Unexplained Recurrent Spontaneous Abortion Research Articles

Beyond Immune Balance: The Pivotal Role of Decidual Regulatory T Cells in Unexplained Recurrent Spontaneous Abortion.

Recurrent spontaneous abortion (RSA) is defined as two or more consecutive pregnancy failures, which brings tremendous stress to women of childbearing age and seriously affects family well-being. However, the reason in about 50% of cases remains unknown and is defined as unexplained recurrent spontaneous abortion (URSA). The immunological perspective in URSA has attracted widespread attention in recent years. The embryo is regarded as a semi-allogeneic graft to the mother. A successful pregnancy requires transition to an immune environment conducive to embryo survival at the maternal-fetal interface. As an important member of regulatory immunity, regulatory T (Treg) cells play a key role in regulating immune tolerance at the maternal-fetal interface. This review will focus on the phenotypic plasticity and lineage stability of Treg cells to illustrate its relationship with URSA.

The safety and efficacy of tumor necrosis factor-alpha inhibitor on pregnancy outcomes in patients with unexplained recurrent miscarriage

ObjectivesAlthough tumor necrosis factor-alpha inhibitor (TNFi) treatment may improve pregnancy outcomes in unexplained recurrent miscarriage (URM) patients, evidence for its efficacy and safety is still insufficient. The goal of this study was to evaluate the efficacy and safety of TNFi on pregnancy outcomes in patients with URM. MethodsThis retrospective study was conducted at a single institution in China, involving 121 patients treated with TNFi for URM from 2019 to 2022. Patients enrolled were divided into treatment group (receiving TNFi and heparin therapy) and control group (receiving heparin therapy). The outcome variables were the 24-week live birth rate, miscarriage rate, ectopic pregnancy rate, neonatal outcomes, and adverse events. ResultsIn our study, patients receiving TNFi treatment exhibited a significant increase in live birth rates, achieving 71.2 % compared to the 50.9 % observed in the control group (OR 2.507, 95 % CI: 1.127–5.579). Concurrently, there was a discernible reduction in the miscarriage rate within the TNFi-treated group, marking 24.2 %, in contrast to 43.6 % in the control group (OR 0.387, 95 % CI: 0.170–0.884). Subgroup analyses further illuminated that those under the age of 35 benefitted remarkably from TNFi treatment, with live birth rates soaring to 62.5 % (OR 2.525, 95 % CI: 1.041–6.125). For patients with a history of two miscarriages, the TNFi regimen significantly augmented the live birth rate to 58.9 % (OR 3.044, 95 % CI: 1.039–8.921). Patients with a normal weight range registered a 58.4 % live birth rate post-TNFi treatment (OR 4.261, 95 % CI: 1.539–11.397). Notably, an evident interaction between BMI and TNFi treatment was identified, suggesting a potential modulatory role of BMI on the therapeutic efficacy of TNFi. About safety assessments, neither the TNFi-treated group nor the control manifested any significant disparities in liver function abnormalities, platelet count anomalies, or other pregnancy-related complications. ConclusionsTNFi, alongside basic therapy, notably enhances the live birth rate in URM patients under 35, with two prior miscarriages or a normal BMI, without increasing adverse event risk. Further prospective studies are essential to validate these observations.

Maternal-fetal immunity and recurrent spontaneous abortion.

Recurrent Spontaneous Abortion (RSA) is a common pregnancy complication, that has multifactorial causes, and currently, 40%-50% of cases remain unexplained, referred to as Unexplained RSA (URSA). Due to the elusive etiology and mechanisms, clinical management is exceedingly challenging. In recent years, with the progress in reproductive immunology, a growing body of evidence suggests a relationship between URSA and maternal-fetal immunology, offering hope for the development of tailored treatment strategies. This article provides an immunological perspective on the pathogenesis, diagnosis, and treatment of RSA. On one hand, it comprehensively reviews the immunological mechanisms underlying RSA, including abnormalities in maternal-fetal interface immune tolerance, maternal-fetal interface immune cell function, gut microbiota-mediated immune dysregulation, and vaginal microbiota-mediated immune anomalies. On the other hand, it presents the diagnosis and existing treatment modalities for RSA. This article offers a clear knowledge framework for understanding RSA from an immunological standpoint. In conclusion, while the "layers of the veil" regarding immunological factors in RSA are gradually being unveiled, our current research may only scratch the surface. In terms of immunological etiology, effective diagnostic tools for RSA are currently lacking, and the efficacy and safety of immunotherapies, primarily based on lymphocyte immunotherapy and intravenous immunoglobulin, remain contentious.

