Analysis of the evolution of chromosome abnormalities in human embryos from Day 3 to 5 using CGH and FISH

Abstract

The use of interphase fluorescent in situ hybridization (FISH) has shown that a large number of human embryos exhibit chromosomal abnormalities in vitro. The most common abnormality is mosaicism which is seen in up to 50% of preimplantation embryos at all stages of development. In this study, comparative genomic hybridization (CGH) was used to analyse 1-2 cells biopsied on Day 3 of development while the rest of the embryo was cultured until Day 5. Embryos were spread on Day 5 and analysed by FISH using probe combinations that varied depending on the CGH result, to investigate the progress of any abnormalities detected on Day 3. A total of 37 frozen-thawed embryos were analysed in this study. One gave no CGH or FISH results and was excluded from analysis. Six embryos failed to give any FISH result as they were degenerating on Day 5. Thirty embryos provided results from both techniques. According to the CGH results, the embryos were divided into two groups; Group 1 had a normal CGH result (13 embryos) and Group 2 an abnormal CGH result (17 embryos). For Group 1, three embryos showed normal CGH and FISH results, while 10 embryos were mosaic after FISH analysis, with various levels of abnormalities. For Group 2, FISH showed that all embryos were mosaic or completely chaotic. The combination of CGH and FISH enabled the thorough investigation of the evolution of mosaicism and of the mechanisms by which it is generated. The main two mechanisms identified were whole or partial chromosome loss and gain. These were observed in embryos examined on both Day 3 and 5.

The Human Cleavage Stage Embryo Is a Cradle of Chromosomal Rearrangements

“…Approximately 30% of the embryos contained a segmental aneuploidy including simple losses or gains of p-or q-terminal chromosome fragments, but again more complex segmental aneuploidies were detected consisting of a deletion of a terminal part of a chromosome and a duplication of the remaining part of that chromosome. Daphnis et al [2008] further analyzed and confirmed part of these segmental aneuploidies by FISH. Rius et al [2010] analyzed 157 blastomeres by a short CGH protocol from 22 day 4 embryos following FISHbased preimplantation genetic aneuploidy screening that was offered to 8 couples with advanced maternal age.…”

“…Low-resolution genome-wide surveys of single human blastomeres, using metaphase comparative genomic hybridization (CGH), for the first time revealed, in addition to whole chromosome aneuploidies, the occurrence of segmental chromosome imbalances in approximately 7-32% of the embryos [Voullaire et al, 2000[Voullaire et al, , 2002Wells and Delhanty, 2000;Wilton et al, 2003;Daphnis et al, 2008;Rius et al, 2010] ( table 1 ). Voullaire et al [2000] detected a postzygotic breakage of chromosome 2 at q31 and of chromosome 10 at q11.2 in 1 out of 12 good quality embryos (8%) yielding cells with reciprocal loss and gain of the 2 fragments from each chromosome.…”

“…Wilton et al [2003] reported the loss of the 9q-arm in 1 blastomere, the nature of the other segmental aberrations were not described. Daphnis et al [2008] biopsied 1 or 2 blastomeres from 37 human embryos 3 days after IVF and analyzed them by single-cell CGH. The embryos, minus 1 or 2 blastomeres, were further developed to day 5 and FISH was performed for a number of the aberrant loci that were detected by single-cell CGH.…”

“…First, most embryos were obtained from patients with fertility problems [Daphnis et al, 2008], including patients with an assumedly increased risk for embryonic aneuploidy due to recurrent implantation failure [Voullaire et al, 2002] and advanced maternal age [Rius et al, 2010]. Hence, little was known about the chromosomal intactness within blastomeres of embryos from normal fertile partners.…”

Environmental Hazard in the Aetiology of Somatic and Germ Cell Aneuploidy

“…Chromosome losses and gains, presumably due to anaphase lagging, are a common feature of mosaicism in human preimplantation embryos [e.g. Daphnis et al, 2008]. Currently, it is still unclear whether mitotic nondisjunction is a normal phenomenon in early human preimplantation development or mainly related to suboptimal culture conditions in assisted reproduction.…”