Genetic testing on embryos: Current benefits from PGS less than hoped.

October 25, 2010Carole 1 Comment »

Has preimplantation genetic testing lived up to the high clinical expectations for genetic testing?- namely to eradicate genetic disease and improve delivery rates? The answer varies depending on whether genetic testing is used for specific gene mutations (PGD) or aneuploidy screening (PGS).  Newer technical methods may improve the clinical pay off from both PGS and PGD. In their paper,  The use of arrays in preimplantation genetic diagnosis and screening, Dr. Joyce Harper and Gary Harton have reviewed the current state of preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS) and how evolving techniques may improve clinical outcomes for IVF patients.

What is the difference between PGD and PGS? PGD is used to diagnose an embryo as a carrier of a specific genetic mutation associated with diseases. It’s not a shot gun approach. With PGD, you need to know what familial disease the parents are at risk of passing on to their children and create a probe that will detect that specific mutation. Depending on how the gene is inherited and how many copies you need to experience the disease, embryos may be carriers of the gene but unaffected themselves or carriers that are affected (and will be sick) or they may not have inherited the mutation at all (normal gene). In PGD for single gene mutation, you don’t discover any other genetic mutations except the ones you are specifically looking for. For instance, an embryo may be free of the Tay Sachs Disease gene based on your TaySachs test, but may still have an undetected extra chromosome 21 causing Downs Syndrome so it is normal for Tay Sachs but abnormal for chromosome 21. Newer genetic tests which amplify and test the whole genome may reveal both structural chromosomal abnormalities and specific gene mutations in the same test.

PGD is used to identify IVF embryos that are affected by specific inherited diseases and these embryos are removed from the transfer pool, leaving normal and/or unaffected embryos for transfer.  Embryos carrying a genetic mutation are not always discarded but may sometimes be donated to embryonic stem cell line research. Cell lines carrying the mutation can be created from these embryos and used to study the disease an perhaps find a cure.

In contrast, PGS is used as a screening test to look for structural abnormalities in chromosome number or size. You are looking for any structural chromosomal abnormality–usually in the number of chromosomes but sometimes also swapped regions that make a particular chromosome abnormally short or long.  For PGS,  fluorescent in situ hybridization (FISH) is used to look at structural abnormalities in the chromosome.  Chromosomes from a single embryonic cell are spread out and displayed on a microscope slide and identified by probes that are specific for specific chromosomes and so can identify missing or extra chromosomes or large swapped pieces of chromosome. FISH analysis can also be used to look for X and Y chromosomes to identify the gender of the embryo to avoid gender linked disease gene transmission  or for social reasons like family balancing.

The low delivery rate in older women and women suffering from recurrent pregnancy losses is in large part due to transferring embryos with abnormal chromosomes. Older women and women with recurrent pregnancy losses are more likely to produce aneuploid embryos. The hope of PGS was that by detecting structural chromosomal abnormalities in embryos, only embryos with the normal number and structure of chromosomes would be transferred eliminating pregnancy losses due to aneuploid embryos. Because no specific genetic disease is tested, this genetic analysis is considered a screening, not diagnostic tool.

FISH analysis has several limitations when used for embryo genetic analysis that have limited its usefulness in increasing the delivery rate. First, FISH was established for analysis of thousands of cells, not the single cell that can be spared for analysis of early embryos, so FISH has certain technical limitations. When only a single cell is analyzed, it is more likely that the single cell may not be representative of the remaining cells in the embryo- a condition called genetic mosaicism. A false normal result due to genetic mosaicism would result in transfer of an abnormal embryo and increased risk of pregnancy loss. Second,  there is no safety margin for technical errors that may arise when you are using a single cell in your assay. You have one chance to get it right. A third technical limitation of FISH is that the miniscule amount of DNA from the single cell must be probed and reprobed several times to try to try to look at most of the chromosomes. The test sample degrades with each cycle of testing so FISH can only examine 10-12 chromosomes, not all 24. Therefore, abnormalities associated with other non-tested chromosomes will not be detected–and embryos with undetected abnormalities will still be transferred and pregnancies lost.

Problems not specific to the FISH technique have also hobbled the promise of PGS.

Biopsy stage. The stage of development from which the sample is removed will also affect the type and quality of genetic analysis that can be performed. For example, if the first polar body of the egg is analyzed, only maternal DNA is analyzed and any mutations introduced by the sperm in the subsequent embryo can not be detected, limiting the usefulness of first polar body biopsy in the detection of abnormal embryos. If the second polar body (released after fertilzation), is also tested, comparison of both can infer the genetic complement of the embryo. Biopsy of the cleavage stage embryo is a better source in some regards because it is clearly entirely of embryonic origin and cleavage stage biopsy allows genetic testing to be completed in time for a fresh transfer on day 5.

Sample Size. Robust clinical tests usually start with enough sample volume to get a clear signal and repeat results if necessary. For traditional genetic testing such as chorionic villus testing or amniocentesis, a large number of cells (thousands? millions? ) are sampled and tested. Odd reactions with one cell’s DNA are washed out in the average of the signal from many cells. In contrast, consider the poor embryo, able to spare only one or at most two cells at the eight cell stage for testing.  As mentioned previously, relying on a single cell opens the possibility that the cell tested is not representative of the remaining embryo due to genetic mosaicism. When the embryo is biopsied at the blastocyst stage, many more cells are analyzed, averaging out any odd ball results and avoiding the hazards of genetic mosaicism. At the blastocyst stage, the entire embryo contains between 50-150 cells and is composed of cells destined to produce the embryo proper (inner cell mass) and cells destined to produce the fetal part of the placenta (trophectoderm cells). The biopsied cells are removed from the trophectoderm.

Proficiency in blastocyst culture and vitrification is often necessary to use the newer techniques and many clinics are wed to day 3 culture and transfer and have mediocre freezing programs. The downside of trophectoderm biopsy which occurs on day 5 (the normal transfer day) is that the genetic analysis takes several days so that results would be received past the window of implantation for the patient to have a fresh transfer. Embryos must be frozen pending test results and preparation of the patient’s uterine lining for a future frozen embryo transfer. Therefore, ART clinics who want to offer trophectoderm biopsy and newer genetic testing methods beyond FISH must also be proficient in day 5 culture of embryos to the blastocyst stage and vitrification of embryos. Clinics who have been reluctant to fully embrace blastocyst culture and vitrification may need to reevaluate their position, or else severely limit their ability to provide state of the art genetic testing.

PGD for single gene mutations has largely lived up to clinical expectations,  allowing patients to virtually eradicate specific genetic diseases from their family tree. Based on their analysis of a number of randomized controlled studies, Harper and Harton conclude that PGS, unlike PGD,  has NOT resulted in the expected clinical outcomes, namely,  improved delivery rates through detection, selection and transfer of embryo with a normal chromosomal profile. Two new techniques, Comparative Genomic Hybridization (CGH) and Single Nucleotide Polymerism (SNP) arrays may be the solution to reach the clinical potential offered by PGS. The next post will examine these new techniques in detail.

© 2010, Carole. All rights reserved.

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