pgt applications and biopsy procedures - cook media workshop- dubai 2014
TRANSCRIPT
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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