Gamete and Embryo Research Laboratory, The Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center, West Orange, NJ 07052, USA
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Abstract |
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Key words: blastocyst/chromosome abnormalities/embryo development/extended culture
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Introduction |
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Another aim of this study was to determine if culture to blastocyst stage could be an effective tool to select against chromosomally abnormal embryos. Embryo selection can be a powerful tool in establishing high implantation rates. The selection can be pro-active, against morphologically, developmentally or genetically abnormal embryos, or indirect, by culturing the embryos as long as possible. This extra challenge appears to arrest a proportion of unsuitable embryos. Many morphological abnormalities observed between the zygote and cleavage stages have been related to chromosome abnormalities (Munné and Cohen, 1998). However, not all morphologically abnormal embryos are chromosomally affected, and aneuploidy is not associated with morphological abnormalities, at least up to day 3 of development. Therefore, it would be of interest to find a link between blastocyst formation and/or morphology and aneuploidy.
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Materials and methods |
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Published scoring criteria (Munné and Weier, 1996) were used to differentiate FISH errors from mosaicism. To classify the different chromosome abnormalities detected by FISH, we used the following criteria. (i) Normal, aneuploid, haploid and polyploid embryos were those where all the cells of the embryo had the same chromosome constitution or <10% abnormal cells. The 10% threshold is included here because it is the approximate FISH error level with this protocol. (ii) Diploid mosaic embryos were those mosaics that had a diploid line or, on average, the number of chromosomes per cell was diploid. Similarly, when the average number of cells was haploid or polyploid they were classified as haploid and polyploid mosaics respectively. However, to simplify the analysis, polyploid and haploid mosaics were grouped with pure polyploid and haploid embryos. (iii) Some embryos showed chaotic chromosome complements, and, while several authors (Harper et al., 1995b; Harper and Delhanty, 1996
) consider such embryos as a separated mosaic category, in the present study, these embryos were counted as mosaics following the above classification (diploid, haploid or polyploid mosaics). (iv) Mosaic embryos were sub-classified into extensive and limited mosaics if they had more or less than 38% (3/8) abnormal cells. This definition was applied because of the high frequency of mosaicism in human embryos, which suggests that low doses of mosaicism (limited) may not be detrimental to embryo development (Munné et al., 1997
). The distinction between extensive and limited mosaicism is not made for haploid and polyploid mosaic embryos because in these, all cells are abnormal by definition. (v) Within the diploid mosaic group, we differentiated those that had a diploid line and a polyploid line (2n/4n mosaics) from the others because the occurrence of polyploid cells in a blastocyst is considered a normal feature of human embryo development (Angell et al., 1987
; Benkhalifa et al., 1993
; Drury et al., 1998
; Evsikov and Verlinsky, 1998
) Therefore, if the proportion of polyploid cells was <38%, they were considered normal.
A 2-test was applied to compare blastocyst formation rates among the different groups.
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Results |
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Of the 83 aneuploid embryos, 23 were monosomics of which only one monosomy 21 and one monosomy X reached the blastocyst stage. Thirty-five trisomies were detected and 37% of those became blastocysts. There were 24 aneuploid embryos that were also extensive mosaics, none of which developed further than the morula stage. The remaining embryo was polyploid mosaic and in addition aneuploid with half the number of chromosomes 21 per cell than the ploidy of each cell (i.e. if the cell was tetraploid, there were two chromosomes 21). Interestingly, this embryo developed to the blastocyst stage. In total, 19% of the aneuploid embryos reached the blastocyst stage, but this low figure is mostly due to arrest of monosomic embryos.
There were 82 diploid mosaics (excluding limited 2n/4n mosaics grouped here with the normal ones) of which nine were extensive 2n/4n mosaics, 33% of which reached blastocyst stage. There were also 24 mosaics with diploid and aneuploid cell lines, and 49 embryos with chaotic cells (Figure 1). Twelve per cent (n = 6) of the chaotic mosaics reached blastocyst stage, but only 8% (n = 2) of the diploid/aneuploid embryos reached that stage. None of the six chaotic mosaic blastocysts had >60 cells (average: 30 cells, range: 1551) while the average number of nuclei per blastocyst found in this study was ~114 (5744/51, range: 19410). Twenty-one per cent of polyploid and none of the haploid embryos developed to blastocyst stage.
