Pre-implantation Genetic Diagnosis (PGD)
1. Aneuploidy Screening
2. Single Gene Disorders
3. Chromosome Translocation
Team Miracle are industry leaders in Pre-implantation Genetic Diagnosis (PGD) and Dr. Firdevs offers one of the most successful gender selection programs anywhere in the world. As well as enabling patients to choose the gender of the future child, PGD also screens for over 400 hereditary diseases.
Aneuploidy Screening
Aneuploidy screening reduces the chance that a transferred embryo has a chromosome abnormality. The most common chromosome abnormalities in miscarriages include: trisomy (three copies of a chromosome), monosomy (one copy of a chromosome), triploidy (three copies of all the chromosomes) and abnormalities of the sex chromosomes.
When does PGD happen in the IVF cycle?
Embryos are created in the laboratory using IVF and are then cultured for three days. (See our IVF in Cyprus page for more information on the IVF process. http://cyprusivfcentres.com/)
On day three, the PGD biopsy is done, and one or two cells are removed from the embryo. The genetic material inside these cells is tested for abnormalities. On day five, the woman returns to Cyprus IVF Centre to see Dr. Firdevs and discuss her PGD test results. Decisions regarding selection of embryos to transfer into the uterus are made with the advice of the Team Miracle medical team.
Is PGD Safe?
Yes. PGD is done before the embryo’s genetic material becomes ‘active’. Since it is done so early, the cells inside the embryo are still all identical and each cell is capable of becoming any part of a baby. Removal of a cell of the early embryo does not alter the ability of that embryo to develop into a complete, normal pregnancy.
How do they get the cells out of the embryo?
By the time of the biopsy the embryos will be made up of 8 cells. Embryos with normal development on day three will have one or two cells removed for testing. The embryos are placed under a powerful microscope and a laser is used to create a tiny opening in the zona pellucida (outer membrane). One or two cells are then aspirated. The little hole that has been created closes up automatically and no cells can ‘fall out’ accidentally.
In order to obtain results of the biopsy, the one or two cells removed must contain a nucleus, as the nucleus contains the genetic information necessary for testing. If the cell removed has no nucleus or if the nucleus breaks open as it is being prepared, testing is unable to be performed on that cell and therefore the embryo will be declared “anucleur”. Anucleur embryos are not necessarily genetically unhealthy. It just means that it was unable to be tested so the gender and health status are unknown.
Additionally, since embryos are actively growing and dividing, sometimes the cell taken out contains two nuclei. This could mean the cell is abnormal or is caught in the process of division. Testing of these cells may be difficult to interpret.
How exactly is the PGD test done?
PGD testing is different than most genetic testing since it is done on only one or two cells and is completed within 48 hours for a fresh embryo transfer by day five. Since standard chromosome analysis takes several days, a different method called fluorescence in-situ hybridization (FISH) is performed.
Each chromosome has unique areas of DNA present only on that chromosome. A small DNA probe is used to recognize these unique patterns and fluoresce, or light up, when it attaches to the chromosome. Each probe shines light in a different colour, allowing several chromosomes to be tested at the same time. This technique is called FISH.
Which chromosomes are tested at Cyprus IVF Centre?
Dr. Firdevs uses 5 probe fluorescent in-situ hybridization (FISH) which screens for chromosomes 13, 18, 21, X and Y because these are the chromosomes that are most commonly abnormal. A normal cell should show two FISH signals (or lights) for each of the numbered chromosomes, and either two X signals for a female or one X and one Y signal for a male. There are only five different colours that can be used. If more than 5 chromosomes were to be tested, the first five chromosomes would be tested, those probes would be washed off and then the remaining chromosomes would be tested. The washing process can affect the integrity of each chromosome and the accuracy of the results and therefore we only test the 5 main chromosomes. For this reason, every chromosome cannot be tested.
What conditions can PGD in Cyprus detect?
If you have PGD for gender selection then your embryos will also be screened for the following conditions. Please note, the list is not exclusive. It just gives some examples out of many.
