OMES & OMICS in ART Moncef Benkhalifa, Ph.D. RBMG. Reproductive Biology & Genetics

1. OMES & OMICS in ART Moncef Benkhalifa, Ph.D. RBMG. Reproductive Biology & Genetics Technical & Development Director UNILABS. France

3. 1. Prélèvement de l’échantillon Organismes / tissues / cellules 3. Amplification (PCR) et marquage fluorescent 2. WGA, RTPCR 4. Pré-hybridation Hybridation Lavage Décontamination 5. Scan des données – Analyse des résultats

4. The “Omes and Omics” From James Poscillico

6. A B q21 q25 0.8 1.2 C der(X) X D E 21.1 25 21.1 25 X X dupXq dupXq Tachdjian G, Aboura A,Benkhalifa M et al De novo interstitial direct duplication of Xq21.1q25 associated with skewed X-inactivation pattern.

7. : Prenatal Diagnosis, October 2005

8. Custom Profiling: Route to Drug Discovery Application in ovarian cancer Chromosome segment 8q Sample 2 Chromosome 13

9. Ladder single cell MDA 600 bp 2 kb Embryon biobsy and single cell MDA 6 -8 hours of amplification

10. 1;2;3 4;5;6 7;8;9 10;11;12 13;14;15 16;17;18 19;20;21 22;X;Y Hellani A, Coskun S, Benkhalifa Met al Multiple displacement amplification on single cell and possible PGD applications.Mol Hum Reprod. 2004 Nov;10(11):847-52.

12. DNA Multi-Typing CELL LYSIS MULTIPLEX PCR AMPLIFICATION “NESTED” PCR AMPLIFICATION MUTATION & FRAGMENT ANALYSIS ddNTP PRIMER EXTENSION or “MINISEQUENCING” AUTOMATED SEQUENCING FLUORESCENT SIZING

13. Beta ThalassemiaCod.39 CTMethods Comparison Sequencing Analysis Minisequencing Normal Allele Mutated Allele Normal Allele Mutated Allele

14. PCR PRODUCT QUANTIFICATION NORMAL MALE KLINEFELTER Peak areas

15. Microarray for point mutation Analysis: Serial Genetics/ATL R&D RNA or DNA DNA probes immobilized preparation amplification reading interpretation A great number of parameters are detected simultaneously with microarrays

16. CFTR microarray: interpretation of results

17. SNP’s Single Genes Viability Genes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y Viable Embryo Trisomy 1 Monosomy 14 DNA Fingerprint CF Deletion Universal Microarray Platform Gardner et al

18. Affymetrix Arrays principle for transcriptome investigation 1 information unit 1,28cm 1 oligonucléotide  Transcriptomes Analysis Human Exon Array : ~1,1 .106 d’exons couverts par ~1,8 .106 PSR (Probeset regions). U133 Plus 2.0 Array : 47,000 transcripts quantified with minimum of 22 séquences (Probeset).

19. Experiment Total RNAs extraction quality control cDNA quality control Data Control Données contrôlées

20. Green colour means downregulated Red colour means upregulated

22. What is Proteomics? • Proteomics is the large-scale study of proteins, particularly their structures and functions. • Relatively little is known regarding the proteome of the human biological cell system • Unlike the genome, the proteome is dynamic constantly changing through its biochemical interactions with the genome and its response to internal and external stimuli

23. CHANGES IN THE CULTURE MEDIA INDICATIVE OF VIABILITY Production Uptake Lactate Glucose Ammonium Pyruvate Amino Acids Amino Acids Enzymes, eg LDH sHLA-G Other Sugars HOXA10 regulator Oxygen PAF Other Peptides & Factors µl drop of defined culture medium [Sakkas and Gardner, Curr. Opin. Obstet. Gynecol. 2005]

24. KEY EVENTS IN EMBYRO DEVELOPMENT Day 1 Day 3 Day 5 EMBRYONIC TRANSCRIPTION maternal mRNA Pyruvate Lactate Glucose

25. PROTEOMICSThe expression of an 8.5-kDa protein biomarker appears to be directly associated with ongoing human blastocyst development. Significant difference in expression [Katz-Jaffe et al. Fertil Steril 2006]

26. Metabolomics Metabolomics can be thought of as the interface between genomics and a functional phenotype The metabolome represents the final products of gene expression Assessed by investigating inventory of small molecule biomarkers (LMW)

27. What is Measured? • Functional Groups • CH • NH • OH • SH • C=C • Constituents • Albumin • Lactate • Pyruvate • Glutamate • Glucose • Clinically • How the embryo modifies its environment • Biologically • Changes in concentrations of:

28. Spent Culture Media Analyses Specific Targets (hypotheses-based) Utilization of media component Targeted secretion products Protein profiling Metabolite profiling Profile Analyses (systems-based)

29. Amino Acid Turnover and Implantation No pregnancy Pregnancy Brison et al ’04;Human Reprod 19:2319

30. Near Infra-Red Analyses of Media Each line is the profile for one embryo Seli et al ’07;Fertil Steril 88:1350-7

31. Viability ScoreMethodology Step 1: Single embryo culture Blank Step 2: Spectral analysis of media sample ratio against the blank Viability Score 0.6 0.2 Step 3: transfer embryo with highest Viabilityscore

32. Distribution of the Viability Score of Individual Embryos of the same Morphology: Clinical Trial: MOL BIO-EYLAU Morphology Grade A (very good) Grade B Grade C Grade D (poor) Day 3 Day 2 32 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1

33. The impact of the ViaTest score on selectingembryos of same morphology from the patient’s cohort: Clinical Trial. MOL BIO-EYLAU <0.207 50% 1 0.208 to 0.288 45% 0.289 to 0.354 40% 2 3 >0.355 35% 30% % Fetal Cardiac Activity Positive 25% 4 20% 15% 10% 5% 0% Grade A B C&D

34. Genomics • Proteomics • Metabolomics • Economics

35. Conclusions • Developments in genomics will serve to improve analysis of entire genome and facilitate genetic fingerprinting • Analysis of the secretome, plausibly combined with carbohydrate utilization and amino acid turnover, and a general analysis of metabolic footprint, will serve to form the base of new assays of human viability of any biological process. • Simplification of proteomics and metabolic analysis through microfluidic devices may serve as an economic methodology in clinical practice in the future.

36. EylauLaboratory/ Unilabs. Paris-Geneva Paul Cohen Bacrie Stéphanie Belloc Martine Dumont Anne Marie Junca Philippe Renard Pr Yyves Menezo Pr Alain Dalleac Gurgan CLINIC. IVF & Genetic Dept . Ankara. Turkey Pr T Gurgan Dr A Demirol T Sari