Epimutations of the <Emphasis Type="Italic">KCNQ1OT1</Emphasis> imprinting center of chromosome 11 in early human embryolethality

Disturbance of the epigenetic status of the H19 and KCNQ1OT1 imprinting centers of chromosome 11 and the CDKN1C imprinted gene in early human embryolethality have been studied. This is the first study to detect hypomethylation of KCNQ1OT1 in the maternal homolog in placental tissues from some (9.5%) spontaneous abortions with impaired cell proliferation during the first trimester of pregnancy. Tissue specificity of the aberrant methylation status of the imprinting center has been found. A hypothesis on the postimplantation origin of epimutations in somatic cells of the developing embryo is put forward. The selective role of epimutations of imprinted genes in early human ontogeny as compared to uniparental disomy is considered; estimation of the epigenetic risk entailed in using assisted reproductive technologies is discussed.     

Association of gene polymorphisms of matrix metalloproteinases with reproductive losses in the first trimester of pregnancy

In the present study, the frequencies of genotypes and alleles of candidate genes with respect to polymorphisms associated with increased pregnancy loss in the first trimester of pregnancy, including MMP1–1607insG, MMP9 A–8202G, and TIMP1 С536T, were reported. The frequency of homozygotes for allele MMP9 A–8202 was increased by a factor of two among women with miscarriage in the first trimester compared to the control. Significant models of interaction of genes MMPs and TIMP1 were revealed. The genotypes of genes MMP1 (rs1799750), MMP9 (rs11697325), and TIMP1 (rs11551797) increasing the risk of pregnancy loss in the first trimester were determined.     

Normal early pregnancy

The objective of this study was to analyze genome-wide differential methylation patterns in maternal leukocyte DNA in early pregnant and non-pregnant states. This is an age and body mass index matched case-control study comparing the methylation patterns of 27,578 cytosine-guanine (CpG) sites in 14,495 genes in maternal leukocyte DNA in early pregnancy (n = 14), in the same women postpartum (n = 14), and in nulligravid women (n = 14) on a BeadChip platform. Transient widespread hypomethylation was found in early pregnancy as compared with the non-pregnant states. Methylation of nine genes was significantly different in early pregnancy compared with both postpartum and nulligravid states (< 10% False Discovery Rate). Early pregnancy may be characterized by widespread hypomethylation compared with non-pregnant states; there is no apparent permanent methylation imprint after a normal term gestation. Nine potential candidate genes were identified as differentially methylated in early pregnancy and may play a role in the maternal adaptation to pregnancy.     

Normal early pregnancy: A transient state of epigenetic change favoring hypomethylation

The objective of this study was to analyze genome-wide differential methylation patterns in maternal leukocyte DNA in early pregnant and non-pregnant states. This is an age and body mass index matched case-control study comparing the methylation patterns of 27,578 cytosine-guanine (CpG) sites in 14,495 genes in maternal leukocyte DNA in early pregnancy (n = 14), in the same women postpartum (n = 14), and in nulligravid women (n = 14) on a BeadChip platform. Transient widespread hypomethylation was found in early pregnancy as compared with the non-pregnant states. Methylation of nine genes was significantly different in early pregnancy compared with both postpartum and nulligravid states (< 10% False Discovery Rate). Early pregnancy may be characterized by widespread hypomethylation compared with non-pregnant states; there is no apparent permanent methylation imprint after a normal term gestation. Nine potential candidate genes were identified as differentially methylated in early pregnancy and may play a role in the maternal adaptation to pregnancy.     

Depression in pregnancy,infant birth weight and DNA methylation of imprint regulatory elements

Depressed mood in pregnancy has been linked to low birth weight (LBW, < 2,500 g), a risk factor for adult-onset chronic diseases in offspring. We examined maternal depressed mood in relation to birth weight and evaluated the role of DNA methylation at regulatory sequences of imprinted genes in this association. We measured depressed mood among 922 pregnant women using the CES-D scale and obtained birth weight data from hospital records. Using bisulfite pyrosequencing of cord blood DNA from 508 infants, we measured methylation at differentially methylated regions (DMRs) regulating imprinted genes IGF2/H19, DLK1/MEG3, MEST, PEG3, PEG10/SGCE, NNAT and PLAGL1. Multiple regression models were used to examine the relationship between depressed mood, birth weight and DMR methylation levels. Depressed mood was associated with a more that 3-fold higher risk of LBW, after adjusting for delivery mode, parity, education, cigarette smoking, folic acid use and preterm birth. The association may be more pronounced in offspring of black women and female infants. Compared with infants of women without depressed mood, infants born to women with severe depressed mood had a 2.4% higher methylation at the MEG3 DMR. Whereas LBW infants had 1.6% lower methylation at the IGF2 DMR, high birth weight (> 4,500 g) infants had 5.9% higher methylation at the PLAGL1 DMR compared with normal birth weight infants. Our findings confirm that severe maternal depressed mood in pregnancy is associated with LBW, and that MEG3 and IGF2 plasticity may play important roles.     

