isivf09 program web

SPONSOR ACKNOWLEDGEMENTWe thank the following companies for their generous support of the Congress by providingunrestricted educational grants:

ELITE SPONSORMerck Serono

PREMIER SPONSORIBSA

CORPORATE SPONSORSchering-Plough

SUPPORTERFerring Pharmaceuticals

Wireless Internet is available for delegates carrying a laptop and can be accessedanywhere inside the CICG. Laptops must be wi-fi enabled.

Username: isivfPassword: isivf

Geneva1

FINAL PROGRAMME & ABSTRACTS

TABLE OF CONTENTSGeneral Information

Committees

Message from the President of ISIVF

About ISIVF

Message from the President of ISIVM

Message from the Congress President

CME Information

Congress Information

Map / Floor Plans

Local Information

Social Programme & Accompanying Persons Tour

Scientific ProgrammeAgenda-at-a-Glance

Instructions to Oral and Poster Presenters

ISIVF/ISIVM Faculty

Industry-Sponsored Symposia

Sunday – April 19 Pre-Congress Courses

Monday – April 20 Programme

Tuesday – April 21 Programme

Wednesday – April 22 Programme

AbstractsMonday

Tuesday

Wednesday

Posters

Exhibit Directory

Exhibit Floor Plan

Exhibit Hours

List of Exhibitors

Exhibitor Details

Presenter Index

Certificate of Attendance

2009 Congress Secretariatc/o IS Event Solutions

1334 Notre-Dame West, 2nd FloorMontréal, Québec, Canada H3C 1K7

Tel.: (514) 392-7703Fax: (514) 227-5083

[email protected]

Printed in Switzerland, April 2009

ISIVF Membership Secretariat2155 Guy Street, 14th Floor

Room 1471Montreal, Quebec, Canada H3H 2R9

Tel.: (514) 843-1556Fax: (514) 843-2880

[email protected]

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April 19-22, 2009 2

15th World Congress on In Vitro Fertilization | 4th World Congress on In Vitro Maturation

COMMITTEESOrganizing CommitteeJEAN-BERNARD DUBUISSON, Chair

BARRY CAPPEL

ANIS FEKI

VICTOR GOMEL

SEANG LIN TAN

Scientific Programme CommitteeJEAN-BERNARD DUBUISSON

ANIS FEKI

VICTOR GOMEL

TIMUR GURGAN

SEANG LIN TAN

Local Scientific CommitteeSE. ANTONARAKISDepartment of Genetic Medicine and Development, University of GenevaMedical School, and University Hospitals of Geneva, Geneva

P. BISCHOFDepartment of Obstetrics and Gynecology,Geneva University Hospitals, Geneva

C. DE GEYTERGynecologic Endocrinology, Universitätsspital Basel, Basel

J.B. DUBUISSON - Department of Obstetrics and Gynecology,Geneva University Hospitals, Geneva

P. FEHREuro Haus, Schaffhausen

A. FEKIDepartment of Obstetrics and Gynecology,Geneva University Hospitals, Geneva

M. GERMONDCentre de Procréation Médicalement Assistée et d'EndocrinologieGynécologique (CPMA), Lausanne

M. HOHLFrauenklinik, Kantonsspital Baden, Baden

O. HOVATTADepartment of Clinical Science, Intervention and Technology, Division ofObstetrics and Gynecology, Karolinska Institutet, Stockholm, Sweden

B. IMTHURNDepartment of Obstetrics and Gynaecology,University Hospital Zurich, Zurich

M. JACONIDepartment of Pathology and Immunology,Faculty of Medicine, Geneva University, Geneva

K.-H. KRAUSEDépartement d'Immunologie et Pathologie,Faculté de médecine de Genève, Geneva

L. MENCAGLIADepartment of Obstetrics and Gynecology,Geneva University Hospitals, Geneva

M. MUELLERUniversitaetsklinik fuer Frauenheilkunde, Inselsspital Bern

P. PETIGNATDepartment of Obstetrics and Gynecology,Geneva University Hospitals, Geneva

P.A. SAPPINOService d'oncologie, Geneva University Hospitals, Geneva

D. STUCKIClinique de gynécologie et d'obstétrique,Hopital Cantonal de Fribourg, Fribourg

S. VAN DER POELWorld Health Organization, Geneva

International Scientific Committee

MICHEL ABOU ABDALLAHBeirut, Lebanon

MOHAMED ABOULGHARCairo, Egypt

AYDIN ARICINew Haven, United States

PHILIPPE BOUCHARDParis, France

KEITH CAMPBELLLoughborough, UK

RI-CHENG CHIANMontreal, Canada

KLAUS DIEDRICHLuebeck, Germany

JACQUES DONNEZBrussels, Belgium

JEAN-BERNARD DUBUISSONGeneva, Switzerland

ANIS FEKIGeneva, Switzerland

RENÉ FRYDMANClamart, France

JUAN A. GARCIA VELASCOMadrid, Spain

DAVID GARDNERMelbourne, Australia

VICTOR GOMELVancouver, Canada

TIMUR GURGANAnkara, Turkey

MILTON LEONGHong Kong, China

RAGAA MANSOURCairo, Egypt

GIANPIERO PALERMONew York, United States

ZEV ROZENWAKSNew York, United States

HASSAN SALLAMSmouha, Egypt

BILL SCHOOLCRAFTEnglewood, United States

LYNETTE SCOTTMiddleton, United States

SEANG LIN TANMontreal, Canada

Scientific Programme Contributers

ALPHA Scientists in Reproductive MedicineAPARTMiddle Eastern Fertility SocietyTurkish Society of Reproductive Medicine

Geneva3


WELCOME MESSAGE

WELCOME FROM THE PRESIDENT OF ISIVF

Dear Colleagues, Ladies and Gentlemen,

It is my particular pleasure and honor to welcome you to the 15th World Congress on IVF,jointly held with the 4th World Congress on IVM here in Geneva. First of all, I would like toexpress my cordial gratitude to the Congress President Prof. Dubuisson, the members of thescientific program committee and the organizing committee for their elaborate and dedicatedcontribution, which is the key to providing a quality programme, leading to overall success of the Congress. Looking atthe marvelously constructed program, I believe that the Congress shall enjoy a great deal of success, giving satisfactionto every participant.

Since the birth of the first IVF baby achieved by Professor Robert Edwards, our Honorary Patron, IVF-related scienceand technology has widened and deepened during the past 30 years in an unexpectedly speedy pace to establish asystematized infertility treatment discipline called assisted reproductive technology (ART). World IVF Congress, foundedby the late Professor Kurt Semm in 1980, has consistently served as the central venue of forum responsible for thereproductive revolution. With these changes occurring in ART worldwide, it was proposed and agreed at the BoardMeeting of the 13th World Congress in Istanbul to establish the International Society for IVF (ISIVF), for innovation inits structure and function as a scientific body, to cover all the disciplines and address concerns of all professionalsassociated with ART.

I wish you a wonderful Congress and a memorable stay in Geneva!

Takahide MoriPresident, International Society for In Vitro Fertilization

ABOUT ISIVFISIVF's primary mission is to promote research and clinical development of in vitro fertilization of human oocytes (IVF)for the treatment of infertility and as an Assisted Reproduction Technology. Objectives include:

1 Promulgating ethical practice and standards of practice of IVF treatment;

2 Fostering collaboration between the various centres in the world that offer IVF treatment;

3 Organizing symposia for the purposes of presentation, discussion, exchange and publication ofresearch, data, knowledge, theories and standards pertaining to IVF,and specifically, a forum under the name “World Congress on In Vitro Fertilization".

April 19-22, 2009 4


WELCOME MESSAGE

Dear colleagues,

On behalf of the Organizing and Scientific Committees itis my pleasure to welcome you to the 4th WorldCongress on In Vitro Maturation. We are delighted to beholding this year’s congress in the beautiful city ofGeneva.

It is especially fitting that Geneva serves as the Europeanheadquarters for the United Nations, a globalorganization that promotes international cooperation andunderstanding. As speakers and delegates from aroundthe world assemble for this year’s congress, the parallelnature of our missions becomes very apparent. We arehere from many nations to share the latest advancementsand to provide new insights into finding the bestpossible ways to treat the growing problem of infertility– a problem that affects all cultures and nationalities.

Because it does not require the use of fertility drugs, IVMhas become an increasingly important method oftreatment and its applications are widening to includefertility preservation, particularly for patients with cancer,autoimmune disorders, severe endometriosis or TurnerMosiac. This year’s scientific program has beendesigned to provide you with a comprehensive overviewand understanding of this procedure as well as toaddress some of the concerns related to it.

However, the learning process is ongoing and keepingup with the latest advances and their attendantcontroversies is a demanding task. The ISIVM Societywas formed to foster the continued exchange of ideasand knowledge and to promote the ethical, standardizedpractice of IVM. If you have not already done so, I urgeyou to join this important organization to share in itsmany benefits. Please visit our website atwww.isivm.com for further information.

In conclusion, I would like to thank the CongressPresident, Professor Jean-Bernard Dubuisson; ProfessorTakahide Mori, President of the International Society forIn Vitro Fertilization; and the members of the Scientificand Organizing Committees for all of their hard work inmaking this meeting possible. My sincere thanks arealso extended to the speakers and session chairs fortheir participation in this year’s program.

It is our sincere hope that you will enjoy the Congressand all of the charm that Geneva has to offer. We hopethat you will take home with you new and valuableinformation about IVM that will assist you in your futurepractice and research.

Seang Lin TanPresident, International Society for In Vitro Maturation

WELCOME FROM THE PRESIDENT OF ISIVM

Geneva5


Dear colleagues,

Geneva is an international business and art centre. It isthe meeting point of world diplomacy; its political,economic and cultural aura combined with an excellenttourist trade. All this, make Geneva a major metropolis ofworld fame. Great international organizations like the RedCross, the International Labour Office, the World HealthOrganization, have their headquarters here. It is ofimportance to mention that Geneva has a long tradition ofwatch making (Baume et Mercier, Charriol, Chopard,Franck Muller, Patek Philippe, Rolex, Raymond Weil,Omega, etc.).

With this small introduction I wish to welcome you to thecity of Geneva for the 15th World Congress on In VitroFertilization, and the 4th World Congress on In VitroMaturation.

The Geneva ISIVF Congress will bring together leadingscientists and clinicians from various areas of infertility,ART, IVM and stem cell research.The inspiration to organise such conference came fromthe importance and the necessity of close and continuous

interaction between the protagonists in these differentfields. To further enhance this interaction and dialogue,the International Board of ISIVF 2009 took the initiative todrive forward this international Congress in Geneva.

Reproduction and stem cell biology are by any measureconsidered amongst the currently most dynamic researchfields in life science. This International Congress willspan several of these areas. Important topics are to reviewthe current advances in the treatment of infertility couples,including controlled ovarian stimulation, natural cycles,endometriosis, low cost IVF and fertility preservation.This Congress will cover the current status of stem cellresearch for potential therapy for the field of reproduction.

Welcome to Geneva, the city of Calvin. I am sure that youwill enjoy these few days in Geneva, the “Capital ofPeace”, the International city of new ideas andhumanitarian laws!

Jean-Bernard DubuissonCongress President

WELCOME MESSAGE

WELCOME FROM THE CONGRESS PRESIDENT

April 19-22, 2009 6


CME INFORMATION

CME CreditsThis event is approved for up to 27 credits by theCentre for Continuing Medical Education (“CME”). TheCentre for CME, Faculty of Medicine, McGill University isfully accredited by the Committee on Accreditation ofCanadian Medical Schools and through the (CACMS) isaccredited to award AMA PRA category 1 credits.

This event is an Accredited Group Learning Activity asdefined by the Maintenance of Certification programme ofthe Royal College of Physicians and Surgeons of Canada.The Centre for CME, Faculty of Medicine, McGillUniversity designates this activity for Category 1 credittowards the AMA Physicians Recognition Award up to themaximum number of credit hours noted above. Eachphysician should claim only those hours of credit thathe/she actually spent at the educational activity.

Delegates requiring CME Credits must sign in atthe Congress Services Desk every day that theyare present at the congress and must collecttheir Attestation CME Certificate on the last dayof their attendance at the congress.

Congress EvaluationA Congress evaluation form is included in your delegatebag. Please complete it and drop it off at the CongressEvaluation mailbox located in the Registration Area.TheCommittee appreciates your constructive feedback.

CONGRESS INFORMATION

Best Abstract Awards(provided by the International Society for In Vitro Fertilization)

Two abstract awards winners, one for poster and one fororal presentation, will be selected. The winners will beannounced in the Closing Ceremonies.

Certificate of AttendanceThe certificate of attendance is included as last page ofthis publication. You can simply remove the certificateand fill in your name.

Learning Objectives

General goals for event:

As a result of participation and interaction in thiscongress, participants will be able to:

• Expand their broad understanding of the currentstate of the art in In Vitro Fertilization and In VitroMaturation

• Gain an in-depth knowledge of selected focusedsubject matter at the leading edge of currentdevelopments in the fields of IVM and IVF

• Interact and network with key individuals in thefield

• Know about research being done at present thatwill impact on their practice in the future

• Become re-energized in their professional lives bya sense of shared experiences and collegiality

Knowledge participants gain:

• Review of all IVF, IVM and ART related topics (all-inclusive programme)

• Update on most recent advances

• Close look at latest stem cell research

• Research content and clinical science will bediscussed

Geneva7


CONGRESS INFORMATION

Delegate Hospitality

• Badge CordGraciously sponsored by Schering Plough

• Delegate BagGraciously sponsored by Merck Serono

• Notepad and PenGraciously sponsored by IBSA Pharmaceuticals

• Coffee BreaksGraciously sponsored by Ferring PharmaceuticalsDaily coffee breaks will be served in the exhibit area. Forexact timing, please refer to the detailed programme.

• Delicious and Affordable Lunches are served at thecafeteria on Level 1. Refreshments and snacks can bepurchased at the Café.

Emergency / First AidFor any emergency or first aid services inside the ConventionCentre, please dial “9112” from any phone.

InformationShould you require any assistance during the Congress,please call the Congress Services desk directly at +41 22791 94 92. The Conference Services Desk is located in theregistration area and is in service for the entire duration of theCongress.

Internet AccessOPTION 1: Internet StationsThe CICG provides 4 Internet stations which are located in theexhibit area. These stations allow for Internet access to checkyour emails via a web browser. You can not print, upload ordownload files from these stations. The terminals will operateduring exhibit hours.

OPTION 2: Wireless Internet (wi-fi)Wireless Internet is available for delegates carrying a laptopand can be accessed anywhere inside the CICG. Laptops mustbe wi-fi enabled.

Username: isivf Password: isivf

Lost and FoundAny lost and found items will be held at theCongress Services desk for the duration of the event.For any unclaimed items after the congress, pleasecontact IS Event Solutions.

LuggageWe recommend that you leave your luggage with theBellman at your hotel. The Convention Centre doesnot have a bellman or concierge service. Theorganizers don't take any responsibility for suitcasesthat are left anywhere at the Convention Centre,including the registration desk.

Poster AreaThe poster area is available at all times towards theback of the exhibit area. Delegates can visit postersat their convenience. Poster presenters areencouraged to be at their posters during break times.

Registration Information

• Registration HoursSunday, April 19 15:00 - 20:00Monday, April 20 07:30 - 18:00Tuesday, April 21 07:30 - 18:00Wednesday, April 22 07:30 - 18:00

• Name BadgesFor security purposes, all congress participantsmust wear their name badges when onsite at theCICG.

• Badge ColoursMagenta Delegates and sponsorsBlue Invited speakersRed Executive Board membersGreen ExhibitorsOrange Single day registrantsYellow GuestsBlack Media representativesWhite Staff

Public NoticeThe Convention Centre (CICG) is a smoke-freeenvironment. Smoking is permitted only outside thebuilding.

April 19-22, 2009 8


Lac de Genève

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Le Léman

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CICG

Aéroportinternational

GareCornavin

Genève

Adresses utiles

Genève Tourisme18 rue du Mt-Blanc - CP 1602CH 1211 Genève 20Tél. +41 (0)22 909 70 00Fax +41 (0)22 909 70 01

Aéroport InternationalCP 100 - CH 1215 Genève 15Tél. +41 (0)22 717 71 11Fax +41 (0)22 798 43 77

Gare de CornavinCH 1200 GenèveTél. 0900 300 300

CCV

EntréeEspace Dunand

Entrée Principale

>

>Les accèsAccess

Coordonnées

Centre International de Conférence Genève (CICG)17 rue de Varembé

t

CH 1211 Genève 20Tél. +41 (0)22 791 91 11Fax +41 (0)22 791 90 64

Centre de Conférencesde Varembé (CCV)9-11 rue de Varembé

t

CH 1211 Genève 20Tél. +41 (0)22 791 91 11Fax +41 (0)22 791 90 64

Geneva9


FLOOR PLANSLocation of Rooms 2-3-4 (Level 0)

Location of Room 18 (Level -1)

April 19-22, 2009 10


LOCAL INFORMATION

About GenevaSome call it the "world's smallest metropolis", others the"peace capital". Whatever image it brings to mind, Genevahas always been a popular destination, known throughout theworld as the United Nations' European headquarters and thehead office of the International Committee of the Red Cross.Geneva is the smallest of the world's major cities. It is a worldapart, with something for everyone. Distinguished by itsunique geographical position in the heart of Europe, state-of-the-art technology, high-quality services, prestige and rankingas a world-class city, coupled with the advantages of a smalltown, Geneva is the ideal venue for our World Congress.Geneva is magnificently situated on the banks of the largestlake in central Europe, at the foot of the Jura mountains.At the gates of the Alps, Geneva has all that is neededto charm you.

The town, whose rich past still comes to life today, is thetrue international capital and can offer visitors a great numberof different aspects. These include its humanitariancommitment, its varied cultural activities, its majorcongresses and exhibitions and its renowned cuisine,beautiful countryside and opportunities for many differentexcursions. The beauty of the site, the close proximity, aprestigious Alpine region and its privileged location along themain axes of the West, make Geneva naturally one of thelargest European centres of tourism.

CommunicationPublic telephones are easy to locate and international callingcards may be used. They can be purchased at mostconvenience stores and newsstands. Major credit cards arealso acceptable billing alternatives.

Credit CardsIn order to report lost or stolen credit cards, these numberswill provide assistance.

American Express +41 44 659 63 33Mastercard 0 800 89 4732Visa 0 800 89 7092

CurrencyThe Swiss franc (CHF). 1 CHF divided into 100centimes is the national currency. Automatic tellermachines and exchange offices are readily available.Most hotels, restaurants and shops accept majorcredit cards.

At press time, the following exchange rates apply(April 2009).1 USD 1.14 CHF1 EURO 1.54 CHF100 JAPAN YEN 1.18 CHF1 CHINA YUAN RENMINBI 0.66 CHF

Getting Around GenevaUnireso is the tariff community covering all ofGeneva.Tickets and day passes must be purchasedbefore boarding the vehicle from automatic vendingmachines located at the stops. Ticketing machinesaccept Swiss and Euro coins. A ticket costs CHF 3.00and a 1-day pass costs CHF 10. Most hotels providea complimentary transportation card for all of Geneva.

Important Telephone NumbersWhen calling from within Switzerland, you alwayshave to dial the area code 22 with a “0” in front of it,even if you're in the same area. To call from withinGeneva or other places in Switzerland, dial 022 + areacode + local number.

To dial a number overseas, press 00 + country code +area code + telephone number.

Hotel InformationInterContinental Geneva (0) 22 919 39 39Hotel Le Royal (0) 22 906 14 14Hotel Auteuil (0) 22 544 22 22Hotel Epsom (0) 22 544 66 66Hotel Kipling (0) 22 544 40 40

Other Important NumbersTaxi Service (0) 22 33 141 33Tourism Geneva (0) 22 909 70 00

Geneva11


LOCAL INFORMATION

ElectricityThe current used throughout Switzerland is 230 Volts(AC), 50 cycles. Most power sockets are designed forthree pin round plugs. The standard continental type plugwith two round pins, applied for many electrical travelproducts, may be used without problems. Adaptors areavailable in most hotels.

LanguagesThe official language of the Conference will be English.The language most frequently spoken in Geneva isFrench. However, you should have no problemconversing in English.

ShoppingGeneva is a shopper's paradise! With a myriad ofboutiques and department stores, Geneva offerssomething for everyone. It's the watch capital of theworld, a center for exquisite jewelry, and a place to findhigh quality Swiss and imported items. Before you leave,don't forget to purchase some chocolate from one ofGeneva's master chocolate-makers. And why not stock upon Swiss army knives? They make the idealgift for anyone - including yourself!

Store Hours:Monday - Wednesday 9 am-7 pmThursday 9 am-9 pmFriday 9 am-7.30 pmSaturday 9 am-6 pmSunday closed

Time DifferenceGMT +1 hour

Taxes and Tax RefundThe VAT you pay on purchased goods in Switzerland is7.6%. You may ask at the shops for your Global RefundCheque and reclaim the VAT. The total purchases in a shopmust amount to CHF 400.00 (including VAT). The touristmust be resident outside Switzerland and the goods must beexported within 30 days.

3 easy steps to claiming your refund in SwitzerlandIN THE STORE:Your total purchases in a shop must amount to CHF 400.00(Including VAT). You must be a resident outside Switzerlandand the goods must be exported within 30 days.

THROUGH CUSTOMS:When leaving Switzerland the Tax-free Shopping Chequeshave to be stamped by Swiss customs authorities after theyhave seen the goods.

COLLECTING THE REFUND:You have several choices: immediate cash at a CashRefund Office, direct crediting to a chosen credit card orbank account, a bank check and even, for certain countries,a cash refund when you return home.

April 19-22, 2009 12


SOCIAL PROGRAMME &ACCOMPANYING PERSONS TOUROfficial Opening Ceremonies & Welcome ReceptionSunday, April 19, 17:00 to 20:00Centre de conférence international de Genève (CICG)(included in full registration fee for delegates and registered accompanying persons)

Dress Code: CasualAfter the initial inauguration of the Congress with light entertainment, delegates will have a chance to meet withcolleagues and exhibitor representatives as we inaugurate the congress exhibit with a welcome reception, where lightcocktails and refreshments will be served.

Accompanying Persons TourTuesday, April 21, 10:00-17:00Starting point: Centre de conférence international de Genève (CICG)The first part of the tour is a city tour which takes you to the International part of Geneva, through the centre of theworld's peacemaking organizations followed by a tour of the main tourist attractions of the city. Get a taste of Geneva'shistory and dig even deeper into the Old Town on a guided walking tour. The day will then continue with a countrysideexcursion which will take you around Lake Geneva and through charming vineyards, castles and wonderfulfortified houses.

Geneva13


SCIENTIFIC PROGRAMME

NOTES

April 19-22, 2009 14

Geneva15


AGENDA AT A GLANCE

April 19-22, 2009 16


INSTRUCTIONS FOR ORALAND POSTER PRESENTERSSpeaker Preparation HoursThe speaker check-in is located in front of room 2.All speakers must visit the speaker check-in in order to upload the presentation and sign required paperwork.

SUNDAY, APRIL 19 14:00-20:00

MONDAY TO WEDNESDAY, APRIL 20-22 07:30-18:00

Speakers MUST check in and submit their PowerPoint presentation at the Speaker Check-in outside room 2 at least24 hours before their scheduled presentation time and revisions may be made up to 3 hours prior to presentation.This is the only way that we can be sure that you are present, and that the Chairs of your session are updated on anylast-minute changes. It is also very important that you ensure the seamless functioning of your data presentation.

A technician will load your presention onto the main network server. Your presentation will be on the network andtherefore available when you arrive in your session room.

Please note that individual laptop computers are not permitted for presentations.

Poster PresentersParticipants will view posters during lunches and breaks, therefore authors are encouraged to be present at theirposters during those times.

Authors are responsible for the setting up and the removal of their posters according to the following schedule:

Mounting time: MONDAY, APRIL 20 07:00-08:30

Removal: WEDNESDAY, APRIL 22 BY 18:00

Posters not removed by the specified time on the last day of their presentation will be removed and discarded byMeeting staff. ISIVF cannot accept liability for lost or damaged posters. ISIVF will not mail posters to authors afterthe meeting.

