Genetics in ART

Jolene Stockton

PGD Scientist

• The process of taking a small biopsy sample from an embryo, and then using a genetic test that allows selection or exclusion of embryos depending on their genetic/chromosomal make-up

What is PGS and PGD?

• PGD (Pre-implantation genetic diagnosis) – Patients affected by or who

are carriers of a known single gene disorder

– Patients who need a HLA match for an affected child

– Patients with translocations or other chromosomal rearrangements

– Patients seeking medical sex selection (e.g. autism)

• PGS (Pre-implantation genetic screening)

– Advanced maternal age

– Recurrent miscarriage

– Failed transfer cycles

Who can benefit from PGD and PGS?

Patient Timeline – IVF/PGD cycle

Genetic Intake

•Confirm details about specific genetic disease

•Identify AFF/carrier individuals

•Discuss workup timeframe and cost

Consults

•Clinical Geneticist

•Nursing/Finance

•Scientist

•Counselor

Workup

•1st Scientist, 2nd Scientist and final validation stage before sign off

PGD (pre-treatment)

PGD/PGS (treatment cycle)

Egg collection/ insemination

Grow to day 5/6

Trophectoderm biopsy

Transfer of unaffected

embryo

Vitrification

• Egg retrieval and insemination (ICSI)

IVF/Embryology

Day 1 (1 cell) Pronuclei

Day 3 (6-8 cells)

• Fertilization and development

The foundation of a successful PGD program is excellent IVF

• GERI – time lapse incubator

• Blastocyst stage biopsy (~5-10 cells)

• Embryo subsequently frozen

• Cells washed and transferred into tubes, then DNA extracted and amplified

Blastocyst biopsy

Single gene disorders

(incl. HLA)

Trans-locations

UPD & breakpoint

analysis

Duplications

Deletions

Medical sex selection

Technologies in PGD and PGS

PGD • Custom linkage

• Next Generation Sequencing

• Custom linkage

• Next Generation Sequencing

• Custom linkage

• karyomapping

• karyomapping

• Custom aCGH

• Custom aCGH

PGS

• Next Generation Sequencing

1. Custom linkage analysis – PCR/STR

• Short Tandem Repeats

• Involves a workup phase (10-12 weeks)

• Requires 2 generations to track inheritance

• Many different disorders, however the approach is the same

• Up to 99% accurate, +/- mutation test (unless de novo)

a b c d

e f g h

Paternal

* *

1 2 3 4

5 6 7 8

Maternal

* *

1 2 3 4

a b c d

*

Embryo

*

3’ Linkage (2MB) 5’ Linkage (2MB)

*

Mutation

CACACACACA allele 1

CACACACACACACACA allele 2

DNA fingerprinting Sequencing

PCR/STR Analysis

CACACACACA allele 1

allele 1 allele 2

CACACACACACACACA allele 2

Case study: GM1 + CDG1p + AGAP2 + PGS

GLB1 c.1354 C>T

ALG11 c.1184 T>C

AGAP2 c.1222 C>T

GLB1 (mutation not found)

ALG11 c.1184 T>C

AGAP2 c.1222 C>T

AFF for GM1

Homozygous ALG11 c.1184

Homozygous AGAP2 c.1222

• 37 years, consanguineous with no history of infertility

• 2 living children with significant developmental delays/seizures (due to ALG11), AGAP2 uncertain clinical significance.

• Couple seeking IVF/PGD for a healthy child

• Custom workup ~ 6 months to complete

• Estimated ~40% (¾ x ¾ x ¾ = 27/64) of embryos to be unaffected by all 3 conditions

