What does PGD / PGS involve?
Genea's Pre-implantation Genetic Diagnosis scientists have helped hundreds of families maximise the potential of a baby. We first launched chromosome screening of embryos in 1996 with testing for inherited single gene disorders introduced in 1998. Since then our PGD techniques have led the world.
Genea is one of the very few centres in Australia with the vital combination of IVF and genetics facilities to perform these sophisticated tests successfully. Genea was the first clinic in the world to perform blastocyst stage biopsy and we introduced it as routine in our labs in 2004. A review of information captured by the Australian and New Zealand Assisted Reproduction Database (ANZARD) between 2004 and 2008 showed our Day 5/6 biopsy approach to PGD resulted in a significant lift in IVF success rates and the number of babies born.
The data review showed blastocyst biopsy for PGD/PGS improves both implantation and live birth rates when compared to biopsy at Day 3. Since then, blastocyst stage biopsy has become a world standard - an innovation led by the Genea team. Learn more about Genea's PGD success rates.
Embryo development and biopsy for PGD and PGS
How does PGD or PGS fit into an IVF cycle?
Because IVF is the starting point for any embryo testing, whether you are having a standard chromosome analysis, a gene specific test or a combination test, all of our processes have the same beginning.
Fertilisation - Intracytoplasmic sperm injection (ICSI) (Day 0)
Because DNA contamination from cumulus cells surrounding the egg or from unsuccessful sperm can contaminate PGD samples and result in misdiagnosis, we usually fertilise the egg using ICSI to reduce the likelihood of this problem occurring. Fertilisation with ICSI involves carefully removing the cumulus cells and then injecting a single sperm - both of which greatly reduces the risk of contamination.
At times there may be sound clinical reasons to use the alternative IVF insemination protocol and if this is the case for you and your partner, be assured your doctor is aiming at achieving the best possible outcome for you as a couple.
Embryology - early embryos (Day 1-3)
We identify correctly fertilised eggs by the presence of pronuclei on Day 1. A normally developing embryo should have 6-8 cells by day 3.
At some point during embryo development, we make a small hole in the outer layer of the embryo (the zona pellucida) using a near infrared laser beam. Called assisted embryo hatching, this creation of a small hole in the zona helps promote the hatching of blastocysts on Days 5-6, a little earlier than would naturally have occurred with IVF.
Genea Assisted Hatching Video
Embryology - Blastocyst Stage (Day 5/6)
The embryo grows to form a blastocyst - an embryo that consists of two types of cells, and ICM (inner cell mass) and the trophectoderm.
The expanding blastocyst, which is now 80-150 cells, hatches through the small breach that was created in the zona.
Genea Day 5 full hatched blastocyst
Embryo Biopsy (Day 5/6)
The hatching blastocysts is held using gentle suction and then a biopsy pipette is used to gently remove approximately five trophectoderm cells (the cells that go on to become the placenta) usually with the assistance of the laser. The inner cell mass, which goes on to from the foetus, is minimally disturbed. The biopsied trophectoderm cells are now ready for testing. After 1-2 hours recovery, the rest of the embryo is frozen to allow time for chromosome analysis and/or inherited genetic disorder testing.
Freezing and transfer
Embryo Vitrification (Day 5/6 Freezing)
Vitrification is a procedure that preserves embryos for future use by partially dehydrating them prior to snap freezing in liquid nitrogen. The vitrification process maximises the survival rate so that the best embryo can be chosen for thawing and transfer once the genetic analysis is complete.
Genetic tests
At Genea our scientific team use two major types of tests or analysis:
Total chromosome analysis
Understanding PGS
PGS testing checks all chromosomes to see if an embryo has a balanced set. The information obtained is useful for:
- aneuploidy (random chromosome abnormalities): a frequent cause of unsuccessful IVF attempts, miscarriages and abnormal live births;
- translocations: looking for unbalanced parts/sets of chromosomes that can result from inherited chromosomal rearrangements;
- medical sex selection: tells us whether the embryo is male or female, which allows genetic disease risk reduction in such cases.
Chromosomes in detail
Humans generally have 46 chromosomes that come as 23 pairs, one of each pair from our Mum and one of each from our Dad. Problems arise when an embryo randomly misses out on a chromosome or picks up an extra one – that’s called a chromosomal abnormality or aneuploidy.
