In order for the genetic makeup of an embryo to be determined prior to the time of embryo transfer there are 2 separate steps, which are important.
The first of these is embryo biopsy. This is the procedure for removing a cell or cells from an embryo to be submitted for testing. An embryo may be biopsied either on the third day following oocyte retrieval (six to eight cell stage) or as we now do it on the fifth day (blastocyst stage). The procedure used is similar to that for Assisted Hatching (AH) where an opening is made in the outer shell of the embryo with a laser. The opening is somewhat larger than the one made for Assisted Hatching because it is necessary to remove entire cells from the embryo for testing. This is an extremely delicate procedure, for the removal of cells from an embryo may result in damage to the embryo that prevents it from developing further.
Once the embryo has been biopsied, the cell or cells are sent to a different laboratory for testing. There are relatively few laboratories in the United States that have the ability to do the testing required once the cells have been obtained. Therefore, it is common for the ART laboratory to transport the cells to the laboratory that it uses for testing. Whereas initially the embryo biopsy procedure was performed with a single cell removed from a day 3 embryo, we now exclusively do the biopsy on day 5. That is because when an embryo is 3 days old, not always are all of the cells genetically identical. This is referred to as mosaicism. Mosaicism can result in an inaccurate diagnosis since the cell removed might be the only abnormal one in an otherwise normal embryo or conversely the only normal cell in an otherwise abnormal embryo. In contrast, an embryo that is 5 days old has hundreds of cells so it is possible to remove 5-10 cells for testing. Not only is there more genetic material (DNA) available but the problem of mosaicism is minimal. We and others have found that this results in a much more accurate diagnosis and therefore better results. The second step in the process is determination of the genetic information in question. There are two types of genetic information that are of interest. The most common test performed is the determination of the chromosomal composition of the embryo. This is referred to as aneuploidy screening so we refer to this type of testing and Preimplantation Genetic Screening (PGS). Each normal embryo contains 46 chromosomes, 23 of which are contributed by the sperm and 23 by the oocyte. An embryo that contains 46 normal chromosomes is called a euploid embryo. An embryo that has more or less than 46 chromosomes is called an aneuploid embryo.
There are two types of chromosomes, the sex chromosomes (X and Y) which determine the gender of the embryo and the autosomes (1-22) which determine almost everything else. The autosomes are normally present in pairs, that is, two each of chromosome 1, two of chromosome 2, two of 3, etc. The sperm contributes one sex chromosome (X or Y) and 22 autosomes. The oocyte contributes one sex chromosome (X only) and 22 autosomes. The other type of genetic information that we can test is the presence or absence of a specific gene in a given embryo. This type of information is useful when one or both of the prospective parents are known to be a carrier of a gene responsible for a particular disease. Because we can therefore diagnose the embryo as having a disease we refer to this type of testing as Preimplantation Genetic Diagnosis (PGD).The number of diseases known to be caused by a single gene abnormality is growing as researchers learn more about the composition of the human genome.
Currently, the most frequently tested genes are those giving rise to diseases such as Cystic Fibrosis, Tay- Sachs, Hemophilia, and Sickle Cell Disease but there are currently more than 1000 diseases that can be diagnosed by this technique. This type of testing is much different than chromosomal testing. Chromosomes are very large compared to single genes. Each gene is composed of a small piece of DNA that in turn is made from combinations of four basic molecules hooked together in a precise sequence. There are many genes present in each chromosome so in order to detect whether or not the gene in question is present, it must be separated from the chromosome and then copied millions of times so that there is enough present to be seen by laboratory detectors. This is done by cutting the gene out of the chromosome by enzymes called restriction endonucleases. These enzymes will dissolve the attachment of the gene to the chromosome in a very precise manner so that only the sequence of DNA that is needed will come out. This piece of DNA corresponding to the gene in question is then put through a process called the Polymerase Chain Reaction (PCR). PCR is a process that makes copy after copy of the DNA sequence in a very short period of time. Millions of copies are needed before they can be detected by probes specific for that gene. Once the necessary amount of DNA has been produced by PCR, the probe for that gene is combined with the DNA and if the gene is present it is then seen by a process which allows visualization of the probe attached to the gene. Therefore, if cell tests positive for the probe specific for the Cystic Fibrosis gene, for example, then the associated embryo is known to be a carrier of that gene and would be withheld from transfer.