presentation on the validation studies of array CGH validation for its use in PGD for the detection of ALL chromosome aneuploidies.

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

6. Validation of aCGH

7. CGH-based DNA Microarray (aCGH) Test DNA Normal DNA 2700 probes Same band resolution as karyotype

9. 46,XX

10. 47,XX+2

11. 44,XX-9-17

12. 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

15. 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?

16. Comparison of platforms FISH CGH array SNP 12 CGH arrays probes quant./qual. Day 3 biopsy/ day 5 results yes no yes yes Parental DNA needed no no no yes Detect gene defects no no no yes Detect polyploidy (errors) yes 0.2% 0.2% yes Detect MII trisomies w/o crossover yes yes yes yes / no Detect mitotic trisomies (errors) yes yes yes yes / 3.4% Error rate 7% 8% 6% unk No Results Rate 3% 6% 0% unk Increased implantation rate + + + + unk unk Reprogenetics data.

18. Santiago Munn é , PhD, Director Jacques Cohen, PhD, Director munne@reprogenetics.com www.reprogenetics.com Pere Colls, PhD Dagan Wells, PhD Tomas Escudero, MS Kelly Ketterson, MS Jill Fischer, MS John Zheng, MD George Pieczenik, PhD Cristina Gutierrez, PhD Piedad Garzon Jorge Sanchez, MS Tim Schimmel, BS Sasha Sadowy, BS Sophia Tormasi, BS Jessica Vega, MS N-neka Esprit-Ngachou, BS Laurie Ferrara Renata Prates, BS Bekka Sellon-Wright 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 South America Paul Lopez, BS Luis Alberto Guzman, BS Francisco Parera, PhD Germany Karsten Held, MD

19. aCGH, CGH cannot detect polyploidy 91,070 embryos analyzed by FISH with 9-12 probes: Polyploid or haploid: 7.7% - plus aneuploidy: 5.9% (detectable by aCGH) - no other abnormalities: 1.8% (not detectable by aCGH) - Arrested or dysmorphic: 1.6% (unlikely replaced) - Good morphology: 0.2% (risk of misdiagnosis)

20. SNP arrays may not detect mitotic trisomies 91,070 embryos analyzed by FISH with 9-12 probes: Complex abnormal mosaics: 26,624 (29%) - with only trisomies: 4,029 - of mitotic origin (76.2%)*: 3,070 (3.4% potential error) * Munne et al. (2002)