Depression in pregnancy,infant birth weight and DNA methylation of imprint regulatory elements

Depressed mood in pregnancy has been linked to low birth weight (LBW, < 2,500 g), a risk factor for adult-onset chronic diseases in offspring. We examined maternal depressed mood in relation to birth weight and evaluated the role of DNA methylation at regulatory sequences of imprinted genes in this association. We measured depressed mood among 922 pregnant women using the CES-D scale and obtained birth weight data from hospital records. Using bisulfite pyrosequencing of cord blood DNA from 508 infants, we measured methylation at differentially methylated regions (DMRs) regulating imprinted genes IGF2/H19, DLK1/MEG3, MEST, PEG3, PEG10/SGCE, NNAT and PLAGL1. Multiple regression models were used to examine the relationship between depressed mood, birth weight and DMR methylation levels. Depressed mood was associated with a more that 3-fold higher risk of LBW, after adjusting for delivery mode, parity, education, cigarette smoking, folic acid use and preterm birth. The association may be more pronounced in offspring of black women and female infants. Compared with infants of women without depressed mood, infants born to women with severe depressed mood had a 2.4% higher methylation at the MEG3 DMR. Whereas LBW infants had 1.6% lower methylation at the IGF2 DMR, high birth weight (> 4,500 g) infants had 5.9% higher methylation at the PLAGL1 DMR compared with normal birth weight infants. Our findings confirm that severe maternal depressed mood in pregnancy is associated with LBW, and that MEG3 and IGF2 plasticity may play important roles.     

Gene expression pattern of IGF2, PHLDA2, PEG10 and CDKN1C imprinted genes in spontaneous miscarriages or fetal deaths

Genomic imprinting is defined as an epigenetic modification that leads to parent-of-origin specific monoallelic expression. Some current research on the fetal control growth has been focused on the study of genes that display imprinted expression in utero. Four imprinted genes, two paternally expressed (IGF2 and PEG10) and two maternally expressed (PHLDA2 and CDKN1C), are well known to play a role in fetal growth and placental development. Pregnancy loss in the general reproductive population is a very common occurrence and other genetic causes beyond chromosomal abnormalities could be involved in spontaneous miscarriages or fetal deaths, such as alteration of expression in imprinted genes particularly those related to fetal or placental growth. Quantitative Real Time PCR was performed to evaluate gene expressions patterns of the four mentioned genes in spontaneous miscarriages or fetal deaths from 38 women. Expression levels of PHLDA2 gene were upregulated in the first trimester pregnancy cases and all four imprinted genes studied were upregulated in the second trimester of pregnancy cases comparing with controls. In third trimester PEG10 was downregulated in fetal samples group. This is the first study presenting data from human imprinted genes expression in spontaneous miscarriages or fetal deaths cases from the three trimesters of pregnancy.     

Deep Bisulfite Sequencing of Aberrantly Methylated Loci in a Patient with Multiple Methylation Defects

NLRP7 is a maternal effect gene as maternal mutations in this gene cause recurrent hydatidiform moles, spontaneous abortions and stillbirths, whereas live births are very rare. We have studied a patient with multiple anomalies born to a mother with a heterozygous NLRP7 mutation. By array-based CpG methylation analysis of blood DNA from the patient, his parents and 18 normal controls on Illumina Infinium HumanMethylation27 BeadChips we found that the patient had methylation changes (delta ß ≥ 0.3) at many imprinted loci as well as at 87 CpGs associated with 85 genes of unknown imprinting status. Using a pseudoproband (permutation) approach, we found methylation changes at only 7-24 CpGs (mean 15; standard deviation 4.84) in the controls. Thus, the number of abberantly methylated CpGs in the patient is more than 14 standard deviations higher. In order to identify novel imprinted genes among the 85 conspicuous genes in the patient, we selected 19 (mainly hypomethylated) genes for deep bisulfite amplicon sequencing on the ROCHE/454 Genome Sequencer in the patient and at least two additional controls. These controls had not been included in the array analysis and were heterozygous for a single nucleotide polymorphism at the test locus, so that allele-specific DNA methylation patterns could be determined. Apart from FAM50B, which we proved to be imprinted in blood, we did not observe allele-specific DNA methylation at the other 18 loci. We conclude that the patient does not only have methylation defects at imprinted loci but (at least in blood) also an excess of methylation changes at apparently non-imprinted loci.     