Geneva17


ISIVF/ISIVM FACULTY

AAbbiirr,, RRoonniittRabin Medical Center, Petah Tikva, Israel

AAbboouu AAbbddaa ll llaahh,, MMiicchhee llMEFS, Beirut, Lebanon

AAhhuujjaa ,, KKaammaa llThe London Women’s Clinic, London, UK

AAllbbee rrtt iinnii,, DDaavviiddKansas Life Science Innovations Center, Kansas City, USA

AAmmbbrroossee tt tt ii,, AAlleexxaannddrraaGeneva University Hospitals, Geneva, Switzerland

BBeenn--YYoossee ff ,, DDaa lliittThe Tel Aviv Sourasky Medical Center, Tel Aviv, Israel

BBeennkkhhaa lliiff aa ,, MMoonnccee ffATL R&D, Reproductive Biology and Genetics, La Verrière, France

BBiisscchhooff ,, PPaauullGeneva University Hospitals, Geneva, Switzerland

BBooiivviinn,, JJaacckkyyCardiff University, School of Psychology, Cardiff, UK

BBoouucchhaa rrdd,, PPhhiilliippppeeHôpital Saint Antoine, Paris, France

BBuucckkee tt tt ,, WWiilllliiaamm McGill University, Royal Victoria Hospital, Montreal, Canada

CCaammppbbee llll,, KKee iitt hhUniversity of Nottingham - School of Biosciences, Loughborough,UK

CChhiiaann,, RRii--CChheennggMcGill University, Royal Victoria Hospital, Montreal, Canada

CChhiillll iikk ,, CCllaauuddiiooMatercell, Buenos Aires, Argentina

DDaa ll CCaannttoo,, MMaa rriiaabbeeaa tt rriicc eeIstituti Clinici Zucchi, Monza, Italy

ddee GGeeyytt ee rr,, CChhrriisstt iiaannUniversity of Basel, Basel, Switzerland

DDeemmiirrooll,, AAyyggüüllGürgan Clinic, Ankara, Turkey

DDiieeddrriicchh,, KKllaauussUniversitätsklinikum Schleswig-Holstein, Luebeck, Germany

DDoonnnnee zz,, JJaaccqquueessSaint-Luc University, Brussels, Belgium

DDoorr,, JJeehhoosshhuuaaThe Joseph Buchman Gynecology and Maternity Center,Sheba Medical Center, Israel

DDuubbuuiissssoonn,, JJeeaann--BBee rrnnaa rrddGeneva University Hospitals, Geneva, Switzerland

FFaaddiinnii,, RRuubbeennssBiogenesi Reproductive Medicine Center, Monza, Italy

FFeekk ii,, AAnniissGeneva University Hospitals, Geneva, Switzerland

FFeenniicchhee ll,, PPaa tt rriicc kkCentre Hospitalo-Universitaire de Nice, Nice, France

FFrryyddmmaann,, NNee llllyyHôpital Antoine Béclère, Clamart, France

FFrryyddmmaann,, RReennééHôpital Antoine Béclère, Clamart, France

FFuullkkaa JJrr..,, JJoossee ffInstitute of Animal Science, Prague, Czech Republic

GGee rrmmoonndd,, MMaa rrccCentre de Procréation Médicalement Assistée et d’EndocrinologieGynécologique (CPMA), Lausanne, Switzerland

GGiillbbee rrtt ,, LLuuccyyMcGill University, Royal Victoria Hospital, Montreal, Canada

GGoommee ll,, VViicc ttoorrUniversity of British Columbia, Vancouver, Canada

GGoouuggeeoonn,, AAllaa iinnCentre hospitalier Lyon-Sud, Lyon, France

GGüürrggaann,, TTiimmuurrGürgan Clinic, Ankara, Turkey

HHaanncckk ,, BBeevvee rrllyyInfertility Awareness Association of Canada, Montreal, Canada

HHaannddyyssiiddee ,, AAllaann The London Bridge Fertility Gynaecology and Genetic Centre,London, UK

HHoollzzee rr,, HHaannaannee llMcGill Reproductive Centre, Montreal, Canada

HHoovvaa tt tt aa ,, OOuutt iiKarolinska Institutet, Stockholm, Sweden

IIsshhiizzuukkaa ,, BBuunnppee iiSaint Marianna University School of Medicine, Kanagawa, Japan

JJoonneess,, KKee iitt hh TT..The University of Newcastle Australia, Newcastle, Australia

JJoossssoo,, NNaa tt hhaa lliieeINSERM, Clamart, France

KKaa tt oo,, OOssaammuuKato Ladies Clinic, Shinjuku-ku, Tokyo, Japan

LLeeddggee rr,, WWiillll iiaamm Jessop Hospital for Women, Sheffield, UK

LLeeoonngg,, MMiilltt oonnHong Kong Sanatorium and Hospital, Hong Kong, China

April 19-22, 2009 18


ISIVF/ISIVM FACULTY

LLeesssseeyy,, BBrruucceeCenter for Women’s Medicine, Greenville, USA

LLeeuunngg,, PPee tt ee rr CC..KK..The University of British Columbia, Vancouver, Canada

LLiimm,, JJiinn HHooMaria Fertility Hospital, Seoul, Korea

LLiinnddeennbbee rrgg,, SSvveenndd Nordica Fertility Clinic, Institut for Human Reproduktion,Copenhagen, Denmark

MMaannssoouurr,, RRaaggaaaaThe Egyptian IVF-ET Center, Cairo, Egypt

MMeennccaagglliiaa ,, LLuuccaaFlorence Centre of Ambulatory Surgery & Infertility, Florence, Italy

MMee tt tt llee rr,, LLiissee lloott tt eeUniversity of Kiel, Kiel, Germany

MMiikkkkee llsseenn,, AAnnnn LLiissHolbæk Hospital, Holbæk, Denmark

MMoorriimmoott oo,, YYoosshhiihhaa rruuIVF Namba Clinic, Osaka, Japan

MMuuee llllee rr,, MMiicchhaaee ll DD..Bern University Hospital, Bern, Switzerland

MMuurrddoocchh,, AAlliissoonnInternational Centre for Life, Newcastle upon Tyne, UK

NNaa rrddoo,, LLuucc iiaannooSt. Mary’s Hospital, Manchester, UK

PPaa llee rrmmoo,, GGiiaannppiiee rrooWeill Cornell Medicine College, New York, USA

PPee tt iiggnnaa tt ,, PPaa tt rriicc kkGeneva University Hospitals, Geneva, Switzerland

PPoouullyy,, JJ.. LLuuccPolyclinique de l’Hotel Dieu, Clermont-Ferrand, France

PPrraassaadd,, SSuuddhhaaMaulana Azad Medical College, New Delhi, India

QQiiaaoo,, JJiieeThe Third School of Clinical Medicine, Beijing, China

RRaa iinnee --FFeennnniinngg,, NNiicchhoollaassUniversity of Nottingham, Nottingham, UK

RReemmoohhíí GGiimmeennee zz,, JJoosséé University of Valencia & Equipo IVI, Valencia, Spain

SSaa llllaamm,, HHaassssaannUniversity of Alexandria and Alexandria Fertility Center, Alexandria, Egypt

SScchheennkkee rr,, JJoosseepphhMadassah Medical Center Hebrew University, Jerusalem, Israel

SSee rrmmoonn,, KKaa rreennUze Universitat Brussel, Brussels, Belgium

SShhoohhaamm,, ZZeeeevvKaplan Hospital, Rehovot, Israel

SSiillbbee rr,, SShhee rrmmaannThe Infertility Center of St. Louis, Saint Louis, USA

TTaakkee ffmmaann,, JJaannee ttMcGill Reproductive Centre, Montreal, Canada

TTaann,, SSeeaanngg LLiinnMcGill Reproductive Centre, Montreal, Canada

TTuurr--KKaassppaa ,, IIllaannReproductive Genetics Institute, Chicago, USA

UUrrmmaann,, BBuulleennttAmerican Hospital of Istanbul, Nisantasi, Istanbul, Turkey

vvaann ddee rr PPooee ll,, SShhee rryyll UNDP/UNFPA/WHO/World Bank, Geneva, Switzerland

vvaann HHee rreennddaaee ll,, BBrruunnoo JJ..Endoscopic Training Centre Antwerp (ETCA), Antwerp, Belgium

VVaasssseennaa ,, RRiitt aaCentre for Regenerative Medicine, Barcelona, Spain

VVee rrhhaaaakk ,, CChhrriissRadboud Universtiy Medical Center, Nijmegen, The Netherlands

WWaa tt rree lloott ,, AAnnttooiinneeCentre de Recherche et d’Etude de la Stérilité, Lyon, France

WWeennggee rr,, JJeeaann--MMaa rriieeGeneva University Hospitals, Geneva, Switzerland

WWooooddrruuff ff ,, TTee rreessaa KK.. Northwestern University Feinberg School of Medicine, Evanston, USA

YYoouunngg,, LLoorrrraa iinneeUniversity of Nottingham, Nottingham, UK

ZZaa llee ll,, YYaa rroonnSheba Medical Center, Tel Hashomer, Israel

ZZoorrnn,, BBrraannkkooUniversity Medical Center Ljubljana, Ljubljana, Slovenia

Geneva19


INDUSTRY-SPONSORED SYMPOSIA

Merck Serono Satellite SymposiumMonday, April 20 | 12:45-14:00Room 2

IBSA Satellite SymposiumTuesday, April 21 | 12:45-14:00Room 2

GONADOTROPINS FUNCTIONAL PROJECT I: UNDERSTANDING THE MOLECULAR BASIS OFGONADOTROPIN ACTION IN THE GONADAL AXISAND THE CLINICAL IMPLICATIONS IN rFSH/rLHPROTOCOLS OF STIMULATION FOR ART

CHAIR: Marc Germond (Switzerland)

SPEAKERS:

MOLECULAR ACTION OF LH ON THE FOLLICLE Claus Yding Andersen (Denmark)

THE ROLE OF rFSH/rLH IN FOLLICULAR DEVELOPMENTAND THE GENETICS OF THE LH RECEPTORDimitris Loutradis (Greece)

CLINICAL EXPERIENCE WITH A NEW COMBINATION OFRFSH AND RLH IN A 2:1 RATIO FOR ART: SPANISHPRELIMINARY RESULTSJose Luis Caballero (Spain)

HELPING YOU TO IMPROVE YOUR SUCCESSRATE

CHAIRMAN: B. Imthurn (Switzerland)

SPEAKERS:

DOES THE hCG DOSE FOR FINAL FOLLICULARMATURATION HAVE AN IMPACT ON THE IVFOUTCOMES?Christian de Geyter (Switzerland)

ARE THESE DIFFERENT HMGS?Carlo Alviggi (Italy)

IVF IN THE REAL WORLD: SOCIAL AND ETHICALCONCERNSGillian Lockwood (UK)

April 19-22, 2009 20


NOTES

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PRE-CONGRESS COURSES

SUNDAY, APRIL 19, 2009 | HOTEL INTERCONTINENTAL GENEVA

08:30-12:00 Morning Courses

Course 1: Endometriosis and Infertility Room: Madrid100.1 Jacques Donnez, Brussels, Belgium100.2 Jean-Bernard Dubuisson, Geneva, Switzerland100.3 Victor Gomel, Vancouver, Canada100.4 Michael Mueller, Bern, Switzerland100.5 Jean-Marie Wenger, Geneva, Switzerland

Course 2: PGD and ART Outcomes Room: Rome101.1 René Frydman, Clamart, France101.2 Alan Handyside, London, UK101.3 Nelly Frydman, Clamart, France101.4 Karen Sermon, Brussels, Belgium

Course 3: Stem Cells and Reprogramming Room: Brussels102.1 Outi Hovatta, Stockholm, Sweden102.2 Anna Veiga, Barcelona, Spain102.3 Anis Feki, Geneva, Switzerland

12:00-13:00 Lunch Break

13:00-16:30 Afternoon Courses

Course 4: Ultrasonography and ART Room: Madrid103.1 Nicholas Raine-Fenning, Nottingham, UK103.2 Ilan Tur-Kaspa, Chicago, USA103.3 Yuron Zalel, Tel Hashomer, Israel

Course 5: Germ Cell and Tissue OvarianPreservation: Impact on IVM Development Room: Rome104.1 Seang Lin Tan, Montreal, Canada104.2 Timur Gurgan, Ankara, Turkey104.3 Moncef Benkhalifa, La Verriere, France

Course 6: Low Cost IVF Room: Brussels105.1 Sheryl van der Poel, WHO, Geneva, Switzerland

WHO and International Initiatives105.2 Lisa Feldman

WHO Primary Health Care Management of the Infertile Couple105.3 Dr. Meserat Mengistu, Addis Ababa and Umeå, Sweden

The Need and Present Situation in Women's Health and InfertilityTreatment in Ethiopia

105.4 Outi Hovatta, Karolinska Institutet, Stockholm, SwedenExperience from a Low Cost IVF Unit in Khartoum, Sudan

April 19-22, 2009 22


MONDAY, APRIL 20, 2009

08:30-10:15 Plenary I Room: Salle 2

Co-Chairs: Gomel, Victor - Vancouver, CanadaPalermo, Gianpiero - New York, USA

200.1 ALPHA KEYNOTE LECTURE - PGD: CURRENT DEVELOPMENT AND FUTURE DIRECTIONSHandyside, Alan - London, UK

200.2 NATURAL CYCLE IVF/IVM Lim, Jin Ho - Seoul, Korea

200.3 ONCOFERTILITY: THE PRESERVATION OF FERTILITY OPTIONS FOR YOUNGPEOPLE WITH CANCER Woodruff, Teresa K. - Evanston, USA

10:15-10:45 Coffee Break, Exhibits and Poster Viewing

10:45-12:30 Concurrent Symposium 1 - Implantation Room: Salle 3

Co-Chairs: Bischof, Paul - Geneva, SwitzerlandChian, Ri-Cheng - Montreal, CanadaPrasad, Sudha - New Delhi, India

201.1 LUTHEAL SUPPORT IN IVF: WHY, WITH WHAT, WHEN TO START AND FOR HOW LONG?Shoham, Zeev - Rehovot, Israel

201.2 ASSESSMENT AND REGULATION OF ENDOMETRIAL RECEPTIVITYLessey, Bruce - Greenville, USA

201.3 CELLULAR AND MOLECULAR MECHANISMS ALLOWING EMBRYO IMPLANTATIONBischof, Paul - Geneva, Switzerland

201.4 ENDOMETRIAL IMAGING AND ART OUTCOMETur-Kaspa, Ilan - Chicago, USA

10:45-12:30 Concurrent Symposium 2 - Ultrasound and Controlled Room: Salle 2Ovarian Stimulation

Co-Chairs: Raine-Fenning, Nicholas - Nottingham, UKTur-Kaspa, Ilan - Chicago, USA

202.1 TRANSVAGINAL HYDROLAPAROSCOPY IN INFERTILITYWatrelot, Antoine - Lyon, France

202.2 3DUS AND UTERINE PATHOLOGIESZalel, Yaron - Tel Hashomer, Israel

202.3 OVARIAN IMAGING & ART OUTCOMERaine-Fenning, Nicholas - Nottingham, UK

202.4 ULTRASOUND UTERINE INVESTIGATION IN ARTQiao, Jie - Beijing, China

DETAILED PROGRAMME

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10:45-12:30 ALPHA Session (Scientists in Reproductive Medicine) Room: Salle 4

Co-Chairs: Handyside, Alan - London, UKBalaban, Basak - Istanbul, Turkey

203.1 EPIGENETICS AND SAFETY IN ARTYoung, Lorraine - Nottingham, UK

203.2 EFFECT OF OVARIAN STIMULATION ON EMBRYO CHROMOSOMES Handyside, Alan - London, UK

203.3 PREGNANCY AND BIRTH OUTCOME FOLLOWING PGD Handyside, Alan - London, UK

12:30-14:00 Lunch Break, Exhibits and Poster Viewing

12:45-14:00 Merck Serono Satellite Symposium (refer to page 19 for details)

14:00-15:45 Concurrent Symposium 3 - Adnexal Surgery and Fertility Room: Salle 3

Co-Chairs: Lebbi, Issam - TunisiaMueller, Michael D. - Bern, SwitzerlandDubuisson, Jean-Bernard - Geneva, Switzerland

204.1 REPRODUCTIVE LIFE AFTER GYNECOLOGICAL CANCERPetignat, Patrick - Geneva, Switzerland

204.2 IS RESECTION OF ENDOMETRIOMAS PRIOR TO IVF/ICSI NECESSARY?Gürgan, Timur - Ankara, Turkey

204.3 OVARIAN ADHESIOLYSIS AND FERTILITYNardo, Luciano - Manchester, UK

204.4 OVARIAN FUNCTION AND REGULATIONMettler, Liselotte - Kiel, Germany

204.5 HYSTEROSCOPY IN INFERTILITY AND IVF/ICSI TREATMENTDemirol, Aygül - Ankara, Turkey

DETAILED PROGRAMME

April 19-22, 2009 24



14:00-15:45 Concurrent Symposium 4 - Premature Ovarian Failure Room: Salle 2

Chair: Tavmergen, Ege - Izmir, Turkey

205.1 ETIOLOGICAL FACTORS AND CLINICAL COURSE IN PATIENTS WITH PREMATURE OVARIAN FAILUREIshizuka, Bunpei - Kanagawa, Japan

205.2 OVARIAN FAILURE AND FERTILITYBouchard, Philippe - Paris, France

205.3 REGULATION OF GRANULOSA CELL APOPTOSIS BY GNRHLeung, Peter C.K. - Vancouver, Canada

205.4 G-CSF AS NON-INVASIVE APPROACH FOR OOCYTE COMPETENCEFrydman, René - Clamart, France

14:00-15:45 Swiss Society of Reproductive Medicine Session - (SSRM) Room: Salle 4Research Prizes of SSRM: The Best Papers

Co-Chairs: Imthurn, Bruno - Zurich, SwitzerlandGalié-Wunder, Dorothea - Lausanne, Switzerland

206.1 HIGH SURVIVAL AND DEVELOPMENTAL RATES OF VITRIFIED MOUSE ZYGOTES FOLLOWINGPOLAR BODY BIOPSY Macas, M. - Zurich, Switzerland

206.2 COMPUTER-ASSISTED ANALYSIS OF HUMAN PRONUCLEAR ZYGOTESUrner, F. - Lausanne, Switzerland

206.3 MATURE OVARIAN FOLLICLES CONTAIN A SUBPOPULATION OF STEM CELLS Kossowska-Tomaszczuk, K. - Basel, Switzerland

206.4 INTERMEDIATE AND PREMUTATION FMR1 ALLELES IN WOMEN WITH OCCULT PRIMARYOVARIAN INSUFFICIENCYStreuli, I. - Geneva, Switzerland

206.5 GENE EXPRESSION PROFILING OF CULTURED ENDOMETRIUM FROM WOMEN UNDERGOINGIN VITRO FERTILIZATION WITH DIFFERENT OUTCOMEBersinger, N. - Bern, Switzerland

206.6 TASK FORCE OF THE FERTILITY AND CANCER NETWORK IN THE FRENCH SPEAKING PART OFSWITZERLAND (RÉSEAU ROMAND CANCER ET FERTILITÉ) Ambrosetti, Alexandra - Geneva Switzerland

DETAILED PROGRAMME

Geneva25



14:00-15:45 Oral Communications 1 Room: Salle 18

Co-Chairs: Petignat, Patrick - Geneva, SwitzerlandGurgan, Timur - Ankara, Turkey

207.1 DETECTION OF OXIDATIVE DNA DAMAGE BY FLOW CYTOMETRYAND ITS ASSOCIATION WITH MALE INFERTILITYNozha, Chakroun Feki - Sfax, Tunisia

207.2 IMPRINTING IN THE HUMAN EMBRYO: EVIDENCE FOR A RECYCLING OF HOMOCYSTEINE IN THE OOCYTEBenkhalifa, Moncef - La Verrière, France

207.3 HIV SERO-CONVERSION OF A SPERM DONOR DURING THE DONATION PERIODSmith, Venessa – London, UK

207.4 PRESENTATION OF INNOVATIVE PATENTED PRODUCTS FOR OVUM PICK UP AND EMBRYO TRANSFERSteiner, Hans-Peter - Graz, Austria

207.5 COMBINING TWO ZWITTERIONIC BUFFERS IN IVF HANDLING MEDIA SUPPORTS MOUSE BLASTOCYSTDEVELOPMENT AND NORMAL HUMAN OOCYTE FERTILIZATION FOLLOWING ICSISwain, Jason - Ann Arbor, USA

207.6 CORRELATION BETWEEN REACTIVE OXYGEN SPECIES (ROS) IN SEMINAL PLASMA AND SPERM VITALITY,MEMBRANE, DNA INTEGRITY, DNA STRAND BREAKS OF INFERTILE MEN BEFORE AND AFTER SEMENPROCESSING BY PURESPERM AND IVF/ICSI OUTCOMESHammadeh, M.E. - Homburg/Saar, Germany


16:15-18:00 Concurrent Symposium 5 - Development of IVM Room: Salle 2

Co-Chairs: Tan, Seang Lin - Montreal, CanadaMikkelsen, Ann Lis - Holbæk, Denmark

208.1 NUCLEOLI IN MAMMALIAN OOCYTES AND EARLY EMBRYOSFulka Jr., Josef - Prague, Czech Republic

208.2 EARLY OVULATION INDUCTION: IS IT A METHOD TO REPLACE IVM AND PREVENT OHSSPouly, J. Luc - Clermont-Ferrand, France

208.3 AN OOCENTRIC VIEW OF FOLLICULOGENESIS AND EMBRYOGENESISAlbertini, David - Kansas City, USA

208.4 IVM FOR REGULAR MENSTRUAL CYCLING WOMENFadini, Rubens - Monza, Italy

208.5 CURRENT CRITICISM ON IVM TREATMENTLeong, Milton - Hong Kong, China

DETAILED PROGRAMME

April 19-22, 2009 26



16:15-18:00 Concurrent Symposium 6 - Laboratory and Management Room: Salle 3

Chair: Gülekli, Bülent - Balcova Izmir, Turkey

209.1 EMBRYO SELECTIONHovatta, Outi - Stockholm, Sweden

209.2 INTRODUCING ISO INTO IVF ACADEMIC UNITSFrydman, Nelly - Clamart, France

209.3 ISO STANDARDS FOR CLINICAL WORKUP - EXPERIENCES WITH DIN EN ISO 9001:2000Imthurn, Bruno - Zurich, Switzerland

209.4 OBSTETRIC AND NEONATAL OUTCOME FOLLOWING IVMBuckett, William - Montreal, Canada

16:15-18:00 Turkish Society of Reproductive Medicine Session Room: Salle 4

Co-Chairs: Gürgan, Timur - Ankara, TurkeyPabuçcu, Recai - Turkey

210.1 GENETIC APROCHES UNVEIL CHECKPOINTS DURING FOLLICULAR MATURATIONBenkhalifa, Moncef

210.2 NOVEL TECHNOLOGIES IN ART LABORATORIES: FACTS OR ARTEFACTS?Fındıklı, Necati

210.3 WHAT TO DO AFTER ART FAILSGöker, Ege

210.4 OFFICE HYSTEROSCOPY PRIOR ICSI/IVF : DOES IT MAKE ANY DIFFERENCE?Demirol, Aygül

210.5 SURGERY BEFORE IVF/ICSI : CAN WE IMPROVE THE SUCCESS RATESTavmergen, Erol

210.6 COMPARISON OF EFFICACY OF REC-FSH AND HIGHLY PURIFIED HUMAN-DERIVED FSH IN GNRHANTAGONIST CYCLES FOR ARTBahçeci, M.

210.7 COMPARISON OF EFFICACY OF REC-FSH AND HIGHLY PURIFIED HUMAN-DERIVED FSH IN GNRHANTAGONIST CYCLES FOR ARTUlug, U.

DETAILED PROGRAMME

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Chair: Holzer, Hananel - Montreal, Canada

211.1 EFFECT OF SEASON ON FERTILIZATION AND EMBRYO QUALITY RATES IN ARTDanfour, Mohamed - Misurata, Libya

211.2 THE ROLE OF IMMUNOHYSTOCHEMISTRY IN COMPLEX MORPHOLOGICAL DIAGNOSIS OF WOMEN STERILITYKostyuchek, Irina - Saint-Petersburg, Russia

211.3 WHAT WE OFFER TO WOMEN BEFORE UNDERGOING SURGICAL TREATMENT FOR MENORRHAGIAYounas, Kinza - Birmingham, UK

211.4 GLYCODELIN ENHANCES FERTILIZATION DURING IVFGneist, Nadja - Dresden, Germany

211.5 A SYSTEMATIC REVIEW OF MANAGEMENT OF INFERTILITY IN WOMEN OVER THE AGE OF 40Marinakis, Gerasimos - London, UK

211.6 ROLE OF DIAGNOSTIC LAPAROSCOPY IN WOMEN WITH NORMAL PELVIC IMAGING AND FAILED SUPEROVULATION AND INTRA UTERINE INSEMINATIONJayakrishnan, Kay - Trivandrum, India

211.7 EFFECTIVE WAY TO CUT THE POST OPERATIVE ANALGESIC REQUIREMENT AFTER FEMALE TUBALSTERILISATIONYounas, Kinza - Birmingham, UK

DETAILED PROGRAMME

April 19-22, 2009 28


NOTES

Geneva29


TUESDAY, APRIL 21, 2009

08:30-10:15 Plenary II Room: Salle 2

Co-Chairs: Dubuisson, Jean-Bernard - Geneva, SwitzerlandCampbell, Keith - Loughborough, UK

300.1 ENDOMETRIOSIS RELATED INFERTILITY: THE NEED FOR A CONSERVATIVE SURGERYDonnez, Jacques - Brussels, Belgium

300.2 NEW CHALLENGES FOR OVARIAN-CELL THERAPY AT THE DAWN OF THE 21ST CENTURYGougeon, Alain - Lyon, France

300.3 IVM AND FERTILITY PRESERVATIONTan, Seang Lin - Montreal, Canada


10:45-12:30 Concurrent Symposium 7 - Controlled Ovarian Stimulation Room: Salle 4

Co-Chairs: Kato, Osamu - Tokyo, JapanTavmergen, Erol - Izmir, TurkeyBouchard, Philippe - Paris, France

301.1 RECURRENT IMPLANTATION FAILURE: A CLINICIAN'S VIEWSchenker, Joseph - Jerusalem, Israel

301.2 PHARMACOLOGICAL REGIMENTS FOR UTERINE PREPARATION FOR ETTur-Kaspa, Ilan - Chicago, USA

301.3 MULTIPLE PREGNANCY, IS IT A FAILURE OF ART?Belaisch-Allart, Joelle - Sèvres, France

301.4 DIAGNOSTIC AND MANAGEMENT OF POOR RESPONDERSGermond, Marc - Lausanne, Switzerland

301.5 LUTEAL SUPPORT IN ARTPrasad, Sudha - New Delhi, India

10:45-12:30 Concurrent Symposium 8 - Fertility Preservation Room: Salle 3

Co-Chairs: Rosenwaks, Zev - New York, USAChian, Ri-Cheng - Montreal, Canada

302.1 CONSEQUENCES OF CHEMOTHERAPY AND/OR RADIOTHERAPY ON FERTILITYDor, Jehoshua - Tel Aviv, Israel

302.2 CRYOPRESERVATION OF OOCYTESChian, Ri-Cheng - Montreal, Canada

302.3 VITRIFICATION OF HUMAN ZYGOTES: CLINICAL RESULTSDiedrich, Klaus - Luebeck, Germany

DETAILED PROGRAMME

April 19-22, 2009 30



10:45-11:25 APART Symposium 1 - Natural Cycle IVF and Mild IVF Cycle Room: Salle 2in Reproductive Medecine

Co-Chairs: Feichtinger, Wilfried - Vienna, AustraliaKamiya, Hirofumi - Sapporo, Japan

303.1 THE ROLE OF MILD STIMULATION IN THE CURRENT IVF PRACTICEBernard, Artur - Budapest, Hungary

303.2 OUTCOME OF THE RECENT IVF PROGRAM TRANSFERRING SINGLE BLASTOCYST RETRIEVED ONMINIMAL STIMULATION CYCLE AND FROZEN BY VITRIFICATION METHODKato, Keiichi - Tokyo, Japan

303.3 CONTROLLED OVARIAN STIMULATION, MINIMAL STIMULATION OR NO STIMULATION FOR IN VITROFERTILIZATION: IS THERE AN OPTIMAL STIMULATION?Zhang, John - New York, USA

11:25-12:30 APART Symposium 2 - Management of PCO Patients Room: Salle 2

Co-Chairs: Barak, Yona - Berzliya-On-Sea, IsraelMorimoto, Yoshiharu - Sapporo, Japan

304.1 THE BENEFIT OF IVM IN PCO PATIENTSBarak, Yona - Berzliya-On-Sea, Israel

304.2 OXYGEN CONSUMPTION BY SCANNING ELECTROCHEMICAL MICROSCOPY (SECM) FOR HUMAN IVM COC& EMBRYO WITH PCO PATIENTSYoshida, Hiroaki - Sendai, Japan

304.3 CLINICAL DATA OF IVM FOR PCO PATIENTSMorimoto, Yoshiharu - Osaka, Japan

304.4 USE OF LETROZOLE IN PCOSShozu, Makio - Chiba, Japan

304.5 PCOS: MANAGEMENT STRATEGIES IN 2009Yelian, Frank - Los Angeles, USA


12:45-14:00 IBSA Satellite Symposium (for details, please refer to page 19)

DETAILED PROGRAMME

Geneva31



14:00-15:45 Concurrent Symposium 9 - Fertility Preservation Room: Salle 4

Co-Chairs: Donnez, Jacques - Brussels, BelgiumDiedrich, Klaus - Luebeck, GermanyIshizuka, Bunpei - Kanagawa, Japan

