ART: Experimental bases for improving outcomes for the benefit of patientsABSTRACT BOOK

Aims

The knowledge on reproductive function dramatically

improved in recent years, thanks to the research aimed

to ameliorate the outcomes of assisted reproductive

techniques. With the contribution of genetics and

metabolomics, many sources of results variability have

been defined and on other complex aspects the studies are

ongoing. The aim of this edition of the Annual Conference

in Reproductive Medicine is to review these scientific

attainments, starting from the physiology of follicle growth to

the methods for studying embryos’ quality, translating this

information in improved cycles planning and management,

to optimize outcomes.

Learning Objectives

This Conference will offer to participants:

• An exhaustive overview of new achievements on

reproductive function physiology

• Updated information on reproductive function in

stimulated cycles

• Recent proposal to improve oocytes and embryo

quality evaluation

• Practical cues to translate scientific achievements into

outcome improvements

Target Audience

This program is targeted to clinicians and scientists working

on Assisted Reproduction Techniques.

Accreditation

Serono Symposia International Foundation has submitted

this program ART: Experimental Bases for Improving

Outcomes for the Benefit of Patients (Istanbul, Turkey –

February 19 - 20, 2010) for accreditation by the European

Accreditation Council for Continuing Medical Education

(EACCME).

All Serono Symposia International Foundation programs are organized solely to promote the exchange and

dissemination of scientific and medical information. No forms of promotional activities are permitted. There may be

presentations discussing investigational uses of various products. These views are the responsibility of the named

speakers, and do not represent an endorsement or recommendation on the part of Serono Symposia International

Foundation. This program is made possible thanks to the unrestricted Educational grant received from: Centre

d’Esclerosi Multiple de Catalunya, Vall d’Hebron University Hospital, ComtecMed, Congrex Sweden, Congrex

Switzerland, Cryo-Save, Datanalysis, Fundación IVI, ISFP International Society for Fertility Preservation, ISMH

International Society of Men’s Health, K.I.T.E., Merck Serono, Meridiano Viaggi e Turismo, Ministry of Health of the State

of Israel, University of Catania.

Venue

CONRAD ISTANBUL HOTEL

Barbaros Blv.Yildiz Cad. Besiktas

Istanbul, 34353, Turkey

http://conradhotels1.hilton.com

Language

The official language of this Conference will be English.

Location

Istanbul, one of the great historical cities of the world, is

the only city in the world located upon two continents with

one arm reaching out to Asia and the other arm to Europe.

Through the city’s heart runs the Bosphorus sea channel,

which reaches north to the Black Sea and south to the

Marmara Sea. The city is divided into three parts - the old

city, the new city and the Asiatic side.

The old town and new town, which encompass the greater

part of the city, are located in Europe. In the old city the

majority of the Istanbul historical monuments can be

found. Amongst the most famous of these monuments

are St. Sophia, Blue Mosque, Hippodrome, The Wall

of Theodosius, Topkapi Palace, Suleymaniye Mosque,

Underground Cistern, St. Eirene, St. Saviour in Chora,

Grand Bazaar and the Spice Market. The new part of

the city displays a modern impression of Istanbul with its

skyscrapers and luxurious shopping centers, but, at the

same time, one is reminded of Istanbul’s history by the

wooden houses bordering the Bosphorus and historical

sites such as Dolmabahce Palace, Ciragan Palace, Galata

Tower, Nusretiye Mosque, Clock Tower, Yildiz Palace, Umeli

Fortress, etc.

The Asiatic side of the city is a mixture of modern houses,

lovely wooden villas, as well as historical sites, such as

Anadolu Fortress, Beylerbeyi Palace, Kucuksu Kasri, Hidiv

Kasri, Leanders Tower, Cinili Mosque, etc.

Scientific Organizer

Georg Griesinger

Department of Obstetrics and Gynecology

University Hospital of Schleswig-Holstein

Lübeck, Germany

Local Organizer

Semra Kahraman

Istanbul Memorial Hospital

ART and Genetics Center

Istanbul, Turkey

Scientific Committee

Timur Gurgan

Department of Obstetrics and Gynecology

Hacettepe University, School of Medicine

Gurgan Clinic

Ankara, Turkey

Erol Tavmergen

IVF Centre

Ege University, School of Medicine

Bornova-Izmir, Turkey

Bülent Urman

Assisted Reproduction Unit

American Hospital of Istanbul

Istanbul, Turkey

Hakan Yarali

Department of Obstetrics and Gynecology

Hacettepe University, School of Medicine

Anatolia IVF Center Ankara, Turkey

List of faculty members

Carlo Alviggi

Department of Obstetrics and Gynecology

IVF Unit, University of Naples “Federico II”

Naples, Italy

Claus Yding Andersen

Laboratory of Reproductive Biology

University Hospital of Copenhagen

Copenhagen, Denmark

Aydin Arici

Department of Obstetrics, Gynecology &

Reproductive Sciences, Yale University School of Medicine

New Haven, CT, USA

Mario Ascoli

Department of Pharmacology

The University of Iowa

Iowa City, IA, USA

Remi Dumollard

Laboratoire de Biologie du Développement

Station Zoologique

Villefranche sur Mer, France

Robert Fischer

Fertility Center

Hamburg, Germany

Tamara Garrido

Fundacion Instituto Valenciano de Infertilidad (IVI)