• Chance of random chromosome error 40-50%

Fragment length

215 150 126 228 100 Mutation 180 180

GM1 STATUS

Label NED VIC VIC FAM VIC GLB1 NED NED

Locus 30.11 30.63 31.99 32.02 32.27 c.1354 33.64 34.07

Marker D3S3547 D3S3727 D3S1759 D3S2423 AFM087XD

3 C>T D3S3518 D3S1619

5792 218/222 149/155 133 242 103/109 C/T 182/184 182/184 CAR

5797 220/224 155/157 129/133 230/234 103/105 C 177/184 182/186 NAD

5827 218/220 149/157 133 234/242 103 C 177/184 182/186 NAD

5966 220/222 155/157 133 242/257 103/109 C/T 177/182 182/184 CAR

Fit for ET*

see 222 not see

149 N/A N/A see 109 C/T see 182 see 184

CAR mat, 2nd choice for

ET*

Fit for ET see 218 See 149 N/A N/A not see

109 C

not see 182

not see 184

NAD

GM1: NAD embryos first choice. Embryos that carry the maternal GLB1 mutation are considered 2nd choice for ET, and any resulting pregnancy to be tested enzymatically prenatally for the presence of functional GM1.

Case study – linkage charts and priority for ET

Fragment length

297 246 Mutation 135 142

ALG11 STATUS

Label FAM VIC ALG11 PET VIC

Locus 51.14 51.90 c.1184 52.60 52.76

Marker D13S284 D13S1325 T>C D13S1305 D13S270

5792 300/315 270/276 T/C 140/148 155/163 CAR

5797 300/304 272/276 T/C 142/144 155/157 CAR

5827 300 275 C 140/144 155 AFF

Fit for ET 304/315 270/272 T 142/148 157/163 NAD

Fit for ET 300/304 272/276 T/C 140/142 155/157 CAR mat

Fit for ET 300/315 270/276 T/C 144/148 155/163 CAR pat

Not fit for ET

300 276 C 140/144 155 AFF

Fragment length

138 Mutation 207 173

AGAP2 STATUS

Label VIC AGAP2 VIC FAM

Locus 57.11 c.1222 58.44 58.94

Marker D12S1644 C>T D12S305 D12S355

5792 137/139 C/T 213/224 176/183 CAR

5797 135/137 C/T 224 181/183 CAR

5827 137 T 224 183 AFF

Fit for ET 135/139 C 213/224 176/181 NAD

Fit for ET 135/137 C/T 224 181/183 CAR mat

Fit for ET 137/139 C/T 213/224 176/183 CAR pat

Not fit for ET 137 T 224 183 AFF

CDG1p: NAD (non-carriers) first choice, carriers second choice. AFF not for ET.

AGAP2: NAD (non-carriers) first choice, carriers second choice. AFF not for ET.

Mutation 5’ Linkage (2MB) 3’ Linkage (2MB)

• 1st cycle, 3 embryos biopsied

– GM1 = 1/3 NAD, 2/3 maternal carrier

– ALG11 = 1/3 x NAD, 1/3 x CAR, 1/3 x AFF

– AGAP2 = 3/3 x CAR

Case study – outcomes

• 2nd cycle, 2 embryos biopsied – no testing for AGAP2

– GM1 = 2/2 x NAD

– ALG11 = 1/2 x CAR, 1/2 x AFF

– 3/3 ABN for chromosomes = no ET

2 x potential for ET after single gene testing

1 x potential for ET after single gene testing

– 1/2 ABN for chromosomes (ABN embryo same as ALG11 CAR) = no ET

• 3rd cycle, 4 embryos biopsied – no testing for AGAP2

– GM1 = 1/4 x NAD, 3/4 x maternal carrier

– ALG11 = 1/4 x NAD, 2/4 x CAR, 1/4 x AFF

– 4/4 NAD for chromosomes

3 x potential for ET after single gene testing

– ET March 2019, pregnancy ongoing (17/40)

• Single nucleotide polymorphisms, ~1 every 300 bp of the human genome

• Potentially greater coverage of a region of interest, +/- mutation test

• Contains a level of anueploidy detection, however inferior to NGS

2. Karyomapping – generic linkage platform

“A” SNP

“B” SNP

Determining informative SNP’s from family samples

Possible SNP

combinations

in embryos

AB, AA

AB, BB

AA, AB

BB, AB

AA

AB

AA, AB, BB

BB

AB

Informative SNPs

for Mother

SNPs that are

not

informative

Informative SNPs

for Father

Key SNP in

embryo

Non-Key SNP in

embryo

Informative

SNP

Non

informative

Allows us to create patterns associated with NAD/AFF/CAR status

• Requires flanking informative key SNPs for high confidence results (up to 99%), closer to the mutation = higher confidence