Maternal Serum Amyloid a Level as a Novel Marker of Primary Unexplained Recurrent Early Pregnancy Loss

Background: Recurrent abortion affects 1-2% of women. Serum Amyloid A belongs to a family of apolipoproteins produced in response to cytokines released by activated monocytes and macrophages, isolated in the last 50 years. Our current study emphasizes human SAA protein as a sensitive biochemical marker for primary unexplained recurrent miscarriage. The aim of the current study is to examine the hypothesis that primary unexplained REPL might be associated with high maternal serum levels of SAA, which in turn could lead to defective trophoblastic invasion into the decidua, and subsequent pregnancy failure and miscarriage. Patients and Method: A prospective study (case control study) in Al-Zahraa Maternity Hospital, Najaf, Iraq, from first of January to the first of December of 2019. The study was conducted among 91 who were divided into two groups Group 1: women with missed miscarriage in the first trimester with at least two consecutive primary unexplained REPLs and no previous live births were enrolled. Group 2: A control group was formed of women with miscarriage no history of REPL who had at least one previous uneventful pregnancy with no adverse outcomes. Serum samples were collected to measure SAA levels. Result: The main outcome was the association between SAA and primary unexplained REPL. A total number of 91 participants. Mean SAA level was significantly higher among women with REPL than among women in the control group (P<0.001). The SAA level was dependent indicator of primary unexplained REPL, P<0.001 Elevated SAA levels found among women with primary unexplained REPL could represent biomarker for this complication of pregnancy. Conclusions: The present findings suggest that SAA is potentially a novel marker for primary unexplained REPL that warrants further investigation. For example, studies could be conducted to compare SAA levels among women with a history of primary unexplained REPL during the pregnant and non-pregnant states, as well as before and after miscarriage. In future, such studies might guide the timing for initiation of new treatments, such as gene therapy or the use of immune-receptor antagonists.

Strong association between angiotensin I-converting enzyme insertion/deletion polymorphism and unexplained recurrent spontaneous abortion of Sudanese women: a case-control study

This study investigated the link between angiotensin I-converting enzyme insertion/deletion (ACE I/D) polymorphism and unexplained spontaneous abortion. This retrospective analytical case-control was conducted at the Omdurman Maternity Hospital in Sudan. The current study contained 230 individuals, including 119 cases (women who had at least three abortions) of unknown cause and 119 controls (healthy women who had at least two full-term deliveries without spontaneous abortion). Patients and controls were provided five ml of ethylenediaminetetraacetic acid blood and answered questionnaires about their demographics, personal lives, and family histories. ACE I/D polymorphisms were assessed using a conventional polymerase chain reaction approach after total genomic DNA was isolated from blood leukocytes using the GF-1 blood DNA extraction kit. Data was analyzed using the Statistical Package for the Social Sciences version 24. ACE I/D polymorphism is strongly linked to unexplained spontaneous abortion, and women with the I/D and D/D genotypes are more likely to have it than those with the I/I genotype. The current study reveals that ACEI/D polymorphism increases pregnancy problems. Sudanese women may have spontaneous abortions due to the ACE I/D polymorphism.

Correlation between hysteroscopic features and specific microbial species in women with chronic endometritis

Objective and rationaleChronic endometritis (CE) has recently been associated with unexplained infertility and recurrent miscarriages. The current gold standard for CE detection is histopathological examination. However, office hysteroscopy and endometrial cultures are also significant, due to the possible link between CE and various microorganisms. Bacterial colonization of the endometrium has been associated with reduced success rates of in vitro fertilisation embryo transfer. Few studies have tried to correlate CE hysteroscopy findings with pathogenic microorganisms. This prospective cohort study sought to establish whether hysteroscopic diagnostic lesions correlate with specific microbial species. MethodsThe study encompassed women undergoing diagnostic tests for a range of subfertility health issues. 189 women completed the standard office diagnostic hysteroscopy (DH). 181 had also endometrial samples taken for microbial culture investigation. Correlation analysis (χ2 and Fisher's exact test) between hysteroscopic findings suggestive of CE and endometrial cultures was carried out. Logistic regression models were also fitted to measure whether a positive endometrial culture could affect CE conditions. ResultsA significant association of E. coli was observed between the hysteroscopically characterized CE + group with focal hyperplasia, when compared to the non–CE group. Logistic regression analysis revealed that women positive for E. coli were 4.423 times more likely to have focal endometrial hyperplasia. No other significant correlations were identified between DH and positive endometrial cultures. ConclusionsThe presence of E. coli in the endometrium was significantly correlated with focal hyperplasia findings from hysteroscopy, emphasizing the importance of microbial cultures in the diagnosis and targeted treatment of CE in women with subfertility.