Chromosomal abnormalities can cause your embryo to fail to implant, miscarry, or they might result in a baby born with developmental problems or a serious genetic condition.
Some couples also have non-random, inherited chromosomal abnormalities. We find that these sort of chromosomal rearrangements are 10 times more common in couples seeking IVF treatment and so this is why your Doctor will suggest that you have a simple blood test called a karyotype before you begin treatment. A karyotype (or picture of your chromosomes) helps us to identify most types of chromosome rearrangements in the parents prior to embryo testing, giving us a head start on the sorts of problems we need to look for in the embryos you create during your IVF cycle.
Analysis
Cell transfer and Whole Genome Amplification (WGA)
After biopsy, the removed cells are washed and transferred into a reaction tube where the DNA is extracted. To provide sufficient DNA for analysis, we use enzymes to duplicate or amplify the three to six copies of the genome into approximately a million copies. This happens as a whole series of small DNA sections - it is important that the whole genome is copied as evenly as possible to obtain a high quality result.
Next Generation Sequencing (NGS)
A portion of the amplified DNA is then subjected to massively parallel sequencing (often referred to as Next Generation Sequencing or simply NGS). After sequencing the end of each DNA fragment, software can identify not only which chromosome the fragment belongs to but where on the chromosome it sits. Our scientists are then able to look at the profile of all 24 chromosomes and see if there are major imbalances between different chromosomes or smaller parts along an individual chromosome. Embryos with abnormal profiles have chromosome sets that are incompatible with either starting a pregnancy or getting to delivery successfully.
Although NGS represents the most advanced PGS technology to date, there are still limits to how small a chromosomal error can be detected by this method. However, you can rest assured that most of the common chromosome copy number changes can be identified.
Image below: Illumina MiSeq sequencer provides NGS data for scientist to analyse
Interpreting results
The NGS profiles of all 24 chromosomes are interpreted and the results reported to the doctor.
Image below: Example of normal male
Image below: Example of abnormal male, with the loss of one chromosome and gain of two others
Embryo transfer
Suitable embryos, that is those that contain a balanced set of chromosomes, may be thawed and transferred. Don’t be concerned about this impacting your chance of success - our studies show that thawed embryos have implantation rates equal to or greater than fresh embryos.
Possible outcomes of Chromosome testing
No biopsy
Unfortunately, not all embryos are suitable for biopsy. However, they may still be suitable for freezing or untested transfer.
No diagnosis
An answer is achieved for most of the embryos tested. If for technical reasons we do not get an answer on an individual embryo, the genetic status of that embryo will be considered as unknown. These embryos may still be suitable for untested transfer, or potential thaw, rebiopsy and refreeze.
No embryos with desired genetics
Embryos tested and deemed abnormal or affected are unsuitable for transfer. You may complete a cycle and not have an embryo suitable for transfer.
Misdiagnosis
The amplification of a few copies of DNA (from the few cells biopsied from an embryo) into millions of copies of DNA is at risk of contamination from extraneous human DNA as well as amplification biases. Despite our stringent precautions, there is always a small risk of contamination/bias resulting in a misdiagnosis.
Mosaicism
Some embryos can be mosaic, where not all cells contain the same number of chromosomes, so the biopsy piece may not represent the whole embryo. NGS can detect mosaicism in a biopsy sample down to a very low level (20%). Where an embryo returns a mosaic result, its suitability for transfer needs to be assessed on a case by case basis.
Image below: Example of mosaic male embryo (where some chromosome shifts indicate less than 100% of cells have lost a chromosome/chromosome segment)
For the above reasons, the accuracy of the test offered is 95 percent.
Prenatal screening
Should you have prenatal diagnostic tests done? As you have read above, PGD does not provide 100 percent accuracy due to the various technical difficulties that can be experienced. We recommend that prenatal diagnostic tests are discussed with your referring doctor.