Investigation Department

Valencia, Spain

Georg Griesinger

Department of Obstetrics and Gynecology

University Hospital of Schleswig-Holstein

Lübeck, Germany

23

Marie Louise Grøndahl

The Fertility Clinic

Hvidovre Hospital

Copenhagen University Hospital

Hvidovre, Denmark

Timur Gurgan

Department of Obstetrics and Gynecology

Hacettepe University School of Medicine

Gurgan Clinic

Ankara, Turkey

Samir Hamamah

ART/PGD Division

Department of Reproductive Medicine and Biology

Hôpital Arnaud de Villeneuve

Montpellier, France

José A. Horcajadas

Instituto Valenciano de Infertilidad (IVI)

University of Valencia

Valencia, Spain

Semra Kahraman

Istanbul Memorial Hospital

ART and Genetics Center

Instanbul, Turkey

William G. Kearns

Shady Grove Center for Preimplantation Genetics,

Rockville, Maryland, USA

Nick S. Macklon

Department of Obstetrics and Gynaecology

Division of Developmental Origins of Health and Disease

University of Southampton, UK

Scientific Secretariat

Serono Symposia International Foundation

Salita di San Nicola da Tolentino, 1/b

00187 Rome, Italy

Senior Project Manager: Francesca Caputo

Tel.: +39-06-420 413 568

Fax: +39-06-420 413 677

E-mail: info@seronosymposia.org

Serono Symposia International Foundation is a Swiss

Foundation with headquarters in 14, rue du Rhône,

1204 Genève, Switzerland

Organizing Secretariat

Meridiano Congress International

Via Mentana, 2/B - 00185 Rome - Italy

Congress Coordinator: Federica Russetti

Phone: +39-06-88595 209

Fax: +39-06-88595 234

E-mail: f.russetti@meridiano.it

Antonis Makrigiannakis

Laboratory of Human Reproduction

Department of Obstetrics and Gynecology

Medical School, University of Crete

Heraklion, Greece

Emre Seli, M.D.

Division of Reproductive Endocrinology and Infertility

Oocyte Donation and Gestational Surrogacy Program

Department of Obstetrics, Gynecology, and Reproductive

Sciences, Yale University School of Medicine

New Haven, CT, USA

Marc-André Sirard

Research Centre of Reproduction Biology

Department of Animal Sciences

Laval University

Ste-Foy Québec, Canada

Erol Tavmergen

IVF Centre

Ege University, School of Medicine

Bornova-Izmir, Turkey

Bülent Urman

Assisted Reproduction Unit

American Hospital of Istanbul

Istanbul, Turkey

Hakan Yarali

Department of Obstetrics and Gynecology

Hacettepe University

Anatolia IVF Center

Ankara, Turkey

45

Session II: Clinical Pharmacology of Ovarian

Stimulation

Chairman: Timur Gurgan (Turkey)

14:45 L7: Granulosa: gene expression and function

Marie Louise Grøndahl (Denmark)

15:15 L8: Follicular cells transcriptomes and oocyte quality

Samir Hamamah (France)

15:45 Coffee Break

16:15 L9: Endometrium gene expression profile in

stimulated cycles

José A. Horcajadas (Spain)

16:45 Discussion

17:30 End of the day

Saturday February 20, 2010

Session III: Oocyte & Embryo Selection: from

Genetics to Metabolomics

Chairmen: Robert Fischer (Germany)

Hakan Yarali (Turkey)

08:30 L10: Genomics: novel methodologies and objectives

William G. Kearns (USA)

09:00 L11: Metabolomics: ready to be used in practice?

Emre Seli (USA)

09:30 L12: Embryo proteomics: a new tool for screening

Tamara Garrido (Spain)

Round Table

Chairmen: Robert Fischer (Germany)

Bülent Urman (Turkey)

10:00 Genomics vs proteomics vs metabolomics:

pros and cons

William G. Kearns (USA)

Emre Seli (USA)

Tamara Garrido (Spain)

10:30 Coffee break

Session IV- Translational Research to Clinical

Practice

Chairmen: Georg Griesinger (Germany),

Erol Tavmergen (Turkey)

11:00 L13: Ovarian stimulation

Carlo Alviggi (Italy)

11:30 L14: Oocyte selection

Marc-André Sirard (Canada)

12:00 L15: Embryo selection

Emre Seli (USA)

12:30 L16: Implantation

Nick S. Macklon (UK)

13:00 End of the Conference and Closing Lunch

Friday February 19, 2010

09:00 Welcome on behalf of Serono Symposia

International Foundation (SSIF)

Robert Fischer (Germany)

09:10 Introduction & Opening

Georg Griesinger (Germany)

Semra Kahraman (Turkey)

Session I: Physiology of Human Reproduction:

New Insights

Chairmen: Georg Griesinger (Germany)

Semra Kahraman (Turkey)

09:30 L1: From follicle recruitment to ovulation: controlling

factors and sources of variability

Claus Yding Andersen (Denmark)

10:00 L2: Gonadotrophins receptors: regulation of their

expression and functions

Mario Ascoli (USA)

10:30 L3: Physiology and metabolism of early mammalian

embryos

Remi Dumollard (France)

11:00 Coffee break

11:30 L4: Intracellular pathways: from stimula to final effects

Claus Yding Andersen (Denmark)

12:00 L5: Physiology of implantation: how gene expression

influences receptivity

Aydin Arici (USA)

12:30 L6: The embryo invasion phase: immune mechanisms

Antonis Makrigiannakis (Greece)

13:00 Discussion

13:30 Lunch

67

Disclosure of Faculty Relationships

Serono Symposia International Foundation adheres to guidelines of the European Accreditation Council for

Continuing Medical Education (EACCME) and all other professional organizations, as applicable, which state that

programs awarding continuing education credits must be balanced, independent, objective, and scientifically

rigorous. Investigative and other uses for pharmaceutical agents, medical devices, and other products (other than

those uses indicated in approved product labeling/package insert for the product) may be presented in the program

(which may reflect clinical experience, the professional literature or other clinical sources known to the presenter).

