Associations of MCM8 rs3761873 and rs16991617 variants with abnormal uterine bleeding induced by copper intrauterine device.

AIM: Intrauterine device (IUD) is a commonly used contraceptive method worldwide. Abnormal uterine bleeding (AUB) is one of the most common side effects of Cu-IUDs. Since AUB varies among Cu-IUD users, changes in the bleeding-related genetic factors may contribute to AUB. This study aimed to determine the genetic risk factors of AUB after Cu-IUD insertion. METHODS: We conducted a case-control study on women who experienced AUB after Cu-IUD insertion (case:control = 62:59). Six candidate variants were genotyped using the Sequenom MassARRAY. Genotype and allele frequencies were analyzed using SHEsisPlus. We performed Pearson's Chi-squared test to analyze categorical data, and ESEfinder to predict the impact on splicing regulation. RESULTS: MCM8 coding sequence variants: rs3761873-A>C was in Exon 7 and rs16991617 A>G was in Exon 12 of all 19 exons, both of which were significantly different between cases and controls (pallele  = 0.039 and pgenotype  = 0.092). rs6022 and rs6029 in F5 gene and rs3761873 and rs16991617 in the MCM8 gene showed strong linkage disequilibrium (R2 > 0.8). ESEfinder indicated that the variants of MCM8 may affect the splicing regulation. CONCLUSIONS: MCM8 rs376187 and rs16991617 were associated with AUB in Cu-IUDs users. MCM8 may play a role in AUB by regulating functions of reproductive organs and primary ovarian insufficiency. Our findings may improve the understanding of the genetic basis of AUB caused by Cu-IUDs.

Epigenetic alternations of microRNAs and DNA methylation contribute to gestational diabetes mellitus.

This study aimed to identify epigenetic alternations of microRNAs and DNA methylation for gestational diabetes mellitus (GDM) diagnosis and treatment using in silico approach. Data of mRNA and miRNA expression microarray (GSE103552 and GSE104297) and DNA methylation data set (GSE106099) were obtained from the GEO database. Differentially expressed genes (DEGs), differentially expressed miRNAs (DEMs) and differentially methylated genes (DMGs) were obtained by limma package. Functional and enrichment analyses were performed with the DAVID database. The protein-protein interaction (PPI) network was constructed by STRING and visualized in Cytoscape. Simultaneously, a connectivity map (CMap) analysis was performed to screen potential therapeutic agents for GDM. In GDM, 184 low miRNA-targeting up-regulated genes and 234 high miRNA-targeting down-regulated genes as well as 364 hypomethylation-high-expressed genes and 541 hypermethylation-low-expressed genes were obtained. They were mainly enriched in terms of axon guidance, purine metabolism, focal adhesion and proteasome, respectively. In addition, 115 genes (67 up-regulated and 48 down-regulated) were regulated by both aberrant alternations of miRNAs and DNA methylation. Ten chemicals were identified as putative therapeutic agents for GDM and four hub genes (IGF1R, ATG7, DICER1 and RANBP2) were found in PPI and may be associated with GDM. Overall, this study identified a series of differentially expressed genes that are associated with epigenetic alternations of miRNA and DNA methylation in GDM. Ten chemicals and four hub genes may be further explored as potential drugs and targets for GDM diagnosis and treatment, respectively.

Epigenome-Wide Association Study Indicates Hypomethylation of MTRNR2L8 in Large-Artery Atherosclerosis Stroke.

