Chromosome analysis of 303 pregnancy losses in Mexico.

BACKGROUND: Chromosomal abnormalities are present in 50 to 60% of miscarriages and in 6 to 19% of stillbirths. Although microarrays are preferred for studying chromosomal abnormalities, many hospitals cannot offer this methodology. OBJECTIVE: To present the results of the cytogenetic analysis of 303 products of conception (POC), which included 184 miscarriages, 49 stillbirths and 17 cases of undefined age. MATERIAL AND METHODS: Karyotyping, fluorescence in situ hybridization, short tandem repeats and microarrays were used, depending on the type of loss and available sample. RESULTS: In 29 POCs we found maternal tissue and were eliminated from the analyses. Informative results were obtained in 250 (91.2 %)/274 cases; the karyotyping success rate was 80.7%; that of single nucleotide polymorphism microarrays, 94.5%; and that of fluorescence in situ hybridization and short tandem repeat, 100%. Cytogenetic abnormalities were observed in 57.6% of miscarriages and in 24.5% of stillbirths; 94% of total anomalies were numerical and 6% were submicroscopic. CONCLUSIONS: Karyotyping with simultaneous short tandem repeat study to rule out contamination of maternal cells is effective for studying miscarriages; in stillbirths, microarrays are recommended.

ANTECEDENTES: Las alteraciones cromosómicas están presentes en 50 a 60 % de los abortos espontáneos y en 6 a 19 % de los mortinatos. Aunque se prefieren los microarreglos para estudiarlos, numerosos hospitales no pueden ofrecerlos. OBJETIVO: Presentar los resultados del estudio citogenético de 303 productos de la concepción (POC), 184 se obtuvieron de abortos espontáneos, 49 fueron mortinatos y en 17 no se identificó la de edad gestacional. MATERIAL Y MÉTODOS: Se empleó cariotipo, hibridación in situ con fluorescencia, secuencias cortas repetidas en tándem y microarreglos, según el tipo de pérdida y la muestra disponible. RESULTADOS: En 29 POC se encontró tejido materno, por lo que fueron eliminados de los análisis. En 250 (91.2 %)/274 casos se obtuvieron resultados informativos; la tasa de éxito del cariotipo fue de 80.7 %; la de los microarreglos de SNP, de 94.5 %; y la de la hibridación fluorescente in situ y la repetición corta en tándem, de 100 %. Se observaron anomalías citogenéticas en 57.6 % de los abortos espontáneos y en 24.5 % de los mortinatos; 94 % de las anomalías fueron numéricas y 6 %, submicroscópicas. CONCLUSIONES: El cariotipo en conjunto con el estudio de secuencias cortas repetidas en tándem para descartar contaminación de células maternas es efectivo para estudiar abortos espontáneos; los microarreglos se recomiendan en los mortinatos.

Cytogenetic abnormalities associated with reproductive failure in Pakistani population: Experience of a tertiary care hospital.

Constitutional chromosomal abnormalities play a significant role in causing reproductive anomalies in individuals of reproductive age. With the rapid advancement of genome engineering techniques, it has now become possible to cure different genetic disorders. However, very limited data is available regarding the prevalence of such aberrations in the Pakistani population. Considering this factor, this retrospective analysis was undertaken to elucidate the type and prevalence rate of such abnormalities in our population. A total of 241 individuals, who were referred to the Liaquat National Hospital, from January 2017 to December 2021, with a history of infertility or miscarriages, were evaluated using the standard GTG banding technique. The results revealed a notably high percentage 44(18.2%) of chromosomal abnormalities in our population. Surprisingly, the frequency of these anomalies was observed to be higher in males than in females. However, further research is needed using a larger sample size to confirm the findings of this investigation.

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.

Increased levels of villus-derived exosomal miR-29a-3p in normal pregnancy than uRPL patients suppresses decidual NK cell production of interferon-γ and exerts a therapeutic effect in abortion-prone mice.