We ask all presenters to provide participants with information about relationships with pharmaceutical or medical

equipment companies that may have relevance to their lectures. This policy is not intended to exclude faculty

who have relationships with such companies; it is only intended to inform participants of any potential conflicts

so participants may form their own judgments, based on full disclosure of the facts. Further, all opinions and

recommendations presented during the program and all program-related materials neither imply an endorsement,

nor a recommendation, on the part of Serono Symposia International Foundation. All presentations solely represent

the independent views of the presenters/authors.

The following faculty provided information regarding significant commercial relationships and/or discussions of

investigational or non-EMEA/FDA approved (off-label) uses of drugs:

Carlo Alviggi: Declared no potential confict of interest.

Claus Yding Andersen: Declared no potential conflict of interest.

Aydin Arici: Declare receipt of grants from MSD Serono and Ortho-McNeil.

Mario Ascoli: Declared receipt of grants and contracts from NIH.

Remi Dumollard: Declared no potential confict of interest.

Robert Fischer: Declared receipt of honoraria or consultation fees from Serono Symposia International Foundation and to be

a member of Serono Symposia International Foundation Scientific Committee.

Georg Griesinger: Declared receipt of grants and contracts from Merck Serono, Ferring, Schering-Plough, IBSA, Kade-

Besins and honoraria or consultation fees from Merck Serono, Ferring, Schering-Plough, IBSA, Kade-Besins.

Samir Hamamah: Declared receipt of grants or contracts from Ferring and honoraria or consultation fees from

Ferring. Prof. Hamamah also disclosed that his presentation includes the use of off-labelled or otherwise non-

approved use of product.

Josè A. Horcajadas: Declared no potential conflict of interest.

Semra Kahraman: Declared no potential conflict of interest.

William G. Kearns: Declared receipt of grants and contracts from Merck Serono and honoraria or consultation fees

from Merck Serono. Declared to have participation in speaker’s bureau sponsored by Merck Serono.

N.S. Macklon: Declared receipt of research grants from Merck Serono, Ferring, Schering-Plough and Anecova and

honoraria or consultation fees from Merck Serono, Schering-Plough and Anecova.

Antonis Makrigiannakis: Declared no potential conflict of interest.

Emre Seli: Declared receipt of honoraria or consultation fees from Molecular Biometrics, Inc. Declared to be a member of

Molecular Biometrics, Inc. advisory board, board of directors or other similar group. He also declared to be a stakeholder in

Molecular Biometrics, Inc. company (including such things as stock ownership, or options to buy, own, or have applied for

patents related to a company’s product, receive royalties for previous activities, employment, consultation services, etc.).

Marc-André Sirard: Declared receipt of honoraria or consultation fees from EMD Serono Canada Inc. for a scientific

presentation.

Bulent Urman: Declared no potential conflict of interest.

Hakan Yarali: Declared no potential confict of interest.

The following faculty have provided no information regarding significant relationship with commercial supporters and/or

discussion of investigational or non-EMEA/FDA approved (off-label) uses of drugs as of January 27, 2010

Tamara Garrido

Marie Louise Grøndahl

Timur Gurgan

Erol Tavmergen

89

L1:From follicle recruitment to ovulation:

controlling factors and sources of variability

Claus Yding Andersen

Laboratory of Reproductive Biology, University Hospital of Copenhagen, Copenhagen, Denmark

A succession of paracrine and endocrine signals direct the initial primordial cell recruitment and the subsequent maturation of the follicles

through to ovulation. The gonadotrophins, LH and FSH, are known to induce cellular proliferation and differentiation, and steroidogenesis in

the ovary. Under their control, mitotically active, oestrogen-secreting immature granulosa cells differentiate into non-dividing, progesterone-

secreting luteinized granulosa cells.

Initially, FSH acts on the immature early antral follicles, via the FSH receptor, stimulating the expression of both aromatase and the LH

cell surface receptor. This allows LH to act on the cells, causing luteinization and progesterone production through the intracellular

actions of the adenylyl cyclase cAMP pathway, the PKC inositol phosphate pathway, the MAPK cascade and ERK1/2. Importantly,

the time period within which LH can produce its effects is finite, leading to the concept of the LH window.

It has, however, proven difficult to compare the responses to LH of the granulosa and theca cells because the cells must first be stimulated

by FSH to induce expression of the LH receptors. Structural features in the stimulated LH receptor responsible for the cAMP-induced

signals, believed to be the cornerstone of steroidogenesis, may be different from the features that mediate the inositol responses. In

addition, it has been suggested that the different effects observed following FSH or LH stimulation may be related to the changes in

densities of their respective receptors during the menstrual cycle. Thus, it is uncertain whether any differences in the responses of granulosa

cells to FSH or LH receptor activation are due to differences in intracellular signalling or development-dependant, FSH-mediated changes in

the expression of steroidogenic enzymes and other regulatory proteins.

L2: Gonadotrophins receptors:

regulation of their expression and functions

Mario Ascoli

Department of Pharmacology, Carver College of Medicine, The University of Iowa, Iowa City, IA, USA

Using the expression of Cyp19a1 as a marker for the adenovirus-mediated expression of the recombinant gonadotrophin

receptors (LHR and FSHR) in immature rat granulosa cells, we showed that the actions of LH and FSH are the same, but

depend on the densities of the expressed receptors. Our data show that at low LHR or FSHR densities, hCG and FSH only

induce cAMP accumulation, a transient, but fast (

L4: Intracellular pathways: from stimula to final effects

Claus Yding Andersen

Laboratory of Reproductive Biology, University Hospital of Copenhagen, Copenhagen, Denmark

LH and hCG share a common receptor. The LH/hCG receptor belongs to the glycoprotein subfamily of the rhodopsin ß2 adrenergic

receptor-like family A of G-protein-coupled receptors. Stimulation of this receptor results in the activation of adenylate cyclase, causing

an increase in the intracellular levels of cAMP and the subsequent stimulation of steroidogenesis in the preovulatory follicle and the

corpus luteum.