• Direct mutation test where possible

Typical single gene region

Target

gene

SNPs

Mutation

5’ SNPs 3’ SNPs

• Analysis of familial samples (“trio”) to check adequate coverage for the gene region of interest

Karyomapping workup phase

Father

(AFF) Mother

(NAD)

Reference

(AFF)

M1

M2

P1

P2

M1

P1

• Familial samples create the backbone of the test

Haploblock chart – workup and embryo testing

M1 M2 P1 P2 P1 M1

NAD AFF AFF P1 M1 P2 M1 P1 M1

AFF AFF NAD

Target locus

• Add embryo results to pre-existing workup case and analyze.

Embryo testing – display of informative SNPs

Unassigned SNPs

Key SNPs

Non-key

SNPs

Paternal

Maternal

REFERENCE

EMBRYO

Target gene

Multiple disorders + HLA matching

Cystic

Fibrosis

Beta

thalassemia

Huntington

Disease

Fragile X

HLA matching

Polycystic

Kidney Disease

Karyomapping: Comparison with PCR/STR analysis

Karyomapping PCR/STR

Feasibility study Off-the-shelf test: 2-4 weeks Highly customized: 8-12 weeks

Complex tests Simple multi loci screens: -HLA matching -Translocation breakpoint -Multiple single gene disorders

Multiple loci require one test per locus to be built; 12+ weeks.

Coverage Limited to SNPs present on bead array; Rare disorders may not be adequetly covered.

Possible to build test for almost any case.

Aneuploidy Limited, uses MDA (not compatible with NGS) Only detects ~50% of trisomies.

Compatible with comprehensive chromosome screening (NGS).

Efficiency Streamlined laboratory workflow: multiple cases per run.

Each case requires a custom test to be run by staff.

Cost High consumables. High staff hours, consumables high for rare disorders.

Single gene disorders

(incl. HLA)

Trans-locations

UPD & breakpoint

analysis

Duplications

Deletions

Medical sex selection

Technologies in PGD and PGS

PGD • Custom linkage

• Next Generation Sequencing

• Custom linkage

• Next Generation Sequencing

• Custom linkage

• karyomapping

• karyomapping

• Custom aCGH

• Custom aCGH

PGS

• Next Generation Sequencing

3. Array CGH

• 60,000 points on chromosomes measured. Can customize to target smaller chromosomal rearrangements or microdeletions/duplications

• All 24 chromosomes covered (vast improvement from FISH)

• Samples compared to both female and male references

Array CGH: Customization

Customization for familial chr9p23 deletion; potential developmental delay.

NAD

AFF

• 46, XX, t(11;18;17)(p15.3;p11.1;q25)

Case Study – Complex 3 way translocation by CGH

• 35 years old, PCOS, 18 months

primary infertility

• Phenotypically normal

• 50% chance of spontaneous

miscarriage

• 20% risk of abnormal liveborn

• Estimated 6% chance of a balanced

arrangement per embryo

• 64 theoretical segregations,

only 2 of those balanced

arrangements

• First approach, 2 x back-to-back stim cycles

– Batched results

– 6 and 9 embryos biopsied

– 14/15 ABN for the translocation

Case Study – IVF/PGD outcomes

1 x NAD = ET (live birth)

1 x NAD = ET (live birth)

• Second attempt, 1 x stim cycle (no batching)

– 8 embryos biopsied

– 5/8 ABN for the translocation, 3 x NAD.