Background and Purpose- Ischemic stroke, a complex and heterogeneous disease, is the second leading cause of death worldwide. Genetic factors and epigenetic modification contribute to the pathogenesis of this disease. However, the effects of epigenetic factors on this disease have not been systematically investigated. Our study was designed to identify methylation alterations in large-artery atherosclerotic stroke. Methods- We conducted an epigenome-wide association analysis of large-artery atherosclerotic stroke using an Infinium HumanMethylation450 array (cases:controls=12:12), and the differentially methylated loci were validated in 2 cohorts (cases:controls, 110:122 and 191:191, respectively) using a Sequenom EpiTYPER assay. Results- In the screening stage, 1012 differentially methylated CpG sites annotated in 672 genes were found to be significantly associated with large-artery atherosclerotic stroke (mean methylation difference >5%, P<0.01). Disease, Gene Ontology, and pathway analysis highlighted the enrichment of these differentially methylated genes in cardiovascular, metabolic, neurological and immune-related functional gene clusters ( P<0.05). We identified a differentially methylated region in the promoter of a humanin gene ( MTRNR2L8, mean methylation difference=-13.01%, P=8.86×10-14). We constructed a diagnostic prediction model that was based on the mean number of significantly changed CpG loci in MTRNR2L8 and showed high diagnostic specificity and sensitivity ( P<0.0001, area under the curve=0.774). Conclusions- Together, these findings demonstrate that DNA methylation plays an important role in large-artery atherosclerotic stroke and that methylation of MTRNR2L8 is a potential therapeutic target and diagnostic biomarker for stroke.

Iron overload triggering ECM-mediated Hippo/YAP pathway in follicle development: a hypothetical model endowed with therapeutic implications.

Disruption of iron homeostasis plays a negative role in follicle development. The dynamic changes in follicle growth are dependent on Hippo/YAP signaling and mechanical forces. However, little is known about the liaison between iron overload and the Hippo/YAP signalling pathway in term of folliculogenesis. Here, based on the available evidence, we established a hypothesized model linking excessive iron, extracellular matrix (ECM), transforming growth factor-ß (TGF-ß) and Hippo/Yes-associated protein (YAP) signal regarding follicle development. Hypothetically, the TGF-ß signal and iron overload may play a synergistic role in ECM production via YAP. We speculate that the dynamic homeostasis of follicular iron interacts with YAP, increasing the risk of ovarian reserve loss and may enhance the sensitivity of follicles to accumulated iron. Hence, therapeutic interventions targeting iron metabolism disorders, and Hippo/YAP signal may alter the consequences of the impaired developmental process based on our hypothesis, which provides potential targets and inspiration for further drug discovery and development applied to clinical treatment.

Predictive plasma biomarker for gestational diabetes: A case-control study in China.

OBJECTIVE: This study aims to find new plasma biomarkers in early pregnancy. DESIGN: The original study enrolled 1219 pregnant women. We investigated protein expression profiles of placental tissues from women with GDM (n = 89) and normal glucose tolerance (NGT) (n = 83). Maternal plasma samples between two groups in early and middle pregnancy were used for validation of candidate biomarkers. METHODS: Differentially expressed proteins (DEPs) were identified by label-free quantitative proteomics from human placenta samples between two groups. Several DEPs were validated in plasma by Luminex assays. An automatic biochemical analyzer was used to detect blood lipid indexes. The associations of GAL-3BP with biochemical indicators were demonstrated by Pearson's correlation analysis. Binary logistic regression was used to model potential predictive indicators in early pregnancy of GDM. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic accuracy of the predictive model and the value of GAL-3BP. RESULTS: 123 DEPs were found in placenta involved in ribosomal function, pancreatic secretion, oxidative phosphorylation, and inflammatory signaling pathway. Plasma GAL-3BP are significantly higher in women with GDM than NGT in the first (p = 0.008) and second (p = 0.026) trimester, but C9 and VWF have no difference. The predictive value of GAL-3BP in the first trimester of pregnancy (AUC 0.64) is better than that in the second trimester (AUC 0.61), and combined predictive model of TG and GAL-3BP at early pregnancy has greater predictive and diagnostic value for GDM (AUC 0.69) than individual GAL-3BP (AUC 0.64). CONCLUSIONS: Plasma TG and GAL-3BP has good predictive and diagnostic value at early pregnancy, suggesting that these two indicators may be used as biomarkers for early prediction and diagnosis of GDM. SIGNIFICANCE: The advantage of this study is that circulating TG and GAL-3BP might differentiate the progress of women with GDM and normal glucose tolerance (NGT) at the early stage of pregnancy. It is the first study to consider the role of GAL-3BP as an early predictive biomarker in the development of GDM during the whole pregnancy. Another advantage is that volunteers in this study were recruited from two provinces in China to eliminate the impacts of environmental confounders. The similar changes of blood glucose/lipid indicators for women with GDM and NGT in both regions was found in the first and second trimester of pregnancy, which added to the reliability of analytical results.

