Genetics in ART

Claire Lillee PGD Coordinator

Website: www.monashivf.com Email: info@monashivf.com

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?

Thank you for your attention

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