Update on Preimplantation Genetic Diagnosis for all chromosome aneuplodies, translocations and gene defects.

3. High rates of Chromosome abnormalities

4. embryos analyzed: 6054. Morphologically normal embryos: 3751. Source: Munn é et al. 2007. Similar results also found by Munne et al 1995, Marquez et al. 2000, Magli et al. 2007. % chromosomally abnormal embryos 56% Maternal age Morphology: The majority of embryos with ‘good’ morphology are chromosomally abnormal

5. PGD Hypothesis PGD may improve ART outcome in women of advanced maternal age Munné et al. (1993)

6. Despite large studies indicating the advantages of aneuploidy screening, the notion that PGS for infertility is beneficial is not yet shared uniformly.

7. Positive effect Gianaroli et al. 1999 Munne et al 1999 Gianaroli et al 2001a Gianaroli et al. 2001b Munne et al. 2003 Gianaroli et al. 2004 Munne et al. 2005 Munne et al 2006 Verlinsky et al. 2005 Colls et al. 2007 Garrisi et al. 2009 Rubio et al. 2009 No effect (small) Werlin et al. 2003 Jansen et al. 2008 Mersereau et al. 2008 Scholcraft et al. 2009 No effect (Large) Staessen et al. 2004 Platteau et al. 2005 Negative effect Mastenbroek et al. 2007 Hardarson et al. 2008 Contradicting PGD results using day 3 biopsy and FISH

9. Mosaicism

10. 592 embryos found abnormal by PGD were reanalyzed and found to be: normal 13 mosaic <49% abnormal 27 mosaic 50-99% abnormal 124 mosaic 100% abnormal 297 homogeneously abnormal 131 Colls et al. (2007) Mosaicism produces <7% misdiagnosis 6.8% 1[13]1[16]2[18]2[21]1[22] 2[13]1[16]2[18]2[21]2[22] 1[13]1[16]2[18]2[21]1[22] 1[16] 2[13]2[16]2[18]2[21]2[22] 2[13]1[16]2[18]1[21]1[22] 2[13]3[18]1[21]1[22] 3[13]1[16]2[18]1[21]3[22] 1[13]1[16]1[18]1[21]1[22] 3[13]1[16]2[18]1[21]3[22]

12. self-correction myths

13. UNIPARENTAL DISOMY: a trisomy correction creates a zygote with 2 chromosomes of one parent, none of the other (trisomy rescue). Theoretical chance : 1/3 EXAMPLE TRISOMY 15: Prognosis is ethal because of imprinting Trisomy 15 in cleavage stage embryos: 1.874% a UPD-15 in newborns: 0.001% b Estimated correction of trisomy 15 to UDP: 1/3 c Trisomy 15 day 3 embryos that self-corrected: 1.874 x 0.001 / 3 = 0.56% a: Munne et al. (2004), b: From: OMIM, c: 3 X UPD Trisomy correction is rare: UPD evidence

14. Fetus seldom self correct: it’s the placenta that becomes abnormal This work questions the assumption that placental confined mosaicism is the result of fetal self-correction. At the contrary, it suggests that normal fetuses may develop abnormal placenta.

15. Sub-optimizal methods (FISH studies, day 3 biopsy)

16. Optimal PGS Questionable PGS Biopsy media with aminoacids simple media Biopsy time / embryo 1 min > 5 min # cells biopsied one two Fixation method Carnoy’s Tween 20 # chromosomes tested ≥ 8  6 # analysts / case 2 1 Use of NRR* yes no Large experience yes no Error rate <10% 10-50% Number of zygotes >5  5 *NRR: No result rescue, or re-testing of dubious chromosome with different probes. Optimal PGS methods

18. “ The data presented here clearly indicates that two cell biopsy significantly impacts clinical outcome. Our previous report providing no arguments in favor of PGS (Staessen et al., 2004) was criticized by others arguing that PGS might have been beneficial if only one cell had been removed (Cohen et al., 2007). In respect to the present findings, this criticism seems justified ”. P < 0.001 Two cell biopsy is detrimental

20. Analysis of remaining cells of embryos previously analyzed by PGD: study technique error rate Baart et al 2004 FISH 50.0% Li et al. 2005 FISH 40.0% Gleicher et al. 2009 FISH 15-20% Munne et al. 2002 FISH-9 7.2% Colls et al., 2007 FISH-9 4.7% Magli et al. 2007 FISH-9 3.7% Munne et al. 2010 array CGH 1.8% Error rate should be <10%