Expression profiles of circular RNA in human placental villus and decidua and prediction of drugs for recurrent spontaneous abortion.

PROBLEM: We aimed to evaluate potential biomarkers and candidate drugs for recurrent spontaneous abortion (RSA) and explore functional circular RNA pathways involved in regulating RSA. METHOD OF STUDY: Expression profiles of placental villus and decidua samples derived from females with RSA and those with healthy pregnancies who underwent induced abortion were analyzed using high-throughput RNA whole transcriptome sequencing. Abnormally expressed circular RNAs in a larger cohort of samples were validated using real-time quantitative polymerase chain reaction. Drug discovery and molecular docking were performed using online databases and the Autodock tool, respectively. RESULTS: In total, 2103 and 2160 circular RNAs were detected in three pairs of villi and three pairs of decidual tissues, respectively. A total of 22 circular RNAs, 58 miRNAs, and 393 mRNAs with significantly different expression patterns were identified. Five circular RNAs were verified, and the expression of hsa_circ_0088485 was significantly upregulated in the RSA group (P = .041) with a high area under the curve value (.727), sensitivity (76.5%), and specificity (64.7%). GO and KEGG enrichment analyses indicated that differentially expressed genes were associated with angiogenesis and cell adhesion. Drug discovery and molecular docking were analyzed based on 93 differentially expressed mRNAs of the ceRNA network. A total of 36 chemicals were identified as putative bioactive molecules for RSA, and one representative chemical was identified for docking with six proteins. CONCLUSIONS: These findings provide novel insights into the mechanism of regulation of RSA by circular RNA and its clinical diagnosis and treatment.

CircFBXW4 regulates human trophoblast cell proliferation and invasion via targeting miR-324-3p/TJP1 axis in recurrent spontaneous abortion.

INTRODUCTION: Increasing evidence has shown that circular RNAs (circRNAs) play vital roles in embryonic development. However, the function of circRNAs in recurrent spontaneous abortion (RSA) is largely unknown. This study aimed to investigate the expression profile of human circRNAs and their functional mechanisms in regulating RSA. METHODS: The profiles of circRNAs in placental villus tissues from women with RSA and healthy pregnancy with induced abortion were investigated using RNA-sequencing and bioinformatics. Nine circRNAs were verified in the 50 placental villus samples. RNase R digestion, actinomycin D treatment, and fluorescence in situ hybridization were performed to characterize circFBXW4. Furthermore, direct binding of circFBXW4 to miR-324-3p was confirmed by dual-luciferase reporter assay. The roles of circFBXW4 were determined by loss- and gain-of-function assays including cell proliferation, invasion, and apoptosis using CCK8 kit, transwell migration assay, and TUNEL kit in vitro, respectively. RESULTS: A total of 417 aberrantly expressed circRNAs was detected. circFBXW4, a circRNA significantly up-regulated in the RSA group, was further evaluated. circFBXW4 showed higher stability than FBXW4 mRNA and was localized in the cytoplasm and nucleus in HTR-8/SVneo cells. MiR-324-3p was lowly expressed in the RSA group, and directly regulated circFBXW4 and TJP1 expression in a targeted manner. Overexpression and knockdown of circFBXW4 and miR-324-3p mimic/inhibitor could increase or decrease HTR-8/SVneo cell proliferation and invasion. circFBXW4 regulated TJP1 expression, cell proliferation, and invasion by sponging miR-324-3p. DISCUSSION: The circFBXW4/miR-324-3p/TJP1 axis is involved in the occurrence and progression of RSA and may be a promising therapeutic target in RSA.

