What is PGS/PGD?
The process of screening an embryo for genetic or chromosomal conditions
prior to implantation
Biopsy
Genetic Screening
IVF
Unaffected embryos
transferred Embryo
IVF
Egg Collection Insemination Fertilisation Culture
2 cell embryo
Early blastocyst
Hatched blastocyst
A hole is drilled in the zona on Day 3. The
embryo is returned to the culture dish and
cultured until Day 5 or Day 6
By Day 5 or 6, the embryo has
differentiated into:
Inner cell mass (body of the embryo) Trophectoderm (placenta)
Trophectoderm cells (~5-10 cells) herniate
from the hole in the zona and can be
collected for analysis
Blastocyst biopsy
Preimplantation Genetic Screening (PGS)
• Aneuploidy screening
Preimplantation Genetic Diagnosis (PGD)
• Approved sex selection
• Chromosome rearrangement testing
• Single gene disorder testing
Genetic testing
Aneuploidy screening (& approved sex selection)
PGS – Aneuploidy
• PGS is used to detect changes in chromosome copy number
• Aneuploidy describes the loss or gain of a specific chromosome
nullisomy (2n-2)
monosomy (2n-1)
trisomy (2n+1)
tetrasomy (2n+2)
• Autosomal aneuploidy generally
causes implantation failure or
spontaneous abortion
− Small proportion of trisomy embryos
for chromosomes 13, 18 or 21 can
result in live birth
• Sex chromosome aneuploidies are
more viable
− Turner syndrome (Monsomy X)
− Klinefelter syndrome (XXY)
− X chromosome polysomy (XXX, XXXX)
− The XYY karyotype
Aneuploid karyotype
Example:
Trisomy 21
(Down syndrome)
PGS is offered to:
• Infertile patients with a poor prognosis for pregnancy
(eg: advanced maternal age, recurrent IVF failure)
• Fertile patients with a history of repeated miscarriage
• Previous chromosomally abnormal pregnancy
• Altered parental karyotype (eg: XXY male)
• Couples requesting sex selection to avoid the transmission of an
X-linked disease
Despite embryo selection by PGS a remarkable percentage of
chromosomally abnormal embryos (50%) can develop normally to
blastocyst stage, therefore morphological analysis is not enough to select
against chromosome abnormalities.
Preimplantation Genetic Screening
Images kindly provided by Illumina
Whole Genome Amplification
Test cells
DNA fragmentation
and sample barcoding Parallel sequencing
Each sequence is aligned to the reference human genome Barcodes used to differentiate
samples post-sequencing
PGS using Embryo Screen
ANALYSIS ‘Normal’ Female Trisomy 13 Female
PGS using Embryo Screen
3 copies
2 copies
1 copy
• The frequency of chromosome abnormalities increases with maternal
age. Older women will be less likely to obtain a chromosomally ‘normal’
embryo
• Data indicates that once a ‘normal’ embryo is identified for transfer
following PGS, there is no significant difference in pregnancy rate
Maternal Age
‘No
rma
l’ e
mb
ryo
s (
%)
0
10
20
30
40
50
60
70
80
<34 34-35 36-37 38-39 40-41 42-43 44-45
Chromosome screening
Chromosome Screening
This testing won’t change the number of pregnancies that a couple will
ultimately achieve.
• Reduce the timeframe to achieve a successful ongoing pregnancy (by preventing the transfer of embryos that contain a chromosome abnormality that would cause implantation failure or miscarriage)
• Reduce the incidence of chromosome abnormalities at birth (by preventing the transfer of embryos that contain a chromosome abnormality that has the potential to result in the birth of a child with a chromosome abnormality)
Weigh up:
Cost of testing embryos
Cost of undergoing multiple transfers of chromosomally
unsuitable embryos
Versus
Chromosome rearrangement testing
• Offered to couples in which one partner carries a chromosome
rearrangement
• Translocations occur when two chromosomes break at the same time and
then re-join with the “wrong” segment
• Carriers are generally have no phentoype caused by the rearrangement
• Carriers can experience difficulty with reproduction due to the generation
of chromosomally unbalanced embryos
PGD for chrom rearrangements
Normal
Chromosomes
Reciprocal
Translocation Robertsonian
Translocation
Hybridise to microarray, wash and scan
Label embryonic DNA green Label 46,XY control DNA red
Whole Genome Amp
Test cells
Loss of fluorescence relative to the control indicates the embryo is missing chromosomal material. Same
fluorescence as the male control indicates the embryo has the normal number of chromosomes. Gain of
fluorescence relative to controls indicates the embryo has extra chromosomal material
Normal/balanced male Unbalanced female
ANALYSIS Example: reciprocal translocation involving chromosomes 3 and 5
PGD using Array-CGH
Loss of chromosome 8 and gain of chromosome 16 (XX)
Array-CGH
Example: Aneuploid array-CGH result for a biopsied embryo
Single gene testing
PGD for single gene disorders
• Offered to patients who are at risk of passing a specific single
gene disorder on to their child
• Examples include Cystic Fibrosis, Huntington disease, Beta
Thalasaemia, Spinal Muscular Atrophy, Fragile X syndrome
• A technology called Karyomapping is used to analyse embryos
• Karyomapping does not test directly for the gene change
involved in the disorder, it uses family samples to track
inheritance
• Karyomapping is not specifically designed to screen for aneuploidy, however, it has the potential to inadvertently detect
some
DNA incubation (WGA2)
Karyomapping
DNA Fragmentation
Wash and Scan Hybridisation to Beadchip Extend and Stain
Whole Genome Amp
Test cells
Images kindly provided by Illumina
Karyomapping
The son inherited theis syndrome from his father. Our analysis indicates that the son inherited the blue chromosome from his father. Therefore, the father’s blue chromosome must be
linked to his affected gene copy and the red chromosome is linked to his unaffected gene copy. If an embryo inherits the blue chromosome at this gene region, it is inferred that the embryo has also inherited the mutation and is affected. Conversely, if the embryo inherits the red chromosome at this gene region, it is inferred that the embryo is unaffected. Analysis of the embryos indicates that embryo 7 is the only one that inherited the red “unaffected” chromosome from the father at this gene region.
Gene of
interest
ANALYSIS
Example: Peutz-Jegher syndrome (Autosomal dominant disorder affecting the father and son)
Father Son E1 E2 E3 E4 E5 E6 E7 Mother
Karyomapping
Example: Aneuploid karyomapping result for a biopsied embryo
Monosomy:
• B-allele frequency chart
AA
AB
BB
A = assumed to be AA
B = assumed to be BB
Loss of AB suggests monosomy for chromosome 17
Some aneuploidy detection?
Because karyomapping simultaneously analyses SNPs on all
chromosomes, some aneuploidy may inadvertently be detected
Monosomy:
• Detailed haploblock chart
Loss of paternal key SNPs suggests monosomy for chromosome 17
Loss of
paternal
key SNPs
Some aneuploidy detection?
Because karyomapping simultaneously analyses SNPs on all
chromosomes, some aneuploidy may inadvertently be detected
Monosomy:
• Log R ratio (measure of fluorescent signal intensity)
Decreased Log R ratio suggests monosomy for chromosome 17
Decreased
Log R
Questions?
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