IS THERE ANY PLACE FOR PGD/PGS IN ART?

1. IS THERE ANY PLACE FOR PGD/PGS IN ART? L. Gianaroli, M.C. Magli, G. Jones, A.P. Ferraretti S.I.S.ME.R. Reproductive Medicine Unit - Via Mazzini, 12 - 40138 Bologna Italy ART Solution- Melbourne- australia www.iiarg.com www.sismer.it

2. S.I.S.ME.R. ISO 9001:2008 PGD - Legal Status in Europe Allowed Forbidden Not Regulated

3. S.I.S.ME.R. ISO 9001:2008 Reimbursement of ART treatments Reimbursed Not reimbursed

4. S.I.S.ME.R. ISO 9001:2008 Reimbursement of PGD Reimbursed Not reimbursed

5. S.I.S.ME.R. ISO 9001:2008 ASSISTED REPRODUCTIVE TECHNOLOGY IN EUROPE: EIM REGISTERS – PGD CONSORTIUM EIM PGD cons Year 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 No. cycles 203893 232443 258460 279267 289690 257682 295081 367066 419037 459170

6. Sex-selection S.I.S.ME.R. ISO 9001:2008 ESHRE PGD CONSORTIUM To December 2006: 21.743 cycles, 3929 children born Translocations Aneuploidy Monogenic disorders X-linked disorders Goossens et al. (2009) ESHRE PGD Consortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007. Hum Reprod 2009;24:1786-1810.

7. ESHRE PGD CONSORTIUM MEAN NUMBER OF CYCLES REPORTED BY CONTRIBUTING CENTERS TO THE 9 DATA COLLECTIONS PUBLISHED UP TO DATE Data collection I II III IV V VI VII VIII IX 1/97-9/98 to 5/2000 to 5/01 5-12/01 2002 2003 2004 2005 2006 No. of centers 16 25 24 36 43 50 45 39 57 No. of cycles 392 926 782 1890 2219 2984 3358 3488 5858

8. S.I.S.ME.R. ISO 9001:2008 ESHRE PGD CONSORTIUM Data 2006: 5.858 cycles, 1206 children born Goossens et al. (2009) ESHRE PGD Consortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007. Hum Reprod 2009;24:1786-1810.

9. S.I.S.ME.R. ISO 9001:2008 ESHRE PGD CONSORTIUM - Data collection IX Autosomal recessive 1997-2006 Clinical pregnancy rate FHB+ (%) per OR per ET Goossens et al. (2009) ESHRE PGD Consortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007. Hum Reprod 2009;24:1786-1810.

10. PGD WHY TO GO FOR IT? S.I.S.ME.R. VISION 2000 Normally fertile couples whose children might inherit - a severe disease - a predisposition to a pathology Normally fertile couples who wish to save a sibling’s life (HLA-typing)

11. PGD WHY TO GO FOR IT? S.I.S.ME.R. VISION 2000 “IVF aims at having a child, PGD aims at having a healthy child and PGD/HLA testing aims at having a healthy and helpful child”. UNESCO’s report on preimplantation genetic diagnosis (PGD) and Germ-Line Intervention, 2003.

13. S.I.S.ME.R. ISO 9001:2008 INDICATIONS FOR PREIMPLANTATION HLA MATCHING  Haematopoietic disorders requiring HLAcompatible HSC donor - Thalassemia - Fanconi anaemia - Wiskott-Aldrich syndrome - Diamond-Blackfand Anemia - X-linked Hyper IgM Syndrome - X-linked adrenoleukodystrophy - X-linked Hypohidrotic Ectodermal Dysplasia with immune deficiency - Aplastic anemia  Diseases like Acute Lymphoid Leukemia, in which HLA matching becomes the primary indication

14. THALASSEMIA 1.5% carriers in the world  400.000 affected 12% carriers  700 affected 12.5% carriers  1.600 affected 7% carriers  1.300 affected