OBJECTIVE: Recurrent pregnancy loss (RPL) patients have higher absolute numbers of decidual natural killer (dNK) cells with elevated intracellular IFN-γ levels leading to a pro-inflammatory cytokine milieu, which contributes to RPL pathogenesis. The main objective of this study was twofold: first to explore the regulatory effects and mechanisms of villus-derived exosomes (vEXOs) from induced abortion patients or RPL patients at the level of intracellular IFN-γ in dNK cells; second to determine the validity of application of vEXOs in the treatment of unexplained RPL (uRPL) through in vitro experiments and mouse models. METHODS: Exosomes were isolated from villus explants by ultracentrifugation, co-cultured with dNK cells, and purified by enzymatic digestion and magnetically activated cell sorting. Flow cytometry, enzyme-linked immunosorbent assays, and RT-qPCR were used to determine IFN-γ levels. Comparative miRNA analysis of vEXOs from induced abortion (IA) and uRPL patients was used to screen potential candidates involved in dNK regulation, which was further confirmed by luciferase reporter assays. IA-vEXOs were electroporated with therapeutic miRNAs and encapsulated in a China Food and Drug Administration (CFDA)-approved hyaluronate gel (HA-Gel), which has been used as a clinical biomaterial in cell therapy for > 30 years. In vivo tracking was performed using 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) labelling. Tail-vein and uterine horn injections were used to evaluate therapeutic effects of the engineered exosomes in an abortion-prone mouse model (CBA/J × DBA/2 J). Placental growth was evaluated based on placental weight. IFN-γ mRNA levels in mouse placentas were measured by RT-qPCR. RESULTS: IFN-γ levels were significantly higher in dNK cells of uRPL patients than in IA patients. Both uRPL-vEXOs and IA-vEXOs could be efficiently internalized by dNK cells, whereas uRPL-vEXOs could not reduce the expression of IFN-γ by dNK cells as much as IA-vEXOs. Mechanistically, miR-29a-3p was delivered by vEXOs to inhibit IFN-γ production by binding to the 3' UTR of IFN-γ mRNA in dNK cells. For in vivo treatment, application of the HA-Gel effectively prolonged the residence time of vEXOs in the uterine cavity via sustained release. Engineered vEXOs loaded with miR-29a-3p reduced the embryo resorption rate in RPL mice with no signs of systemic toxicity. CONCLUSION: Our study provides the first evidence that villi can regulate dNK cell production of IFN-γ via exosome-mediated transfer of miR-29a-3p, which deepens our understanding of maternal-fetal immune tolerance for pregnancy maintenance. Based on this, we developed a new strategy to mix engineered vEXOs with HA-Gel, which exhibited good therapeutic effects in mice with uRPL and could be used for potential clinical applications in uRPL treatment.

Risk Factors of Thrombophilia-Related Mutations for Early and Late Pregnancy Loss.

