New tools to benchmark the best New tools to benchmark the best stimulation modalilities: stimulation modalilities:

Focus on transcriptomis Focus on transcriptomis

Dr Moncef Benkhalifa, Ph.D. RBMG. Dr Moncef Benkhalifa, Ph.D. RBMG. Reproductive Biology & GeneticsReproductive Biology & Genetics

Technical & Development DirectorTechnical & Development Director

Eylau Laboratory. UNILABS Eylau Laboratory. UNILABS PARIS-GENEVAPARIS-GENEVA

Questions ???Effect of FSH regimens and Ovarian stimulation

on Oocye selection and growth Index

Oocyte Pool depletionCumulus oocyte complex maturation & Competence

Emryo quality & Clinical Pregnancy

0ocyte quality after ovarian stimulation can be variable mainly at maturation (genomic

maturation) and competence (epigenetic and transcripts quality) levels to ensure the early

development until the entire genome expression at day 3 embryo.

Correct messaging from cumulus cells is mandatory for the completion of oocyte meiosis

Factors affecting oocyte competence The study of the transcriptome of cumulus cells and dialogue with oocyte can contribute to the elucidation of oocyte maturation and competence mechanism.

Oocyte maturation and Oocyte maturation and competencecompetenceHow can we explain it?How can we explain it?

Oocyte maturation is accompanied by a complex Oocyte maturation is accompanied by a complex network of translational activation and repression network of translational activation and repression of dormant maternal mRNAsof dormant maternal mRNAs

These maternal mRNAs drive the oocyte's reentry These maternal mRNAs drive the oocyte's reentry into meiosis and control the rate of mitosis during into meiosis and control the rate of mitosis during the early cleavage divisions of the embryothe early cleavage divisions of the embryo. .

The first step in translational activation of stored The first step in translational activation of stored maternally inherited mRNAs is cytoplasmic maternally inherited mRNAs is cytoplasmic extension of their poly(A) tails: translational extension of their poly(A) tails: translational activationactivation

The cytoplasmic polyadenylation element (CPE) The cytoplasmic polyadenylation element (CPE) (consensus sequence UUUUUA1–2U), is necessary (consensus sequence UUUUUA1–2U), is necessary

for cytoplasmic polyadenylationfor cytoplasmic polyadenylation

The Development of Nano Technology (NT)The Development of Nano Technology (NT)opened a new area in clinical pathology and medical care opened a new area in clinical pathology and medical care

practice practice

The Nucleic Acid Chip Technology (NACT) will be a new The Nucleic Acid Chip Technology (NACT) will be a new potential for genomic screening , analysis and drug effect potential for genomic screening , analysis and drug effect

discoverydiscovery

Exemple: Exemple: Microarrays application in cumulus cells and Oocyte Microarrays application in cumulus cells and Oocyte Transcriptome profilingTranscriptome profiling

The human cumulus oocytecomplex gene expression profile The human cumulus oocytecomplex gene expression profile Assou SAssou S et al Hum Reprod 2006 et al Hum Reprod 2006

Cumulus gene expression as a predictor of human oocyte Cumulus gene expression as a predictor of human oocyte fertilisation, embryo development and competence to fertilisation, embryo development and competence to

establish a pregnancy.establish a pregnancy.Anderson RA et al. Reproduction. 2009 Anderson RA et al. Reproduction. 2009

Gene expression microarray profiles of cumulus cells in lean Gene expression microarray profiles of cumulus cells in lean and overweight-obese polycystic ovary syndrome patients.and overweight-obese polycystic ovary syndrome patients.

Kenigsberg S et al. Mol Hum Reprod. 2009 Kenigsberg S et al. Mol Hum Reprod. 2009

Superoxide dismutase expression in human cumulus Superoxide dismutase expression in human cumulus oophorus cellsoophorus cells

Matos L L et al . Mol Hum Reprod. 2009 et al . Mol Hum Reprod. 2009

002

2001

2002

2002

In this study we investigate the effect on the whole transcriptome of cumulus cells following follicular stimulation with regimens differing in their FSH isoforms composition triggering

hCG, 10’000 IU

Standard longDown regulation

Puregon Fostimon

Fostiom or Puregon

Day 6

225 IU

Stimulation

Least acidic (Puregon)vsSequential (Fostimon + Puregon)vsAcidic (Fostimon)

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

Log Probe Intensity Log Probe Intensity

ARN UniverselFostimon +

Puregon Purego

n Fostim

on

Green colour means downregulated

Red colour means upregulated

Involved processes mRNA

transcription Protein targeting

and localization Endocytosis Intracellular

signaling cascade Nucleoside,

nucleotide and nucleic acid metabolism

mRNA transcription regulation

Ion transport

Biological processes

It’s clearly showen that, even if the pathaway is not affected , PP + Fostimon is down regulating whereas Puregon only is up regulating

Biological processes

Conclusions

1.Better effect of Fostimon +Puregon Vs fostimon.

2.Better effect of Fostimon Vs Puregon

3.No significant difference between Fostimon + Puregon Vs Puregon

Biological processes

Deacetylase

Homeobox transcription factor

Acting binding cytoskeletal protein

G-Protein

Reductase

Oxidoreductase

Molecular functionInvolved Function

It’s clearly showen that even if the pathaway is not affected Fostimon is down regulating whereas Puregon is up regulating

Molecular Function

Molecular function

Conclusions

1.Better affect of Fostimon +

Puregon Vs Fostimon 2.Better Effect of Fostimon +

Puregon Vs Puregon3.No difference between Fostimon

Vs Puregon

Based on the comparison of the global effect at biological and molecular levels and on the clinical outcomes we ranked the efficacy of the 3 protocols as follows:

Summary

1.Fostimon + Puregon,

2.Fostimon 3.Puregon

Conclusions

More molecular investigations or the metabolomics of

different fluids and culture medias need to be done to

elucidate better the effect of different FSH glycoforms and Mix in the complex dialogue

between the oocyte and cumulus cells during the

follicular and luteal phases.

Pr Gurgan TimurPr Gurgan TimurDr Aygul Demirol Dr Aygul Demirol

& & the embryology Teamthe embryology Team

Women’s Health Women’s Health Clinic. Clinic.

IVF & Genetics Centre. IVF & Genetics Centre. Ankara. Ankara. TurkeyTurkey

Pierre Delagrange Shili OnsJilani Nej

MontJean DebbieDavid Gentien

Pr Yves menezo

ATL R&D. Reproductive Biology

& Genetics Laboratory. Paris

We Would Like to thank IBSA for the financial Support And specially

Dr Maurizio Dattilo