Aarsog’s syndrome
Adrenoleukodystrophy
Aicardi Syndrome
ALD
Alport’s syndrome
Amyotrophic Lateral Sclerosis
Androgen insensitivity syndrome
Barth syndrome
Becker Muscular Dystrophy
Beta Thalassemia
Blue Cone Monochromacy
Bruton’s agammaglobulinemia
Central Core Disease
Centronuclear (Myotubular) Myopathy
Cerebellar Ataxia
Charcot-Marie-Tooth disease (CMTX2-3)
Chondrodysplasia Punctata
Coffin-Lowry syndrome (CLS)
Colour blindness
Complete androgen insensitivity syndrome
Congenital Aganglionic Megacolon
Congenital aqueductal stenosis (hydrocephalus)
Conradi-Hunnerman Syndrome
Cystic Fibrosis
Down syndrome
Duchenne Muscular Dystrophy
Edward’s Syndrome
Fabry’s disease
Factor IX Deficiency
Factor VIII Deficiency
Familial Spastic Paraparesis
Fragile X Syndrome
Friedrich’s Ataxia
Gardener Syndrome
Giuffrè Tsukahara syndrome
Glucose-6-phosphate dehydrogenase deficiency
Glycogen Storage Disease
Goltz syndrome
Happle Syndrome
Haemophilia A and B
Hunter syndrome
Huntington’s Disease
Hypohidrotic ectodermal dysplasia, presenting with hypohidrosis, hypotrichosis, hypodontia
Idiopathic hypoparathyroidism
Incontinentia pigmenti
Inherited nephrogenic diabetes insipidus
Kabuki syndrome
Kennedy disease
Klinefelter’s Syndrome
Lesch-Nyhan syndrome
Lowe Syndrome
Marfan syndrome
Menkes disease
Muscular dystrophy
Nasodigitoacoustic syndrome
Nonsyndromic deafness and X-linked nonsyndromic deafness
Norrie disease
Occipital horn syndrome
Ornithine carbamoyltransferase deficiency
Ornithine transcarbamylase deficiency
Prostate Cancer
Retinitis Pigmentosa
Rett syndrome
Sickle Cell Anaemia
Siderius X-linked mental retardation syndrome
Simpson-Golabi-Behmel syndrome
Spinal muscular atrophy caused by UBE1 gene mutation
Tay-Sachs Disease
Turner syndrome
Vitamin D resistant rickets: X-linked hypophosphatemia
Von Willebrand Disease
Wiskott-Aldrich syndrome
X-linked agammaglobulinemia (XLA)
X-linked hypophosphatemia
X-linked ichthyosis
X-linked Severe Combined Immunodeficiency (SCID)
X-linked sideroblastic anaemia
How are embryos chosen for transfer?
Embryos that have both a normal test result and appearance can be transferred. Sometimes embryos that have normal genetic tests will have a physical problem that prevents them from growing normally such as fragmentation. Sometimes embryos that have abnormal genetic tests will appear to be physically normal. The combination of normal genetic testing with normal physical appearance indicates the highest chance of that embryo developing into a healthy pregnancy.
On your embryo transfer day in Cyprus, Dr. Firdevs will explain the results to you and give her advice on how many to transfer. Normally we recommend transferring three or four blastocysts for the maximum chance of pregnancy but the decision will always be left to you.
What if all the healthy embryos are not used?
If you have any leftover embryos, you can freeze them for 2 years.
(See our Cryopreservation page for more information. http://cyprusivfcenters.com/)
PGD for Single Gene Disorders
Although PGD can be used for selecting the gender of the next child or checking for common genetic problems, it can also be used for couples who carry a high risk of transmitting an inherited disease to their offspring.
In the past, for couples carrying a single gene disorder, the only option was to have prenatal diagnosis to detect the disorder in the foetus. However, if the results showed a genetically abnormal baby the only options would be to continue with the pregnancy and give birth to a child with a genetic disease or to terminate the pregnancy. Both are difficult, traumatic and life changing decisions to make, especially when the pregnancy is in advanced stages.
PGD in cyprus is now available as an alternative to prenatal diagnosis. It increases the options available for couples who have a known genetically transmittable disease and it gives comfort for those couples who want to have a baby without worrying about whether it will be affected or not. PGD can establish whether a specific embryo is affected by the genetic disease whilst it is still in the laboratory, meaning that only healthy embryos unaffected by the mutation will be selected for transfer.
