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.

METTL14 depletion induces trophoblast cell dysfunction by inhibiting miR-21-5p processing in an m6A-dependent manner.

Spontaneous abortion (SA) is a devastating, but common outcome for expectant parents and their families. However, the mechanism of SA occurrence remains mostly unknown. Herein, we examined human SA villi samples and found decreased N6-methyladenosine (m6A) levels and methyltransferase-like protein 14 (METTL14) expression compared with those in healthy women. Knockdown of METTL14 in trophoblast HTR8 cells induced cellular dysfunction. We identified candidate differentially expressed microRNAs and found that METTL14 accelerated miR-21-5p processing by modulating its m6A modification level. Exogenous miR-21-5p expression attenuated METTL14 knockdown-induced cellular dysfunction. Subsequently, we found that SMAD family member 7 (SMAD7) expression is inhibited by miR-21-5p and that knockdown of SMAD7 rescued the trophoblast cell dysfunction induced by miR-21-5p inhibitors. Then, we revealed that METTL14 can regulate the SMAD7 pathway by modulating miR-21-5p. Finally, we found that exposing pregnant mice to an m6A inhibitor caused embryo loss and reduced expression levels of Mettl14 and miR-21-5p while increasing Smad7 levels. Taken together, this study establishes the involvement of m6A in SA and identified a novel SA signaling pathway. These results reveal the underlying molecular mechanisms of trophoblast cell dysfunction induced by m6A modification and provide new strategies to identify and mitigate SA.

Factors affecting biochemical pregnancy loss (BPL) in preimplantation genetic testing for aneuploidy (PGT-A) cycles: machine learning-assisted identification.

PURPOSE: To determine the factors influencing the likelihood of biochemical pregnancy loss (BPL) after transfer of a euploid embryo from preimplantation genetic testing for aneuploidy (PGT-A) cycles. METHODS: The study employed an observational, retrospective cohort design, encompassing 6020 embryos from 2879 PGT-A cycles conducted between February 2013 and September 2021. Trophectoderm biopsies in day 5 (D5) or day 6 (D6) blastocysts were analyzed by next generation sequencing (NGS). Only single embryo transfers (SET) were considered, totaling 1161 transfers. Of these, 49.9% resulted in positive pregnancy tests, with 18.3% experiencing BPL. To establish a predictive model for BPL, both classical statistical methods and five different supervised classification machine learning algorithms were used. A total of forty-seven factors were incorporated as predictor variables in the machine learning models. RESULTS: Throughout the optimization process for each model, various performance metrics were computed. Random Forest model emerged as the best model, boasting the highest area under the ROC curve (AUC) value of 0.913, alongside an accuracy of 0.830, positive predictive value of 0.857, and negative predictive value of 0.807. For the selected model, SHAP (SHapley Additive exPlanations) values were determined for each of the variables to establish which had the best predictive ability. Notably, variables pertaining to embryo biopsy demonstrated the greatest predictive capacity, followed by factors associated with ovarian stimulation (COS), maternal age, and paternal age. CONCLUSIONS: The Random Forest model had a higher predictive power for identifying BPL occurrences in PGT-A cycles. Specifically, variables associated with the embryo biopsy procedure (biopsy day, number of biopsied embryos, and number of biopsied cells) and ovarian stimulation (number of oocytes retrieved and duration of stimulation), exhibited the strongest predictive power.

Direct embryonic biopsy with transcervical embryoscopy is an effective method for karyotyping and morphology assessment in miscarriages.

PURPOSE: The main purpose of this study is to compare the validity of transcervical embryoscopy method with standard uterine evacuation method in detecting more accurate karyotypes in miscarriages below tenth week of pregnancy. Additionally, the frequency and distribution of fetal morphological abnormality were evaluated. METHODS: A prospective study was carried out at the Gazi University Faculty of Medicine, Department of Obstetrics and Gynecology. Patients with missed abortions between sixth and tenth gestational weeks were included in the study group, and fetal morphological examination and direct embryonic biopsy were performed by transcervical embryoscopy. The control group consisted of patients who experienced miscarriage and genetic material obtained from routine uterine evacuation between February and October 2023. RESULT: A total of 60 patients in the study group and 189 patients in the control group were evaluated. The median ages, previous miscarriage numbers, median gravida numbers, and median gestational weeks were comparable between groups. Chromosomal abnormality was detected in 24 (42.8%) and 52 embryos (29.9%) in the study and control groups, respectively (p = 0.004). Culture failure rates were 6.6% (n = 4) and 7.9% (n = 15) in the study and control groups, respectively. In the study group, 12 embryos had a morphological abnormality in which 6 of them had normal karyotype. CONCLUSION: Direct embryonic biopsy with transcervical embryoscopy is an effective method to exclude maternal decidual cell contamination and placental mosaicism in miscarriages for karyotype analysis. In addition, detecting anomalies in morphology might contribute our understanding in the process of miscarriages which arises independent from structural/numerical chromosomal abnormalities.

