Project description:Genome wide DNA methylation analysis using blood leukocyte DNA was performed on 15 patients with genomic imprinting disorders (13 Beckwith–Wiedemann syndrome [BWS], two Silver–Russell syndrome [SRS]), and four controls. The Illumina HumanMethylation850 BeadChip was used to obtain DNA methylation profiles at a more than 850,000 CpGs.
Project description:Genome wide DNA methylation profiling of whole blood at birth (Guthrie blood spots) and adulthood of individuals conceived by assisted reproductive technology (ART) and matched non-ART controls. The Illumina Infinium MethylationEPIC BeadChip was used to obtain DNA methylation profiles across approximately 850,000 CpGs in guthrie cards and whole adult blood.
Project description:Children conceived using Assisted Reproductive Technologies (ART) have a higher incidence of growth and birth defects, attributable in part to epigenetic perturbations. Both ART and germline defects associated with parental infertility could interfere with epigenetic reprogramming events in germ cells or early embryos. Mouse models indicate that the placenta is more susceptible to the induction of epigenetic abnormalities than the embryo, and thus the placental methylome may provide a sensitive indicator of ‘at risk’ conceptuses. Our goal was to use genome-wide profiling to examine the extent of epigenetic abnormalities in matched placentas from an ART/infertility group and control singleton pregnancies (n=44/group) from a human prospective longitudinal birth cohort, the 3D Study. Principal component analysis revealed a group of ART outliers. The ART outlier group was enriched for females and a subset of placentas showing loss of methylation of several imprinted genes including GNAS, SGCE, KCNQT1OT1 and BLCAP/NNAT. Within the ART group, placentas from pregnancies conceived with IVF/ICSI showed distinct epigenetic profiles as compared to those conceived with less invasive procedures (ovulation induction, intrauterine insemination). Male factor infertility and paternal age further differentiated the IVF/ICSI group, suggesting an interaction of infertility and techniques in perturbing the placental epigenome. Together, the results suggest that the human placenta is sensitive to the induction of epigenetic defects by ART and/or infertility, and we stress the importance of considering both sex and paternal factors and that some but not all ART conceptuses will be susceptible.
Project description:Assisted reproductive therapies (ART) have become increasingly common worldwide and up to ~6% of children currently born in developed countries were conceived employing these technologies. Numerous retrospective studies have suggested that ART-conceived children are more likely to develop the overgrowth syndrome Beckwith-Wiedemann (BWS). In bovine, the use of ART can induce a similar overgrown condition, which is referred to as large offspring syndrome (LOS). Both BWS and LOS involve dysregulation of imprinted genes. However, it remains largely unknown whether aberrant gene expression and DNA methylation occur at non-imprinted loci and to what extent these molecular alterations can contribute to these syndromes. Here we examined the transcriptome of skeletal muscle, liver, kidney, and brain of control and LOS bovine fetuses and found that different LOS fetuses exhibit different severity of the transcriptome alterations and different tissues have distinct gene pathways disturbed in LOS fetuses. Particularly for skeletal muscle, multiple pathways involved in myoblast proliferation and fusion into myotubes are misregulated in LOS fetuses. Further, characterization of the methylome of skeletal muscle demonstrates numerous local methylation differences; however, global DNA methylation is comparable between all individuals regardless of bodyweight. Importantly, only a small percent of differentially expressed genes (DEGs) including the imprinted gene IGF2R can be linked to the neighboring differentially methylated regions (DMRs). In summary, we not only show that misregulation of non-imprinted genes in addition to loss-of-imprinting contributes to the ART-induced overgrowth syndrome but also demonstrate that most of the aberrant gene expression is not associated with DNA methylome epimutations.
Project description:Transcriptome analysis of mouse embryonic stem cell lines derived from embryos cultured in optimal and suboptimal conditions compared to cell lines derived from control embryos. The use of assisted reproductive technologies (ART) such as in vitro fertilization (IVF) has resulted in the birth of more than 5 million children. While children conceived by these technologies are generally healthy, there is conflicting evidence suggesting an increase in adult-onset complications like glucose intolerance and high blood pressure in IVF children. Animal models indicate similar potential risks. It remains unclear what molecular mechanisms may be operating during in vitro culture to predispose the embryo to these diseases. One of the limitations faced by investigators is the paucity of the material in the preimplantation embryo to test for molecular analysis. To address this problem, we generated mouse embryonic stem cells (mESC) from blastocysts conceived after natural mating (mESCFB) or after IVF, using optimal (KSOM + 5% O2; mESCKAA) and suboptimal (Whitten’s Medium, + 20% O2, mESCWM) conditions. We analyzed three female cell lines per group for a total of nine mouse embryonic stem cells on Affymetrix MoGene 1.0 ST Arrays.
