What does PGD / PGS involve?

PGD at Genea

Genea's Pre-implantation Genetic Diagnosis scientists have helped hundreds of families achieve their dream of taking home a baby. We first launched chromosome testing of embryos in 1995 with gene testing 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 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.

 

Chromosomes in detail

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 may 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.
 

Genea-karyotypes

Background to PGD/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.

Genea-icsi-day-0

Embryology - early embryos (Day 1-3)

We identify correctly fertilised eggs by the presence of pronuclei on Day 1.

 

Genea-embryo-day-1-to-3

At some point during embryo development, we make a small hole in the outer layer of the embryo (the zona pellucida) using a delicate near infrared laser beam. Called assisted embryo hatching, this creation of a small slit (breach) in the zona helps promote the hatching of blastocysts on Days 5-6, a little earlier than would occur with IVF.

embryo-hatching-day-3

Genea Assisted Hatching Video

Embryology - Blastocyst Stage (Day 5/6)

The embryo grows to form a blastocyst - a clump of cells (inner cell mass) inside a hollow ball of trophectoderm cells.
The expanding blastocyst, which is now 120-150 cells, hatches through the small breach that was created in the zona.
 
blastocyst-stage-day-5and6

Genea Day 5 full hatched blastocyst

Embryo Biopsy (Day 5/6)


We then hold the hatching blastocysts with gentle suction and use a biopsy pipette to gently remove three to six trophectoderm cells (the cells that go on to become the placenta) usually with the assistance of the laser. These biopsied trophectoderm cells are now ready for testing. For total chromosome testing, the rest of the embryo is frozen to allow time for NGS or combined PCR/NGS analysis. If it is a PCR only test, then your Doctor will discuss with you your options to have either a fresh transfer after testing or a freeze-thaw transfer at a later time.
 

Embryo Vitrification (Day 5/6 Freezing)

Vitrification is a freezing procedure that preserves embryos for future use.
To maximise survival, the embryos are partially dehydrated then snap frozen in liquid nitrogen.

Genetic tests

At Genea our scientific team use two major types of tests or analysis:
 

Total chromosome analysis

This test checks all chromosomes to see if an embryo has a balanced set. It looks for:
  • aneuploidy (chromosome abnormality): 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

Analysis

Cell transfer and Whole Genome Amplification (WGA)

The biopsied cells are washed and transferred into a reaction tube. We then extract the DNA and 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 represented in as even a profile as possible.

The amplified DNA is then modified to make it suitable for massively parallel sequencing (often referred to as Next Generation Sequencing or simply as 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. Using mathematical comparisons, our software is then able to look and see if there are major imbalances amongst chromosomes or smaller parts along the chromosome. There is a limit to how small a piece can be analysed by this method but most of the common chromosome copy number changes can be identified. The software then plots the chromosome results onto a graph that can be interpreted by Scientists and reported to your Doctor. A profile of all 24 types of chromosome will assist the scientists to identify and avoid transferring any embryos with chromosome sets incompatible with either starting a pregnancy or getting to delivery successfully.
 
Image below: Illumina MiSeq sequencer provides NGS data for scientist to analyse
 
Illumnia sequencer
 

Interpreting results

The chromosomes results are interpreted and reported to the doctor.
 
Image below: Example of normal male 
Results normal male
Image below: Example of abnormal male
Genea abnormal chromosome male results
 
 

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. They may however, 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

Not all cells in an embryo may contain the same number of chromosomes, so the biopsy piece may not represent the whole embryo. NGS cannot detect low level Mosaicism (<50 per cent).
 
Image below: Example of mosaic male embryo (less than 100% of cells have lost a chromosome/chromosome segment)
Results mosaic male

For the above reasons, the accuracy of the test offered is 95 per cent.
 

Prenatal screening

Should you have prenatal diagnostic tests done?

As you have read above, PGD does not provide 100 per cent accuracy due to the various technical difficulties that can be experienced. We recommend that prenatal diagnostic tests are discussed with your referring doctor.
 

