PGD Procedure

Removing the Cells
At this time the embryo is still surrounded by a glycoprotein "jelly coat" of the zona pellucida, so to remove any cells this jelly coat must first be cut open. This can be done either using acidified culture medium that "dissolves" the zona pellucida, or more conveniently can be done with a simple laser to drill the exact size hole to allow a glass micro-tool to be pushed through the hole and extract a cell. The hole that is drilled is usually made a little smaller than the cell itself, and this helps maintain the integrity of the embryo within the jelly coat during further development in the IVF lab. The embryo during manipulation on the inverted microscope is held in a warm culture medium that helps to allow the cells to be removed with a minimum of trauma to the overall embryo. The removal of up to a quarter of an embryo is not known to be deleterious to the further development of that embryo, as the embryo at this early stage of development can compensate for this loss of material. All cells at this stage are still totipotent, meaning, all are fully capable of directing further embryonic development.

Analysis
Once a single cell (a blastomere) is removed, the cell is either fixed on a glass slide for chromosomal analysis, or placed in a small tube of chemical buffer for single gene diagnosis. The cells are then analyzed using techniques called fluorescence in situ hybridization (FISH) or DNA analysis. During the genetic analysis, the embryos are usually grown to the fifth day of development at which time they may either be at the morula or blastocyst stage. Those embryos found to be free of genetic abnormalities are then placed into the uterine cavity.

Other Issues

Misdiagnosis
Misdiagnosis can occur due to mosaicism within the embryo. Some embryos may contain blastomeres (cells produced by the cleavage [division] of a fertilized egg) which are genetically normal and, within the same embryo, other blastomeres which are abnormal. This is called mosaicism. For this reason, a diagnosis may be incorrect. This may result in the transfer of an embryo carrying a chromosome abnormality or the failure to transfer a normal embryo.

Experimental error can also account for a misdiagnosis. Improper cell fixation techniques, DNA denaturation errors, allelic drop-out or amplification of contaminated DNA can lead to a wrong diagnosis.

A recent report of the European Society of Human Reproduction and Embryology (ESHRE) documented the PGD results from 25 consortium members from 1999 to 2001. There were 8 confirmed misdiagnoses from 451 PGD tested embryos; 1% (3/305) for chromosome analyses and 3.4% (5/146) for single gene disorders.

Are there risks associated with PGD?
The micromanipulation techniques used for blastomere biopsy are safe with little risk to the embryo. The risk of accidental damage to the embryo during biopsy is less than 1%. There is no risk to the embryo following chromosomal or single gene defect analysis because the analyzed cells are not put back into the embryo. There may be a slightly lower likelihood of implantation after embryo biopsy compared to an embryo not having been biopsied. Other risks may become apparent over time, but to date appear quite limited and need to be weighed against the potential benefits for each couple.

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