Pyroptosis is involved in the immune microenvironment regulation of unexplained recurrent miscarriage.

Unexplained recurrent miscarriage (URM) is a common pregnancy complication with few effective therapies. Moreover, little is known regarding the role of pyroptosis in the regulation of the URM immune microenvironment. To address this issue, gene expression profiles of publicly available placental datasets GSE22490 and GSE76862 were downloaded from the Gene Expression Omnibus database. Pyroptosis-related differentially expressed genes were identified and a total of 16 differentially expressed genes associated with pyroptosis were detected, among which 1 was upregulated and 15 were downregulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the functionally enriched modules and pathways of these genes are closely related to immune and inflammatory responses. Four hub genes were identified: BTK, TLR8, NLRC4, and TNFSF13B. BTK, TLR8, and TNFSF13B were highly connected with immune cells, according to the correlation analysis of four hub genes and 20 different types of immune cells (p < 0.05). The four hub genes were used as research objects to construct the interaction networks. Chorionic villus tissue was used for quantitative real-time polymerase chain reaction and western blot to confirm the expression levels of hub genes, and the results showed that the expression of the four hub genes was significantly decreased in the chorionic villus tissue in the URM group. Collectively, the present study indicates that perhaps pyroptosis is essential to the diversity and complexity of the URM immune microenvironment, and provides a theoretical basis and research ideas for subsequent target gene verification and mechanism research.

Correlation Between Long non-coding RNA SOX2OT rs9839776 C&gt;T Polymorphism and Unexplained Recurrent Miscarriages

Correlation Between Long non-coding RNA SOX2OT rs9839776 C&gt;T Polymorphism and Unexplained Recurrent Miscarriages

Common mental disorders and risk of spontaneous abortion or recurrent spontaneous abortion: A two-sample Mendelian randomization study

BackgroundThe direct causal relationships between common mental disorders (anxiety disorders, broad depression, major depressive disorder (MDD), bipolar disorder, and insomnia) and miscarriage or recurrent spontaneous abortion (RSA) are unclear. Therefore, this study aimed to explore these, using Mendelian randomization. MethodsGenome-wide association studies (GWAS) meta-analyses with the largest sample size possible and selected independent single individuals of European ancestry were selected. Inverse variance weighted (IVW) was the main analysis method. The heterogeneity of the instrumental variables (IVs) was assessed using IVW and MR-Egger, and the horizontal pleiotropy of the IVs was assessed using MR-Egger and MR-PRESSO. ResultsBased on IVW results, the four mental disorders were found to be causally associated with spontaneous abortion (anxiety disorder: OR (95%CI), 1.230 (1.063–1.420), P = 0.0050; major depressive disorder: 1.690 (1.239–2.307), P = 0.0009; bipolar disorder: 1.110 (1.052–1.170), P = 0.0001; insomnia: 1.292 (1.076–1.552), P = 0.0060). Furthermore, no causal relationship was observed between broad depression and spontaneous abortion. Five common mental disorders were not causally associated with the RSA. Limitations(1) Our analysis was limited to the European population; (2) the duration of mental disorders was not analyzed, as no information was available; and (3) it was difficult to completely detect genetic pleiotropy. ConclusionsAnxiety disorders, MDD, bipolar disorder, and insomnia may contribute to spontaneous abortion. Therefore, we should focus on the mental and sleep health of pregnant women. Future studies may be required on whether mental disorders directly lead to RSA, especially unexplained RSA.

The Ratio of Peripheral Blood Natural Killer Cells is not a Solid Surrogate Immune Index in Unexplained Recurrent Miscarriage.