Process
The couple must undergo in vitro fertilisation treatment with Dr. Firdevs if they would like PGD regardless of their fertility status. The mother will take stimulation injections which will allow collection of multiple eggs. The eggs are then fertilized using the father’s sperm and the resulting embryos are transferred to an incubator. After three days developing, the embryos should consist of eight cells (unless they are developing slowly). Once the embryos are made up of 8 cells, the Team Miracle embryologist will remove (biopsy) one or two cells from the embryo. If the cell is found to be unaffected by the mutation then the embryo that the cell was removed from will also be healthy. Embryos that are found to be unaffected by the inherited disease can be transferred to the mother, ultimately producing unaffected babies.
Can gender selection be carried out on the biopsied cell in addition to single gene testing?
Unfortunately not. Testing of the biopsied cells destroys them because their membranes must be broken open to release the DNA. Because of this, they cannot then be used for any other purpose.
Preparation Phase
A huge variety of genetic diseases can be analysed using the PGD procedure, however, first Team Miracle’s specialist genetic professors must develop, optimise and validate the specific probes that will be used for the testing. The probes that we build for our families are uniquely designed for each individual treatment. Cyprus IVF Centre PGD lab is known globally for the ability to test for some of the rarest genetic conditions.
To begin, Dr. Firdevs will require a blood sample from both parents for mutation verification and informativeness for the polymorphic markers included in each assay. In some circumstances we may also need blood from additional family members such as siblings or we may also collect cheek cells.
Before we can proceed with the IVF treatment, the genetic professor will validate a diagnostic single cell PCR protocol, by undertaking extensive preclinical trials on single lymphocytes or cheek cells. This is for evaluation of single cell amplification efficiencies and ADO rates for all the primers to be used in the procedure.
For some of the most common single gene diseases, the genetic professor can perform the probe creation and validation in approximately two weeks. However, for genetic diseases with variable mutations, the preparation phase can take four to six weeks.
Your Team Miracle coordinator will talk you through each stage and the timeframe that we will be working towards.
Genetic Analysis
One the probe has been created and the genetic professor confirms that it is ok to proceed; Dr. Firdevs will perform the IVF process. Once the embryos have been biopsied, the cells are analysed using a technique called the polymerase chain reaction (PCR). PCR is a simplified reproduction of the process of DNA replication. Taking advantage from the chemical properties of DNA and the availability of thermostable DNA polymerases, PCR allows for the enrichment of a DNA sample for a certain sequence.
PCR is used to amplify the DNA to a detectable level and provides the possibility to obtain a large quantity of copies of a particular stretch of the genome, making further analysis possible. It is a highly sensitive and specific technology, which makes it suitable for all kinds of genetic diagnosis, including PGD.
Once amplification has been accomplished scientists can use a variety of techniques to screen an individual gene for abnormalities.
Reaching a Diagnosis
Finally, the genetic professor will advise Dr. Firdevs which cells were affected and which were unaffected. The appearance of a single allele that indicates an affected genotype is considered sufficient to diagnose the embryo as affected and embryos that have been diagnosed with a complete unaffected genotype are preferred for transfer.
PGD for Chromosome Translocation
We are all made up of tiny building blocks called cells. These cells are controlled by information stored in long thin strands of DNA (Deoxyribonucleic Acid) which are stored in the nucleus (control centre) of the cell. The DNA controls the cells using signals from about 30,000 genes. Each gene is a specific piece of DNA that contains particular genetic instructions for our body to grow and develop.
The DNA is normally seen in 46 strands called chromosomes which are arranged into 23 pairs. Scientists have numbered these pairs from 1-22 according to their size, with chromosome 1 being the longest and chromosome 22 the shortest. The two chromosomes in each of these pairs are usually identical. The 23rd pair are called the sex chromosomes which determine whether we are male or female. Females have two X chromosomes and males have an X and a Y chromosome. Each chromosome has a short arm and a long arm, separated by a narrow area in the middle. Scientists only need a small amount of blood to look at a person’s chromosomes under the microscope.
