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OMES & OMICS in ARTOMES & OMICS in ART
Moncef Benkhalifa, Ph.D. RBMG. Moncef Benkhalifa, Ph.D. RBMG.
Reproductive Biology & GeneticsReproductive Biology & Genetics
Technical & Development DirectorTechnical & Development Director
UNILABS. France UNILABS. France
4. Pré-hybridationHybridation
LavageDécontamination
5. Scan des données – Analyse des résultats
1. Prélèvement de l’échantillonOrganismes / tissues / cellules
2. WGA, RTPCR 3. Amplification (PCR) et marquage fluorescent
The “Omes and Omics”
From James Poscillico
X der(X)
A
D
B
E
X dupXq
21.1
X
21.1
dupXq
25
25
C
q21
q25
0.8 1.2
Tachdjian G, Aboura A, Benkhalifa M et al De novo interstitial direct duplication of Xq21.1q25 associated with skewed X-inactivation pattern.
: Prenatal Diagnosis, October 2005
Custom Profiling: Route to Drug Custom Profiling: Route to Drug Discovery Discovery Application in ovarian cancerApplication in ovarian cancer
Sample 2Sample 2
Chromosome Chromosome segment 8qsegment 8q
Chromosome 13Chromosome 13
Embryon biobsy and single cell MDA
2 kb
600 bp
Lad
der
single cell MDA
6 -8 hours of amplification6 -8 hours of amplification
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 M et al Multiple displacement amplification on single cell and possible PGD applications.
Mol Hum Reprod. 2004 Nov;10(11):847-52.
DNA Multi-Typing DNA Multi-Typing CELL LYSISCELL LYSIS
MULTIPLEX PCR AMPLIFICATION MULTIPLEX PCR AMPLIFICATION
FLUORESCENT SIZING
FLUORESCENT SIZING
AUTOMATED SEQUENCING
AUTOMATED SEQUENCING
ddNTP PRIMER EXTENSION or
“MINISEQUENCING
”
ddNTP PRIMER EXTENSION or
“MINISEQUENCING
”
“NESTED” PCR AMPLIFICATION “NESTED” PCR AMPLIFICATION
MUTATION & FRAGMENT ANALYSIS
MUTATION & FRAGMENT ANALYSIS
Sequencing AnalysisSequencing Analysis MinisequencMinisequencinging
Beta Thalassemia Cod.39 CBeta Thalassemia Cod.39 CTTMethods ComparisonMethods Comparison
NormaNormal l
AlleleAllele
MutateMutated d
AlleleAllele
NormaNormal l
AlleleAllele
MutateMutated d
AlleleAllele
PCR PCR PRODUCT PRODUCT QUANTIFICATIONQUANTIFICATION
NORMAL MALENORMAL MALE KLINEFELTERKLINEFELTER
Peak Peak areasareas
A great number of parameters are detected A great number of parameters are detected simultaneously with microarrayssimultaneously with microarrays
preparation
amplification
reading
interpretation
DNA probes immobilized
RNA or DNA
Microarray for point mutation Analysis: Serial Microarray for point mutation Analysis: Serial Genetics/ATL R&DGenetics/ATL R&D
CFTR microarray: interpretation of results
Universal Microarray PlatformUniversal Microarray Platform
Trisomy 1 Monosomy 14
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
Single Genes
CF Deletion
SNP’s
DNA Fingerprint
Viability Genes
Viable Embryo
Gardner et al
Affymetrix Arrays principle for Affymetrix Arrays principle for transcriptome investigation transcriptome investigation
1,28cm
1 oligonucléotide
1 information
unit
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).
Données contrôlées
ExperimentExperiment
cDNA qualitycontrol
Data Control
Total RNAs extraction quality control
Green colour means downregulated
Red colour means upregulated
What is Proteomics?What is Proteomics?
Proteomics is the large-scale study of Proteomics is the large-scale study of proteins, particularly their structures and proteins, particularly their structures and functions. functions.
Relatively little is known regarding the Relatively little is known regarding the proteome of the human biological cell proteome of the human biological cell systemsystem
Unlike the genome, the proteome is Unlike the genome, the proteome is dynamic constantly changing through its dynamic constantly changing through its biochemical interactions with the genome biochemical interactions with the genome and its response to internal and external and its response to internal and external stimuli stimuli
UptakeUptake ProductionProductionGlucose
Pyruvate
Oxygen
Other Sugars
Lactate
Ammonium
Enzymes, eg LDH
sHLA-G
Other Peptides &
Factors
Amino Acids
µl drop of defined culture medium
HOXA10 regulator
PAF
Amino Acids
[Sakkas and Gardner, Curr. Opin. Obstet. Gynecol. 2005]
CHANGES IN THE CULTURE MEDIA INDICATIVE OF VIABILITY
EMBRYONIC TRANSCRIPTIONmaternal mRNA
GlucosePyruvateLactate
KEY EVENTS IN EMBYRO DEVELOPMENTKEY EVENTS IN EMBYRO DEVELOPMENT
Day 1 Day 3 Day 5
PROTEOMICSPROTEOMICSThe expression of an 8.5-kDa protein The expression of an 8.5-kDa protein biomarker appears to be directly biomarker appears to be directly associated with ongoing human blastocyst associated with ongoing human blastocyst development.development.
