Patient information Pre-implantation genetic screening (PGS)

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1 Page 1 of 5 Patient information Pre-implantation genetic screening (PGS) SUMMARY Pre-implantation genetic screening (PGS) analyses the genetic constitution of an egg or embryo to determine whether the genetic material is normal or abnormal. It does this by comparing the DNA of the test specimen with a defined normal profile. There are 2 main approaches to the testing. The first method involves removing cells from an advanced embryo (blastocyst), and testing the DNA of these cells. This reflects the status of the whole embryo. The other method involves testing the polar body (which is a waste product) of the egg and this reflects the status of the egg. In theory, by excluding abnormal eggs and embryos, the procedure should increase the chances of a successful pregnancy. Of course, it also increases the chances of having no embryos to transfer in that cycle if all the eggs/embryos are chromosomally abnormal. Historically, evidence supporting the concept that PGS is beneficial is not robust and it is now clear that the earlier forms of PGS showed little benefit. However, the newer, more comprehensive approaches using the technique called array comparative genomic hybridisation (array CGH) should be of more benefit, and the latest evidence from several clinics has been very encouraging. BACKGROUND What are chromosomes? Chromosomes are minute structures of DNA that exist in nearly every cell of the body. Apart from sperm and eggs, each cell should have exactly 46 chromosomes. Sperm and eggs should each have 23 chromosomes so that when fertilization occurs, the resulting single cell (which goes on to form the embryo) has 46 chromosomes. The chromosomes carry the genes, the genetic material defining the individual, and they also provide the instructions that tell the embryo how to develop into a baby. Genes can be thought of as blueprints, describing how to build a body out of all the different types of tissues. Why look at chromosomes in eggs and embryos? Research has suggested that IVF/ICSI success rates might be improved if embryos are screened for chromosome abnormalities, with only embryos that have the correct chromosome constitution being transferred to the womb. What happens if an embryo has an incorrect number of chromosomes? If a chromosome is lost, or if one of them is duplicated, the genetic instructions no longer make sense and the embryo will be unable to form a healthy baby. It would be like trying to build a complex machine with a page from the blueprints missing. With very few exceptions, an embryo with the wrong number of chromosomes will not produce a baby. Most chromosomallyabnormal embryos either fail to implant in the womb or miscarry during pregnancy. How common are chromosome abnormalities in embryos? Research has shown that chromosome abnormalities are very common in eggs and embryos. Furthermore, the risk of having an embryo with the wrong number of chromosomes is greatly affected by the age of the mother. For women in their early 30 s, about 25-50% of the eggs and embryos have an abnormal number of chromosomes. However, for women over 40 years old, the proportion is even higher, such that around 75-85% of the embryos produced are likely to be abnormal (i.e. the older the woman is, the higher this proportion).

2 Page 2 of 5 Is it a good idea to test IVF/ICSI embryos for chromosome abnormalities? In theory, embryos with a correct number of chromosomes should have a better chance of producing a baby than embryos with an abnormal set of chromosomes. If they could be identified and given priority for transfer to the womb, pregnancy rates are predicted to be increased. It is also likely that the risk of miscarriage and Down syndrome would be reduced, since these problems are caused by chromosomally abnormal embryos. In reality, the story is more complex than this logical picture presents, as most normal embryos carry individual cells which have abnormal chromosome constitutions making the testing more complex than we would like. The first series of PGS trials showed no clinical benefit (no increase in pregnancy rate), but the technology at the time was limited to only testing for a few chromosomes and not all 23 pairs of chromosomes (see below). Are chromosomes routinely tested during IVF/ICSI procedures? In most cases the answer to this question is No. In the lab, the embryologists look at the embryos down a microscope and grade them based on their physical appearance ( morphology ) at that moment in time. The assumption is that the better the appearance, the better the quality of the embryo and hence the more likely it is to implant. In these circumstances the embryologists cannot look at the number of chromosomes in each embryo and we know that just because an embryo looks good, it doesn t mean that it is good i.e. it can still have an abnormal number of chromosomes. Conversely, an embryo that doesn t look very good, by traditional (morphological) assessment, might have the normal number of chromosomes, and hence be more likely to implant. PRE-IMPLANTATION GENETIC SCREENING (PGS) What is pre-implantation genetic screening (PGS)? PGS is testing the egg or embryo for the total number of chromosomes. A normal embryo should have 46 chromosomes (23 pairs). PGS requires that the woman goes through the ovarian stimulation process and egg retrieval. The chromosome constitution can then be tested in a number of ways. Polar body analysis The polar body of each mature egg is removed. The chromosome constitution of the polar body is then tested, and this reflects the chromosome constitution of the egg from which it came. This approach is especially useful in older women because the likelihood of a chromosome problem in the egg is increased. In order to perform the polar body biopsy, we have to strip the eggs of their cumulus cells. Hence, to allow subsequent fertilization with a sperm, ICSI is used to fertilize the eggs (this involves injecting the egg with a sperm). Trophectoderm biopsy of the blastocyst There is an alternative method which is not currently performed at GCRM-BELFAST where several small cells are removed from the outer part (the trophectoderm) of the developing blastocyst (day 5 embryo). The small selection of cells can be tested for their chromosome

