Embryo Genetic Testing (PGD)
Chromosome Screening
What are chromosomes and why are they important in 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 carry two X chromosomes, whereas males carry an X and a Y chromosome (refer to Figure
1).
Figure 1: An example of a set of female chromosomes.
Unlike the other cells in the body, egg and sperm cells only contain 23 individual chromosomes, one
of each of the 22 autosomes and one of the sex chromosomes (either X or Y). When the egg and
sperm unite during conception, these chromosomes combine. This results in the formation of an
embryo with the usual 46 chromosomes, arranged in pairs.
Errors in cell division in the developing egg, sperm or embryo can result in the formation of embryos
with an abnormal number of chromosomes (ie: missing or extra chromosome/s). This is known as
chromosomal aneuploidy. Embryos with a chromosomal aneuploidy will fail to implant, miscarry, or
result in the birth of an affected child (eg: a child with Down syndrome). Approximately 50% of
spontaneous abortions are caused by chromosomal aneuploidy.
How common are chromosome abnormalities in embryos?
It is well documented that the frequency of chromosomal abnormalities in embryos increases with
maternal age. Therefore, older women will be less likely to obtain a “normal” embryo for transfer
compared with younger women (refer to Figure 2). However, once a “normal” embryo is identified for
transfer, the pregnancy rate in older women is not significantly different from that of younger women.
Figure 2: Percentage of ‘normal’ embryos on Day 3 versus Day 5 of embryo development.
80
“Normal” embryos (%)
70
60
50
Day 3
40
Day 5
30
20
10
0
27
29
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33
35
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45
Maternal Age
What is chromosome screening?
Preimplantation Genetic Diagnosis (PGD) with chromosome screening can be used to screen IVF
embryos for chromosome abnormalities prior to implantation. Only embryos that have the correct
number of chromosomes 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.
Traditionally, PGD tests have only been able to analyse a limited number of chromosomes at a time.
This has restricted the amount of genetic information that can be obtained from embryos and has
therefore limited the diagnostic capability and benefits of PGD. Newer PGD technologies, such as
array-CGH and SNP arrays, are capable of simultaneously analysing all human chromosomes in a
single test. These technologies have enabled IVF clinics to more confidently predict whether an
individual embryo is chromosomally normal, resulting in the transfer of embryos that have a higher
chance of implantation and development to term. Other PGD techniques that only test some of the
chromosomes are at risk of missing important genetic information. Since each embryo is precious,
accuracy is critical to avoid discarding normal embryos or transferring abnormal embryos.
How is chromosome screening done?
Step 1: Genetic counselling in PGD clinic
Prior to the commencement of an IVF cycle, the couple attend an appointment with a 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.
Step 2: IVF and Embryo biopsy
All couples undergoing chromosome screening 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. 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 3).
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).
Figure 4: Day 5/6 embryo biopsy
In the majority of cases, 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 3: Genetic testing
The biopsied cells are transferred to a small test tube for genetic testing. Monash IVF currently offers
chromosome screening using two different test types, called Array-CGH and SNP array testing. More
information on each of these test types is included below:
1.
Array-CGH (performed using 24Sure technology)
In this procedure, 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 the copy number of each chromosome in the
biopsied cell/s (Figure 5).
Figure 5: 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 chromosome
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 chromosome
2.
SNP array testing (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 which chromosome/s the embryo inherited
from each parent (Figure 6). In addition to determining chromosome copy number, this testing is
capable of identifying the source of any additional or missing chromosomes (ie: maternal or
paternal origin). This information may be useful to assist the patient with future reproductive
decision making.
Figure 6: 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
A
T
G
C
G C
A
T
G
C
G G
A
T
G
C
C C
A
T
G
C
A T
A
T
G
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T A
A
T
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A T
A
T
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C
C A
A
T
G
C
A C
A
T
G
C
A A
A
T
G
C
G G
A
T
G
C
C G
A
T
G
C
C G
Chromosomes inherited by embryo
Step 4: Embryo transfer
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
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 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.
What type of chromosome screening is best for me?
There are differences in the testing capabilities of array-CGH compared with SNP arrays. SNP array
testing (with parental support) has the diagnostic advantage of being capable of detecting a wider
range of chromosome abnormalities than array-CGH (Table 1). However, as array-CGH is charged
on a per embryo basis, this testing may appeal to patients with small embryo numbers who would
otherwise elect not to proceed with 24 chromosome screening.
Genetic counselling is an important step to help patients understand the differences between these
two types, so that they make an informed decision regarding which test type 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.
Table 1: Detection capabilities of array-CGH compared with SNP arrays.
Array-CGH
SNP
arrays


Some

Some

x

Determine the parental origin of chromosome abnormalities
x

Confirm genetic parentage
x

Detect extraneous DNA contamination
x

Criterion
Screen 24 chromosomes
Detect haploidy
(a missing set of all chromosomes, which results in implantation failure)
Detect triploidy
(an extra set of all chromosomes, which results in implantation failure or miscarriage)
Detect uniparental disomy
(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).
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 +613 9543 2833.