IVF cycle summary:

• Medication is used to stimulate the production of multiple eggs.
• Egg retrieval is performed using an ultrasound-guided needle.
• Eggs are mixed with the partner’s sperm in the IVF Laboratory and placed in the incubator for fertilization and embryo growth.
• When the embryo reaches the four- to 12-cell stage, one or two cells are biopsied from the embryo and PGD is performed.
• Normal embryos are transferred to the mother’s uterus on day four to six following egg retrieval.
  

Embryo Biopsy

In order to screen a human embryo before it is transferred to the mother’s uterus, one or two cells from the early multi-celled embryo are removed for genetic analysis. In routine genetic analysis there are usually hundreds of cells available for processing. However, with embryo biopsy only one or two cells are commonly available,  and they must contain a nucleus with chromosomes present to determine the genetic status of the rest of the embryo.

The biopsy method is a straightforward but not easy procedure. Embryos are typically biopsied at the preimplantation stage on day three of development. At this point the embryo is composed of between four and 12 cells that are still distinct from each other. By day three or day four at the latest, the embryo begins to compact, a process whereby the individual cells lose their clear outline and seem to fuse together with the other cells to form what is called the morula stage embryo. On the third day single cells can be individually removed without disrupting the adjacent cells in the embryo.

Figure: Embryo Biopsy

Removing the Cells 

At this time the embryo is still surrounded by a glygoprotein or "jelly coat" of the zona pellucida or membrane that forms around an ovum. To remove cells for study this jelly coat must first be cut open by an acidified culture medium that "dissolves" the zona pellucida or with a simple laser that drills the exact size hole. This allows a glass micro-tool to be pushed through the hole and extract a cell. The hole that is drilled is usually a little smaller than the cell itself. This helps maintain the integrity of the embryo within the jelly coat during further development in the IVF laboratory.

During its manipulation on the inverted microscope, the embryo is held in a warm culture medium that allows the cells to be removed with a minimum of trauma to the embryo. The removal of up to a quarter of an embryo is not known to be injurious to its further development, as the embryo at this early stage of development can compensate for this loss of material. All cells at this stage are still totipotent, they all are fully capable of directing further embryonic development.

Analysis

Once a blastomere or single cell is removed, it is either fixed on a glass slide for chromosomal analysis or placed in a small tube of chemical buffer for single gene diagnosis. Cells are analyzed using fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR).

During genetic analysis, embryos are usually grown to the fifth day of development, at the morula or blastocyst stage. Those embryos found to be free of genetic abnormalities are then placed into the mother’s uterine cavity.

Fluorescence In Situ Hybridization (FISH)

FISH is a technique used in testing chromosomal abnormalities to determine the proper structure for specific chromosomes. A probe or “tag” attaches to a specific chromosome by matching the sequence of the probe with the sequence of the DNA being tested. The probe has fluorescence and “lights up” under a microscope. Embryos with too much or too little fluorescence have an abnormality that can prevent successful implantation. Such pregnancies result in miscarriages or an abnormal fetus. To aid in the diagnosis of recurrent miscarriage, repeated IVF failure, or age-related chromosomal abnormalities, chromosomes 13, 15, 16, 17, 18, 21, 22, X and Y are most commonly tested; however up to 12 chromosones can be analyzed.  For couples who carry a known chromosomal abnormality, a unique probe tailored to their chromosomal make-up is made to study their embryos.

Polymerase Chain Reaction (PCR)

PCR is a DNA analysis technique for specific single gene disorders, such as cystic fibrosis or Tay Sachs disease. This technique uses an enzyme that makes millions of copies of the cellular DNA. This allows for easier evaluation because of the abundance available DNA to study. The DNA is tested for the specific gene disorder by looking at the sequence of the DNA base pairs, the “DNA code” for the gene. This analysis identifies those embryos that carry the normal and abnormal gene, or mutation.

Other Issues

Misdiagnosis

Mosaicism within the embryo may cause misdiagnosis. Some embryos may contain blastomeres, cells produced by  the cleavage or 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.

Other causes of incorrect diagnosis may be due to experimental error, improper cell fixation techniques, DNA denaturation errors, allelic drop-out, or amplification of contaminated DNA.

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 percent. There is no risk to the embryo following chromosomal or single gene defect analysis because the analyzed cells are not returned into the embryo. There may be a slightly lower likelihood of implantation after embryo biopsy compared to an embryo that was not biopsied. Other risks may become apparent over time, but they are limited and need to be weighed against the potential benefits for each couple.

In June 2007 a study presented to the European Society of Human Genetics documented a group of 563 PGD babies born in Belgium. They had a comparable birth weight to those conceived with the help of other reproductive technologies. At two months, and again at two years of age, these children appeared to be equally healthy.

Related Website Links

• Reprogenetics
  

Related Articles

• Genetics 101
• Indications for PGD
• Inherited Genetic Disorders
• PGD & In Vitro Fertilization (IVF)
• Scheduling Tests for PGD