Immunotherapies targeting peripheral natural killer (pbNK) cells in unexplained recurrent miscarriage (uRM) remain controversial. We hypothesized that the change in pbNK cell count might be a result of innate immune responses rather than a cause. To explore whether the pbNK count is significantly different in women testing positive than those testing negative for commonly studied autoimmune markers. Peripheral blood samples were collected from 302 eligible patients with uRM for the antinuclear antibody (ANA) testing determined by the enzyme-linked immunosorbent assay (ELISA), anti-thyroid peroxidase antibody (TPO-Ab) testing and anti-thyroglobulin antibody (Tg-Ab) testing determined by the chemiluminescent immunoassay, and pbNK cell testing determined by flow cytometry. The patients were divided into two groups according to the pbNK normal range, and the comparative analysis entailed an examination of the prevalence rates of autoantibodies within the high pbNK group and the normal pbNK group, followed by a comprehensive investigation into the potential correlations between autoantibodies and pbNK cells. There was a positive association between TPO-Ab positivity and high pbNK cells (p=0.016, OR=5.097, 95% CI 1.356-19.159), while there was a negative association between ANA positivity and high pbNK cells (p=0.013, OR=0.293, 95% CI 0.111-0.773). TPO-Ab-positive patients had a higher pbNK cell count compared with TPO-Ab-negative patients, while ANA-positive patients had a lower pbNK cell count compared with ANA-negative patients. The change in pbNK cell count may be a consequence of immune responses, and there should be careful consideration in applying it as an immunotherapeutic index.

Exposure to high dose of polystyrene nanoplastics causes trophoblast cell apoptosis and induces miscarriage

BackgroundWith rapid increase in the global use of various plastics, microplastics (MPs) and nanoplastics (NPs) pollution and their adverse health effects have attracted global attention. MPs have been detected out in human body and both MPs and NPs showed female reproductive toxicological effects in animal models. Miscarriage (abnormal early embryo loss), accounting for 15-25% pregnant women worldwide, greatly harms human reproduction. However, the adverse effects of NPs on miscarriage have never been explored.ResultsIn this study, we identified that polystyrene (PS) plastics particles were present in women villous tissues. Their levels were higher in villous tissues of unexplained recurrent miscarriage (RM) patients vs. healthy control (HC) group. Furthermore, mouse assays further confirmed that exposure to polystyrene nanoplastics (PS-NPs, 50 nm in diameter, 50 or 100 mg/kg) indeed induced miscarriage. In mechanism, PS-NPs exposure (50, 100, 150, or 200 µg/mL) increased oxidative stress, decreased mitochondrial membrane potential, and increased apoptosis in human trophoblast cells by activating Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3 signaling through mitochondrial pathway. The alteration in this signaling was consistent in placental tissues of PS-NPs-exposed mouse model and in villous tissues of unexplained RM patients. Supplement with Bcl-2 could efficiently suppress apoptosis in PS-NPs-exposed trophoblast cells and reduce apoptosis and alleviate miscarriage in PS-NPs-exposed pregnant mouse model.ConclusionsExposure to PS-NPs activated Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3, leading to excessive apoptosis in human trophoblast cells and in mice placental tissues, further inducing miscarriage.Graphical

Cryptochrome 2 Suppresses Epithelial-Mesenchymal Transition by Promoting Trophoblastic Ferroptosis in Unexplained Recurrent Spontaneous Abortion

Unexplained recurrent spontaneous abortion (URSA) is a serious reproductive issue that affects women of childbearing age. Studies have shown a close association between disrupted circadian rhythm and impaired epithelial-mesenchymal transition (EMT) process in trophoblasts during URSA, though the underlying mechanism has not been elaborated. This study aims to investigate the regulatory relationship between circadian rhythm gene cryptochrome 2 (CRY2) and ferroptosis on the migratory ability of trophoblast cells. Cell proliferation experiments, wound healing assays, and expression of related markers were conducted to verify the EMT efforts. Trophoblastic ferroptosis was confirmed by the expressions of malondialdehyde, glutathione, mitochondrial membrane potential, divalent iron ions, and expression level of related genes. The results showed significant increased expression of CRY2 and decreased expression of brain and muscle ARNT-like protein 1 (BMAL1) in the URSA villous tissues, accompanied by iron-dependent oxidative changes and abnormal expression of ferroptosis related proteins. It was observed that CRY2 and BMAL1 was co-localized and function as a feedback loop, which regulated the dynamic changes of EMT-related markers in trophoblast cells. CRY2 promoted trophoblastic ferroptosis, while BMAL1 had the opposite effect. Particularly, the ferroptosis inhibitor (Ferrostatin-1) effectively reversed the trophoblastic ferroptosis and EMT inhibition caused by CRY2 overexpression. Collectively, these results suggest that CRY2 regulates trophoblastic ferroptosis and hinders cellular EMT and migratory ability by suppressing BMAL1 expression.