305.1 MANAGEMENT OF OVARIAN TUMORS IN CONTEXT OF FERTLITY PRESERVATIONGilbert, Lucy - Montreal, Canada

305.2 FRESH AND FROZEN OVARY TRANSPLANTATIONSilber, Sherman - Saint Louis, USA

305.3 PRESERVING FERTILITY IN CANCER PATIENTS AND FOLLICLE OVARIAN RESERVEDonnez, Jacques - Brussels, Belgium

305.4 OOCYTE VITRIFICATION FOR CANCER AND NON-CANCER FERTILITY PRESERVATIONTan, Seang Lin - Montreal, Canada

14:00-15:45 Concurrent Symposium 10 - IVM Room: Salle 3

Chair: Leong, Milton - Hong Kong, China

306.1 REGULATION OF SPINDLE AND CHROMATIN DYNAMICS DURING OOCYTE MATURATIONSmith, Gary - Ann Arbor, USA

306.2 FOLLICULAR DEVELOPMENT IN VIVO AND IN VITROAbir, Ronit - Petah Tikva, Israel

306.3 ULTRASTRUCTURE OF IN VITRO MATURED OOCYTESMorimoto, Yoshiharu - Osaka, Japan

306.4 GENOMIC IMPRINTING IN ARTPalermo, Gianpiero - New York, USA

14:00-14:50 APART Symposium 3 - ART for Fertility Preservation Room: Salle 2

Co-Chairs: Kaali, Steven - New York, USAUtsunomiya, Takafumi - Oita, Japan

307.1 FERTILITY PRESERVING OPTIONS OF REPRODUCTIVE AGE CANCER PATIENTSKovacs, Peter - Budapest, Hungary

307.2 THE CURRENT APPROACH TO OOCYTE VITRIFICATION FOR CANCER PATIENTS IN JAPANUtsunomiya, Takafumi - Oita, Japan

307.3 SUCCESSFUL VITRIFICATION OF HUMAN OOCYTESKuwayama, Masashige - Tokyo, Japan

307.4 CRYOPRESERVATION OF PREPUBERTAL TESTICULAR TISSUEYoshida, Atsumi - Tokyo, Japan

DETAILED PROGRAMME

April 19-22, 2009 32



14:50-15:45 APART Symposium 4 - Male Infertility Room: Salle 2

Co-Chairs: Zech, Herbert - Bregenz, AustriaTanaka, Atsushi - Fukuoka, Japan

308.1 DOES A COMBINATION OF CHINESE HERBS AND ACUPUNCTURE TREATMENT AFFECT SPERMCHARACTERISTICS IN INFERTILE COUPLEVelleman, Ramon - Tel Aviv, Israel

308.2 IMPROVEMENT OF A PHOTODYNAMIC SYSTEM FOR OBSERVATION OF SEMINIFEROUS TUBULESIN MICRODISSECTION-TESTICULAR SPERM EXTRACTION (MD-TESE)Tanaka, Atsushi - Fukuoka, Japan

308.3 A NOVEL CRYOPRESERVATION TECHNIQUE FOR VERY FEW MOTILE SPERM FROM SEVERELY INFERTILE MENKyono, Koichi - Sendai, Japan

308.4 ARGUMENTS TO IMPLEMENT THE SELECTION OF SPERMATOZOA AT HIGH MAGNIFICATION BEFORE ICSIZech, Herbert - Bregenz, Austria


Chair: Germond, Marc - Lausanne, Switzerland

309.1 SEARCH FOR MECHANISMS UNDERLYING IMPRINTING DEFECTS IN SPERMNaumova, Anna K. - Montreal, Canada

309.2 EFFECT OF MATURATION IN VITRO ON SPINDLE MORPHOLOGY IN HUMAN OOCYTESMunk, Mette - Copenhagen, Denmark

309.3 PATERNAL AGE AFFECTS SPERM DNA FRAGMENTATION BUT NOT ITS PACKAGINGBelloc, Stéphanie - Paris, France

309.4 THE LOCALIZATION OF SPECIFIC PROTEIN KINASE C ISOTYPES IN MOUSE OVARYTepekoy, Filiz - Antalya, Turkey

309.5 IMPACT OF A GNRH ANTAGONIST ON THE OUTCOME OF CONTROLLED OVARIAN HYPERSTIMULATION (COH) IN WOMEN WITH POLYCYSTIC OVARY SYNDROME: A PROSPECTIVE AND RANDOMIZED STUDYStadtmauer, Laurel - Norfolk, USA

309.6 OVARIAN AGING: PROGNOSTIC FACTORS, BIOLOGICAL FACTORS AND OUTCOME OF ARTGenazzani, Andrea Riccardo - Pisa, Italy

309.7 FIRST TIME EVIDENCE FOR INCREASED PLACENTAL GROWTH FACTOR MRNA IN ENDOMETRIUM OFPATIENTS WITH SUCCESSFUL IMPLANTATIONSanti, Alessandro - Bern, Switzerland


DETAILED PROGRAMME

Geneva33




Co-Chairs: Tan, Seang Lin - Montreal, CanadaAlbertini, David - Kansas City, USA

310.1 EFFICIENCY OF IVM EGG COLLECTIONGülekli, Bülent - Izmir, Turkey

310.2 FSH PRIMING FOR IVMMikkelsen, Ann Lis - Holbæk, Denmark

310.3 HCG PRIMING FOR IVMSon, Weon-Young - Montreal, Canada

310.4 ENDOMETRIAL PREPARATION IN IVM TREATMENTLindenberg, Svend - Copenhagen, Denmark

16:15-18:00 Concurrent Symposium 12 - PGD Room: Salle 2

Co-Chairs: Frydman, Nelly - Clamart, FranceAntonarakis, Stylianos - Geneva, Switzerland

311.1 PRE IMPLANTATION GENETIC DIAGNOSIS (PGD) FOR CANCER PREDISPOSITION GENESYaron, Yuval - Tel Aviv, Israel

311.2 PGD IMPROVED PGD TESTS AND CLINICAL APPLICATIONSFrydman, René - Clamart, France

311.3 PGD VS PRE-GENETIC SCREENING (PGS): WHERE IS THE LIMIT?Gianaroli, Luca - Bologna, Italy

DETAILED PROGRAMME

April 19-22, 2009 34




Co-Chairs: Benkhalifa, Moncef – La Verrière, FranceGougeon, Alain - Lyon, France

312.1 ULTRASOUND GUIDED VERSUS BLIND TOUCH EMBRYO TRANSFER: THE EGYPTIAN EXPERIENCETaha, Tamer Fouad - Cairo, Egypt

312.2 A MICROSYSTEM WITH WHITE LIGHT OPTICAL SENSOR TO QUALIFY THE CYTOPLASM MATURITYOF HUMAN OOCYTESRoux, Christophe - Besançon, France

312.3 HYSTEROSCOPIC ENDOMETRIAL EMBRYO DELIVERY (HEED): TRANSFER OF DAY 2-5 EMBRYOSKamrava, Michael - Beverly Hills, USA

312.4 NOVEL METHOD OF HAND FREE ULTRASOUND QUIDED ETHaloob, A. Rahim - Basildon, UK

312.5 CERVICAL MUCUS STATUS CAN BE ACCURATELY ESTIMATED BY TRANSVAGINAL ULTRASONOGRAPHYDURING FERTILITY EVALUATIONWolman, Igal - Tel-Aviv, Israel

312.6 ULTRASOUND MONITORING AND HORMONAL PROFILES IN SERUM AND FOLLICULAR FLUID FORUNSTIMULATED IVF-CYCLES IN RELATION TO ÌEMPTY FOLLICLE SYNDROMEÎ.Tang-Pedersen, Mikael - Odense, Denmark

312.7 A STUDY OF EPIGENETIC ALTERATIONS ASSOCIATED WITH PREMATURE OVARIAN FAILUREGhahremani, Manda - Vancouver, Canada

DETAILED PROGRAMME

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WEDNESDAY, APRIL 22, 2009

08:30-10:15 Plenary III Room: Salle 2

Co-Chairs: Feki, Anis - Geneva, SwitzerlandJaconi, Marisa - Geneva, Switzerland

400.1 DERIVATION AND CHARACTERIZATION OF HUMAN EMBRYONIC STEM CELL LINES Hovatta, Outi - Stockholm, Sweden

400.2 REPROGRAMMING SOMATIC CELLS: FUTURE PERSPECTIVESCampbell, Keith - Loughborough, UK

400.3 TOOLS FOR STEM CELL RESEARCH AND CLINICAL APPLICATIONS: THE HUMAN EMBRYONIC STEM CELL REGISTRY (HESCREG) AND THE ISSCR GUIDELINES FOR THE TRANSLATION OF STEM CELL RESEARCHVeiga, Anna - Barcelona, Spain

10:45-12:30 Concurrent Symposium 13 - Controlled Ovarian Stimulation Room: Salle 3

Co-Chairs: Frydman, René - Clamart, FranceSallam, Hassan - Alexandria, Egypt

401.1 OUTCOME OF ASSISTED REPRODUCTION AFTER OVARIAN HYPERSTIMULATION AND AFTERCRYOPRESERVED EMBRYO TRANSFERde Geyter, Christian - Basel, Switzerland

401.2 OHSS PREVENTION AND TREATMENTSallam, Hassan - Alexandria, Egypt

401.3 MINIMAL STIMULATION IN IVFKato, Osamu - Tokyo, Japan

401.4 MATRIX METALLOPROTEINASE ACTIVITY DURING THE MENSTRUAL CYCLEAbdallah, Michel Abou - Beirut, Lebanon

10:45-12:30 Concurrent Symposium 14 - Human ES and Reproductive Medicine Room: Salle 2

Co-Chairs: Jaconi, Marisa - Geneva, SwitzerlandFeki, Anis - Geneva, SwitzerlandErenus, Mithat - Turkey

402.1 PROMISES AND CHALLENGES TOWARDS THE CLINICAL USE OF PLURIPOTENT STEM CELLSFeki, Anis - Geneva, Switzerland

402.2 EMBRYONIC STEM CELLS DERIVED FROM PGD-AFFECTED EMBRYOS FOR MODELING HUMANGENETIC DISORDERSBen-Yosef, Dalit - Tel Aviv, Israel

402.3 THE STEM CELL NICHE IN THE TESTIS: POTENTIAL FOR REGENERATIVE AND REPRODUCTIVE MEDICINEVassena, Rita - Barcelona, Spain

DETAILED PROGRAMME

April 19-22, 2009 36



10:45-12:30 Concurrent Symposium 15 - Surgery for Reproductive Medicine Room: Salle 4

Co-Chairs: Dubuisson, Jean-Bernard - Geneva, SwitzerlandNardo, Luciano - Manchester, UK

403.1 LAPAROSCOPIC TREATMENT OF UTERUS BICORNISvan Herendael, Bruno J. - Antwerp, Belgium

403.2 DEEP PELVIC ENDOMETRIOSIS AND FERTILITYDubuisson, Jean-Bernard - Geneva, Switzerland

403.3 ROLE OF HYSTEROSCOPY IN INFERTILITY AND IVFMencaglia, Luca - Florence, Italy

403.4 REPRODUCTIVE SURGERY AND IVFGomel, Victor - Vancouver, Canada


14:00-15:45 Concurrent Symposium 16 - Egg Donation Room: Salle 3

Co-Chairs: Murdoch, Alison - Newcastle upon Tyne, UKChian, Ri-Cheng - Montreal, CanadaBoivin, Jacky - Cardiff, UK

404.1 SUCCESSFUL EGG DONATION PROGRAMMurdoch, Alison - Newcastle upon Tyne, UK

404.2 OOCYTE SHARING AS TREATMENT OF INFERTILITYAhuja, Kamal - London, UK

404.3 OPTIMIZING TREATMENT WITH OOCYTE DONATIONChillik, Claudio - Buenos Aires, Argentina

404.4 LUTHEAL PHASE SUPPORT, AN EVIDENCE-BASED APPROACHAboulghar, Mohamed - Cairo, Egypt


405.1 MECHANISM OF OOCYTE MATURATIONJones, Keith T. - Newcastle, Australia

405.2 SELECTION OF PATIENTS AND OOCYTE COLLECTION FOR IVMHolzer, Hananel - Montreal, Canada

405.3 PGD AND EMBRYO STATUS: ETHICAL AND MORAL ASPECTSDal Canto, Mariabeatrice - Monza, Italy

405.4 AMH/MIS AS A MARKER OF OVARIAN RESERVE AND PREDICTOR OF OVARIAN RESPONSELedger, William - Sheffield, UK

DETAILED PROGRAMME

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14:00-15:45 Concurrent Symposium 18 - Male Reproduction and ART Room: Salle 4

Chair: Palermo, Gianpiero - New York, USA

406.1 NEW GENETIC APPROACHES FOR THE SUCCESS OF ARTBenkhalifa, Moncef – La Verrière, France

406.2 IMPACT OF SPERM PREINCUBATION AND ACROSOME REACTION FOR ICSI OUTCOMEMansour, Ragaa - Cairo, Egypt

406.3 HOW TO STUDY AND SELECT THE BEST SPERM FOR ICSIZorn, Branko - Ljubljana, Slovenia

406.4 MALE GAMETE EMPOWERMENTPalermo, Gianpiero - New York, USA

14:00-15:45 Oral Communications 5 Room : Salle 18

Co-Chairs: Smith, Gary - Ann Arbor, USADemirol, Aygül - Ankara, Turkey

407.1 STUDY ON EFFECTS OF MALE REPRODUCTIVE TRACT INFECTIONS WITH AEROBIC BACTERIAON SPERM QUALITYKaluarachchi, Athula - Colombo, Sri Lanka

407.2 SEMINAL MORPHOLOGY AND THE OUTCOME OF ASSISTED REPRODUCTION TECHNIQUESKaluarachchi, Athula - Colombo, Sri Lanka

407.3 UTERINE SEPTUM DECREASES THE IMPLANTATION RATE IN IVF / ICSI CYCLESTomazevic, Tomaz - Ljubljana, Slovenia

407.4 A NOVEL PROCEDURE FOR SEVERE CASES OF ADENOMYOSIS – SURGICAL TREATMENT BY THETRIPLE-FLAP METHOD FOR RECONSTRUCTION OF THE UTERINE WALLOsada, Hisao - Tokyo, Japan

407.5 PREVALENCE OF MARKERS FOR THROMBOPHILIA AND IMMUNOLOGICAL DISORDERS IN PATIENTSHAVING IN VITRO FERTILISATION AND EMBRYO TRANSFERMettler, Liselotte - Kiel, Germany

407.6 ASSESSMENT THE OVARIAN RESERVE FOLLOWING CHEMOTHERAPY AMONG A COHORT OF CANCERPATIENTS WHO UNDERWENT UNILATERAL OOPHORECTOMY VERSUS PARTIAL OOPHORECTOMY FOROVARIAN TISSUE CRYOPRESERVATIONAzem, Foad - Tel-Aviv, Israel

407.7 VASCULAR COMPLICATIONS OF OVARIAN HYPERSTIMULATION (OHS)Dreyfus, Jean-Michel - Lyon, France


DETAILED PROGRAMME

April 19-22, 2009 38



16:15-18:00 Concurrent Symposium 19 - AMH and Fertility Room: Salle 3

Co-Chairs: Remohí Gimenez, José - Valencia, SpainBischof, Paul - Geneva, SwitzerlandKadayifci, Oktay - Turkey

408.1 ANEUPLOIDIES IN OVARIAN STIMULATIONRemohí Gimenez, José - Valencia, Spain

408.2 BIOLOGY AND BIOCHEMISTRY OF AMH/MISJosso, Nathalie - Clamart, France

408.3 AMH/MIS AS A MARKER FOR THE PRESENCE OF TESTICULAR TISSUE AND AS A PARAMETER IN THEWORK-UP OF MALE INFERTILITYFenichel, Patrick - Nice, France

408.4 AMH IN PCO PATIENTSFanchin, Renato - Clamart, France

16:15-18:00 Concurrent Symposium 20 - Psychological and Social Aspect Room: Salle 4of ART Treatment

Co-Chairs: van der Poel, Sheryl - Geneva, SwitzerlandBoivin, Jacky - Cardiff, UK

409.1 LOW COST ASSISTED REPRODUCTION IN LOW-RESOURCE SETTINGSvan der Poel, Sheryl - Geneva, Switzerland

409.2 PSYCHOLOGICAL AND SOCIAL ASPECT OF ART TREATMENTVerhaak, Chris – The Netherlands

409.3 SECURING GOVERNMENT FUNDING FOR IVF TREATMENT: THE CANADIAN EXPERIENCEHanck, Beverly - Montreal, Canada

409.4 PSYCHOLOGICAL COMPLEXITY SURROUNDING ENDING IVF TREATMENTTakefman, Janet - Montreal, Canada

409.5 IMPACT OF STRESS ON IVF OUTCOMEBoivin, Jacky - Cardiff, UK

DETAILED PROGRAMME

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Co-Chairs: Jaconi, Marisa - Geneva, SwitzerlandAboulghar, Mohamed - Cairo, Egypt

410.1 EFFECTS OF L-GLUTAMINE AND STRAW SIZE, FREEZING RATE AND THAWING RATE UPON POST-THAWQUALITY OF HUMAN SPERMATOZOAAsherkaci, Hussian - Misurata, Libya

410.2 IN VITRO FERTILIZATION IN NATURAL CYCLE FOR CONCURRENT TRANSFER OF FRESH ANDFROZEN/THAWED EMBRYOSSmirnova, Anna - Moscow, Russia

410.3 N-ACETYL-CYSTEIN IMPROVES RESULTS OF LONG-TERM CULTURE OF FROZEN / THAWED HUMANOVARIAN TISSUEFabbri, Raffaella - Bologna, Italy

410.4 VITRIFICATION OF DAY 3 EMBRYOS IMPROVES THE POST THAW PREGNANCY RATESPai, Hrishikesh - Mumbai, India

410.5 HIGH SURVIVAL AND PREGNANCY RATE AFTER SINGLE EMBRYO TRANSFER USING CRYOTOPBLASTOCYST VITRIFICATION METHODOkubo, Tsuyoshi - Shinbashi Minato-ku, Japan

410.6 COMPARISON OF VITRIFICATION VERSUS SLOW COOLING FOR BLASTOCYST CRYOPRESERVATIONIN AN IVF PROGRAMDorfmann, Andrew - Fairfax, USA

410.7 FROZEN EMBRYO TRANSFER: THE EFFECT OF NUMBER TRANSFERD, STAGE TRANSFERDAND SYNCHRONISATION ON THE CLINICAL OUTCOMESalman, Tarique - Rochdale, UK

DETAILED PROGRAMME

April 19-22, 2009 40


NOTES

Geneva41

ORAL ABSTRACTS – MONDAY

200.2 – NATURAL CYCLE IVF/M

Jin-Ho Lim, Maria Infertility Hospital, Seoul, Korea.

The first live birth following in vitro fertilization (IVF) was produced from anatural cycle IVF (1). However, this was slowly replaced by ovary stimulatedIVF, because it was believed that the number of oocytes retrieved relates tothe embryos available for transfer and this directly affects the probability ofsuccessful pregnancy (2-4). At the beginning, the relatively inexpensiveclomiphene citrate was used to stimulate ovaries for producing multiplefollicles, but currently the ovarian stimulation protocols use the much moreexpensive gonadotropin-releasing hormone (GnRH) agonist or antagonist incombination with gonadotropin to generate multi-follicles in the ovaries.

Some women are extremely sensitive to stimulation with exogenousgonadotropin and are at an increased risk of developing ovarianhyperstimulation syndrome (OHSS), sometimes, a life-threatening condition(5). In addition, there is an anxiety that the long-term side effects ofrepeated ovarian stimulation may increase the risk of ovarian, endometrialand breast cancers (6). Although these problems are not encountered innatural cycle IVF treatment, a number of other problems arise, including anincreased risk of no oocyte retrieved during collection and no embryoavailable for transfer. Nevertheless, there is resurgence of interest in naturalcycle IVF treatment in recent years, because the efficiency of IVF technologyhas been improved markedly (7,8).

Immature oocyte retrieval followed by in vitro maturation (IVM) of theseoocytes is an attractive infertility treatment for women with infertility. Incomparison with ovary stimulated IVF treatment, the major advantages ofIVM treatment include avoidance of the risk of OHSS, reduced cost, andsimplified treatment. Immature oocyte retrieval followed by IVM of theseoocytes was initially shown to be a successful treatment for infertile womenwith polycystic ovary syndrome (PCOS), because there are numerous antralfollicles within the ovaries in this group of patients (9). Immature oocyteretrieval followed by IVM might be useful in up to approximately 30% ofwomen undergoing IVF treatment who have large numbers of antral folliclesincluding patients with PCOS (10). Additionally because of the low cost oftherapy, it is important to identify candidates for IVM technology for womenwith various causes of infertility. This strategy has been explored usingnatural IVF combined with IVM for women without PCOS (11-13).Therefore, it is important to evaluate the efficiency of natural cycle IVFcombined with IVM as a clinical treatment for infertile women.

As the protocol, the first line is to select the patients for natural cycle IVF/Mtreatment for all patients who came to our infertility clinic, if a total of morethan 7 follicles existed in both sides of ovaries at baseline ultrasound scanon day 3-5 of the menstrual cycle. The second ultrasound scan wasperformed on day 7-9 to ensure the size of follicles and endometriumthickness. Human chorionic gonadotropin (HCG; 10,000 IU) was given whenthe size of the leading follicles reached to 12-14 mm in diameter and thethickness of endometrium is >6 mm, and oocyte retrieval was performed 36hours later. The leading and the small follicles were aspirated bytransvaginal ultrasound-guided needle connected with aspiration pump.After oocyte collection, the mature oocytes from the leading follicles weresubjected insemination by intracytoplasmic sperm injection (ICSI)immediately, and the immature oocytes were transferred to IVM-Medium forculture. The immature oocytes were assessed for its maturity 24 hrs afterincubation. If any oocytes matured, they were inseminated by ICSI. Theresulted embryos from each patient were pooled together for transfer on day3-4 following oocyte retrieval. For the preparation of the endometrium, thepatients were given estradiol daily starting on the day of oocyte retrieval.Luteal support was provided progesterone daily starting on the day of ICSI.

We obtained approximately 40% clinical pregnancy rate, and more than50% of patient population can be treated with natural cycle IVF/M or IVMalone, indicating that natural cycle IVF/M is an efficient alternative ofinfertility treatment. We propose the new concept, namely, natural cycleIVF/M, which is an efficient infertility treatment for all indications, especiallyfor women under 35 years of age. In conclusion, the results from our datademonstrate that natural cycle IVF/M together with IVM alone can be usedto treat more than a half of population of infertile patients with acceptablepregnancy rate.

Main References:

Steptoe PC, Edwards RG. Successful birth after IVF. Lancet 1978; 312: 366.

Lopata A, Brown JB, Leeton JF, et al. In vitro fertilization of preovulatoryoocytes and embryo transfer in infertile patients treated with clomipheneand human chorionic gonadotropin. Fertil Steril 1978; 30: 27-35.

Johnston I, Lopata A, Speirs A, et al. In vitro fertilization: the challenge ofthe eighties. Fertil Steril 1981; 36: 699-706.

Jones HW Jr, Jones GS, Andrews MC, et al. The program for in vitrofertilization at Norfolk. Fertil Steril 1982; 38: 14-21.

Beerendonk CCM, van Dop PA, Braat DDM, Merkus JMWM. Ovarianhyperstimulation syndrome: facts and fallacies. Obst Gynecol Surv 1998; 53:439-449.

Brinton LA, Moghissi KS, Scoccia B, Westhoff CL, Lamb EJ. Ovulationinduction and cancer risk. Fertil Steril 2005; 83: 261-271.

Nagund G, Waterstone J, Bland JM, et al. Cumulative conception and livebirth rates in natural (unstimulated) IVF cycles. Hum Reprod 2001; 16: 258-262.

Lukassen HGM, Kremer JA, Lindeman EJM, et al. A pilot study of theefficiency of intracytoplasmic sperm injection in a natural cycle. Fertil Steril2003; 79: 231-232.

Chian RC, Buckett WM, Tulandi T, Tan SL. Prospective randomized study ofhuman chorionic gonadotrophin priming before immature oocyte retrievalfrom unstimulated women with polycystic ovarian syndrome. Hum Reprod2000; 15: 165-170.

Buckett WM, Bouzayen R, Watkin KL, et al. Ovarian stromal echogenicity inwomen with normal and polycystic ovaries. Hum Reprod 1999; 14: 618-621.

Chian RC, Buckett WM, Abdul Jalil AK, et al. Natural-cycle in vitrofertilization combined with in vitro maturation of immature oocytes is apotential approach in infertility treatment. Fertil Steril 2004; 82: 1675-1678.

Lim JH, Park SY, Yoon SH, et al. Combination of natural cycle IVF with IVMas infertility treatment. In Chapter 27: In-vitro Maturation of Human Oocytes,basic science to clinical application edited by SL Tan, RC Chian, WMBuckett. Informa Healthcare Press, London, UK. 2007; Pp 353-360.

Lim JH, Yang SH, Chian RC. New alternative to infertility treatment forwomen without ovarian stimulation. Reprod BioMed Online 2007; 14: 547-549.

200.3 – ONCOFERTILITY: THE PRESERVATION OF FERTILITY OPTIONS FORYOUNG PEOPLE WITH CANCER

Teresa K. Woodruff, The Feinberg School of Medicine, NorthwesternUniversity, Chicago, IL, USA.

Cancer is now a disease with a variety of treatment options, which areleading to longer and more productive lives by survivors. Globally, there are10 million people diagnosed with cancer. 10% of these newly diagnosedmen and women are under the age of 45 years old. Infertility can be aconsequence of many of the more aggressive chemo- and radiationtherapies that prolong and save lives. The ability to easily preserve spermprior to cancer treatment provides hope at the time of diagnosis and familieslater in life for male survivors. A notable example is Tour de France winnerLance Armstrong who has three children conceived using sperm frozen daysbefore he underwent the massive chemo- and radiation therapy that savedhis life. Unlike sperm, the female germ cell, the oocyte or egg must beretrieved surgically. Moreover, the vast majority of collected oocytes will beimmature and cannot be used immediately by a woman who is ready to starta family. The overall hypothesis of the program is that effective fertility-extending options can be provided to young women undergoinglife-preserving cancer treatment. The purpose of our work is to bringphysicians, medical ethicists, social scientists and basic scientists togetherto develop new strategies for fertility preservation for female cancersurvivors under the new discipline of oncofertility. And even as the lexicon isbeing established, complex bioethical issues face both providers andparents. At the basic science level, complex issues of ovarian function andpreservation must be addressed including the problem of follicle growth anddevelopment in vitro. Our investigative group has pioneered the developmentof a 3-dimensional system that supports follicle development, largely, webelieve, because the links between the egg and its surrounding cells are

April 19-22, 2009 42


maintained. Using a tissue-engineered approach, we have developed an invitro follicle growth system that supports the maturation of the enclosedoocyte, which can be fertilized and results in live, healthy and reproductivelycompetent mice. The goal of our program and the broader OncofertilityConsortium is to explore and expand the reproductive options available toyoung people facing a fertility-threatening but life-preserving cancertreatment.

This work is supported by NIH/NICHD Structure-Function Relationships inReproductive Science, U54 HD041857; and, the Oncofertility Consortium,UL1DE019587 and RL1HD05829 http://oncofertility.northwestern.edu/

202.1 – FERTILOSCOPY BEFORE ART: A COMPLEMENTARY TOOL

A. Watrelot, J.M. Dreyfus. Centre de Recherche et d’Etude de la Stérilité(CRES®), Lyon, France.

We have described the technique of fertiloscopy in 1997.

Since then , we have demonstrated its efficiency, and its accuracy comparedto laparoscopy in a randomized prospective manner (the FLY study- Hum;Reprod.2003,18:834-839).

After performing more than 2000 procedures, it seems now obvious thatFertiloscopy practised early in the infertile work-up represent a valuable toolto decide which kind of ART will be the most effective in infertile patientswith no obvious pathology.

If every thing is normal including the tubal mucosa, then Intra UterineInsemination (IIU) is the preferred option. When there is no pelvicpathology, systematic salpingoscopy and microsalpingoscopy allowshowing abnormal tubal mucosa in 22% of cases even if the tubes seempatent and macroscopically normal. In these cases, In vitro fertilization hasto be proposed directly.