23. CGH and array CGH

24. Kallioniemi et al. (1992), applied to single cells by Wells et al. (1999) Normal DNA Test DNA Comparative Genome Hybridization (CGH) Normal Trisomy Monosomy

25. Array CGH Test DNA Normal DNA 2700 probes Same band resolution as karyotype

26. 46,XY

27. 47,XY+2

28. 44,XY-9-17

30. 46,XY-10 +16 aCGH detected 50% more abnormalities than FISH-12 and 20% more abnormal embryos (Colls et al. 2009) Detection of abnormalities: aCGH vs FISH-12 Detectable by FISH

31. aCGH validation: no results Embryos undiagnosed: biopsy on day 3 : 2% (16/724) biopsy on day 5 : >2% (0/54) Method: Bluegnome array, Sureplex amplification.

33. aCGH results on day 3: validation data Cells from the same embryo: X O , + 2, - 4p, + 11, - 13, - 18, - 22 XX, - 2, - 4p, + 4q31, - 13, - 18, - 22 XX X , + 2, - 4p, - 4q31, + 11, - 13, - 18, - 22 X O , + 2, + 4p, - 4q31, - 11, - 13, - 18, - 22 Who said mosaicism was a FISH artifact?

34. Day 3 biopsy, day 5 transfer and array CGH Cycles performed: 151 Maternal age (av.) 37.2 Pregnancy Rate Ongoing Pregnancy Rate Per Cycle Per ET Per Cycle Per ET Control 38% 38% 31% 31% PGD 46% 59% 42% 54% NS < 0.001 NS < 0.001 * Expected from each center SART data, controlled by age Data from 24 centers. Munné et al. (2010) PGDIS, and unpublished data

36. Effect of embryo vitrification and Replacement on next cycle

39. CGH on blastocyst biopsies: Implantation is independent of age Patients <43 who are eligible for blastocyst transfer have a >95% change of having normal embryos available for transfer

42. PGD for translocations and 24 chromosome abnormalities

43. aCGH for translocations and 24 chromosome aneuploidy Patient: 46,XY,t(3;11)(q22.2;q23.3)

44. Comparison of different approaches

45. aCGH vs. SNP arrays: Genome coverage # of probe genome probes size covered aCGH 4,000 x 150,000 kb = 600.0 Mb (25%) SNPs 300,000 x 50 kb = 1.5 Mb (>0.1%)

46. aCGH vs. SNP arrays: Resolution More resolution, more counseling nightmares: In pre-natal diagnosis the minimum resolution reported is 20 MB: Smaller abnormalities are not well understood and difficult to council At 1.5 Kb resolution there are >300,000 CNPs described: Difficult to differentiate a new polymorphism from an abnormality

50. Comparison of platforms FISH-12 CGH a array SNP probes a CGH a arrays Day 3 biopsy/ day 5 results yes no yes yes Parental DNA needed no no no yes Aneuploid embryos detected 89% 100% 100% 97% Detect gene defects no no no yes Detect polyploidy (errors) yes (1.8%) (1.8% ) yes Detect MII trisomies w/o crossover yes yes yes no if qual. Detect mitotic trisomies (errors) yes yes yes (3%) Error rate (cell lines analysis) 2% 1% 0% d 4% b Error rate (embryo reanalysis) 7% 8% 2% unk Embryos with no results 3% 6% 2% 8% b Increased implantation rate (day 3) + n/a ++ unk Increased implantation rate (day 5) unk +++ +++ +++ c a Reprogenetics data, b Jonhson et al. (2010), c Scott et al. (2010, PCRS), d Mamas et al (submitted)