Study on the immunomodulatory mechanism of vitamin D in patients with unexplained recurrent spontaneous abortion

BackgroundTo investigate the mechanism of vitamin D level on the regulation of peripheral blood lymphocyte subsets and serum Th1/Th2 cytokines in patients with unexplained recurrent spontaneous abortion (URSA). MethodsEighty female patients with URSA attending Sichuan Jinxin Xinan Women's and Children's Hospital from January 2020 to May 2021 were selected as the study group, and 30 age-matched women with a history of healthy deliveries were chosen as the control group, and peripheral blood lymphocyte subpopulations and serum Th1/Th2 cytokines of people with different levels of vitamin D were detected in the study group by flow cytometry, respectively. The results of immune factors before and after supplementation were analyzed in 40 of these patients with low vitamin D levels. The results of lymphoid subpopulations and Th1/Th2 cytokines in 19 patients with normal pregnancy before and after vitamin D supplementation and after normal pregnancy were also analyzed comparatively. Results(1) Serum 25(OH)D in the study group was lower than in the control group; peripheral blood Th cells, B cells and NK cells in the study group were higher than in the control group; IL-2, TNF-α, IFN-γ and IL-6 in the study group were higher than in the control group, while IL-4 and IL-10 in the study group were lower than in the control group (P < 0.05). (2) Th cells, B cells and NK cells of URSA patients in the vitamin D low level group were higher than those in the vitamin D normal group; serum cytokines IL-2, TNF-α and IFN-γ of patients in the vitamin D low level group were higher than those in the vitamin D normal group (P < 0.05); (3) Th cells, B cells and NK cells in URSA patients after vitamin D supplementation were lower than before vitamin D supplementation; serum cytokines IL-2, TNF-α and IFN-γ after vitamin D supplementation were lower than before vitamin D supplementation, IL-4 and IL-10 after vitamin D supplementation were higher than before vitamin D supplementation (P < 0.05), and there was no significant difference in IL-6 before and after vitamin D supplementation. (4) Th cells, B cells and NK cells in patients with normal pregnancy after vitamin D supplementation and after pregnancy were lower than those before vitamin D supplementation; serum cytokines IL-2, TNF-α and IFN-γ after vitamin D supplementation and after pregnancy were lower than those before vitamin D supplementation, and serum cytokines IL-4 and IL-10 after vitamin D supplementation and after pregnancy were higher than those before vitamin D supplementation, TNF -α, IFN-γ after pregnancy were lower than after vitamin D supplementation (P < 0.05), IL-6 was not significantly different before and after vitamin D supplementation and after pregnancy. ConclusionVitamin D deficiency rate was high in URSA patients. Th、B、NK cells and IL-2, TNF-α, IFN-γ, IL-6 cytokines were high, while IL-6 and IL-10 were low in URSA patients. IL-2, TNF-α, IFN-γ cytokines and Th, B, NK cells were increased in vitamin D deficient URSA patients, and Vitamin D deficiency may be an important cause or aggravating factor of immune dysfunction in URSA patients. Vitamin D has an immunomodulatory effect on URSA patients, promoting successful pregnancy by down-regulating peripheral blood Th, B, and NK cells and IL-2, TNF-α, and IFN-γ cytokines, while up-regulating IL-4 and IL-10.