Upregulation of GPR133 expression impaired the phagocytosis of macrophages in recurrent spontaneous miscarriage.

Decidual macrophages are the second-largest immune cell group at the maternal-foetal interface. They participate in apoptotic cell removal, and protect the foetus from microorganisms or pathogens. Dysfunction of decidual macrophages gives rise to pregnancy complications such as preeclampsia and recurrent spontaneous miscarriage (RSM). However, the mechanisms by which decidual macrophages are involved in the occurrence of adverse pregnancy outcomes have not been elucidated. Here we integrated DNA methylation and gene expression data from decidua macrophages to identify potential risk factors related to RSM. GPR133 was significantly hypomethylated and upregulated in decidual macrophages from RSM patients. Further demethylation analysis demonstrated that GPR133 expression in decidual macrophages was significantly increased by 5-Aza-dC treatment. In addition, the influence of GPR133 on the phagocytic ability of macrophages was explored. Phagocytosis was impaired in the decidual macrophages of RSM patients with increased GPR133 expression. Increased GPR133 expression induced by demethylation treatment in the decidual macrophages of healthy control patients led to a significant decrease in phagocytic function. Importantly, knockdown of GPR133 resulted in a significant improvement in the phagocytic function of THP-1 macrophages. In conclusion, the existing studies have shown the influence of GPR133 on the phagocytic function of decidual macrophages and pregnancy outcomes, providing new data and ideas for future research on the role of decidual macrophages in RSM.

Copy number variation sequencing for the products of conception: What is the optimal testing strategy.

BACKGROUND: Copy number variation sequencing (CNV-seq) is crucial in prenatal diagnosis, but its limitations in detecting polyploidy, maternal cell contamination (MCC), and uniparental disomy (UPD) restrict its application in the analysis of products of conception (POCs). This study aimed to investigate an optimal genetic testing strategy for POCs in the era of CNV-seq. METHODS: CNV-seq and quantitative fluorescent polymerase chain reaction (QF-PCR) were performed in all 4,211 spontaneous miscarriage cases. Different testing strategies were compared and the optimal testing strategies were proposed. RESULTS: Of the 4,211 cases, 2561 (60.82%) exhibited clinically significant chromosomal abnormalities. CNV-seq alone, without QF-PCR, might misdiagnose 311 (7.39%) cases, including 278 polyploidy, 13 UPD, and 20 MCC. In 20 MCC cases identified by QF-PCR, CNV-seq successfully pinpointed the cause of miscarriage in 13 cases. Furthermore, in cases where QF-PCR suggested polyploidy, CNV-seq improved the diagnostic accuracy in 54 (1.28%) hypo/hypertriploidy cases. After comparing four different strategies, the sequential approach (initiating with CNV-seq followed by QF-PCR if necessary) emerged as advantageous, reducing approximately 70% of the cost associated with QF-PCR while maintaining result accuracy. CONCLUSIONS: We propose an initial CNV-seq followed by QF-PCR if needed-an efficient and cost-effective strategy for the genetic analysis of POCs.