Also in selected cases, reconstructive surgery per laparoscopy or perFertiloscopy may be offered if the tubal mucosa is normal

Therefore, using this mini-invasive approach, the best option is chosenwithout delay which decreases the time for patient to conceive

204.4 – HIGH PREVALENCE OF MARKERS FOR THROMBOPHILIA ANDIMMUNOLOGICAL DISORDERS IN PATIENTS HAVING IN VITROFERTILIZATION

L. Mettler, A. Salmassi, A. Schmutzler. Department of Obstetrics andGynaecology – Campus Kiel, University Clinics Schleswig-Holstein, Kiel,Germany.

Synopsis: In spite of the current disagreement among reproductiveimmunologists, the study showed a complex pattern of coagulation andimmunologic disorders impairing the outcome of IVF (66.7% of patients hadpathological assays).

Objective: As birth rates after IVF remain low at 26 %, the hypothesis of thehigh incidence of subclinical autoimmune and hematologic abnormalities inIVF populations [1] is investigated.

Methods: In a first limited retrospective study we analysed 123 unselectedpatients undergoing IVF. Blood samples were determined using a selectedpattern of hereditary (protein-S, -C-deficiency, factor V Leiden), acquiredthrombophilia (antiphospholipid antibodies) and immunological parameters(ANA, complement factor 4). We tested for only one antiphospholipidantibody although we are aware that other APAs may, in fact, be moreprevalent that ACA-Abs.

Results: No patient displayed clinical evidence for an active immunologicalor hypercoagulative disease. Prevalence of protein S deficiency (11.2 %),positive antiphospholipid antibodies (32.5 %) and ANA (30 %) weresignificantly higher than in the normal population (< 1 %;p < 0.005; ~2 %;p< 0.005; ~5 %;p < 0.005). Overall 82/123 (66.7%) patients were positive forany pathological marker, 31/123 (25.2%) patients had a combination ofparameters of hereditary and/or acquired thrombophilia and/orautoantibodies. Women with multiple abnormal tests were found to have astrong tendency towards a lower pregnancy rate after IVF, although thedifference did not reach statistical significance (38.7 % vs. 51.2 %respectively; p > 0.005).

Conclusions: In an unselected population of patients undergoing IVF, asubstantial share (66.7 %) demonstrates pathological assays forthrombophilia or immunological disorders without symptoms of a clinicallyactive disease. Every fourth (25.2 %) patient displays combinations of theselaboratory parameters. These patients show a strong tendency towardsdecreased pregnancy rates after IVF.

205.1 – ETIOLOGICAL FACTORS AND CLINICAL COURSE IN PATIENTS WITHPREMATURE OVARIAN FAILURE

Bunpei Ishizuka, Saint Marianna University School of Medicine, Kanagawa,Japan.

In an analysis of 130 consecutive cases with spontaneous prematureovarian failure (POF), we found X chromosomal abnormalities in 15% andFMR-1 premutation in 2.5% of the cases. About half of the cases showed atleast one type of autoantibody tested and about half of the cases withpositive autoantibodies had clinical autoimmune diseases.

In the present study, we analyzed the clinical course of POF of these patientswith different etiological factors.

205.3 – REGULATION OF GRANULOSA CELL APOPTOSIS BY GnRH

Peter C.K. Leung, University of British Columbia, Vancouver, BC, Canada.

In view of the wide applications of GnRH analogs in assisted humanreproduction, it is important to understand how GnRH exerts its effects indifferent tissues. The expression of GnRH-I, GnRH-II and their commonreceptor (GnRHR) has been demonstrated by immunohistochemistry in thehuman ovary. Our analysis of the 5’-flanking regions of the human GnRHRgene has indicated a differential usage of specific promoter regions invarious tissues. Mutational analysis demonstrates the importance of threeclosely spaced CCAAT/enhancer binding protein and GATA motifs, whichfunction cooperatively in regulating GnRHR gene transcription in humangranulosa cells. This granulosa cell-specific GnRHR promoter region furthersupports the importance of an autocrine GnRH-GnRHR system in thecontrol of ovarian follicle development in humans.

Recent studies in our laboratory have demonstrated the direct effect ofGnRH-II in regulating ovarian steroidogenesis. Like GnRH-I, treatment ofhuman granulosa cells with GnRH-II results in down-regulation of hCG-stimulated progesterone production. This inhibitory effect of GnRH-II canbe abolished by the GnRH-I antagonist Antide, suggesting a commonreceptor. Similar to GnRH-I, GnRH-II does not interfere with the hCG-stimulated cAMP production in the granulosa cells. Rather, these hormonesdown-regulate the mRNA levels of FSH and LH receptors. Treatment withGnRH-I produces a biphasic response in its own mRNA level such that highconcentrations decrease the GnRH-I mRNA level, whereas lowconcentrations increase GnRH-I gene expression. In contrast, treatmentwith GnRH-II results in a homologous down-regulation of its mRNA level atall concentrations. The expression of GnRHR is differently regulated byGnRH-I and GnRH-II. Also, the expressions of GnRH-I and GnRH-II areregulated differentially by FSH and hCG, such that the gonadotropinsincrease the mRNA level of GnRH-II but decrease that of GnRH-I in hGCs.Both GnRH-I and GnRHR mRNA levels have been shown to be down-regulated by estradiol . By contrast, estradiol increased GnRH-II mRNAlevels in granulosa cells.

We have further observed that GnRH-I and -II induce apoptosis in humangranulosa cells through GnRH-I receptors, which mediate the proteolyticcaspase cascade involving caspase-8 (the initiator) and caspase-3 and 7(the effectors). GnRH-I and II induced TUNEL-positive apoptotic cells andincreased cleavage activities of caspase-8, 3 and 7 by 48 hours and peakedat 72 hours. Antide effectively blocked these TUNEL-positive changes andexpression levels of caspase-3 induced by GnRH-I or II. Activation ofcaspase-8, 3 and 7 was inhibited by the corresponding caspase inhibitor.Caspase-8 inhibitor also abolished cleavages of caspase-3 and 7 induced byGnRH-I and II. Interestingly, FSH protects human granulosa cells fromapoptosis induced by GnRH I or II. This raises potentially important roles ofGnRH-I and GnRH-II, together with FSH, in regulating ovarian follicledevelopment and atresia.

[Supported by the Canadian Institutes for Health Research]

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206.1 – HIGH SURVIVAL AND DEVELOPMENTAL RATES OF VITRIFIED MOUSEZYGOTES FOLLOWING POLAR BODY BIOPSY

E. Macas, X. Min, R. Stiller, S.G. Merki-Feld, P. Pelczar, B. Imthurn. Division ofReproductive Endocrinology, University Hospital Zurich, Zurich, Switzerland.

Introduction: Pre-implantation genetic diagnosis (PGD) on polar bodies is one ofthe most technically complex procedures within the field of assisted reproductivetechnology. Numerous specific problems can arise during any phase of thisprocedure, resulting in either an incomplete genetic diagnosis or none at all. Forinstance, the actual biopsy itself poses serious technical difficulties, and even asmall error made here may lead to the loss or damage of the polar bodies.Subsequent factors such the extensive micromanipulation of the polar bodyrequired during hypotonic treatment and fixation, as well as errors made duringfluorescence in-situ hybridization, may also seriously affect the safety andoutcome of the entire biopsy procedure. Because of these problems, the need foran effective freezing programme is particularly important in order to avoidrepetition of polar body biopsy if the excess zygotes are found to bechromosomally normal. However, studies conducted on biopsied zygotesfollowing application of conventional slow-freezing protocols are limited, and thefew results obtained thus far have been rather disappointing.

In the present study, therefore, before introducing it into routine practice of theDivision of Reproductive Endocrinology, University of Zurich, cryopreservation byvitrification was tested using a mouse zygote. Applying this model the effect oftwo different methods of polar body biopsy followed by vitrification on thesurvival and development to blastocyst stage was examined.

Material and Methods: Prior to vitrification, a total of 119 and 124 mousezygotes were subjected to polar body biopsy using either laser-assisted or partialzona dissection (PZD) techniques, respectively. Vitrification was also applied to122 zona-intact zygotes that served as a control group.

Results: Following vitrification, no differences in the rate of zygote survival(95.8%, 91.9% and 94.2%) or in the rate of development to expanded blastocyststage (82.3%, 79.8% and 81.9%) were observed between the two groups ofbiopsied zygotes, or between the biopsied zygotes and control zygotes. The meantotal number of cells comprising the blastocysts of controls (77.1 ± 4.7) wascomparable to the mean cell number recorded in the laser (66.4 ± 4.7) and PZD(69.7 ± 5.3) groups. Blastocysts developed from laser-treated zygotes hatchedmuch earlier than blastocysts developed from the control and PZD groups ofzygotes (P < 0.001).

Conclusion: The data obtained in the present study demonstrate that,irrespective of the biopsy method used prior to vitrification, mouse zygotessurvive and develop to blastocysts upon warming in proportions similar to thoseof non-biopsied zygotes.

206.2 – COMPUTER-ASSISTED ANALYSIS OF HUMAN PRONUCLEAR ZYGOTES

F. Urner1, A. Beuchat2, C.O.S. Sorzano2, P. Thevenaz2, M. Unser2, T. Ebner3, A.Chanson1, M. Germond1, A. Senn1. 1FABER Foundation and Centre of MedicallyAssisted Procreation, Lausanne; 2Biomedical Imaging Group, Swiss FederalInstitute of Technology of Lausanne; 3Women’s General Hospital, IVF-Unit. Linz,Austria

Introduction: To decrease the number of multiple pregnancies without affectingpregnancy rates after in vitro fertilisation, a low number of embryos withpresumably high chances of implantation is usually transferred. In this situation,the accurate prediction of implantation is crucial. The assessment of zygotemorphology is one of the available tools to predict implantation when embryoselection is not permitted, as in Switzerland. The aims of the present study are toprovide a software tool able to objectively measure several morphologicalfeatures of the pronuclear zygote and to identify which of these features areuseful to predict embryo implantation.

Materials and Methods: To objectively analyse pronuclear zygote digital images,a computer-assisted method was developed by creating a plug-in of the imageprocessing ImageJ (http://rsb.info.nih.gov/ij). Images of zygotes were captured atthe pronuclear stage 16-20 h after insemination under Hoffman contrast by usingan Octax camera (MTG, Herborn, Germany). Images of 350 pronuclear zygotes,corresponding to transferred embryos with known individual implantationoutcome, were provided by two different IVF centres (Lausanne and Linz). Theywere then analysed by using the ImageJ plug-in and by subjective scoring asdescribed previously by Senn et al. (Human Reproduction 21 :234-239, 2006).

The accuracy of the different zygote features to predict implantation wasdetermined by ROC analysis. The area under the ROC curve (AUC) was used todetermine the global performance of each feature to predict implantation.

Results: Zygote images coming from the two IVF centres were successfullyanalysed with the software, resulting in a series of precise measurements of 24variables for each zygote. Following this analysis, the only feature presenting asignificant difference between implanted (n=99) and non-implanted (n= 251)zygotes was nucleolar precursor bodies (NPB) distribution quantified bymeasuring the mean distance between NPB and the line separating the twopronuclei (13.37±3.4 microns versus 14.39±3.03 microns, p=0.001).Subjectively scored NPB distribution was also significantly different betweenimplanted and non-implanted zygotes (2.08 ± 0.56 versus 1.89±0.59, p=0.008).According to ROC, the computer-assisted measured NPB distribution presentedan AUC of 0.61 (95% CI 0.56-0.66) that indicates a poor prognostic efficiency ofthis parameter. However, all the other parameters presented lower AUC values. Atan optimal threshold value (≤ 11 microns), specificity was high (88%) butsensitivity was low (33%), indicating that implantation was correctly predicted foronly 33% of implanted zygotes. Compared to computer-assisted measurement,subjective scoring of NPB distribution resulted in a lower AUC (0.58, 95%CI 0.52-0.63) and a significantly lower sensitivity (20 %, p=0.000) at a specificity of 87%.

Conclusions: The morphology of human pronuclear zygotes can be objectivelycharacterised by using an advanced image analysis tool. Among all the featuresanalysed, NPB distribution appears as the most useful marker to predictimplantation. However, its predictive value remains limited, probably becauseother factors, not related to zygote morphology, play a critical role in embryodevelopment and implantation. When embryo selection cannot be performed forlegal constraints, the choice of zygotes on the basis of the distribution of theirNPB remains an alternative that contributes to increase the implantation ratesafter fresh transfer.

206.3 – MATURE OVARIAN FOLLICLES CONTAIN A SUBPOPULATION OF STEMCELLS

K. Kossowska-Tomaszczuk1, H. Zhang1,2, A. Scherberich2, I. Martin2, Ch. DeGeyter1,2. 1Woman’s Hospital; 2Department BioMedicine ; University Hospital ofBasel, Basel, Switzerland.

Introduction: Luteinzing granulosa cells (GCs) reside in mature Graafian folliclesand are considered to be terminally differentiated destined to undergo apoptosiswithin several days. We now demonstrate that a subpopulation of cells collectedfrom Graafian follicles possess all characteristics of multipotent stem cells.

Material and Methods: Luteinizing GCs, collected from infertile women treatedwith assisted reproduction, were identified and sorted with a fluorescence-activated cell sorter (FACS) based upon the presence of the follicle-stimulatinghormone receptor (FSHR). These sorted cells were then cultured in a culturemedium either supplemented with the leukemia inhibing factor (LIF) or not.

Results: Luteinizing GCs cultured in the absence of LIF invariably becameapoptotic after approximately 10 days. However, in the presence of LIF luteinizingGCs remained viable for up to three months and obtained many of thecharacteristics of multipotent stem cells, as given by the expression of varioussurface markers such as CD29, CD44, CD90, CD105, CD117 and CD166. Duringprolonged culture in the presence of LIF the sorted cells progressively lost theirability to express both FSHR and aromatase. In contrast, luteinizing GCscontinued to express OCT4, a marker of multipotent stem cells, but not vasa,nanog nor stellar, markers of the germ-stem cell line. The multipotency of thesecells was then demonstrated by their differentiation into the neuronal,osteoblastic and chondrogenic lineages when incubated in various specificculture media. In addition, we were able to demonstrate that multipotent stemcells developed from luteinizing GCs in the presence of LIF grew intochondrogenic and osseous tissue after transplantation into the back of immuno-incompetent nude mice.

Conclusion: For the first time we were able to demonstrate that mature Graafianfollicles contain a subpopulation of multipotent stem cells. This novel finding mayhave important implications for current concepts on follicular recruitment, thedevelopment of ovarian endometriosis and the pathogenesis of ovarian cancer.

April 19-22, 2009 44


206.4 – INTERMEDIATE AND PREMUTATION FMR1 ALLELES IN WOMEN WITHOCCULT PRIMARY OVARIAN INSUFFICIENCY

I. Streuli, T. Fraisse, V. Ibecheole, I. Moix, M. Morris, D. de Ziegler. HôpitauxUniversitaires de Genève et Centre Hospitalier Universitaire Vaudois, Geneva,Switzerland.

Introduction: The FMR1 premutation is now an established cause of primaryovarian insufficiency (POI) and it is currently recommended to test women withovert ovarian insufficiency. These recommendations led infertility specialists inGeneva to request FMR1 testing more frequently from early 2005. Forty-fourwomen were referred by infertility specialists for FMR1 testing between January2005 and September 2006. Most women referred had signs of occult ovarianinsufficiency (oPOI, defined as FSH levels >10 UI/L, AMH levels < 7 pmol/L and/ora resistance to ovarian stimulation in infertile women with either regular orirregular cycles under the age of 40 years) but did not meet the criteria for overtPOI. Whether women oPOI are at risk of carrying expansions in the FMR1 gene iscurrently not clear.

The primary aim of our study was to establish whether intermediate andpremutation alleles are more frequent in the population of infertile women withoPOI than in a control population.

Material and Methods:We compared the incidence of upper normal,intermediate and premutation FMR1 alleles in women with occult primary ovarianinsufficiency (oPOI) and in controls. In the study group we included 27 womenwith oPOI and a normal karyotype referred by infertility specialists for FMR1testing in 2005-2006 because of unexplained poor response to COH or alteredhormonal profiles. In the control group we included 32 women undergoinggenetic testing for conditions unrelated to mental retardation or ovarian function.

Results: In the group of women with oPOI, 4/54 alleles were in the upper normalrange (35-40 repeats), 4/54 were in the intermediate zone (41-60 repeats) and3/54 were premutated (61-199 repeats). In the control group, 1/64 allele was inthe upper normal range, 1/64 in the intermediate range and none waspremutated. We compared the prevalence of alleles with 41-199 repeats andalleles with 35-60 repeats between the groups. The differences were statisticallysignificant with p=0.02 et p=0.015 respectively.

The analysis was also conducted at the level of individuals. One woman carriedan intermediate and a premutation allele, and another carried two upper normalrange alleles. Therefore, in the oPOI group, 7/27 women had alleles ranging from35-60 repeats and 6/27 alleles ranging from 61-199 repeats. In the control group1/32 woman had an upper normal range allele and 1/32 an intermediate allele.There is a relative risk of 7 of having an allele with 41-199 repeats (intermediateand premutation range) in women with oPOI compared to controls with p=0.037.At the individual level, the difference between groups for alleles between 35-60repeats is not significant with p = 0.065.

Conclusion: The results of our study show an increased incidence of alleles inthe intermediate to premutation range in patients with oPOI compared to thegeneral population. For this reason, the increased screening in Geneva seemsjustified. In light of our findings, further efforts should be put into studying theincidence of FMR1 expansions in all forms of ovarian insufficiency includingotherwise unexplained infertility. We suggest that FMR1 testing has a role in thecomprehension of the aetiology of ovarian insufficiency and in the prevention oftransmission of Fragile X syndrome and should be considered before infertilitytreatments are initiated.

206.5 – GENE EXPRESSION PROFILING OF CULTURED ENDOMETRIUM FROMWOMEN UNDERGOING IN VITRO FERTILISATION (IVF) WITHDIFFERENT OUTCOME

N.A. Bersinger, M.D. Mueller, M.H. Birkhäuser, D.M. Wunder. Klinik undPolikliniken für Gynäkologie und Geburtshilfe, Universitätsfrauenklinik InselspitalBern, Bern, Switzerland.

Introduction: Successful embryo implantation is only possible during a shorttime window within the luteal phase. Estradiol and progesterone are crucial forthe acquisition of receptivity and the change in transcriptional activity of targetgenes. Genomic microarrays have been used since 2002 to study theimplantation window. Comparing mid- to early luteal phase transcriptionalactivity in the endometrium a considerable number of genes was found to bedifferentially expressed. For many of these a role in the context of endometrialreceptivity or even fertility had not been previously described, but one of the

strongest transcriptional upregulations was observed for glycodelin A (PP14)which is known to be a progestagen-dependent, immunosuppressive, secretoryepithelial endometrial product with high serum levels towards the end of theluteal phase. Later the same technique was used to compare gene expressionprofiles from endometrium after controlled ovarian hyperstimulation with tissueobtained in natural cycles. Again, differential expression was found for >200genes, and it was concluded that current ovarian stimulation for IVF treatmentwas far from optimal. Gene expression profiles in patients with recurrentmiscarriage in comparison to ongoing, uneventful pregnancy are not wellinvestigated to date. The aim of this study was to differentiate the transcriptionalregulation of genes in the endometrium of patients with recurrent implantationfailure (IF) versus those who became pregnant after IVF treatment, andparticularly between miscarried (M) and ongoing clinical pregnancies (OP).Moreover, the effect of embryo-derived factors on endometrial transcriptionalactivity was studied.

Material and Methods: Nine women with known IVF outcome (IF,M, OP) andundergoing hysteroscopy were enrolled. An endometrial tissue sample wasbiopsied using a soft suction curette. Mean patient age was 33.7 ± 4.5 yearswithout difference between the groups. All tissue samples were taken during themidluteal phase (day 23.6 ± 1.8 of a 30.0 ± 1.6 day cycle). Explant suspensioncultures were set up in order to maintain an intact three-dimensionalenvironment. After culture in presence of embryo-conditioned IVF media, totalRNA was extracted using a method which included on-column DNAse treatment.Three micrograms of total RNA per culture condition were submitted to reversetranscription, target cDNA synthesis, biotin labelling, fragmentation, andhybridisation using the Affymetrix Human Genome U133A 2.0 Chip which covers18,400 transcripts and variants including 14,500 characterised genes. Using theautomated ranking of differential expression and their relevance in reproductivefunction, genes were selected for specific investigation with quantitative, real-time polymerase chain reaction (PCR) using the Taqman assay-on-demandprotocol. Results were calculated as threshold cycle (Ct) differences between theIVF outcome groups and normalised to GAPDH and �-actin.

Results: Automatic screening of the raw hybridisation data identified the geneswhich were found to be differentially up- or downregulated by a factor of 2 ormore between the different IVF outcome conditions. When comparing endometrialtissue from OP with M, 1875 up- and 1807 downregulated genes were returned.Real-time PCR analysis confirmed upregulation for somatostatin, PLAP-2, mucin4, and CD163, and downregulation of glycodelin, IL-24, CD69, leukaemiainhibitory factor, and prolactin receptor. When the different embryo conditionedmedia were compared, no significant differential regulation could bedemonstrated.

Conclusions: Microarray profiling may currently not be sensitive enough forstudying the effects of embryo-derived factors on the endometrium. But given theobserved differences between M and OP, we believe that it will become aninteresting tool for the identification of fertility and obstetrically relevant markersproduced by the endometrium in natural and stimulated cycles.

207.1 – DETECTION OF OXIDATIVE DNA DAMAGE BY FLOW CYTOMETRY ANDITS ASSOCIATION WITH MALE INFERTILITY

Chakroun Feki Nozha1, Zribi Nassira1, Eleuch Henda2, Gdoura Radouane3,Sellami Afifa1, Bahloul Ali4 Hammami Adnene3, Gargouri Jalel2, Rebai Tarek1,Keskes Ammar Leila1. 1Laboratoire d’Histologie Embryologie, Faculté deMédecine de Sfax; 2Centre de transfusion sanguine, Sfax; 3Laboratoire deMicrobiologie, Faculté de Médecine de Sfax; 4Unité de recherche infertilitémasculine UR07US0011; Tunisie.

In the last two decades, the expanding research interest on the impact ofsperm DNA damage on the ability of sperm to fertilize and on the pregnancyoutcome, has led to the development of various techniques evaluatinggenome integrity of spermatozoa. DNA oxidation has been considered asone of the important factors which affect sperm quality and increase the riskof genetic defects. Our aim with this work was to evaluate oxidative damagein human spermatozoa of infertile Tunisian men using the 8-oxo-guaninemeasurement by flow cytometry and to correlate results sperm DNAoxidation with semen parameters. Our work was performed on 15 semenspecimen coming from 15 infertile men; semen aliquots obtained bymasturbation were analyzed according to WHO guidelines. Leukocyteconcentration was determined by peroxidase staining using aheamocytometric method. We distinguished arbitrary 2 groups according tothe value of leucocytospermia [< 250000/ml (G1) and ≥ 250000/ml (G2)].

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We measured sperm DNA oxidation by flow cytometry using the Oxi-DNAassay and correlated it with semen parameters and leucocytospermia. Weused linear regression to correlate DNA oxidation, leukocyte concentrationand semen parameters.

Interestingly, sperm DNA oxidation was correlated to leukocyteconcentration in semen (p=0,006, r=0,7) and was significantly higher in G2(p=0,03). These results suggest that low rates of leukocytes in semeninduce DNA oxidation and that the 8-oxo-guanine flow cytometry test can beconsidered a sensitive biomarker of oxidative stress in human spermatozoa.

207.2 – IMPRINTING IN THE HUMAN EMBRYO: EVIDENCE FOR A RECYCLINGOF HOMOCYSTEINE IN THE OOCYTE

Moncef Benkhalifa, Debbie Montjean, Paul,Cohen-Bacrie, Yves Menezo.UNILABS, Laboratoire d’Eylau, Paris, Ile de france, France.

Introduction: Assisted reproductive technology, especially with poor qualitysperm has been recently in the centre of controversies concerningeprgenetic risks. Epigenetic plays an important role in many cancers thatappear late in life and may have roots in epigenetic variation established inearly development. One of the very early epigenetic regulations in life is DNAmethylation strongly involved in imprinting, catalysed by DNAmethyltransferase and requiring S-Adenosyl-Methionine (SAM) as a methyldonor. Imprinting which also regulates implantation. Homocysteine (Hcy),released after méthylation, is a well known inhibitor of methylation

Material and Methods: We have used microarrays, Affymetrix HG U-133plus 2.0 chips, in order to determine the expression of the pathwaysremoving or recycling homocystein, in 6 pools of GV oocyte collected at thetime of ICSI

Results: We confirm our previous data indicating that SAM synthesis isactive: There is a high level of expression for methionineadenosyltransferase (MAT) II, alpha and MAT II beta. The S-Adenosyl-homocysteine hydrolase which releases homocysteine from SAH is stronglyexpressed: this leads to high level of formation of Hcy post methylation.

All the enzymes involved in recycling of Hcy are highly expressed: 5, 10methylene tetrahydrofolate reductase (MTHFR) and methionine synthase(MTR). We found also that methionine can be form using the betainehomocysteine pathway (BHMT2). However we were unable to detect anysignal for cystathionine beta synthase (CBS), which allows recyclinghomocysteine towards cysteine, contrarily to what is currently observed inanimals.

Conclusions: If we consider the biochemical pathways present andconsidering that at least a part of the mRNAs detected are translated, thehuman oocytes is able to keep the Hcy level in check via re-methylation. Thisaspect is important as recent works have shown that controlled ovarianhyperstimulation affects the homocysteine concentration in the follicularfluid. This recycling may alleviate, the imprinting inhibitory effect of Hcy; yetthis can be modulated by maternal age and culture conditions it has to bementioned here that these two methionine recycling pathways are alsopresent in the testis

ADA: Adenosine de-aminase, AK: adenosine kinase, CBS: cystathionine betasynthase, MAT: méthionine adénosyltransférase, MTAses: various methylasesMTR: Methionine synthase, MTHFR: Methylene tetra hydrofolate reductase,SAHH: SAH hydrolase

207.3 – HIV SERO-CONVERSION OF A SPERM DONOR DURING THE DONATIONPERIOD

Venessa Smith, Geetha Venkat, Eric Simons, Shailaja Nair, Kamal Ahuja.London Womens Clinic, London, UK.

Background: An incident of HIV sero-conversion of a donor during thedonation period is documented. We also report the subsequent efforts madeby our clinic to ensure the safety of patients and staff and to rule out othersamples which might have been exposed to the contaminated material.

The recruitment policies of the donor bank are based on recommendationsby BAS, BFS, NICE, BICA and the HFEA Code of Practice. The sperm donorsuccessfully passed initial serum and STD screening and joined the programin August 2005. During the subsequent 5 months his donations declined inquality, as a result few were stored. His inclusion was then terminated.

Routine serology screening was repeated in order to assess the suitability ofhis samples for patient treatment. The “signal response” to the HIV test waspositive. A repeat test excluded a false positive result.

Methods: Immediate action was taken to isolate all samples that may havebeen in contact with the contaminated material. The HFEA were immediatelyinformed and the donor offered immediate consultation and counseling.There were 3 areas of concern, direct recipient infection, indirectcontamination of adjacent samples and risk of infection of laboratory staff.A risk assessment of other samples within the same storage vessel becameimportant.

