Abnormal methylation of KCNQ1OT1 and differential methylation of H19 imprinting control regions in human ICSI embryos.

Summary To evaluate the integrity of genomic imprinting in embryos that failed to develop normally following intracytoplasmic sperm injection (ICSI), we analysed the methylation profile of H19 and KCNQ1OT1 imprinting control regions, H19DMR and KvDMR1 respectively, in high-grade blastocysts and in embryos that exhibited developmental anomalies. Significant hypomethylation of KvDMR1 was specifically observed in 5/5 atypical blastocysts graded BC, which probably reflected the vulnerability of the imprint in the inner cell mass during the methylation remodelling phase in the early embryo. In addition, KvDMR1 was hypermethylated in 2/5 CC graded atypical blastocysts and in 2/8 embryos that exhibited developmental delay. H19DMR appeared differentially methylated in all groups of embryos. DNA methyltransfersase 1 (DNMT1) expression was similar in most of the tested embryos and could not account for the abnormal methylation patterns of KvDMR1 observed.

The primate-specific microRNA gene cluster (C19MC) is imprinted in the placenta.

Imprinted genes play crucial roles in mammalian development and disruption of their expression is associated with many human disorders including tumourigenesis; yet, the actual number of imprinted genes in the human genome remains a matter of debate. Here, we report on the unexpected finding that the chromosome 19 microRNA cluster (C19MC), the largest human microRNA gene cluster discovered so far, is regulated by genomic imprinting with only the paternally inherited allele being expressed in the placenta. DNA methylation profiling identified a differentially methylated region (C19MC-DMR1) that overlaps an upstream CpG-rich promoter region associated with short tandem repeats. It displays a maternal-specific methylation imprint acquired in oocytes and generates a complex population of large, compartimentalized non-coding RNA (ncRNA) species retained in close proximity to the C19MC transcription site. This occurs adjacent to, but not within, a poorly characterized nuclear Alu-rich domain. Interestingly, C19MC maps near another imprinted gene, the maternally expressed ZNF331 gene, and therefore may define a novel, previously unrecognized large imprinted primate-specific chromosomal domain. Altogether, our study adds C19MC to the growing list of imprinted repeated small RNA gene clusters and further strengthens the potential involvement of small ncRNAs in the function and/or the evolution of imprinted gene networks.

Vitrification at the germinal vesicle stage does not affect the methylation profile of H19 and KCNQ1OT1 imprinting centers in human oocytes subsequently matured in vitro.

OBJECTIVE: To evaluate the integrity of genomic imprinting in oocytes vitrified at the germinal vesicle (GV) stage and in vitro matured (IVM) after thawing. DESIGN: Clinical research and application. SETTING: University-based fertility center. PATIENT(S): Immature oocytes were donated for research by patients who were included in an intracytoplasmic sperm injection program. INTERVENTION(S): Immature oocyte retrieval after ovarian stimulation, followed by oocyte vitrification, thawing, and IVM. MAIN OUTCOME MEASURE(S): Methylation profile of H19 and KCNQ1OT1 imprinting control regions, H19DMR and KvDMR1, respectively. RESULT(S): Among 184 vitrified GV oocytes, 102 survived thawing (55.4%), 77 (75.5%) of which reached the meiosis II (MII) stage after IVM. One hundred twenty control GV oocytes were only subjected to IVM; 70.8% reached the MII stage. GV vitrified as well as control oocytes acquired full imprint at KvDMR1 after IVM and generally retained the unmethylated state of H19DMR. CONCLUSION(S): For the first time, we show that oocyte vitrification does not affect the methylation profile of H19DMR and KvDMR1: during their IVM, vitrified GV oocytes acquire DNA methylation in the maternally imprinted KCNQ1OT1 gene with the same efficiency as fresh GV oocytes; the vitrification process does not alter the unmethylated state of the paternally imprinted H19 gene.

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

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 (P<0.05).