15. S.I.S.ME.R. ISO 9001:2008 Nested PCR for specific amplification of HLA antigen gene exons 2 and 3 was performed using gene-specific outer primers Asp5 (5'-GCCCCGAACCCTC(CT)TCCTGCTA-3') and Asp3 (5'-CCGTGGCCCCTGGTACCCGT-3'),20 followed by 4 separate second-round PCR with allele-specific inner primers 085 (5'-TCCTCGTCCCCAGGCTCT-3') and 98 (5'-GCAGGGTCCCCAGGTCCA-3') for A2 allele,21 140 (5'-GGTTCTCACACCATCCAGATA-3') and 142 (5'-CAGGTATCTGCGGAGCCCG-3') for A1 allele,21 140 (5'-GGTTCTCACACCATCCAGATA-3') and 126 (5'-CCACTCCACGCACGTGCCA-3') for A3 allele,21 and 118 (5'-TCCATGAGGTATTTCTACACC-3') and 145 (5'-GCAGGGTCCCCAGGTTCG-3') for allele A26.21 As haplotype analysis for father, mother, and the affected child showed different polymorphic short-tandem repeat (STR) marker (GAAA)n (C2_4_4) located between HLA-A and HLA-B and in the HLA-E to HLA-C region (Figure 2), a hemi-nested PCR system was designed to study the number of repeats in blastomeres from different embryos.22 The first-round amplification cocktail for this system contained outer primers P1-1 (5'-GGCTTGACTTGAAACTCAGAG-3') and P1-3 (5'-TATCTACTTATAGTCTATCACG-3'), while the second round PCR used in addition to P1-1, the inner primer P1-2 (5'-CTTCAAACAATACGCAATGACA-3'). The nested PCR system for HLA-B allele discrimination included outer primers Bout 1 (5'-GAGGGTCGGGCGGGTCTCAG-3') and Bout 2 (5'-TGGGGGATGGGGAGTCGTGAC-3') for the first round of amplification. The second round of amplification for HLA B35 was performed using inner PCR primers CG4 (5'-GACGACACCCAGTTCGTGA-3') and 35in (5'-GAAGATCTGTGTGTTCCGG-3'). Accordingly, the second round of amplification of HLA B41 was performed with primers CG3 (5'-CTCTGGTTGTAGTAGCCGC-3') and 41up (5'-CCACGAGTCCGAGGAAGG-3'), and HLA B44 with primers 41up and GC2 (5'-GCTCTGGTTGTAGTAGCGGA-3').23 Of 30 embryos tested in 4 IVF attempts, 6 were homozygous affected and 24 were unaffected. Five of these embryos were also found to be HLA-compatible, of which 2 were transferred in the first and 1 in each of the other 3 cycles, resulting in a pregnancy and birth of an unaffected child in the last cycle

16. Reproductive Genetics Institute rgi@flash.net Overall Results and Outcome of Preimplantation Diagnosis for Single Gene Disorders & Preimplantation HLA testing RGI Experience 39.4 delivery rate / patients *ongoing pregnancies Rechitsky and Kuliev, RBM Online, 20:S1, 2010

17. S.I.S.ME.R. ISO 9001:2008 2093 139 couples  284 cycles  165 transfers (58%)  45 term pregnancies (27%)  51 babies born 10 successful HSC transplantations Van de Velde et al., Hum Reprod 2009

18. S.I.S.ME.R. VISION 2000 Trophectoderm biopsy for developmental competence Biopsy for genetic diagnosis Transfer to patient Blastocyst 8-cell 4-cell Inner cell mass Embryos 2-cell Transfer to patient ICSI Use in regenerative medicine (eg. diabetes, Parkinson’s Disease, stroke, respiratory disease, cardiac damage, quadriplegia) IVF Female infertility Idiopathic infertility few eggs Many eggs Immature eggs recovered in natural cycle (no fertility drugs) Fertility drugs given to women to make many mature eggs Reproductive Medicine  Therapeutic Medicine

19. EU hESC line Registry (hESCReg) Primary objective: provide information about all human ESC lines derived and used in Europe which are available to the scientific community. Specific Support Action funded by VI FP European Commission (1.048.000 €, 2007 - 2010) S.I.S.ME.R. VISION 2000 Coordinated by: Joeri Borstlap (BCRT - Technical Coord.) Anna Veiga (CRMB - Scientific Coord.)

