Preimplantation Genetic Diagnosis, IVF PGD in Los Angeles, California.

Preimplantation Genetic Diagnosis - PGD

Advanced Maternal Age and PGD

It has been well known that women experience an age related decline in the fertility rate starting at 35-36 years of age that carries across the type of treatment performed. Ultimately, the decline in fertility observed in women over 40 years of age results in less than one half the fertility rate compared to women under 40 as a group. At the same time, the incidence of miscarriage increases from about 25% at age 35, to 33% at age 40 and 50% at age 45. Better known are the statistics related to genetic abnormality of pregnancies as women age. At age 35, the total risk for chromosomal abnormality as demonstrated by amniocentesis is 1/132; at age 40 it is 1/51, and by age 45 has increased to 1/12. Until recently, we could only assume that these were related occurrences, resultant from an increase in the percentage of genetically abnormal oocytes caused by prolonged exposure to environmental mutogens.

PGD Research

PGD in IVF under the microscopeSeveral fertility clinics have accumulated data from embryo biopsy procedures that now validates our assumptions. It is now possible to remove a single cell from an eight cell embryo, and analyze the chromosomal composition within hours using a procedure called Fluorescent In Situ Hybridization (FISH)*. This allows determination of genetic normalcy of all embryos prior to transfer. From this data came several interesting observations. Firstly, in women over 40, 90 % of the embryos biopsied were found to be genetically abnormal. This for the first time explains the decrease in fertility rate and the increase in miscarriage and genetic abnormality rate of older women. It was also found that morphological criteria currently used for assessment of embryo quality might in some cases be misleading. In other words, some of the embryos that appeared perfect in form were found to be genetically abnormal while some very unattractive embryos were in fact normal.

The Future of PGD

This has tremendous implications for the future of ART. In the same way that ICSI was first performed only in a few fertility clinics worldwide and after dissemination of expertise and experience has now become routine in nearly every ART laboratory, embryo biopsy and PGD may become routine in the next few years. This would mean that for a particular individual, if it was found that the majority of her embryos were abnormal for instance, that individual would have powerful objective information that could lead her to seek an egg (oocyte) donor, thus sparing the expense and disappointment of repeated IVF failures. It would also allow physicians to transfer only normal embryos, which would maximize the chances of a successful outcome irrespective of age. Clearly, this is one of the most exciting developments in the field of reproductive medicine.

PREIMPLANTATION GENETIC DIAGNOSIS IN IVF

PGD is actually a number of procedures that combined have the ability to determine the genetic makeup of an embryo while it is at the 8-cell to blastocyst stage. This information allows for decision making prior to embryo transfer so that abnormal embryos may be prevented from becoming ongoing intrauterine pregnancies. In this way, later decisions which are morally and ethically difficult and become necessary when a genetically abnormal pregnancy is detected by traditional testing such as Amniocentesis or Chorionic Villus Sampling (CVS) are eliminated.

Watch Dr. Anderson's news segment on PGD pros and cons (ca. 2:16 minutes).

HOW IS PGD PERFORMED?

In order for the genetic makeup of an embryo to be determined prior to the time of embryo transfer there are two separate steps, which are important. The first of these is embryo biopsy. Most commonly, an embryo is biopsied on the third day following oocyte retrieval when it is at the six to eight cell 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 micropipette. The opening is somewhat larger than the one made for Assisted Hatching because it is necessary to remove one or two cells from the embryo for testing. This is an extremely delicate procedure, for the removal of cells from an embryo at this stage may result in damage to the embryo that prevents it from developing further. Once the embryo has been biopsied, there is about 48 hours for the testing to be completed before the embryo must be transferred. The embryo is allowed to develop to the blastocyst stage during this 48-hour period. There are relatively few cytogenetic 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 cytogenetic laboratory that it uses for testing. The testing is easily performed during the 48 hours that the embryo is developing to the blastocyst stage. Recently, some groups have also removed cells from embryos at the blastocyst stage. This allows more cells to be used for testing but significantly decreases the time available for the results to be obtained before the embryo transfer must be performed.

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 a Aneuploidy testing. 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. Currently, it is possible to test for some but not all of the chromosomes present in an embryo. This is done by exposing the cell removed from the embryo to specific probes that have the ability to attach themselves to the chromosome in question. Each probe is a molecule that is composed of two important parts, a site that attaches itself only to one specific chromosome and another part that when exposed to certain laboratory conditions gives off a fluorescence of a unique color. Therefore, each chromosome present that is exposed to its probe will be detected by visualization of one spot of light of a certain color. For instance, if the X chromosome has a probe that gives off a blue light and the Y chromosome has a probe that gives off a red light, an cell from a normal male embryo would have one blue and one red spot of light when tested, since it would contain one X and one Y chromosome. A cell from a normal female embryo would have two blue spots when tested since it would contain two X chromosomes. If chromosome 21 has a probe that gives off a yellow light, then a cell that has 3 yellow spots when tested would be known to contain three copies of chromosome 21 which is diagnostic of Down's Syndrome. Currently, cells are tested with probes that detect twelve of the 23 chromosome pairs: 8, 13, 14, 15, 16, 17, 18, 20, 21, 22, X and Y. This means that a cell from a normal embryo would have 24 spots of light detected when tested.
If more or less of these spots are seen, the embryo is known to be genetically abnormal and the color of the spots will allow determination of which chromosome is giving rise to the abnormality. This process of testing is called Fluorescent In Situ Hybridization (FISH)*.

The second type of genetic information obtained from PGD is the presence or absence of a specific gene in a given embryo. This type of information is useful when one of the prospective parents is known to be a carrier of a gene responsible for a particular disease. 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 among others.

