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Z. P. Nagy, H. I. Kort, J. B. Massey, C. W. Elsner, T. Taylor, A. Jones; Reproductive Biology Associates, Atlanta, GA
Objective: Application of preimplantation genetic diagnosis (PGD) is rapidly growing and provides important advantages compared to the alternative method of the prenatal diagnosis. Most of the cases performed in the world have been for the indication of aneuploidy both for diagnostic and for treatment purposes. Today the standard method of PGD requires the removal of a cell from a day 3, 6-8 cell cleavage stage IVF embryo, followed by fixation of the cell and FISH analysis. Although there are some minor variations in the different PGD protocols, all of them require the use/biopsy of an intact and living cell. The objective of the present study was to investigate if it was possible to augment the PGD protocol in a way that it can be used for FISH analyses using degenerated/lysed embryonic cells instead of intact/living cells.
Design: Experimental research protocol.
Materials and Methods: A total of ten embryos (with a total of 52 cells) donated to research were involved in this study. A standard slow freezing and rapid thaw protocol was applied. Both intact and degenerated cells were biopsied from thawed, disaggregated and fixed to a glass slide using a modified Tarkowski’s protocol. Both live and degenerated cells were hybridized with 5 chromosomal probes (13, 16, 18, 21, 22) and were evaluated by fluorescent microscope.
Results: The ten embryos contained a total of 52 blastomeres from what 20 survived intact and 32 cells degenerated. All of the intact cells provided results on all chromosomes, while 85% of the lysed cells after thawing provided results (NS) including one embryo where all degenerated cells uniformly failed to provide signals (4 of the 6 cells). In 7 embryos the 13 degenerated cells displayed normal diploid set for the five chromosomes screened corresponding to the 15 living cells from the same embryos. One embryo appeared to be a haploid mosaic with one live cell showing a monosomy for chromosome 21, and the remaining lysed cells showing at least one monosomy for all other chromosomes examined. The final embryo was a diploid mosaic with two live cells both showing a monosomy and trisomy for chromosomes 16 and 21 respectively, while the remaining 3 degenerated cells were disomic for all chromosomes analyzed.
Conclusion: The results of the present study demonstrate that it is possible to use degenerated/lysed blastomeres for PGD. It has also been shown that fixation and hybridization is similarly efficient when performed on degenerated cells compared to live/intact cells using an augmented PGD protocol. This new approach is especially important for cryo-ET cycles, because it does not require the removal of live blastomere(s) from the embryo which in return would assist to preserve a higher viability and developmental potential of the embryo.
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