Embryo Genetic Testing (PGD)
Chromosome Rearrangement (eg translocation)
Testing
What are chromosome rearrangements and how do they impact fertility?
An individual’s genetic information is packaged into strings of DNA called chromosomes. Normal
individuals contain 46 chromosomes, which are arranged in 23 chromosome pairs. These
chromosome pairs are labelled 1 to 22 (the autosomes) and X and Y (the sex chromosomes).
Females have two X chromosomes, whereas males have one X and one Y chromosome (refer to
Figure 1).
Figure 1: An example of a set of female chromosomes.
Rather than carrying 46 regular chromosomes, some individuals carry a chromosome rearrangement.
These chromosome rearrangements can be classified according to how the chromosome material has
been rearranged (Figure 2):
 A reciprocal translocation occurs when two chromosomes break and then rejoin with the
wrong segment. This results in the formation of two translocated chromosomes.
 A Robertsonian translocation occurs when two chromosomes break, and the long arms of
each of the chromosomes join together. This results in the formation of a large “translocated
chromosome” in the place of two regular chromosomes.
 A pericentric inversion occurs when one chromosome breaks twice, once on each arm of the
chromosome. The chromosomal material located between these breakpoints rotates 180o and
reinserts, with the breakpoints reuniting.
 A paracentric inversion occurs when one chromosome breaks twice, and both breakpoints are
on the same arm of the chromosome. The chromosomal material located between these
breakpoints rotates 180o and reinserts, with the breakpoints reuniting.
Figure 2: Different types of chromosome rearrangements.
Regular
Chromosomes
Reciprocal
Translocation
Robertsonian
Translocation
Pericentric
Inversion*
Paracentric
Inversion*
* Inverted regions are indicated by cross shading
Carriers of these chromosome rearrangements are considered “balanced” because all their genetic
information is present. They often experience no symptoms and are not aware that they carry the
chromosome rearrangement. However, they may experience difficulties with reproduction due to the
generation of chromosomally unbalanced embryos. This may result in difficulty to conceive, recurrent
miscarriage or the birth of a child with a chromosome abnormality.
ormal
What is chromosome
rearrangement PGD testing?
Chromosomes
PGD may be available to couples where one partner carries a balanced chromosomal rearrangement,
such as a translocation. PGD with chromosome rearrangement testing can be used to screen IVF
embryos to distinguish between normal/balanced embryos (which have the potential to produce a
healthy baby) and unbalanced embryos (which would fail to implant, miscarry or result in an affected
baby). PGD testing cannot distinguish between normal embryos and embryos that are balanced for
the chromosome rearrangement.
In addition to testing for the chromosome rearrangement, in some cases it may be possible to analyse
all of the remaining 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). Only normal/balanced embryos will be selected for transfer to the uterus. This
testing will help to ensure that the embryo that is selected for transfer has the best possible chance of
developing into a healthy baby.
How is chromosome rearrangement PGD testing done?
Step 1: Chromosome testing on each partner
In order to undertake PGD, one or both partners must have been found to carry a chromosome
rearrangement (ie: translocation or inversion). The PGD team needs to know the precise
chromosome rearrangement in order to develop a PGD test.
Step 2: Genetic counselling in PGD clinic and PGD test selection
Once the specific chromosome rearrangement has been identified, the couple attend an appointment
with a clinical geneticist and/or genetic counsellor. During this appointment the couple will be
provided with information relating to PGD and will have an opportunity to have any questions
answered. This appointment is free of charge and enables couples to decide if PGD is the right option
for them.
Monash IVF currently offers chromosome rearrangement testing using three different test types,
called SNP array testing, Array-CGH and Fluorescent in-situ hybridisation (FISH). Each of these tests
is capable of detecting some, but not all, chromosome rearrangements. Therefore, the test type/s
available will depend on the specific chromosome rearrangement that has been identified in each
couple. 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: Comparison of the different PGD test types for chromosome rearrangement testing.
SNP array
ArrayCGH


Screen 24 chromosomes


x
Detect haploidy

Some
x

Some
x
(the presence of two copies of a given chromosome from one parent and
none from the other. This can result in particular genetic syndromes or
medical, cognitive or physical disabilities).

