Diapositive 1

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Transcript Diapositive 1

OMES & OMICS in ART
Moncef Benkhalifa, Ph.D. RBMG.
Reproductive Biology & Genetics
Technical & Development Director
UNILABS. France
1. Prélèvement de l’échantillon
Organismes / tissues / cellules
4. Pré-hybridation
Hybridation
Lavage
Décontamination
2. WGA, RTPCR
3. Amplification (PCR)
et marquage fluorescent
5. Scan des données – Analyse des résultats
The “Omes and Omics”
From James Poscillico
A
B
q21
q25
A
0.8
der(X)
X
1.2
C
B
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.
: Prenatal Diagnosis, October 2005
Custom Profiling: Route to Drug Discovery
Application in ovarian cancer
Chromosome
segment 8q
Sample 2
Chromosome 13
Ladder
Embryon biobsy and single cell MDA
2 kb
600 bp
single cell MDA
6 -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
CELL LYSIS
MULTIPLEX PCR AMPLIFICATION
“NESTED” PCR AMPLIFICATION
MUTATION &
FRAGMENT
ANALYSIS
AUTOMATED
SEQUENCING
FLUORESCENT
SIZING
ddNTP PRIMER
EXTENSION or
“MINISEQUENCING”
Beta Thalassemia Cod.39 CT
Methods Comparison
Sequencing Analysis
Minisequencing
Normal Mutated
Allele
Allele
Normal
Allele
Mutated
Allele
PCR PRODUCT QUANTIFICATION
NORMAL MALE
Peak
areas
KLINEFELTER
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
CFTR microarray: interpretation of results
Universal Microarray Platform
1 2
Trisomy 1
3 4 5 6
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y Viability Genes SNP’s
Monosomy 14
Viable
Embryo
DNA
Fingerprint
Single Genes
CF Deletion
Gardner et al
Affymetrix Arrays principle for transcriptome investigation
1 information
unit
1,28cm
 Transcriptomes Analysis
1 oligonucléotide
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).
Experiment
Total RNAs
extraction
quality control
cDNA
quality
control
Données
contrôlées
Data
Control
Green colour
means
downregulated
Red colour
means
upregulated
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
CHANGES IN THE CULTURE MEDIA INDICATIVE OF
VIABILITY
Uptake
Lactate
Glucose
Production
Ammonium
Pyruvate
Amino Acids
Amino Acids
Enzymes, eg LDH
sHLA-G
Other Sugars
HOXA10 regulator
Oxygen
PAF
µl drop of defined culture
medium
Other Peptides
&
Factors
[Sakkas and Gardner, Curr. Opin. Obstet. Gynecol. 2005]
KEY EVENTS IN EMBYRO DEVELOPMENT
Day 1
Day 3
maternal mRNA
Pyruvate
Lactate
Day 5
EMBRYONIC
TRANSCRIPTION
Glucose
PROTEOMICS
The 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]
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)
What is Measured?

Clinically
– How the embryo modifies its environment

Biologically
– Changes in concentrations of:
Functional Groups
•CH
•NH
•OH
•SH
•C=C
Constituents
•Albumin
•Lactate
•Pyruvate
•Glutamate
•Glucose
Spent Culture Media Analyses
Specific Targets
(hypotheses-based)
Utilization of media component
Targeted secretion products
Protein profiling
Metabolite profiling
Profile Analyses
(systems-based)
Amino Acid Turnover and Implantation
No pregnancy
Pregnancy
Brison et al ’04;Human Reprod 19:2319
Near Infra-Red Analyses of Media
Each line is the profile for one embryo
Seli et al ’07;Fertil Steril 88:1350-7
Viability Score Methodology
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 Viability score
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
Day 3
Day 2
(poor)
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
3
2
The impact of the ViaTest score on selecting
embryos of same morphology from
the patient’s cohort: Clinical Trial. MOL BIO-EYLAU
% Fetal Cardiac Activity Positive
50%
<0.207
1
45%
40%
3
35%
0.208 to
0.288
0.289 to
0.354
2
>0.355
30%
25%
20%
4
15%
10%
5%
0%
Grade A
B
C&D
Genomics
Proteomics
Metabolomics
Economics
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.
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