Preimplantation Genetic Diagnosis: An Overview Dr. Laila Bastaki , MD

1. Preimplantation Genetic Diagnosis: An Overview Dr. LailaBastaki, MD Consultant of Medical Genetics Director of KMGC

2. The development of PGD is one of the most exciting and important milestones in the history of Assisted Reproductive Technology

3. Preimplantation Genetic Diagnosis(PGD) • PGD is a state-of-the-art procedure used in conjunction with In Vitro Fertilization (IVF)in which the embryo is tested for certain conditions prior to being placed in the womb of the woman. • PGD was first reported in 1990. • PGD combines the recent advances in molecular genetics and in assisted reproductive technology

4. Indications for PGD • Chromosomal Disorders • Numerical • Chromosomal aneuploidy • Structural • Inversions • Translocations • Deletions and duplications • Gender determination for severe X-linked diseases with unknown gene • Severe monogenic diseases (cystic fibrosis, ß thalassaemia, sickle cell anemia, fragile X syndrome, myopathies) • PGD for HLA-typing (to allow selection of embryos that are histocompatible with live siblings)

5. HOW IS PREIMPLANTATION GENETIC DIAGNOSIS PERFORMED? • Technically demanding • Very Complex • Requires special skills

6. How is PGD performed? Ovarian Stimulation IVF Blastomere Biopsy on Day 3 Transfer of Unaffected Embryo Genetic Analysis (FISH or Molecular) Outcome Clinically Normal Baby

7. The Methods of Preimplantation Genetic Diagnosis • Remove a single cell from the 6-8-cell embryo using a fine glass needle to puncture the zonapellucida and aspirate the cell • In skilled hands, this generally does not harm the developing embryo. • Each cell is called a blastomere.

8. Blastomere Biopsy Video

9. The PGD process provides two categories of analysis • Fluorescence In Situ Hybridization (FISH). • Gene Chip array • Polymerase Chain Reaction (PCR)

10. Fluorescence In Situ Hybridization (FISH) • Using fluorescent probes specific for each chromosome. • useful for identifying aneuploidies (incorrect chromosome numbers) and translocations • procedure destroys the tested cell • limited number of chromosomes can be checked simultaneously • some abnormalities undetectable

11. Screening aneuploids with multiple probes Aneuploidy is the most frequent cause of spontaneous abortions

12. Gene chip array (Array CGH Analysis)

13. Array-CGH allows the laboratory to determine if the correct number of each chromosome is present in the egg or embryo • This technology simultaneously tests for all 24 chromosomes (1-22, X and Y) What is array-CGH analysis?

14. With array-CGH, the amount of DNA present for each chromosome is compared to that of a normal standard, enabling us to detect monosomies (missing chromosomes), trisomies (extra chromosomes), and other abnormalities What is array-CGH analysis?

15. What is array-CGH analysis?

16. Genetic testing for specific disease loci (PCR) • Polymerase chain reaction (PCR) • The gene causing the disorder should be confirmed and tested in the couple • Amplification of DNA specific to a gene of interest (family history guides choice of genes) • Second round PCR used for specific exonic sequencing and/or linkage analysis (Fragment analysis)

17. Fragment analysis for HLA matching

18. Sequence analysis for a specific familial mutation

19. Examples of genetic disorders detectable via PCR-based tests: • Tay Sachs (autosomal recessive) • Cystic fibrosis (autosomal recessive) • Huntington’s disease (autosomal dominant) • Thalassemias (autosomal recessive blood disorder) • Duchenne muscular dystrophy (X-linked recessive) • Spinal muscular atrophy (X-linked recessive) • As more genetic tests are developed as diagnostic tools, more will be used for predictive purposes in PDG.

20. Limitations of PCR-based tests: • Both alleles may not amplify equally (allele dropout), leading to misdiagnosis or inconclusive results • PCR-based tests only detect disorders at target loci; other mutations may exist elsewhere • To accommodate these limitations, prenatal amniocentesis or chorionic villus sampling is usually recommended as a supplement to PGD.

21. Benefits of PGD Reduction in the Chance of Having a Child with Aneuploidy Reduces the possibility of pregnancy termination following a prenatal diagnosis of a genetic disorder.

22. Risks • Embryo damage Oocyte and Embryo Biopsy are invasive procedures • Misdiagnosis The accuracy of the PGD for translocation is 90%. 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.

23. Human Error • Mislabeling, misidentification, misinterpretation • Wrong embryo transfer • Incorrect probes or primers • Technical • Probe or primer failure • Contamination (maternal, paternal, operator, carry-over) • Intrinsic (embryo) • Mosaicism • Allele drop out • UniparentalDisomy Causes of Misdiagnosis

24. European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium, 2003 Major malformations: 2.6% • Phocomelia and pulmonary deficiency, chylothorax, congenital hip dislocation, abdominal cystic mass, pesequinivarus, exencephaly Minor malformations: 1.4% • syndactyly, hydrocele testis, ASD, mongolian spot, sacral dimple Liebaers et al, Belgium 2010 Major malformations: 2.1% vs ICSI: 3.4% • chylothorax, VSD, oeasophageal atresia, cataract, umbilical hernia, ichthyosis, cardiopathy PGD & Malformations

25. Alternatives to PGD • Conceive naturally and have prenatal diagnosis during pregnancy

26. Future of PGD • Efforts continue to be focused on improving methods to obtain an accurate diagnosis. • PGD holds great promise for the future as techniques and genetic tests are perfected. • PGD may become routine in the next few years.

27. Conclusions • For couples at risk for producing offspring with either debilitating monogenic disorders or chromosomal abnormalities IVF/PGD represents a major scientific advance

28. Complications, both before and after birth, are no different in type or number from those found in a comparable ICSI population • Other parameters such as birth weight and length, are also similar to an ICSI population • PGD appears to be a safe method to avoid the birth of children with genetic defects Conclusions

29. 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 Conclusions

30. Thank You