51. PGD for gene defects and 24 chromosome abnormalities

52. PGD for gene disorders Disease tested: Acetil Co Oxidase type I defficiency, Adrenoleucodistrophy, Alpha-thalassemia, Alport syndrome, Autosomal Dominant Polycystic Kidney Disease (ADPKD), Autosomal Recesive Polycystic Kidney Disease (ARPKD), Beta-thalassemia, Branchio-Oto-Renal syndrome (BOR), BRCA1 breast cancer predisposition, BRCA2 breast cancer predisposition, CanavanCharcot-Marie-Tooth type IA (CMT1a), Choroideremia, Congenital adrenal hyperplasia (CAH), Congenital neutropenia, Connexin 26 hearing loss, Cystic fibrosis, Duchenne/Becker Muscular Dystrophy (DMD), Ectrodactyly, Ectodermal dysplasia, and Cleft lip/palate syndrome (EEC1), Fabry Disease, Familial adenomatous poliposis coli (FAP), Familial dysautonomia, Familial intrahepatic cholestasis 2, Fanconi anemia, Fragile site mental retardation , Gangliosidosis type 1 (GM1), Gaucher disease, Glomuvenous malformations (GVM), Glycogen-storage disease type I (GSD1), Glycosylation type 1C, Hemoglobin SC disease, Hemophilia A, Hemophilia B, Hereditary nonpolyposis colon cancer (HNPCC), Hereditary pancreatitis, HLA matching Huntington disease, Hurler syndrome, Hypophosphatasia, Incontinential pigmenti, Krabbe disease (Globoid cell leukodystrophy), Long QT syndrome, Marfan syndrome, Meckle gruber, Metachromatic leukodystrophy (MLD), Methylmalonic aciduria cblC type (MMACHC), Myotonic Dystrophy 1, Myotubular myopathy, Neurofibromatosis 1, Neurofibromatosis 2, Niemann-Pick Disease, Noonan syndrome, Oculocutaneous albinism 1 (OCA1), Ornithine carbamoyltransferase deficiency (OTC), Osteogenesis Imperfecta 1, Rapp Hodgkin ectodermal dysplasia, Retinitis pigmentosa, Retinoblastoma, Sickle Cell Anemia, Smith-Lemli-Opitz syndrome (SLOS), Spinal bulbar muscular atrophy (SBMA), Spinal Muscular Atrophy Type 1 (SMA1), Tay Sachs, Tuberous sclerosis 1 (TSC1), Tuberous sclerosis 2 (TSC2), Von Hippel-Lindau Syndrome (vHL), X-linked dominant Charcot–Marie–Tooth (CMTX), etc…… (see review Gutierrez et al. (2008)) We can do PGD for any disease with known mutation

53. Mutation site Polymorphic site Conventional approach to PGD of gene defects to avoid Allele Drop Out (ADO) accurate amplification of both loci ADO affecting normal allele ADO affecting mutation site

54. PGD of gene defects using SNP array Karyomapping Δ 508 Father: Δ 508 Mother: Slide adapted from A.Handyside Monosomy 11 Trisomy 21 Carrier Δ 508

55. Example: First Clinical Application of Karyomapping for PGD of Gaucher Disease Combined with 24 Chromosome Screening Handyside A, Grifo, J, Gabriel A, Thornhill A, Griffin D, Ketterson K, Prates R, Tormasi S, Fischer J, Munné S (2010) PGDIS Embryo # Gene defects Chromosome defects 5 Affected normal 7 Affected monosomy 22 8 Carrier monosomy 10, 21,22 9 Affected monosomy 22 2 unaffected Monosomy 22 10 Affected Monosomy 9,17,19,22, trisomy 14 Results confirmed by a-CGH and by PCR

56. Conclusions

58. Studies with improved results differ from those that show no improvement in that: 1) Reduce biopsy damage (1 cell, experience, blast?) 2) Low error rate (fixation, NRR, 2 analyzers) 3) Analyze 16,15,21,22 chromosomes + ≥4 more 4) Extensive experience 5) Appropriate population (≥5 embryos, ≥ 35 y. old) Conclusions: FISH studies

61. Integration with e IVF

66. Santiago Munné, PhD, Director Jacques Cohen, PhD, Director munne@reprogenetics.com www.reprogenetics.com Pere Colls, Ph.D Dagan Wells, Ph.D George Pieczenik, Ph.D Cristina Gutiérrez, Ph.D Jorge Sanchez, PhD John Zheng, MD Tomas Escudero, MS Kelly Ketterson, MS Jill Fischer, MS Jessica Vega, MS Tim Schimmel Sasha Sadowy Sophia Tormasi N-neka Goodall Renata Prates Piedad Garzon Laurie Ferrara Bekka Sellon-Wright Maria Feldhaus USA Spain Mireia Sandalinas, MS Carles Giménez, PhD César Arjona, MS Ana Jiménez, PhD Elena Garcia, MS Japan Tetsuo Otani, MD Muriel Roche Miho Mizuike UK Dagan Wells, PhD Elpida Fragouli, PhD Samer Alfarawati, MS Michalis Konstantinidis South America Paul Lopez Luis Alberto Guzman Germany Karsten Held, MD