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Pre-Implantation Testingby Luba Djurdjinovic, Genetic counselor, Ferre Institute, Utica, NY [12] "Is there a way to prevent the VHL gene from being passed on to future generations?" The advances in the last 10 years in reproductive medicine and rapid identification of genes through the efforts of the Human Genome Project are changing the answers to that question. The choices that families with known inherited conditions have today are better, but they continue to be associated with challenging questions related to technology as well ethics. The following description of preimplantation genetic testing (PGT) hopes to provide an overview of this technology that has already been offered to many families with various genetic conditions, including cystic fibrosis, Duchenne muscular dystrophy, Charcot Marie Tooth, hemophilia and rare rearrangements of the chromosomes. PGT attempts to diagnose a fertilized egg before the pregnancy is implanted in the mother’s uterus. The first PGT was reported in 1992 to assist in identifying embryos that carry the X (female determining) chromosome or Y (male determining) chromosome.6 This was very exciting news for families where the genetic condition is passed on through the X chromosome affecting boys and usually sparing the girls. For genetic conditions where the chromosome location of the gene is known, or more specifically, if the gene sequence (code) has been identified, the potential for PGT exists. The PGT procedure requires that embryos be retrieved for molecular analysis before implantation. Fertilization of the egg by the sperm occurs in the upper section of the fallopian tube. The fertilized egg travels down the tube and after several days drops into the uterus. It usually takes several more days to implant in the uterus and initiate the pregnancy process. During the travel of the egg within the fallopian tube, the fertilized egg undergoes cell division, so that by the time the egg gets to the uterine wall this conception will resemble a ball of cells. This is called the blastocyst. The TechnologyCollection of fertilized eggs. There are two egg collection approaches in use by PGT Centers. One method involves collecting the fertilized egg when it drops into the uterus by flushing out the uterine cavity (lavage). To assure that there will be a suitable fertilized egg, PGT centers also require the use of fertility medication that will cause superovulation, meaning the ovary will produce more than one egg with the potential of being fertilized. It is important to remember that each egg has its own separate risk for inheriting the gene. Thus, the availability of more than one egg is critical to be able to choose an unaffected egg and initiate implantation. This method does have a drawback, in that there has to be very careful timing of the lavage procedure in order to offer the best chance of retrieving an egg. There is always the possibility that the timing of this process can be unsuccessful and that an unscreened egg may implant, carrying the risk associated with that condition. It has been reported that embryos are successfully collected by lavage only 40% of the time.4 The method most often used for egg retrieval is in vitro fertilization (IVF). This procedure also requires the woman to take fertility medications that increase the number of available eggs. The eggs are retrieved 36 hours after administration of hCG. This hormone facilitates the release of the mature eggs from the ovary’s surface. The eggs are fertilized in the lab with the partner’s sperm. The fertilized eggs are then closely watched as they undergo cell division and develop into a blastocyst. This procedure provides more control over egg collection.11 Testing the fertilized egg. Once the fertilized egg has reached a 4 to 8 cell stage, one or two cells are removed from the surface of this blastocyst. One approach in cell collection is holding the embryo up "against the tip of a pipette while a fine needle is used to make a small slit in the zona (outer layer) and a cell (or two) removed by gentle suction".3 The removed cell is then subjected to a molecular analysis. This requires the removal of the genetic material— DNA. This minuscule amount of DNA is amplified, meaning multiple copies are made through a molecular process known as PCR (polymerase chain reaction). These copies are then subjected to a molecular analysis that assists in identifying the sequence (code) that will determine the inheritance of the gene in question. VHLThe VHL gene has been found to be on the short arm of the number 3 chromosome. The gene directs the making of a protein that functions as a tumor suppressor gene. Persons with VHL have changes (mutations) to the genetic code that inactivates the altered copy of the VHL gene. Testing for the mutation in this gene is possible in most individuals. Identification of the mutation in the gene and tracing the gene within a family is possible for up to 80% of VHL families.5 The optimal testing situation would be to directly read the code in the VHL gene. This is not always possible, and sometimes the gene and its chromosome is traced in a