KLF4 regulates trophoblast function and associates with unexplained recurrent spontaneous abortion.

BACKGROUND: Recurrent spontaneous abortion (RSA) is defined as two or more consecutive spontaneous abortions before 20 weeks with the same spouse [1]. However, approximately 50% of RSA cases of unknown cause are classified as unexplained recurrent spontaneous abortion (URSA). Potential factors include decreased trophoblast cell migration and invasion, leading to impaired placental implantation and maintenance of the normal maternal-fetal interface. However, the mechanism of this pathogenesis remains unknown. In this study, we investigated the potential role and mechanism of KLF4 in regulating URSA by influencing the invasion and migration ability of trophoblast cells. METHODS: We firstly identified 817 differentially expressed genes by performing a difference analysis of the dataset GSE121950 [2] related to recurrent abortion, and intersected the top 10 genes obtained respectively by the three algorithms: DMNC, MNC, and EPC using Venn Diagram.To detect the expression levels of core genes, villi samples were obtained from normal pregnant women and patients with URSA. RT-qPCR analysis revealed a significant difference in KLF4 mRNA expression and KLF4 was then analyzed. Trophoblast cell lines HTR8 and JEG3 were used to investigate the effect of KLF4 on trophoblastic function. Wound healing and transwell assays was performed to detect the invasion and migration of trophoblast cells. The expression of epithelial-mesenchymal transition(EMT) molecules were detected by RT-qPCR and western blot. Promoter detection and epigenetic modification were detected by chromatin immunoprecipitation (ChIP) assay. Molecular nuclear localization was detected by immunofluorescence and subcellular fractionation. Miscarried mice model was used to study the effects of KLF4 on URSA induced by reduced trophoblast invasion and migration. RESULTS: KLF4 is highly expressed in the villi of patients with URSA. KLF4 inhibits the expression level of H3R2ME2a in trophoblast cells by regulating the transcriptional level and nuclear translocation of PRMT6, thereby inhibiting the possible regulatory mechanism of trophoblastic invasion and providing a potential treatment strategy for URSA in vivo. CONCLUSIONS: The KLF4/PRMT6/H3R2ME2a axis regulates mechanisms associated with unexplained recurrent spontaneous abortion by regulating trophoblast function.

Low-dose aspirin and heparin treatment improves pregnancy outcome in recurrent pregnancy loss women with anti-ß2-glycoprotein I/HLA-DR autoantibodies: a prospective, multicenter, observational study.

Introduction: Anti-ß2-glycoprotein I (ß2GPI)/human leukocyte antigen (HLA)-DR antibodies may be a risk factor for recurrent pregnancy loss (RPL). The therapeutic modality for women with RPL and anti-ß2GPI/HLA-DR antibody positivity has not been evaluated. This prospective, multicenter, observational study aimed to assess whether low-dose aspirin (LDA) and/or heparin therapies improve pregnancy outcomes in women with RPL who tested positive for anti-ß2GPI/HLA-DR antibodies. Methods: Between August 2019 and December 2021, 462 women with RPL underwent anti-ß2GPI/HLA-DR antibody measurements and risk assessments for RPL. Each attending physician decided the treatment modality for women with RPL who tested positive for anti-ß2GPI/HLA-DR antibodies, and their pregnancy outcomes were followed up until December 2023. Finally, 47 pregnancies in 47 women with RPL and anti-ß2GPI/HLA-DR antibody positivity were included in the analysis and were divided into two groups regarding whether they were treated with LDA and/or unfractionated heparin (UFH) (LDA/UFH group, n = 39) or with neither of them (non-LDA/non-UFH group, n = 8). The rates of live birth and pregnancy complications (i.e., preeclampsia and preterm delivery before 34 gestational weeks due to placental insufficiency) were compared between the two groups. Results: The live birth rate in the LDA/UFH group was higher than that in the non-LDA/non-UFH group (87.2% vs 50.0%, p = 0.03). The pregnancy complication rate in the LDA/UFH group was significantly lower than that in the non-LDA/non-UFH group (5.9% vs 50.0%, p = 0.048). Among 21 women who tested positive for anti-ß2GPI/HLA-DR antibodies and had no other risk factors for RPL, the live birth rate in the LDA/UFH group (n = 14) was much higher than that in the non-LDA/non-UFH group (n = 7) (92.9% vs 42.9%, p = 0.03). Discussion: This study, for the first time, demonstrated that LDA and/or UFH therapies are effective in improving pregnancy outcomes in women with RPL and aß2GPI/HLA-DR antibody positivity.

Unexplained Recurrent Pregnancy Loss: Clinical Application of Immunophenotyping.

