1. In Vitro Fertilization and Preimplantation Genetic Diagnosis
Adrianna Vlachos, MD
DBA Camp 2015

2. In Vitro Fertilization and Pre-Implantation Genetic Diagnosis
Goal:
Child unaffected by a genetic (hereditary) illness defined by a known genetic mutation
Secondary goal:
“Creating” a child unaffected by the genetic illness who is a transplant donor for the affected child

3. In Vitro Fertilization and Pre-Implantation Genetic Diagnosis
IVF
Hormonal therapy to the mother to get many eggs for ovulation
Number of eggs depends on age of mother
Side effects of these medications to mother
Eggs harvested (under anesthesia)
Eggs are fertilized with sperm outside of mother (“in the test tube”)

4. In Vitro Fertilization and Pre-Implantation Genetic Diagnosis
PGD
Fertilized egg reaches 8-cell stage
One cell taken for RP/HLA testing
“RP neg, HLA matched” fertilized egg implanted in mother (1/8 chance)
Hormonal therapy to continue pregnancy, usually until weeks
CVS (10 wks) or Amnio (18 wks) for confirmation

5. Pre-natal vs Pre-implantation diagnosis
Dr. I. Souter, MGH Fertility Center

6. Pre-natal Diagnosis
Amniocentesis
Chorionic Villus Sampling (CVS)

7. Pre-implantation Diagnosis
Introduced initially in 1990
Biopsy of a single cell per embryo
followed by its genetic diagnosis through different techniques
the subsequent replacement to the patient of those embryos classified by genetic diagnosis as unaffected

8. PGD Indications Primary Goal Procedure is offered to couples:
With known single gene disorders that can be detected by PGD - DBA
With known chromosomal abnormalities that can be detected by PGD
requesting sex selection for X-linked disorders – DBA

9. PGD Indications Secondary Goal
Requesting PGD for HLA-typing (to allow selection of embryos that are histocompatible with live siblings)

10. Single Gene Disorders

11. PGD Process Ovulation Induction Retrieval Fertilization
Embryo Bx on Day-3
Genetic Analysis
Embryo Transfer

12. Ovulation induction

13. Oocyte Retrieval

14. Fertilization Conventional Insemination
Intracytoplasmic Sperm Injection (ICSI)

15. Embryo Culture

16. Day 3/Cleavage Stage Embryo

17. Cleavage Stage Biopsy

18. Genetic Analysis/PCR DNA amplification Mutation Characterization
sequence harboring the mutation
Mutation Characterization
FISH
PCR
HLA Matching

19. Embryo Transfer

20. Early Pregnancy

21. Risks
Embryo damage
Oocyte and Embryo Biopsy are invasive procedures
Misdiagnosis
False negative result
False positive result
The chance for NO result
The chance for mosaicism
IVF Risks
Not Achieving Pregnancy
There may not be any normal embryos available for transfer.
The embryos may not implant and develop even if they do not have the defect.
The workup for PGD is expensive and labor intensive
PGD can only detect a specific genetic disease in an embryo. It cannot detect many genetic disorders at a time and cannot guarantee that the fetus will not have an unrelated birth defect. .
The Risk of Embryo Biopsy While PGD is a relatively new procedure in IVF, the micromanipulation or biopsy techniques required to perform the procedure have been in use for many years. The risk of accidental damage to an embryo during removal of the cell(s) is very low %.
Concerns have been expressed regarding the development and viability of the embryo after biopsy
Studies have shown that biopsy at the 8 cell stage has no detrimental effects on further growth The balance between potential biopsy damage and beneficial effects of PGD seems to be positive.
The other risks include those common to all IVF treatments - and these include: OHSS ( ovarian hyperstimulation syndrome), ectopic pregnancy and multiple pregnancy
Misdiagnosis
Mosaicism is defined as the embryo having cells with different chromosome make up. Typically, all cells of the embryo have the same chromosomal make up as they originate from the same fertilized egg. However , it is possible for cells of the same embryo to have differing numbers of chromosomes.
. If we analyze a cell that has normal chromosomal content, but another cell has an extra chromosome, we erroneously diagnosed that embryo as being chromosomally normal. Due to the chance of misdiagnosis as well as the presence of anueploidy for which we do not test, we recommend prenatal testing as stated earlier.
What Is The Reliability of PGD? There is a small error rate with PGD due to the complexity of testing a single cell in the laboratory.. It must be stressed that this technology is still considered experimental. For this reason, prenatal testing by chorionic villus sampling or amniocentesis is required by the PGD laboratory to confirm their diagnosis. PGD can only detect a specific genetic disease in an embryo. It cannot detect many genetic disorders at a time and cannot guarantee that the fetus will not have an unrelated birth defect.
Patients opting for PGD have to be aware of the limitations
Conventional prenatal diagnosis still needs to be recommended to patients to confirm PGD results by amniocentesis or CVS.
they have to go through IVF even though they may be fertile, the workup of new diagnosis for PGD is labour intensive, expensive and may take some time, Single cell PCR is a technically challenging procedure as it is at risk of contamination and allele dropout (ADO). Since only 1 cell is amplified, if another cell or piece of DNA enters the tube, it will also be amplified. Therefore ICSI is used to ensure that there are no excess sperm present (paternal contamination) and all of the cumulus cells have to be carefully removed (maternal contamination). Stringent conditions have to be employed to ensure that no other contamination results
No part of the future fetus will be lacking because one or two cells are removed form the embryo approximately 2 days after fertilization. All the cells of the embryo remain totipotent until after the fourth day which means having all the potential into growing into a whole fetus.
The accuracy of the PGD for translocation is 90%.

22. Summary
Before PGD is performed, genetic counseling must be provided to ensure that patients fully understand the
risk for having an affected child
the impact of the disease
the available options
the multiple technical limitations including the possibility of an erroneous result
Prenatal diagnostic testing is strongly encouraged to confirm the results of PGD

23. Conclusions It is a personal choice!!!
This requires identification of your family’s DBA gene.
Obstacles
Practical
Religious
Ethical
Financial
There are other options available
Adoption
Sperm/egg donation
Just having a baby