20. S.I.S.ME.R. VISION 2000

21. RGI’s Repository of Human Embryonic Stem Cells S.I.S.ME.R. VISION 2000 Reproductive Genetics Institute As of 10/2008

22. S.I.S.ME.R. ISO 9001:2008 ESHRE PGD CONSORTIUM PROPORTION OF PGS OVER TOTAL PGD CYCLES % Data collection I+II III IV V VI VII VIII IX

23. S.I.S.ME.R. ISO 9001:2008 ESHRE PGD CONSORTIUM To December 2006: 13053 PGS cycles PGS INDICATIONS % AMA RIF RPL SMF AMA+RIF Other AMA+RPL No indication AMA: advanced maternal age RPL: recurrent pregnancy loss RIF: repeated IVF failures SMF: severe male factor Goossens et al. (2009) ESHRE PGD Consortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007. Hum Reprod 2009;24:1786-1810.

24. S.I.S.ME.R. ISO 9001:2008 ESHRE PGD CONSORTIUM To December 2006: 13053 PGS cycles REMARKS Mean no. of retrieved oocytes Mean no. of biopsied embryos Data collection I+III IV V VI VII VIII IX Goossens et al. (2009) ESHRE PGD Consortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007. Hum Reprod 2009;24:1786-1810.

25. S.I.S.ME.R. ISO 9001:2008 ESHRE PGD CONSORTIUM To December 2006: 13053 PGS cycles REMARKS Goossens et al. (2009) ESHRE PGD Consortium data collection IX: cycles from January to December 2006 with pregnancy follow-up to October 2007. Hum Reprod 2009;24:1786-1810.

26. S.I.S.ME.R. ISO 9001:2008

27. S.I.S.ME.R. ISO 9001:2008 SELECT ION 13 15161821 22 IVF ICSI

28. 3x16 PB1 MII S.I.S.ME.R. ISO 9001:2008 Is PB1 testing for chromosomes a reliable procedure? Is PB1 the mirror image of MII according to FISH results? 1x16 Gianaroli et al., Hum Reprod. Accepted for publication.

29. 19 Euploid PB1 19 Euploid MII 99 diagnosed 104 MII 80 Aneuploid PB1 76 Aneuploid MII 3 Euploid MII 1 Aneuploid MII – different abnormality S.I.S.ME.R. ISO 9001:2008 Correspondence between PB1 and oocyte Full concordance: 96% Gianaroli et al., Hum Reprod. Accepted for publication.

30. S.I.S.ME.R. ISO 9001:2008 Overall results for clinical outcome of cycles in which chromosomal analysis of PB1 was performed - 1 aP=0.00001 bP=0.009 cP=0.049 Gianaroli et al., Hum Reprod. Accepted for publication.

31. S.I.S.ME.R. ISO 9001:2008 Overall results for clinical outcome of cycles in which chromosomal analysis of PB1 was performed - 2 aP<0.005 bP<0.05 Gianaroli et al., Hum Reprod. Accepted for publication.

32. S.I.S.ME.R. ISO 9001:2008 Overall results for clinical outcome of cycles in which chromosomal analysis of PB1 was performed - 3 *2 after prenatal diagnosis, normal karyotype aeP<0.025 bdmP<0.001 cP<0.05 fP<0.005 gP=0.047 Gianaroli et al., Hum Reprod. Accepted for publication.

33. S.I.S.ME.R. ISO 9001:2008 Oocyte chromosomal status as detected by PB1 analysis Missing chromatids Extra chromatids Missing chromosomes Extra chromosomes % Complex abnormalities Gianaroli et al., Hum Reprod. Accepted for publication.

34. S.I.S.ME.R. ISO 9001:2008 Incidence of aneuploidy in relation to maternal age a b ab % 35 36-37 38-39 40-42 43 years aP<0.001 bP<0.01 Gianaroli et al., Hum Reprod. Accepted for publication.

35. Aneuploidy Other abnormalities S.I.S.ME.R. VISION 2000 9.96-12.07life PGD FOR ANEUPLOIDY CHROMOSOMAL ABNORMALITIES ACCORDING TO AGE n=5352 P<0.001 % CROMOSOMALLY ABNORMAL EMBRYOS 35 36-37 38-39 40-42 43 years

36. Indications to FISH analysis Hormonal stimulation Type of infertility Reproductive history Response to stimulation S.I.S.ME.R. ISO 9001:2008 Factors affecting aneuploidy in human oocytes Gianaroli et al., Hum Reprod. Accepted for publication.