High Incidence of CPLANE1-Related Joubert Syndrome in the Products of Conceptions from Early Pregnancy Losses

Background: The fetal monogenic causes of early pregnancy losses (EPLs) are mainly unknown, with only a few articles on the subject published. In our previous study of EPLs using whole-exome sequencing analysis, we confirmed a genetic diagnosis of CPLANE1-related Joubert syndrome (JS) in three EPLs from two couples and identified a relatively common CPLANE1 allele among our population (NM_001384732.1:c.1819delT;c.7817T>A, further after referred as "complex allele"). Pathogenic variants in the CPLANE1 (C5orf42) gene are reported to cause JS type 17, a primary ciliopathy with various system defects. Aims: To examine the hypothesis that the CPLANE1 "complex allele," whether homozygous or compound heterozygous, is a common cause of EPLs in our population. Study Design: Cohort study/case-control study.ontrol study. Methods: In this study, we used polymerase chain reaction-based methods to screen for CPLANE1 "complex allele" presence among 246 euploid EPLs (< 12 gestational weeks) from families in North Macedonia. We also investigated the impact of this allele in 650 women with EPLs versus 646 women with no history of pregnancy loss and at least one livebirth, matched by ethnic origin. Results: We found a high incidence of JS in the total study group of EPLs (2.03%), with a considerably higher incidence among Albanian families (6.25%). Although not statistically significant, women with EPLs had a higher allele frequency of the CPLANE1 "complex allele" (AF = 1.38%) than the controls (AF = 0.85%; p = 0.2). Albanian women had significantly higher frequency of the "complex allele" than the Macedonians (AF = 1.65% and 0.39%, respectively; p = 0.003). Conclusion: To the best of our knowledge, this is the highest reported incidence of fetal monogenic disease that might cause EPLs. Targeted screening for the CPLANE1 "complex allele" would be warranted in Albanian ethnic couples because it would detect one JS in every 16 euploid EPLs. Our findings have a larger impact on the pathogenesis of pregnancy loss and contribute to a better understanding of the pathogenicity of the variants in the CPLANE1 gene.

Genetic Factors Associated With Adverse Pregnancy Outcomes in Chronic Pancreatitis.

INTRODUCTION: The effects of genetic factors on pregnancy outcomes in chronic pancreatitis (CP) patients remain unclear. We evaluated the impacts of clinical features and mutations in main CP-susceptibility genes ( SPINK1 , PRSS1 , CTRC , and CFTR ) on pregnancy outcomes in Chinese CP patients. METHODS: This was a prospective cohort study with 14-year follow-up. The sample comprised female CP patients with documented pregnancy and known genetic backgrounds. Adverse pregnancy outcomes were compared between patients with and without gene mutations. Univariate and multivariate analyses were performed to determine the impact factors for adverse pregnancy outcomes. RESULTS: Totally, 160 female CP patients with a pregnancy history were enrolled; 59.4% of patients carried pathogenic mutations in CP-susceptibility genes. Adverse pregnancy outcomes occurred in 38 patients (23.8%); the prevalence of adverse outcomes was significantly higher in those harboring gene mutations than those without (30.5% vs 13.8%, P = 0.015). Notably, the rates of preterm delivery (12.6% vs 3.1%, P = 0.036) and abortion (17.9% vs 4.6%, P = 0.013) were remarkably higher in patients with gene mutations (especially SPINK1 mutations) than those without. In multivariate analyses, both CP-susceptibility gene mutations (odds ratio, 2.52; P = 0.033) and SPINK1 mutations (odds ratio, 2.60; P = 0.037) significantly increased the risk of adverse pregnancy outcomes. Acute pain attack during pregnancy was another risk factor for adverse pregnancy outcomes. DISCUSSION: Pathogenic mutations in CP-susceptibility genes, especially SPINK1 , were independently related to adverse pregnancy outcomes in CP patients. Significant attention should be paid to pregnant females harboring CP-susceptibility gene mutations (ClinicalTrials.gov: NCT06055595).

Epigenetic role of LINE-1 methylation and key genes in pregnancy maintenance.