The expression of the LH/hCG receptor on the cell surface varies during different physiological states of the ovary throughout

the menstrual cycle. The receptors are up-regulated in response to FSH stimulation, with transient receptor down-regulation

occurring in response to the endogenous preovulatory LH surge or by the administration of a pharmacological dose of hCG.

This transient down-regulation of the LH/hCG receptor occurs in parallel with the disappearance of its mRNA transcripts.

Indeed, evidence suggests that the expression of the LH/hCG receptor mRNA (along with other ß2 adrenergic receptors,

c-fos and c-myc) during follicle growth, ovulation and luteinization is highly regulated, at least in part, by intercellular mRNA

degradation. In addition to mRNA degradation, receptor expression is governed by various trans-acting proteins that can

interact with the LH/hCG receptor mRNA. In particular, the LH binding protein (LHBP) attaches to the coding region of the LH/

hCG receptor mRNA with high affinity, enabling it to specifically reduce the translation of the LH/hCG receptor mRNA into fully

functional cell surface receptors.

L3: Physiology and metabolism of early mammalian embryos

Rémi Dumollard1, John Carroll2, Michael Duchen2 and Karl Swann3

1Laboratoire de Biologie du Développement, UMR 7009, Station Zoologique, Villefranche sur Mer, France2Department of Physiology, University College London, London, UK3Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Cardiff, UK

The metabolism of mammalian oocytes and early embryos is transformed during ovulation and at fertilization. From ovulation

up to implantation, the oocyte and embryo rely solely on internal metabolism to supply all their energy demands and to set

the intracellular redox state. At fertilization, sperm entry triggers intracellular calcium signals that stimulate both embryonic

development and the ATP production required to support its development. ATP production in the oocyte and early embryo is

provided by the metabolism of exogenous substrates through mitochondrial oxidative phosphorylation. However, embryos are

extremely sensitive to the deleterious ROS generated during metabolism.

By monitoring cytosolic and mitochondrial calcium and ATP levels, together with the intracellular redox state, we assessed the

metabolism of single oocytes or embryos at fertilization and during pre-implantation development. We found that ATP supply and

demand are closely coupled in early embryos to allow for the minimal stimulation of mitochondrial oxidative metabolism in order to

minimize ROS production. Furthermore, we characterized the metabolism of the major metabolic substrates present in culture media

and found that embryos use very specific substrates, with pyruvate being the main source of energy production. Conversely, glucose

is poorly metabolized and lactate is metabolized only to set the intracellular redox state.

The importance of such critically balanced metabolism is reflected in the high sensitivity of early embryos to metabolic

stress (generated internally or by the environment), leading to the discovery that oocytes and early embryos with a ‘quieter’

metabolism have a much better potential for development. During cleavage stages, metabolism may be assayed non-

invasively by measuring oxygen consumption, intracellular redox state or by amino-acid turnover profiling. These studies have

all concluded that embryos showing lower metabolic activity have a better developmental potential, probably because they

can circumvent intracellular oxidation arising from aerobic metabolism more effectively. If oxygen consumption is difficult to

measure accurately at the single embryo level, amino-acid turnover seems a promising, non-invasive technique that can be

used on human embryos to predict their capacity to develop to the blastocyst stage.

1213

L6: The embryo invasion phase: immune mechanisms

Antonis Makrigiannakis

Department of Obstetrics and Gynaecology, Medical School, University of Crete, Heraklion, Greece

Successful embryo implantation in mammals requires the coordinated development of a competent blastocyst and an

adhesive endometrium. Given the indispensable role of implantation for the furtherance of the species, a number of molecular

mechanisms have evolved to regulate the process. A variety of molecules, produced by embryonic as well as maternal tissue,

participate in the cross-talk between the implanting blastocyst and the endometrium. The interplay between the various

molecules and the different pathways they take to produce their effect are now being elucidated.

Because impaired implantation represents the most important limiting factor in the establishment of pregnancy, it is believed

that research in the field will allow clinicians to improve the respective rates. Here we will review certain groups of molecules

that are considered to play key roles in the mechanisms of implantation.

L5: Physiology of implantation: how gene expression influences receptivity

Aydin Arici M.D.,

Department of Obstetrics, Gynecology & Reproductive Sciences, Yale University School of Medicine,

New Haven, CT, USA

Implantation is the process by which the blastocyst becomes intimately connected with the maternal endometrium. The independently

developing blastocyst then becomes dependent on the maternal environment for its continued development. Although the factors involved

in the regulation of blastocyst implantation are incompletely understood, recent studies have advanced our knowledge and understanding

of the communication between maternal and fetal cells during this critical period.

A fundamental requirement for successful implantation is that there must be a synchrony between the development of the embryo and

the uterus. Under the influence of ovarian hormones, the uterine lining reaches a transient receptive stage at which an embryo that has

reached the blastocyst stage can attach. Overall, estrogen and progesterone control the cyclic changes that occur in the endometrium

in preparation for pregnancy. In the endometrium, growth factors (i.e., CSF, EGF, TGF-) and cytokines (i.e., LIF, IL-1) function to mediate

the actions of steroids to promote endometrial receptivity, directly enhance embryo viability and growth, and induce decidual changes

that control the trophoblastic invasion of the embryo. Several adhesive glycoproteins have been identified in the endometrium, including

integrins, cadherins, and carbohydrate-rich membrane-bound glycoproteins. Currently, it is thought that each of these molecules has a role

in the implantation cascade.