37. S.I.S.ME.R. ISO 9001:2008 Statistical evaluation • - A multivariate regression analysis corrected for semiquantitative data was performed to investigate the determinants of aneuploidy, expressed as the proportion of euploid oocytes over the number of diagnosed oocytes. • The weight of each different indication to FISH analysis on PBs and cause of infertility were estimated by calculation of X variables marginal contribution. Relationships were evaluated for significance using inferential analysis of the regression coefficients of the equation that define relationships. • The goodness of fit test was applied to evaluate whether aneuploidy within the same oocyte is randomly or not randomly generated. Gianaroli et al., Hum Reprod. Accepted for publication.

38. Relationships between the proportion of aneuploid oocytes calculated over the number of diagnosed oocytes and the studied variables S.I.S.ME.R. ISO 9001:2008 No correlation Negative correlation P<0.01 Positive correlation P<0.01 Aneuploid oocytes

39. S.I.S.ME.R. ISO 9001:2008 Is there a correlation between the analyzed variables and the type of meiotic error? - Chromatid predivision - Chromosome non-disjunction - Combined chromatid and chromosome errors - Aneuploidy per single chromosome Stepwise regression analysis Gianaroli et al., Hum Reprod. Accepted for publication.

40. S.I.S.ME.R. ISO 9001:2008 Stepwise regression analysis of the studied variables in regard of independent variable Gianaroli et al., Hum Reprod. Accepted for publication.

41. S.I.S.ME.R. ISO 9001:2008 SELECT ION 13 15161821 22 IVF ICSI Should other chromosomes be tested?

42. S.I.S.ME.R. ISO 9001:2008 Aneuploidy of chromosomes 1, 4 and 6 Study design 85 patients with a normal karyotype that had undergone an assisted conception cycle with chromosomal analysis of first polar bodies (PB1) for chromosomes 13, 15, 16, 18, 21 and 22 (first panel) • 43 pregnant • 42 not pregnant Each pregnant patient was matched to the non pregnant patient having similar characteristics in terms of age, cause of infertility, and indication to chromosomal analysis Magli et al., Fertil Steril 2010. In press.

43. S.I.S.ME.R. ISO 9001:2008 Aneuploidy of chromosomes 1, 4 and 6 Study design After conclusion of clinical pregnancies to delivery or abortion, PB1s were reanalyzed for chromosomes 1, 4 and 6 (second panel) 1st panel 2nd panel 131516 18 21 22 1 46 Magli et al., Fertil Steril 2010. In press.

44. S.I.S.ME.R. ISO 9001:2008 Aneuploidy events per single chromosome in PB1 flo ekn bhm dj ag ci mno % ghijkl abcdef aefhklP<0.001 bdP<0.005 cjoP<0.01 gimnP<0.025 Magli et al., Fertil Steril 2010. In press.

45. S.I.S.ME.R. ISO 9001:2008 Aneuploidy of chromosomes 1, 4 and 6 following PB1 reanalysis of the transferred embryos  The incidence of aneuploidy for the 2nd panel chromosomes was higher in the nonpregnant patients.  There is no difference between term pregnancies and spontaneous abortions. Magli et al., Fertil Steril 2010. In press.

46. PGS BY MICROARRAYS CGH

47. PGS BY MICROARRAYS CGH The ESHRE PGS Task Force has decided to start a proof of principle study using their methods with the aim of determining whether biopsy of the first and second polar body, followed by subsequent analysis of the complete chromosome complement of these polar bodies using an array-based technique, enables a timely identification of the chromosomal status of an oocyte. The specific objectives of this study are: (i) To show that the analysis of both polar bodies can be completed within a time period that allows for fresh transfer; (ii) To ensure the reliable identification of the chromosomal status of an oocyte in at least 90% of polar body biopsy attempts; (iii) To test the feasibility of a multicentre randomized trial based on the technology used in the pilot study.

48. PGS BY MICROARRAYS CGH Objective 1 is fulfilled Results are obtained in 13.5 hrs (even less)

49. PGS BY MICROARRAYS CGH Objective 2 To ensure the reliable identification of the chromosomal status of an oocyte in at least 90% of polar body biopsy attempts.

50. PGS BY MICROARRAYS CGH The study aims to include 500 oocytes, independent from the number of patients. Assuming that 300 oocytes will be fertilized after ICSI, 600 polar bodies will be processed for chromosomal diagnosis. Expecting a 50% aneuploidy rate, another 150 aneuploid oocytes will be processed for array analysis to verify the data obtained from the polar bodies. In addition, undiagnosed oocytes will also be analysed but only if the resulting embryos are not transferred.