Genetics in ART
Claire Lillee
PGD Coordinator
Website: www.monashivf.com
Email: [email protected]
What is PGS/PGD?
The process of screening an embryo for genetic or chromosomal conditions
prior to implantation
Biopsy
Genetic Screening
Embryo
IVF
Unaffected embryos
transferred
IVF
Egg Collection
Insemination
Fertilisation
Culture
2 cell embryo
Early blastocyst
Hatched blastocyst
Blastocyst biopsy
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
Genetic testing
Preimplantation Genetic Screening (PGS)
• Aneuploidy screening
Preimplantation Genetic Diagnosis (PGD)
• Approved sex selection
• Chromosome rearrangement testing
• Single gene disorder 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)
• 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)
tetrasomy (2n+2)
− The XYY karyotype
Aneuploid karyotype
Example:
Trisomy 21
(Down syndrome)
Preimplantation Genetic Screening
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.
PGS using Embryo Screen
Whole Genome Amplification
DNA fragmentation
and sample barcoding
Parallel sequencing
Test cells
Each sequence is aligned to the reference human genome
Barcodes used to differentiate
samples post-sequencing
Images kindly provided by Illumina
PGS using Embryo Screen
‘Normal’ Female
ANALYSIS
Trisomy 13 Female
3 copies
2 copies
1 copy
Chromosome screening
• The frequency of chromosome abnormalities increases with maternal
age. Older women will be less likely to obtain a chromosomally ‘normal’
embryo
‘Normal’ embryos (%)
80
70
60
50
40
30
20
10
0
<34
34-35
36-37
38-39
40-41
42-43
44-45
Maternal Age
• Data indicates that once a ‘normal’ embryo is identified for transfer
following PGS, there is no significant difference in pregnancy rate
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
Versus
Cost of undergoing multiple
transfers of chromosomally
unsuitable embryos
Chromosome rearrangement
testing
PGD for chrom rearrangements
• Offered to couples in which one partner carries a chromosome
rearrangement
Normal
Chromosomes
Reciprocal
Translocation
Robertsonian
Translocation
• 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 using Array-CGH
Whole Genome Amp
Label embryonic DNA green
Label 46,XY control DNA red
Hybridise to microarray,
wash and scan
Test cells
ANALYSIS
Example: reciprocal translocation involving chromosomes 3 and 5
Normal/balanced male
Unbalanced female
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
Array-CGH
Example: Aneuploid array-CGH result for a biopsied embryo
Loss of chromosome 8 and gain of chromosome 16 (XX)
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
Karyomapping
Whole Genome Amp
DNA incubation (WGA2)
DNA Fragmentation
Extend and Stain
Hybridisation to Beadchip
Test cells
Wash and Scan
Images kindly provided by Illumina
Karyomapping
ANALYSIS
Example: Peutz-Jegher syndrome (Autosomal dominant disorder affecting the father and son)
Father
Mother
Son
E1
E2
E3
E4
E5
E6
E7
Gene of
interest
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.
Karyomapping
Example: Aneuploid karyomapping result for a biopsied embryo
Monosomy:
• B-allele frequency chart
BB
AB
AA
B = assumed to be BB
A = assumed to be AA
 Loss of AB suggests monosomy for chromosome 17
Some aneuploidy detection?
Because karyomapping simultaneously analyses SNPs on
chromosomes, some aneuploidy may inadvertently be detected
all
Monosomy:
• Detailed haploblock chart
Loss of
paternal
key SNPs
 Loss of paternal key SNPs suggests monosomy for chromosome 17
Some aneuploidy detection?
Because karyomapping simultaneously analyses SNPs on
chromosomes, some aneuploidy may inadvertently be detected
all
Monosomy:
• Log R ratio (measure of fluorescent signal intensity)
Decreased
Log R
 Decreased Log R ratio suggests monosomy for chromosome 17
Questions?
Thank you for
your attention
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