Cryo-damaged single blastomeres may be used efficiently for preimplantation genetic diagnosis (PGD) for the purpose of screening for single gene defect

Application of preimplantation genetic diagnosis (PGD) is rapidly growing and provides important advantages compared to the alternative method of the prenatal diagnosis. The cases performed in the world have been for the indication either of aneuploidy or for preventing the inheritance of commonly occurring single gene defects (such as cystic fibrosis). 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 transferring the cell into a PCR tube, performing the PCR reaction and analyzing the results. 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 apply the PGD protocol in a way that it can be used for PCR analyses using lysed embryonic cells instead of intact cells. Experimental research protocol. A total of 6 embryos 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 transferred to a PCR tube. A total of 24 blastomeres were removed from the embryos; and they were separated according to intactness. PCR tubes were marked correspondingly if they contained a single living or a single degenerated/lysed blastomere. Both live and degenerated cells were submitted to fluorescent polymerase chain reaction (PCR) and subsequent restriction enzyme digestion and fragment analysis on an automated sequencer. The test was designed to analyze the region corresponding to the most frequently occurring mutation represented by deletion of 3 basepairs, at the amino acid position 508 of the putative product of the CF gene. A total of 24 blastomeres were separated from the frozen-thawed embryos. Twelve of them were completely intact and therefore considered to be alive while the other 12 blastomeres were displaying all the signs of cryodamage therefore were considered as degenerated. All the 12 intact blastomeres provided clear signals (100%) on the gene region that was amplified corresponding to a non-mutated sequence, as expected. Equally, all the 12 degenerated blastomeres provided clear signals (100%) to the same region corresponding to a non-mutated sequence. The results of the present study demonstrate that it is possible to use degenerated/lysed blastomeres for the purpose of preimplantation genetic diagnosis to detect alterations in the gene sequences. It has also been shown that amplification of genetic material obtained from a single cell is similarly efficient when performed on degenerated cells compared to live/intact cells using a non-altered protocol. As the results demonstrated, fragmentation of nuclear DNA in the degenerated cells may not present a high risk (that would interfere with amplification efficiency) depending perhaps on the timing of blastomere separation relative to the thaw procedure. 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. Extension of the present study to test different gene sequences is warranted in order to demonstrate that this new approach is well applicable for screening various types of single gene defects.