Project description:Assisted reproductive technologies (ART) account for 1-6% of live births in developed countries. While most children conceived using ART are healthy, increases in birth and genomic imprinting defects have been reported; such abnormal outcomes have been attributed to underlying parental infertility and/or the ART used. Here, we assessed whether paternal genetic and lifestyle factors, that are associated with male infertility and affect the sperm epigenome, can influence ART outcomes. We examined how paternal factors, Dnmt3L haploinsufficiency and/or diet-induced obesity, in combination with ART (superovulation, in vitro fertilization, embryo culture and embryo transfer), could adversely influence embryo development and DNA methylation patterning in mice. While male mice fed high-fat diets (HFD) gained weight and showed perturbed metabolic health, their sperm DNA methylation was minimally affected by the diet. In contrast, Dnmt3L haploinsufficiency induced a marked loss of DNA methylation in sperm; notably, regions affected were associated with neurodevelopmental pathways and enriched in young retrotransposons, sequences that can have functional consequences in the next generation. Following ART, placental imprinted gene methylation and growth parameters were impacted by one or both paternal factors. For the embryos conceived by natural conception, the abnormality rates were similar for WT and Dnmt3L+/- fathers. In contrast, paternal Dnmt3L+/- genotype, as compared to WT fathers, resulted in a 3-fold increase in the incidence of morphological abnormalities in embryos generated by ART. Together, the results indicate that embryonic morphological and epigenetic defects associated with ART may be exacerbated in offspring conceived by fathers with sperm epimutations.
Project description:Transcriptome analysis of mouse embryonic stem cell lines derived from embryos cultured in optimal and suboptimal conditions compared to cell lines derived from control embryos. The use of assisted reproductive technologies (ART) such as in vitro fertilization (IVF) has resulted in the birth of more than 5 million children. While children conceived by these technologies are generally healthy, there is conflicting evidence suggesting an increase in adult-onset complications like glucose intolerance and high blood pressure in IVF children. Animal models indicate similar potential risks. It remains unclear what molecular mechanisms may be operating during in vitro culture to predispose the embryo to these diseases. One of the limitations faced by investigators is the paucity of the material in the preimplantation embryo to test for molecular analysis. To address this problem, we generated mouse embryonic stem cells (mESC) from blastocysts conceived after natural mating (mESCFB) or after IVF, using optimal (KSOM + 5% O2; mESCKAA) and suboptimal (Whitten’s Medium, + 20% O2, mESCWM) conditions.
Project description:The in-vitro analysis of the hypomethylation of the imprinting control region 1 (ICR1) within the IGF2/H19 locus is challenged by the mosaic distribution of the epimutation in tissues from children with Silver-Russell syndrome (SRS). For excluding mosaicism, clonal cultures of skin fibroblasts from four children with SRS and three controls were analyzed. Cell proliferation, IGF-II secretion, and expression of IGF2 and H19 were measured. Microarray expression analysis was performed. Single cell expansion established severely ICR1 hypomethylated clones (SRShypo) and normomethylated clones (SRSnormo) from patients and controls (Cnormo). IGF2 expression was below the detection limit of the qRT-PCR assay, while H19 expression was detectable, without differences between fibroblast clones. Cell count-related IGF-II release was comparable in supernatants of SRShypo and Cnormo. Cell proliferation was diminished in SRShypo compared to Cnormo (p=.035). Microarray analysis revealed gene expression changes in SRS clones predicting a decrease of cell proliferation and a delay of mitosis. The analysis of severely ICR1 hypomethylated clonal fibroblasts did not reveal any functional differences towards the normomethylated clones with respect to IGF2 and H19 expression. Furthermore, a clear difference to the clones from healthy individuals was not detected except from a lower proliferation rate arisen from impaired cell cycle progression. 16 samples: 8 SRShypo, 4 SRSnormo, 4 Cnormo
Project description:Genomewide methylation analysis in Silver Russell syndrome patients compared to healthy controls Bisulphite converted DNA from the 24 samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip