Triple colour fluorescent in-situ hybridization for chromosomes X,Y and 1 on spare human embryos

Abstract

The potential for implantation of human embryos obtained by in-vitro fertilization is presumably determined to a large extent by their chromosomal constitution but cytogenetic analysis of preimplantation embryos has been hampered by a number of practical and technical problems. With the advent of fluorescent in-situ hybridization (FISH) a practical method for numerical chromosomal analysis has become available. A limited amount of data has been obtained with FISH on human embryos using probes binding to chromosomes X, Y, 16, 18 and 13/21 combined or for chromosomes X and Y or 1 and 17. It was our purpose to extend these data by the combined analysis of chromosomes X, Y and 1 in spare human embryos. A short fluorescent in-situ hybridization procedure involving the simultaneous use of three deoxyribonucleic acid probes detected with red, green, and a mixture of red and green was used to determine chromosomal abnormalities in 116 spare embryos with a poor morphological score and/or displaying one or more multinucleated blastomeres. The majority of the embryos was obtained by intracytoplasmic sperm injection. Less than half of the embryos (n = 54) were diploid and only 39 of them were uniformly XY11 or XX11; two embryos showed a non-disjunction and 13 embryos were aneuploid. Of the remainder, 22 were mosaic, nine were either haploid, triploid or tetraploid and 12 embryos were classified as chaotic. The latter pattern was particularly frequent in multinucleated blastomeres. Our data are comparable with those obtained with FISH using other chromosomal probes and confirm that the majority of preimplantation embryos carry a numerical chromosomal defect. Aneuploidy for chromosome 1 does not appear to be more common in preimplantation embryos than is reported for other chromosomes. Although the high incidence of chromosomal anomalies is presumably biased by the fact that only embryos with a poor morphological score were analysed, it nevertheless indicates that natural selection is the foremost reason for the low implantation rates of human preimplantation embryos in in-vitro fertilization (IVF) programmes.

Application of FISH method for preimplantation genetic diagnostics of reciprocal and Robertsonian translocations

“…Indeed, the risk of having unbalanced sperm is 7-16% [12,[24][25][26] and the risk of having unbalanced oocytes 32-36% [13,27] in transformation carriers as demonstrated by FISH method, reflecting higher maternal impact on the embryo [16,22]. Combined with factors such as maternal age [28], abnormal embryo morphology [20,29], lack of cell cycle check-points in the early embryonic mitotic divisions [30], all this issues cumulate and result in very high proportion of aneuploid embryos.…”

Insights on blastomere nuclearity

“…Studies on the chromosomal status of MN embryos indicate that most of them resulted in chaotic or abnormal complement numbers [17,24]. Kligman et al [8] found 74.5% of chromosomal abnormalities in MN embryos versus 32.3% on mono-nucleated embryos using five FISH probes.…”

Cytoplasmic fragmentation in activated eggs occurs in the cytokinetic phase of the cell cycle, in lieu of normal cytokinesis, and in response to cytoskeletal disorder

“…In human embryos, extensive cytoplasmic fragmentation is almost always accompanied by other cytoplasmic and nuclear anomalies, for instance, blastomere multinucleation and chromosomal mosaicism (Pellestor et al, 1994;Kligman et al, 1996;Laverge et al, 1997;Marquez et al, 2000). Collectively, these abnormalities can explain the abnormal pre-and post-implantation development often seen in fragmented embryos (Jackson et al, 1998;Alikani et al, 1999Alikani et al, , 2000Hardy et al, 2003;Racowsky et al, 2003;Van Royen et al, 2003), though it is important to keep in mind that such embryos are not necessarily non-viable, and that even if non-viable, they may still contain viable cells (Alikani and Willadsen, 2002).…”