27 - 30 May 2005

IMPRINTING AND RISK FOR ART CHILDREN De Rycke M [1], Bonduelle M [1], Geuns E [1], Van Steirteghem A [2] en Liebaers I [1] Centre for Medical Genetics [1] and Centre for Reproductive Medicine [2] University Hospital and Medical School Vrije Universiteit Brussel (Brussels Free University) Laarbeeklaan 101 B- 1090 Brussels Belgium

Many couples rely on assisted reproductive technologies (ART) to overcome infertility problems. Concern raised after recent reports about a possible higher incidence of two rare imprinting syndromes, Beckwith-Wiedemann (BWS) and Angelman syndrome (AS), in children born after ART.

Genomic imprinting is an epigenetic mechanism of gene expression which involves differential epigenetic modifications, “imprints’, leading to the preferential or exclusive expression of one of two parental alleles of a gene. Although BWS and AS may be due to genetic as well as epigenetic defects, it is remarkable that only epigenetic defects (maternal methylation loss) have been found in the reported cases after ART. In our follow-up study, three BWS cases have been reported so far; one BWS with omphalocoele and another with a small umbilical hernia and macroglossia in the ICSI group and one case of omphalocoele in the IVF group.

It is not known which specific factor or mechanism is responsible for the imprinting defects, but it has been suggested that the culture and manipulations of gametes and embryos during ART would disturb the imprinting process. In vitro embryo culture may interfere with the maintenance of maternal methylation patterns at imprinted loci in preimplantation embryo or with maternal imprint resetting in case of genes which are only reset during late oogenesis (i.e. later than the germinal vesicle stage). Another hypothesis is that the hormonal stimulation of the ovaries interferes with maternal imprint resetting. The basic processes of imprint resetting and maintenance have not been well studied in the human.

We studied the timing of maternal imprint establishment by analysis of methylation patterns of selected imprinted regions in different stages of oogenesis (germinal vesicles (GV), metaphase I (MI) oocytes and MII oocytes) using bisulphite sequencing. Imprint maintenance during the preimplantation period was analysed in human ICSI embryos that were donated for research. We studied the methylation status of the imprint control regions (ICR) of LIT1 (associated with BWS) and SNRPN (associated with Prader-Willi Syndrome/AS) which are both maternally methylated regions. The intergenic (IG) region of DLK1-GTL2 was selected as paternally methylated region. The results for SNRPN and LIT1 showed that the maternal methylation marks are already established in the germinal vesicle stage and that mainly unmethylated patterns are present in spermatozoa. Embryo analysis for these specific regions indicated that the differential methylation patterns of the gametes are stably maintained during the preimplantation period.

The methylation imprints for the IG region are differentially set in human gametes with methylation of the paternal allele. These differential methylation patterns are inherited in the embryos but they are only maintained till the 4-cell-stage. The finding of imprinting relaxation with intermediate methylation patterns at later embryonic stages corresponds with the results in somatic cells.

Overall, the finding of similar methylation patterns in embryos and somatic cells suggests that the process of imprint maintenance is not disturbed for the three regions under study.

List of abstracts from the 3rd International Conference on the Female Reproductive Tract