Preimplantation Genetic Diagnosis (PGD) II. AS3323/5621 Lecture 8 Sept 21, 2017







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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

3. Fertilization:

Standard IVF vs. ICSI


4. Embryo transferSelective 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

Biopsy Nucleus

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


Robertsonian Translocations

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

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


Balanced Translocations

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



Punnett Square:

Bb X


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 4


and 20







n problems

HaploidGenes 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 (

AS 5621


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)

1st PCR


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



Social Sexing

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

Fanconi's anemia

(1 in 350,000 births)

Autosomal recessive

Both parents need to be carriers to have an affected child



Limitations of PGD

- Biological

Embryo number & quality (age-related), mosaicism (2~5% misdiagnosis), complex disorders.

- Technical

Labor intensive, single cell, detection failure (false results)

- Economical

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 differenceStudy 3:

Parents: decision-making, considerations and motivations, moral issues, inner conflicts, experiences and satisfaction with PGD, most are happyStudy 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

Carrier screening

Late onset disease screening

Cancer susceptibility (BRCA1 mutations


Other diseases ?

Designer babies?

Where do you stop?



Don’t be confused: Amniocentesis

Pre-natal genetic testing





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