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.

Because of the little room for error that PGD affords, it is not a procedure that should be offered by all ART laboratories at the present time. In order to have a successful PGD program, an ART laboratory must have someone on site that is especially skilled in micromanipulative procedures. PGD is much more technically challenging than Intracytoplasmic Sperm Injection (ICSI) or Assisted Hatching, so this technician must have special abilities to insure that the embryos can survive the biopsy procedure. Secondly, there must be an affiliation with a cytogenetic laboratory capable of performing FISH and PCR. Mechanisms for the transport of cells to this laboratory must be in place. Our ART laboratory utilizes the Reprogenetics laboratory in San Francisco directed by Dr. Santiago Munne for Aneuploidy testing and the Genesis Genetics laboratory of Dr. Mark Hughes in Detroit for single gene testing. Lastly, since the embryos must be placed in extended culture for 48 hours following embryo biopsy to allow enough time for the testing to be completed, the ART laboratory must be experienced in the culturing of embryos to the blastocyst stage.

WHY PGD?

PGD has been performed around the world for about ten years. With the development of extended culture media allowing blastocyst stage embryos to be transferred, the previously described techniques have become clinically useful in a few fertility clinics for the past several years. During this time, data has accumulated that reveals the importance of this new technology. Researchers have found that in women who have a poor prognosis for success following in Vitro Fertilitzation, PGD is helpful because it allows selection of the best embryos for transfer. For instance, in one series, women over age 36 were found to have 64% of their embryos aneuploid when tested by FISH. Transfer of genetically normal embryos in this group resulted in more than double the ongoing implantation rate compared to same age controls that did not have PGD by FISH. Women with three or more IVF failures were found to have 54% of their embryos aneuploid when tested by FISH. Transfer of genetically normal embryos in this group resulted in nearly double the ongoing implantation rate compared to same age untested controls. Finally, women known to have an abnormal genetic makeup of their own were found to have 62% of their embryos aneuploid when tested by FISH. Transfer of genetically normal embryos resulted in more than triple the ongoing implantation rate compared to same age untested controls.

In another report, investigators found that even younger women have a large percentage of abnormal embryos when tested by FISH. Women aged 20-34 years were found to have 50% of their embryos aneuploid when tested, even when the embryos looked normal by microscopic examination. The rate of aneuploidy was even higher in older women in this series. 64% of normal appearing embryos were aneuploid in women 35-39 and 71% of embryos were aneuploid in women over 40. In all age groups in this series, embryos that had arrested in development or were slow to develop were found to have even higher rates of aneuploidy compared to same age women with embryos that looked normal. As more chromosomes are becoming testable by PGD, experimental evidence is beginning to accumulate that the use of PGD has led to an increase in the pregnancy rates of some groups of women. However, PGD is not to be used indiscriminately for all cases where IVF is needed, as some are doing. There are still limitations to the technology such as misdiagnosis, embryo damage and the presence of mosaicism (more than one cell line in the same embryo) which make it prudent for PGD to be used responsibly in the situations where it is most likely to be of benefit.

Therefore, PGD has allowed us to learn that many failures in IVF can be attributed to the transfer of genetically abnormal embryos and that these embryos can develop from oocytes obtained from women of any age. In general, the rate of genetic abnormality increases with increasing age. This information corresponds well with clinical data that we have been aware of for years, which shows a decrease in fertility rate, increase in miscarriage rate and increased incidence of genetic abnormality in ongoing pregnancies with increased maternal age.

HOW SHOULD WE USE PGD?

Now that we have this new technology, we have to determine when and how to use it. This may prove to be more challenging that it appears at first glance. At the present time, it seems prudent to consider using this technology for the testing of individuals who are at risk for the development of a genetic abnormality in their offspring. These would include known carriers of a disease caused by a single gene disorder, older women, individuals who are known to have an abnormal genetic makeup of their own and women with recurrent miscarriages who have an otherwise normal evaluation. Whereas it would be desirable to also test women undergoing IVF who have had repeated unexplained failures, the experimental evidence to date does not yet support the expectation of higher pregnancy rates in this group when PGD is used. It is important to counsel the couples who are offered PGD that since only 12 of the 23 chromosome pairs can be tested and there remain significant factors which limit its effectiveness, the risks of the procedure must be carefully weighed against the potential benefits before deciding to use PGD as part of their treatment. When it is possible to test for all 23 chromosome pairs, it may then become justifiable to use PGD for aneuploidy testing on a more routine basis. The routine use of PGD for aneuploidy testing in all women at the present time is not supported by the clinical data in the medical literature. Hopefully someday PGD will allow us to maximize the success of IVF and lower the incidence of multiple pregnancy by transferring fewer but all genetically normal embryos.

However, as PGD becomes more widespread, there will become the possibility for its use in more debatable situations. Whereas no one would dispute the withholding of genetically abnormal embryos from transfer, some have suggested that PGD could be used for sex selection. This raises some concern in those who believe that destruction of normal embryos simply because of their gender is an ethical or moral abuse of this technology. As we learn more about the composition of the human genome and can determine which traits are determined by particular genes, the potential to design the makeup of an individual by removing or inserting genes becomes a possibility. This is an area that will become extremely complicated both ethically and morally. It is the responsibility of the medical community to insure that this technology is put to use in the most well thought out manner. These difficult issues will be the subjects of much debate in the years to come. It is safe to say, however, that the development of PGD is one of the most exciting and important milestones in the short history of Assisted Reproductive Technology.

Harper, J.C. and Delhanty, J.D.A. (1996) Detection of chromosomal abnormalities in human preimplantation embryos using FISH. J. Assist. Reprod. Genet., 13, 137–139