Preimplantation Genetic Diagnosis (PGD) in Medicine

1. Preimplantation Genetic Diagnosis (PGD) in Medicine Dr. Hazem Al-Rumaih – FRCOG , MD Consultant OBGYN & reproductive Medicine

2. Introduction • The past 100 yrs have given birth to the most profound changes in society, medicine & technology the world have ever witnessed. • Genetics is one such field that have enjoyed a meteoric rise during this time.

3. History of Genetics • Progressing from Mendelian genetics in the 1950s to the discovery of DNA in the 1960s with the consequent discoveries of genetic (hereditary) aetiotogy of many diseases, to the ability to diagnose genetic defects of embryos before birth in the 1980s. • In 1990 the first PGD test done worldwide in the UK. • Reaching the ability to sequence the whole human genome in 2002.

4. The Consequences • This magnificent development in genetics have shifted medicine completely from only diagnosing to preventing hereditary disorders.

5. What is PGD / PGS • It is genetic testing done to cell(s) extracted from day 3 / 5 embryos in the lab before transfer to the uterus as part of IVF treatment. • This is done to diagnose or screen for genetic status &/or disorders.

8. Intracytoplasmic Sperm Injection (ICSI)

9. d2 Dobson et al, 2004; R Reijo Pera,2010

10. Blastomere Biopsy

16. Comparison of embryo-biopsy strategies

17. D3 vs D5/D6 biopsy VS.

18. Aim of PGD • Offers couples at risk the chance to have an unaffected (desired) child , without facing termination of pregnancy.

19. Uses of PGD • Diagnose & avoid embryos with chromosomal aberrations. • Diagnose & avoid single gene disorders. • Diagnose & avoid X-linked diseases (specific embryo gender). • Human leucocyte antigen (HLA) typing for stem cell transplant for an affected offspring. • First & second polar body testing can be done to study maternal genetic contribution.

20. Cleavage stage biopsy Day-3 Preimplantation Genetic Screening (PGS) Comprehensive Chromosome Screening (CCS) • Advanced maternal age (≥38 yrs; >40 yrs) • Implantation failure (≥ 3 IVF attempts) • Recurrent miscarriage (≥ 2 miscarriages) • Severe male factor Polar Body biopsy Blastocyst biopsy Day-0 Day-1 Day-5

21. Professional Pre Requisites • Parents counseled & accepted. • ≥ 5 grade A or 1 embryos. • Both IVF & genetic labs are trained & prepared to do PGD.

22. PGD Techniques • FISH (Fluorescent In Situ Hydridization) : for cytogenetic diagnosis of 5-7 chromosomes: (13,16,18,21,22,X &Y). Now obsolete. • PCR (Polymerase Chain Reaction): for molecular diagnosis of specific diseases. More recent & more accurate: • CGH-Array (Comparative Genomic Hydridization): 24 chromosones are tested.

23. FISH in Detection of Chromosomal Abnormality Triploid Chromosome 13 and normal other chromosomes

24. CCS with CGH arrays: 24-chromosomes screening Results <24hours Biopsy Sample 1 Sample 2 Cell (s) loading Amplification (~ 3 hrs) (98.3%) Labelling (2 hrs) Cy5 Cy3 DNA precipitation (~ 1 hrs) Hybridisation (5–12 hrs) 24sure BlueGnome 2684 clones 1Mb coverage Washing (~ 1/2 hr) BlueFuse Multi software Scanning

25. Euploid embryo Loss of chromosome 8

26. Gain chromosome 21 Partial gain chromosome 2p

27. Complex aneuploidy Chaotic pattern

28. Ethical & Legal Issues • Couple’s Informed consent. • Should not be done on social grounds. • Should be done on professional grounds only. • Should be provided only by well trained teams

29. Maternal Age and Infertility ≈38% SocietyforAssistedReproductiveTechnology 2011 (www.sart.org)

30. Results: Incidence of chromosomal aneuploidies Pearson’s correlation (p<0.05)

31. RCT Advanced Maternal Age 41-44 yrs (2009-2011) 13, 16, 18, 21, 22 15, 17, X,Y Additional rounds: TEL and LSI * Two-sides Fisher´s test Rubio et al., FS 2013 Tel 16qx2 16 16 INITIAL DIAGNOSIS Monosomy 16 FINAL DIAGNOSIS Normal

32. Day-3 RCT using CGH arrays (May 2012- Sept 2013) VS.

33. RCT RepetitiveImplantationFailure (2004- 2011) * One-side Fisher´s test Rubio et al., Fertil Steril 2013

34. CLINICAL RESULTS using CGH in different indications

35. Single Gene Disease Screening(Monogenic Diseases)

36. PCR DNA DOUBLE HELIX CHROMOSOME

37. Single Gene Disease Screening

38. Single Gene Disease Screening • Mutation report and requisition form. • 5 ml blood (EDTA tube) or buccal swab. • Primer development (3 weeks common and 6 weeks for rare mutation). • Day 5 biopsy for Single Gene disease plus Aneuploidy. • Report 2 days after receiving the samples, i.e. freeze embryos & transfer later (FET).

39. Children Follow up • PGD Children follow up showed similar outcome to IVF / ICSI without PGD.

40. Near Future of PDG Next Generation Sequencing (NGS) Rapid Accurate More efficient Scan for more genes

41. Work in progress: NGS Illumina Sept 2013

42. Work in progress: NGS Life Tech November 2013

43. Conclusions • PGD is currently easier & faster to do, cheaper, more comprehensive & accurate than before. • It is now an integral part of ART. • More advances in the technology is coming soon. • It should be use within clear regulations. • It has a great potential in reducing incidence of chromosomal & genetic disorders.

44. Future of Genetics • Hopefully in the near future we will wetness the use of knowledge & technologies in genetics to repair or correct genetic defects.

45. Thanks Finally I would like to thank Prof. Carlos Simon Scientific Director of IVI & Igenomix Valencia , Spain For allowing me to share some of his results in this lecture.

46. Thank you