Spontaneous abortion is a pregnancy complication characterized by complex and multifactorial etiology. About 5% of childbearing women are globally affected by early pregnancy loss (EPL) and most of them experience recurrence (RPL). Epigenetic mechanisms and controlled inflammation are crucial for pregnancy maintenance and genetic predispositions may increase the risk affecting the maternal-fetal crosstalk. Combined analyses of global methylation, inflammation and inherited predispositions may contribute to define pregnancy loss etiopathogenesis. LINE-1 epigenetic regulation plays crucial roles during embryo implantation, and its hypomethylation has been associated with senescence and several complex diseases. By analysing a group of 230 women who have gone through pregnancy interruption and comparing those experiencing spontaneous EPL (n = 123; RPL, 54.5%) with a group of normal pregnant who underwent to voluntary interruption (VPI, n = 107), the single statistical analysis revealed significant lower (P < 0.00001) LINE-1 methylation and higher (P < 0.0001) mean cytokine levels (CKs: IL6, IL10, IL17A, IL23) in EPL. Genotyping of the following SNPs accounted for different EPL/RPL risk odds ratio: F13A1 rs5985 (OR = 0.24; 0.06-0.90); F13B rs6003 (OR = 0.23; 0.047-1.1); FGA rs6050 (OR = 0.58; 0.33-1.0); CRP rs2808635/rs876538 (OR = 0.15; 0.014-0.81); ABO rs657152 (OR = 0.48; 0.22-1.08); TP53 rs1042522 (OR = 0.54; 0.32-0.92); MTHFR rs1801133/rs1801131 (OR = 2.03; 1.2-3.47) and FGB rs1800790 (OR = 1.97; 1.01-3.87), although Bonferroni correction did not reach significant outputs. Principal Component Analysis (PCA) and logistic regression disclosed further SNPs positive/negative associations (e.g. APOE rs7412/rs429358; FGB rs1800790; CFH rs1061170) differently arranged and sorted in four significant PCs: PC1 (F13A, methylation, CKs); PC3 (CRP, MTHFR, age, methylation); PC4 (F13B, FGA, FGB, APOE, TP53, age, methylation); PC6 (F13A, CFH, ABO, MTHFR, TP53, age), yielding further statistical power to the association models. In detail, positive EPL risk association was with PC1 (OR = 1.81; 1.33-2.45; P < 0.0001) and negative associations with PC3 (OR = 0.489; 0.37-0.66; P < 0.0001); PC4 (OR = 0.72; 0.55-0.94; P = 0.018) and PC6 (OR = 0.61; 0.46-0.81; P = 0.001). Moreover, significant inverse associations were detected between methylation and CKs levels in the whole group (rIL10 = - 0.22; rIL17A = - 0.25; rIL23 = - 0.19; rIL6 = - 0.22), and methylation with age in the whole group, EPL and RPL subgroups (r2TOT = 0.147; r2EPL = 0.136; r2 RPL = 0.248), while VPI controls lost significance (r2VPI = 0.011). This study provides a valuable multilayer approach for investigating epigenetic abnormalities in pregnancy loss suggesting genetic-driven dysregulations and anomalous epigenetic mechanisms potentially mediated by LINE-1 hypomethylation. Women with unexplained EPL might benefit of such investigations, providing new insights for predicting the pregnancy outcome and for treating at risk women with novel targeted epidrugs.

Evaluation of Combined p57KIP2 Immunohistochemistry and Fluorescent in situ Hybridization Analysis for Hydatidiform Moles Compared with Genotyping Diagnosis.

Immunostaining with p57KIP2 is a widely used diagnostic technique to differentiate complete hydatidiform moles (CHMs) from partial hydatidiform moles (PHM) and non-molar hydropic abortion. However, distinguishing between PHMs and non-molar hydropic abortions using histopathology alone is often challenging. This study aimed to evaluate the technical validity and additional benefits of using fluorescence in situ hybridization (FISH) in combination with p57KIP2 immunostaining to diagnose molar and non-molar conceptuses. The study involved 80 specimens, which underwent genetic diagnosis using short tandem repeat analysis, including 44 androgenetic CHMs, 20 diandric monogynic PHMs, 14 biparental non-molar hydropic abortions, 1 monoandric digynic triploid abortion, and 1 vaginal specimen of gestational trophoblastic neoplasia. Two pathologists independently diagnosed the cases based on morphology and p57KIP2 immunostaining while the clinical information was masked. FISH analysis was performed using 3 probes (CEP17, CEPX, and CEPY), which revealed that all androgenetic CHM and biparental diploid non-molar hydropic abortion specimens were diploid. Among the 20 diandric monogynic PHM cases examined by analyzing short tandem repeat polymorphisms, 18 were triploid, and the remaining 2 were diploid. These two specimens were possibly androgenetic/biparental mosaics based on FISH analysis, where the three-signal ratios counting 50 cells were clearly within the diploid ranges. Eight of the 20 genetic PHMs and 2 of the 14 genetically confirmed non-molar hydropic abortions that were falsely diagnosed based on morphology and immunohistochemistry by at least 1 pathologist were correctly diagnosed as PHM and non-molar hydropic abortion, respectively, by FISH analysis. However, 1 monoandric digynic villus was classified as triploid by FISH analysis, leading to a false PHM diagnosis. In conclusion, the combination of FISH analysis with p57KIP2 immunostaining helps in diagnosing molar and non-molar conceptuses in numerous cases; nevertheless, exceptional cases should be considered.