Background and Objectives: This retrospective cohort study investigates the role of genetic thrombophilia in pregnant women experiencing early pregnancy loss compared to those with late pregnancy loss. Materials and Methods: Participants were categorized into early and late pregnancy loss groups based on gestational age. A total of 156 patients were included, out of which 103 had early-trimester pregnancy losses and 96 had multiple miscarriages. Results: The study revealed a synergistic effect of Factor V Leiden (FVL G1691A) and Methylenetetrahydrofolate Reductase (MTHFR C677T) mutations (coefficient 3.42). Prothrombin (PT) G20210A and ß-Fibrinogen 455 G>A mutations exhibited a significant interaction (coefficient 1.98). Additionally, MTHFR A1298C and Plasminogen Activator Inhibitor-1 (PAI-1 4G/5G) mutations showed a significant interaction (coefficient 1.65). FVL G1691A and Endothelial Protein C Receptor (EPCR) allele A1/A2 mutations also demonstrated a significant association (coefficient 2.10). Lastly, MTHFR C677T and Glycoprotein IIb/IIIa T1565C mutations interacted significantly (coefficient 1.77). Risk factor analysis identified several mutations associated with early pregnancy loss, including PAI-1 4G/5G homozygous (OR 3.01), FVL G1691A heterozygous (OR 1.85), and MTHFR A1298C heterozygous (OR 1.55). Both homozygous and heterozygous MTHFR C677T mutations were significant risk factors (OR 2.38; OR 2.06), as was PT G20210A homozygous mutation (OR 1.92). The PAI-1 4G/4G homozygous variant posed a risk (OR 1.36). Late pregnancy loss was associated with MTHFR A1298C homozygous mutation (OR 3.79), ß-Fibrinogen 455 G>A heterozygous mutation (OR 2.20), and MTHFR A1298C heterozygous mutation (OR 2.65). Factor XIII G1002T heterozygous mutation (OR 1.18) and PAI-1 4G/5G homozygous mutation (OR 2.85) were also significant risk factors. EPCR allele A1/A2 (OR 1.60) and A2/A3 (OR 1.73) mutations were identified as significant risk factors for late pregnancy loss. Furthermore, FVL G1691A homozygous mutation, PT G20210A homozygous mutation, MTHFR C677T heterozygous mutation, MTHFR A1298C heterozygous mutation, and EPCR allele A1/A2 were identified as significant risk factors for multiple miscarriage. Conclusions: This study highlights significant interactions and risk factors related to genetic thrombophilia mutations in different types of pregnancy loss, contributing valuable insights for miscarriage management guidelines.

Elevated sperm DNA fragmentation is correlated with an increased chromosomal aneuploidy rate of miscarried conceptus in women of advanced age undergoing fresh embryo transfer cycle.

Background: Male sperm DNA fragmentation (SDF) may be associated with assisted reproductive technology (ART) outcomes, but the impact of SDF on the occurrence of aneuploid-related miscarriage remains controversial. Methods: Genome-wide single-nucleotide polymorphism-based chromosomal microarray analysis was performed on 495 miscarried chorionic villus samples undergone IVF/ICSI treatment from the Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University. SDF was assessed using sperm chromatin structure assay. Patients were divided into four groups according to embryo transfer cycle type and maternal age, and the correlation between SDF and chromosome aberration was analyzed. A receiver operating characteristic (ROC) curve was utilized to find the optimal threshold. Results: Total chromosomal aneuploidy rate was 54.95%, and trisomy was the most common abnormality (71.32%). The chromosomally abnormal group had higher SDF than the normal group (11.42% [6.82%, 16.54%] vs. 12.95% [9.61%, 20.58%], P = 0.032). After grouping, elevated SDF was significantly correlated with an increasing chromosome aneuploidy rate only in women of advanced age who underwent fresh embryo transfer (adjusted odds ratio:1.14 [1.00-1.29], adjusted-P = 0.045). The receiver operating characteristic curve showed that SDF can predict the occurrence of chromosomal abnormality of miscarried conceptus in this group ((area under the curve = 0.76 [0.60-0.91], P = 0.005), and 8.5% was the optimum threshold. When SDF was ≥ 8.5%, the risk of such patients increased by 5.76 times (adjusted odds ratio: 6.76 [1.20-37.99], adjusted-P = 0.030). Conclusion: For women of advanced maternal age undergoing fresh embryo transfer, older oocytes fertilized using sperm with high SDF in IVF/ICSI treatment might increase the risk of chromosomal abnormality in miscarried conceptus.

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.

HLA-G isoforms, HLA-C allotype and their expressions differ between early abortus and placenta in relation to spontaneous abortions.