The prevalence of antiphospholipid antibodies and effects on the outcomes of in vitro fertilization

Introduction: The problem of infertility is common worldwide. IVF remains the most widely used technique of assisted reproductive technology. Embryo implantation failure is a common cause of unexplained infertility and recurrent miscarriages, thus the relationship between antiphospholipid antibodies and embryo implantation failure after IVF and embryo transfer is an important problem in fertility medicine that must be studied. Patients and methods: A retrospective and prospective (bidirectional) cohort study were conducted at the Laboratory Department of the Faculty of Medicine, and Hospital from June 2017 to June 2021. The sample size was 1000 participants, 900 of them were retrospective (2017–2020), and 100 were prospective (2020–2021). Data was collected, including patients’ demographics, type and duration of fertility, and antiphospholipid antibody values. Inclusion criteria-Women &lt;40 years old, undergoing IVF, and in the retrospective study, patients who undergo IVF and who have antibody data, positivity of phospholipids, and/or anticardiolipin, and/or B2 glycoprotein I, and the presence of the gestational sac on ultrasound as a criterion for pregnancy. We performed a PTT LA for the control group in a cohort study (20 healthy women), then a PTT LA test, and the blending test were performed, and the values were considered positive if the correction was not made. The PTT LA test is a screening test. Results: The average age of the women was 31.91, the success rate of assisted fertilization in inducing pregnancy in the last trial was 52.8%, the prevalence was 4.4% of anticardiolipin IgG, 2.6% of anticardiolipin IgM, and 4% of Lupus anticoagulant. No relation was found between the antiphospholipid value and the age of the women, the number of times the experiment was conducted, the rate of success and failure, the type of infertility, and the number of oocytes induction, while the duration of sterility was affected by positive values of IgG and lupus coagulant antibodies. Conclusion: There is no need for a routine antiphospholipid assay for female candidates for IVF, and when it is confirmed, treatment is required by Anticoagulants.

HOTAIR/miR-1277-5p/FBN2 signaling axis is involved in recurrent spontaneous abortion by regulating the growth, migration, and invasion of HTR-8/SVneo cells.

This study aimed to explore the specific pathways by which HOX transcript antisense intergenic RNA (HOTAIR) contributes to the pathogenesis of unexplained recurrent spontaneous abortion (URSA). Real-time quantitative PCR (RT-qPCR) was employed to assess the differential expression levels of HOTAIR in chorionic villi tissues from URSA patients and women with voluntarily terminated pregnancies. HTR-8/SVneo served as a cellular model. Knockdown and overexpression of HOTAIR in the cells were achieved through siRNA transfection and pcDNA3.1 transfection, respectively. Cell viability, migration, and invasion were evaluated using cell counting kit-8 (CCK-8), scratch, and Transwell assays, respectively. The interaction among the HOTAIR/miR-1277-5p/fibrillin 2 (FBN2) axis was predicted through bioinformatics analysis and confirmed through in vitro experiments. Furthermore, the regulatory effects of the HOTAIR/miR-1277-5p/FBN2 signaling axis on cellular behaviors were validated in HTR-8/SVneo cells. We found that HOTAIR was downregulated in chorionic villi tissues from URSA patients. Overexpression of HOTAIR significantly enhanced the viability, migration, and invasion of HTR-8/SVneo cells, while knockdown of HOTAIR had the opposite effects. We further confirmed the regulatory effect of the HOTAIR/miR-1277-5p/FBN2 signaling axis in URSA. Specifically, HOTAIR and FBN2 were found to reduce the risk of URSA by enhancing cell viability, migration, and invasion, whereas miR-1277-5p exerted the opposite effects. HOTAIR promotes URSA development by targeting inhibition of miR-1277-5p/FBN2 axis.

Association of inherited thrombophilia mutations and their combinations among palestinian women with unexplained recurrent miscarriage