PROBLEM: Recurrent pregnancy loss (RPL) is defined as the failure of two or more pregnancies and affects approximately 5% of couples, often without a clear cause. The etiologies of RPL include factors such as maternal age, endocrine dysfunction, uterine abnormalities, chromosomal abnormalities, thrombophilias, infections, and autoimmune disorders. However, these conditions account for only 50%-60% of RPL cases. Research has explored whether an altered immune system, compared to the physiological state, may be linked to RPL. This review aims to determine whether specific immunophenotypes are associated with unexplained Recurrent Pregnancy Loss (uRPL) and whether targeted therapies addressing specific immunophenotypic alterations can improve pregnancy outcomes. METHODS: A literature review was conducted using Pubmed/Medline, Scopus, and Embase databases, analyzing data from 95 articles published between 2001 and 2023. The roles of various cells of the immune system (B lymphocytes, T lymphocytes, natural killer cells, macrophages) in different tissues (peripheral blood, menstrual blood) were specifically investigated in women with uRPL. DISCUSSION AND CONCLUSION: Specific immunophenotypes have been demonstrated to be associated with this condition. However, there is a need to standardize immunophenotyping assays and conduct more trials to stratify RPL risk and improve potential therapeutic strategies.

Biochemical markers for prediction of the first half pregnancy losses: a review.

Objective: 26% of all pregnancies end in miscarriage, and up to 10% of clinically diagnosed pregnancies, and recurrent pregnancy loss is 5% among couples of childbearing ages. Although there are several known causes of pregnancy loss in the first half, including recurrent pregnancy loss, including parental chromosomal abnormalities, uterine malformations, endocrinological disorders, and immunological abnormalities, about half of the cases of pregnancy loss in its first half remain unexplained. Methods: The review includes observational controlled studies (case-control or cohort, longitudinal studies, reviews, meta-analyses), which include the study of biochemical factors for predicting pregnancy losses in the first half, in singlet pregnancy. The Newcastle-Ottawa Scale (NOS) was used to assess the research quality. Results: Finally, 27 studies were included in the review, which has 134904 examined patients. The results of the review include estimates of ß-human chorionic gonadotropin, progesterone, pregnancy-associated protein - A, angiogenic vascular factors, estradiol, α-fetoprotein, homocysteine and CA-125 as a predictors or markers of the first half pregnancy losses. Conclusion: It may be concluded that to date, research data indicate the unavailability of any reliable biochemical marker for predicting pregnancy losses in its first half and require either a combination of them or comparison with clinical evidence. A fairly new model shall be considered for the assessment of α-fetoprotein in vaginal blood, which may have great prospects in predicting spontaneous miscarriages.

Comparison of the predictive value of four insulin resistance surrogates and hyperuricemia in women with recurrent pregnancy loss: A cross-sectional study.

BACKGROUND: Insulin resistance (IR), hyperuricemia (HUA), and recurrent pregnancy loss (RPL) elevate the risk of cardiovascular disease and metabolic disorders, while also impacting reproductive health. The relationship between IR, HUA, and RPL has not been thoroughly investigated. This study investigates the relationship between four IR surrogates and the risk of HUA in RPL patients. METHODS: Data from a real-world study on RPL in China were analyzed using multivariable regression to determine the relationship between HUA and triglyceride and glucose (TyG) index, triglyceride glucose-body mass index (TyG-BMI), triglyceride to high-density lipoprotein cholesterol (TG/HDL-c) ratio, and metabolic score for insulin resistance (METS-IR). The predictive ability of these surrogates for detecting HUA in RPL patients was evaluated using the area under the curve and receiver operating characteristic analysis. Sensitivity analysis was performed using bootstrapping resampling. RESULTS: The study included 769 patients with a mean age of 30 ± 4 years old, 8.32% of whom had HUA. Four IR surrogates were closely related to HUA in patients of RPL after adjusting for age, menstrual cycle, creatinine, alanine transaminase, aspartate transaminase, total cholesterol, homocysteine, and low-density lipoprotein, with area under the curve values of TyG index (OR = 0.693, 95% confidence interval [CI]: 0.626, 0.759), TyG-BMI (OR = 0.731 95% CI: 0.657, 0.805), TG/HDL-C (OR = 0.703, 95% CI: 0.641, 0.764), and METS-IR (OR = 0.728, 95% CI: 0.655, 0.799). Bootstrap resampling yielded similar results. CONCLUSIONS: The TyG index, TyG-BMI, TG/HDL-c, and METS-IR significantly correlated with HUA in patients with RPL. The TyG-BMI had the highest predictive value of the four IR surrogates.

Causality between neuroticism personality clusters and female reproductive diseases in European population: a two-sample bidirectional mendelian randomization study.

BACKGROUND: The causality between neuroticism, a personality trait characterized by the tendency to experience negative emotions, and female reproductive diseases remains unclear. To provide evidence for the development of effective screening and prevention strategies, this study employed Mendelian randomization (MR) to investigate the causality between neuroticism clusters and female reproductive diseases. METHODS: Instrumental variables were obtained from large-scale genome-wide association studies of populations of European descent involving three neuroticism clusters (depressed affect, worry, sensitivity to environmental stress, and adversity [SESA]) in the Complex Trait Genetics database and six female reproductive diseases (infertility, polycystic ovary syndrome [PCOS], spontaneous abortion, recurrent spontaneous abortion, endometriosis, and uterine fibroids) in the FinnGen database. The bidirectional two-sample MR analysis was conducted using the inverse variance-weighted, weighted median, and MR-Egger methods, whereas the sensitivity analysis was conducted using the Cochran's Q-test, MR-Egger intercept, and leave-one-out analysis. RESULTS: In the forward analysis, genetically predicted depressed affect and worry components of neuroticism significantly increased the risk of infertility (depressed affect: odds ratio [OR] = 1.399, 95% confidence interval [CI]: 1.054-1.856, p = 0.020; worry: OR = 1.587, 95% CI: 1.229-2.049, p = 0.000) and endometriosis (depressed affect: OR = 1.611, 95% CI: 1.234-2.102, p = 0.000; worry: OR = 1.812, 95% CI: 1.405-2.338, p = 0.000). Genetically predicted SESA component of neuroticism increased only the risk of endometriosis (OR = 1.524, 95% CI: 1.104-2.103, p = 0.010). In the reverse analysis, genetically predicted PCOS was causally associated with an increased risk of the worry component of neuroticism (Beta = 0.009, 95% CI: 0.003-0.016, p = 0.003). CONCLUSIONS: The MR study showed that the three neuroticism personality clusters had definite causal effects on at least one specific female reproductive disease. Moreover, PCOS may increase the risk of the worry component of neuroticism. This finding suggests the need to screen for specific female reproductive diseases in populations with high neuroticism and assess the psychological status of patients with PCOS.

Discussion on the evaluation of the therapeutic efficacy of uterine artery blood flow parameters and serum PLGF and sFlt-1 in patients with recurrent spontaneous abortion.

OBJECTIVE: To investigate the effects of different drug treatments on uterine artery blood flow parameters, serum placental growth factor (PLGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and sFlt-1/PLGF in patients with recurrent spontaneous abortion and to explore the predictive value of uterine artery blood flow parameters, serum PLGF, sFlt-1, and sFlt-1/PLGF for pregnancy outcomes. METHODS: This retrospective cohort study included 173 patients who experienced recurrent spontaneous abortion and 100 control patients. Patients with recurrent spontaneous abortion were divided into an aspirin group (75 patients), aspirin combined with low molecular weight heparin (LMWH) group (68 patients), and non-drug group (30 patients) based on different drug treatments. Uterine artery blood flow parameters at gestational weeks 30-31+6 were monitored for the four groups, and serum samples were collected at gestational weeks 30-31+6 to measure the levels of serum PLGF and sFlt-1 and calculate the sFlt-1/PLGF ratio. RESULTS: 1. Uterine artery blood flow parameters at gestational weeks 30-31+6 were significantly greater in the non-drug group than in the aspirin group, combined drug group, and control group (p<0.05). 2. Serum PLGF levels and the sFlt-1/PLGF ratio at gestational weeks 30-31+6 were significantly lower in the non-drug group than in the aspirin group, combined drug group, and control group, while serum sFlt-1 levels were significantly greater in the non-drug group than in the aspirin group, combined drug group, and control group (p<0.05). 3. Serum PLGF, sFlt-1, and sFlt-1/PLGF had lower diagnostic efficiency for predicting hypertensive disorders during pregnancy than the combined diagnostic efficiency of serum PLGF, sFlt-1, and sFlt-1/PLGF with uterine artery blood flow parameters at gestational weeks 30-31+6. CONCLUSION: Aspirin and aspirin combined with LMWH can upregulate serum PLGF and decrease serum sFlt-1 levels in patients with recurrent spontaneous abortion, reduce the miscarriage rate, and significantly improve pregnancy outcomes. The combination of serum PLGF, sFlt-1, sFlt-1/PLGF, and uterine artery blood flow parameters can effectively predict hypertensive disorders during pregnancy.

MNSFß promotes LPS-induced TNFα expression by increasing the localization of RC3H1 to stress granules, and the interfering peptide HEPN2 reduces TNFα production by disrupting the MNSFß-RC3H1 interaction in macrophages.

Abnormally elevated tumor necrosis factor-α (TNFα) levels at the maternal-fetal interface can lead to adverse pregnancy outcomes, including recurrent miscarriage (RM), but the mechanism underlying upregulated TNFα expression is not fully understood. We previously reported that the interaction between monoclonal nonspecific suppressor factor-ß (MNSFß) and RC3H1 upregulates TNFα expression, but the precise mechanisms are unknown. In this study, we found that MNSFß stimulated the LPS-induced TNFα expression by inactivating the promoting effect of RC3H1 on TNFα mRNA degradation rather than directly inhibiting the expression of RC3H1 in THP1-Mϕs. Mechanistically, the 81-326 aa region of the RC3H1 protein binds to the 101-133 aa region of the MNSFß protein, and MNSFß facilitated stress granules (SGs) formation and the translocation of RC3H1 to SGs by interacting with RC3H1 and fragile X mental retardation 1 (FMR1) in response to LPS-induced stress. The SGs-localization of RC3H1 reduced its inhibitory effect on TNFα expression in LPS-treated THP1-Mϕs. The designed HEPN2 peptide effectively reduced the LPS-induced expression of TNFα in THP1-Mϕs by interfering with the MNSFß-RC3H1 interaction. Treatment with the HEPN2 peptide significantly improved adverse pregnancy outcomes, including early pregnancy loss (EPL) and lower fetal weight (LFW), which are induced by LPS in mice. These data indicated that MNSFß promoted TNFα expression at least partially by increasing the localization of RC3H1 to SGs under inflammatory stimulation and that the HEPN2 peptide improved the adverse pregnancy outcomes induced by LPS in mice, suggesting that MNSFß is a potential pharmacological target for adverse pregnancy outcomes caused by abnormally increased inflammation at early pregnancy.