x
x
Determine the parental origin of chromosome abnormalities

x
x
Confirm genetic parentage

x
x
Detect extraneous DNA contamination

x
x
Cost for feasibility testing
x
x

1 month
1 month
3 - 6 months
ICSI
ICSI
Standard
insemination
or ICSI
per cycle
per embryo
per cycle
D3 or D5/6
D3 or D5/6
D3 only
Frozen
Frozen
Fresh or
Frozen
Criterion
Detect unbalanced chromosome rearrangement
(Reciprocal and Robertsonian translocations, Pericentric inversions)
(a missing set of all chromosomes, which results in implantation failure)
FISH

(if targeted)
Detect triploidy
(an extra set of all chromosomes, which results in implantation failure or
miscarriage)
Detect uniparental disomy
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 chromosome rearrangement. The
purpose of feasibility testing is to determine which test type/s is appropriate for the chromosome
rearrangement in question. The feasibility test will also ensure that the final PGD test can adequately
distinguish between normal/balanced embryos (suitable for transfer) and all possible unbalanced
embryos (not suitable for transfer). The feasibility process is slightly different for each test type:
1. SNP array testing (performed using “parental support”)
Feasibility testing will require a copy of the carrier’s genetic report. Based on this genetic
report, the Genetics team will determine whether or not SNP array testing is capable of
distinguishing between embryos which are normal/balanced for the chromosome
rearrangement and embryos which are unbalanced for the chromosome rearrangement.
Assuming PGD is feasible, a repeat chromosome test will be performed to ensure that the
final PGD test is targeting the correct chromosome rearrangement. There is no charge for this
feasibility testing process.
2. Array-CGH (performed using 24Sure technology)
Feasibility testing is performed using the same process as outlined for the SNP array testing.
3. Fluorescent in situ hybridisation (FISH)
Feasibility testing will require a blood sample from both partners. Cells will be extracted from
this blood sample and used for test development. The PGD scientists will identify a set of
fluorescent DNA probes which are capable of distinguishing between embryos which are
normal/balanced for the chromosome rearrangement and embryos which are unbalanced for
the chromosome rearrangement. Once an accurate test has been developed, it will be
validated on cells from embryos which have been donated for succumbed research and
training. This will help the Monash IVF PGD laboratory to ensure that the final test is sensitive
enough to provide a result from a single cell from an IVF embryo. There is a non-refundable
fee for this feasibility test.
A feasibility report outlining the results of the feasibility testing will be sent to the couple’s IVF doctor
and genetic counsellor. The IVF doctor or genetic counsellor will contact the couple to go through the
results of the feasibility testing process. In some instances it may not be possible to develop an
accurate test and PGD may not be available.
Step 4: IVF and embryo biopsy
Once feasibility testing has been completed and PGD is feasible, the couple are able to undertake an
IVF/PGD cycle. 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. If the couple are planning to undertake SNP array testing or array-CGH, the
embryos must be created using a fertilisation method called Intracytoplasmic Sperm Injection (ICSI).
ICSI involves the injection of a single sperm into the egg and is specifically used for these test types to
reduce the risk of misdiagnosis due to the presence of additional sperm around the egg/embryo. If
the couple are planning to undertake FISH testing, they can elect to have ICSI or standard fertilisation.
This decision will be made in consultation with their IVF clinician.
After fertilisation, 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 potentially be
performed at two different stages during an embryo’s development. The stage of embryo biopsy will
be influenced by the PGD test type chosen (Refer to Table 1):
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 3). This type of embryo
biopsy can be performed for SNP array testing, array-CGH or FISH.
Figure 3: 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 4). This type of embryo biopsy is only
available for SNP array testing or array-CGH (ie: Day 5/6 biopsy is not available for FISH
testing).
Figure 4: Day 5/6 embryo biopsy
Where possible, Monash IVF recommends Day 5/6 biopsy. This is due to the following reasons:
 Randomised control trials have shown that Day 5/6 biopsy is better for the embryo compared with
Day 3 biopsy (Scott et al, 2013).
 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).
 Day 5/6 embryos are more likely to be chromosomally normal then Day 3 embryos (as some of
the embryos that are 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.
 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 genetic testing can proceed. The genetic testing process will depend on
the PGD test type being performed:
1. SNP array testing (performed using “parental support”)
In this procedure, the biopsied cells are transferred to a small test tube for genetic testing.
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 normal/balanced
or unbalanced for the chromosome rearrangement. An analysis of all chromosomes is also
performed to determine how many chromosome copies are present in each embryo (Figure
5). Only embryos which are diagnosed as normal/balanced, have the correct number of
chormosomes are are developing normally will be considered suitable for transfer. Embryos
which are unbalanced and/or have an abnormal number of chromsomes (ie: missing or extra
chromosome/s) will not be considered suitable for transfer.
Figure 5: 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
T
G
C
A T
A
T
G
C
A T
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T
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C
G C
A
T
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C
G G
A
T
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C
C C
A
T
G
C
A T
A
T
G
C
T A
A
T
G
C
A T
A
T
G
C
C A
A
T
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C
A C
A
T
G
C
A A
A
T
G
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G G
A
T
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C
C G
A
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G
C
C G
Chromosomes inherited by embryo
2. Array-CGH (performed using 24Sure technology)
In this procedure, the biopsied cells are transferred to a small test tube for genetic testing.
The DNA from the embryonic cells is multiplied thousands of times (to generate enough DNA
for testing) and is then fluorescently labelled. Once labelled, the DNA sample is placed on a
microarray platform. This microarray platform consists of a matrix of thousands of different
DNA probes which are specific to each chromosome. The fluorescently labelled DNA from
the embryo biopsy sample binds to the DNA probes on the microarray platform. By
comparing the fluorescent intensity of the embryo biopsy sample with that of a control male
and control female sample, it is possible to determine whether each embryo is
normal/balanced or unbalanced for the chromosome rearrangement. An analysis of all
chromosomes is also performed to determine how many chromosome copies are present in
each embryo (Figure 6). Only embryos which are diagnosed as normal/balanced, have the
correct number of chormosomes are are developing normally will be considered suitable for
transfer. Embryos which are unbalanced and/or have an abnormal number of chromsomes
(ie: missing or extra chromosome/s) will not be considered suitable for transfer.
Figure 6: Array-CGH procedure
+
Test
cell/s
Test DNA
46,XY (male)
Control DNA
46,XX (female)
Control DNA
Loss of fluorescence relative to controls indicates the embryo is missing a portion of one
of the translocation chromosomes
Same fluorescence as controls indicates the embryo has the normal number of chromosomes
Gain of fluorescence relative to controls indicates the embryo has an extra portion of one of the
translocation chromosomes
3. FISH testing
In this procedure, embryo biopsy must be performed on Day 3 after egg collection. The
biopsied cell/s are fixed to a microscope slide for testing. Fluorescent dyes are used to tag
the specific chromosomes involved in the chromosome rearrangement. When analysing the
cell under a fluorescent microscope, these tags show as coloured dots. The number of
coloured dots present in the cell/s is used to indicate whether the embryo is normal/balanced
for the chromosome rearrangement or carries an unbalanced form of the chromosome
rearrangement (Figure 7). Normal/balanced embryos should have two tags for each
chromosome region, whereas unbalanced embryos will have extra or missing tags. This
testing will only provide an analysis of the chromosomes involved in the chromosome
rearrangement, and will not provide any information on the remaining chromosomes in the
embryo.
Figure 7: Example of chromosome rearrangement testing using FISH. Normal/balanced
cells should have two red signals, two green signals and two aqua signals.
Parental Profile
(Carrier)
Embryo 1
(Normal/Balanced)
Embryo 2
(Unbalanced)
Embryo 3
(Unbalanced)
Step 6: Embryo transfer
The time of embryo transfer will also 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 normal/balanced 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
successful pregnancy and significantly reduce the risk of miscarriage.
Surplus
normal/balanced embryos will remain in storage. These embryos may be used in a
subsequent cycle. Chromosomally abnormal embryos will be removed from storage and
allowed to succumb.
2. Array-CGH (performed using 24Sure technology)
Embryo transfer is performed using the same process as outlined for the SNP array testing.
3. FISH testing
Final results are usually obtained 24 hours after biopsy. The embryo is kept in culture while
testing of the biopsied cell/s proceeds. Embryos identified as being normal/balanced for the
chromosome rearrangement can be transferred fresh on Day 5 of development. A PGD
scientist/Embryologist will discuss the PGD results with the couple prior to transfer. The
couple’s nurse will organise a pregnancy test to be performed on Day 16 of the cycle. This
process should increase the chance of a successful pregnancy and significantly reduce the
risk of miscarriage. When a number of normal/balanced embryos are identified,
morphological criteria are also used to select the best embryo/s for transfer. Surplus
normal/balanced embryos which are not transferred but which continue to develop
satisfactorily to the blastocyst stage may be frozen. These embryos may be used in a
subsequent IVF cycle. Chromosomally abnormal embryos are allowed to succumb.
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.