Embryo Genetic Testing (PGD)
Single Gene Testing
What is a single gene disorder?
Single gene disorders are genetic conditions which are caused by specific gene change/s in a
person’s DNA. Single gene disorders are heritable and often run in families. Individuals with a family
history of a single gene disorder may be at risk for passing the condition onto their children.
Examples of single gene disorders include Cystic Fibrosis, Huntington Disease, Fragile X and
Myotonic Dystrophy.
What is PGD with single gene testing?
PGD is a viable option for couples at risk of passing on a specific single gene disorder to their child.
PGD can be used to screen IVF embryos for the single gene disorder prior to implantation, with the
aim of distinguishing between unaffected embryos and affected embryos. Only unaffected embryos
will be selected for transfer to the uterus.
In addition to testing for the single gene disorder, it may be possible to analyse the chromosomes in
the embryo (ie. chromosome screening). Some embryos can have an abnormal number of
chromosomes (ie: missing or extra chromosome/s) due to errors in cell division in the developing egg,
sperm or embryo. This is known as chromosomal aneuploidy. An aneuploid embryo will fail to
implant, miscarry, or result in the birth of an affected child (eg: a child with Down syndrome). This
additional testing will help ensure that the embryo that is selected for transfer has the best possible
chance of developing into a healthy baby.
Monash IVF has developed PGD tests for numerous single gene conditions, some of which are
outlined in Table 1. The Monash IVF PGD team is highly skilled in developing new PGD technologies
and is able to develop tests for other genetic disorders upon request. If the disorder you are
interested in is not listed please contact Monash IVF for further information.
Table 1: Tests have previously been developed for these single gene disorders
Achondroplasia
Hypophosphatemic Rickets
Adrenoleukodystrophy
Infantile Batten Disease
Alagille Syndrome
Incontinentia Pigmenti
Albright Hereditary Osteodystrophy
Juvenile Retinoschisis
Alpers Syndrome
KELL Blood Group
Alpha thalassaemia
Laing Distal Myopathy
Amyloidosis
Larsen Syndrome
Aniridia
Leri-Weill Syndrome
Antithrombin III
Lesch Nyhan Syndrome
Ataxia Telangiectasia
Lissencephaly
Autosomal dominant polycystic kidney disease
Loeys Dietz Syndrome
Autosomal recessive polycystic kidney disease
Long QT Syndrome
Becker Muscular Dystrophy
Lymphoproliferative Syndrome
Beta-Thalassaemia
Lynch Syndrome
Bethlem Myopathy
Marfan Syndrome
BrCa1
Medium Chain Acyl CoA Dehydrogenase Deficiency
BrCa2
Metachromatic Leukodystrophy
Bruton’s X-linked Agammaglobulinaemia
Multiple Endocrine Neoplasia, Type 1
Bullous Ichthyosiform Erythroderma
Multiple Endocrine Neoplasia, Type 2a
Cadasil
Multiple Osteochondromas (Exotoses)
Cardiomyopathy
Myotonic Dystrophy
Carpenter syndrome
Myotonic Dystrophy Type 2
Cartilage hair hypoplasia dysplasia
Myotubular Myopathy
Central core disease
Neurofibromatosis, Type 1
Charcot-Marie-Tooth, Type 1A
Neurofibromatosis, Type 2
Charcot-Marie-Tooth, Type 1B
Neurofibromatosis, Type 3
Charge syndrome
Ocular Albinism Type 1
Chronic Granulomatous Disease
Optic Atrophy
Congenital Amegakaryocytic Thrombocytopenia
Opitz GBBB Type 1
Congenital insensitivity to pain
Osteogenesis Imperfecta
Crouzon Syndrome
Peutz-Jeghers Syndrome
Cystic Fibrosis
Phaeochromoctoma
Deafness (connexin 26)
Polycystic Kidney Disease
Diastrophic Dysplasia
Pontocerebellar hypoplasia
Duchenne Muscular Dystrophy
Presenilin 1
Elastin gene disorder
Propionic Acidemia
Escobar Syndrome
Retinitis Pigmentosa
Facioscapulohumeral muscular dystrophy
Retinoblastoma
Familial Adenomatous Polyposis
Rhesus D Blood Group
Familial Hemophagocytic Lymphohistiocytosis
Saethre Chotzen Syndrome
Familial Motor Neuron Disease
Severe Combined Immune Deficiency
Fragile X
Smith Lemli Opitz Syndrome
Gastric Cancer
Spinal Muscular Atrophy (SMA)
Glycogen Storage Disease Type IV
Spinocerebellar Ataxia, Type 1
Haemophilia A
Spinocerebellar Ataxia, Type 2
Haemophilia B
Spinocerebellar Ataxia, Type 3
Hereditary Diffuse Gastric Cancer
Spinocerebellar Ataxia, Type 6
Hereditary Multiple Exostoses
Spinocerebellar Ataxia, Type 7
Hereditary Non-Polyposis Colorectal Cancer
Tay Sachs Disease
Hereditary Sensory Neuropathy
Treacher Collins Syndrome Type 1
Holt-Oram Syndrome
Trimethylaminuria
Huntington’s Disease
Tuberous Sclerosis
Hydrocephalus
von Hippel-Lindau Disease
Hypertrophic Obstructive Cardiomyopathy
Wiskott-Aldrich Syndrome
Hypohidrotic Ectodermal Dysplasia
Zellweger Syndrome
Hypophosphatasia
How is PGD with single gene testing done?
