Preimplantation Genetic Diagnosis
AS3323/5621 Lecture 8 Sept 21, 2017
IVF steps(compare to bovine MOET and OPU-IVP)
•1. Ovarian stimulation
•2. Egg retrieval
– Standard IVF vs. ICSI
•4. Embryo transfer – Selective assisted hatching
•5. Cryopreservation (embryo freezing)
Basic Steps of IVF-PGD
1. Regular IVF (can be for fertile couples)
2. Embryo biopsy: day 3/5 (what stage?)
3. PGD by cellular/molecular analyses
- FISH and PCR
- chromosome number, structure, single gene, social sexing
Aneuploidy Analysis with FISH
fixation Addition of probes
13 = red 16 = aqua 18 = blue 21 = green 22 = yellow Denaturation of probes/nucleus Hybridization (4 hrs / overnight) Wash to eliminate non-specific hybridization Visualization
with proper filters
Re-hybridize with X = yellow Y = aqua 15 = green 17 = orange 22 16 13 17 Y 15 9 probes: X, Y, 13, 15, 16, 17, 18, 21, 22 21 18 X Wash off probes
– Involve the acrocentric chromosomes (13, 14, 15, 21, and 22)
– The short arm is made up of satellite DNA, which is lost and the chromosomes join at their centromere.
– Balanced Robertsonian translocation: two chromosomes are fused giving rise to 45 chromosomes rather than 46.
– The most common translocation involves chromosomes 13 and 14, 1/1,300
Normal, carrier, affected
– Reciprocal translocations, breaks occur in two chromosomes which re-join incorrectly.
– Exchange of material between non-homologous chromosomes
– Reciprocal translocations are found in about 1 in 600 newborns.
– Usually harmless for carriers
Balanced reciprocal translocation
Both type of translocations are balanced
Only balanced carriers exist
They don’t know they are carriers until their
chromosomes are examined
Phenotypically normal except fertility
Why is there no one with imbalanced
What are the genotypes and phenotypes of the gametes and offspring ?
% of each genotype, % of each phenotype?
50% offspring: Physically normal 25% carrier 25%: monosomy 25%: Down’s syndrome Mother 14, 21 14-21, 21 14, 0 14-21, 0 Father 14, 21 A A B B A B b
HW3: Chromosomal translocation
of the 4th
n problems– Haploid – Genes missing
PGD example #1
Chromosome Structural Analysis
• Balanced translocation
Cornell First Healthy Baby from PGD
for Balanced Translocation
• 27 year old (April, 1997).
• During 1/95-9/96 (21 months) 5 spontaneous miscarriages & 1 ectopic pregnancy.
A healthy girl was born in Dec, 1997.
WCP 4 WCP 11 46,XX,t(4;11)(q21;q13) 46,XX (Amniocyte) 4 4 11 11 PGD 4 11 der(11) der(4)
Metaphase spreads: chromosomes? chromatin?
PGD example #2
Single Cell PCR & Genetic Analysis
• A particular fragment of DNA is amplified in test tubes
•Sensitive, detection of single copy
• the amplified DNA products are analyzed with available mutation detection methods.
Biopsy Single cell loading
Break cell to components
PGD for Sickle Cell Anemia (
The A to T substitution abolishes a restriction site (Dde I)
Autosomal recessive (11p). A to T mutation causes Glu. to Val. on the 6th codon of hemoglobin -chain.
4 3 2 A/S A/A S/S 364 291 201 90 74 571 bp F-1 R-1 F-2 R-2 364 bp Exon - 1 Exon - 2 90 bp 201 bp 74 bp
A nested-PCR to amplify DNA from single cells, followed by RFLP (Dde I)
“S” allele (A to T), “A” allele (A)
Xu et al., 1999; JAMA
PGD example #3
Sex Linked Disorders
over 400 X-linked diseases
If only transfer female or male embryos ? % carriers, ? % affected
Inheritance of Recessive X-linked
Embryo Sexing (sperm sexing)Female Male M X Y 18 X,Y,18 probes 18 X X 18 18 X
Selection of desired sex by PGD or sperm sexing
Allowed in the US
Banned in the UK and many other countries
International IVF coupled with tourism
Baby Conceived to Provide Cell Transplant
for His Dying Sister
•Human leukocyte Antigen (HLA) matching
(1 in 350,000 births)
•Both parents need to be carriers to have an affected child
Limitations of PGD
Embryo number & quality (age-related), mosaicism (2~5% misdiagnosis), complex disorders.
Labor intensive, single cell, detection failure (false results)
Cost (IVF & PGD) - Ethical
Embryo manipulation, sex selection for social reasons? HLA-matching for cord blood, etc.
Safety of PGD: a Concern,
Particularly the Long-term Safety
Crucial questions are: Is biopsy safe? Would it affect embryo survival?
Would it affect the children born from this new technique? For PGD, all laboratory procedures are the same as
regular IVF and ICSI, except one or a few cells are removed from a Day 3/5 embryo.
On-going study in the Netherlands from 2012
• Study 1:
– all pregnancies after PDG implemented (1995-2014)
– No difference in number of children, miscarriage, preterm birth, (very) low birth weight and congenital defects to non-PGD
• Study 2:
– 51 5-yr old from IVF-PGF: controls: 52 IVF, 35 no IVF but family history of genetic defects
– do not differ in physical nor neuropsychological measurement
– At 8, age appears to predict future cardiovascular health: no difference • Study 3:
– Parents: decision-making, considerations and motivations, moral issues, inner conflicts, experiences and satisfaction with PGD, most are happy • Study 4:
– interviewed ten families in which PGD was performed for Huntington’s disease in one parent, psychosocial problems related to the symptomatic
parent in the child 26
Ethical Concerns of PGD
Late onset disease screening
Cancer susceptibility (BRCA1 mutations
Other diseases ?
Where do you stop?
Don’t be confused: Amniocentesis
Pre-natal genetic testing