Analysis of H19 methylation in control and abnormal human embryos, sperm and oocytes

Abstract

ART is suspected to generate increased imprinting errors in the lineage. Following an intra cytoplasmic sperm injection (ICSI) procedure, a certain number of embryos fail to develop normally and imprinting disorders may be associated to the developmental failure. To evaluate this hypothesis, we analysed the methylation profile of H19DMR, a paternally imprinting control region, in high-graded blastocysts, in embryos showing developmental anomalies, in the matching sperm and in oocytes of the concerned couples when they were available. Significant hypomethylation of the paternal allele was observed in half of the embryos, independently of the stage at which they were arrested (morula, compacted morula, pre blastocyst or BC-graded blastocysts). Conversely, some embryos showed significant methylation on the maternal allele, whereas few others showed both hypomethylation of the paternal allele and abnormal methylation of the maternal allele. The matching sperm at the origin of the embryos exhibited normal methylated H19 patterns. Thus, hypomethylation of the paternal allele in the embryos does not seem inherited from the sperm but likely reflects instability of the imprint during the demethylating process, which occurred in the early embryo. Analysis of a few oocytes suggests that the defect in erasure of the paternal imprint in the maternal germ line may be responsible for the residual methylation of the maternal allele in some embryos. None of these imprinting alterations could be related to a particular stage of developmental arrest; compared with high-grade blastocysts, embryos with developmental failure are more likely to have abnormal imprinting at H19 (Po0.05).

Methylation Status of H19/IGF2 Differentially Methylated Region in in vitro Human Blastocysts Donated by Healthy Couples

“…In contrast to our results and the aforementioned studies [21,33] , Chen et al [27] reported higher degree of DNA methylation (49.4%±9.7% as compared to our value 37.85±4.87%) in human embryos. However, the main difference between our study and Chen et al, The number of clones is shown in the raw of "Seq.…”

“…This observation, to a certain degree, is in concordance with a previous report from the Lefèvre group [21] . Using genome-wide analysis of DNA methylation of early human blastocyst embryo, Okae et al [33] also reported an average of methylation levels with a median of around 40% for gDMRs, which is close to the value reported in this study and Lefèvre group's study [21] . It is important to note that Okae and colleagues [33] did not specifically report the methylation level of ICR1.…”

“…To our knowledge, there is no other study to assess the DMR in high-quality human blastocysts with specific attention to gDMRs or ICR1. Therefore, the values obtained in this study and those reported by others [21,33] , which are close to each other, can be considered as reference value for future studies.…”

“…The first-round PCR product (2 µL) was used as DNA input for amplification in the second-round nested PCR mix with the following conditions: 95°C for 10 min, 39 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 20 min. Four independent nested PCRs were performed per template to avoid amplification bias, which may lead to preferential amplification of maternal or paternal DMRs [21] . PCR products were sub-cloned into a pTZ57R/T cloning vector (InsTAcloneTM PCR Cloning Kit, Fermentas, Lithuania) according to the manufacturer's protocol.…”

“…Although assisted reproductive techniques (ARTs) are now well-established and globally applied, several epidemiological studies have shown the association of ART with the increased incidence rate of certain imprinting disorders such as BWS and RSS [19] . This phenomenon has been mainly attributed to the coincidence between gamete and embryo in vitro manipulations events and the normal pattern of epigenetic reprogramming, which begins at fertilization and continues during pre-implantation embryo development [6,20,21] . Despite several investigations in the gametic DMRs (gDMRs) in human pre-implantation embryos, there is still space for further studies.…”