Trophoblast-derived miR-410-5p induces M2 macrophage polarization and mediates immunotolerance at the fetal-maternal interface by targeting the STAT1 signaling pathway.

BACKGROUND: Macrophages phenotypic deviation and immune imbalance play vital roles in pregnancy-associated diseases such as spontaneous miscarriage. Trophoblasts regulate phenotypic changes in macrophages, however, their underlying mechanism during pregnancy remains unclear. Therefore, this study aimed to elucidate the potential function of trophoblast-derived miRNAs (miR-410-5p) in macrophage polarization during pregnancy. METHODS: Patient decidual macrophage tissue samples in spontaneous abortion group and normal pregnancy group (those who had induced abortion for non-medical reasons) were collected at the Reproductive Medicine Center of Renmin Hospital of Wuhan University from April to December 2021. Furthermore, placental villi and decidua tissue samples were collected from patients who had experienced a spontaneous miscarriage and normal pregnant women for validation and subsequent experiments at the Shenzhen Zhongshan Obstetrics & Gynecology Hospital (formerly Shenzhen Zhongshan Urology Hospital), from March 2021 to September 2022. As an animal model, 36 female mice were randomly divided into six groups as follows: naive-control, lipopolysaccharide-model, agomir-negative control prevention, agomir-410-5p prevention, agomir-negative control treatment, and agomir-410-5p treatment groups. We analyzed the miR-410-5p expression in abortion tissue and plasma samples; and supplemented miR-410-5p to evaluate embryonic absorption in vivo. The main source of miR-410-5p at the maternal-fetal interface was analyzed, and the possible target gene, signal transducer and activator of transcription (STAT) 1, of miR-410-5p was predicted. The effect of miR-410-5p and STAT1 regulation on macrophage phenotype, oxidative metabolism, and mitochondrial membrane potential was analyzed in vitro. RESULTS: MiR-410-5p levels were lower in the spontaneous abortion group compared with the normal pregnancy group, and plasma miR-410-5p levels could predict pregnancy and spontaneous abortion. Prophylactic supplementation of miR-410-5p in pregnant mice reduced lipopolysaccharide-mediated embryonic absorption and downregulated the decidual macrophage pro-inflammatory phenotype. MiR-410-5p were mainly distributed in villi, and trophoblasts secreted exosomes-miR-410-5p at the maternal-fetal interface. After macrophages captured exosomes, the cells shifted to the tolerance phenotype. STAT1 was a potential target gene of miR-410-5p. MiR-410-5p bound to STAT1 mRNA, and inhibited the expression of STAT1 protein. STAT1 can drive macrophages to mature to a pro-inflammatory phenotype. MiR-410-5p competitive silencing of STAT1 can avoid macrophage immune disorders. CONCLUSION: MiR-410-5p promotes M2 macrophage polarization by inhibiting STAT1, thus ensuring a healthy pregnancy. These findings are of great significance for diagnosing and preventing spontaneous miscarriage, providing a new perspective for further research in this field.

Defective Homologous Recombination Repair By Up-Regulating Lnc-HZ10/Ahr Loop in Human Trophoblast Cells Induced Miscarriage.