Step 1: Genetic testing
One or both partners must have had previous genetic testing to determine the exact gene change/s
causing the genetic condition in their family. If the causative gene change/s has not yet been
identified, the couple should be referred to meet with a clinical geneticist or genetic counsellor. These
genetic specialists will be able to organise this genetic testing. The PGD team needs to know the
causative gene change/s in order to proceed with developing a PGD test.
Step 2: Genetic counselling in PGD clinic
Once the specific gene change/s has been identified, the couple meets with a clinical geneticist or
genetic counselor to discuss PGD. During this appointment the couple will be provided with
information relating to the PGD process at Monash IVF and will have an opportunity to have any
questions answered. If the couple wishes to proceed with PGD testing, the genetic counselor will
arrange for the collection of blood/DNA samples for feasibility testing.
Monash IVF currently offers single gene testing using two different test types, called SNP array testing
and Polymerase Chain Reaction (PCR). The main difference between these two test types is that
SNP array testing will allow simultaneous analysis of chromosome copy number, whereas PCR
testing will only provide an analysis of the single gene disorder. The test type/s available to each
couple will depend on the specific gene change/s that have previously been identified. Genetic
counselling is an important step to help patients understand the differences between the tests, so that
they make an informed decision regarding which test is best in their case. The PGD testing option
that is performed will be decided by the patient in consultation with their IVF specialist and Genetic
Counsellor. A comparison of the different PGD test types is provided in Table 1.
Table 1: Detection capabilities of SNP arrays compared to PCR testing.
Criterion
SNP array
PCR
Screen for targeted gene change/s


Screen 24 chromosomes

x
Confirm genetic parentage

Some
Detect extraneous DNA contamination

Some
Cost for feasibility testing


2-3 months
3-6 months
ICSI
ICSI
per cycle
per cycle
D3 or D5/6
D3 or D5/6
Frozen
Fresh (D3 biopsy)
Frozen (D5/6 biopsy)
Estimated time frame for feasibility
Fertilisation method
(Standard insemination or Intracytoplasmic sperm injection)
Embryo biopsy fee charged
Day of embryo biopsy
Embryo transfer
Step 3: Feasibility testing
Prior to commencement of an IVF/PGD cycle, it is necessary for the couple to undergo a feasibility
test in order to determine if PGD will be possible for their particular gene change/s. The feasibility test
will ensure that the final PGD test can adequately distinguish between unaffected embryos (suitable
for transfer) and affected embryos (not suitable for transfer). Feasibility testing will require a copy of
the carrier’s genetic report/s and a blood sample from both partners. In most cases, a blood or DNA
sample will also be required from one of the following:
- Both partner’s parents
- The couple’s child
- A DNA sample from prenatal testing in a previous pregnancy
In some cases, a sperm sample from the male partner may also be requested. These additional
samples are used to track the inheritance of the particular gene change/s within the family.
A feasibility report outlining the results of the feasibility testing is sent to the IVF doctor and genetic
counsellor. The IVF doctor or genetic counsellor will contact the couple to go through the results of the
feasibility test. In some instances it may not be possible to develop an accurate test and PGD may
not be available. There is a non-refundable fee for the feasibility test. If feasibility has been
confirmed, IVF/PGD may proceed.