Results: All stored vials from this donor were tested for HIV viral load. 4 ofthe 7 stored semen samples did express viral copies of 1 HIV gene or more.However, the viral load was only significant in one sample. This sample wasthe first stored immediately after the initial negative HIV serology test andyet 9470 viral copies/ml with 3 HIV genes were found.

Within that storage tank there were samples from 34 different donors, ofwhich 3 were stored within the same canister as the HIV infected donor.Random selection of these samples were also tested HIV viral load, using 4methods. No HIV was detected. However, all the samples were discarded.

Discussion: Investigation was complicated by the difficulty of obtaining clearadvice regarding the contamination risk to staff and the frozen samples inthe same dewar. Various scientific bodies and individuals were questionedbut without any contribution of value. Literature searches confirmed thatcross contamination of samples through liquid nitrogen has never beendocumented and this case emphasizes that contamination does not seem tooccur.

Although this was essentially a fact finding exercise we have modified ourstorage arrangements to reduce the impact of a recurrence. This includesmore diligent analysis of any change in sample quality and more regularassessment of risk behavior. The HFEA were supportive and helpful at alltimes. Their system of ‘alerts’ is recommended. We feel though that thisdoes not supersede the established scientific practice of early recourse topublication and dialogue.

207.4 – PRESENTATION OF INNOVATIVE PATENTED PRODUCTS FOR OVUMPICK UP AND EMBRYO TRANSFER

Hans-Peter Steiner, Institute for In-Vitro-Fertilization and Endocrinology,Graz, Upper Austria, Austria.

The following innovative devices for IVF/ET are products of the author`s 20-years experience in IVF.

The current worldwide ovum pick up technique in in vitro maturation (IVM)is using a single lumen needle without flushing the follicles. In order to flushthe needle system accordingly repeated withdrawal and reinsertion of theneedle into ovaries (up to 15- 20 times) was necessary so far. Thisprocedure is painful, time consuming and there is a high risk forcontamination of the cumulus-oocyte-complex with blood. STEINER Needle,on the other hand, is 18 Gauge having a double lumen (starting 7cmproximal of needle tip) allowing for flushing of one follicle at a time within afew seconds without removal of the needle and, thus, minimizing thecontamination with blood. In addition, no filters are needed.

In times of mild IVF and IVM flushing follicles has a revival. STEINER Flushis the first mechanical flushing pump worldwide activated by the physician´s foot via a cable pull and it enables to choose the amount, velocity andpressure of flushing medium.

ETFLEX Steiner was particularly invented for difficult embryo transfers aswell as mock Transfers (it has a cm scale). The metallic outer catheter with aspiral tip (up to 5.5cm) can be used as a disposable or reusable device. Thespiral tip is clearly seen at ultrasound.

The STEINER Pistol enables the embryologist and physician to aspirate andinject a reproducible amount of fluid during Embryo Transfer. Per click 5 or10µl can be aspirated or transferred.

Short videos sequences and computer animations will complete thepresentation.

April 19-22, 2009 46


207.5 – COMBINING TWO ZWITTERIONIC BUFFERS IN IVF HANDLING MEDIASUPPORTS MOUSE BLASTOCYST DEVELOPMENT AND NORMALHUMAN OOCYTE FERTILIZATION FOLLOWING ICSI

Jason E. Swain1, Virginia Ord2, Darleen Taylor2, Veronica Sossamon2,Thomas B. Pool2. 1Center for Reproductive Medicine, University of Michigan,Ann Arbor, MI; 2Fertility Center of San Antonio, San Antonio, TX; USA.

Introduction: Maintenance of stable pH is important for optimizing gameteand embryo culture. This is especially true when handling the denudedoocyte, which lacks robust pH regulatory mechanisms. One method tostabilize pH entails using zwitterionic buffers in handling media used outsidethe laboratory incubator. Current handling media utilize single buffers, suchas MOPS or HEPES. However, use of a single buffer limits the ability toadjust optimal buffering capacity. For example, the pKa, or optimalbuffering of MOPS at 37°C is 7.02, which is below the traditional pH rangeused to handle oocytes and embryos of ~7.3. Furthermore, potentialtoxicity concerns exist with elevated concentrations of buffers, such asHEPES, required in mono-buffered handling media. Therefore, traditionalhandling media utilizing a single buffer may not be ideal.

Materials and Methods: Media was prepared, varying the type and amountof zwitterionic buffer. Media containing HEPES, MOPS, or HEPES+MOPSwere tested. All other parameters, including pH and osmolarity werecontrolled. An acid/base challenge, using incremental addition of 0.1M HCLand NaOH, followed by pH measurements, was used to examine bufferingcapacity of various media. Additionally, blastocyst development and cellnumber of frozen/thawed 1-cell mouse embryos grown in respective mediaat 37°C in room atmosphere was used to validate efficacy. Finally,fertilization and embryo development following microinjection of humanoocytes in our standard HEPES-buffered medium was compared to aHEPES/MOPS-buffered medium. Participants included all patients (n=12)undergoing ICSI with at least 6 mature eggs. Half of the oocytes wereinjected in a 10mM HEPES-buffered Tyrodes medium. The remaining matureoocytes were injected in media containing a 1:1 ratio of MOPS/HEPES (5mMof each). Treatment groups were alternated in respect to their order ofinjection to avoid bias. All other conditions besides holding media used formicroinjection remained consistent between treatments. Data were analyzedusing Chi-Square.

Results: We confirmed the ability of a double-buffered handling media,containing both HEPES and MOPS, to allow adjustment of pKa value andoptimal buffering capacity compared to media containing HEPES or MOPSalone. This allowed for the subsequent reduction of individual bufferconcentrations, while maintaining overall buffering capacity. This double-buffered media also yielded similar rates of mouse blastocyst developmentand total cell numbers compared to media containing only a single buffer.Though no significant differences exist, injection of human oocytes indouble-buffered media with HEPES/MOPS (n=69) tended to have greaternormal fertilization (71.0% vs. 62.7%) and lower abnormal fertilization(4.3% vs. 11.9%) than oocytes injected in HEPES alone (n=67). Nodifferences were apparent in rates of multi-nucleation on day 3 betweenMOPS/HEPES and HEPES alone (12.2% vs. 16.7%). Continued experimentsto increase patient number may allow data to reach significance.

Conclusion: Use of a double-buffered handling medium for ICSI is at least aseffective as a traditional HEPES-only buffered medium. This approach ofcombining multiple organic buffers to regulate media pH offers the potentialadvantages of reducing individual buffer concentration and resulting toxicityconcerns, as well as the ability to fine-tune and optimize maximal bufferingcapacity at a specific pKa value at a specific temperature.

207.6 – CORRELATION BETWEEN REACTIVE OXYGEN SPECIES (ROS) INSEMINAL PLASMA AND SPERM VITALITY, MEMBRANE, DNAINTEGRITY, DNA STRAND BREAKS OF INFERTILE MEN BEFORE ANDAFTER SEMEN PROCESSING BY PURESPERM AND IVF/ICSI OUTCOME

M.E. Hammadeh, C. Fischer-Hammadeh, A.S. Amer, M.F. Hamad, M. Deryal.Department of Obstetrics and Gynecology, University of Saarland,Homburg/Saar, Germany.

Introduction: the purpose of this study was to determine the effect of ROS inseminal plasma of infertile men undergoing IVF/ICSI therapy on the spermvitality, membrane integrity, DNA integrity and DNA strand breaks before

and after semen processing by Pure Sperm gradients centrifugation and itseffect on fertilization and pregnancy rate.

Material and Methods: Semen sample of patients (n= 87) attending ourclinic were collected from men undergoing IVF (n=36) or ICSI (n=51)therapy after minimum 3 days of sexual abstinence. A routine semenanalysis was performed according to WHO guideline (1999). Semensamples were prepared using two layers (90/45) of PureSperm gradientcentrifugation. Each aliquot of liquefied semen was layered on top of thegradient and centrifuged at 250xg for 20 min. The sperm pellet was used foroocytes insemination or injection. Semen vitality was evaluated using Eosintest and the membrane integrity was evaluated by performing Hypo osmoticswelling test (HOS-test). Besides, the DNA integrity of individualspermatozoa was determined using chromomycine staining CMA3 (Sigma).Whereas, DNA stand breaks was evaluated using TUNEL-Kit (Hoechst,company). ROS was measured using a commercial kit (Randox; company).

Results: Sperm vitality, membrane, DNA integrity and DNA stand breaks infresh semen samples were (41.9±24.2%; 57.6±21.5%; 44.4±24.3%; and51.4±22.9% respectively) and the corresponding values after semenprocessing were (53.9±21.2%;69.8±18.8%; 64.5±20.8%; and70.5%±18.9% respectively). In both groups ROS concentration in the nativesemen samples was 46.4±67.86 µmol/l. ROS in seminal plasma correlatenegatively, but not significant, with vitality and membrane integrity beforeand after semen processing. Whereas, a significant negative correlationswere observed between ROS and DNA integrity (r=-0.295; p=0.006) andDNA Stand breaks (r= -0.253; p=0.008) not only before, but also aftersemen processing (r=-0.283; p= 0.008 and r=-0.213; p=0.047). Besides; anegative correlation was shown between ROS concentration in seminalplasma and fertilization and pregnancy rates (r=-0.095; p=0.382 and r=-0.125; p=0.267 respectively).

Conclusion: ROS concentration in seminal plasma affects spermatozoa’squality but can not affect the fertilization and pregnancy rate in IVF/ICSIprogramme.

208.5 – CURRENT CRITICISM ON IVM TREATMENT

Milton Leong, Hong Kong, China.

IVM is a laboratory technique initially used to rescue germinal vesicleoocytes and make them suitable for fertilization. In 1968, Edwards was thefirst to demonstrate IVM in mouse oocytes; and then applied to humanoocytes, and from research to treatment form in the ensuing decades.However, it wasn’t until the end of the 20th Century that consistentpregnancy rates were obtained, and probably not until the last 5 years that itbecame an established clinical modality, largely through the efforts fromScandinavia, Korean, Asian groups, and especially the works from MontrealCanada.

One must not forget that IVM is not a stand-alone fertility treatment, butmerely a technique to prepare the oocytes for fertilization, and that IVF-ET isstill involved. Strictly speaking, it should be termed IVM/IVF.

At the present day, a good, experienced center should be able to achieve andIVM rate of 70-80%, with similar fertilization rates. Pregnancy rates shouldreach 30-40% per cycle, if 3 embryos are replaced. There have been over2000 babies born in the world. These results make IVM an alternatetechnique to conventional ovulation stimulation and IVF, especially in thepatients suffering from PCOS as well as the hyper-responders. However,controversy exists as to whether IVM should be accepted as a validtreatment for infertile couples, and whether it can be a truly universallyapplicable technique.

Criticism on IVM treatment will be discussed in the following headings:

A. Technical:i. ?Secret recipe?ii. FSH primingiii. HCG loadingiv. Culture systemsv. Identifying oocytes

B. Clinical:i. choice of patientsii. FSH priming

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iii. Significance of endometrial thicknessiv. ISCI – always?v. Egg collection techniques

C. Outcome: i. inconsistent pregnancy ratesii. Miscarriage ratesiii. Baby Registry and follow up birth defects and other effectsiv. Epigenetic changes

Conclusion: IVM is a valid technique giving more flexibility to clinicaltreatment of infertility, and also for those who desires fertility preservation.There are problems to be solved as long-term results are yet to be realized,and its benefit should be weighed against probably undesirable effects andto be used with prudence as with any other clinical modality

209.3 – ISO STANDARDS FOR CLINICAL WORKUP - EXPERIENCES WITH DINEN ISO 9001:2000

Bruno Imthurn, Division of Reproductive Endocrinology, Department ofObstetrics and Gynaecology, University Hospital, Zurich, Switzerland.

The introduction of a quality management (QM) system into an institutionusually goes along with some scepticism on the part of staff. However,although perhaps successful regarding pregnancy rates as a matter of factmany centres suffer under a chaotic organisation, unstructuredadministration and little defined training processes for their staff.

Among the different QM systems DIN EN ISO 9001:2000 especially fits theneeds of an ART clinic, as it is internationally acknowledged, successfullyapplied by many European IVF centres and widely known by the public.

The following main issues are covered by DIN EN ISO 9001:2000: 1. Generalprocedures; 2. Patient satisfaction; 3. Clinical aspects; 4. Laboratorymethods; 5. Staff training and development.

Based on our own experiences the presentation intends to give othercolleagues hints and tricks, how to introduce a QM system into an ARTcentre.

As a consequence DIN EN ISO 9001:2000 resulted in our unit (1. positive) ina more structured organisation, less improvisation and higher pregnancyrates, but also (2. negative) in more paperwork and higher costs.

211.1 – EFFECT OF SEASON ON FERTILIZATION AND EMBRYO QUALITY RATESIN ART

M.A. Danfour, W.M Elmahaishi, H.M Asherkaci, O.A Elsraite, M.SElmahaishi. Faculty of Medicine – 7th October University and MisurataInfertility Centre, Misurata, Libya.

Several studies have investigated seasonal variations during IVF. As knowncauses for reducing the rate of success of in-vitro fertilization (IVF). Theirresults are contradictory, especially concerning fertilization and pregnancyrates. The aim of the present study was to re-evaluate these parametersusing a large number of IVF cycles. METHODS: A total of 2250 ICSI cycles(first trial ICSI 1411) conducted in Misurata IVF Center, Misurata-Libyabetween February 2002 and august 2006 were retrospectively analysed. Toavoid a bias in the evaluation of the fertilization rate, the second and thirdICSI trial for some patients were considered for analysis. After adjusting forconfounding by the age of the woman, type of infertility, indication for IVFand year of aspiration, some seasonal variation was observed in the oocytematuration, oocyte quality, fertilization rate, embryo quality. RESULTS: Therewere statistically significant seasonal differences in Libya (Misurata) thatinfluenced the outcome of IVF treatment. The highest fertilization andquality-A embryo rates were observed during the spring and the lowest inthe autumn. These changes correlated with the absolute number of lighthours and humidity and its increment over time, but not with thetemperature. CONCLUSION(S): Seasonality seems to have a significantinfluence on the fertilization process and on the quality of the humanembryos that are obtained in vitro, possibly because of the light/darkvariations over time and humidity. More studies required to confirm thisfinding. Once it confirmed, these seasonal changes should be taken intoaccount when evaluating infertility data and in everyday clinical practice.

211.2 – THE ROLE OF IMMUNOHYSTOCHEMISTRY IN COMPLEXMORPHOLOGICAL DIAGNOSIS OF WOMEN STERILITY

Irina N. Kostyuchek1, Sergey V. Nikitin2, M. Kleshchov1, Kiryl I.Prashchayeu3, Viktoria O. Polyakova1 , N. Balashova1, Igor M. Kvetnoy 1. 1OttInstitute of Obstetrics and Gynecology Russian Academy of MedicalScience, Saint-Petersburg; 2 Clinica «Andromeda», Saint-Petersburg;3Belgorod State University, Belgorod; Russia.

Introduction: Inflammation, endocrine disorders and resistance ofendometrial cells lossed hormonal receptors are the most common reasonsof sterility. The aims of this study were to characterize estrogen andprogesterone receptor status , identify immune cells in endometrium andcompare this data with histological features of uterine mucosa in sterilitywomen.

Materials and Methods: 16 endometrial biopsies from 21-41 years oldsterility women (middle age 30 yo) was investigated on 22-24 day ofmenstrual cycle. After histological study immunohistochemical identificationof ER, PR, CD4, CD8, CD16, CD57, CD20 and HLA-DR was carry out.Quantitative analysis of immunohistochemical expression was done byusing computer image analisator «Video-Test, Morphology-5».

Results: Chronical endometritis, which characterized by limphocyticinfiltration and stromal fibrosis of endometrium was observed in 6 cases.CD16 and CD57 were positive in NK-cells, CD20 – in B lymphocytes, CD 4and CD8 – in T lymphocytes. In 2 cases autoimmune inflammation wasprobable (high level of HLA-DR positive cells and negative microbiologicalinvestigation of endometrium). In 6 biopses luteal deficiency wereobserved.Morphologically it’s appears by disoders in endometrial secretorytransformation, decrease of PR expression and comparative increase of ERexpression in endometrial glands and stroma. In one patient combineddisease took place: chronical endometritis and luteal deficiency.Inflammation and immature regression of corpus lutea was in one case.Typical endometrial hyperplasia with increase expression of ER and low levelof PR was in one biopsy.

Conclusion: Immunohistochemical methods accompaning classicalhistologocal examination elaborate causes of sterility and useful for choiceof treatment.

211.3 – WHAT WE OFFER TO WOMEN BEFORE UNDERGOING SURGICALTREATMENT FOR MENORRHAGIA

Kinza Younas, A. Khan, S. Younas, R. Settatree. Obstetrics & GynaecologySolihull Hospital Heartofengland NHS Trust; Birmingham HeartlandsHospital, Birmingham, UK

Aims/Standards: To determine whether women with menorrhagia areundergoing surgical treatment unnecessarily without trying all possiblemedical methods. To see the treatment outcomes of Mirena coil.

Nice guideline for the management of heavy menstrual bleeding no.44Jan.2007. RCOG guidelines for the management of menorrhagia.

Background: Excessive menstrual bleeding, which interferes with women’sPhysical, social, emotional and quality of life. In early 1990s 60% of womenwith menorrhagia underwent hysterectomy while this trend is declining now.There are recommendations for the medical management of menorrhagia.

Methods and Materials: Random, prospective questionnaire survey atSolihull and Birmingham Heartlands Gynaecology unit, wards and the clinic.

52 completed questionnaires analysed.

Results: Duration of menorrhagia with the mean 9 years, mode 3,4 yearsand median 5 years. Mirena coil was offered in 41% of patients only and(7/21) 13% tried the treatment while 2 stopped using it because of sideeffects. Tranexamic acid was tried in 11(52%) while Mafenamic acid wastried in 7 (13%). Endometrial ablation was tried in 11 cases (21%).Norethisterone was tried in 5 (10%). Mirena coil was offered by hospitaldoctor in 18 cases as compare to only 01 case by the GP.

Recommendations: •Primary care services should be encouraged to followthe guidelines by the NICE and RCOG. •Try all the available resources for themanagement before the surgical options.

Conclusion:•General practice should be encouraged to offer the Mirena as aprinciple treatment for menorrhagia. •More data is required to come to aconclusion and re-audit the practice in due course of time.

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211.4 – GLYCODELIN ENHANCES FERTILIZATION DURING IVF

Nadja Gneist, Gudrun Keck, Ina,Trinkaus, Birgit Leuchten, Katrin Hanseroth,Evelyn Gouma, Wolfgang Distler; Klinik fur Frauenheilkunde undGeburtshilfe, Dresden, Saxony, Germany.

Introduction: During the first day of IVF oocyte morphology and itsdevelopmental potential can not be evaluated as prognostic value, only theexpansion of the cumulus oophorus complex (COC) gave a hint for theoocyte maturity. Previous investigations of our study group aboutGlycodelin (Gd) in cumulus cells derived from denudation of ICSI-cyclesshowed a higher Gd-level correlated to mature eggs. In this present studywe investigated cumulus cells from IVF cycles for Gd and correlated theresults first to oocyte and COC maturity, respectively, and second to thefertilization outcome, which was of special interest because of the assumedselective potential of the cumulus oophorus. As described in literature,cumulus cells are able to modify the uptaken exogenous Gd-isoforms GdAand GdF to GdC; thereafter spermatozoa-zona pellucida binding becomesstimulated.

Material and Methods: We investigated the morphology of 125 COC derivedfrom 21 patients undergoing IVF (6,0± 2,3 per patient) after follicularaspiration. All patients gave signed informed consent. A compact COC wasvalued as “immature”. During maturation the cumulus expanses, theoocytes becomes visible. Expanded COCs have been valued as “mature”. Inaddition we defined a borderline stage, which corresponds to the beginningof cumulus expansion. Each of the COC’s have been grouped into one ofthese stages.

16 till 18 hours after insemination we performed PN-scoring. The oocytesbecame denuded and were transferred into fresh culture media. Thefertilization outcome has been assessed. The remaining cumulus cell- spermsuspension has been prepared using cytospin centrifugation. The Gd-immunostaining and semiquantification followed.

Results: Gd has been found in cumulus cells after IVF. The staining showedvariations that were found being correlated to oocyte maturity of thecorresponding oocyte. While the COC maturity increases, the Gd-level of thecumulus cells rises (p<0,05). The PN-scoring showed a regular 2PN-patternbeing accompanied with an increased Gd-level of the COC (p<0,05).

Conclusions: First, these results show a clear correlation of ooctye maturityand cumulus Gd, and second the necessity of Gd in the COC. The isoformGd C is described having a spermatozoa- zona pellucida binding enhancingcapacity. It can be hypothesized, that only in a mature COC the interactionsbetween the Gd-isoforms and the spermatozoa are advancing thefertilization process.

211.5 – A SYSTEMATIC REVIEW OF MANAGEMENT OF INFERTILITY INWOMEN OVER THE AGE OF 40

Gerasimos Marinakis, Dimitrios Nikolaou. Imperial College of Science,Technology and Medicine, Chelsea and Westminster Campus, OvarianAgeing and Fertility Clinic, London, UK.

Introduction: The current systematic review looked at the latest data fromnational statistics on fertility trends in the 40s, as well as the most up-to-date results of IUI and IVF, and the effectiveness of additional interventionsand adjuvant treatments in this age-group.

Materials and Methods: Data on natural fertility in the forties were retrievedfrom National Statistics bureaux from UK and USA, as well as Eurostat. Thelatest results of Assisted Reproduction Treatments were also retrieved fromthe HFEA. Finally, the effectiveness of additional interventions such as AZH,blastocyst transfer and PGS, as well as various adjuvant treatments, wasexamined in published meta-analyses, as well as primary searches inMEDLINE and EMBASE

Results: Natural fertility in the 40s has remained low despite all the changesin lifestyle and medical advances. There is a well documented steadyincrease in the number of women having fertility treatment in the 40s. Thesuccess rates of IVF and IUI are low. HFEA data from 2006 showed that thelive- rate from IVF in the UK was 11% in the age-group 40-42, 4.6% in theage-group 43-44 and 4% in women over 44. Meta Analysis of (28 RCTs)showed an increase in clinical pregnancy rate (OR 1.29, 95% CI 1.12 to1.49) and significantly increased multiple pregnancy rates per woman after

AZH. Meta analysis suggests that the probability of live-birth after fresh IVFis higher after blastocyst-stage embryo transfer compared to cleavage-stageembryo but there are not recent studies specifically for women over 40.Recent RCTs did not show an increase in live birth rate after PGS in relationto advanced maternal age. In the first trial the implantation rate was greaterin the PGS group (17.1% v 11.5%) but was statistically not significant.Oocyte donation appears the most successful option for women withadvanced reproductive age as the success rates do not alter with therecipient’s age.

Conclusions: Despite many women’s expectations, systematic review of allavailable evidence suggests that the Assisted Reproduction Technologycontinues to have very low live-birth rates in women over 40. Analysis oftrials showed that assisted hatching may increase the chance of pregnancyin women with poor history. The application PGS for selection of goodquality oocytes in elderly women have not increased the success rates. Themanagement of the infertile couple over 40 needs to focus on the exclusionof other factors apart for age, such as male or tubal factor, as well aseducation on normal ageing and fertility and psychological support. Anevidence-base approach will protect infertile couples form investigations orinterventions that are not proven worthwhile. In terms of population strategythe key to the management of infertility in women over 40 is prevention.

211.6 – ROLE OF DIAGNOSTIC LAPAROSCOPY IN WOMEN WITH NORMALPELVIC IMAGING AND FAILED SUPER OVULATION AND INTRAUTERINE INSEMINATION

Kay Jayakrishnan, Aby Koshy, Raju, RKJK Hospital, Fertility Research andGynaec Centre, Trivandrum, Kerala, India.

Introduction: Though the advent of laparoscopy had revolutionized the waywe diagnose and treat women with infertility, there are areas of debate onthe role of diagnostic laparoscopy in infertility. Laparoscopy has long beenused in the evaluation of patients with infertility where other diagnosticmethods have failed to come up with a cause. It also has the advantage ofbeing a see and treat modality. Though a considered a safe procedure,laparoscopy is associated with complications inherent to the surgicalprocedure and the anaesthesia administered and these risks should not bedisregarded. The costs incurred and the risks involved with the procedureneeds to be balanced with the therapeutic benefit. Though improvements inimaging technologies have reduced the requirement for diagnosticlaparoscopy, it is still considered the gold standard for the evaluation of thepelvis. We therefore undertook a retrospective study over an 8 year periodto evaluate the role of diagnostic laparoscopy and hysteroscopy in womenwith normal ultrasound findings who failed to conceive after 3 or morecycles of super ovulation and intra uterine insemination (IUI).

Materials and Methods: A retrospective study of patients who underwentdiagnostic laparoscopy between January 2001 and December 2008 wasperformed. Those patients who had no detectable pathology based onhistory, physical examination and 3 D ultrasound and had treatment for 3 ormore cycles in the form of super ovulation and IUI were included in thestudy. Moderate and severe male factor infertility was excluded in the study.All patients underwent diagnostic laparoscopy and hysteroscopy undergeneral anaesthesia. Patient profile, intra operative findings and operativecomplications were noted. Statistical analysis was performed using SPSS16.

Results: 127 patients underwent diagnostic laparoscopy and hysteroscopyin the study period. The mean age of the patients was 29.45 ± 4.34 years.The mean duration of infertility was 5.09 ± 3.03 years and a mean of 4.96 ±2.4 cycles of super ovulation and IUI were performed prior to the surgicalprocedure. There were no major intra or post operative complications.12.6% (n=16) patients had normal findings on laparoscopy andhysteroscopy with bilateral patent tubes. 77.2 % had endometriosis (AFS -minimal (37.8%, n=48), mild (33.1%, n=42) and moderate (6.3%, n=8)) and5.5% (n=7) had pelvic inflammatory disease (PID). One patient with PID hadtuberculosis diagnosed on histopathological examination. One patient withmoderate endometriosis was diagnosed to have an adenocarcinoma arisingfrom endometriotic lesions in the pouch of Douglas. 82.7% (n=105) ofpatients had patent tubes, 13.4% (n=17) had unilateral tubal bock and 2.9%(n=5) had bilateral tubal block on chromopertubation. 3.1% (n=4) womenhad unilateral and 2.4% (n=3) had bilateral peritubal adhesions.

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Conclusions: The most common findings in women who had a negativehistory, normal examination findings and a normal pelvic ultrasound andhad failed to conceive with super ovulation and IUI was endometriosis.Though the benefit of treatment for AFS class I (minimal) and II (mild)endometriosis has been questioned, significant pelvic pathology was seen in26.8% (n=34) of women. They included moderate endometriosis (AFS 3),PID or tubal pathology. Use of laparoscopy was of significant benefit, as atleast 1 in 4 women had conditions where treatment was proven to improveher chances of future fertility. Where resources are limited, it seems logicalto treat patients with normal pelvic imaging for 3 or more cycles of superovulation and IUI before proceeding to laparoscopy.

211.7 – EFFECTIVE WAY TO CUT THE POST OPERATIVE ANALGESICREQUIREMENT AFTER FEMALE TUBAL STERILISATION.

Kinza Younas, T. Nash, S. Ekladios, T. Abo Kaseem, Ralph Settatree. Royal College of Obstetrics & Gynaecology London; Sketty Swansea,Swansea; UK.