Chronic endometritis and recurrent reproductive failure: a systematic review and meta-analysis.

Background: The endometrium holds a crucial role in reproduction by supporting blastocyst adhesion, cytotrophoblast invasion and fetal development. Among the various uterine disorders, endometritis, particularly chronic endometritis (CE), has gained attention due to its association with adverse reproductive outcomes (recurrent pregnancy loss (RPL), recurrent implantation failure (RIF), and infertility). The association between CE and adverse reproductive outcomes stresses the necessity for comprehensive diagnostic and therapeutic strategies to optimize fertility outcomes and support individuals in their journey towards parenthood. Aim: To explore the relationship between CE and reproductive disorders. Methods: Following PRISMA guidelines, a systematic review and meta-analysis using published data from 1990 to 2024 were carried out. Results: A population of 1,038 women was included. Regarding CE-infertility association, a positive correlation was found, with 19.46% CE rate in infertile women compared to 7.7% in controls (OR: 2.96, 95% CI 1.53-5.72, p 0.001). No significant association was observed between RIF and CE (OR: 1.10, 95% CI 0.26-4.61, p 0.90), CE rates in both groups were relatively comparable, with 6.35% in women with RIF and 5.8% in controls. On the opposite, a strong association between CE and RPL was found, reporting a CE rate of 37.6% in RPL cases compared to 16.4% in controls (OR: 3.59, 95% CI 2.46-5.24, p < 0.00001). Conclusions: CE appears to be associated to infertility and RPL, while no significant association was noted in cases of RIF. Systematic review registration: https://www.crd.york.ac.uk/prospero/#recordDetails PROSPERO, identifier CRD42024541879.

Clinical efficacy and adverse effects of LMWH combined with ASA in the treatment of RSA: A meta-analysis and systematic review.

BACKGROUND: The incidence of recurrent spontaneous abortion (RSA) in the clinic shows an increasing trend year by year, and the coagulation status of this group of patients is mostly relatively abnormal. Currently, commonly used drugs in clinical practice include Aspirin (ASA) and low molecular weight heparin (LMWH), but their optimal treatment remains controversial. We aimed to evaluate the clinical efficacy and adverse effects of LMWH combined with ASA in the treatment of RSA. METHODS: Randomized controlled trials of LMWH combined with ASA for RSA were searched in the databases of PubMed, EMBASE, Cochrane Library, China National Knowledge Infrastructure, Wanfang, VIP, and Chinese Biomedical Literature Service System from the construction of the database to June 2024. Data were analyzed using Review Manager 5.3 and Stata software. Dichotomous variables were analyzed using relative risk (RR) and 95% confidence interval (CI) as their statistics. The included literature was assessed for bias and risk of bias of eligible studies using Cochrane risk of bias tool. The risk of bias was evaluated based on the evaluation criteria recommended by the Cochrane Guidance Manual for Systematic Evaluation. RESULTS: A total of 32 papers with a total of 3397 patients with RSA were finally included. LMWH combined with ASA treatment significantly improved the live birth rate (RR = 1.31, 95% CI: [1.19, 1.45], P < .00001), the rate of preterm stillbirths (RR = 0.23, 95% CI: [0.13, 0.40], P < .00001), rate of term delivery (RR = 1.55, 95% CI: [1.43, 1.67], P < .00001), rate of miscarriage (RR = 0.42, 95% CI: [0.36, 0.48], P < .00001), incidence of petechiae (RR = 0.44, 95% CI: [0.26, 0.72], P = .001), and incidence of thrombocytopenia (RR = 0.61, 95% CI: [0.39, 0.96], P = .03). In contrast, the incidence of preterm live births (RR = 1.07, 95% CI: [0.90, 1.28], P = .44), adverse reactions (RR = 0.77, 95% CI: [0.59, 1.00], P = .05), gingival bleeding (RR = 1.12, 95% CI: [0.65, 1.93], P = .69), and gastrointestinal reactions (RR = 0.87, 95% CI: [0.64, 1.17], P = .35) were not significant. CONCLUSION: LMWH combined with ASA treatment might improve pregnancy outcomes and reduces the incidence of adverse events in patients with RSA.

Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) gene polymorphisms and five related serum molecular levels in 2587 patients: Associated differentially with adverse pregnancy.

