Genetics in ART

Claire Lillee Genetics Coordinator

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

What is Genetics in ART?

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

• The presence of two or more cell lines with different chromosome

constitutions in the one individual

General mosaicism: Two or more cell lines are present throughout an entire individual

Confined placental mosaicism: The placenta has a different chromosome constitution to the embryo

• The incidence of mosaicism decreases as gestational age increases

• There is no significant difference in the frequency of mosaicism at birth

following IVF compared with natural conception

Mosaicism

20-30% Preimplantation Embryos

(irrespective of maternal age)

Day 5/6

0.2 – 0.25% Amniotic Fluid

Samples

~16 weeks

0.8 - 2% Chorionic Villus

Samples

~12 weeks

What can PGS detect?

• Not all mosaics will be detected following PGS. We are only

sampling ~5 cells from the trophectoderm

• The likelihood of detecting mosaicism will be influenced by:

- The cells sampled for PGS

- The chromosome constitution of

each cell line and the proportion of each cell line in the biopsy sample

- The quality of the PGS data

What can PGS detect?

• Even if mosaicism is detected, the underlying genetic basis

of the result remains unclear

Example:

Sample diagnosed as mosaic trisomy 21 with a 20% gain (~2.2), Embryologist noted

~5 cells biopsied

Significant diagnostic limitation :

• The clinical outcome of these two scenarios may be quite different

• A mosaic embryo may be completely aneuploid

• Result may or may not be indicative of the mosaicism level in the remaining TE or

ICM

• Not possible to predict how the mosaicism will progress past day 5/6 of

development

= 11/5 = 2.2 or

• There has been a reluctance to transfer mosaic embryos due to a

perception that these embryos may display an increased risk of

congenital abnormalities at birth

• However, we have been transferring untested mosaic embryos all

the time! (~30%)

• Mosaic embryos are associated with an increased risk of

implantation failure and miscarriage, a small proportion do have the potential to result in healthy live births (Fragouli et al, 2015;

Fiorentino et al, 2016; Spinella et al, 2017)

• Monash IVF may transfer mosaic embryos provided certain criteria

are met

Mosaic embryos

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 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

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

PGD using Embryo Screen

ANALYSIS

‘Normal’ Female ‘Unbalanced’ Female

46,XY,t(2;9)(p24;q33)

PGD using Embryo Screen

2 copies

3 copies

1 copy

Gain of chromosome 2 and loss of chromosome 9

Further information

• Interested to learn more about PGD using Embryo Screen

• This study has recently been published in Human Reproduction

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 Peutz-Jegher syndrome from his father. SNP 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 Peutz-Jegher 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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Monash IVF Policy

• The embryo must have only a single mosaicism detected (<70%)

(complex mosaic embryos are not eligible for transfer)

• The patient must have met with Monash IVF Clinical Geneticist

- Provide an individual risk assessment for that particular embryo

based on the chromosome involved and the level of mosaicism

detected

- Explain to the patient the residual uncertainty regarding the

clinical significance of a mosaic result following PGS

- Counsel the patient to undergo amniocentesis if pregnancy is

achieved

• The patient must sign a specific mosaic embryo transfer consent form

• The mosaic embryo must be transferred in a single embryo transfer

Monash IVF Policy

NOT recommended for transfer:

Mosaicism Justification (predicted clinical outcome)

• Mosaic trisomy for 13, 18 or 21

• Mosaic monosomy for X

Widely reported to result in viable pregnancies

associated with disability/anomalies

• Mosaic trisomy for 7, 11, 14 or

15

Trisomy rescue resulting in maternal or

paternal UDP of these chromosomes is

associated disability/anomalies

• Mosaic trisomy for 2 or 16 Confined placental mosaicism associated

with high risk of fetal demise/IUGR/premature

delivery

All other mosaics will are assessed on a case-by-case basis