Whole exome sequencing identifies genetic variants in Chinese Han pregnant women with venous thromboembolism.

OBJECTIVES: Venous thromboembolism (VTE) is a common health problem, causing considerable morbidity and mortality. The incidence of VTE is higher in pregnant women than in those who are not pregnant. However, genetic factors for VTE in pregnant women are largely unknown. METHODS: We performed a large-scale prospective cohort study of 65,138 pregnancies. 24 pregnant patients diagnosed with VTE and 24 matched pregnant women without VTE were enrolled and sequenced by whole exome sequencing. We investigated the occurrence of known VTE risk variants, damaging variants found in thrombophilia genes and rare damaging variants possibly related to VTE in pregnancies. An exome-wide association study was also performed using a case-control design. RESULTS: Our findings suggest that VTE in pregnancies may be mainly related to (i) the presence of known pathogenic variants in affected individual like F2, (ii) possibly damaging variants found in major thrombophilia gene GCKR, and (iii) 1 pathogenic and 13 likely pathogenic rare damaging variants associated with genetic errors. In exome-wide association stage, 42 variants at 34 genes may show suggestive associations with VTE in pregnancies (the lowest P = 3.5 × 10-7). ZFP41 (rs6558339, P = 8.85 × 10-5) in 24 patients were the only exonic missense variant. CONCLUSION: The combined findings suggest that some genes may be involved in the mechanism of basement membranes, sterol accumulation and atherosclerosis, lipid metabolism and coagulation deficiency. These identified risk variants may improve the understanding of VTE pathogenesis in pregnancies and provide potential biomarker for development of clinical diagnosis.

Placental DNA methylation changes in gestational diabetes mellitus.

In this study, we investigated the association between altered methylation in the maternal placenta and hyperglycaemia and explored the epigenetic mechanisms underlying gestational diabetes mellitus (GDM). Reduced representation bisulphite sequencing (RRBS) and RNA sequencing (RNA-seq) were performed on placental tissues obtained from women with GDM and healthy controls. Further, pyrosequencing, correlation analyses, and linear regression analyses were performed to valuate relationships between aberrantly methylated-differentially expressed genes and clinical parameters. The EMBOSS and JASPAR databases were used for a computational analysis of CpG islands and transcription factor-binding sites in the TRIM67 promoter region. A CpG island with a length of 264 bp in the placental TRIM67 promoter region in the GDM group exhibited significant hypermethylation at four CpG sites. The hypermethylation of the TRIM67 promoter region in the maternal placenta showed a significant, positive correlation with the 1 h and 2 h oral glucose tolerance test (OGTT) values and a negative correlation with lipoprotein(a). Placental DNA methylation levels in the TRIM67 promoter region were markedly elevated in GDM and were associated with blood glucose and lipid levels during healthy pregnancy.

Whole-exome sequencing reveals novel variants associated with abnormal uterine bleeding caused by copper intrauterine device.

Aim: This study aimed to explore the genetic risk factors and validate variants of abnormal uterine bleeding after copper intrauterine device insertion. Methods: Whole-exome sequencing was performed and several variants were validated by Sequenom MassARRAY. Results: Eight variants showed potential clinical damage according to American College of Medical Genetics and Genomics criteria. By combined analysis of screening and validation, NFASC RS2802808 C>G p.Ile971Met (Pallele = 0.009 and Pgenotype = 0.027) and PIGR RS2275531 C>T p.Gly365Ser (Pallele = 0.009 and Pgenotype = 0.013) variants were identified as significantly associated with abnormal uterine bleeding with a false discovery rate <0.05. NFASC and PIGR may play a role in abnormal uterine bleeding by regulating coagulation fibrinolysis and endometrial epithelium inflammation functions. Conclusion: These findings provide a genetic basis for clinical individualization and precision of intrauterine device implantation.