[Katz-Jaffe et al. Fertil Steril 2006]
Significant difference in expression
MetabolomicsMetabolomics Metabolomics can be thought of as Metabolomics can be thought of as
the interface between genomics and the interface between genomics and a functional phenotypea functional phenotype
The metabolome represents the final The metabolome represents the final products of gene expressionproducts of gene expression
Assessed by investigating inventory Assessed by investigating inventory of small molecule biomarkers (LMW)of small molecule biomarkers (LMW)
What is Measured?What is Measured?
ClinicallyClinically– How the embryo modifies its How the embryo modifies its
environmentenvironment
BiologicallyBiologically– Changes in concentrations of:Changes in concentrations of:
Constituents•Albumin•Lactate•Pyruvate•Glutamate•Glucose
Functional Groups•CH•NH•OH•SH•C=C
Spent Culture Media Spent Culture Media AnalysesAnalyses
Specific TargetsSpecific Targets(hypotheses-based)(hypotheses-based)
Profile AnalysesProfile Analyses(systems-based)(systems-based)
Utilization of media componentTargeted secretion products
Protein profilingMetabolite profiling
Brison et al ’04;Human Reprod 19:2319
Amino Acid Turnover and ImplantationAmino Acid Turnover and Implantation
No pregnancy Pregnancy
Near Infra-Red Analyses of MediaNear Infra-Red Analyses of Media
Seli et al ’07;Fertil Steril 88:1350-7
Each line is the profile for one embryo
0.6 0.2Step 3: transfer embryo with highest Viability score
Viability Score
Viability Score Methodology
Blank
Step 1: Single embryo culture
Step 2: Spectral analysis of media sample ratioagainst the blank
32
Distribution of the Viability Score of Individual Embryos of the same Morphology: Clinical Trial: MOL BIO-EYLAU
-0.4
-0.2
0 0.2
0.4
0.6
0.8
1
Grade A(very good)
Grade C
Grade D(poor)
Grade B
Morphology
Day 3Day 2
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
B C&D
The impact of the ViaTest score on selectingThe impact of the ViaTest score on selectingembryos of same morphology from embryos of same morphology from
the patient’s cohort: Clinical Trial. MOL BIO-EYLAUthe patient’s cohort: Clinical Trial. MOL BIO-EYLAU
% F
eta
l C
ard
iac
Act
ivit
y P
osi
tive
1
23
4
<0.2070.208 to 0.2880.289 to 0.354
>0.355
Grade A
GenomicsGenomicsProteomicsProteomicsMetabolomicsMetabolomicsEconomicsEconomics
ConclusionsConclusions Developments in genomics will serve to improve Developments in genomics will serve to improve
analysis of entire genome and facilitate genetic analysis of entire genome and facilitate genetic fingerprinting fingerprinting
Analysis of the secretome, plausibly combined with Analysis of the secretome, plausibly combined with carbohydrate utilization and amino acid turnover, and carbohydrate utilization and amino acid turnover, and a general analysis of metabolic footprint, will serve to a general analysis of metabolic footprint, will serve to form the base of new assays of human viability of any form the base of new assays of human viability of any biological process.biological process.
Simplification of proteomics and metabolic analysis Simplification of proteomics and metabolic analysis through microfluidic devices may serve as an economic through microfluidic devices may serve as an economic methodology in clinical practice in the future.methodology in clinical practice in the future.
EylauLaboratory/ EylauLaboratory/ Unilabs.Unilabs.
Paris-Geneva Paris-Geneva
Paul Cohen BacriePaul Cohen BacrieStéphanie BellocStéphanie BellocMartine DumontMartine DumontAnne Marie JuncaAnne Marie JuncaPhilippe RenardPhilippe Renard
Pr Yyves MenezoPr Yyves MenezoPr Alain DalleacPr Alain Dalleac
Gurgan CLINIC. IVF & Genetic
Dept . Ankara. Turkey
Pr T GurganDr A Demirol
T Sari