INTRODUCTION: Spontaneous abortion (SAB) affects approximately 10% of clinically recognized pregnancies. Fetal trophobalst invasion and remodeling of maternal spiral arteries is reported to be dependent on crosstalk between HLA-C/HLA-G expressed on extra villous trophoblast (EVTs)and Killer cell Immunoglobin like receptors (KIRs) of decidual NK (dNK). Immune dysfunction in decidua contributes to early miscarriage. METHODOLOGY: The study used mother neonate paired cord blood and term placenta samples (n = 46), elective abortus (n = 17,gestational age = 10-12 weeks of pregnancy) and SAB abortus (n = 24, gestational age = 12-15 weeks of pregnancy) for HLA-G, KIR2D and HLA-C. In addition, term placenta was collected from women with history of spontaneous pregnancy loss (n = 24) and women with history of live birth (n = 32). SSP-PCR was used for genotyping, RT-PCR for gene expression, copy number variation (CNVs) and HLA-C allotyping and ELISA for protein expression studies. RESULTS: Membrane bound HLA-G4 isoform proportion was higher 39.28%, p = 0.02) in term placenta. SAB abortus had higher proportion of HLA-G3 (50%),while elective abortus exhibited higher proportion of soluble isoforms (HLA-G5, = 5.9, HLA-G6 = 5.9%, HLA-G7 = 11.8%). Higher inhibitory KIR2DL1 content and copy numbers with lower HLA-C2 in SAB contrasted with higher copy numbers of KIR2DS1(p = 0.001), KIR2DS1+/2DL1+- HLA-C2 combined genotype in healthy placenta. Elevated KIR2D protein levels (p = 0.001), and concurrently, HLA-C levels were upregulated in healthy placenta. CONCLUSION: Our data supports lower cognate receptor ligand KIR2DS1+/2DL1+ HLA-C2 together with predominance of HLA-G3 isoform in SAB as confounding factors in spontaneous pregnancy loss. HLA-G isoforms and expression differed between first trimester abortus and term placenta suggesting temporal modulation and marks novelty of the study.

The interrelation between the high expression level of MIR34a and the trisomic abortion materials.

AIMS: The underlying mechanism and constitution of spontaneous abortions are complicated and heterogeneous. Many factors, including epigenetic scenarios like micro-ribonucleic acids (miRNAs, MIRs), can additively affect the progression of pregnancy losses. This study aimed to evaluate whether the expression levels of placental inhibitor and/or activator miRNAs had a difference between the numerically abnormal and normal karyotyped spontaneous abortions. METHODS: The case-control study included 100 spontaneous abortion materials consisting of trophoblastic tissues with 42 disomies (controls), 43 aneuploidies (including trisomy 16, 21, 22, and monosomy X), and 15 triploidies. Disomic abortion materials with XX normal karyotypes were omitted from the study to exclude possible maternal decidual cell contamination. Total RNA isolation was performed with TRIzol™ reagent directly from frozen trophoblastic tissues, and the mature miRNAs were obtained by reverse transcription via quantitative real-time polymerase chain reaction (qRT-PCR). Then, the expression levels of placental activators MIR378a-5p, MIR376c, MIR195, and inhibitors MIR34a and MIR210 were relatively evaluated using MIR130 as a reference. RESULTS: The expression level of placental inhibitor MIR34a was detected to be high in trisomic abortion materials (trisomy 16 and 21) when compared to the disomic ones (p = 0.0324). MIR195 (p = 0.0484) and MIR34a (p = 0.0346) expression levels were increased in numerically abnormal cases with advanced maternal age compared to the disomic ones within all maternal ages. CONCLUSIONS: It seems likely that the high expression level of MIR34a and the coexistence of trisomic abortion materials are quite interrelated with the additive effect of advanced maternal age.

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

BACKGROUND: The 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. METHODS: Genome-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. RESULTS: Based 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. CONCLUSIONS: Anxiety 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.

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.

Miscarriage risk assessment: a bioinformatic approach to identifying candidate lethal genes and variants.