Abnormal uterine bleeding (AUB) after Cu intrauterine device (Cu-IUD) insertion is the most common side effect of Cu-IUD use. AUB is a multifactorial process that relates to endometrial-related genetic factors, ovulatory function-related genetic factors, coagulation, the fibrinolytic system, contraction of the uterine arteries and endometritis inflammatory factor. This is the first study to explore the underlying genetic mechanisms of AUB related to the use of Cu-IUDs by whole-exome sequencing in the Chinese Han population. The authors found that variants of NFASC and PIGR genes were significantly associated with AUB in women using Cu-IUDs. NFASC and PIGR may be involved in coagulation fibrinolysis and endometrial epithelium inflammation functions, indicating its potential functions in AUB. This study could provide a genetic basis for studies on the individualization and precision of IUD use in the future.

Integrated single-cell RNA-seq and DNA methylation reveal the effects of air pollution in patients with recurrent spontaneous abortion.

BACKGROUND: Maternal air pollutants exposure is associated with a number of adverse pregnancy outcomes, including recurrent spontaneous abortion (RSA). However, the underlying mechanisms are still unknown. The present study aimed to understand the mechanism of RSA and its relationship with air pollution exposure. We compared data of decidual tissue from individuals with induced abortions and those with RSA by bulk RNA sequencing (RNA-seq), reduced representation bisulfite sequencing (RRBS), and single-cell RNA sequencing (scRNA-seq). Differentially expressed genes (DEGs) were verified using RT-qPCR and pyrosequencing. A logistic regression model was used to investigate the association between air pollutants exposure and RSA. RESULTS: We identified 98 DEGs with aberrant methylation by overlapping the RRBS and RNA-seq data. Nineteen immune cell subsets were identified. Compared with normal controls, NK cells and macrophages accounted for different proportions in the decidua of patients with RSA. We observed that the methylation and expression of IGF2BP1 were different between patients with RSA and controls. Furthermore, we observed significant positive associations between maternal air pollutants exposure during the year prior to pregnancy and in early pregnancy and the risk of RSA. Mediation analyses suggested that 24.5% of the effects of air pollution on the risk of RSA were mediated through IGF2BP1 methylation. CONCLUSION: These findings reveal a comprehensive cellular and molecular mechanism of RSA and suggest that air pollution might cause pregnancy loss by affecting the methylation level of the IGF2BP1 promoter.

Tandem mass tag-based quantitative proteomic profiling of the serum of patients with abnormal uterine bleeding associated with copper intrauterine device.

OBJECTIVE: To investigate changes in the level of protein in serum and uncover the underlying pathogenesis of abnormal uterine bleeding (AUB) associated with copper intrauterine devices (Cu IUD). METHODS: Protein profiles were investigated via tandem mass tag (TMT)-based quantitative proteomics and bioinformatics technology. Quantification and characterization of candidate proteins were further performed in 33 controls and 45 cases by Luminex assay and enzyme-linked immunosorbent assay. RESULTS: In total, 842 proteins were identified via TMT coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the serum of individuals with IUDs. Among them, 25 differentially expressed proteins (p < 0.05) were observed, including eight upregulated proteins and 17 downregulated proteins. Ten proteins were verified, and Alpha-1-Antitrypsin (a1AT) had a significantly elevated expression in women with AUB associated with the Cu IUD compared with healthy controls (p = 0.026) and a high area under the curve value (0.656), as well as sensitivity (64.9%) and specificity (71.9%). CONCLUSION: This is the first study to explore changes in serum protein and the underlying mechanisms of AUB associated with the Cu IUD via TMT technology. a1AT with biomarker potential was validated. These findings might provide an experimental basis for the early diagnosis or treatment of AUB associated with the Cu IUD.