BackgroundInherited thrombophilia (IT) has a complex pathophysiology and is associated with recurrent miscarriage (RM) by causing placental insufficiency and inhibiting fetal development. However, thrombophilia screening in unexplained RM cases is still questionable. This study aimed to investigate the association between the common eight IT mutations and their combinations among Palestinian women with unexplained RM.MethodsThis is an unmatched case-control study with 200 women (100 unexplained RM cases, 100 controls). Eight common IT mutations namely Factor V Leiden (FVL), prothrombin gene (FII) G202120A, Methylenetetrahydrofolate Reductase (MTHFR) gene (C677T and A1298C), B-fibrinogen gene − 455G > A, FV HR2 A4070G, Plasminogen activator inhibitor 1 (PAI1) 5G/4G and Factor XIIIA (FXIIIA) V34L; were analyzed. The first five mutations were analyzed by Restriction Fragment Length Polymorphism PCR and the other three mutations were analyzed using Amplification Refractory Mutation System PCR.ResultsThe prevalence of the eight IT mutations among the control group was in the order PAI1 5G/4G (69%), MTHFR C677T (53%) and A1298C (47%), BFG − 455G > A (35%), FVL and FV HR2 (each 18%), FXIIIA V34L (16%) and FII G20210A (3%). Patients had a higher percentage of MTHFR A1298C (heterozygotes and mutant homozygote) compared to controls (p = 0.016). Frequencies of mutant alleles MTHFR A1298C (p < 0.001) and FXIIIA V34L (p = 0.009) were higher among patients compared to controls. No significant differences were observed for all other mutations or mutant alleles. Most patients (75%) and controls (75%) have 2–4 mutant alleles out of 8 mutant alleles studied, while 1% of patients and 2% of controls have zero mutant alleles. None of the combinations of the most often studied mutations (FVL, FII G20210A, MTHFR C1677T, and MTHFR A1298C) showed a significant difference between patients and controls.ConclusionsThere was a significant association between unexplained RM and the mutant alleles of MTHFR A1298C and FXIIIA V34L. No significant association was observed between unexplained RM and the combination of both mutant alleles for the mutations studied. This study is the first Palestinian report that evaluates eight inherited thrombophilia mutations and their alleles’ combinations in unexplained RM cases.

Bioinformatics analysis of a disease-specific lncRNA-miRNA-mRNA regulatory network in recurrent spontaneous abortion (RSA).

This study investigated the molecular mechanisms of long non-coding RNAs (lncRNAs) in RSA using the lncRNA-miRNA-mRNA regulatory network. The present study obtained expression datasets of long non-coding RNAs (lncRNAs), messenger RNAs (mRNAs), and microRNAs (miRNAs) from blood samples of individuals with unexplained recurrent spontaneous abortion (RSA) and healthy controls. Differentially expressed lncRNAs (DELs), mRNAs (DEMs), and miRNAs (DEmiRs) were identified. A regulatory network comprising lncRNA, miRNA, and mRNA was constructed, and Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted to analyze the biological functions of DEM. Also, a protein-protein interaction (PPI) network was made and key genes were identified. A total of 57 DELs, 212 DEmiRs, and 301 DEMs regarding RSA were identified. Later analysis revealed a lncRNA-miRNA-mRNA network comprising nine lncRNAs, 14 miRNAs, and 65 mRNAs. Then, the ceRNA network genes were subjected to functional enrichment and pathway analysis, which showed their association with various processes, such as cortisol and thyroid hormone synthesis and secretion, human cytomegalovirus infection, and parathyroid hormone synthesis. In addition, ten hub genes (ITGB3, GNAI2, GNAS, SRC, PLEC, CDC42, RHOA, RAC1, CTNND1, and FN1) were identified based on the PPI network results. In summary, the outcomes of our study provided some data regarding the alteration genes involved in RSA pathogenic mechanism via the lncRNA-miRNA-mRNA network and reveal the possibility of identifying new lncRNAs and miRNAs as promising molecular biomarkers.

FTO protein regulates the TGF-β signalling pathway through RNA N6-methyladenosine modification to induce unexplained recurrent spontaneous abortion.

Recent studies have revealed the involvement of RNA m6A modification in embryonic development; however, the relationship between aberrant RNA m6A modification and unexplained recurrent spontaneous abortion (URSA) remains unclear. In this study, we analysed the level of RNA m6A modification in trophoblasts using dot blot, RNA m6A quantification, and MeRIP assays. By integrating data from the GEO database, RNA-Seq, and MeRIP-Seq, we examined the aberrant expression of m6A methyltransferases and their downstream molecules in chorionic villus (placental) tissues. RNA pull-down, RIP, and electrophoretic mobility shift assay were used to analyse the binding relationship between the YTHDC1 protein and MEG3. Additionally, RNA stability and BrU immunoprecipitation chase assays were utilised to elucidate the regulation of MEG3 stability by YTHDC1. ChIP and DNA pull-down RNA experiments were performed to elucidate the mechanism by which MEG3 targets EZH2 to the TGF-β1 promoter. The results showed that the expression of the m6A demethylase FTO protein was significantly increased in URSA trophoblasts, leading to inhibition of the MEG3 m6A modification and weakening of the stabilising effect of the m6A binding protein YTHDC1 on MEG3. Furthermore, MEG3 was found to bind simultaneously with the EZH2 protein and the TGF-β1 gene promoter, enabling the localisation of EZH2 protein to the TGF-β1 gene promoter and subsequent inhibition of TGF-β1 gene expression. In summary, our findings elucidate the mechanism by which FTO protein regulates the MEG3-TGF-β signalling pathway, thereby suppressing trophoblast invasion and proliferation in URSA trophoblast cells. These findings provide new insights for the treatment of URSA.