Human trophoblast cells are crucial for healthy pregnancy. However, whether the defective homologous recombination (HR) repair of dsDNA break (DSB) in trophoblast cells may induce miscarriage is completely unknown. Moreover, the abundance of BRCA1 (a crucial protein for HR repair), its recruitment to DSB foci, and its epigenetic regulatory mechanisms, are also fully unexplored. In this work, it is identified that a novel lnc-HZ10, which is highly experssed in villous tissues of recurrent miscarriage (RM) vs their healthy control group, suppresses HR repair of DSB in trophoblast cell. Lnc-HZ10 and AhR (aryl hydrocarbon receptor) form a positive feedback loop. AhR acts as a transcription factor to promote lnc-HZ10 transcription. Meanwhile, lnc-HZ10 also increases AhR levels by suppressing its CUL4B-mediated ubiquitination degradation. Subsequently, AhR suppresses BRCA1 transcription; and lnc-HZ10 (mainly 1-447 nt) interacts with γ-H2AX; and thus, impairs its interactions with BRCA1. BPDE exposure may trigger this loop to suppress HR repair in trophoblast cells, possibly inducing miscarriage. Knockdown of murine Ahr efficiently recovers HR repair in placental tissues and alleviates miscarriage in a mouse miscarriage model. Therefore, it is suggested that AhR/lnc-HZ10/BRCA1 axis may be a promising target for alleviation of unexplained miscarriage.

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.

FTO protein regulates the TGF-ß 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-ß1 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-ß1 gene promoter, enabling the localisation of EZH2 protein to the TGF-ß1 gene promoter and subsequent inhibition of TGF-ß1 gene expression. In summary, our findings elucidate the mechanism by which FTO protein regulates the MEG3-TGF-ß signalling pathway, thereby suppressing trophoblast invasion and proliferation in URSA trophoblast cells. These findings provide new insights for the treatment of URSA.

Genetic analysis of chorionic villus tissues in early missed abortions.

Chromosomal abnormalities are the most common etiology of early spontaneous miscarriage. However, traditional karyotyping of chorionic villus samples (CVSs) is limited by cell culture and its low resolution. The objective of our study was to investigate the efficiency of molecular karyotyping technology for genetic diagnosis of early missed abortion tissues. Chromosome analysis of 1191 abortion CVSs in early pregnancy was conducted from August 2016 to June 2021; 463 cases were conducted via copy-number variations sequencing (CNV-seq)/quantitative fluorescent-polymerase chain reaction (QF-PCR) and 728 cases were conducted using SNP array. Clinically significant CNVs of CVSs were identified to clarify the cause of miscarriage and to guide the couples' subsequent pregnancies. Among these, 31 cases with significant maternal cell contamination were removed from the study. Among the remaining 1160 samples, 751 cases (64.7%) with genetic abnormalities were identified, of which, 531 (45.8%) were single aneuploidies, 31 (2.7%) were multiple aneuploidies, 50 (4.3%) were polyploidies, 54 (4.7%) were partial aneuploidies, 77 (6.6%) had submicroscopic CNVs (including 25 with clinically significant CNVs and 52 had variants of uncertain significance), and 8 cases (0.7%) were uniparental disomies. Our study suggests that both SNP array and CNV-seq/QF-PCR are reliable, robust, and high-resolution technologies for genetic diagnosis of miscarriage.

Predicting risk of blastocyst aneuploidy among women with previous aneuploid pregnancy loss: a multicenter-data-based multivariable model.