Polymerase Chain Reaction (PCR)


This test tracks the inheritance of specific small chromosomal regions associated with:
  • single gene disorders;
  • HLA matching to detect antigens (genetic markers);
  • Translocations and uniparental disomy (UPD);
  • sex identification.

PCR in depth

Polymerase chain reaction (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. Most PCR testing is done in combination with chromosome analysis (NGS), but PCR can also be done in isolation on 'fresh' embryos.
 

Analysis

Cell transfer and PCR reaction

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, PCR enzymes are used to duplicate or amplify specific small segments of the genome into millions of copies.
 

Test workup

For single gene disorders, HLA matching and translocations we create or establish a unique PGD-PCR test for you as a family.

The screening process identifies informative linkage markers which we use to distinguish 'bad' mutation carrying chromosomes from the 'good' non-mutated chromosomes. Linkage markers are put together to form a mini DNA fingerprint that tells us whether the embryo has inherited the DNA mutation.

The primary test for all cases uses linkage markers which are small highly variable regions of DNA very near to the gene.

test-workup

Where possible, a direct mutation test will also be included as backup confirmation.

PGD-PCR test workups take time to complete and must be finished before a cycle can commence.

Once the workup is completed, a PGD-PCR test can be used for the following:

Single Gene Disorders

  • Family Linkage – Recessive Disorders. For an embryo to be affected, a recessive disorder requires two copies of the mutated gene, one from each parent (as illustrated by the black peaks shown in the DNA linkage pattern below).
 
recessive-disorders
 
  • Family Linkage – Dominant Disorders. A dominant disorder requires just one copy of the mutated gene, inherited from an affected parent.
  • Family Linkage – X-linked Disorders. An X-linked disorder requires just one copy of the mutated gene, inherited from an affected parent (males are affected and females are typically unaffected carriers).

Translocations

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.

Aneuploidy / Medical Sex Selection

The status for chromosomes X, Y and 21 of an embryo can be determined by looking at the DNA inheritance from both parents.
 

Possible outcomes of PCR testing

No biopsy

Unfortunately, not all embryos are suitable for biopsy. They may however, 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 transfer, freezing for future use, or if already frozen can potentially be thawed, rebiopsied 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. PCR will copy any DNA and 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. 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.
 

Technical limitations

Preferential Amplification and Allele Drop-out

DNA may copy unevenly which could cause difficulty in interpreting the results for certain linkage markers. This is why the DNA fingerprint looks at multiple regions around the gene.

preferantial-amplification-allele-drop-out

Recombination

Chromosomes naturally rearrange their DNA during egg and sperm formation and this may affect the interpretation of the result. If a direct mutation test was able to be included, it may help resolve any interpretation problem.

recombination
 

Transfer or freezing

Embryo transfer

After a PCR-only test is completed, a suitable embryo may be transferred later that day or frozen and then thawed for transfer at a future time, depending on what has been decided in conjunction with your Doctor. Embryos undergoing combined PCR/NGS testing will all be frozen for use in a future transfer.

Embryo vitrification

Excess suitable embryos or those undergoing combined PCR/NGS testing can be preserved for future use by vitrification. Vitrification is a special freezing procedure during which we partially dehydrate the embryos before snap freezing them in liquid nitrogen.

Prenatal screening - Should you have prenatal diagnostic tests done?

As you’ve read above, PGD/PGS does not provide 100 per cent accuracy due to the various technical difficulties that can be experienced. We recommend that prenatal diagnostic tests are discussed with your referring doctor.
 
New technologies now mean we can combine both PCR and Chromosome testing 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.

As with total chromosome testing, embryos are vitrified after biopsy while the dual analysis is underway. Embryos that are identified as being disease gene free and chromosomally balanced are available for transfer on a subsequent freeze/thaw cycle.

Refer to Total Chromosome Analysis and Polymerase Chain Reaction (PCR) for an in-depth explanation.

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 on average three to six 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 conduct 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:

Biopsy process

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.

Biopsy process

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, so you can have confidence in this process.

Embryos that we find have a normal chromosome complement can then 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 a technique called Polymerase Chain Reaction (PCR).

In the PCR process, 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 sample carries the affected gene.