PREIMPLANTATION GENETIC DIAGNOSIS - PGD

Preimplantation genetic diagnosis (pgd) is a state-of-the-art procedure used in conjunction with in vitro fertilization (IVF). Your physician may recommend PGD if there is a likelihood that your embryos will be affected by certain chromosomal conditions. These conditions can prevent implantation of embryos, lead to pregnancy loss or result in the birth a child with physical problems and/or mental retardation. PGD can help prevent these adverse outcomes by identifying affected embryos as they are developing in the laboratory and before they are transferred during IVF.  It is important to note that some of our protocols are experimental and are supervised by the Internal Review Boards. This includes PGD for translocations.

 
PREIMPLANTATION GENETIC DIAGNOSIS (PGD) FOR TRANSLOCATION

PGD involves testing an embryo for certain conditions prior to being placed in the womb of the woman. Under the direction of Santiago Munné, Ph.D., Director of PGD; and Dr. Jacques Cohen, Scientific Director, the PGD team is a world leader in both research and clinical application of this technique. Jill M. Fischer, M.S., is the PGD Program Coordinator and Genetic Counselor for Reprogenetics.

 
CHROMOSOMES

Chromosomes are string-like structures found in the center of the cell, the nucleus. They contain genes that are made of DNA. Therefore, our inherited information is housed on the chromosomes. Normal human cells (embryo, fetus, baby or adult) contain 46 chromosomes, or 23 pairs. We receive 23 chromosomes from each parent.

The first 22 pairs of chromosomes are the same for men and womenand are labeled largest to smallest from 1 through 22. The 23rd pair determines our sex. A female has two "X" chromosomes whereas a male has an "X" and a "Y." As such, the woman can only pass an X to her child in her egg. The man passes either the X or the Y in the sperm therefore determining the sex of the child.

 
CHROMOSOME TRANSLOCATIONS

A translocation is a change in chromosome structure in which chromosomes are attached to each other or pieces of different chromosomes have been interchanged. An individual with a translocation is unaffected if there is no extra or missing chromosome material and if the break in the chromosome did not disrupt gene function. If there is no additional or missing chromosome material, the translocation is considered to be "balanced." A translocation is "unbalanced" if there is extra or missing material.

Individuals with balanced translocations typically have no medical issues though some do have fertility concerns, such as reduced fertility. The concern regarding having a balanced translocation is that, though the individual is healthy, the egg or sperm of that individual can have an unbalanced chromosome make-up that leads to the resultant embryo or pregnancy being unbalanced. The presence of an unbalanced translocation can lead to an embryo not implanting, a pregnancy being lost or a child being born with mental and physical problems. Individuals with a translocation may, therefore, experience multiple pregnancy losses or have a child affected with physical and mental problems that may be lethal.

RECIPROCAL TRANSLOCATIONS
Approximately one in 625 individuals has a reciprocal translocation. These translocations involve any of the chromosomes. Reciprocal defines the translocation as one in which chromosomes have swapped material. Breaks occur anywhere in the chromosomes allowing for pieces to be interchanged between them.

ROBERTSONIAN TRANSLOCATIONS
Approximately one in 900 individuals has a Robertsonian translocation. These translocations involve chromosomes 13, 14, 15, 21 or 22. These chromosomes have a unique structure in that they are primarily made of a bottom half. This translocation results from fusion of two of these chromosomes such that the two bottoms are attached.

 
PGD – THE PROCEDURE

To analyze an egg or embryo, we first have to biopsy it. Two procedures allow this to be done. The PGD team of doctors, geneticists and embryologist will decide which procedure to use depending on the type of translocation and other considerations.

Biopsy of Polar Bodies
When the person with the translocation is female, we may be able to analyze the polar body. The ripening egg produces two small cells called polar bodies that degenerate after fertilization. The chromosomal content of these cells allows us to infer the chromosomal content of the egg. If one is testing the polar body, an opening is made in the covering of the egg and the polar body is removed with a pipette. The polar body is then analyzed while the egg is placed in an incubator. By analyzing polar bodies, we obtain information from only the mother. Chromosome abnormalities that may occur after fertilization, when the sperm meets the egg, will not be detected.

Biopsy of Blastomeres
We analyze blastomeres when the male has the translocation and, in certain cases, when the female has the translocation. A blastomere is a cell from an embryo. To test the blastomere, an opening is made in the covering of the embryo during its third day of development when the embryo has 8-10 cells. A blastomere is removed via aspiration with a pipette. The embryo is placed in an incubator while the cell is analyzed.

Analysis
The biopsied cells are analyzed using a technique called fluorescence in-situ hybridization or FISH. This technique uses probes, small pieces of DNA that are a match for the chromosomes we want to analyze, to study the chromosomes present. Each probe is labeled with a different fluorescent dye. These fluorescent probes are applied to the biopsied cell and attach to the chromosomes. Probes attach to specific areas of the chromosome or can be used to color the whole chromosome. Under a fluorescent microscope, balanced and unbalanced chromosomal make-up can be identified in that cell. The geneticist, therefore, can distinguish normal cells from cells with an unbalanced translocation.

Testing of the cells destroys them because they must be glued to a glass slide and repeatedly heated and cooled. As such, one cannot use them for another purpose or return them to the embryo. The slides are kept for future reference. This analysis causes no extra inconvenience to the patient as it is accomplished in one day.