Analysis of Inherited Genetic Disorders
Testing for genetic disorders tracks the inheritance in a family of specific small chromosomal regions that are associated with:
- single gene disorders;
- HLA matching to detect antigens (genetic markers);
- Translocation carrier status and uniparental disomy (UPD)
Workup phase
Tracking of inheritance in embryos involves a technique called “linkage analysis”. Linkage analysis uses natural variations between the genomes of individuals to track the inheritance of chromosomal regions from one generation to the next. At Genea we offer two different technologies by which linkage analysis can be achieved; Karyomapping and PCR/STR analysis. These 2 approaches permit us to offer testing for the widest possible range of genetic disorders and ensures that the best approach is utilized for your individual situation. Where possible, we will also develop a direct mutation test as backup confirmation of the linkage analysis.
Karyomapping
Karyomapping is an advanced technology that delivers rapid test development for families requiring analysis to prevent the inheritance of single gene disorders. Karyomapping employs single nucleotide polymorphisms (SNPs) near known disease gene regions to provide linkage information for PGD applications.
This platform allows for multiple disorders and HLA matching to be added to embryo testing without extending the waiting time for commencement of a cycle.
Where possible this test is offered in conjunction with direct detection of any identified familial mutation to deliver a highly accurate means of selecting embryos without the inherited condition.
What is the process to get started?
Once you are under the care of a Genea IVF specialist we will collate all the relevant genetic history for your family (including any genetic mutation report) and organise for the appropriate family members to provide a DNA sample.
To confirm that karyomapping is suitable for your family we will perform a preliminary test development which will take approximately 2-4 weeks from receipt of your samples.
Once this has been completed an IVF PGD cycle can commence and a report on your embryos will be issued to you within 2-3 weeks post embryo biopsy.
If you do not qualify for karyomapping then we will need to develop a custom linkage method for you that requires a 10-12 week test development phase (please see PCR/STR information below). The fees you have paid for a karyomapping evaluation will be incorporated to the test development fee for a custom PCR/STR work up.
In addition to tracking inheritance of genetic disorders, Karyomapping is able to detect some chromosome abnormalities which may be associated with implantation failure, miscarriage, or abnormalities at birth. While the test is not specifically designed to screen for chromosome errors and there is no guarantee that all chromosome abnormalities will be detected, this does represent a significant improvement compared to no chromosome testing.
Image below: Illustrates how the SNP data from karyomapping is used to map the inheritance of a mutated gene region from the parents (top row) to embryos (bottom row). This example documents the outcomes for a recessive condition where both parents carry a genetic mutation.
Custom STR linkage using Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) utilizes simple tandem repeats (STRs) to provide an alternative means of linkage analysis. PCR is a molecular technique which amplifies a single copy or a few copies of a specific piece of DNA, generating thousands to millions of copies of a particular DNA sequence. PCR/STR testing has been used reliably for many years for the analysis of single gene defects and continues to provide an important alternative approach when patients do not qualify for karyomapping.
Image below: Illustrates how the pattern of STR linkage markers can be used to select embryos that have not inherited a mutated “e,f,g,h” region from the Father.
Combined PCR/STR
Although PCR/STR analysis does not provide any information on chromosome abnormalities, we can combine both single gene detection by PCR/STR and comprehensive chromosome testing by NGS on the one biopsy piece taken from your embryo - a second biopsy is not required to perform both analyses. As a result we can not only eliminate the risk from the inherited disorder but also ensure we exclude embryos that are chromosomally abnormal. This will allow better selection of embryos for transfer and help us achieve a healthy ongoing pregnancy faster.
Analysis for Single gene disorders
Cell transfer and single gene testing
We wash the cells biopsied from the embryo and transfer them into a reaction tube before extracting their DNA. Because the biopsied cells do not contain enough DNA to analyse directly, the sample undergoes a whole genome amplification step to generate a million copies of the embryo DNA. Portions of these embryo samples are then used for the karyomapping or PCR/STR linkage analysis described above.
Translocations and other inherited chromosomal rearrangements
DNA linkage patterns can be used to check if an embryo is balanced for a specific translocation, is affected by UPD and potentially determine if balanced embryos are carriers of a translocation or not. Translocation patients can also receive comprehensive chromosome analysis of their embryos to screen for general chromosomal abnormalities in addition to those that arise due to their translocation.