The picture below shows what our chromosomes look like under the microscope if they are arranged according to their size.
How are chromosomes inherited?
When eggs or sperm are made, the pairs of chromosomes separate and one from each pair goes into each egg and sperm. Therefore, eggs and sperm only have 23 chromosomes. All eggs have an X sex chromosome and each sperm has either an X or a Y sex chromosome. When the egg is fertilised by the sperm, the resulting baby inherits half of its chromosomes, and therefore its genetic material, from its mother and half from its father.
We need to have the correct amount of genetic material for normal development. At any stage during this process, mistakes can happen including:
• The wrong number of chromosomes (either extra or missing chromosomes). A common example is Down’s syndrome, where individuals have three copies of chromosome 21.
• Mistakes in the structure of the chromosomes such as translocations.
What is a chromosome translocation?
A translocation means that a piece of one chromosome has broken off and stuck on a different chromosome. Chromosome translocations cannot be repaired. If a translocation has not resulted in any genetic material being lost, it is known as a balanced translocation. The picture below shows how a balanced translocation happens.
Balanced translocations do not usually cause any medical problems. This is because all the genetic material is present, even though it is in a slightly different arrangement. Balanced translocations are relatively common and occur in 1 in 625 people.
Having Children…
The main concern for people with a balanced translocation is that they may have a child with missing or extra genetic material, which can cause medical problems. For each pregnancy, the outcome depends on whether the sperm or the egg from the parent who has the balanced translocation contains both, one or none of the chromosomes that was involved in that translocation.
For example, if the father has a balanced translocation between chromosomes 3 and 7 and the mother has normal chromosomes, there are different possibilities as shown in the picture below.
However, it is not possible to determine the likelihood of each of these possibilities.
The baby can inherit:
(A) Neither of the chromosomes from the father that were involved in the balanced translocation. This will result in a normal healthy baby as they have inherited a normal set of chromosomes.
(B) Both chromosomes from the father that were involved in the balanced translocation. This does not usually cause any medical problems, but the baby will be a carrier of the balanced translocation just like their father.
(C) One chromosome from the father that was involved in the balanced translocation and normal copies of the other chromosomes. This means that the baby has missing or extra genetic material (i.e. the translocation is now unbalanced). This is likely to result in physical or mental disability. The type and severity of the disability depends on the amount of genetic material that is missing or extra. If a large amount of genetic material is missing or extra, the pregnancy is likely to end in a miscarriage.
For each pregnancy, a person with a balanced translocation has an increased risk of miscarriage and an increased risk of the baby being born with a disability.
What can Team Miracle do to help?
Couples who have PGD will undergo an IVF cycle to create embryos. Genetic analysis will then be performed on cells from each embryo prior to transfer into the woman’s uterus. To analyse an embryo, we biopsy the embryo around the third day of its development when the embryo has approximately eight cells. One or two cells are taken from the embryo. The embryo is incubated until testing is complete.
The biopsied cells are analyzed using a technique called fluorescence in-situ hybridization (FISH). This technique uses small pieces of DNA that are a match for the chromosomes we want to analyze, to count the chromosomes present.
For couples undergoing IVF and PGD for translocations, the embryos will first be tested for unbalanced translocations. If technically possible, we will also perform a second test for chromosomes 13, 18, 21, X, and Y which are those chromosomes most likely to result in a live born child with a chromosome abnormality. The FISH analysis involves two rounds of testing for each cell.
Testing of the cells destroys them because they must be glued to a glass slide and repeatedly heated and cooled. As such, one cannot use them for another purpose or return them to the embryo. This analysis causes no extra inconvenience to the patient as it is accomplished in one day.
Aneuploidy
The biopsied cells are first fixed to a microscope slide and then the cellular material is digested away leaving the nucleus, which contains the DNA, in a spread out and decondensed form. These cells are then hybridised with DNA probes, labelled with different fluorochromes. Each of these probes is specific for part of a chromosome; they will only attach to their exact DNA match on a particular chromosome. Excess probe is washed off, and the cell is examined under the fluorescent microscope. We then count the number of chromosomes of each type (colour) there are in that cell. The geneticist therefore can distinguish normal cells from cells with aneuploidy.
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