Aims and Standards: We have evaluated the effiectiveness of application of2% lignocaine gel to Filshie clips and the direct infiltration of mesosalpinxwith Bupivacain to relieve postoperative pain as against the control groupwhere no local analgesic was used. National evidence based guidelines formale and female sterilisation Jan.2004.

Background: Laparoscopic sterilisation is more painful than diagnosticlaparoscopy probably because of local tissue necrosis and ischaemia at thesite of tubal interruption.

Methods and Materials: 77 healthy women not allergic to local anaestheticused had laparoscopic tubal occlusion under general anaesthesia at theSolihul and Birmingham Heartlands hospitals, UK. Survey forms were filledin the ward by the nursing staff at 01 hr.post operative to assess pain scoreagainst 10 point numeric rating scale (NRS), at the request of analgesia andat time of discharge.

Results: Flishie clips covered with 2% lignocaine gel were used in 24/77while direct infilteration of mesosalpinx in 13/77 cases. 75/77 patients hadskin infiltration at the site of incisions. Pain scores remain similar in 2%lignocaine gel and Bupivacain group and had significantly longer time to firstanalgesia, less pain at 1 hour and shorter recovery time(P value <0.0001) ascompare to the control group. There was no case of failed sterilisation oradverse reaction to local analgesics.

Recommendation: The application of local anaesthetic gel to Filshie clips is asafe, non-invasive, and effective method of relieving postoperative painduring laparoscopic tubal sterilisation as compare to the direct tubalinfilteration which may be associated with haemotoma formation or risk ofbleeding,more time consuming and expertise required for this techni que.

Conclusion: As a safe and quick method Filshie clips soaked with lignocainegel should be used to reduce the postoperative analgesic requirement.

More work in the form of large scale audit is needed to support these resultsin future.

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ORAL ABSTRACTS – TUESDAY

300.2 – NEW CHALLENGES FOR OVARIAN-CELL THERAPY AT THE DAWN OFTHE 21ST CENTURY

Alain Gougeon1, Dominique de Ziegler2. Inserm U865, Lyon; 2Gynécologie-Obstétrique II, GH Cochin-St Vincent de Paul, Paris; France

Our lecture will provide an overview of the most salient findings that wererecently made in the emerging domain of cell therapy. This will constitutethe stronghold on which projections will be drawn for elaborating thepossible new developments that can reasonably be foreseen for the nextdecade or so. Expending upon existing data and elaborating from theexpected future accomplishments to be made in this field, particularemphasis will be put on the possible practical applications of ovarian celltherapy. In doing so, we’ll primarily focus on depicting how this novelapproach may help in the future women who suffer from major reproductivefailure and assist them in their quest for conceiving and becoming pregnant.

The reproductive failures at stake that are possible candidate for ovarian celltherapy include premature menopause, premature ovarian failure (POF)occurring either spontaneously or triggered by past chemo- and/or radiationtherapy for cancers that affected women of reproductive age. In the longrun, cell therapy may also prove helpful for treating other forms of infertility,including the variants that are unexplained or insufficiently explained(associated with contributing factors but no true cause).

Recently, sets of ever increasing efforts in basic research have profoundlyimproved our found of knowledge on how genes and local factors – thoseinvolved in oogenesis, follicle growth and oocyte maturation – ultimatelyinteract and cross-communicate. Prior work has also thought us somefundamentals on how mammalian oocytes orchestrate the rate of ovarianfollicular development trough a continuous cross-talk process that takesplace between granulosa and theca cells.

Taken together, these new achievements are likely to soon spearhead somemajor improvements in the field of ovarian cell culture. High on the list ofthese impending achievements are the emerging and most promisingdomains of in vitro folliculogenesis and in vitro oocyte maturation (IVM),which are meant to soon reliably provide mature and fertilizable oocytes.Finally, the possibility of regenerating the intra-ovarian pool of resting orprimordial follicles will also be examined in the light of pluripotent stem celltechnologies and confronted to the challenging results of neo-oogenesis.Conversely however, the transfer of fetal primordial germ cells will not beconsidered because of the lack of ethical consensus regarding this issue.

300.3 – IN-VITRO MATURATION AND OOCYTE VITRIFICATION FOR THETREATMENT OF INFERTILITY AND THE PRESERVATION OF FERTILITY

Seang Lin Tan, Jack Yu Jen Huang, Ri-Cheng Chian, Hananel Holzer, EzgiDermitas, Lucy Gilbert. McGill University Health Center (MUHC), Montreal,QC, Canada.

Fertility declines with increasing age,1, 2 and is markedly acceleratedfollowing gonadotoxic therapy for cancer and other non-oncologicconditions,3 such as SLE or other auto-immune disorders,4 and followingsurgery for endometriosis5 and ovarian neoplasm,6 as a consequence ofgenetic disorders such as Turner syndrome7, 8 and Fragile X pre-mutation.Finally, many women whose current circumstance precludes a pregnancyfind themselves facing potential childlessness1. ASCO and ASRM haveendorsed IVF and embryo cryopreservation (EC) as the only method offemale fertility preservation. 9, 10 However, this requires 2-5 weeks tocomplete, produces relatively high estradiol levels, which may be deleteriousin certain malignancies, and requires a male partner. Cryopreservation of anentire ovary has not been successfully performed in humans.11 Ovariantissue cryopreservation with orthotopic transplantation requires twooperations,12 has produced only five published live births,13-16 and in cancerpatients carries a small but finite risk of re-introducing malignant cells.17

Oocyte cryopreservation represents the least invasive option for patientswithout a male partner. Conventional slow-freezing is consideredexperimental because of the low survival and pregnancy rates.9, 18 Recentadvances in vitrification techniques have markedly improved the efficacy ofoocyte cryopreservation.19-28 In a clinical trial at the McGill ReproductiveCenter (MRC) involving 38 infertile women, oocyte vitrification (OV) usingthe McGill Cryoleaf resulted in a mean survival rate of 81% post-thawing, a76% fertilization rate, a CPR/C of 45%, an LBR/C of 40%, and 22 healthy

babies.29 In a review of 165 pregnancies and 200 infants conceived followingOV, the birth weight and the incidence of congenital anomalies (2.5%) werecomparable to those following spontaneous conception or IVF treatment.30 Anovel fertility preservation strategy involves immature oocyte retrieval in anunstimulated menstrual cycle31 or from ovarian tissue biopsies,32 followedby in-vitro maturation (IVM) and OV or EC. IVM has become an effectivetreatment option for many infertile women,33-37 resulting in the birth of over500 healthy infants38 without any increase in fetal abnormality ormiscarriage rates in comparable patients.39, 40 In a pilot study at the MRC onIVM-OV, an LBR/C of 20 % per cycle was achieved, including the world’sfirst four live births from vitrified IVM oocytes.29, 41 Advantages of IVM-OV orEC include: 1) eliminating expensive drugs and monitoring; 2) completingtreatment within 2 to 10 days;31 3) avoiding hormone-sensitive tumors; and4) retrieving oocytes at any phase of the menstrual cycle.42 To date, the MRChas provided fertility preservation to over 130 patients with breast,43 hema-tological, brain, soft tissue, colorectal and gynecological cancers.Primary-care physicians and oncologists need to be made aware of thefertility preservation options and to provide early discussion with thepatients followed by referral, if desired, to an IVF center that offers the fullrange of fertility preservation options.

References1. Heffner LJ. Advanced maternal age—how old is too old? N Engl J Med 2004;351(19):1927-9.

2. Tan SL, Royston P, Campbell S, et al. Cumulative conception and livebirth rates after in-vitrofertilisation. Lancet 1992;339(8806):1390-4.

3. Gidoni Y, Holzer H, Tan SL, Tulandi T. Fertility Preservation in non-oncologic patients. ReprodBiomed Online 2008;16:792-800.

4. Elizur SE, Chian R, Pineau CA, et al. Fertility preservation treatment for young women withautoimmune disease facing treatment with gonadotoxic agents. Rheumatology 2008;47:1506-9.

5. Elizur SE, Chian RC, Holzer HEG, Gidoni Y, Tulandi T, Tan SL. Cryopreservation of oocytes in ayoung woman with severe and symptomatic endometriosis: A new indication for fertilitypreservation. Fertil Steril:In Press.

6. Huang JY, Buckett WM, Gilbert L, Tan SL, Chian RC. Retrieval of immature oocytes followed by invitro maturation and vitrification: a case report on a new strategy of fertility preservation in womenwith borderline ovarian malignancy. Gynecol Oncol 2007;105(2):542-

7. Huang JY, Tulandi T, Holzer H, et al. Cryopreservation of ovarian tissue and in-vitro maturedoocytes in a female with mosaic Turner syndrome Hum Reprod 2008;23(2):336-9.

8. Lau NM, Huang JYJ, MacDonald S, et al. Feasibility of fertility preservation in young females withTurner syndrome. Reprod Biomed Online;In press.

9. Lee SJ, Schover LR, Partridge AH, et al. American Society of Clinical Oncology recommendationson fertility preservation in cancer patients. J Clin Oncol 2006;24(18):2917-

10. The Ethics Committee of the American Society of Reproductive Medicine. Fertility preservationand reproduction in cancer patients. Fertil Steril 2005;83(6):1622-8.

11. Wang X, Chen H, Yin H, Kim SS, Tan SL, Gosden RG. Fertility after intact ovary transplantation.Nature 2002;415(6870):385.

12. Oktay K, Buyuk E, Veeck LL, et al. Embryo development after heterotopic transplantation ofcryopreserved ovarian tissue. Lancet 2004;363(9451):837-40.

13. Donnez J, Dolmans MM, Demylle D, et al. Livebirth after orthotopic transplantation ofcryopreserved ovarian tissue. Lancet 2004;364(9443):1405-10.

14. Meirow D, Levron J, Eldar-Geva T, et al. Pregnancy after transplantation of cryopreserved ovariantissue in a patient with ovarian failure after chemotherapy. N Engl J Med 2005;353(3):318-21.

15. Demeestere I, Simon P, Emiliani S, Delbaere A, Englert Y. Fertility Preservation: SuccessfulTransplantation of Cryopreserved Ovarian Tissue in a Young Patient Previously Treated forHodgkin’s Disease. Oncologist 2007;12(12):1437-42.

16. Andersen CY, Rosendahl M, Byskov AG, et al. Two successful pregnancies followingautotransplantation of frozen/thawed ovarian tissue. Hum Reprod 2008;23(10):2266-72.

17. Meirow D, Hardan I, Dor J, et al. Searching for evidence of disease and malignant cellcontamination in ovarian tissue stored from hematologic cancer patients. Hum Reprod2008;23(5):1007-13.

18. The Practice Committee of the American Society of Reproductive Medicine. Ovarian tissue andoocyte cryopreservation. Fertil Steril 2004;82(4):993-8.

19 .Chian RC, Kuwayama M, Tan L, Tan J, Kato O, Nagai T. High Survival Rate of Bovine OocytesMatured In Vitro Following Vitrification. The Journal of Reproduction and Development2004;50(6):685-96.

20. Huang JYJ, Chen HY, Tan SL, Chian RC. Effect of choline-supplemented sodium-depleted slowfreezing versus vitrification on mouse oocyte meiotic spindles and chromosome abnormalities.Fertil Steril 2007;88(4, Suppl 1):1093-100.

21. Huang JYJ, Chen HY, Park JYS, Tan SL, Chian RC. Comparison of spindle and chromosomeconfiguration in in vitro- and in vivo-matured mouse oocytes after vitrification. Fertil Steril2008;90(4 (Suppl 1)):1424-36.

22. Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril2002;78(3):449-54.

23. Oktay K, Cil AP, Bang H. Efficiency of oocyte cryopreservation: a meta-analysis. Fertil Steril2006;86(1):70-80.

24. Katayama KP, Stehlik J, Kuwayama M, Kato O, Stehlik E. High survival rate of vitrified humanoocytes results in clinical pregnancy. Fertil Steril 2003;80(1):223-4.

25. Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservationof human oocytes. Reprod Biomed Online 2005;11(3):300-8.

26. Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos:The Cryotop method. Theriogenology 2007;67(1):73-80.

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27. Lucena E, Bernal DP, Lucena C, Rojas A, Moran A, Lucena A. Successful ongoing pregnanciesafter vitrification of oocytes. Fertil Steril 2006;85(1):108-11.

28. Cobo A, Kuwayama M, Perez S, Ruiz A, Pellicer A, Remohi J. Comparison of concomitantoutcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method.Fertil Steril 2008;89:1657-64.

29. Chian RC, Huang JYJ, Gilbert L, et al. Obstetric outcomes following vitrification of in-vitro and in-vivo matured oocytes. Fertil Steril:In press.

30. Chian RC, Huang JYJ, Tan SL, et al. Obstetric and perinatal outcome in 200 infants conceivedfrom vitrified oocytes. Reprod Biomed Online 2008;16(5):608-10.

31. Rao GD, Chian RC, Son WS, Gilbert L, Tan SL. Fertility preservation in women undergoing cancertreatment. Lancet 2004;363(9423):1829-30.

32. Huang JY, Tulandi T, Holzer H, Tan SL, Chian RC. Combining ovarian tissue cryobanking withretrieval of immature oocytes followed by in vitro maturation and vitrification: an additionalstrategy of fertility preservation. Fertil Steril 2008;89(3):567-72.

33. Chian RC, Gülekli B, Buckett WM, Tan SL. Priming with human chorionic gonadotropin beforeretrieval of immature oocytes in women with infertility due to the polycystic ovary syndrome. NEngl J Med 1999;341(21):1624-6.

34. Chian RC, Buckett WM, Tulandi T, Tan SL. Prospective randomized study of human chorionicgonadotrophin priming before immature oocyte retrieval from unstimulated women withpolycystic ovarian syndrome. Hum Reprod 2000;15(1):165-70.

35. Jurema MW, Nogueira D. In vitro maturation of human oocytes for assisted reproduction. FertilSteril 2006;86(5):1277-91.

36. Al-Sunaidi M, Tulandi T, Holzer H, Sylvestre C, Chian R-C, Tan SL. Repeated pregnancies and livebirths after in vitro maturation treatment. Fertility and Sterility 2007;87(5):1212.e9-.e12.

37. Son W-Y, Chung J-T, Chian R-C, et al. A 38 h interval between hCG priming and oocyte retrievalincreases in vivo and in vitro oocyte maturation rate in programmed IVM cycles. Hum Reprod2008;23:2010-1016.

38. Edwards RG. Foreword. In: Tan SL, Chian RC, Buckett WM, eds. In vitro maturation of humanoocytes Basic science to clinical application. London: Informa; 2006:xv-xx.

39. Buckett WM, Chian RC, Holzer H, Dean N, Usher R, Tan SL. Obstetric outcomes and congenitalabnormalities after in vitro maturation, in vitro fertilization, and intracytoplasmic sperm injection.Obstet Gynecol 2007;110(4):885-91.

40. Buckett WM, Chian R-C, Dean NL, Sylvestre C, Holzer HEG, Tan SL. Pregnancy loss inpregnancies conceived after in vitro oocyte maturation, conventional in vitro fertilization, andintracytoplasmic sperm injection. Fertil Steril 2008;90:546-50.

41. Chian RC, Gilbert L, Huang JYJ, et al. Live birth after vitrification of in vitro matured humanoocytes. Fertil Steril:In press.

42. Demirtas E, Elizur S, Holzer H, et al. Immature oocyte retrieval in the luteal phase to preservefertility in women with cancer facing imminent gonadotoxic therapy: it is worth a try. ReprodBiomed Online 2008;17(4):520-3.

43. Oktay K, Demirtas E, Son WY, Lostritto K, Chian RC, Tan SL. In vitro maturation of germinalvesicle oocytes recovered after premature luteinizing hormone surge: description of a novelapproach to fertility preservation. Fertil Steril 2008;89(1):228.

301.4 – DIAGNOSIS AND MANAGEMENT OF POOR RESPONDERS

Marc Germond, Centre de Procréation Médicalement Assistée (CPMA) andFABER Foundation, Lausanne, Switzerland.

The definition of a poor response to ovarian stimulation varies according towhether an Ovulation Enhancement (OE) prior to timed sexual intercourse or aControlled Ovarian Stimulation (COS) for oocyte recovery and in vitrofertilisation is considered. As a result, there is no universal definition. Mostoften, an absence of follicular development is considered as a poor responsein the first case, and the recovery of less than 5 metaphase II oocytes in thesecond one.

The poor response can have a genetic, immunological, iatrogenic orbiochemical origin. In most cases, the age of the patient is implicated in thisphenomenon, which would remain undiagnosed if no treatment wereundertaken.

Echographic and hormonal diagnostic tools have been evaluated innumerous studies. A consensus tends to involve the ovarian volume and thenumber of antral follicles evaluated by ultrasound, an AMH level of less than3 IU/l, a FSH >12 IU/l, as well as a low E2 on the 3rd day of the cycle;however, no ideal combination of these factors could be established. Astimulation trial constitutes the ultimate step able to confirm the clinicalprognosis.

The literature agrees on a maximum dose of 450 IU that can beadministered per day. The time at which treatment can be started (luteal orfollicular phase), the concomitant administration of LH, GnRH agonists orantagonists are still the subjects of many publications.

We report on our experience based on 3’561 oocyte recoveries performedon patients aged 35 to 45 years. The results were analysed retrospectively inrespect to the oocyte and zygote numbers, as well as to the implantationand delivery rates. These data were related to the number of FSH unitsadministered and re-evaluated in the light of the patient’s age.

Our conclusions confirm that the therapeutic response remains the best

prognostic tool allowing to decide whether the cost efficiency of a treatmentis adequate or not. The objective of such an attitude is to start or to abandontreatment and, through appropriate counselling, to optimise the medicalcare. Patients may be offered other alternatives, such as oocyte donation oradoption, in case the parental project is not abandoned.

301.5 – LUTEAL SUPPORT IN ARTS

Sudha Prasad, Maulana Azad Medical College, New Delhi, India.

Luteal phase support is an integral part of assisted reproductive treatment.The luteal phase, as a continuation of the follicular phase, depends on thephysiological sequence of events related to follicular maturation.

Defective luteal phase in assisted reproductive cycles has been attributed toadverse effects of controlled ovarian hyperstimulation, suppression of thepituitary LH release by GnRH analogues, and depletion of granulosa cellsduring follicle aspiration. Supra-physiological levels of steroids secreted bya high number of corpora lutea in stimulated IVF cycles directly inhibit theLH release via negative feedback actions at the hypothalamic-pituitary axislevel. This seems to be the primary cause of the luteal phase defect duringthe early luteal phase.

Controlled ovarian hyperstimulation (COH) has been shown to advanceendometrial maturation thus disrupting the delicate mechanism of embryo-endometrial interaction. During COH, estradiol concentrations aresupraphysiological due to multifollicular maturation. Furthermore,immediately after ovulation, estradiol concentrations decrease to a greaterextent due to follicular aspiration and early progesterone rise. The decreaselevel of estradiol is more pronounced due to formation of multiple corporalutea in response to stimulation with HCG from granulosa-lutein cellsaspirated during oocyte retrieval in agonist or antagonist cycles.

Implantation is a complicated process that involves adequate preparation ofthe endometrium. An important contributor to this preparatory process isthe corpus luteum. The primary function of which is to secrete progesteroneto induce secretory transformation of the endometrium to facilitateimplantation. During the early stages of its development necessary supportmay be required for the continuation of pregnancy. The corpus luteumrequires continual stimulation by luteinizing hormone (LH) to maintainadequate production of progesterone which is produced by the action ofhuman chorionic gonadotropin (HCG) on the LH receptor during pregnancy.

Stimulated IVF cycles are associated with luteal phase defect. In order toovercome this, different doses, durations and types of luteal phase support(LPS) have been evaluated. There is still no agreement regarding the optimalsupplementation scheme.

HCG is a time honored hormone that has been used for luteal phasesupport. Due to the increased risk of hyperstimulation, it has largely beenreplaced by progesterone.

Progesterone can be administered orally, vaginally, or intramuscularly. Itleads to higher serum progesterone levels and better pregnancy rates.

Dydrogesterone (DG) was introduced to support the luteal phase ofstimulated IVF cycles. DG, a retroprogesterone is a biologically activemetabolite of progesterone. Its anti-estrogenic effect on the endometriumhelps in achieving the desired secretory transformation.

The addition of estradiol (E2) seems to be beneficial in long GnRH agonistprotocol but not in the short GnRH agonist and GnRH antagonist protocol.The randomized trials showed a beneficial effect of use of estrogen in termsof pregnancy rates (1). However, the latest meta-analysis published in 2008could not reveal any beneficial effect of E2 co-administration in stimulatedIVF cycles.

Several other agents have been mainly used as adjuncts to progesterone.These are HCG, estradiol, GnRH agonists, aspirin, and various others.Estrogen has been advocated as an adjuvant to progesterone for LPS.Estrogen can be administered either orally or transdermally. While studiesfailed to corroborate the effective results.

Several adjutants together with mainly progesterone have been administeredduring the luteal phase with the aim to increase the implantation rate. Theaddition of ascorbic acid or prednisolone has not been found to be beneficial(2, 3).

Aspirin has been advocated both to increase ovarian responsiveness and

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implantation. Although a very recent meta-analysis of the prospectiverandomized studies showed that aspirin did not increase pregnancy anddelivery rates in the ART setting (4).

Despite the widely adopted practice of luteal phase support especially inART cycles there is still the need for properly designed and adequatelypowered randomized studies to determine the agents/s that are associatedwith higher implantation rates.

References:

Farhi J, Weissman A, Steinfeld Z, Shorer M, Nahum H, Levran D. Estradiolsupplementation during the luteal phase may improve the pregnancy rate inpatients undergoing in vitro fertilization-embryo transfer cycles. Fertil Steril,2000; 73: 761-6.

Griesinger G, Franke K, Kinast C, Kutzelnigg A, Riedinger S, Kulin S KaaliSG, and Feichtinger W. Ascorbic acid supplement during luteal phase in IVF.J Assist Reprod Genet, 2002; 19: 164-8.

Ubaldi F, Rienzi L, Aniballo S, Iacobelli M, Cobellis L, Greco E. Low doseprednisolone administration in routine ICSI patients does not improvepregnancy and implantation rates. Hum Reprod, 2002; 17: 1544-1547.

Gelbaya T, Kyrgiou M, Stern C, Nardo L. Low dose aspirin for in vitrofertilization: a systematic review and meta-analysis. Hum Reprod Update,2007; 13: 357-364.

302.2 – CRYOPRESERVATION OF OOCYTES

Ri-Cheng Chian, McGill Reproductive Center, Department of Obstetrics andGynecology, McGill University, Montreal, Canada.

Improving success rate associated with the use of cryopreserved humanoocytes would be an important advance in human assisted reproductivetechnology (ART). The objectives of this presentation are to evaluate thesurvival rates and pregnancies of cryopreserved human oocytes followingvitrification. As a clinical trial, a total of 38 women were included in thestudy, in which women underwent ovarian stimulation, their mature andimmature oocytes collected and vitrified. The mean age of women was31.5±0.5 years. The vitrified oocytes were thawed and inseminated usingintracytoplasmic sperm injection (ICSI), and the resulting embryos weretransferred in a subsequent menstrual cycle. The Hospital Research EthicsBoard (REB) has approved the protocol. The patients were given fullyexplanation for the procedures and a written consent forms were obtained.As the procedure of vitrification, the oocytes were suspended inequilibration medium (7.5% ethylene glycol and 1,2-propanediol) for 5 min,and then the oocytes were transferred to vitrification medium (15% ethyleneglycol and 1,2-propanediol + 0.5 M sucrose) for 45-60 seconds at roomtemperature. The oocytes were loaded on a special designed device, McGillCryoleaf, and immediately plunged it into liquid nitrogen for storage. Forthawing, the McGill Cryoleaf was directly inserted into 37ºC thawing medium(1.0 M sucrose) for 1 minute. The thawed oocytes were transferred to 0.5 Mand 0.25 M sucrose medium for 3 minutes respectively, and then washedtwice with culture medium for insemination using intracytoplasmic sperminjection (ICSI) method. As the results, a total of 463 oocytes were vitrifiedfrom these 38 women. Of those, 383 (82.7%) oocytes were survived post-thaw. Following insemination by ICSI, 287 oocytes were fertilized normally(75.0%), and 17 patients became pregnant (44.7%) after transferring theresulted embryos (3.7±1.1). Implantation rate was 18.1% (24/133).Obstetric and perinatal analysis of pregnancies and live births indicates thereis no adverse outcome associated with oocyte vitrification. This resultindicates that human oocytes can be cryopreserved successfully with theprocedure of vitrification and that vitrification method may be efficientlyused for a program of cryopreservation of human oocytes in ART. Oocytevitrification technology may offer to women for fertility preservation.

303.1 – THE ROLE OF THE MILD STIMULATION IN THE CURRENT IVFPRACTICE

Artur Bernard, Kaali Institute, IVF Center, Budapest Hungary.

The birth of the first IVF child was achieved from a natural cycle./Steptoe andEdwards 1978/. The aim of the ovarian stimulation has been to produce, retrieveand fertilize more oocytes, and select afterwards the best embryos for transfer.Therefore in the 80’s most centers used high dose stimulation to increaseoocyte/embryo yield, and therefore to improve pregnancy rates. In 1991

transvaginal ultrasound guided follicle aspiration was introduced, whichsimplified the retrieval and made it more effective. .In 1992 the introduction ofICSI improved dramatically the fertilizations rate. Assisted hatching, theavailability of new transfer media and blastocyst stage transfer also improved theimplantations rate significantly.. Therefore fewer follicles and oocytes wereenough to achieve success in IVF treatment. This brought up the issue of mildstimulation again (R.G. Edwards 1996). The discovery and application the GnRHantagonists enhanced the use of the milder stimulation procedures. Due to thesuccessful application of ICSI in the treatment of the male infertility, the numberof the healthy and young IVF patients is increasing. The single embryo transfer-toavoid the multiple pregnancies- is also more widely used. Because of thesefacts, mild stimulation in the current IVF practice has an increasing importance.Clinical research has to focus on this kind of stimulation, which is safe, effectiveand cost saving.

303.2 – OUTCOME OF THE RECENT IVF PROGRAM TRANSFERRING SINGLEBLASTOCYST RETRIEVED ON MINIMAL STIMULATION CYCLE ANDFROZEN BY VITRIFICATION METHOD

Keiichi Kato, Kato Ladies Clinic, Tokyo, Japan

Introduction: One of the major concerns of the modern assisted reproductivetechnology (ART) is to reduce the number of oocytes retrieved and transferredkeeping pregnancy rate high. Ovarian hyperstimulation impose physical andfinancial burden on patients and transfer of multiple embryos cause multiplepregnancy, which cause maternal complications and neonatal morbidity andmortality. To overcome these issues, we have implemented a new IVF program inwhich small number of oocytes were retrieved under the clomiphene-basedminimal stimulation cycle and frozen by vitrification method, and then followed bysingle blastocyst transfer under hormone-controlled or un-stimulated naturalcycle. The purpose of this presentation is to report the most recent results of theprogram in which more than 10,000 ET cycles were performed in 2007.

Materials and Methods: In 2007, 19,305 IVF cycles of minimal stimulation and10,115 cycles of embryo transfer were performed. All cycles that the IVF protocolspecified below were completed included in this study. The mean age of patientwas 39.4±4.8 years old. There was no exclusion criterion on cause of infertility.