Genea also offers customization of Comparative Genome Hybridization (CGH) arrays for detection of unbalanced embryos arising from translocations, duplications, or deletions that may be carried by your family, but which are too small to be detected by our standard NGS chromosome screening assay. In many cases the customized array approach can also be supplemented with a linkage test to provide confirmation of the result.
Possible outcomes of embryo gene testing
No biopsy
Unfortunately, not all embryos are suitable for biopsy.
No diagnosis
An answer is achieved for most of the embryos tested. If, for technical reasons, we do not get an answer on an individual embryo, the genetic status of that embryo will be considered as unknown. These embryos can potentially be thawed, re-biopsied and refrozen.
No embryos with desired genetics
Embryos tested and deemed abnormal or affected are unsuitable for transfer. You may complete a cycle and not have an embryo suitable for transfer.
Misdiagnosis - contamination
While we take stringent precautions to avoid DNA contamination, there is always a small risk that it may occur and if it does, it could result in a misdiagnosis. Whole genome amplification reactions and PCR will copy all DNA, so any contamination present will contribute to the answer.
Misdiagnosis – technical factors
Other technical factors may contribute to loss of genetic information that might result in misinterpretation of the genetic status of the embryo. In particular DNA may copy unevenly which could cause difficulty in interpreting the results for certain linkage markers. Technical limitations can also lead to a reduction in test accuracy. If this occurs during your treatment it will be discussed with you by your PGD scientist.
Recombination
Chromosomes naturally rearrange their DNA during egg and sperm formation and this may affect the interpretation of embryo linkage results. If a direct mutation test was able to be included, it may help resolve any interpretation problem.
Prenatal screening - Should you have prenatal diagnostic tests done?
As you’ve read above, PGD/PGS does not provide 100 percent accuracy due to the various technical difficulties that can be experienced. We recommend that prenatal diagnostic tests are discussed with your referring doctor.
Further information
Embryo biopsy
To conduct PGD or PGS, we biopsy or remove a small number of cells from each embryo for analysis. Don’t worry, this does not damage your precious embryos - at Genea, our advanced embryo culture techniques allow us to wait until the embryos have reached the blastocyst stage of development (Day 5 or 6). This means the embryos have a hundred or more cells and we need to remove approximately five cells.
By waiting until embryos have reached the blastocyst stage, Genea scientists can select cells from the trophectoderm, the part of the embryo that will go on to form the placenta. The inner cell mass, the part that will become the baby, is not touched.
In contrast, many other clinics have until recently conducted the biopsy at Day 3 of the embryos' development when they consist of just six to eight cells. They remove one or two cells which may impact on the potential for the embryo to successfully implant in the uterus.
This explains the process at Genea:
1. At one point of the embryo's development, a small hole is made in the outer layer of the embryo (the zona pellucida) using a delicate laser beam.
2. The embryo continues development until Day 5 or 6 when it becomes a blastocyst, characterised by the separation of cells into trophoblasts (which go on to become the placenta) and the inner cell mass (which goes on to become the fetus).
3. Trophoblasts are drawn out through the hole using a hollow suction tube called a biopsy pipette. The required cells are separated from the others using the laser and collected separately.
4. The remaining cells quickly realign realign and seal the area from which the biopsied cells were removed.
Analysis
Genea scientists use a number of different methods to analyse the biopsied cells. For chromosome analysis, we use a technique called Next Generation Sequencing (NGS) to analyse biopsied cells.
To begin, the biopsied cells are subjected to whole genome amplification (WGA), a process that results in the production of multiple copies of all the chromosomes present.
Because NGS involves an extensive analysis procedure which takes some time, we vitrify or freeze all embryos undergoing chromosome analysis. Genea have been vitrifying Day 5/6 blastocysts clinically since January 2006 and there is no difference in success rates between fresh and frozen embryo transfers.
Embryos that are found to have a normal chromosome complement can be transferred in subsequent frozen embryo cycles.
If the problem or condition we’re looking for is at a gene level rather than chromosome then we will use one of the linkage techniques currently available called karyomapping or PCR/STR analysis.
Using these gene testing processes, we make millions of copies of a particular section of a gene region which allows us to see whether this part of the DNA in the embryo sample carries the affected gene.