Preimplantation Genetic Diagnosis/Screening - PGD/PGS
Advanced Maternal Age and PGS
It is well known that women experience an age related decline fertility starting in their early 30's. 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 1/38, and by age 45 has increased to 1/12. Until recently, we could only assume that these were related occurrences, resulting from an increase in the percentage of genetically abnormal oocytes with increasing age.
PGS Research
It has now been 20 years since the procedure involving embryo biopsy and subsequent genetic testing has been available. Initially, testing was accomplished by a procedure called Fluorescent In Situ Hybridization (FISH)*. The limitation of this particular technology was that it wasn't possible to test for all 24 chromosomes due to technical limitations. At first, only 5 chromosomes could be tested. With time, this was increased to 7, then 9 and ultimatley 12. Even though at best only half of the chromosomes from a biopsied embryo could be tested, several interesting findings emerged. First, in women over 40, 90 % of the embryos biopsied were found to be genetically abnormal. This finding lent support to our assumptions regarding the cause of the decrease in fertility rate and the increase in miscarriage and genetic abnormality rates of older women. Second, 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. Yet the inability of FISH to provide complete genetic information for each embryo biopsied limited its effectiveness. In fact, there was little data supporting the use of FISH to improve the success of IVF and only limited data supporting its use to improve outcome in women with recurrent miscarriages.
Recently, a newer technology has emerged that has appears much more promising in its ability to deliver complete genetic informtion about an embryo in question. This technique is referred to as Comparative Genomic Hybridization (CGH). In contrast to FISH, CGH is able to provide information about all 24 chromosomes from a single cell which has been removed from a 3 day old embryo. It is likely that CGH will replace FISH for this reason and hopefully will actually be able to improve the efficiency of IVF while limiting the occurence of miscarriages.
The Future of PGS
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 PGS 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 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/SCREENING IN IVF
PGD/PGS 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/PGS 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 laser. 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 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. The testing is easily performed during the 48 hours that the embryo is developing to the blastocyst stage. Alternatively, cells can be removed from embryos at the blastocyst stage. This allows more cells to be used for testing but requires freezing of the embryos after biopsy since the results of the genetic testing could not 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. As previously mentioned there are two techniques for testing the chromosomes of a biopsied embryo.
The procedure which has been used the longest is called Fluorescent In Situ Hybridization (FISH). With this technique 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.
The process of screening embryos for genetic abnormality has become much more useful because of a new technology called Comparative Genomic Hybridization (CGH). It is now possible to determine the complete chromosomal composition of an embryo,( that is all 24 ) from testing a single cell. Each chromosome is composed of two strands of DNA that are chemically attracted to each other in a way that holds them together for their entire length. These two strands of DNA are referred to as complementary strands and it is the chemical attraction between complementary strands that is the basis for testing chromosomes with CGH. CGH is done by first making many copies of the DNA obtained from the nucleus of the cell which has been removed from the embryo to be tested. The two strands of DNA are then separated and only one strand from each chromosome is used for testing. The DNA is then broken up into very small pieces and then stained with a dye of a certain color. DNA obtained from a cell which is known to have normal chromosomes is then broken up into the exact same small pieces and stained with a dye of a different color. A glass slide is then coated with the pieces of DNA that are from the complementary strand of the stained pieces of DNA to be tested. These pieces of DNA are from a cell with normal chromosomes and are arranged in a pattern on the slide referred to as a microarray. Because of their ability to attract the complementary strand, pieces of DNA on the microarray will attract the stained pieces of DNA when it is added. The stained pieces of DNA from the test cell and the control cell are then added to the microarray. Since each cell has been stained a different color, it is possible to see how much DNA from each cell has been attracted to the pieces of DNA on the microarray by determining how much of each color is present. The pieces of DNA on the microarray represent all of the chromosomes in a normal cell and the location of all of the pieces that make up each chromosome are known. Therefore it is possible to determine how many copies of each chromosome are present by looking at the amount of each color that is present on all of the pieces of DNA that make up a particular chromosome on the microarray. For example, if the test cell has two copies of a particular chromosome then it is normal. So when the microarray is examined, there would be an equal amount of each color present for that chromosome since the control cell also has two copies of each chromosome. If however, the test cell has three copies of a particular chromosome, then it is abnormal and referred to as a trisomy. This is the case in the condition known as Down Syndrome where there are three copies of chromosome 21. The microarray from a trisomy would have more of the color from the test cell than the color from the control cell therefore.
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.
WHY PGD/PGS?
PGD/PGS has been performed around the world for about twenty years. During this time, data from FISH 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, PGS 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 PGS 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 the number of chromosomes testable by FISH increased to 12, evidence accumulated that the use of PGS may lead to an increase in the pregnancy rates of some groups of women. Hopefully as data becomes available for CGH it will be possible to apply this technology to even more women. However, PGS is not to be used indiscriminately since 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 PGS to be used responsibly in the situations where it is most likely to be of benefit.
Therefore, PGS 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/PGS?
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 PGS is used. Perhaps now that all 24 chromosomes can be tested with CGH, future results will be improved. It is important to counsel the couples who are offered PGS that the risks of the procedure must be carefully weighed against the potential benefits before deciding to use it as part of their treatment. The routine use of PGS for aneuploidy testing in all women at the present time is not supported by the clinical data in the medical literature. Hopefully, with the newer technology someday PGS 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 PGS becomes more widespread, there will become the possibility for its use in more debatable situations. 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 PGS 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