Step 4: IVF and embryo biopsy
All couples undergoing PGD testing must undertake an IVF cycle to stimulate the woman’s ovaries to
produce a number of eggs. These eggs are collected and fertilised using the male partner’s sperm.
Embryos are created using a fertilisation method called Intracytoplasmic Sperm Injection (ICSI), which
involves the injection of a single sperm into the egg. ICSI is specifically used in these cases to reduce
the risk of misdiagnosis due to the presence of additional sperm around the egg/embryo. The
resulting embryos are cultured in the laboratory and their growth is monitored on a daily basis.
Embryo biopsy (sampling cell/s from the embryo for genetic testing) can be performed at two different
stages during an embryo’s development:
1. Day 3 embryo biopsy is performed 3 days after fertilization, when the embryo is at the
cleavage stage and is typically composed of 6 to 8 cells. Embryos that have developed to at
least 5 cells on Day 3 are suitable for biopsy. A hole is drilled in the outer shell of the embryo
and 1 or 2 cells are removed for genetic analysis (refer to Figure 1).
Figure 1: Day 3 embryo biopsy
2. Day 5/6 embryo biopsy is performed 5 or 6 days after fertilization. By this time, the embryo
should have developed to the blastocyst stage, and should be comprised of an inner cell mass
(which will go on to form the fetus) and trophectoderm cells (which will go on to form the
placenta). Embryos need to have a clear inner cell mass and a suitable number of healthy
trophectoderm cells to be considered suitable for biopsy. Approximately 5 trophectoderm cells
are removed for genetic analysis (Refer to Figure 2).
Figure 2: Day 5/6 embryo biopsy
In the majority of cases, Monash IVF recommends Day 5/6 biopsy. This is due to the following
reasons:
1. Randomised control trials have shown that Day 5/6 biopsy is better for the embryo compared with
Day 3 biopsy (Scott et al, 2013).
2. The embryo has more cells on Day 5/6 of development (~100 to 150 cells) compared with Day 3
of development (~6 to 8 cells). This means:
- More cells can be biopsied for genetic testing on Day 5/6 of development compared with Day
3 of development (ie: approximately 5 cells versus 1-2 cells, respectively). The availability of
more cells improves the accuracy of the PGD test results.
- Despite biopsying more cells from Day 5/6 embryos, a smaller percentage of cells is removed
from the embryo following a Day 5/6 biopsy compared with a Day 3 biopsy (ie: we are
removing approximately 5/100 (5%) cells on Day 5/6, compared with 1/6 (17%) cells on Day
3).
3. Day 5/6 embryos are more likely to be chromosomally normal then Day 3 embryos (as some of
the embryos that are chromosomally abnormal on Day 3 do not have the developmental capacity
to grow to Day 5/6 in culture). Therefore, growing the embryos to Day 5/6 before performing the
PGD testing provides an element of natural selection.
4. Day 5/6 embryo biopsy enables the patient to confirm that their embryos are capable of
developing to an advanced stage in culture before proceeding with PGD testing. The alternative
is to perform PGD testing on Day 3 with the knowledge that the embryos may not continue to
develop and therefore may not be suitable for transfer from an Embryology perspective.
Step 5: Genetic testing
Following embryo biopsy, the biopsied cells are transferred to a small test tube for genetic testing.
The genetic testing process will depend on the PGD test type being performed:
1. SNP array (performed using “parental support”)
In this procedure, the DNA from the embryonic cells is multiplied thousands of times (to
generate enough DNA for testing) and is placed on a microarray platform. This microarray
platform contains probes for over 300,000 different DNA sites. The DNA from the embryo
biopsy sample binds to the DNA probes on the microarray platform. Following binding, it is
possible to “read” the DNA code at each of these DNA sites. By screening a blood sample
from each partner in parallel with the embryonic cells, it is possible to determine whether each
embryo is unaffected or affected by the specific single gene disorder. An analysis of all
chromosomes is also performed to determine how many chromosome copies are present in
each embryo (Figure 3). Only embryos which are identifed as being unaffected by the
disorder, have the correct number of chromosomes and are developing normally will be
considered suitable for transfer. Embryos which are affected and/or have an abnormal
number of chromosomes (ie: missing or extra chromosomes) will not be considered suitable
for transfer.