family. This tracing of affected members through the generations is called linkage. To date, there have not yet been any reported cases of births where VHL preimplantation testing occurred. Laboratories involved in PGT are open to working with families with documented gene diagnosis. Families choosing this option in the immediate future need to understand that they are participating in the development of a specific application of PGT. This then carries a certain level of uncertainty and cost for development of molecular strategies. The cost of PGT is dependent on the collection approach (IVF or lavage) and the technical difficulty in identifying the gene. The cost for this procedure is approximately $10,000 per each cycle.9 It is important that in selecting a PGT Center that a couple investigate the center’s previous experience and the number of successful births as compared to attempted cycles and implantation. In 1992 there was only an approximate 15% rate of successful implantation that results in a live birth with an IVF procedure;10 today some clinics have success rates as high as 50%.13 Finally, it can take, on average, two to three cycles to achieve a pregnancy. Safety and accuracyThe safety of PGT has not been established. This question has been of great interest since a cell is removed from the early dividing fertilized egg. To date, there have been only about 100 live births using PGT. This number is still too small to determine if the procedure carries an increased risk of birth defects over the general population. There have been no reported cases of birth defects resulting from the procedure of PGT.7,13 The accuracy of the preimplantation genetic diagnosis is highly dependent on the sensitivity of the gene analysis procedure available for the specific genetic condition. There are reported cases of misdiagnosis with this procedure.8 Many centers are offering prenatal testing, CVS or amniocentesis to confirm the fetus gene status.14 The experience of PGT can also bring with it psychological challenges. There are feelings of loss that one has to turn to this level of technology to protect future generations. The couple has lost a sense of invincibility around reproduction that most unaffected couples experience. There are periods of anxiety since the technology carries a great deal of uncertainty. Finally, the financial cost of this procedure is burdensome, particularly when families are looking to have more than one child. Technology such as PGT offers hope to some couples who wish to build families but are very eager to reduce the risk of passing on the genetic condition that has burdened the family. For VHL families the opportunities are there, but they are still in development. If someone wishes to explore this option it is important to identify a PGT Center. Learn as much as possible about this center and their approach. Arrange a consultation to discuss what you hope is available and learn what they feel can be achieved. Take your time in choosing a center. Resources 1. The American Society for Reproductive Medicine, 409 12th Street SW, Washington, DC 20024. +1 (202) 863 4985. http://www.asrm.com 2. Ferre Institute, Inc., 258 Genesee Street, Utica, NY 13502, +1 (315) 724- 4348. http://members.aol.com/ferreinf/ferre.html Bibliography 3. Black SH (1997) "Preimplantation genetic diagnosis and sperm separation." http:/www.givf.com 4. Carson SA (1990) "Recovery of blastocysts by uterine lavage following superovulatory drugs." J in Vitro Fert Embryo Transfer Vol. 7:187. 5. Decker HJ et al (1997) "The von Hippel-Lindau tumor suppressor gene." Cancer Genetics Cytogenet Vol. 93, pp.74-83. With advances in the technology and better equipment, some labs are finding an even higher percentage of mutations. Ask each lab for its percentage success rate. 6. Handyside AH et al (1992) "Birth of a normal girl after in vitro fertilization and preimplantation diagnosis for cystic fibrosis." NEJM Vol.327:13, pp.905-909. 7. Lopez G (1995) "Preimplantation genetic diagnosis." Prenatal Diagnosis Lab. Newsletter. 8. Pergament E and A Bonnicksen (1994) "Preimplantation genetics: A case for prospective action." Am. J. of Med. Gen. Vol.52, pp. 151-157. 9. Schulman JD et al (1996) "Preimplantation genetic testing for Huntington Disease and certain other dominantly inherited disorders." Clinical Genetics, Feb 1996. 10. Simpson JL and Carson SA (1992) Editorial: "Preimplantation genetic diagnosis." NEJM Vol. 327:13 pp.951-953. 11. Singer N (1995) "Embryo genetic disorder analysis demonstration project: A model protocol." J of Gen Counseling Vol.4:2, pp.139-146. 12. Zilberstein M and Seibel MM (1995) "Preimplantation genetic diagnosis: What can be done?" Faulkner Ctr. for Reproductive Medicine. Forum. Boston, MA. 13. Correspondence with Dr. Robert Gagel, M.D. Anderson Cancer Research Center, Houston. 14. Pre-natal testing is testing of the fetus before birth. Two principal methods are used: amniocentesis and CVS. See index on page 5 for related stories. q As printed in the VHL Family Forum 6:1, March 1998. For permission to reprint, please contact VHL Family Alliance, info@vhl.org. |
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