Leukocytes form a substantial proportion of the cells of human endometrium, accounting for approximately 7% of stromal cells in the

proliferative endometrium, and increasing to more than 30% in early pregnancy, characteristic of an immunologically active tissue. However,

organization of the endometrial lymphoid system is atypical in several aspects. Large granular lymphocytes, resemble natural killer cells,

form the most abundant lymphoid cell population in human endometrium in the late luteal phase and in early pregnancy. Their increase

around the days of expected implantation, and their persistence in the first trimester of pregnancy, followed by a rapid decline, have

suggested that these cells may play a role in implantation and placental development. During its penetration into the decidua, the human

embryo elicits a response from the maternal immune system as a result of its paternal antigens. Despite this, the embryo is capable of

surviving in a host that is immunologically hostile owing to the presence of nonspecific suppressor cells in the decidua or to the presence of

an embryo-associated immunosuppressor factor.

Trophoblasts selectively express antigens that are recognized by effector immune cells. They do not synthesize major histocompatibility

complex (MHC) class II antigens, but specific subpopulations express unusual MHC class I molecules. Human trophoblasts do not

express classic MHC class I molecules (HLA-A, B, and C), but express a nonclassic molecule, HLA-G, that exhibits limited polymorphism.

Expression of HLA-G has been shown to protect against killing by natural killer cells.

A range of proteins and peptides that are the products of homeobox genes have been found to be very important for early embryonic

development. It has recently been speculated that homeodomains of maternal origin may positively regulate other transcriptional activators

and thus have a crucial role in implantation.

As additional research is performed to understand the molecular aspects of endometrial receptivity and implantation, new markers may

emerge which can be used to assess the implantation process.

1415

L7: Granulosa: gene expression and function

Marie Louise Grøndahl

Laboratory of Reproductive Biology and Fertility Clinic, University Hospital of Copenhagen, (Rigshospitalet)

Copenhagen, Denmark

During folliculogenesis from primordial follicle to ovulatory follicle, the granulosa cells differentiate and proliferate from a mono-

layer of 10–20 cells to about 1000 cumulus cells and more than 1 million mural granulosa cells. It has been established that

the oocyte secures this differentiation of the somatic cell compartment into cumulus and granulosa cells in order for the oocyte

itself to undergo optimal growth and maturation. Little is known about the mechanisms behind the bidirectional communication

between the oocyte and the somatic cells. This is especially true at the molecular level where there is a paucity of information

on the changes that characterize the developmental stages of folliculogenesis, as well as how these stages may be influenced

by different physiological conditions.

The last stage of folliculogenesis was investigated using isolated cumulus and mural granulosa cells from pre-ovulatory follicles.

These follicles were harvested at the time of ovum pick-up, after COS, in women undergoing IVF/ICSI treatment. Isolated cells

were immediately frozen to avoid any in vitro influence on the gene expression. Later, mRNA was extracted and amplified for

whole genome expression microarray analysis.

We have focused on the gene expression profile of cumulus and mural granulosa cells of the pre-ovulatory follicles in relation

to age of the women, the stimulatory regimen used for COS and the differences in the expression profile between the two

compartments of cells. This presentation will review our results.

L8: Follicular cells transcriptomes and oocyte quality

Samir Hamamah

ART/PGD Division, Department of Reproductive Medicine, Aranud de Villeneuve Hospital

Montpellier, France

In ART, pregnancy and birth rates following IVF remain low. Subjective morphological parameters are still a primary criterion for

selecting healthy embryos for use in IVF and ICSI programmes. However, such criteria do not truly predict the competence of

an embryo. Many studies have shown that a combination of several different morphologic criteria (early embryonic cleavage,

number and size of blastomeres on day 2 or 3, fragmentation percentage and the presence of multi-nucleation at the 4- or

8-cell stage) lead to more accurate embryo selection. However, in order to increase IVF success rates, reduce the number of

embryos required for fresh replacement, lower multiple pregnancy rates and lower early pregnancy losses, there is still a need

for further improvements in the accuracy of embryo selection. For these reasons, several biomarkers for embryo selection are

currently being investigated.

Genomics are providing vital information about the genetic and cellular function during embryo development. The emergence

of DNA chips for transcriptomic study offers the possibility to exhaustively catalogue the genes expressed in any given tissue

at any given time. A number of studies suggest that changes in gene expression in oocytes (GDF9, BMP15) and in cumulus

cells that surround the egg (PTX3, BCL2L11, PCK1, NFIB) can be monitored for selecting the best oocytes for fertilization and

embryos for implantation.

Some recent studies have identified non-invasive methods for oocyte and embryo viability, by analysing human follicular fluid

or culture media. However, an important technical drawback is the number of metabolites and the concentration that each

metabolite must reach to enable detection. Therefore, a few studies in human species have been conducted by studying

cumulus cells to identify biomarkers for oocyte quality and competence, or for early embryo development. In our group, we

perform a non-invasive ‘G-test’ by studying the differential gene expression between cumulus cells from different oocytes and

matching them with their subsequent pregnancy outcomes. This analysis is a novel concept, and provides new biomarker

candidates to assess the embryo potential for pregnancy outcome. These markers include BCL2L11, PCK1 and NFIB.