Figure 3: PGD testing method using SNP arrays.
+
Test
cell/s
DNA from
male partner
Test DNA
DNA from
female partner
A
T
G
C
T
T
A
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A T
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A T
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G C
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G G
A
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C C
A
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C
A T
A
T
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T A
A
T
G
C
A T
A
T
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C
C A
A
T
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A C
A
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A A
A
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G
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G G
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C G
A
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G
C
C G
Chromosomes inherited by embryo
2. Polymerase chain reaction (PCR)
In this procedure, the DNA from the embryonic cells is multiplied thousands of times in a
targeted manner using a procedure called PCR. This generates millions of copies of the gene
of interest. The product from the PCR reaction is tested for the presence or absence of the
known parental gene change/s using a range of genetic techniques and a genetic analyser
(Figure 4). In most cases, one or two linked markers are included in the final test in order to
increase the accuracy of the results.
Figure 4: Example of single gene PGD results. Affected gene copies are indicated by an
asterisk.
c.2027 C to A
gene change
D7S1825
linked marker
D7S1513
linked marker
Mother (normal)
Father (affected)
Embryo 1 (normal)
Embryo 2 (affected)
Step 6: Embryo transfer
The time of embryo transfer will be dependent upon the PGD test type performed, as follows:
1. SNP array testing (performed using “parental support”)
Because of the time taken to perform the genetic testing, the embryos must be frozen
following biopsy. Final results are usually available 2 to 3 weeks after biopsy. If available,
one or two unaffected embryos can be thawed for use in a frozen embryo transfer cycle. A
PGD scientist/Embryologist will discuss the PGD results with the patient prior to transfer.
The patient’s IVF nurse will organise a pregnancy test to be performed on Day 16 of the
frozen embryo transfer cycle. This process should increase the chance of a healthy
pregnancy and significantly reduce the risk of miscarriage. Surplus unaffected embryos will
remain in storage. These embryos may be used in a subsequent cycle. Affected embryos
will be removed from storage and allowed to succumb.
2. PCR testing
Embryos identified as being unaffected by the disorder are considered suitable for transfer.
 If a Day 3 biopsy has been performed, unaffected embryo/s can be transferred fresh on
Day 5/6 of development. When a number of unaffected embryos are identified,
morphological criteria are also used to determine the best embryo/s for transfer. Surplus
unaffected embryos which are not transferred but which develop satisfactorily to the
blastocyst stage may be frozen. These embryos may be used in a subsequent IVF cycle.
Affected embryos will not be considered suitable for freezing and will be allowed to
succumb.
 If a Day 5/6 biopsy has been performed, the embryos must be frozen following biopsy.
Final results are usually obtained within 1 week after biopsy. If available, one or two
unaffected embryos can be thawed for use in a frozen embryo transfer cycle. Surplus
unaffected embryos will be kept in storage. These embryos may be used in a subsequent
IVF cycle. Affected embryos will be removed from storage and allowed to succumb.
A PGD scientist/Embryologist will discuss the PGD results with the couple prior to transfer.
The patient’s IVF nurse will organise a pregnancy test to be performed on Day 16 of the
cycle. This process should increase the chance of a healthy pregnancy.
Why choose Monash IVF for PGD?
Monash IVF has offered PGD as a clinical service since 1996 and is one of the few centres in
Australia that specialises in this area of reproductive medicine. In 1996 we were proud to report the
birth of Australia’s first PGD babies and since then we have performed over 3,000 PGD cycles with
proven high success rates. Our specialised genetics team contains highly qualified experts in PGD,
ensuring the best quality of care for patients.
The genetics team at Monash IVF is responsible for providing a specialised PGD service not only to
our own patients, but also to patients undergoing IVF cycles at fourteen different IVF clinics
throughout Australia and New Zealand. While the main PGD laboratory is located in Clayton,
Melbourne, Australia, embryo biopsy can be performed away from the genetics laboratory and the
embryonic cells sent by courier to Clayton. Centralising the genetic testing enables patients to access
the highest levels of expertise without having to leave their home state.
Where can I get more information?
If you would like further information regarding the PGD program at Monash IVF, please feel free to
contact a member of the Genetics team on +61 3 9543 2833.