Baicalin ameliorates deficient decidualization in URSA by regulating mitochondrial fission induced necroptosis

Unexplained recurrent spontaneous abortion (URSA) is a common complication of pregnancy that affects the health of pregnant women. Deficient endometrial decidualization has been associated with URSA. However, the underlying mechanism is poorly understood. This study aims to explore the mechanisms of mitochondrial fission induced necroptosis in deficient decidualization in URSA, and explore the regulation of baicalin on this mechanism. Initially, decidual tissues were collected from patients with URSA and health controls. Subsequently, in vitro induced decidualization model of Telomerase-Immortalized Human Endometrial Stromal Cells (T-hESCs) was constructed. Additionally, murine models of URSA (CBA/J × DBA/2) and normal pregnancy (CBA/J × BALB/c) were established, respectively. The level of decidualization, necroptosis, and mitochondrial fission of decidual tissues from clinical samples were detected. The function of mitochondrial fission on necroptosis during decidualization in T-hESCs was assessed by enhancing or inhibiting mitochondrial fission or necroptosis. Finally, CBA/J × DBA/2 pregnant mice were administrated with different doses of baicalin or saline, and the expression of mitochondrial fission, necroptosis, and decidualization markers were verified. The results of the study demonstrated a significant decrease in decidualization markers in the decidual tissues of URSA patients (P < 0.05), along with an increase in the incidence of cell necroptosis (P < 0.05) and hyperactive mitochondrial fission (P < 0.05). In vitro experiments, LPS was induced to trigger necroptosis of T-hESCs during induced decidualization, and decidualization markers IGFBP1 and PRL were subsequently decreased (P < 0.05). Besides, the mitochondrial fission agonist Tyrphostin A9 was found to promote the level of necroptosis (P < 0.05) and induced deficient decidualization (P < 0.05), which could be rescued by mitochondrial fission inhibitor Mdivi-1 and necroptosis inhibitor Nec-1 (P < 0.05). In addition, baicalin was shown to reduce hyperactive mitochondrial fission (P < 0.05), necroptosis (P < 0.05) and ameliorate deficient decidualization in vitro and in URSA murine models (P < 0.05). Collectively, baicalin shows potential in ameliorating deficient decidualization in URSA by inhibiting mitochondrial fission-triggered necroptosis.

GRIM-19 deficiency promotes macrophage polarization to the M1 phenotype partly through glycolysis in unexplained recurrent spontaneous abortion†.

The occurrence of unexplained recurrent spontaneous abortion (URSA) is closely related to immune system disorders, however, the underlying mechanisms remain unclear. The purpose of this study was to investigate the expression of GRIM-19 in URSA and the possible pathogenesis of URSA according to macrophage polarization. Here, we showed that GRIM-19 was downregulated in the uterine decidual macrophages of patients with URSA and that GRIM-19 downregulation was accompanied by increased M1 macrophage polarization. Furthermore, the expression levels of glycolytic enzymes were substantially enhanced in the uterine decidual macrophages of URSA patients, and glycolysis in THP-1-derived macrophages was further enhanced by the downregulation of GRIM-19. Additionally, the increase of M1 macrophages resulting from the loss of GRIM-19 was significantly reversed in cells treated with 2-deoxy-D-glucose (2-DG, an inhibitor of glycolysis). To provide more direct evidence, GRIM-19 deficiency was shown to promote macrophage polarization to the M1 phenotype in GRIM-19+/- mouse uteri. Overall, our study provides evidence that GRIM-19 deficiency may play a role in regulating macrophage polarization in URSA, and that glycolysis may participate in this process.