STUDY QUESTION: Can blastocyst aneuploidy be predicted for patients with previous aneuploid pregnancy loss (PAPL) and receiving preimplantation genetic testing for aneuploidy (PGT-A)? SUMMARY ANSWER: Multivariable logistic regression models were established to predict high risk of blastocyst aneuploidy using four identified factors, presenting good predictive performance. WHAT IS KNOWN ALREADY: Aneuploidy is the most common embryonic chromosomal abnormality leading to pregnancy loss. Several studies have demonstrated a higher embryo aneuploidy rate in patients with PAPL, which has suggested that PGT-A should have benefits in PAPL patients intending to improve their pregnancy outcomes. However, recent studies have failed to demonstrate the efficacy of PGT-A for PAPL patients. One possible way to improve the efficacy is to predict the risk of blastocyst aneuploidy risk in order to identify the specific PAPL population who may benefit from PGT-A. STUDY DESIGN, SIZE, DURATION: We conducted a multicenter retrospective cohort study based on data analysis of 1119 patients receiving PGT-A in three reproductive medical centers of university affiliated teaching hospitals during January 2014 to June 2020. A cohort of 550 patients who had one to three PAPL(s) were included in the PAPL group. In addition, 569 patients with monogenic diseases without pregnancy loss were taken as the non-PAPL group. PARTICIPANTS/MATERIALS, SETTING, METHODS: PGT-A was conducted using single nucleotide polymorphism microarrays and next-generation sequencing. Aneuploidy rates in Day 5 blastocysts of each patient were calculated and high-risk aneuploidy was defined as a rate of ≥50%. Candidate risk factors for high-risk aneuploidy were selected using the Akaike information criterion and were subsequently included in multivariable logistic regression models. Overall predictive accuracy was assessed using the confusion matrix, discrimination by area under the receiver operating characteristic curve (AUC), and calibration by plotting the predicted probabilities versus the observed probabilities. Statistical significance was set at P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: Blastocyst aneuploidy rates were 30 ± 25% and 21 ± 19% for PAPL and non-PAPL groups, respectively. Maternal age (odds ratio (OR) = 1.31, 95% CI 1.24-1.39, P < 0.001), number of PAPLs (OR = 1.40, 95% CI 1.05-1.86, P = 0.02), estradiol level on the ovulation trigger day (OR = 0.47, 95% CI 0.30-0.73, P < 0.001), and blastocyst formation rate (OR = 0.13, 95% CI 0.03-0.50, P = 0.003) were associated with high-risk of blastocyst aneuploidy. The predictive model based on the above four variables yielded AUCs of 0.80 using the training dataset and 0.83 using the test dataset, with average and maximal discrepancies of 2.89% and 12.76% for the training dataset, and 0.98% and 5.49% for the test dataset, respectively. LIMITATIONS, REASONS FOR CAUTION: Our conclusions might not be compatible with those having fewer than four biopsied blastocysts and diminished ovarian reserves, since all of the included patients had four or more biopsied blastocysts and had exhibited good ovarian reserves. WIDER IMPLICATIONS OF THE FINDINGS: The developed predictive model is critical for counseling PAPL patients before PGT-A by considering maternal age, number of PAPLs, estradiol levels on the ovulation trigger day, and the blastocyst formation rate. This prediction model achieves good risk stratification and so may be useful for identifying PAPL patients who may have higher risk of blastocyst aneuploidy and can therefore acquire better pregnancy outcomes by PGT-A. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China under Grant (81871159). No competing interest existed in the study. TRIAL REGISTRATION NUMBER: N/A.

FKBP5 regulates trophoblast-macrophage crosstalk in recurrent spontaneous abortion through PI3K/AKT and NF-κB signaling pathways.

FK506-binding protein 5 (FKBP5) contributes to many diseases; However, it remains unclear whether FKBP5 is relevant to recurrent spontaneous abortion (RSA) and the mechanisms by which it is involved in maternal-fetal immunological tolerance. Placental tissue was collected in women with normal pregnancy and RSA and examined for FKBP5 expression. Human trophoblast cell lines and THP-1-derived M0 macrophages were used to explore the role of FKBP5 in RSA and its mechanism. The role of FKBP5 on pregnancy outcomes was assessed using a mouse model of miscarriage. This study found that upregulation of FKBP5 at the placental interface is involved in the pathogenesis of RSA by depressing trophoblast function and promoting M1-type macrophage polarization. First, FKBP5 expression was upregulated in the villi of RSA, and FKBP5 regulated trophoblast function by inhibiting HAPLN1 expression through suppression of PI3K/AKT signaling. In addition, FKBP5 inhibited trophoblast IL-6 secretion by suppressing PI3K/AKT signaling, thereby promoting macrophage polarization toward the M1 phenotype. Meanwhile, FKBP5 was significantly elevated in decidual macrophages from patients with RSA and promoted M1 macrophage polarization via ROS/NF-κB signaling and further inhibited trophoblast function. Finally, FKBP5 inhibitors improved embryo resorption rate in miscarried mice. In conclusion, FKBP5 is essential in maintaining pregnancy and trophoblast-macrophage crosstalk in the maternal-fetal interface, which may be a potential target for diagnosing and treating RSA.

Double Trisomy 16 and 22 Clinically Mimic Partial Hydatidiform Mole in a Case of Subsequent Pregnancy Loss.

