714 is necessary to warrant proactive counseling and treatment against RSA. [ABSTRACT FROM AUTHOR]

Immunological mechanisms have been proposed to underlie the pathogenesis of recurrent spontaneous abortion (RSA). Vitamin D has a potent immunomodulatory effect, which may affect pregnancy outcome. The objective of this study was to investigate 25-hydroxyvitamin D [25(OH) D] concentration and vitamin D receptor (VDR) expression in the decidual tissues of RSA patients. Thirty women with RSA (RSA group) and thirty women undergoing elective abortion (control group) were recruited during 2016 from gynecology outpatient clinics. We measured 25(OH) D, interleukin (IL)-17, IL-23, transforming growth factor β (TGF-β), VDR and 1-α-hydroxylase (CYP27B1) in decidual tissues collected during the abortion procedure. In the RSA group, 25(OH) D and TGF-β were significantly decreased while IL-17 and IL-23 were significantly increased compared with the control group. VDR expression was significantly decreased in the RSA group compared with the control group. Logistic regression analysis showed a significant negative correlation between 25(OH) D in decidual tissues and RSA. These results indicated that vitamin D concentrations in the decidua are associated with inflammatory cytokine production, suggesting that vitamin D and VDR may play a role in the etiology of RSA.

Abstract: Aim: The aim of this study was to investigate the association of microRNA polymorphisms (miR-146aC>G, miR-149T>C, miR-196a2T>C, and miR-499A>G) in Korean patients with recurrent spontaneous abortion (RSA). Methods: We conducted a case–control study of 564 Korean women: 330 patients with at least two unexplained consecutive pregnancy losses and 234 healthy controls with at least one live birth and no history of pregnancy loss. Results: RSA patients exhibited significantly different frequencies of the miR-196a2CC (TT+TC vs. CC; adjusted odds ratio [AOR], 1.587; 95% confidence interval [CI], 1.042–2.417) and miR-499AG+GG genotypes (AOR, 1.671; 95% CI, 1.054–2.651) compared with the control group. The combination of miR-196a2CC and miR-499AG+GG showed synergistic effects (AOR, 3.541; 95% CI, 1.645–7.624). Conclusion: miR-196a2CC, miR-499AG+GG, and the miR-196a2CC/miR-499AG+GG combination are significantly associated with idiopathic RSA in Korean women. [Copyright &y& Elsevier]

Background: Th1-type cytokines secretion such as IFN-γ, and Th2 cytokines such as IL-10, have been shown to exert deleterious effects on pregnancy, inhibiting fetal growth and development . Aim: Measurement of the locally concentrations of selected Th1 and Th2 cytokines in women with a history of recurrent spontaneous abortion (RSA) at the time of abortion using in situ hyperdization technique. Methods: A total of one hundred and nineteen women, ranged from the mean age (23.9 - 28.5)years, were enrolled in the current study and were further classified into three categories: Group A- Recurrent spontaneous abortion (RSA): n= 62 women, with a mean age of (28.5 + 0.68);Group B- non- recurrent spontaneous abortion (non-RSA): n= 34 women, with a mean age of (26.4 ± 0.85)and group C- Control (successful pregnancy): n= 23 women, with a mean age of (23.9 ± 0.88). From each patient and control, placental tissues were collected. Trophoblasts tissues (an image for the local microenvironment) were screened to determine their in situ levels of IL-10 and IFN-γ based on cDNA probes (for in-situ hybridization, ISH). Results: There was a significant increase in the level of IL-10 within trophoblast tissues biopsies exclusively from women with successful pregnancies (group C) (p < 0.001). On the other hand, IFN-γ was found predominantly expressed in trophoblast tissue biopsies of patients with RSA whether IHC or ISH were conducted (p< 0.05). Accordingly, only trophoblast tissues biopsies from patients with RSA revealed a significant increase in the ratio of IFN-γ/IL-10 levels expressed as determined by in situ hybridization in comparison to the same ration calculated from trophoblasts tissues of women with successful pregnancies (group C) (p< 0.001). as marker for Th2 immune response, during successful pregnancies. Furthermore, the current study failed to demonstrate a significant difference in the tissue levels of IL-8 between RSA and control group (p> 0.05) and no significant different between non-RSA and control (p>0.05) , (always p < 0.05). Conclusion: These outcomes may further support the possible exisance of an immune response that orchestrates abortive phenomena and the possible protective role of IL-10. Keywords: Recurrent spontaneous abortion (RSA), in-situ hybridization, ISH, cytokine,IL- 8,IL-10,IFN γ. [ABSTRACT FROM AUTHOR]