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Pre-implantation genetic diagnosis (PGD or PIGD) comprises a series of techniques used to diagnose and exclude genetic abnormalities of embryos and oocytes prior to their return to the body during an IVF treatment cycle. Newcastle is one of very few centres in Australia that possess the combination of advanced IVF and genetics facilities to perform these sophisticated tests.
Types of genetic disorders
1. Mutations within a gene
2. Chromosome errors (numeric or rearrangement)
3. Errors within the mitochondrial DNA
4. Errors in more than one gene
Detectable genetic disorders
Any genetic disorder that can be diagnosed in an adult or by pre-natal diagnosis can be diagnosed by PGD.
Who will benefit from PGD
Any patient who might consider pre-natal diagnosis can benefit from PGD. In particular, genetic disorders in a previous child or family member such as
- Cystic Fibrosis
- Down’s syndrome or another numeric chromosomal disorder
- balanced chromosomal translocations (or prior pregnancy with an unbalanced translocation)
- women over the age of 35
- women with a history of recurrent miscarriage
may benefit from various types of PGD.
PGD Methodologies
Embryo Biopsy
PGD requires the patient to undergo an IVF cycle during which eggs are collected and embryos created. Embryos can be biopsied at 2 stages of development.
Cleavage Biopsy
Three days after egg collection, embryos usually consist of six to eight cells. Each embryonic cell has the potential to continue to grow to establish pregnancy. One or two cells are biopsied. The embryo will usually continue to develop normally. The removed cells are analysed for specific genetic problems by one of the methods outlined below and then those embryos that are determined to be normal are transferred back to the uterus at the blastocyst stage of embryonic development. Removing cells at this stage means taking away 12-25% of the embryo in total. A large number of the biopsied embryos will not make it on to the blastocyst stage as they do not have the correct developmental potential. Sometimes the cells studied are genetically different from the rest of the embryo and may therefore not accurately represent the embryo’s overall chromosomal state- we call this phenomena mosaicism.
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Cleavage stage embryo biopsy |
Blastocyst Biopsy
When embryos are 5 or 6 days old they contain many cells (usually over 100). It is possible to remove some of the cells from the outside of the embryo (trophoectoderm) and test them just as we do the cleavage stage cells (see below). Removing cells from the blastocyst has several advantages:
- More cells are obtained: 3-9 usually
- Much smaller fraction of cells removed from embryo
- The cells that go on to form the baby (inner cell mass) are not removed
- Only those embryos with the best developmental potential are biopsied
- A greater percentage of blastocysts are chromosomally normal than cleavage stage embryos
Blastocyst stage embryo biopsy
Genetic analysis
PCR
For errors within a gene (mutations) it is usually possible to design a test using Polymerase Chain Reaction (PCR) techniques that will show whether an embryo has the normal gene or the mutant gene. It helps if the mutation is known. Both dominant (eg myotonic dystrophy) and recessive (eg cystic fibrosis) conditions can be detected. When the error is not understood, it is usually possible to offer PGD by examining patterns in the DNA (polymorphisms or linked markers) in that part of the chromosome where the gene is known to be. The position of the affected gene must always be known.
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Fluorescent in situ hybrisdiation |
Molecular karyotyping |
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Comparative genomic hybridisation |
Gel chromotography |
FISH
Testing for both aneuploidy (numeric chromosome abnormalities such as Down’s syndrome) and sex determination is undertaken by fluorescent in situ hybridisation (FISH). Probes are designed that will attach to part of a chromosome. Each probe has a fluorescent signal of a unique colour attached. One set of probes called five colour FISH consists of probes with different coloured signals, designed to hybridise to chromosomes 13, 18, 21, X and Y. Normally we should see two signals of each colour. In trisomy 21 (Down syndrome) for example, we would expect to see three signals of one colour as the embryo has an extra chromosome.
In the case of X linked disorders (eg haemophilia and Duchene muscular dystrophy) the first step is to determine the sex of each embryo and then to determine, by PCR, which of the male embryos are affected and which are not, and which of the female embryos are carriers and which are not.
Methods for detecting disorders by PGD
Polymerase Chain Reaction (PCR)
• Gene mutations
• Gene deletions
• Mitochondrial DNA disorders
Fluorescent In Situ Hybridisation (FISH)
• Aneuploidy screening (e.g. screeing for Down syndrome)
• Chromosomal Translocations
• Sex linked diseases and
• Sex selection
Comparative Genomic Hybridisation (CGH)
• Multiple chromosome analysis
A new technique not available yet but may be helpful in the future to analyse all chromosomes.
Can all genetic problems be found using PGD?
Unfortunately not. In many cases of genetic disease the exact gene structure and location on a particular chromosome is not known. With the completion of the Human Genome project the range of genetic disorders that can be detected is rapidly increasing.
Using PGD to improving IVF pregnancy rates
PGD permits the development and transfer of embryos during an IVF treatment cycle with no identified genetic abnormality. As a result, in the future we expect this to result in a higher rate of embryo implantation and a reduced rate of miscarriage.
PGD in Newcastle