3 Page 3 of 5 constitution. This, in turn, will reflect the chromosome constitution of the embryo. This approach is potentially useful in women who have previously been able to conceive naturally but have had repeated miscarriages or women who have IVF or ICSI but have repeated implantation failure. Who might benefit from PGS? Logic dictates that couples with a greater likelihood of a chromosome problem in the embryo are the people who are most likely to benefit. These are: Women who have had repeated IVF/ICSI attempts, but implantation has repeatedly failed or they have miscarried at an early stage of pregnancy. Women who have conceived naturally but have had repeated miscarriages. Older women (over 37 years old) who are predicted to produce good numbers of eggs but don t conceive. This is because the rate of abnormal chromosome numbers in eggs increases with advancing female age. Women who wish to reduce their chance of having a child with abnormal chromosomes. Have any studies shown the chromosome tests to be beneficial for IVF/ICSI patients? Some studies have suggested that the use of PGS increases pregnancy rates, but other research has provided contradictory data. It is clear that the initial approaches were not beneficial. However, these studies used a technique called fluorescence in situ hybridization (FISH), which could only test about one third of the chromosomes in each embryo i.e. not all 23 pairs of chromosomes were tested. We now use a PGS method called array-cgh, which assesses all of the chromosomes. So far, there have only been a few PGS trials using this technology. However, the first trials conducted in the United States are encouraging, with initial evidence indicating that the chance of a transferred embryo becoming a live-birth can be increased by more than 50%. The chance of having no embryo transfer was also increased; this is to be expected as no embryo may be deemed normal. The number of individual studies published so far is small but the results are very encouraging. Nevertheless, more robust clinical trials are needed to determine whether or not PGS can significantly increase live birth rates. How is the PGS test done? To have PGS, the woman must go through the ovarian stimulation process and preferably have at least 5 eggs retrieved. PGS can be performed on fewer eggs but there is a higher risk of having no embryos to transfer due to failure of the PGS process or because all the embryos are chromosomally abnormal. Polar body biopsy We know that at least 85% of all chromosome problems in the embryo and fetus arise because of a chromosome problem in the egg. If polar body biopsy is performed, then a fresh embryo transfer can take place, providing there is at least one chromosomally normal embryo. Day 2/Day 3 (cleavage stage) biopsy These are no longer performed because the studies showed that biopsying at this stage reduced pregnancy rates. Does PGS have any risks to me or the embryos?