Oocytes were retrieved on the minimal stimulation cycle: clomiphene citrate (50-100 mg/day) was administered from day 3 up to one days before the oocyteretrieval with or without hMG or rFSH (50-150 IU/every other day, from day 8 orlater), to elaborate 1-4 follicles. LH surge was induced by nasal GnRH agonist(Sprecure®, 300 μg) when the size of the leading follicle is more than 21-24mm). 34-36 hr later, oocytes were retrieved and inseminated conventionally orby ICSI cultured to blastocysts. Fertilized oocytes were cultured individually in 20μl of Cleavage medium (SAGE, USA) from day 1 to day 3, and then in Blastocystmedium (SAGE) from day 4 to day 6. In 4449 cycles, embryo (mostly day 5blastocysts) were frozen by vitrification method using Cryotop® as describedelsewhere. Single ET was performed using thawed embryos on the next cycle orlater (4,449 cycles), or using fresh embryo (mostly day2 embryo) (5,666 cycles).For frozen-thaw transfer, uses of hormonal replacement cycle were a choice.

Results: The average number of oocyte picked up per cycle was 1.35 (26,100 /19,305 cycles). At least one blastocyst was achieved in 54.6% of 19305 cycles.Average number of ET performed was 1.01. Overall survival rate of frozenblastocyst was 95.0%., comparable to that of cleavage stage (98%).

The pregnancy rates achieved frozen-thawed single blastocyst transfer were41.3% (per ET), which were significantly higher than that obtained by fresh day2-embryo transfer (19.8%). Pregnancy rate showed age-dependent decrease inboth frozen-thawed ET as in fresh ET, the positive impact of frozen ET wasenhanced in the older age group (≥35 y.o.) compared to the younger group (<34y.o.).

The overall multiple pregnancy rates were as low as 1.3%, almost comparable tothat of normal Japanese population. Ectopic pregnancy rate was 0.3%.

Conclusions: The clomiphene-based minimal stimulation IVF programs gavesatisfactory outcomes, in terms of pregnancy rate, comparable to that achievedby standard controlled ovarian hyperstimulation methods in Japan, patientcompliance and singleton pregnancy. Use of blastocyst ET also reduced ectopicpregnancy rate to the level of non-IVF pregnancy.

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303.3 – CONTROLLED OVARIAN STIMULATION, MINIMAL STIMULATION OR NOSTIMULATION FOR IN VITRO FERTILIZATION: IS THERE AN OPTIMALSTIMULATION?

John Zhang, Osamu Kato. New Hope Fertility Center, New York NY, USA and KatoLadies Clinic, Tokyo, Japan.

The success of IVF treatment is assessed by four criteria consisting of pregnancyoutcome, safety, patient comfortableness and cost. With improvement of embryopreservation with vitirfication and promotion of single embryo transfer it is thetime to reassess various stimulation protocols in terms of safety and efficiency. InUnited States of American almost 95% of IVF cycles are performed through dailygonodotropine injection with GnRH analogs or antagonists to control prematureovulation (conventional IVF). Since 2004 we have elected to use primarily a mildstimulation protocol with clomiphene citrate (CC) in combination with a smalladdition of human menopausal gonadotropin (hMG) in our IVF cycles (minimalstimulation IVF).

Clomiphene citrate (50 mg) was initiated orally each day, beginning on Day 3.Subcutaneous administration of 75–150 IU of hMG every 48 hours was begun onDay 5 or 8 depending on Day 3 FSH level. For patients with Day 3 FSH level ismore than 15 no Clomiphene citrate will be give and patient will only undernatural cycle IVF. A gonadotropin releasing hormone agonist (GnRHa) nasal spray(Synarel) was administered to trigger an endogenous LH surge for finalmaturation of the oocytes. Oocyte retrieval was performed 34 hours after theadministration of the spray

In this presentation we wish to compare various protocols of ovarian stimulationand would like to propose a concept of optimal ovarian stimulation which isbased on daily FSH levels. Traditionally daily dosing of stimulation is tailored topatient age, ovarian reserve and how follicular development and estrogenhormone rising. A FSH level based stimulation will provide an opportunity to givethe least medicine need to generate certain number eggs in each IVF cycles tominimizing hyperstimlulation and save the cost of medications.

304.1 – THE BENEFIT OF IVM IN PCOS PATIENTS

Yona Barak, Olga Sikorska, Tomasz Rokicki, Aneta Karwacka, Tomasz Dworniak,Ricardo Faundez. InviMed, European Centre of Motherhood, Warsaw, Poland.

Objective: In vitro maturation (IVM) was established as a new method for treatinginfertility. The procedure was applied in our centers in patients showingPolycystic Ovary Syndrome (PCOS).

Design. Rate of maturation, fertilization, embryo scoring and pregnancies werecompared in patients with PCOS, undergoing IVM procedure (study group) withpatients undergoing IVF due to female mechanical cause of infertility (controlgruop).

Materials and Methods: Twenty nine patients determined as PCOS underwent32 IVM cycles. The control group (n=28) underwent conventional ICSI procedure.IVM patients were mildly stimulated using 150 I.U. Gonal-F for 3-4 days. IVM wasperformed according to the procedure of MediCult IVM System (MediCult,Denmark). ICSI was performed 30 hours (Day 0) after IVM medium (MediCult,Denmark). Injected oocytes were then cultured in MediCult ISM1 medium.Patients of control group were stimulated in the short protocol using 75 IU ofFostimon with GnRH antagonist Cetrotide. Ovulation was induced by Ovitrelle.Following ICSI, oocytes cultured identically as PCOS oocytes.

Number of retrieved oocytes, rates of fertilization, cleavage, quality of embryosand pregnancies were compared with PCOS and control group, by Yate’scorrected �2 test. Results. Mean number of aspirated cumulus enclosed oocytes(OCCs) per patient was 8,8 and was significantly lower in comparison to thecontrol (17,0). A significant difference in the rate of OCCs which reached the MIIstage was observed after 30h in the PCO group in comparison to the control(138/257 (53,7%) vs 409/509 (79,4%), respectively, p<0,01). No differenceswere observed in fertilization rate (122/156 (78,2%) vs 337/377 (89,4%)however a significant difference was found in cleavage rates (101/119 (84,9%)vs 177/184 (96,2%), p<0.05). A higher value of the mean fragmentation grade of4-5cell stage embryos was observed on day 2 in the PCO group in comparison tothe control group (1,6 vs 1,3 respectively; p<0.01). Nine pregnancies out of 26embryo transfers (ETs; 34,6%) were achieved in the PCO group; 23 ETsperformed in the control group resulted in 13 pregnancies (46,4%; mean of 2.3vs 2.0 embryos per patient, respectively).

Conclusions: Although conventional procedure in mechanical infertility patientsis still superior, IVM was found to be an efficient method in patients with PCOsyndrome. Since our results are based on a small number of patients, furtherstudies are still ongoing.

304.2 – OXYGEN CONSUMPTION BY SCANNING ELECTROCHEMICALMICROSCOPY (SECM) FOR HUMAN IVM COC & EMBRYO WITH PCOPATIENTS

Hiroaki Yoshida, Yoshida Ladies Clinic, Reproductive Research Center, Sendai,Japan

Introduction: Recently, in vitro maturation (IVM) has been used for humanassisted reproductive technology (ART) for women with PCO. IVM-IVF wasperformed for patients with PCOS in 2008. Among a total of 186 retrievedoocytes, the maturation rate was 60.7%, the pregnancy rate was 29.4%. As theboth maturation and pregnancy rates were considered to be unsatisfactory low,the simultaneous use of nuclear and cytoplasmic maturation systems wasconsidered in order to clarify the relationship between cytoplasmic maturationand mitochondrial distribution or function. Furthermore the mitochondrialfunction of IVM oocytes and the mitochondrial membrane potential wereobserved using transmission electro microscopy (TEM) and scanningelectrochemical microscopy (SECM). As oxygen consumption is an ideal indicatorof metabolic activity. SECM measuring systems have been demonstrated tosuccessfully non-invasively the measure respiration activity of single embryosfrom several species. It has been reported that the bovine embryos with higheroxygen consumption are better candidates for further development of goodquality embryos and yielded higher pregnancy rates. Despite this apparentcorrelation between respiration activity and embryo quality, .oxygenconsumption in human IVM oocytes and embryos has yet to be evaluated.

Materials and Methods: Cumulus oocyte complexes (COC) and oocytes weretransferred into a plate filled with HFF99 medium. A microelectrode was used toscan along the z-axis from the edge of the sample and the oxygen consumptionrate was calculated based on the spherical diffusion theory using customsoftware. Measurements of each oocyte were performed rapidly (within 1 min).Subsequently, a sample of each oocyte was collected for observation by TEM.

Results: We classified morphologically the COC and oocyte size from grade1 to5. Our observation suggested that. Oocyte maturation rate was decreased fromgrade 1 to 5. The consumption for a single oocyte was as follows: GV 0.49, MI0.47, MII 0.41 Fx1014/mols-1. Weak correlation with COC size and oxygenconsumption rate before and after 26 hrs culture was observed; however therewas no significant difference between oocytes before and after culture. Thesefindings suggested that the oxygen consumption rate is representative of theCOC respiration activity and that it can be used to indicate adequate oxygenconsumption. There were no significant difference in the mean of oxygenconsumption at each cleavage in embryos (0.2�~0.56×1014/mols-1).

Conclusion: The COC mitochondria which were enlarged and showed elongatedmorphology. Good quality oocytes have rich mitochondria and have gap junctionbetween cumulus cells. IVM COC was found to be important for oocytematuration and embryo development and it is strongly related to oocytes via gapjunctions. There was no significant difference embryo consumption whichmoderate respiration rate of embryos showed high developmental rate to theblast cyst. Further study of cytoplasmic maturation and mitochondrial functionfrom IVM oocytes and embryos is required.

304.3 – CLINICAL DATA OF IVM FOR PCO PATIENTS

Yoshiharu Morimoto, The Centre of Reproductive Medicine and Infertility, IVFJapan, Osaka, Japan.

Polycystic ovary syndrome (PCO) is a disease including ovulation dysfunction ofovaries or amenorrhea, hirsutism and obesity. It is one of the most importantorigins of infertility and is difficult to be treated. In the treatments of PCO,ovulation induction, birth control pills, operative procedures such as wedgeresection of ovary and ovarian drilling under laparoscopy have been applied.Recently insulin sensitizing medications such as Metformin, Piogritazone andRosiglitazone have been added as an option.

In Vitro Maturation (IVM) has been first reported by Cha et al. (1991) as one oftreatments for PCO and nowadays spread worldwide. In the initial stage, theclinical success rate of IVM was low, however, it has been improved remarkablyby the development of media specified for IVM and puncture needle. Hence, IVM

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may be a preferable option to be selected in terms of prevention of ovarianhyperstimulation syndrome (OHSS)and accuracy.

We had the first success in IVM procedure in 1999 in Japan. The first baby wasborn next year, and first baby by frozen embryo transfer by IVM was born in2001.

Our indication for IVM procedure is mainly for PCO. But we apply this procedurefor the patients with normal menstrual cycles, rarely for the cases of poor qualityembryo.

We start follicle monitoring from Day 7 of the menstrual cycle. At the day we canrecognize at least two follicles of the diameter of over 7mm, immature oocytesare collected. The program should be cancelled when we see a dominant follicleor a ovarian cyst. HCG priming by 10000 iu is done 36 hours before oocyteretrieval. Follicles are aspirated by IVF OSAKA IVM needle (Kitazato BiopharmaCo.ltd, Tokyo, Japan). The IVF OSAKA IVM needle is designed by ourselves and iscomposed of two segments of puncture needle and holding needle. MediCult IVMmedium is arranged with 10% patient’s serum and HCG and FSH. Retrievedoocytes are cultured for maturation for 24 hours and all matured oocytes areinseminated by ICSI.

Up to 2007, we had 705 IVM cases and 91 pregnancies. The live birth and on-going pregnancy rate was 78% and the rate of abortion and stillbirth was 23.1%.We had only one case of malformation which was Goldenhar syndrome.

From these data, we strongly recommend to select IVM option for the treatmentsof PCO patients as a first choice.

304.4 – USE OF LETROZOLE IN POLYCYSTIC OVARY SYNDROME (PCOS)

Makio Shozu, Tomoya Segawa2, Satoshi Kawachiya2, Osamu Kato2. Department ofReproductive Medicine, Graduate School of Medicine, Chiba University, Chiba;2Kato Ladies Clinic; Shinjuku, Tokyo; Japan.

An aromatase inhibitor, letrozole, has recently been introduced into variablesituations of ovarian stimulation. The major application of the drug issuperovulation in IVF or IUI protocols, in which letrozole is administratedcombined with gonadotropins to increase the number of oocytes ovulated and theresulting pregnancy rate. Letrozole is also used for PCOS in an attempt to achievesingle ovulation and singleton pregnancy. This is because, in contrast toclomiphene citrate (CC), letrozole potentially allows natural selection of adominant follicle. An additional advantage of theoretical interest is seen withletrozole use for PCOS compared to CC. The shorter half-life of letrozole favorsrapid recovery of endometrial thickening, which may enhance implantation rate.Ovarian hyperstimulation syndrome may also be avoided by reducing the numberof oocytes. These advantages of letrozole over CC have been postulated based onclinical studies in infertile women with heterogenous etiologies, including PCOS,and the advantages in PCOS remain inconclusive. The effectiveness of letrozole ininfertile patients with PCOS (2003 ESHRE/ASRM criteria) was thus assessed. Over400 PCOS patients treated with or without letrozole were included and reviewedfor this analysis. With concomitant use of FSH (75 or 150 IU/every other day, days8 - 12), letrozole (2.5 mg/day, days 3 - 7) was as successful for ovulationinduction as CC (100 mg/day, days 3 - 7), with ovulatory rates of 89% and 83%,respectively. Total dose of FSH was significantly lower in the letrozole+FSH group(mean, 242 IU; range, 75 - 1050 IU) compared to the letrozole+CC group (mean,406 IU; range, 100 - 1200 IU). Single ovulation rate was significantly higher in theletrozole+FSH group (68%) compared in the CC+FSH group (38%). Clinicalpregnancy rate tended to be higher in the letrozole+FSH group (44%) than in theCC+FSH group (34%), although this difference was not significant. Letrozole isthus as effective as CC in terms of ovulation induction and may be superior to CCin terms of single ovulation even when administered with FSH.

304.5 – PCOS: MANAGEMENT STRATEGIES IN 2009

Frank Yelian, LIFE Clinic, Los Angeles, CA, USA.

Polycystic ovarian syndrome is one of the most common endocrine disorders inreproductive women. It is often presents with oligomenorrhea, androgen excess,insulin resistance, and obesity. The short and long term consequences of PCOSinclude ovulatory dysfunction and infertility; metabolic syndrome and type IIdiabetes; endometrial hyperplasia and cancer; dyslipidemia and cardiovasculardiseases. PCOS is now considered to be a complex genetic disorder. It has beensuggested that the disorder is a result of one of many intrinsic variant genetictraits that interact with other congenital or environmental factors to cause theendocrine dysfunction. The pathophysiology and clinical symptoms of the

condition are caused by abnormal pituitary function; abnormal steroidogeneis inovary and adrenal gland; and abnormal tissue respond to various hormones. Dueto the complexity of the disorder, the management strategies should be designedto tailor individual patient. Based on each patient’s presentation and the goal ofseeking for medical care, we should form a short and long term treatment plan.This presentation will address the current management strategies on androgenexcess; ovulatory dysfunction; insulin resistance; endometrial protection; obesityand metabolic syndrome. Various options in ovulation induction and ART inmanagement of PCOS will also be discussed.

306.3 – HUMAN OOCYTE ULTRASTRUCTURE IN IVM

Yoshiharu Morimoto, IVF Namba Clinic, The Centre for ReproductiveMedicine and Infertility, Osaka, Japan.

Introduction: In vitro maturation technique (IVM) has been introduced worldwide. In order to improve the technique toward better result, theunderstanding of the oocyte maturation, especially of cytoplasm, isimportant. The ultrastructural investigation for oocyte in IVM may open theblack box of maturation mechanism.

Oocyte maturation: Human primordial germ cells in the fourth week ofembryonic development migrate to the coelomic epithelium of the gonadalridges and develop to oogonia. Oogonia forms and exists as a manner ofnest. The first follicles are recruited by the action of neurotrophic factorssuch as brain derived neurotrophic factor (BDNF), nerve growth factor andneurotrophins(NT). On the way of its process, one layer of follicular cellscovers oocyte and follicle development comments. For the transformation,Kit system and anti-mullerian hormone has important roles on the process.

Oocyte maturation process is divided into several categories, such asnuclear, cytoplasmic, zona pellucida and follicular cell maturation. In orderto acquire the competence for fertilization and further development,accomplishment and harmony between each factor are needed.

Nuclear maturation: Meiosis is a reducing process of chromosomes fromdiploid to haploid in gametes. At the time of migration to the coelomicepithelium of the gonadal ridges, primordial germ cell undergoes mitoticdivision. The first meiotic division is a transition from oogonia to oocytes.The completion of nuclear maturation occurs with germinal vesiclebreakdown by the action of maturation promoting factor and finishes atextrusion of polar body.

Cytoplasmic maturation: Cytoplasmic maturation of oocyte may be anessential phenomenon for the oocyte to acquire competence to fertilize andcleave. Oocyte undergoes its maturation in harmonization with maturation ofnucleus and cytoplasm. A plenty of mRNA including maternal genes areaccumulated in the cytoplasm. The fact is shown as increase anddifferentiation of organella.

Ultrastructure of the ooplasm in maturing process: Immature and maturedoocytes were donated by PCO patients aged from 27 to 33 years whounderwent IVM program in the two centers of infertility treatments in Osakaafter informed consent. Oocytes were cultured in IVM medium NG 1.3 (Medicult, Denmark), 10% patient serum, 100 IU/L human chorionicgonadotropin and 75 IU/L follicle stimulating hormone under theatmosphere of 5% CO2, 5% O2 and 90% N2. Those oocytes were fixed andultrathin sectioned and observed by electron microscope at 0, 6, 12 and 24hours of culture.

Immature oocyte which is still in GV stage cultured in vitro for 6 hoursshowed that granulosa cells surrounding oocyte looked very active andincluded a plenty of lipid droplet. Mitochondria were dispersed in cytoplasmand cortical granules were prominent on the edge of the plasmalemma.After 24 hours of in vitro culture, the ultrastructure showed remarkablechange. In the nucleus, germinal vesicle was broken down and corticalgranules disappeared on the edge of the cell. More noteworthy character isthat of mitochondria. They have migrated over into the center of the cell,increased in number and aggregated. Furthermore microvilli on the surfaceof the oocyte showed morphological remarkable development.

Conclusion: Despite during the short period of 24 hours, the cellconstruction has dramatically changed in its ultrastructure. The remarkabledevelopment of microvilli on the surface of the oocyte indicates the activecommunication from the inside to the outside through plasma membrane.The purpose of the oocyte maturation is defined as preparation forfertilization and cleavage. The movement and aggregation of the activated

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mitochondria indicates the competence acquirement in preparation forcoming big events by charging up cell activity.

Those characteristic changes in ultrastructure may be a part of reality ofoocyte cytoplasmic maturation.

307.1 – FERTILITY PRESERVING OPTIONS OF REPRODUCTIVE AGE CANCERPATIENTS

Peter Kovacs, Steven G. Kaali. Kaali Institute IVF Center, Budapest, Hungary.

Over the past few years the number of reproductive age survivors of cancertreatment has increased as a result of newer surgical methods and improvedchemotherapeutic and radiation therapy regimens. Survival rates of hematologicmalignancies (most lymphomas, leukemias are diagnosed in patients under theage of 40) have increased to 50-90%. Ten to forty percent of gynecologicmalignancies are also diagnosed in reproductive age women. Less destructivesurgical options, a wider availability of medical options and improved assistedreproductive technology all offer hope to a significant proportion of these women.

Because of these developments the fertility concerns of these women have to beaddressed. As most treatment methods will still severely compromise or evendestroy ovarian function the pre cancer treatment and post cancer treatmentoptions need to be reviewed with the patient when appropriate. There aresurgical, medical and ART options that may be utilized to maintain fertility.

Surgical options should be considered for patients diagnosed with gynecologicmalignancies. They include conservative surgery for certain type and stageovarian cancers, radical trachelectomy for early stages of cervical cancer andadnexal lateral position in case abdominal radiation is planned.

Medical options (GnRH agonist use) may help to reduce the toxic effects ofchemotherapy. Progestin therapy is an alternative to surgery when well-differentiated, localized, early stage endometrial cancer is diagnosed.

ART is playing an increasingly important role in the management of thesepatients. For a long time embryo cryopreservation meant the only option. While itis still considered the most effective technique it cannot be used in all cases. Inrecent years oocyte freezing and ovarian tissue cryopreservation offer furtheroptions.

Finally, alternative options like donor egg use or adoption need to be mentionedto complete the list of choices these patients have.

During the presentation the role of the above-mentioned surgical, medical, ARTmethods will be reviewed including our own cases as examples. We wish tostress the importance of appropriate counseling of these patients which oftenrequires a multidisciplinary approach including, psychiatrists, internists,surgeons, gynecologist, oncologists and fertility experts to address the problem.

307.2 – THE CURRENT APPROACH TO OOCYTES VITRIFICATION FOR CANCERPATIENTS IN JAPAN

Takafumi Utsunomiya1, Osamu Kato2, Hirofumi Kamiya3, Hiroaki Yoshida4, YasuhitoMichikura5, Masanori Ochi6, Yuji Fujino7, Kohji Yano8, Yasuyuki Mio9, ShokichiTeramoto10. 1St Luke Clinic, Oita; 2Kato Ladies Clinic, Tokyo; 3Kamiya LadiesClinic, Sapporo; 4Yoshida Ladies Clinic, Sendai; 5Towako Ladies’ Clinic, Komatsu;6Yume Clinic Nagoya, Nagoya; 7Fujino Ladies Clinic, Osaka; 8Yano MaternityClinic, Matsuyama; 9Mio Fertility Clinic, Yonago, 10Shinbashi Yume Clinic, Tokyo;Japan.

Objective: Aggressive chemotherapy and radiotherapy have greatly enhancedthe life expectancy of young cancer patients, but these treatments cause massivedestruction of the ovarian reserve resulting in infertility or sterility. However,oocyte cryopreservation can preserve their fertility of these patients after cancertreatment. We applied a minimal ovarian stimulation protocol using clomiphenecitrate (CC) to retrieve mature oocytes, and the cryotop vitrification method tocryopreserve them in Japan.

Materials and Methods: Thirty four unmarried hematopoietic defect patientswith informed consent who underwent the CC cycle from January, 2007 toOctober, 2008. Fifty mg CC was administered from cycle day 3 and 75 IUrecombinant FSH was administered every other day from day 8 until the leadingfollicle developed to 18 mm in diameter. Administration of CC was then stopped,and 300μg GnRH-agonist (buserelin) was given as a maturation trigger. Oocyteswere retrieved from 30 to 36 hrs following the administration of the GnRH-agonist using a 22 gauge needle with local anesthesia (Teramoto, 2007). Theretrieved oocytes were denuded before vitrification. The cryotop method

(Kuwayama 2005) was used to vitrify the oocytes. The oocytes were equilibratedin 7.5% ethylene glycol and 7.5% DMSO in modified medium 199 (M-199) for15min before being transferred into the vitrification solution (VS) for 30 sec.Oocytes were then transferred into onto the cryotop with minimum volume, andimmediately submerged into liquid nitrogen.

Results: Oocyte retrieval and cryopreservation was successful in 89% (54/61) ofthe patients. No patients had any adverse side effects using the minimal ovarianstimulation protocol and aspiration with a 22 gauge needle under localanesthesia. The mean age of the patients was 26.0 (±4.8, S.D.). The meannumbers of oocyte retrieval cycles per patient was 1.79, and the mean number ofretrieved oocytes per patient was 7.7, and per cycle was 4.3. The mean numberof morphologically normal cryopreserved oocytes per patient was 6.4, and percycle was 3.5. The type of cancers included acute and chronic leukemia,malignant lymphoma, aplastic anemia, and myelodysplastic syndrome.

Conclusion: Our data showed that the minimal ovarian stimulation protocol usingCC for oocyte retrieval with a 22 gauge needle and with local anesthesia was asafe, simple, effective method of preserving fertility for unmarried cancerpatients.

307.3 – SUCCESSFUL VITRIFICATION OF HUMAN OOCYTES

Masashige Kuwayama, Advanced Medical Research Institute of Reproduction,Kato Ladies’ Clinic, Tokyo, Japan.

Recent drastic advances in cryobiology have made it possible to preserve varioustypes of reproductive cells with relatively little loss of viability. Ultra-rapidvitrification, the alternative cryopreservation method seems to be a powerful toolto any biological specimens, which cannot be preserved by the conventional slowfreezing and previous vitrification. Ultra-rapid vitrification realized the successfulclinical use of vitrification not only for human PN zygotes, cleavage stageembryos and blastocysts but also for oocytes.

The purpose of this lecture is first to introduce the history of vitrification ofhuman IVF, and the mechanism of vitrification of the cells, how to maintain thehigh viability of the oocytes and embryos under liquid nitrogen temperature, foryour basic knowledge.

To provide evidence for the potential significance of vitrification, achievementswith the Cryotop technology, an advanced version of the “minimal volumeapproaches” is analyzed. This technology alone has resulted in more healthybabies after cryopreservation of any stages of embryos than any othercryopreservation technique, and more successful human oocyte vitrificationresulting in normal births than any other cryopreservation method. The value ofthis method is also demonstrated by achievements in the field of domesticanimal embryology.

Cryotop method has been applied to more than 100,000 clinical cases of oocytesand embryo cryopreservation for these 8 years, and is now used in more than800 IVF faculties in 12 countries producing excellent clinical results (nearly 100% of post-thaw survival rates for PN zygotes, 4-cells stage embryos, blastocystsand oocytes) especially the method actually realized the oocytes cryopreservationas an effective protocol in Human IVF. More than 450 of healthy babies have beenalready delivered from Cryotop oocytes vitrification.

Human oocytes banks for unmarried young cancer patients and for egg donationprogram have also established and been giving dream and courage of life for thepatients to improve their quality of lives as women.

307.4 – CRYOPRESERVATION OF PREPUBERTAL TESTICULAR TISSUE

Atsumi Yoshida, Yoko Kurotaki, Miho Tanaka, Hiroki Suzuki, Kiba Park Clinic, Kiba,Koto-Ku, Tokyo, Japan.

For the progress of recent advances in cancer treatments, cure rates of childhoodcancers are very high. However, these treatments may prove toxic to the testis.Fertility preservation is becoming an important issue in the management of thequality of life of prepubertal boys undergoing gonadotoxic treatment. As thesepatients do not yet produce spermatozoa for freezing, the only theoretical optionfor preservation of fertility in these boys is the preservation of the spermatogonialstem cells for autologous intratesticular stem cell transplantation or in vitroculture of spermatogonia. Currently, spermatogonial stem cell transplantation isconsidered to be the most promising tool for fertility restoration in young cancerpatients. The spermatogonial stem cells are the male germline stem cells. Theycan self renew to maintain the stem cell population and produce large numbersof differentiating cells of the spermatogenic line. Spermatogonial stem cell

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transplantation was firstly introduced in the mouse by Brinster et al. In animals,the initial method for transplantation was intratubular microinjection in theseminiferous tubules. In human, the most common infusion technique for germcells is ultrasound guided injection into the rete testis. Most studies oncryopreservation of testicular tissue aimed at preserving sperms forintracytoplasmic sperm injection. Testicular sperm freezing is carried out using inmany instances. The simple rapid method of suspending straws in theuncirculated liquid nitrogen, whereas in freezing of spermatogonial stem cells, itis necessary to accomplish freezing by lowering slowly the temperature bymeans of a programmed freezer as is the case with round spermatids and latespermatids. At this institution, we make it a rule at present to perform freezing oftesticular tissues in a programmed freezer, using a cryoprotectant solutioncontaining sucrose and ethylene glycol. However, for prepubertal testiculartissues, it is considered necessary to re-evaluate the cryoprotectant and theprogram of freezing, taking account of their being unlike adult testicular tissues.Moreover, testicular tissue from cancer patients may be contaminated withcancerous cells. It would be of importance to restore, with discretion in variousrespects, testicular cells collected from a pediatric cancer patient into thatpatient after completion of cancer treatment.