Reduction in the Chance of Having a Child with the Translocation
Our personnel have performed the most procedures of PGD of translocations, 295 cycles up to August 2003. Normal or balanced embryos were available to be transferred to the patient in the majority of cycles. Pregnancy occurred in up to 40% of the cycles with transfer, depending on the Fertility Center were the cycle was performed. None of the delivered babies has been found to have an unbalanced translocation.

Reduction in Pregnancy Losses
The PGD procedure significantly reduces the chance of pregnancy loss. The patients who achieved pregnancy after PGD had experienced miscarriage in the majority (>90%) of their previous pregnancies. When these same patients underwent PGD, fewer than 10% of pregnancies were miscarried. This is a significant reduction in pregnancy losses.

 
ISSUES OF PREIMPLANTION GENETIC DIAGNOSIS

PGD is not void of risks but these appear to be outweighed by the benefits described above.

The Risk of Embryo Biopsy
While PGD is a relatively new procedure in in vitro fertilization (IVF), the micromanipulation techniques required to perform it have been in use for many years, and the risk of accidental damage to an embryo during removal of the cell(s) is very low - 0.6%. Procedures such as intracytoplasmic sperm injection (ICSI), fragment removal and assisted hatching are all performed by making openings in the zona pellucida. These techniques have been found to have usually positive or at least neutral effects on embryo development and implantation.

Removal of Cells from the Embryo
No part of the future fetus will be lacking because one or two cells are removed from the embryo approximately two days after fertilization. All the cells of the embryo remain totipotent until about the fourth day. Totipotent means having "all potential." These cells have not differentiated yet, meaning that each cell by itself can grow into a whole and perfect fetus. The procedure merely delays continued cell division for a few hours, after which the embryo reaches the same number of cells as before and continues its normal development.

Normal development has been seen many times in humans and other mammals after cell loss due to embryo freezing. One or more cells may fail to survive thawing, yet the embryos from such develop into normal offspring. An unanswered question is whether biopsied embryos implant less than untouched ones. Data regarding such is incomplete. Embryo biopsy may lower implantation rates slightly while selection of chromosomally normal embryos via PGD may increase it. The balance between potential biopsy damage and beneficial effects of PGD seems to be positive.

Misdiagnosis
The accuracy of PGD for translocation is approximately 90%. This means that the error rate is 10%. Within this chance of misdiagnosis, there is a false negative rate, a false positive rate, the chance for no result and the chance for mosaicism. A mosaicism is defined as the embryo having cells with different chromosome make-up. Typically, all cells of the embryo have the same chromosomal make-up as they originate from the same fertilized egg. However, it is possible for cells of the same embryo to have differing numbers of chromosomes.

If we analyze a cell that has normal chromosomal content, but another cell has an extra chromosome, we erroneously diagnosed that embryo as being chromosomally normal. Due to the chance of misdiagnosis as well as the presence of other chromosome conditions for which we do not test, we recommend prenatal testing via chorionic villious sampling or amniocentesis.

Few Eggs Produced or No Normal Embryos for Transfer
For regular infertility patients in our clinic, we consider it appropriate to transfer three embryos in a woman younger than 35 and possibly four or five embryos in patients older than 35. However, female translocation carriers usually produce fewer eggs than other patients.

We have found that translocation patients produced an average of 9.5 mature eggs compared to 13 in non-translocation patients. The proportion of abnormal embryos found from translocation carriers has ranged from 0 to 100%, with an average of 65% abnormal embryos. In approximately 22% of cycles, all the embryos were chromosomally abnormal. Therefore, it is possible that less than three embryos (or even none) will be available for transfer, which may lead to lower pregnancy rates than for non-translocation patients.

 
PRELIMINARY ANALYSES

For reciprocal translocations, we request blood from the individual with the translocation. This allows for preliminary analyses to be performed with probes to confirm that PGD can be done. The patient must provide us with a deposit that covers the cost of the probes and the preliminary work. Once the blood and deposit are received, probes are ordered and the preliminary work can be performed.

If the individual with the translocation is a male, we recommend FISH analyses of sperm prior to undergoing PGD. Determination of the percentage of unbalanced sperm will allow for estimates of the percentage of embryos that will be unbalanced, and therefore, determination of whether PGD is the best option.

 
PERFOMANCE OF THE PGD PROCEDURE

Dr. Santiago Munné, Ph.D., PGD Program Director, and Dr. Jacques Cohen, Scientific Director, have been involved in PGD since the start of the technique. Dr. Munné heads a team of scientists at Reprogenetics. This team has developed most of the techniques involved in PGD of translocations and has performed the most procedures of  PGD of translocations.  Two papers obtained the Prize Paper of the Society for Assisted Reproductive Technology in the 50th (1994) and 51st (1995) Annual Meetings of The American Fertility Society. More recently their work received the General Program Prize at the American Society for Reproductive Medicine (ASRM) in 2000.

Preimplantation genetic diagnosis for translocations and other indications, with the exception of aneuploidy, is an experimental procedure and will be performed under Internal Review Board approved protocols.


PGD OLLOW-UP PROGRAM

All patients who achieve pregnancy after IVF with PGD are asked to participate in our follow-up program. Information regarding pregnancy, pregnancy outcome and child development will be gathered.