Fluorescent in-situ hybridization on human embryos showing cleavage arrest after freezing and thawing

Abstract

Our current freezing-thawing policy is to transfer only embryos that cleave further in the 24 h following thawing. The purpose of our study was to investigate the incidence of numerical abnormalities for chromosomes X, Y and 1 in blastomeres of human preimplantation embryos that survived cryopreservation but did not cleave further after thawing. A total of 63 embryos surviving a freezing-thawing cycle but not cleaving further within 24 h after thawing were screened. Of the 63 screened embryos that showed cleavage arrest 24 h after thawing, 56 embryos (88.9%) remained arrested 48 h after thawing and slightly more than half of these (29/56; 51.8%) showed further deterioration in morphological quality. Seven embryos (11.1%) showed signs of further cleavage; five embryos showed additional cleavage of one blastomere and two developed a blastocoelic cavity. Fluorescent in-situ hybridization (FISH) with three specific probes for simultaneous detection of chromosomes X, Y and 1 was performed and was successful in 60 out of 63 embryos. Of these successfully labelled embryos, 26 (43.3%) were in the diploid range: 12 (20%) were uniformly diploid for the chromosomes X, Y and 1; three embryos showed aneuploidy in all their blastomeres (two were XXY-karyotype and one was monosomy 1) and in 11 embryos nondisjunction was detected. Thirteen embryos were categorized as being either haploid, triploid, tetraploid or hexaploid. Nine embryos were classified as mosaic and 12 as being highly abnormal or chaotic. These preliminary results suggest that a large proportion of embryos that do not cleave further after freezing and thawing carry chromosomal aberrations. This finding supports our policy of not transferring cryopreserved embryos which do not cleave further 24 h following thawing.

Embryonic death and the creation of human embryonic stem cells

“…Cleavage arrest most often reflects severe genetic abnormalities but -and this is the crucial point -not all cells of arrested embryos need be abnormal for arrest to occur. In a study by Laverge et al, out of 166 frozen embryos thawed for further growth, 78 embryos remained arrested at 24 hours after thawing, and 71 showed no sign of further cleavage at 48 hours (12). Fluorescence in situ hybridization with probes for chromosomes X, Y, and 1 was performed on all blastomeres of 63 arrested embryos, and 80% showed genetic abnormalities.…”

Chromosome abnormalities in human embryos

“…Since most polyploid embryos arrest before the onset of genome activation, which occurs around the 4-to 8-cell stage (Braude et al, 1988), oocyte quality or culture conditions may be the cause of their arrest. Interestingly, embryos that at the time of freezing were not arrested but that were not cleaved after 24 h in culture after thawing are rarely chromosomally normal (20%; Laverge et al, 1998) and show high rates of polyploidy and mosaicism. This reinforces the hypothesis that cytokinesis arrest, at any stage, results in polyploidy in all (pure polyploidy) or some cells (diploid/polyploid mosaicism).…”

“…This means that arrested embryos cannot be analysed at all, and implies that mosaicism is severely underestimated. Fluorescence in-situ hybridization (FISH) has been used with much higher efficiencies (85-95%) to study the chromosome constitution of cleavage-stage human embryos, arrested or not (Griffin et al, 1992;Benkhalifa et al, 1993;Munné et al, , 1994aMunné et al, , 1995aHarper et al, 1994a;Munné and Weier, 1996;Laverge et al, 1998). FISH with multiple probes can differentiate polyploidy from aneuploidy and also haploidy from monosomy, and when most or all cells of an embryo are analysed, mosaicism can be differentiated from FISH or fixation failure, as well from aneuploidy (Munné et al, 1994b,c).…”

A high degree of aneuploidy in frozen-thawed human preimplantation embryos

“…An impressive body of information is available on morphologically abnormal embryos and cleavage arrested embryos as well as for embryos with abnormal fertilisation, showing a high degree of aneuploid embryos (Bergere et al 1995;Coonen et al 1994;Kligman et al 1996;Laverge et al 1997;Laverge et al 1998;Munne et al 1994;Palermo et al 1994;Staessen and Van Steirteghem 1997). However, only a limited amount of data has so far been obtained for chromosomal abnormalities in normally developing human preimplantation embryos.…”