A case of double trisomy 16 and 22 in the second pregnancy loss is presented. DNA analyses (short tandem repeats genotyping) of miscarriage specimen was indicated because of ultrasound suspicion of partial hydatidiform mole. After the partial hydatidiform mole exclusion, further DNA analyses focused on the most common aneuploidies causing pregnancy loss, detected double trisomy 16 and 22 in the product of conception. The couple was referred to clinical genetic consultation and normal parental karyotypes were proved. For further explanatory purposes, archived material from the first pregnancy loss was analyzed and trisomy of chromosome 18 was detected. By comparison of allelic profiles of the mother, father, and both losses, the maternal origin of all aneuploidies was proven what can be attributed to frequent meiosis errors, probably due to advanced maternal age (44 years at the first loss and 45 years at the second loss). In conclusion, aneuploidies can mimic partial hydatidiform mole. Genetic analysis is helpful on the one hand to rule out partial hydatidiform mole and on the other hand to identify aneuploidies and in this way to determine the cause of miscarriage.

PGT-A: Houston, we have a problem.

Preimplantation genetic testing for aneuploidy (PGT-A) is a common add-on to IVF cycles. As it is presently performed, PGT-A relies on whole genome amplification of small amounts of DNA from cells removed from the trophectoderm (TE) of a blastocyst for determination of gain or loss of chromosomal material by next-generation sequencing. Whole genome amplification may introduce artifacts such as allele dropout and loss of heterozygosity in up to 25% of cases. In addition, the high prevalence of mosaicism in human embryos is a complicating factor in interpreting the results of PGT-A screening. In the presence of mosaicism, biopsy of TE cells cannot provide accurate results regarding the chromosomal make-up of the inner cell mass. The available clinical data suggest that PGT-A is probably harmful when IVF outcomes are analyzed by intention to treat or by live birth rate per cycle started rather than per embryo transfer, especially in women with three or fewer blastocysts. In addition, hypothesized advantages of reduced spontaneous abortion rate and reduced time to conception may be modest at best.

Associations between obesity, smoking behaviors, reproductive traits and spontaneous abortion: a univariable and multivariable Mendelian randomization study.

Background: The correlation between potential risk factors such as obesity (leg fat percentage (left), arm fat percentage (left), waist circumference, body fat percentage, trunk fat percentage), smoking behaviors (past tobacco smoking, smoking initiation, smoking/smokers in household, current tobacco smoking) and reproductive traits (age first had sexual intercourse (AFS), age at menarche (AAM), and age at first birth (AFB)) have been linked to the occurrence of spontaneous abortion (SA). However, the causal associations between these factors and SA remain unclear. Methods: We conducted univariable and multivariable Mendelian randomization (MR) analyses to evaluate the associations of obesity, smoking behavior and reproductive traits with SA. To select appropriate genetic instruments, we considered those that had reached the genome-wide significance level (P < 5 × 10-8) in their corresponding genome-wide association studies (GWAS) involving a large number of individuals (ranging from 29,346 to 1,232,091). SA was obtained from the FinnGen consortium, which provided summary-level data for 15,073 SA cases and 135,962 non-cases. Results: Assessed individually using MR, the odds ratios (ORs) of SA were 0.728 (P = 4.3608×10-8), 1.063 (P = 0.0321), 0.926 (P = 9.4205×10-4), 1.141 (P = 7.9882×10-3), 5.154 (P = 0.0420), 1.220 (P = 0.0350), 1.228 (P = 0.0117), 0.795 (P = 0.0056), 1.126 (P = 0.0318), for one standard deviation (SD) increase in AFS, AAM, AFB, smoking initiation, smoking/smokers in household, arm fat percentage (left), leg fat percentage (left), waist circumference and body fat percentage, 0.925 (P = 0.4158) and 1.075 (P = 0.1479) for one SD increase in past tobacco smoking, trunk fat percentage for one SD increase in SA. In multivariable MR (MVMR), only AFS (OR = 0.802; P = 0.0250), smoking initiation (OR = 1.472, P = 0.0258), waist circumference (OR = 0.813, P = 0.0220) and leg fat percentage (left) (OR = 4.446, P = 0.043) retained a robust effect. Conclusion: Smoking behaviors, reproductive traits and obesity-related anthropometric indicators are potential causal factors for SA. Higher leg fat percentage; smoking initiation; and lower waist circumference and AFS may increase the risk of SA. Understanding the causal relationship for SA may provide more information for SA intervention and prevention strategies.