PURPOSE: Miscarriage, often resulting from a variety of genetic factors, is a common pregnancy outcome. Preconception genetic carrier screening (PGCS) identifies at-risk partners for newborn genetic disorders; however, PGCS panels currently lack miscarriage-related genes. In this study, we evaluated the potential impact of both known and candidate genes on prenatal lethality and the effectiveness of PGCS in diverse populations. METHODS: We analyzed 125,748 human exome sequences and mouse and human gene function databases. Our goals were to identify genes crucial for human fetal survival (lethal genes), to find variants not present in a homozygous state in healthy humans, and to estimate carrier rates of known and candidate lethal genes in various populations and ethnic groups. RESULTS: This study identified 138 genes in which heterozygous lethal variants are present in the general population with a frequency of 0.5% or greater. Screening for these 138 genes could identify 4.6% (in the Finnish population) to 39.8% (in the East Asian population) of couples at risk of miscarriage. This explains the cause of pregnancy loss in approximately 1.1-10% of cases affected by biallelic lethal variants. CONCLUSION: This study has identified a set of genes and variants potentially associated with lethality across different ethnic backgrounds. The variation of these genes across ethnic groups underscores the need for a comprehensive, pan-ethnic PGCS panel that includes genes related to miscarriage.

MiR-142-3p Regulates ILC1s by Targeting HMGB1 via the NF-κB Pathway in a Mouse Model of Early Pregnancy Loss.

OBJECTIVE: Innate lymphoid cells (ILCs) are a class of newly discovered immunocytes. Group 1 ILCs (ILC1s) are identified in the decidua of humans and mice. High mobility group box 1 (HMGB1) is predicted to be one of the target genes of miR-142-3p, which is closely related to pregnancy-related diseases. Furthermore, miR-142-3p and HMGB1 are involved in regulating the NF-κB signaling pathway. This study aimed to examine the regulatory effect of miR-142-3p on ILC1s and the underlying mechanism involving HMGB1 and the NF-κB signaling pathway. METHODS: Mouse models of normal pregnancy and abortion were constructed, and the alterations of ILC1s, miR-142-3p, ILC1 transcription factor (T-bet), and pro-inflammatory cytokines of ILC1s (TNF-α, IFN-γ and IL-2) were detected in mice from different groups. The targeting regulation of HMGB1 by miR-142-3p in ILC1s, and the expression of HMGB1 in normal pregnant mice and abortive mice were investigated. In addition, the regulatory effects of miR-142-3p and HMGB1 on ILC1s were detected in vitro by CCK-8, Annexin-V/PI, ELISA, and RT-PCR, respectively. Furthermore, changes of the NF-κB signaling pathway in ILC1s were examined in the different groups. For the in vivo studies, miR-142-3p-Agomir was injected in the uterus of abortive mice to evaluate the abortion rate and alterations of ILC1s at the maternal-fetal interface, and further detect the expression of HMGB1, pro-inflammatory cytokines, and the NF-κB signaling pathway. RESULTS: The number of ILC1s was significantly increased, the level of HMGB1 was significantly upregulated, and that of miR-142-3p was considerably downregulated in the abortive mice as compared with the normal pregnant mice (all P<0.05). In addition, miR-142-3p was found to drastically inhibit the activation of the NF-κB signaling pathway (P<0.05). The number of ILC1s and the levels of pro-inflammatory cytokines were significantly downregulated and the activation of the NF-κB signaling pathway was inhibited in the miR-142-3p Agomir group (all P<0.05). CONCLUSION: miR-142-3p can regulate ILC1s by targeting HMGB1 via the NF-κB signaling pathway, and attenuate the inflammation at the maternal-fetal interface in abortive mice.

Anti-proliferation and apoptosis induced via the mTOR/PGC-1α signaling pathway in trophoblast cells of miscarriage.

Miscarriage is a common complication during early pregnancy and affects approximately 10%-15% of all pregnant women. Several studies have reported that the abnormal expression of mitochondrial oxidative stress-related genes might be involved in the occurrence and progression of miscarriage. The present study attempted to uncover the role of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in miscarriage chorionic villous tissue. The hypothesis that PGC-1α is crucial for glycolysis and oxidative phosphorylation during early pregnancy was tested. The results showed that the mRNA and protein levels of PGC-1α were significantly increased in the miscarriage chorionic villous tissues compared with the artificial selective abortion group, and that the expression was regulated by mTOR in knockdown and overexpression of mTOR in HTR8 cell lines. PGC-1α also promoted mitochondrion oxidative phosphorylation but inhibited glycolysis process. In addition, PGC-1α could drive ROS production, reduce mitochondrial membrane potential and block NADPH synthesis, resulting in cell cycle arrest and cell apoptosis, eventually leading to miscarriage. These results suggested that the aberrant expression of PGC-1α is involved in the etiology of early miscarriage, providing new perspectives regarding the mechanisms of miscarriage and a potential therapeutic target for miscarriage.

