Preimplantation Genetic Testing

Ahmad Mustafa Metwalley, MSc, Molecular Pathology

ART Lab. DirectorAl Baraka Fertility Hospital

PGT, is a technique used to identify genetic defects

in embryos.

Chromosomes• PGS: Preimplantation Genetic Screening

• CCS: Comprehensive Chromosomal Screening

Genes• PGD: Preimplantation genetic Diagnosis

• PGH: Preimplantation Genetic Haplotyping

1968 1978 1990 1993 1996 2000 2004 2008 2009 2012

History of PGT

Sexing Rabbit1st Sexing baby, single gene amplification Single cell amplification, Hellani et al. Array-CGHIonTorrent/Next Generation Sequencer

Genetic Study

A- Preimplantation Genetic Diagnosis (PGD)

PGD is performed to differentiate affected, carrier or

healthy embryo for transfer from abnormally genotyped

parents.

Step to avoid abnormality inheritance……….

• Single gene disorders

– Recessive, dominant, X-linked: Sickle cell, cystic fibrosis..

– Triplet repeat disorders:  fragile X syndrome

– Late onset disorders: Cancers

• Chromosome abnormalities

– Translocations: Robertsonian Translocations

– Inherited chromosome abnormalities:

• X linked disease

– Specific diagnosis: DMD, Hemophilia..

– Sex selection

Genetic Study

B- Preimplantation Genetic Haplotyping

1%

Chromosomal Study

A- Preimplantation Genetic Screening(PGS)

•PGS are screening techniques for aneuploidy from normal

genetic parents.

•To improve IVF take home baby.

1. Advanced maternal age (>35/38)

2. Severe male factor

3. Recurrent IVF failure (2/3 or more)

4. Recurrent miscarriage (Normal karyotype)

Solution for a daily problem

Solution for a daily problem

IVF/PGT Setup

1. IVF unit

2. Successful PGD Preparation

3. Basis of selection of procedures

IVF Unit

Clinician

Embryologist

IVF Lab.

Successful PGD Preperation

Post-EC

Post - ET

• Documentation• Updating Genetic unite: Genetist/Embryologist• Patients Instructions: • Counter Check

Pre -EC

• Patient: proper counseling• Plan: type, • Proper coordination: with Genetic Unit

• Biopsy Practitioner: Biopsy, Fixation, Tubing, • D5 facility: Incubator, Manipulations Practice,• Cryo service:

Basis of selection of procedures

• Accurate

• Sensitive

• Fast

Diagnostics

PGT

Genetic

PCR FISH CGH

PGD/PGS

Chromosomal

Polymerase Chain Reaction (PCR)

Multiple copies of specific DNA sequence‘Molecular photocopying’

Fluorescence in situ hybridization(FISH)

G G

C

T

T

C

C G C

A

A

G G

C GCC

T

GG CG

G

C G

T

T

C

GCT G

C

GG

GG

T

T

A

AA

T

A

C

C

G

A

C

C

C CA

Chromosome on slide / …Fixation

Fluorescent Probes

….Coloring Chromosomes

Sex Selection

Chromosome X Chromosome Y Chromosome 16

Normal Female Normal Male

Microarrays Array-Comparitive Genomic Hybridization

(a-CGH)

It is ultra sensitive and accurate technology to read 24

chromosomes together.

&

Amplify DNA WGA

Biopsied cell(s)

Control DNA

Combine labelled DNA

Blue Probes

Biopsied cell DNA

Limitations and Misdiagnosis• Amplification failure

– Degenerate nucleus– Un-Nucleated Blastomere– Multi- nucleated Blastomere– Fragments

• Mosaicism– TE biopsy

• Contamination– Cumulus cells: Eggs denudation

– Sperm: ICSI

– Practitioner

Biopsy

Techniques: Biopsy

Polar Body Biopsy

Cleavage

D3- Biopsy

Blastocyst

D5-BiopsyTime

Maternal Only

Many Cells

Freezing

Single Cell

Mosaicism

Days: 0/1,3 or 4 & 5

D3 Cleaving Biopsy

D5 Blastocys

t Biopsy

Drilling

Acid Tyrodes solution, pH 2.2 Chemical Drilling

Sharp NeedleMechanical

Drilling

Compact diode 1.48m laser

Laser Drilling

Blastomere Extraction

2 holes, Injection media, control?!!

Displacement

1 hole, Easy, fast, I like it??!!Extrusion

1 hole, blastomere lysisAspiration

Ca. Mg free medium Arrest Embryos

Washout Biopsy Media

To avoid Embryo ArrestTemperature

Proper Labeling Numerical Sequence

SurfaceOil

Stripping

CO2

StressTiming

Post Biopsy

Embryo Biopsy: Troubleshooting

Defragmentation

Non/ Multi Nucleated

blastomers

Compaction

Cumulus

cells

Considerations

EnsureBiopsy medium will warmedWorking with 3D

Embryo stripper 300

LASER from OUT to IN

Space between droplets

AvoidCO2 with biopsy mediaBlastomere Stripper < 130Drill wide hole

Big droplets

10 % HEPES NOT Ca Mg FreeMedia

1 hole, Easy, fast, I like it??!!LASER

1 hole, blastomer lysisMechanical

Blastocyst BiopsyTrophoectoderm

Considerations

If not expanded

do not biopsy

Differentiate ICM

Hatching better than

hatched

Biopsy MediaVitrification Immediate

Practice

Summary

Type and method of biopsy will dependent on:

•Testing required – maternal/paternal/ postzygotic

•Time required for diagnosis

•Successful cryopreservation programmed?

•Number of cells required

•Efficiency of diagnosis

•Equipment available

•Skills of biopsy practitioner

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