1617

L9: Endometrium gene expression profile in stimulated cycles

José A. Martínez-Conejero1,2, Carlos Simón1, Antonio Pellicer1 and José A. Horcajadas1,2

1Fundación IVI-Instituto Universitario IVI-University of Valencia, Spain,2iGenomix, Valencia, Spain

Controlled ovarian stimulation (COS) used in assisted reproductive techniques (ART) results in lower implantation rates per

transferred embryo compared with natural and ovum donation cycles, suggesting suboptimal endometrial development. Over

the last few decades, endometrial alterations have been investigated using histological and biochemical techniques.

Recent developments in functional genomics have provided objective tools to analyse the endometrium in natural cycles and

evaluate the impact of COS protocols on endometrial development. COS cycles using GnRH agonists and antagonists, and

natural cycles have been analysed in detail during the implantation window to establish any differences in their respective

endometria.1–5 Following these analyses, it has been demonstrated that the endometria from natural cycles follow different

genomic patterns compared with endometrial from COS cycles in the transition from the pre-receptive phase (days LH/hCG+1

until LH/hCG+5) to the receptive phase (day LH+7/hCG+7). Specifically, a 2-day delay in the activation/repression of two gene

clusters, the 218 and 133 genes on day hCG+7 versus LH+7, has been shown.6

In this presentation we review, from the molecular point of view, the results obtained in different studies to elucidate the

changes induced by the different protocols used for ovarian stimulation in an attempt to evaluate their potential clinical

implications.

1. Mirkin S, Nikas G, Hsiu JG, Diaz J, Oehninger S. Gene expression profiles and structural/functional features of the peri-implantation endometrium in natural and gonadotropin-stimulated cycles. J Clin Endocrinol Metab 2004;89:5742–5752.

2. Horcajadas JA, Riesewijk A, Polman J et al. Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles. Mol Human Reprod 2005;11:195–205.

3. Simón C, Bellver J, Vidal C et al. Similar endometrial development in oocyte donors treated with high- or low-dose GnRH-antagonist compared to GnRH-agonist treatment and natural cycles. Human Reprod 2005;12:3318–3327.

4. Liu Y, Lee KF, Ng EH, Yeung WS, Ho PC. Gene expression profiling of human peri-implantation endometria between natural and stimulated cycles. Fertil Steril 2008;90:2152–2164.

5. Haouzi D, Assou S, Mahmoud K et al. Gene expression profile of human endometrial receptivity: comparison between natural and stimulated cycles for the same patients. Hum Reprod 2009;6:1436–1445.

6. Horcajadas JA, Mínguez P, Dopazo J et al. Controlled ovarian stimulation induces a functional genomic delay of the endometrium with potential clinical implications. J Clin Endocrinol Metab 2008;11:4500–4510.

L10: Preimplantation Genetics

Genomics: novel methodologies and objectives

William Kearns

Shady Grove Center for Preimplantation Genetics

Rockville, Maryland, USA

Preimplantation genetic diagnosis (PGD) identifies genetic abnormalities in preimplantation embryos prior to embryo transfer.

PGD is an exciting technology that may improve the likelihood of a successful pregnancy and birth for five distinct patient

groups: 1) those with infertility related to recurrent miscarriages or unsuccessful in vitro fertilization (IVF) cycles, 2) those

with unexplained infertility, 3) advanced maternal age, 4) severe male factor infertility and 5) couples at risk for transmitting a

hereditary disease to their offspring. PGD is always performed following an IVF cycle where multiple oocytes are retrieved and

fertilized.

Sophisticated techniques such as single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH)

microarrays and multi-probe and multi-color fluorescence in situ hybridization (FISH) are used to test single cells for structural

or numerical chromosome abnormalities, while the polymerase chain reaction (PCR), linkage analysis and DNA sequencing are

used to analyze single cells for disease specific DNA mutations.

PGD allows one to transfer only those embryos identified as being free of genetic abnormalities, thus potentially increasing

the implantation rate and decreasing the miscarriage rate. These technologies identify embryos free of specific genetic

abnormalities and may increase the likelihood of achieving the patients’ goal: the birth of a healthy baby.

This presentation will discuss cell types used for PGD and compare and contrast the different methodologies used for

preimplantation genetic diagnostics.

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L11: Metabolomics: ready to be used in practice?

Emre Seli

Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine

New Haven, CT, USA

The high success rates seen following in vitro fertilization (IVF) are attained in many cases through the simultaneous transfer

of multiple embryos at the expense of multiple pregnancies. Multiple pregnancies, in turn, are associated with significant

morbidity and mortality, primarily due to their propensity to result in preterm birth. Consequently, decreasing multiple

gestations, while maintaining or improving overall pregnancy rates, remains the most significant contemporary goal in the

treatment of infertility. In order to achieve this goal, an improvement over our current embryo assessment strategies largely

based on embryo morphology and cleavage rates would be useful.

In 2007, we reported the results of a proof-of-concept study where we collected day 3 spent culture media of individually

cultured embryos with known pregnancy outcome and analyzed the samples using near infrared (NIR) and Raman

spectroscopy (Seli et al. Fertil Steril 2007). The mean spectrum for embryos that resulted in live birth was determined and

compared to the mean spectrum obtained from embryos that failed to implant. Using a mathematical model, the regions

in the spectrum most predictive of pregnancy outcome were identified. Next, based on these regions in the spectrum, an

algorithm to calculate a viability index for each individual embryo reflective of its reproductive potential was developed. In this

initial study, using both NIR and Raman spectroscopy, the mean viability index of embryos that implanted and resulted in a

live birth was significantly higher (p< 0.01) compared to the mean viability index of embryos that failed to implant. Raman and

NIR spectroscopies achieved a sensitivity of 76.5% and 83.3%, and a specificity of 86% and 75%, respectively. Moreover, the

test was rapid (less than 1 minute per sample) and required a very small sample volume (less than 15 µl). Subsequently, the

algorithm described above was tested in prospective blinded trial (Scott et al. 2008) and successfully predicted the pregnancy

outcome for embryos transferred on day 3 and day 5 (p

complement.11 Although more data are required, proteomic analysis of secreted proteins shows great promise as an additional

screen to morphology for assessing the viability and competence of pre-implantation embryos. This presentation provides an

overview of the current evidence for the viability of screening pre-transfer embryos by proteomic analysis of secreted proteins

and discusses the potential benefits that this technology could provide in ART.