BACKGROUND: The aim of the study is to investigate the relationship between Methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR) polymorphisms, 5 serum related molecular levels and the risk of adverse pregnancies in different genders. METHODS: Patients aged from 22 to 38 with a history of adverse pregnancy treated in our genetic eugenics clinic of Henan Provincial People's Hospital are selected. The controls aged from 20 to 34 undergoing eugenics examinations in our genetic eugenics clinic that had no history of adverse pregnancy and at least one healthy child are selected. Sanger sequencing and Chemiluminescence Microparticle Immuno Assay (CMIA) are used for detecting the mutations of MTHFR and MTRR and the 5 serum molecular serum levels. RESULTS: In the female group, MTHFR 677 C > T is associated with Recurrent spontaneous abortion (RSA) (P = 0.0017), Chromosomal abnormality (CA) (P = 0.0053), Cleft lip and palate (CLP) (P = 0.0326) and Brain dysplasia (BD) (P = 0.0072); MTHFR 1298 A > C is associated with Infertility (P = 0.0026) and BD (P = 0.0382); MTRR 66 A > G is associated with CLP (P = 0.0131). In the male group, MTHFR 677 C > T is associated with RSA (P = 0.0003), Infertility (P = 0.0013), CA (P = 0.0027) and BD (P = 0.0293). In the female group, the genotype of MTHFR 677 C > T is associated with RSA (P = 0.0017), CA (P = 0.0014) and BD (P = 0.0021); MTHFR 1298 A > C is associated with Infertility (P = 0.0081) and MTRR 66 A > G is associated with Infertility (P = 0.0309). In the male group, the genotype of MTHFR 677 C > T is associated with RSA (P = 0.0008), Infertility (P = 0.0096) and CA (P = 0.0165) and MTRR 66 A > G is associated with Infertility (P = 0.0158) and congenital heart disease (CHD) (P = 0.0218). In the male group, there is statistically significant difference of the serum Homocysteine (Hcy) levels (P < 0.0001) between adverse pregnancy group and controls. In the female group, there is statistically significant difference of the serum vitamin D levels (P = 0.0015) between adverse pregnancy group and controls. CONCLUSIONS: Polymorphic variants in MTHFR and MTRR, serum Folic acid (FA), Hcy and B12 levels in the male group and vitamin D levels in the female group are associated differentially with adverse pregnancy.

Surface Immune Checkpoints as Potential Biomarkers in Physiological Pregnancy and Recurrent Pregnancy Loss.

Due to the genetic diversity between the mother and the fetus, heightened control over the immune system during pregnancy is crucial. Immunological parameters determined by clinicians in women with idiopathic recurrent spontaneous abortion (RSA) include the quantity and activity of Natural Killer (NK) and Natural Killer T (NKT) cells, the quantity of regulatory T lymphocytes, and the ratio of pro-inflammatory cytokines, which indicate imbalances in Th1 and Th2 cell response. The processes are controlled by immune checkpoint proteins (ICPs) expressed on the surface of immune cells. We aim to investigate differences in the expression of ICPs on T cells, T regulatory lymphocytes, NK cells, and NKT cells in peripheral blood samples collected from RSA women, pregnant women, and healthy multiparous women. We aim to discover new insights into the role of ICPs involved in recurrent pregnancy loss. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation from blood samples obtained from 10 multiparous women, 20 pregnant women (11-14th week of pregnancy), and 20 RSA women, at maximum of 72 h after miscarriage. The PBMCs were stained for flow cytometry analysis. Standard flow cytometry immunophenotyping of PBMCs was performed using antibodies against classical lymphocyte markers, including CD3, CD4, CD8, CD56, CD25, and CD127. Additionally, ICPs were investigated using antibodies against Programmed Death Protein-1 (PD-1, CD279), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3, CD366), V-domain Ig suppressor of T cell activation (VISTA), T cell immunoglobulin and ITIM domain (TIGIT), and Lymphocyte activation gene 3 (LAG-3). We observed differences in the surface expression of ICPs in the analyzed subpopulations of lymphocytes between early pregnancy and RSA, after miscarriage, and in women. We noted diminished expression of PD-1 on T lymphocytes (p = 0.0046), T helper cells (CD3CD4 positive cells, p = 0.0165), T cytotoxic cells (CD3CD8 positive cells, p = 0.0046), T regulatory lymphocytes (CD3CD4CD25CD127 low positive cells, p = 0.0106), and NKT cells (CD3CD56/CD16 positive cells, p = 0.0438), as well as LAG-3 on lymphocytes T (p = 0.0225) T helper, p = 0.0426), T cytotoxic cells (p = 0.0458) and Treg (p = 0.0293), and cells from RSA women. Impaired expression of TIM-3 (p = 0.0226) and VISTA (p = 0.0039) on CD8 cytotoxic T and NK (TIM3 p = 0.0482; VISTA p = 0.0118) cells was shown, with an accompanying increased expression of TIGIT (p = 0.0211) on NKT cells. The changes in the expression of surface immune checkpoints indicate their involvement in the regulation of pregnancy. The data might be utilized to develop specific therapies for RSA women based on the modulation of ICP expression.

Association of MTR and MTRR polymorphisms with recurrent pregnancy loss: a case control study.