Polymorphic Imprinting of <Emphasis Type="Italic">SLC38A4</Emphasis> Gene in Bovine Placenta

Imprinted genes are characterized by monoallelic expression that is dependent on parental origin. Comparative analysis of imprinted genes between species is a powerful tool for understanding the biological significance of genomic imprinting. The slc38a4 gene encodes a neutral amino acid transporter and is identified as imprinted in mice. In this study, the imprinting status of SLC38A4 was assessed in bovine adult tissues and placenta using a polymorphism-based approach. Results indicate that SLC38A4 is not imprinted in eight adult bovine tissues including heart, liver, spleen, lung, kidney, muscle, fat, and brain. It was interesting to note that SLC38A4 showed polymorphic status in five heterogeneous placentas, with three exhibiting paternal monoallelic expression and two exhibiting biallelic expression. Monoallelic expression of imprinted genes is generally associated with allele-specific differentially methylation regions (DMRs) of CpG islands (CGIs)-encompassed promoter; therefore, the DNA methylation statuses of three CGIs in the SLC38A4 promoter and exon 1 region were tested in three placentas (two exhibiting paternal monoallelic and one showing biallelic expression of SLC38A4) and their corresponding paternal sperms. Unexpectedly, extreme hypomethylation (<?3%) of the DNA was observed in all the three detected placentas and their corresponding paternal sperms. The absence of DMR in bovine SLC38A4 promoter region implied that DNA methylation of these three CGIs does not directly or indirectly affect the polymorphic imprinting of SLC38A4 in bovine placenta. This suggested other epigenetic features other than DNA methylation are needed in regulating the imprinting of bovine SLC38A4, which is different from that of mouse with respect to a DMR existence at the mouse’s slc38a4 promoter region. Although further work is needed, this first characterization of polymorphic imprinting status of SLC38A4 in cattle placenta provides valuable information on investigating the genomic imprinting phenomenon itself.     

A maternal hypomethylation syndrome presenting as transient neonatal diabetes mellitus

The expression of imprinted genes is mediated by allele-specific epigenetic modification of genomic DNA and chromatin, including parent of origin-specific DNA methylation. Dysregulation of these genes causes a range of disorders affecting pre- and post-natal growth and neurological function. We investigated a cohort of 12 patients with transient neonatal diabetes whose disease was caused by loss of maternal methylation at the TNDM locus. We found that six of these patients showed a spectrum of methylation loss, mosaic with respect to the extent of the methylation loss, the tissues affected and the genetic loci involved. Five maternally methylated loci were affected, while one maternally methylated and two paternally methylated loci were spared. These patients had higher birth weight and were more phenotypically diverse than other TNDM patients with different aetiologies, presumably reflecting the influence of dysregulation of multiple imprinted genes. We propose the existence of a maternal hypomethylation syndrome, and therefore suggest that any patient with methylation loss at one maternally-methylated locus may also manifest methylation loss at other loci, potentially complicating or even confounding the clinical presentation.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Multilocus epimutations of imprintome in the pathology of human embryo development

Genomic imprinting is one of the key epigenetic phenomena involved in embryonic development of eutherians and humans. Molecular mechanisms of imprinting disturbances in the pathology of prenatal and postnatal onthogenesis are to a great extent related to methylation abnormalities of the imprinted genes. Over recent years, data are accumulating on multiple abnormalities of methylation simultaneously in several imprinted loci in the development of various pathologies that raises the issue of deciphering the structural and functional organization of imprintome and the interaction of imprinted genes. The present work analyzes DNA methylation of 51 imprinted genes in the placental tissues of human spontaneous abortions. We revealed multiple epimutations in from four to 12 imprinted genes in every embryo. Most of the epimutations (78%) were of a postzygotic origin. It has been established for the first time that the total incidence of methylation disturbance in maternal and paternal alleles of the imprinted genes leading to embryo development suppression is significantly higher than the incidence of epimutations, which stimulate embryogenesis. This fact supports at the epigenetic level the hypothesis of parent-offspring conflict that describes the occurrence of a monoallelic expression of imprinted genes in mammalian evolution.     