4 Page 4 of 5 For the woman, the risks of an IVF/ICSI cycle and performing array-cgh on the eggs or embryos are exactly the same as those in a standard IVF/ICSI cycle. Performing array-cgh does not add to the risks to the woman. For the eggs and embryos, there are theoretical risks of excessive damage caused by the removal of cells prior to testing. The evidence from some series suggests that this effect is minor, while some reports suggest that there is a clear detrimental effect. The series using CGH appear to indicate that the damage is minimal when performed at the correct stage of embryo development. All the embryologists who perform the biopsies at GCRM-B have been fully trained to perform the biopsies. If the egg or embryo is damaged by the biopsy it will be unable to produce a baby. Specifically, for eggs the risk is very low - around 1%. What happens next? What is array CGH? The polar body of the egg is subjected to DNA analysis using array CGH. Array CGH, (also known as Chromosomal Microarray Analysis, Microarray-based comparative genomic hybridization, acgh, or Virtual Karyotype) is a technique that compares sections of the whole genome DNA of the polar body with normal (reference) DNA. DNA from the biopsy sample and a normal reference sample are labelled using different fluorescent markers and hybridized (bound together) to constitute several thousand identifier probes. The probes are derived from most of the known genes on the 24 different chromosomes and the signals from the test sample are compared to the signal from the known, normal sample. The ratio of the signals (the test signal vs. the reference signal) should be 1:1 and the signals should cancel each other out. However, if there is a disparity in the comparisons, this suggests that either there is an extra chromosome in the test sample (too much signal) or a missing chromosome in the test sample (too little signal). It should be noted that if polar body biopsy is performed, the eggs must be subjected to ICSI to achieve fertilization. For cost reasons, only those embryos that have fertilized normally on the day after injection will have CGH performed on the polar body that came from that egg. Equally, if CGH is performed on the trophectoderm only viable embryos will be sampled. If an abnormality is detected in the test tissue, it is inferred that the egg or embryo from which it came is similarly abnormal, and that embryo cannot be transferred. Only chromosomally-normal embryos would be transferred; chromosomally-abnormal embryos will never be transferred. Is the array CGH procedure safe for the embryos? Thousands of babies have been born following polar body biopsy and many thousands have been born after blastomere biopsy. To date there is no evidence that it is detrimental to a developing embryo and fetus. What sort of results can I expect following array-cgh testing of my eggs and how accurate are the tests? A report, giving details of the results for each of the eggs tested, is produced. In most cases, some will be found to have the correct number of chromosomes and some will be found to be abnormal. However, it is possible that the test may reveal that none of the embryos is healthy, in which case there will be no embryo to transfer. The likelihood that this will happen is dependent on your age and the number of eggs and embryos produced. Additionally, about 10% of the eggs tested fail to give a result. Eggs that have been biopsied but which have failed to give a result can still be transferred, but it will not be possible to say whether or not they have the correct number of chromosomes. The potential advantages of array-cgh will therefore not apply to such embryos. There is still a risk of misdiagnosis. The PGS result may suggest an abnormal number of chromosomes and yet the egg was normal. However, this is only thought to occur in less than

5 Page 5 of 5 5% of cases. Likewise, PGS does not replace the need for invasive antenatal testing. The studies show that in about 1% of the tests that give a reassuring result (i.e. a normal embryo), the fetus has an abnormal chromosome complement. As such, it is still recommended that you consider having screening tests at the antenatal clinic and, should the result suggest a high risk of a chromosome abnormality, that you consider an amniocentesis as this will give a definitive diagnosis. PGS is very expensive and there are also considerable emotional costs, especially if it doesn t work. This must always be borne in mind, and we encourage patients to speak with our counsellor before embarking on treatment. As with all IVF/ICSI procedures, there is no guarantee that any embryos will be suitable for freezing (vitrification) to be used at a later date. Furthermore, just because an embryo is chromosomally normal, it doesn t guarantee that it will implant or that a miscarriage won t happen as there are so many other factors, over which we have no influence, that affect normal embryo development. What tests are required prior to PGS? Before you decide to have PGS with array-cgh, it is recommended that you have a consultation with a doctor or nurse who specialises in these procedures, so you can discuss the test and have any questions answered. If you wish to be referred to a genetic counsellor, please let us know. Array-CGH should not be considered a substitute for invasive prenatal testing (see above). Prior to embarking on PGS, we strongly recommend that the chromosome complement (karyotype) of the male and female partner is checked to see that they are normal. This involves a blood test from each partner. The results take a few weeks to be reported, so these tests must be performed some weeks prior to planned ovarian stimulation. In the unlikely event that one or other is abnormal, then specialist genetic counselling is required. Costs The costs quoted are for polar body biopsy and based on up to 8 polar bodies being tested. Each subsequent polar body that is tested is individually priced and the costs are available from our website. Retention of samples The polar bodies which are tested are destroyed during the process of the array-cgh analysis. They cannot be returned to the embryo or used for any other purpose. DNA from these cells will be stored for a minimum of one year. This is to allow referral back to the sample should there be a discrepancy between the baby s chromosome complement and the report issued by ReproGenetics. If embryos are frozen, GCRM-BELFAST will tell ReproGenetics which samples they should retain and advise them when those frozen embryos have subsequently been transferred. They will then keep those samples for a year after the frozen embryo transfer. Further questions If you have further questions, please write them down and bring them with you to a consultation and we will do our best to answer them.

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