BACKGROUND: In light of several epidemiological studies, the etiology of recurrent pregnancy loss is complex. One of the most frequent causes of women experiencing inexplicable recurrent pregnancy loss is maternal thrombophilia. Hence, the association between genetic polymorphisms causing thrombophilia and recurrent pregnancy loss needs to be explored. AIM: Is to study the relation of polymorphisms affecting folate pathway mainly, 5-Methytetrahydrofolate-Homocysteine Methyltransferase (MTR A2756G) and 5-Methytetrahydrofolate-Homocysteine MethyltransferaseReductase (MTRR A66G) with recurrent pregnancy loss. METHODS: It is a case-control study. Four hundred participants were enrolled. Two hundred participants with unexplained recurrent pregnancy loss (case group) and two hundred healthy fertile participants (control group). All participants were screened for (MTR A2756G) and (MTRR A66G). DNA was extracted using salting out method followed by genotyping via Real-time PCR. RESULTS: Mutant homozygous genotype (GG) in MTRR A66G was statistically significantly among RPL group in comparison to controls. (GG vs. AA) had odds ratio and confidence interval of 1.22(1.12-2.23), P = 0.012. (GG) increased the liability 1.2 folds for recurrent pregnancy loss. Mutant homozygous genotype (GG) in MTR A2756G was not correlated with the risk of recurrent pregnancy loss. (GG vs.AA) = (1.13(0.56-2.29)), P = 0.7 CONCLUSION: MTRR A66G increases susceptibly for recurrent pregnancy loss among Egyptian women.

Optimizing evaluation of endometrial receptivity in recurrent pregnancy loss: a preliminary investigation integrating radiomics from multimodal ultrasound via machine learning.

Background: Recurrent pregnancy loss (RPL) frequently links to a prolonged endometrial receptivity (ER) window, leading to the implantation of non-viable embryos. Existing ER assessment methods face challenges in reliability and invasiveness. Radiomics in medical imaging offers a non-invasive solution for ER analysis, but complex, non-linear radiomic-ER relationships in RPL require advanced analysis. Machine learning (ML) provides precision for interpreting these datasets, although research in integrating radiomics with ML for ER evaluation in RPL is limited. Objective: To develop and validate an ML model that employs radiomic features derived from multimodal transvaginal ultrasound images, focusing on improving ER evaluation in RPL. Methods: This retrospective, controlled study analyzed data from 346 unexplained RPL patients and 369 controls. The participants were divided into training and testing cohorts for model development and accuracy validation, respectively. Radiomic features derived from grayscale (GS) and shear wave elastography (SWE) images, obtained during the window of implantation, underwent a comprehensive five-step selection process. Five ML classifiers, each trained on either radiomic, clinical, or combined datasets, were trained for RPL risk stratification. The model demonstrating the highest performance in identifying RPL patients was selected for further validation using the testing cohort. The interpretability of this optimal model was augmented by applying Shapley additive explanations (SHAP) analysis. Results: Analysis of the training cohort (242 RPL, 258 controls) identified nine key radiomic features associated with RPL risk. The extreme gradient boosting (XGBoost) model, combining radiomic and clinical data, demonstrated superior discriminatory ability. This was evidenced by its area under the curve (AUC) score of 0.871, outperforming other ML classifiers. Validation in the testing cohort of 215 subjects (104 RPL, 111 controls) confirmed its accuracy (AUC: 0.844) and consistency. SHAP analysis identified four endometrial SWE features and two GS features, along with clinical variables like age, SAPI, and VI, as key determinants in RPL risk stratification. Conclusion: Integrating ML with radiomics from multimodal endometrial ultrasound during the WOI effectively identifies RPL patients. The XGBoost model, merging radiomic and clinical data, offers a non-invasive, accurate method for RPL management, significantly enhancing diagnosis and treatment.

Mass spectrometry of water-soluble vitamins to establish a risk model for predicting recurrent spontaneous abortion.

The adverse pregnancy outcomes, including recurrent spontaneous abortion (RSA), are strongly correlated with water-soluble vitamins, but how to predict RSA occurrence using them remains unsatisfactory. This study aims to investigate the possibility of predicting RSA based on the baseline levels of water-soluble vitamins tested by ultra-liquid chromatography-tandem mass spectrometry. A total of 918 pregnant women was consecutively enrolled in this cross-sectional study. According to the miscarriage numbers, they were divided into normal first pregnancy (NFP, n = 608), once spontaneous abortion (OSA, n = 167), and continuous spontaneous abortion (CSA, n = 143) groups. The Cox proportional-hazards regression model was employed to establish a risk model for predicting RSA. The RSA occurrence was 6.54% in overall pregnant women, with a prevalence of 12.57% in the OSA group and 27.27% in the CSA group. Significant differences were observed in baseline deficiencies of vitamin B3, B5, B6, and B9 among NFP, OSA, and CSA groups (χ2 = 12.191 ~ 37.561, all P < 0.001). Among these vitamins, B9 (HR = 0.89 and 0.88, all P < 0.001) and B6 (HR = 0.83 and 0.78, all P < 0.05) were identified as independent factors in both the OSA and CSA groups; whereas B5 was identified as an additional independent factor only in the CSA group (HR = 0.93, P = 0.005). The Cox proportional-hazards model established using these three vitamins exhibited poor or satisfactory predictive performance in the OSA (Sen = 95.2%, Spe = 39.0%) and CSA (Sen = 92.3%, Spe = 60.6%) groups, respectively. However, B5, B6, and B9 compensatory levels were not associated with RSA occurrence (all P > 0.05). Our study presents a highly sensitive model based on mass spectrometry assay of baseline levels in B vitamins to predict the RSA occurrence as possible.

