Genetic technologies used in PGD

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Genetic technologies used in PGD

• Prof. Dr. K. Sermon

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What is PGD?

• An early form of prenatal diagnosis
• For couples at high risk ? PGD
• E.g. monogenic disease, chromosomal abnormalities
• For couples at low risk ? PGS
• To improve IVF results in e.g. patients with
  increased maternal age

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ART and PGD

• More aggressive stimulation to obtain more
  embryos
• Usually ICSI
• To avoid contamination in PCR
• To avoid fertilisation failure
• Biopsy of one or two blastomeres
• Alternatives polar bodies or blastocysts

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ICSI
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Evolution of the embryo
18h
D2
D2
D5
D3
D4
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Biopsy at cleavage stage
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Blastomere spreading

• Blastomere is put on glass slide
• Cytoplasm and nuclear membrane are removed with
• HCl/Tween20 solution (Coonen et al.)
• Methanol/acetic acid (Tarkowski method)
• Further treatments
• Pepsin to remove proteins
• Paraformaldehyde to fixate proteins

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Principle of FISH
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Sexing with 5-colour FISH
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FISH for aneuploidy screening

• FISH for chromosomes X, Y, 13, 14, 15, 16, 18,
  21, 22
• found in miscarriages or live born with
  chromosomal aberrations
• In order to increase the pregnancy rates after
  IVF
• In patients with advanced maternal age (gt37)
• With recurrent miscarriages (gt3)
• With repetitive failed IVF (gt3)
• Other indications

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FISH for structural aberrations


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FISH for structural aberrations
Scriven PN, Handyside AH, Mackie Ogilvie C.
Prenat Diagn. 1998 Dec18(13)1437 Slide courtesy
P. Scriven
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Problems with translocations

• Robertsonian translocations 35 transferable
  embryos
• Reciprocal translocations 22 transferable
  embryos!
• Poor pregnancy result
• 19 per OR for Robertsonian
• 12 per OR for reciprocal
• Data from ESHRE PGD Cons report VI, Hum Reprod.
  2007 Feb22(2)323-36.

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Sexing by FISH

• Only useful for X-linked recessive diseases
• Half of the discarded males are healthy
• Half of the transferred females are carriers
• Patients usually prefer DNA-specific diagnosis if
  possible
• 3/4 embryos are healthy
• Healthy males can be selected
• Carrier females can be identified and not
  transferred if wanted

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Polymerase Chain Reaction
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Factors influencing PGD

• Efficiency and accuracy of PCR at single cell
  level
• Use of multiplex PCR
• Biopsy of one vs. two cells

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Factors in single cell PCR

• Efficiency of PCR and ADO same pitfalls
• Lysis method (water vs. ALB)
• PCR method (denaturation T, DNA polymerase)
• Detection method (EtBr vs fluorescent PCR)
• Cell type (lymphocytes vs. blastomeres)
• Contamination important factor in inexperienced
  hands and labs with many cycles

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Single cel PCR methods
Blastomere is tubed
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Single cel PCR methods
PCR mix is prepared in DNA-free laminar flow
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Efficiency of PCR-ADO

• Efficiency of more than 90 should be aimed at
• Pre-clinical tests on single cells (lymphocytes,
  fibroblasts, amniocytes,)
• At least 50 cells in total
• Lower efficiency coincides with higher ADO and
  preferential amplification
• ADO is most influenced by PCR method
  (conventional vs. fluorescent)
• ESHRE PGD Consortium guidelines, Hum Reprod. 2005
  Jan20(1)35-48

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Multiplex PCR
Provides control for contamination and ADO E.g.
cystic fibrosis
4 possible offspring
mother
father
p.F508del
IVS17BTA
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PGD for DM1
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Comparative genomic hybridisation
Figure courtesy L. Wilton
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Comparative genomic hybridisation
Figure courtesy L. Wilton
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Principle of MDA

• Devised for circular DNA
• Isothermal
• Random priming
• Polymerase makes strand and displaces other
  strand, e.g. F29 polymerase
• Whole genome amplification

Spits et al., 2006, Nature Protocols, Vol 1(4)
1965-1970
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MDA and PGD

• Use for haplotyping in PGD for monogenic disease
• But high ADO rate, so many markers to be repeated
• Use for array-CGH for PGS or chromosomal
  aberrations
• Shorter protocol, better resolution
• Combine both the above

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CGH with microarrays
Test DNA or cDNA
Control DNA or cDNA
Slide courtesy of J. Harper