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.

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).

Experience of copy number variation sequencing applied in spontaneous abortion.

PURPOSE: We evaluated the value of copy number variation sequencing (CNV-seq) and quantitative fluorescence (QF)-PCR for analyzing chromosomal abnormalities (CA) in spontaneous abortion specimens. METHODS: A total of 650 products of conception (POCs) were collected from spontaneous abortion between April 2018 and May 2020. CNV-seq and QF-PCR were performed to determine the characteristics and frequencies of copy number variants (CNVs) with clinical significance. The clinical features of the patients were recorded. RESULTS: Clinically significant chromosomal abnormalities were identified in 355 (54.6%) POCs, of which 217 (33.4%) were autosomal trisomies, 42(6.5%) were chromosomal monosomies and 40 (6.2%) were pathogenic CNVs (pCNVs). Chromosomal trisomy occurs mainly on chromosomes 15, 16, 18, 21and 22. Monosomy X was not associated with the maternal or gestational age. The frequency of chromosomal abnormalities in miscarriages from women with a normal live birth history was 55.3%; it was 54.4% from women without a normal live birth history (P > 0.05). There were no significant differences among women without, with 1, and with ≥ 2 previous miscarriages regarding the rate of chromosomal abnormalities (P > 0.05); CNVs were less frequently detected in women with advanced maternal age than in women aged ≤ 29 and 30-34 years (P < 0.05). CONCLUSION: Chromosomal abnormalities are the most common cause of pregnancy loss, and maternal and gestational ages are strongly associated with fetal autosomal trisomy aberrations. Embryo chromosomal examination is recommended regardless of the gestational age, modes of conception or previous abortion status.

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.

Causal association between rheumatoid arthritis and pregnancy loss and intrauterine growth retardation: A bidirectional two-sample Mendelian randomization study.

OBJECTIVE: To investigate the causal relationship between rheumatoid arthritis (RA) and pregnancy loss and intrauterine growth retardation (IUGR) using Mendelian randomization (MR). METHODS: Genetic variants associated with RA (12,555 cases and 240,862 controls), miscarriage (1475 cases and 149,622 controls), and IUGR (3558 cases and 207,312 controls) were obtained from the FinnGen consortium, and supplementary data on RA (5201 cases and 457,732 controls) and miscarriage (7069 cases and 250,492 controls) were obtained from the Medical Research Council Integrated Epidemiology Unit (MRC-IEU). 47 Single nucleotide polymorphisms (SNPs) associated with RA were screened as instrumental variables (IV). The causal relationship between RA and pregnancy loss and IUGR were assessed by 5 MR methods, mainly inverse variance weighting (IVW). Sensitivity analyses were also performed to test the stability of the results. RESULTS: Bidirectional MR showed that genetically predicted RA was causally associated with pregnancy loss and IUGR in forward MR analyses, and that RA significantly increased pregnancy loss [odds ratio (OR) = 1.13, 95% confidence interval (CI): 1.00-1.33, P = .03] and IUGR (OR = 1.08, 95% CI: 1.01-1.15, P = .019). In the reverse MR, there was no causal association between pregnancy loss (P = .15) and IUGR (P = .87) and RA. CONCLUSION: This study found a significant genetic association between RA and pregnancy loss and IUGR. RA is considered to be a high-risk factor for adverse maternal outcomes. Pre-pregnancy prophylaxis and intra-pregnancy control of patients should be emphasized to reduce the incidence of adverse pregnancy outcomes such as pregnancy loss and IUGR.

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.