Polycystic ovarian syndrome (PCOS) and recurrent spontaneous abortion (RSA) are associated with the PI3K-AKT pathway activation.

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.

Exposure to BaA inhibits trophoblast cell invasion and induces miscarriage by regulating the DEC1/ARHGAP5 axis and promoting ubiquitination-mediated degradation of MMP2.

Benz[a]anthracene (BaA), a hazardous polycyclic aromatic hydrocarbon classified by the EPA, is a probable reproductive toxicant. Epidemiological studies suggest that BaA exposure may be a risk factor for recurrent miscarriage (RM). However, the underlying mechanisms are not well understood. This study identified DEC1 as a key gene through RNA-seq and single-cell RNA sequencing analysis. DEC1 expression was found to be downregulated in villous tissues from women with RM and in primary extravillous trophoblasts (EVTs) exposed to BaA. BaA suppressed DEC1 expression by promoting abnormal methylation patterns. Further analysis revealed that ARHGAP5 is a direct target of DEC1 in EVTs, where DEC1 inhibits trophoblast invasion by directly regulating ARHGAP5 transcription. Additionally, BaA destabilized matrix metalloproteinase 2 (MMP2) by activating the aryl hydrocarbon receptor (AhR) and promoting E3 ubiquitin ligase MID1-mediated degradation. In a mouse model, BaA induced miscarriage by modulating the DEC1/ARHGAP5 and MID1/MMP2 axes. Notably, BaA-induced miscarriage in mice was prevented by DEC1 overexpression or MID1 knockdown. These findings indicate that BaA exposure leads to miscarriage by suppressing the DEC1/ARHGAP5 pathway and enhancing the MID1/MMP2 pathway in human EVTs.

Effect of IVIG therapy on pregnant women with unexplained recurrent spontaneous abortion: a systematic review and meta-analysis.

Objective: To assess the effect of intravenous immunoglobulin (IVIG) therapy on unexplained recurrent spontaneous abortion (URSA). Methods: We retrieved all randomized controlled trials (RCTs) related to the effect of IVIG therapy on URSA in the following databases: PubMed, Embase, Web of Science, and Cochrane Central Register of Controlled Trials before April 30, 2023, according to the PRISMA statement. The therapeutic effect of IVIG was measured by live birth rates. Quality assessment was conducted independently by two reviewers, based on the Newcastle-Ottawa scale. For the meta-analysis, we used odds ratios (random effects model and fixed effects model). The between-study heterogeneity was assessed by the Q test. Publication bias was assessed by funnel plots. Results: A total of 12 studies with 751 participants were included in this meta-analysis. There was no statistical significance [OR = 1.07, 95%CI (0.65, 1.75), P=0.80] between the IVIG group and the non-IVIG group, including low molecular weight heparin (LMWH) plus low-dose aspirin (LDA), intralipid, multivitamins, albumin, and normal saline. A subgroup analysis was conducted according to the different treatment regimens of the non-IVIG group. Compared to the placebo group, including multivitamins, albumin, and saline, the live birth rate of the IVIG group is superior, but there was no statistical significance [OR =1.43, 95%CI (0.99, 2.07), P=0.05]. Another subgroup analysis was performed according to URSA with positive for antiphospholipid antibodies (aPLs). Results showed the live birth rate of IVIG on URSA with positive for aPLs is inferior to that of LMWH plus LDA [OR = 0.25, 95%CI (0.11, 0.55), P=0.0007]. Conclusions: IVIG didn't increase the live birth rate of URSA compared to placebo. Conversely, compared with the IVIG, the LMWH plus LDA treatment schedule can increase the live birth rate of URSA with positive for aPLs.

A nomogram and risk stratification to predict subsequent pregnancy loss in patients with recurrent pregnancy loss.