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Confirmation of PGD diagnosis
A total of 198 embryos with previous PGD results were reanalysed in day 5. The most serious error classified three embryos as normal by PGD when they should have been considered as abnormal based on the criteria applied in this study. However, all three embryos had a diploid line, being extensive mosaics for chromosome 16, chromosomes 16, 21 and 22 plus polyploidy and finally an extensive mosaicism for polyploidy (considered as an abnormal, and therefore unsuitable for transfer in this study). Fourteen embryos were false abnormal, being seven false monosomies, two false trisomies and five double false aneuploidies. In three of the cases, embryos were limited mosaics for the chromosomes diagnosed as aneuploid by PGD. The total error rate of false normal and abnormal in this study was 8.6%.
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Discussion |
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Our data suggest that the modest developmental rates through extended culture are attributable, at least in part, to chromosomal abnormalities. A decrease in embryo survival from cleavage stage to blastocyst is observed in all types of human embryos. Moreover, the results seem to suggest that there is a strong developmental block at compaction, with 65% (120/184) of chromosomally abnormal embryos arresting before compaction, compared with only 28% (9/32) of normal embryos (2-test, P < 0.001). The other suggested block, around cavitation (Janny and Ménézo, 1996
) was less evident, with 19% (35/184) chromosomally abnormal embryos arresting around morula stage compared with 6% (2/32) of normal embryos (not significant).
The selection against chromosomally abnormal embryos is not the same in all groups. Monosomies, with the exception of monosomy X and 21, haploidies and aneuploidies, combined with extensive mosaicism never developed to blastocyst. Interestingly, the types of chromosome abnormalities observed in blastocysts have a striking resemblance to those found in first trimester conceptuses, that is, trisomies, polyploidy, monosomy X and 21, and limited mosaics (Simoni et al., 1986; Simpson, 1990
; Wolstenholme, 1996
). A recent report by (Evsikov et al., 2000
) also indicated that embryos with unbalanced translocations, commonly found in first trimester conceptuses, also easily reached blastocyst stage.
The specific arresting stages of unique chromosomal groups require further investigation, particularly as far as developmental anomalies are concerned. For instance, do monosomic embryos arrest when the embryonic genome activates or do they fail to form structural and functional junctions after genomic activation? Of these abnormalities, the least understood is mosaicism. This is a common finding in cleavage-stage human embryos (Munné et al., 1994c; Delhanty et al., 1997) as well as human blastocysts (Clouston et al., 1997
; Evsikov and Verlinsky, 1998
; Bergers-Janssen et al., 1999
; Veiga et al., 1999
; Magli et al., 2000
; Ruangvutilert et al., 2000
). We found that 70% of the embryos analysed, independently of the type of ploidy, were mosaics. However, not all types of mosaicism and proportion of abnormal cells have the same impact on embryo development. Although polyploid cells in normal embryos are considered a normal feature of blastocyst formation in all mammalian species studied (Barlow and Sherman, 1972
; Hare et al., 1980
; Long and Williams, 1982
; Murray et al., 1986
; Angell et al., 1987
), a high proportion of polyploid cells may be detrimental. In this study, 2n/4n mosaics with <38% abnormal cells developed 78% of the time to blastocyst stage compared with only 33% of those with >38% abnormal cells (P = 0.021). Mosaicism due to aneuploid cells seems somehow to interfere with embryo development for reasons that are not entirely clear. Only 11% of diploid mosaic embryos develop to blastocyst, but we did not find statistical differences according to the percentage of abnormal cells (17% for limited versus 6% for extensive mosaicism). However, aneuploidy combined with extensive mosaicism may have a stronger effect as, in this study, none developed to blastocyst. Some chaotic mosaic embryos developed to blastocyst, but were probably developmentally compromised, since they never had more than 60 cells. Though the observations relating blastocyst morphology to chromosomal status are preliminary, we have observed many chromosomally abnormal embryos with abnormalities at the blastocyst stage, including multi-cavitation, extensive exclusion of cells, and an irregularly formed trophectoderm.
In conclusion, while an early developmental block seems to prevent full differentiation of chromosomally abnormal embryos, extended culture does not universally select against all anomalies. The data also imply that the decrease in implantation with advanced maternal age is partially due to the arrest of chromosomally abnormal embryos prior to blastocyst formation.
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Acknowledgements |
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Notes |
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References |
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Submitted on November 6, 2000; accepted on May 31, 2001.