Whole-exome sequencing reveals novel candidate single nucleotide variations for preventing adverse effects of levonorgestrel implantation.

Aim: To identify novel genes associated with adverse effects of levonorgestrel (LNG) implants based on comparative whole-exome sequencing. Materials & methods: A cohort comprising 104 participants, including 52 controls and 52 women with LNG-related adverse effects, was recruited. Seven cases and eight controls were selected for whole-exome sequencing. We verified 13 single nucleotide variations (SNVs) related with integrin-mediated signaling pathway and cell proliferation using the MassARRAY platform. Results: Finally, we screened 49 cases and 52 controls for analyses. Two SNVs (rs7255721 and rs1042522) were located in ADAMTS10 and TP53, respectively, and significantly different between two groups. These two SNVs lead to changes in protein structure and physicochemical parameters. Conclusion: Here, we defined two pathogenic mutations related to adverse LNG effects.

Association of Sperm Methylation at LINE-1, Four Candidate Genes, and Nicotine/Alcohol Exposure With the Risk of Infertility.

In this study, we examined whether smoking and drinking affect sperm quality and the DNA methylation of the repetitive element LINE-1, MEST, P16, H19, and GNAS in sperm. Semen samples were obtained from 143 male residents in a minority-inhabited district of Guizhou province in southwest China. Quantitative DNA methylation analysis of the samples was performed using MassARRAY EpiTYPER assays. Sperm motility was significantly lower in both the nicotine-exposed (P = 0.0064) and the nicotine- and alcohol-exposed (P = 0.0008) groups. Follicle-stimulating hormone (FSH) levels were higher in the nicotine-exposed group (P = 0.0026). The repetitive element LINE-1 was hypermethylated in the three exposed groups, while P16 was hypomethylated in the alcohol and both the alcohol and nicotine exposure groups. Our results also show that alcohol and nicotine exposure altered sperm cell quality, which may be related to the methylation levels of MEST and GNAS. In addition, MEST, GNAS, and the repetitive element LINE1 methylation was significantly associated with the concentration of sperm as well as FSH and luteinizing hormone levels.

Polymorphisms in CYP450 Genes and the Therapeutic Effect of Atorvastatin on Ischemic Stroke: A Retrospective Cohort Study in Chinese Population.

PURPOSE: Ischemic stroke (IS) is one of the most common neurologic diseases and is the main cause of death and disability in the Chinese population. This retrospective cohort study was performed to elucidate the relationship between single nucleotide polymorphisms (SNPs) in cytochrome P450 genes and the therapeutic effect of atorvastatin. METHODS: A total of 192 cases of IS were enrolled in the study. All patients were treated with atorvastatin, and their lipid levels and proportions were measured. Six SNPs in 4 cytochrome P450 genes (CYP2C19, CYP2D6, CYP3A4, and CYP4F2) related to drug metabolism were selected to be genotyped and analyzed. FINDINGS: Data were analyzed for 192 patients (sex, male/female, 122/70; mean age, 69.81 [9.35] years; Hypertension, 163[84.90%]; Cigarette smoking, 34[17.71%]). Among the 192 patients with IS treated with atorvastatin, it was found that the G allele of rs1065852 (CYP2D6) had a better effect on lowering of ΔLDL (P < 0.001), ΔLDL/LDL (P < 0.001), Δ(LDL/HDL) (P < 0.001), and Δ(LDL/HDL)/(LDL/HDL) (P < 0.001). We also found that rs2242480 (CYP3A4) showed marginal association with ΔLDL (P = 0.049) under the dominant model. In addition, rs2242480 and rs1065852 exhibited cumulative effects on the lipid-lowering (ΔLDL, ΔLDL/LDL, and Δ[LDL/HDL]) efficacy of atorvastatin (P < 0.001). IMPLICATIONS: The results suggest that CYP2D6 and CYP3A4 affect treatment with atorvastatin in patients with IS.