STUDY QUESTION: Could the risk of subsequent pregnancy loss be predicted based on the risk factors of recurrent pregnancy loss (RPL) patients? SUMMARY ANSWER: A nomogram, constructed from independent risk factors identified through multivariate logistic regression, serves as a reliable tool for predicting the likelihood of subsequent pregnancy loss in RPL patients. WHAT IS KNOWN ALREADY: Approximately 1-3% of fertile couples experience RPL, with over half lacking a clear etiological factor. Assessing the subsequent pregnancy loss rate in RPL patients and identifying high-risk groups for early intervention is essential for pregnancy counseling. Previous prediction models have mainly focused on unexplained RPL, incorporating baseline characteristics such as age and the number of previous pregnancy losses, with limited inclusion of laboratory and ultrasound indicators. STUDY DESIGN, SIZE, DURATION: The retrospective study involved 3387 RPL patients who initially sought treatment at the Reproductive Immunology Clinic of Renji Hospital, Shanghai Jiao Tong University School of Medicine, between 1 January 2020 and 31 December 2022. Of these, 1153 RPL patients met the inclusion criteria and were included in the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: RPL was defined as two or more pregnancy losses (including biochemical pregnancy loss) with the same partner before 28 weeks of gestation. Data encompassing basic demographics, laboratory indicators (autoantibodies, peripheral immunity coagulation, and endocrine factors), uterine and endometrial ultrasound results, and subsequent pregnancy outcomes were collected from enrolled patients through initial questionnaires, post-pregnancy visits fortnightly, medical data retrieval, and telephone follow-up for lost patients. R software was utilized for data cleaning, dividing the data into a training cohort (n = 808) and a validation cohort (n = 345) in a 7:3 ratio according to pregnancy success and pregnancy loss. Independent predictors were identified through multivariate logistic regression. A nomogram was developed, evaluated by 10-fold cross-validation, and compared with the model incorporating solely age and the number of previous pregnancy losses. The constructed nomogram was evaluated using the AUC, calibration curve, decision curve analysis (DCA), and clinical impact curve analysis (CICA). Patients were then categorized into low- and high-risk subgroups. MAIN RESULTS AND THE ROLE OF CHANCE: We included age, number of previous pregnancy losses, lupus anticoagulant, anticardiolipin IgM, anti-phosphatidylserine/prothrombin complex IgM, anti-double-stranded DNA antibody, arachidonic acid-induced platelet aggregation, thrombin time and the sum of bilateral uterine artery systolic/diastolic ratios in the nomogram. The AUCs of the nomogram were 0.808 (95% CI: 0.770-0.846) in the training cohort and 0.731 (95% CI: 0.660-0.802) in the validation cohort, respectively. The 10-fold cross-validated AUC ranged from 0.714 to 0.925, with a mean AUC of 0.795 (95% CI: 0.750-0.839). The AUC of the nomogram was superior compared to the model incorporating solely age and the number of previous pregnancy losses. Calibration curves, DCAs, and CICAs showed good concordance and clinical applicability. Significant differences in pregnancy loss rates were observed between the low- and high-risk groups (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: This study was retrospective and focused on patients from a single reproductive immunology clinic, lacking external validation data. The potential impact of embryonic chromosomal abnormalities on pregnancy loss could not be excluded, and the administration of medication to all cases impacted the investigation of risk factors for pregnancy loss and the model's predictive efficacy. WIDER IMPLICATIONS OF THE FINDINGS: This study signifies a pioneering effort in developing and validating a risk prediction nomogram for subsequent pregnancy loss in RPL patients to effectively stratify their risk. We have integrated the nomogram into an online web tool for clinical applications. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (82071725). All authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Impact of sperm DNA fragmentation in couples with unexplained recurrent pregnancy loss: A cross-sectional study.

OBJECTIVE: Recurrent pregnancy loss (RPL) has a multifactorial etiology, with a majority of cases remaining unexplained. To account for these unexplained cases, possible male factors are being explored. Conventional semen analysis lacks a qualitative assessment of sperms and information regarding sperm DNA integrity. Sperm DNA fragmentation (SDF) has diagnostic value in unexplained RPL, and it may account for a number of unexplained cases. Hence, we planned a study to explore and evaluate the impact of sperm DNA fragmentation in couples with unexplained recurrent pregnancy losses. STUDY DESIGN: Analytical cross-sectional study was conducted at a tertiary-level referral facility in India between August 2021 and July 2023. Participants (n = 70) were divided into two groups-male partners of couples with unexplained RPL (following spontaneous conceptions) (n = 35) and men with at least one previous live birth (spontaneous or following fertility treatments for female factor infertility such as ovulation induction or intrauterine insemination) as controls (n = 35). Neither of the two groups of couples recruited for this study had undergone ART as fertility treatment. Primary outcome assessed was mean DNA fragmentation index (DFI). Secondary outcomes included differences in semen parameters such as sperm concentration, progressive sperm motility and morphology, proportion of men with high (≥30%) and low DFI in the two groups, and the association between various semen parameters and DFI. RESULTS: Univariate logistic regression revealed that sperm DNA fragmentation was higher in men with unexplained RPL (30.0; IQR (interquartile range) 19.0, 46.0) as compared to controls (22.0; IQR 14.0, 30.0) although it was not statistically significant (OR, odds ratio, 1.02; 95% CI 1.0-1.1, p = 0.08). A higher proportion of men with unexplained RPL had DFI ≥30% compared to controls (54.2% vs. 25.7%; OR 3.43 (95% CI 1.2-9.4); p = 0.02). No statistically significant differences were observed in semen volume, sperm concentration, progressive motility, and morphology between the two groups. Sperm DNA fragmentation index also showed a weak but significant inverse relationship with sperm morphology (r = -0.336, p = 0.004). CONCLUSION: The current study did not show any significant difference in the mean sperm DNA fragmentation levels in male partners of couples with unexplained RPL compared to controls. However, a higher proportion of men with DFI ≥30% were observed in unexplained RPL population when compared to controls.