• Increase in patients opting for PGS

• Requirement for high throughput, lower cost generic anueploid detection platform

– Compatible with single gene customized linkage tests

• Improved quality of result

– Clearer profiles, enhanced detection of mosaicism

4. NGS – Veriseq

CGH – log ratio

NGS - lineal

NGS: VeriSeq workflow

Library building

•Unique index sequences added during library preparation identify each sample

•Semi-automated

Massively parallel sequencing

•~25 million reads per run = 500k reads per sample after filtering.

Data processing

•Demultiplexing and alignment against reference genome

•Number of reads are proportional to copy number

NGS: Profiles

• Software corrects for biases, 5-10 MB detection limit • Internal copy number comparison for each sample

– no detection of triploid 69XXX

• Random chromosome error increases with maternal age (oocytes main source of error)

NGS: Enhanced detection of mosaicism

• Mosaicism independent of age, ~10% samples with current reporting guidelines

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

<35 35-37 38-39 40-42 43+

N = 764

• Limited data worldwide regarding clinical outcomes following transfer of mosaic embryos – Reports of mosaic embryos creating healthy, ongoing pregnancies – Increased risk of failed implantation and miscarriage

• Biopsy sample limited to the pre-placental cells – Abnormality confined to the placenta? – Abnormality worse in the fetal cells? – Similar mix of normal/abnormal cells in fetal cells?

Challenges with enhanced detection of mosaicism

? ?

• Healthy euploid babies were born from mosaic ET’s

• No difference between type of mosaicism present

• Extent of mosaicism influenced outcome

– Higher percentage of abnormal cells resulted in significantly lower IR and live birth rate.

Recent literature on clinical outcomes of mosaic ET’s

Spinella et al, Extent of chromosomal mosaicism influences the clinical outcome of in vitro

fertilization treatments. Fertility and Sterility Vol. 109, No. 1, January 2018

Munne et al, Detailed investigation into the cytogenetic constitution and pregnancy outcome of replacing mosaic blastocysts detected with the use of high-resolution next-generation sequencing. Fertility and Sterility® Vol. 108, No. 1, July 2017

NAD – no mosaicism

30% - low level

50% - high level

Approach to transfer of mosaic embryos – May 2018

• TF-A to D ranking system

Transfer Category Geneticist

consult?

Mosaic shift

observed

Chromosomes involved

TF-A, 95% NAD Not required None N/A

TF-B, 95% NAD

Variable genomic

profile Not required ≤40% of sample

All whole chromosomes All segmental shifts

TF-C, 95% Mosaic

*Not required >40% - <80%

Whole chromosomes including: 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 16, 17, 19, 20, 22

All segmental shifts

TF-D, 95% Mosaic Mandatory >40% - <80% Whole chromosomes including: 6, 7, 13, 14, 15, 18, 21, X, Y

ABN NA ≥80%

All whole chromosomes All segmental shifts

*Follow up with Geneticist recommended by Counsellor if appropriate on a case by case basis.

• Some patients consider further prenatal testing for reassurance

– 95% accuracy for PGS

• NIPS – safe and fast screening process, as early as 10 weeks gestation

– Trisomy 21 (Down Syndrome)

– Trisomy 13 (Patau Syndrome)

– Trisomy 18 (Edwards Syndrome)

– Monosomy X (Turners Syndrome)

– XX and XY determination of fetus

Non-Invasive Prenatal Screening

• Rapid changes in technology – Clearer results

– Ability to automate processes

• “Off the shelf” products – Lower cost

– Faster results

– Wider availability to patients

• Presents new challenges and opportunities – Mosaicism: more knowledge but limited clinical data.

Overload of information?

Genetics in ART – an evolving technological field

• Morphology of embryo

• Time lapse

• PGD/S biopsy

– Trophectoderm (TE)

– Blastocoel fluid (BF)

– Spent media (SBM)

• Metabolomics for embryo viability

PGD/S – part of a multi faceted selection process

Capalbo. Noninvasive embryo genetic testing.

Fertil Steril 2018.

F

i

g

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Uyar. Metabolomic assessment of embryo viability. Semin Reprod Med 2014

Thank you