Multilocus epimutations of imprintome in the pathology of embryo development

Genomic imprinting is one of the most significant epigenetic phenomena, which is involved in the support of eutherians and human embryo development. Molecular mechanisms of imprinting disturbance in the pathology of pre- and postnatal ontogeny are related to a considerable degree to aberrant DNA methylation of imprinted genes. At present time data about multiple abnormalities of DNA methylation arising simultaneously in several imprinted loci are accumulated. This fact brings up the problem of interpretation of imprintome structural and functional organization, as well as interaction of imprinted genes. At present study DNA methylation analysis of 51 imprinted genes in placental tissues of human spontaneous abortions was performed. The presence of several epimutations affected from four to 12 imprinted genes was observed in each embryo. Majority of epimutations (78%) had a postzygotic origin. It was shown for the first time that the total incidence of abnormal DNA methylation of maternal and paternal alleles of imprinted genes, which lead to suppression of embryo development, is significantly higher than the incidence of epimutations, which can lead to stimulation of ontogenesis processes. This fact supports at the epigenetic level the "sex conflict" hypothesis, which explains the appearance of monoallelic imprinted genes expression in the evolution of mammals.     

Epigenetic inactivation of the <Emphasis Type="Italic">RB1</Emphasis> gene as a factor of genomic instability: A possible contribution to etiology of chromosomal mosaicism during human embryo development

The methylation status of the cell cycle control gene RB1 has been studied in placental tissues of spontaneous abortions of the first trimester of pregnancy with mosaic variants of numerical chromosomal abnormalities verified by a molecular cytogenetic examination. Aberrant methylation of the gene promoter region has been revealed for the first time in 20% of embryos with chromosomal mosaicism that died in utero. A maximum frequency of epimutations was recorded in a group of embryos with a low level of abnormal cells for which mitotic errors are most likely to determine the formation of mosaic aneuploidy in primary euploid zygotes. It has been suggested that aberrant epigenetic genomic modifications at early stages of human embryonic development can be one of the mechanisms promoting genomic instability realized in the form of mosaic abnormalities of the karyotype that are incompatible with the normal course of embryogenesis.     

Association between <Emphasis Type="Italic">TNF,IL1B,IL6, IL10</Emphasis> and <Emphasis Type="Italic">IFNG</Emphasis> polymorphisms and recurrent miscarriage: a case control study

Background

Approximately half of recurrent miscarriages have unexplained etiology. Recent evidences suggest that cytokines are important determinants in pregnancy maintenance and as such, cytokine gene polymorphisms, which can affect cytokine production and/or functionality, could play a role in the disorder. Thus, we aimed to investigate the association of selected cytokine gene polymorphisms with risk of recurrent miscarriage among Chinese.

Methods

TNF -238G > A, TNF -308G > A, IL1B -511 T > C, IL1B 3954C > T, IL6 -174G > C, IL6 -634C > G, IL10 -1082A > G and IFNG 874A > T polymorphisms were genotyped on 775 women with idiopathic recurrent miscarriage and 805 healthy parous control women. Logistic regression analysis was performed to determine the odds ratios (ORs) of the association between the polymorphisms and recurrent miscarriage risk.

Results

Among the eight polymorphisms studied, only the IL1B -511 T > C and IL6 -634C > G polymorphisms showed statistically significant associations with recurrent miscarriage risk. For the former, a significantly increased risk of recurrent miscarriage was observed for the mutant (CC) genotype (OR: 1.377; 95% CI: 1.039–1.824; P?=?0.026). However, for the IL6 -634C?>?G polymorphism, a decreased recurrent miscarriage risk was observed for the heterozygous (CG) genotype (OR: 0.614; 95% CI: 0.493–0.765; P < 0.001) and the mutant (GG) genotype (OR: 0.414; 95% CI: 0.251–0.684; P?=?0.001).

Conclusions

The IL1B -511 T > C polymorphism may serve as important risk factor for recurrent miscarriage while the IL6 -634C > G polymorphism may protect against the risk of recurrent miscarriage.

     

Manipulation of cytokine production profiles as a therapeutic approach for immunologic pregnancy loss

Pregnancy is not as successful as one might think; it can be compromised by several complications such as recurrent spontaneous miscarriage, pre-term delivery, pre-eclampsia etc. Much attention has been paid to the possibility of the maternal immune system mediating deleterious effects on pregnancy. Research conducted during the last two decades has shed much light on cell-mediated immunologic effectors that might underlie these pregnancy complications. Of particular interest are the effects that pro-inflammatory and anti-inflammatory cytokines have on the foetus and placenta, and thus on the success and failure of pregnancy. This review presents evidences that certain cytokine profiles are associated with recurrent miscarriage and pre-term delivery and discusses possible pathways of effector function of cytokines in pregnancy loss and the redirection of cytokine profiles from one that is antagonistic to pregnancy towards one that is conducive to the success of pregnancy. Among the promising agents for the modulation of the Th1/Th2 balance are progestogens like progesterone and dydrogesterone; this review also discusses recent evidence that progestogens are capable of modulating cytokine production patterns in pregnancy loss.     