1. Ebner T, Moser M, Sommergruber M et al. Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development: a review. Hum Reprod Update

2003;9:251–262.

2. Katz-Jaffe MG, Schoolcraft WB, Gardner DK. Analysis of protein expression (secretome) by human and mouse preimplantation embryos. Fertil Steril 2006;86:678–685.

3. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril. 1:3-17, 1950.

4. DeSouza L, Diehl G, Yang EC, Guo J, Rodrigues MJ, Romaschin AD, Colgan Tj, Siu KW. Proteomic analysis of the proliferative and secretory phases of the human endometrium: protein identification

and differential protein expression. Proteomics 2005; 5:270-281.

5. Dominguez F, Garrido-Gomez T, López JA, Camafeita E, Quiñonero A, Pellicer A, Simón C. Proteomic analysis of the human receptive versus non-receptive endometrium using differential in-gel

electrophoresis and MALDI-MS unveils stathmin I and annexin A2 as differentially regulated. Human Reproduction 2009; 24:2607-2617.

6. Katz-Jaffe MG, Linck DW, Schoolcraft WB, Gardner DK. Proteomic analysis of mammalian preimplantation embryonic development. Reproduction 2005;130:899-890.

7. Katz-Jaffe MG, Gardner DK, Schoolcraft WB. Proteomic analysis of individual human embryos to identify novel biomarkers of development and viability. Fertil Steril 2006;85:101-107.

8. Gutstein HB, Morris JS, Annangudi SP et al. Microproteomics: analysis of protein diversity in small samples. Mass Spectrom Rev 2008;27:316–330.

9. Dominguez F, Gadea B, Esteban FJ, Horcajadas JA, Pellicer A, Simón C. Comparative protein-profile analysis of implanted versus non-implanted human blastocyst. Human reproduction 2008; 12:1-

8.

10. Dominguez F, Gadea B, Mercader A, Esteban FJ, Pellicer A, Simón C. Embryologic outcome and secretome profile of implanted blastocysts obtained after coculture in human endometrial epithelial

cells versus the sequential system. Fertil and steril 2008; 2.

11. Katz-Jaffe MG, Fagouli E, Fillipovits J et al. Relationship between the human blastocyst secretome and chromosomal constitution. Fertil Steril 2008;90:S80.

L13: Ovarian stimulation

Carlo Alviggi, Roberto Clarizia and Giuseppe De Placido

Department of Obstetrics and Gynecology, IVF Unit, University of Naples “Federico II”

Naples, Italy

An increasing body of evidence clearly supports the idea that personalising controlled ovarian stimulation (COS) regimens

optimises the outcome of in vitro fertilisation (IVF) procedures. Availability of recombinant products allows independent use

of the two gonadotrophins, giving the opportunity for evaluating the impact of LH and different FSH:LH ratios on ovarian

response. On these bases, LH supplementation has been tested in different subgroups of patients. Available data indicate

that recombinant LH (r-hLH) is able to improve the ovarian response and the outcome of IVF in women identified as “hypo-

responders” to r-hFSH monotherapy. In particular, the concept of “hypo-response” to COS has been proposed to identify

normogonadotrophic women who have normal estimated ovarian reserve but, when stimulated with standard GnRH-a long

protocol require high amounts of FSH to obtain an adequate number of oocytes retrieved (De Placido et al., 2001, 2004,

2005; Ferraretti et al., 2004; Mochtar et al., 2007; Devroey et al., 2009). These women seem to be distinct from classical poor

responders because they have normal ovarian reserve, but unexpectedly show sub-optimal response when stimulated with

standard regimens. Conversely, specific adjustments of classical protocols, including the use of higher doses of r-FSH and/or

r-hLH supplementation seem to optimise their ovarian response.

On the basis of the current literature, it is possible to argue that hypo-response is related to genetic characteristics. More

specifically, it has been found that this condition is associated with an increased frequency of a common and less bioactive LH

polymorphism (v-LH).

Moreover, a polymorphic variant of the FSH receptor (FSH-R) in which aminoacid asparagine (Asn) at position 680 is replaced

by Serine (Ser) has been associated with higher FSH basal levels and increased number of antral follicles during the early

follicular phase. Recent studies have also proven that this common polymorphism is associated with a higher consumption of

exogenous FSH during COS for IVF/ICSI cycles.

These lines of research suggest that ovarian resistance (hypo-response) to exogenous FSH can be related to specific gene

polymorphisms. In addition, these data support the idea that testing women for these genetic characteristics may allow

tailored gonadotrophins administration. In fact, women with FSH-R polymorphism may benefit from higher r-hFSH starting

dose, whereas those having v-LH may be treated with appropriate LH supplementation. A pharmacogenomic approach to

COS may lead to develop new tailored and cost-effective treatments.