Frequencies of killer immunoglobulin-like receptor genotypes influence susceptibility to spontaneous abortion

Natural killer (NK) cells are the most abundant lymphocyte population in the decidua. These cells express killer immunoglobulin-like receptors (KIRs), which upon recognition of HLA class I molecules on trophoblasts may either stimulate NK cells (activating KIRs) or inhibit them (inhibitory KIRs) to produce soluble factors necessary for the maintenance of pregnancy.KIR genes exhibit extensive haplotype polymorphism; individuals differ in both the number and kind (activating vs. inhibitory) ofKIR genes. This polymorphism affects NK cell reactivity and susceptibility to diseases, including gynecological disorders. Therefore weKIR-genotyped 149 spontaneously aborting women and 117 control multiparae (at least 2 healthy-born children). Several genotypes (i.e. combinations of variousKIR genes) were differently distributed among the patients and control subjects. Differences were observed in the numbers and the ratios of activating to inhibitory KIRs between patients and healthy women: (i) genotypes containing 6 activatingKIR genes were less frequent and those containing 6 inhibitoryKIR genes were more frequent in patients than in control subjects, and (ii) an excess of inhibitory KIRs (activating-to-inhibitoryKIR gene ratios of 0.33 to 0.83) was associated with miscarriage, whereas ratios close to equilibrium (0.86–1.25) seemed to be protective. In addition, the results suggest for the first time that sporadic and recurrent spontaneous abortions as well as miscarriage in the presence or absence of autoantibodies may have differentKIR genotypic backgrounds.     

Recurrent spontaneous abortion and polycystic ovarian disease: comparison of two regimens to induce ovulation.

OBJECTIVE--To determine whether pituitary suppression before induction of ovulation reduces the rate of spontaneous abortion in women with polycystic ovarian disease and primary recurrent spontaneous abortions. DESIGN--Closed, randomised, sequential trial. Pairs of women were allocated to each treatment by the toss of a coin. SETTING--Supraregional clinic for women who had had recurrent spontaneous abortions. SUBJECTS--Forty two women with polycystic ovarian disease and primary recurrent spontaneous abortions. INTERVENTIONS--Ovulation was induced by clomiphene or pituitary suppression with buserelin followed by pure follicle stimulating hormone. MAIN OUTCOME MEASURES--Preference for a particular treatment was noted. A preference occurred when one woman in a pair had a successful pregnancy (defined as one of over 12 weeks'' gestation) and one had a spontaneous abortion; the preference was for the treatment resulting in the successful pregnancy. RESULTS--Spontaneous abortions occurred in 11 of 20 women given clomiphene compared with two of 20 who had pituitary suppression. Eleven preferences were found for buserelin and two for clomiphene. In seven pairs both women had successful pregnancies. One pair was discarded because one of the women did not become pregnant. The ratio of luteinising hormone concentration to follicular diameter was found to be a possible diagnostic indicator of spontaneous abortion. CONCLUSION--Pituitary suppression before induction of ovulation significantly reduces the risk of spontaneous abortion in women with polycystic ovarian disease and primary recurrent spontaneous abortions.     

Parent-of-origin effects on gene expression and DNA methylation in the maize endosperm

Imprinting describes the differential expression of alleles based on their parent of origin. Deep sequencing of RNAs from maize (Zea mays) endosperm and embryo tissue 14 d after pollination was used to identify imprinted genes among a set of ~12,000 genes that were expressed and contained sequence polymorphisms between the B73 and Mo17 genotypes. The analysis of parent-of-origin patterns of expression resulted in the identification of 100 putative imprinted genes in maize endosperm, including 54 maternally expressed genes (MEGs) and 46 paternally expressed genes (PEGs). Three of these genes have been previously identified as imprinted, while the remaining 97 genes represent novel imprinted maize genes. A genome-wide analysis of DNA methylation identified regions with reduced endosperm DNA methylation in, or near, 19 of the 100 imprinted genes. The reduced levels of DNA methylation in endosperm are caused by hypomethylation of the maternal allele for both MEGs and PEGs in all cases tested. Many of the imprinted genes with reduced DNA methylation levels also show endosperm-specific expression patterns. The imprinted maize genes were compared with imprinted genes identified in genome-wide screens of rice (Oryza sativa) and Arabidopsis thaliana, and at least 10 examples of conserved imprinting between maize and each of the other species were identified.