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L14: Oocyte selection

Marc-Andre Sirard1, Melanie Hamel1, Isabelle Dufort1, Claude Robert1, Marie-Claude Leveille2 and Arthur Leader2

1Centre de recherche en biologie de la reproduction, Université Laval, Quebec, Canada2Ottawa Fertility Centre, Ottawa, Canada

The use of embryo selection in human IVF procedure is still impaired by low pregnancy rates with single embryo transfer (SET). The use of

non invasive markers is showing promises as they become useful tools to predict embryo quality. We believe that there is a functional link

between the oocyte and follicular cells which is essential to achieve developmental competence. Differences in the gene expression from

follicle leading to pregnancy could provide useful markers of oocyte developmental competence.

Cells and follicular fluid were recovered by individual follicle puncture. Expression levels of potential markers were assessed by quantitative

PCR with an intra-patients and inter-patients analysis approaches. Gene expression predictor model of ongoing pregnancy has been

investigated.

Five genes were found to be associated with pregnancy across patients: 3ß-HSD-1, adrenodoxin, SERPINE2, CYP19A1 and cdc42. By

intra-patients analysis, phosphoglycerate kinase 1 (PGK1) and regulator of G-protein signalling 2 (RGS2) showed significant difference from

follicles leading to a pregnancy compared to embryos with developmental failure. Two more genes, Pleckstrin homology-like domain family

A member 1 (PHLDA1) and UDP-glucose pyrophosphorylase 2 (UGP2) were associated with embryos with normal morphology that lead to

pregnancy compared to similar embryos that failed.

Both inter-patients and intra-patients approaches must be taken into consideration to delineate gene expression variations in the context of

follicular competence. Predictor model using biomarkers could improve the efficiency of predicting developmental competence of oocyte.

These new approaches provide useful tool in the context of embryo selection and could lead to improved pregnancy rates with SET.

L15: Embryo selection

Emre Seli

Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine

New Haven, CT, USA

A key step in ART is the assessment of embryo viability in order to identify the embryo(s) most likely to result in pregnancy.

Currently used embryo assessment systems are largely based on morphology and cleavage rate. While these systems have

been pivotal in improving implantation and pregnancy rates and reducing multiple gestations, their precision is still insufficient.

The limitations of strategies based on morphology have led to the investigation of adjunctive technologies for non-invasive

assessment of embryo viability in ART. These include the measurement of glucose, pyruvate or amino acid levels in the embryo

culture media, assessment of oxygen consumption by the embryo, genomic and proteomic profiling and, most recently,

analytical examination of the embryonic metabolome. As the number of ART cycles increases worldwide, improvements in

the ability to quickly and non-invasively identify the best embryos for transfer becomes an increasingly important goal for

reproductive medicine.

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Notes:L16: Implantation

Nick S. Macklon

Department of Obstetrics and Gynaecology, Division of Developmental Origins of Health and Disease, University of Southampton, UK.

Compared to most mammalian species, human reproduction can only be described as inefficient. Monthly fecundity rates

(MFRs) in fertile couples are low, on average 20%. This is largely attributable to the high incidence of embryo loss, estimated

to be 30% prior to implantation (pre-implantation loss) and a further 30% before 6 weeks gestation (pre-clinical/biochemical

pregnancy loss). In addition, in excess of 10% of clinical pregnancies result in miscarriage, mostly prior to 12 weeks gestation,

and 1-2% of couples experience recurrent pregnancy loss (RPL), defined as failure of 3 or more consecutive pregnancies

Large-scale structural chromosomal imbalances, caused predominantly by mitotic non-disjunction, are considered to be the

primary cause of human embryo wastage. Array-based analysis has shown that less than 10% of cleavage-stage IVF embryos

have a normal karyotype in all blastomeres, approximately half have no normal cells at all, and the remainder are mosaic.

The phenomenal incidence of gross chromosomal rearrangements in human preimplantation embryos implies that selection

mechanisms must exist to limit implantation of compromised embryos.

The endometrial luminal epithelium is not normally receptive and must transiently acquire this phenotype to allow attachment

and invasion of a blastocyst. In humans, the putative ‘implantation window’ opens 6 days after the postovulatory progesterone

surge and is thought to last no longer than 2-4 days. From a teleological perspective, a confined period of endometrial

receptivity synchronizes implantation events in species with multiple gestations and ensures that pregnancy occurs under the

right maternal conditions. For example, many species are capable of delaying implantation by temporarily suspending embryo

development, a process termed ‘diapause’, which is reversed when the endometrium signals an optimal metabolic and

hormonal intrauterine milieu. Since there is no evidence that human embryos are able to uncouple pre- and post-implantation

development, and multiple gestations are relatively rare, a restricted period of endometrial receptivity may serve to prevent

implantation of chromosomally chaotic but highly invasive embryos.

Once the luminal epithelium of the endometrium is breached, the implanting mammalian embryo elicits a decidual response,

characterised by transformation of stromal fibroblast into secretory, epitheloid-like decidual cells, influx of specialized uterine

immune cells and vascular remodelling [8,9]. The human situation, however, is again quite distinct from most mammals in that

decidual programming is primarily under maternal control and initiated in the mid-secretory phase of each cycle, irrespectively

of whether pregnancy has occurred or not.

Our group has characterized a number of soluble factors involved in crosstalk between decidualizing ESCs and the implanting

embryo using this co-culture model. We found that ESCs selectively recognize and respond to the presence of a compromised

embryo but only upon differentiation into decidual cells. We propose that cyclic decidualization in humans limits implantation

of compromised embryos and, conversely, that failure to express an adequate decidual phenotype disables this process of

embryo recognition and selection, leading to implantation but subsequent pregnancy failure. In this lecture, this novel embryo

selection hypothesis for human implantation is further discussed.

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