308.1 – DOES A COMBINATION OF CHINESE HERBS AND ACUPUNCTURETREATMENT AFFECT SPERM CHARACTERISTICS IN INFERTILECOUPLES? A PILOT STUDY

Ramon Velleman1,Tal Belo1, Ilya Barr, Guy Cassuto3, Shai Davids1, Yona Barak4.1Kibbutzim College of Education, Tel-Aviv, Israel; 2Israeli Fertility Center, Israel;3Laboratory Drouot , France; 4Dr Yona Barak Laboratories LTD, Israel.

Objective: Classic therapies are known to have a limited effect in the treatmentof patients with poor sperm characteristics. The aim of this study was todetermine the effect of the combination of Chinese herbs and acupuncture onthese males, who failed to conceive in their previous IVF-ICSI attempts

Design: Preliminary prospective analysis

Material and Methods: Sperm samples of 9 couples who failed to conceive in atleast 3 previous IVF-ICSI attempts, were analyzed by light microscope, accordingto WHO criteria. Herbs formulas were daily administrated. Acupuncturetreatments, were done weekly. Sperm analysis were performed before the treatment, 1 month and 2 monthsafter the combined acupuncture treatments and herbs formulas started. Acomparison of the following sperm parameters: volume of ejaculate, PH, spermconcentration, sperm motility and morphology, was performed before and duringthe study. These parameters were also compared with sperm criteria of 12patients, who underwent 2 sperm analyses within a 4 months period of time,without any conventional treatments, or any alternative treatment (control group).

Results: Out of the 9 couples, 3 conceived during 3 months time; two couplesconceived following IVF-ICSI treatment and the other following intrauterineinsemination. A tendency of 0.06 was noticed in a comparison of the rate ofnormal forms before and after 1 month of treatments (14.7%±6.3 vs 28%±13.4,respectively). No change was noticed in the rate of normal forms in the controlgroup (28.67±12.3 vs 31.0±13.12%).

Conclusion: The combination of acupuncture and Chinese herbs may be auseful, no traumatic supporting treatment for males of couples which failed toconceive in IVF, and intend to undergo further fertility treatments. Since our pilotstudy is based on a small number of patients further investigation is still takingplace.

308.2 – IMPROVEMENT OF A PHOTODYNAMIC SYSTEM FOR OBSERVATION OFSEMINIFEROUS TUBULES IN MICRODISSECTION-TESTICULAR SPERMEXTRACTION (MD-TESE)

Atsushi Tanaka, Motoi Nagayoshi, Shoichiro Awata, Norio Himeno, Izumi Tanaka.Dept. of Obstet. and Gynecol., Saint Mother Hospital, Fukuoka, Japan.

Objective: Operating microscopes have long been used for microdissection-testicular sperm extraction (MD-TESE). Although “white, thick, and twisted”seminiferous tubules have been targeted in TESE, it is necessary to observe theinside of the seminiferous tubules through the walls for more accurateidentification of sperm and sperm cells. From this point of view, we hereinvestigated the potential of a newly modified photodynamic system.

Methods: (1) Ophthalmology contact type lenses (2.0~3.0X, BlumenthalSuturelysis lens, VOLK OPTICAL INC. USA) were placed at the focus on the light

axis of the microscope. The light axis and focus distance were appropriately fine-tuned and it was possible to firmly fix the lenses placed at the front with anadditional flexible arm for the surgical microscope. Magnification was improvedby attaching a contact-type lens flexible arm for ophthalmology use to a surgicalmicroscope. In addition, it was possible to clearly observe inner structure of thetesticular tube by obtaining an appropriate lighting angle and height with anexternal light source (fiber-type). To fix the operational distance, “the directcontact method” was employed, and three-dimensional views were obtained bybinocular vision. (2) By attaching ophthalmology lenses as an adaptor,magnification and stability of the attachment were improved. (3) An appropriatelighting angle and proper luminous intensity were obtained with an external fiberlight source.

Results: 1. Sperm or spermatid were found totally in 35.3% (54/153) of nonobstructive azoospermic men using this optic system. 2. In 85% (54/64) of TESEtrials, spermatids and sperm were found when thick seminipherous tubules werefilled with homogenous fine and whitish granules that did not move so muchwithin. 3. No spermatogenic cells (primary spermatocyte, spermatid) were foundand sperms were absent (0/25) when seminipherous tubules were filled withlower density, yellowish, coarse granules. Yellowish substances were debris. 4.Sperm or spermatogenic cells were likely to be found more frequently when thewhitish granules in the seminipherous tubules were not moving so muchcompared to those when they were moving more. 5. Pregnancy rates andmiscarriage rates after microfertilization using testicular sperm and spermatidwere [38.9 % (7/18), 28.6% (2/7)], [22.2% (8/36), 37.5% (3/8)] respectively.

Conclusions:With this system, it was possible to three-dimensionally visualizethe structure of seminiferous tubules and objectively evaluate the degree ofattachment of sperm cells, cellular density, cellular sizes, and color from the wallthickness of the seminiferous tubules. Clinical results with this new photodynamic system will be improved.

308.3 – A NOVEL CRYOPRESERVATION TECHNIQUE FOR VERY FEW MOTILESPERM FROM SEVERELY INFERTILE MEN

K. Kyono, H. Hattori, C. Nishinaka, M. Doshida, K.Yasuda, S. Kanto. Kyono ARTClinic, Honcho, Aobaku, Sendai, Miyagi, Japan.

Sperm of severe oligozoospermia and azoospermia frequently demand freezingfor future fecundity. However, no reliable protocol for freezing very low numbersof sperm has been established. Therefore, we evaluated two new freezingprocedures for sampling very low numbers of sperm.

We used some ejaculated semen from infertile men. Sperm was retrieved byputting a few sperm into a 2 μl droplet of cryoprotectant using a manipulatorunder a reverse microscope. The droplet was diluted by mixing 0.7 of SpermFreezeTM (FertiPro,N.V., Belgium)(SF) and 1.0 of HTF (Irvine, USA). Each includedsome sperm a 2μl droplet placed on 60 mm petri dish (Falcon1006, USA) wascovered with mineral oil and wrapped in Saran wrap. It was left for 10 minutes atroom temperature(RT), for 15 minutes at 4�, for 30 minutes on liquid nitrogenvapor, and immersed into LN2.

On the other hand, in the tip group, 2 μl droplets were aspirated into a CryotipR

(Kitasato Biopharma, Tokyo, Japan). It was left for 10 minutes at RT after closingthe tip point, for 5 minutes on LN2 vapor, and respectively and immediatelyimmersed into LN2.

In thawing procedure of the Dish group, the dish was thawed at RT until dropletswith sperm were completely thawed, and sperm was retrieved by a manipulator.In thawing procedure of the Tip group, the cryotip was incubated in water of 37 �for 20 seconds, and sperm was retrieved by a manipulator. We comparedretrieval rates of sperm (RR), survival rates (SR) and available useful rates (AUR)for ICSI between the Dish group and the Tip group.

In the Dish group, RR was 97.3% (299/309), and SR was 30.0% (82/273), andmotility rate after thawing was 2.9% (8/275), and reaction of hypo osmoticswelling test (Host), positive was 27.9% (74/265), and AUR was 26.5% (82/309).In comparison, in the Tip group, RR was 67.7% (239/353), and SR was 15.9%(36/226), and motility rate after thawing was 3.0% (7/232), and reaction of Host,positive was 13.2% (29/219), and AUR was 10.2% (36/353). RR, SR, and AURwere significantly different between the two groups (P<0.01).

In conclusion, it has been suggested that the freezing methods using a dish is ahighly useful protocol for very low numbers of sperm. However, to raise thecollection rate and the survival rate we need to try other kinds of cryoprotectantand improve the cryopreservation.

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308.4 – ARGUMENTS TO IMPLEMENT THE SELECTION OF SPERMATOZOA ATHIGH MAGNIFICATION BEFORE ICSI

H. Zech1, A. Stecher A1, M. Bach1 , T. Neyer1, M. Zinst1, P. Vanderzwalmen1,2, N.Zech1. 1Institue for Reproductive Medicine and Endocrinology, Bregenz, Austria;2Centre Hospitalier Inter Regional Cavell, Bruxelles – Braine l alleud, Belgium.

Selection of spermatozoa during ICSI procedure is performed at a rather lowmagnification at 200-400X with Hoffman Modulation interferential contrast(HMC) optics. Using such optical tools, selection of spermatozoa has severelimitations. This has been one of the major concerns related to ICSI as the spermselection process, occurring during normal IVF in presence of cumulus cells, istotally bypassed. The changing of HMC by Nomarski differential interferentialcontrast and examination at high magnification, has allowed a better observationof sperm cells in real time. Using “motile-sperm organelle-morphologyexamination” MSOME, “normal spermatozoa” exhibit a large panel of nucleusmalformations in terms of shape, size and presence of vacuoles are detected andnormally not selected. However, a more acute selection of spermatozoa meansthat it is not always possible to find spermatozoa morphologically completelynormal. Several studies reported that the existence of large vacuoles in thenuclei of spermatozoa dramatically reduces the proportion of good qualityembryos reaching the blastocyst stage and logically the pregnancy (PR) andimplantation (IR) rates. More over higher rates of abortion is also noticed.

As a consequence, the pending and crucial question concerns their meaning,origin and the further impact on embryo quality and downstream consequenceson embryo development and more concerning, the progeny.

Can we assume that there is a relationship between chromatin defect - DNAdamage and the presence of nuclear vacuoles? Two recent papers (Garolla 2008and Franco 2008), reinforce the concept that an association between largevacuoles and secondary and tertiary DNA structure damages in sperm nucleusexists.

What are the consequences of DNA damage? In the light of the reports of Aitken(2007), the negative effect of sperm DNA fragmentation may affect the nextgeneration (Aitken, 2007). Furthermore a recent work of Fernandez-Gonzalez(2008) in the mouse model demonstrated that DNA-fragmented spermatozoa inICSI can generate effects that emerge during later life, such as aberrant growth,premature aging, abnormal behaviour, and mesenchymal tumors.

Even though there is a strong “in vivo” selection after embryo transfer. we mustbe cautious, in order to lower the potential risks mentioned here.

In fine, the one of the most frequent questions regarding IMSI relates to itsindications: should we perform IMSI to all the patients? Another debate raises thequestion of what should we do for patients carrying 100% large vacuoles in theirsperm samples. Even though there is no real proof in the human species on theabnormal outcome generated by spermatozoa carrying vacuoles, this ultra-morphology technique has to be added as an additional tool for ICSI knowing theconsequence of possible DNA damage for offspring (Carrell, 2008). Shall wecontinue the attempt with his sperm or propose the option of sperm donor? Theestablishment of new classification criteria, based on an assessment system,seems a valuable approach to determine a threshold limit for making the righttherapeutic decision. In conclusion, sperm selection before ICSI seems more andmore unavoidable whatever the screening technique (hyaluronique binding test),in order to lower the potential risks detailed here. Analysis of semen samples onan ultramorphologic scale for the presence of vacuoles should therefore berecommended to patients before ICSI.

309.1 – SEARCH FOR MECHANISMS UNDERLYING IMPRINTING DEFECTS INSPERM

Anna K. Naumova1,2,5, Sanny Moussette1, Aabida Saferali2, Nicola Dean1,Donovan Chan, Jacquetta Trasler2,3,4,5, Taiping Chen6, Teruko Taketo7, RimaRozen2,3,5 , Seang Lin Tan1,5. Departments of 1Obstetrics and Gynecology,2Human Genetics, 3Pediatrics, 4Pharmacology and Therapeutics,7Department of Surgery and Biology, McGill University, Montreal, QC,Canada; 5Research Institute of the McGill University Health Centre, Montreal,QC, Canada; 6Novartis Institutes for BioMedical Research, Cambridge, MA,USA.

Introduction: Genomic imprinting is a parent-of-origin dependent epigeneticmarking of chromosomes. During gametogenesis, genomic imprints areerased, and reestablished depending upon the sex of the individual.

Abnormal imprint establishment has been observed in the sperm of menwith defective spermatogenesis. However, the exact mechanism underlyingthis phenomenon remains elusive. The goals of the present study are 1) todetermine if the patient population in Montreal has imprinting defects insperm DNA and 2) to elucidate the mechanisms underlying imprintingdefects in infertile men. We hypothesized that imprinting defects in humansperm DNA may result from partial loss of function mutations in genesinvolved in imprint establishment in the germ line.

Materials and Methods: Patients with normal and defective spermatogenesiswere recruited in the McGill University Reproduction Centre. DNAmethylation pattern of three imprinted regions (H19, SNRPN and IGdifferentially methylated regions (DMR)) were investigated in 15 patients.Six of these patients had male factor infertility and 9 had normal spermparameters. In parallel, to test our hypothesis, we tested the efficiency ofimprint establishment in the germ line of mice that carry heterozygousmutations in the genes known to be involved in imprint resetting and/orspermatogenesis. We explored the effect of mutations in the 5,10-methylenetetrahydrofolate reductase (Mthfr), DNA methyltransferase 1(Dnmt1), Dnmt3a, Dnmt3b and mutS homolog 5 (E. coli) (Msh5) genes. Wehave also tested the effect of folate deficiency on DNA methylation in sperm.

Results: incomplete methylation at the H19 DMR and partial methylation ofthe SNRPN DMR were found in 3 and 4 patients, respectively. Consistentwith reports from other groups, the same defects were found in both maleswith defective and normal spermatogenesis: among the patients with normalspermatogenesis, two had partial methylation of the SNRPN DMR and onehad incomplete methylation of the H19 DMR. Among the patients withabnormal spermatogenesis, two had defects in H19 methylation and one hadpartial methylation of the SNRPN DMR. Some of the mouse mutations wereassociated with incomplete imprint establishment at the H19 locus andshowed aberrant methylation patterns similar to those observed in thesperm of human males.

Conclusions: Abnormal methylation at imprinted regions is not necessarilyassociated with abnormal spermatogenesis in human males. Methylationpatterns reminiscent of those found in humans were observed for some ofthe mouse mutations, suggesting that partial deficiency for enzymesinvolved in DNA methylation and/or meiosis may be the underlyingmechanism of human imprinting defects.

This work was supported by funds from the McGill Reproductive Center(MRC) and the National Sciences and Engineering Research Council ofCanada (NSERC).

309.2 – EFFECT OF MATURATION IN VITRO ON SPINDLE MORPHOLOGY INHUMAN OOCYTES. A COMPARATIVE STUDY ON PATIENTS WITH MALEFACTOR

M. Mette Munk, E. Novella-Maestre, F.B. Lindenberg, S.S. Jensen, S.Lindenberg. Section for Reproductive Biology and ART, Nordica FertilityClinic, Copenhagen, Denmark; Instituto Valenciano de Infertilidad (IVI),University of Valencia, Spain.

Introduction: Incomplete cytoplasmic maturation of in vitro matured (IVM)oocytes has been known to cause microtubule and filament alterations,which may results in abnormal pronuclear formation and later failedembryogenic development. In the present study we examined the influencesof in vitro maturation of human ova to the MFII stage in a standardized invitro maturation medium compared to in vivo matured MFII stage ova priorto the ICSI procedure.

Material & methods: A total of 44 patients admitted for IVM and 44 patientsadmitted for IVF/ICSI were analyzed using a polscope for evaluating thepresence and placement of the spindle in relation to the polar bodies in allMFII oocytes produced after egg collection prior to ICSI or after 28 h in vitromaturation following the IVM protocol.

Results: We found the same mean number of oocyte retrieved (IVM 4,9oocyte versus ICSI 5,9 oocyte), the mean number of MFII oocytes aftercollection of IVF/ICSI oocytes were 5,1 versus 2,1 for the IVM oocytes after28 h maturation (P<0,01) and finally we found a significantly higher rate ofdisplacement of the spindle apparatus in the in vivo matured oocytescompared to the in vitro matured oocytes. (P > 0.05).

Conclusions: These findings indicate that in vitro matured human oocyteseither mature from MF I to MFII normally having a normal spindle

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appearance after 28 hours in vitro maturation or detoriate completely. Thisindicate, that IVM matured oocytes if they reach the MFII are normal, andthe obvious lower implantation rate in IVM might be due to other factorssuch impaired endometrial development during the IVM procedure

309.3 – PATERNAL AGE AFFECTS SPERM DNA FRAGMENTATION BUT NOT ITSPACKAGING

Stéphanie Belloc, André Hazout, Martine Cohen-Bacrie, Moncef Benkhalifa,Martine Dumont, Anne Marie Junca, Yves Menezo, Paul Cohen-Bacrie.Laboratoire Eylau Unilabs, Paris, France.

Introduction: Basic parameters such as concentration, motility andmorphology are of limited value in determining the “embryotrophic”potential of sperm, i.e. the ability of sperm cell to establish a full termpregnancy. DNA integrity, including secondary and tertiary structure i.e.DNA fragmentation and decondensation are now mandatory to approachthis sperm functional aspect.

Material and Methods: Paternal age has only recently been considered(Klonoff-Cohen and Natarajan, 2004, Belloc et al. 2008), as affecting thepotential of sperm. We have tested on more than 1000 patients the effects ofage on sperm DNA fragmentation (measured with TUNEL) anddecondensaion measured with aniline blue (AB). AB stains lysine-richhistone proteins, leaving the arginine-rich protamine proteins unstained andthus is more specific of sperm maturity.

Results: Sperm DNA fragmentation increases with age (p<0.001) but notwith the percentage of atypical forms. In contrast, decondensationdecreases, but not significantly, with increased age (p=0.063), yet the qualityof DNA packaging is negatively associated with the percentage of atypicalforms (p<0.006). These two observations fit with the obvious lessresistance towards Reactive Oxygen Species with age. ROS are involved inDNA fragmentation but also in condensation through protamines crosslinking. DNA damages affect the outcome of ART procedures. If the impactof fragmentation is evident, anomalies in the tertiary structure of sperm DNAdo affect, sooner or later, normal embryonic development even if fertilizationis not impaired (Rousseaux et al. 2008).

Conclusion: These two parameters should not be neglected irrespective ofthe age of the male partner, yet the age of the female partner can bebalancing aspect as the oocyte is able to repair partly fragmentation decayand more weakly some tertiary structure anomalies (Ménézo et al. 2007).

309.4 – THE LOCALIZATION OF SPECIFIC PROTEIN KINASE C ISOTYPES INMOUSE OVARY

Filiz Tepekoy, Zeliha Sahin, Gokhan Akkoyunlu. Department of Histology andEmbryology, Medical Faculty, Akdeniz University, Antalya, Turkey.

Introduction: The mammalian protein kinase C (PKC) consists of a family oftwelve distinct members. They can be subdivided into three subfamilieswhich are classified on the basis of sequence similarities and their modes ofactivation. The conventional PKCs (α, β1, β2, γ) are all activated byphospholipids, in particular phosphatidyl serine, DAG (diacylsglycerol) andCa2+ where the novel PKCs (δ, ε, η, θ, µ) do not require Ca2+for its action.The atypical PKCs (λ, ι, ξ) respond neither to DAG nor to Ca2+ but they stillrequire phospholipids. The PKCs are suggested being critical in theregulation of follicular development, ovulation and luteinization. Although itis hypothesized to control different cellular processes, the specificlocalization of these PKCs has not been enlightened in ovary up today. Theresults of this study will suggest the specific localization of PKC isotypes innormal female mouse ovary.

Materials & Methods: Ovary samples were obtained from normal adultfemale mouse (n= 6). The localization of PKC isotypes was determined oncryosections by using immunohistochemical techniques.

Results: PKCα expression was present in all different maturation stage ofoocytes in developing follicules. The PKCα expression was also seen ingranulosa cells of Graaf follicules and steroidogenic luteal cells.

The PKCδ expression was still apparent in luteal cells of corpora lutea.However, it was less obvious in the oocytes of Graaf follicules compared toPKCα expression and was negative in granulosa cells.

The PKCε expression was showed a similar expression pattern with PKCδ in

oocytes and granulosa cells. Hence, its expression in corpora lutea waslocalized on luteal cell membranes.

Conclusions: Our data demonstrate that PKC isotypes are differentiallyexpressed in normal mouse ovary. The mode of action of DAG, Ca2+,phospholipid dependent PKCα seems to be regulating the granulosa cellactivity and luteal cell functions. Oocyte maturation may also be maintainedvia PKCa. However Ca2+ independent PKCδ and ε seems to regulate onlyluteal cell functions. Although we did not differentiate between theexpressions of large and small luteal cells, localization of the novel PKCisotypes PKCδ and ε support their role in luteolysis. We suggest that theovary took the advantage of differentially stimulated PKCs where they canregulate different follicle stages and corpus luteum functions by processingdifferent PKC isotypes.

Acknowledgement: The study was supported by Akdeniz University,Scientific Research Fund.

309.5 – IMPACT OF A GnRH ANTAGONIST ON THE OUTCOME OF CONTROLLEDOVARIAN HYPERSTIMULATION (COH) IN WOMEN WITH POLYCYSTICOVARY SYNDROME: A PROSPECTIVE AND RANDOMIZED STUDY

Laurel A. Stadtmauer1, Silvina Bocca1, Hind Baydoun2 , Beth Pultz1, SergioOehninger1. 1The Jones Institute for Reproductive Medicine, Department ofObstetrics and Gynecology, Eastern Virginia Medical School; 2Department ofEpidemiology, Eastern Virginia Medical School; Norfolk, VA, USA.

Introduction: The study was conducted to evaluate the efficacy of theGonadotropin releasing hormone (GnRH) antagonist Ganirelix® when usedas adjuvant to gonadotropin stimulation with recombinant folliclestimulating hormone (Follistim®) in PCOS patients undergoing ovulationinduction and IUI.

Materials and Methods: A prospective randomized controlled study ofanovulatory women with PCOS undergoing ovulation induction cycles withIUI (n=138). The 3 groups were: Group 1, Follistim® alone; Group2,Follistim® with Ganirelix®, initiated at a follicle size ≥13 mm in diameter;Group 3,Follistim® with Ganirelix from day 1 of stimulation. The primaryoutcome measure was the clinical pregnancy rate per cycle of treatment.Secondary outcomes measures included the total gonadotropin dose percycle, number of days of treatment, peak serum estradiol levels in eachcycle, and premature luteinization rate and live birth rate per completedcycle..

Results: The results show a trend towards an improvement in the clinicalpregnancy rates per cycle initiated in patients treated with Ganirelix®(Group 2, 17/48, 35%), compared with Groups 1 (10/48, 22%) or Group 3(8/42, 20%) with p=0.1. All except 2 pregnancies were singletons (2 twins inGroup 2). In the absence of Ganirelix® (Group 1), premature luteinizationoccurred more often (25% vs. 0% vs. 2.% p<0.05) and the LH levels on theday of hCG were significantly higher than in Groups 2 or 3. Group3 had thehighest cancellation rate and cost per cycle. Ganirelix® did not have anyeffect on peak estradiol levels, total vials of r-FSH used or days ofstimulation.

Conclusions: Premature luteinization occurred commonly in women withPCOS undergoing ovulation induction with Follistim ® alone and this isassociated with lower pregnancy rates. There may be a benefit in theaddition of a GnRH antagonist. The higher, although not significant,pregnancy rate in Group 2 patients suggests that the flexible Ganirelixregimen could be the treatment of choice for PCOS undergoinggonadotropin stimulation and IUI.

309.6 – OVARIAN AGING: PROGNOSTIC FACTORS, BIOLOGICAL FACTORS ANDOUTCOME OF ART

Andrea R. Genazzani, P. Monteleone, O.M. Di Berardino, G. Simi, P.G. Artini.University of Pisa, Pisa, Italy.

Numerous evidences support the idea that the chief regulator of femalereproductive aging is the ovary. Female ageing the most significantdeterminant of success in IVF. Ovarian functional decline with aging hasbeen so far studied in depth in terms of accelerated depletion of the ovarianfollicle pool and reduced ability to produce oocytes competent forfertilization and development. Few studies have addressed the questionabout potential causal factors of ovarian aging. According to the most

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relevant concept of ageing, age-associated malfunction results fromphysiological accumulation of irreparable damage to biomolecules as anunavoidable side effect of normal metabolism. Moreover, it was suggestedthat an important environmental factor responsible for oocyte senescencemight be represented by a reduced oxygen supply to the leading follicle, acondition dependent on a compromised perifollicular vascularisation. Futureinvestigation of age-related molecular damage in the different ovariancomponents is imperative in order to develop strategies to possibly save orrescue the developmental potential of aged oocytes.

309.7 – FIRST TIME EVIDENCE FOR INCREASED PLACENTAL GROWTHFACTOR MRNA IN ENDOMETRIUM OF PATIENTS WITH SUCCESSFULIMPLANTATION

Alessandro Santi, Niklaus A. Bersinger, Dorothea Galié-Wunder, Micheal D.Mueller. University Hospital Berne, Berne, Switzerland.

Introduction: The aim of this prospective study was to analyse the in vivovascularisation of the endometrium as described at hysteroscopy and todetermine a possible relationship with angiogenic factors and theimplantation rate.

Material and Methods: Consecutively admitted infertile patients with aplanned hysteroscopic evaluation for infertility were asked to participate inthe study. The study protocol was approved by the local ethical committee.All patients had a pre-operative transvaginal sonography (TVS). To evaluatethe vascularisation of the endometrium at hysteroscopy the procedure wasperformed in the secretory phase of the menstrual cycle. The surgeon wasblinded to the TVS-findings. The quality of the endometrium was evaluatedaccording to the Sakumoto-Masamoto grading (“good” vs. “poor”) at thetime of hysteroscopy and an endometrium biopsy was taken at the end ofthe procedure using a soft curette. An aliquot of the biopsied tissue wassubjected to total RNA extraction, reverse transcription, and quantitativepolymerase chain reaction (QPCR) for several angiogenic factors. The datawere analysed in order to find a possible relationship between thevascularisation of the endometrium, the implantation rate (spontaneouspregnancy, intrauterine insemination by husband and IVF/embryo transfer)and the transcription rate (RT-QPCR) of the angiogenic factors. Forstatistical evaluation a t-student test was applied using Instat Graph Padversion 4.0 for windows.

Results: One hundred and sixty-two infertile patients with a median age of36.4 years (range 26-43) were included in the study. One hundred and eight(66.7%) were classified endoscopically as having “good” mid-secretoryendometrium and 54 patients (33.3%) as “poor”. There were no differencesin the distribution pattern of the infertility causes between these two groups,the age of the patients, and the delay of infertility. The overall pregnancy ratewas 37.0% (60 patients). Endometrial biopsy samples from 16 patients witha successful pregnancy and a “good” endometrium (group A) werecompared with biopsies from 10 patients with a “bad” endometrium andwithout pregnancy (group B). The patients of group A presented asignificantly higher level of mRNA for Placenta Growth Factor (PLGF) mRNAin the biopsy than those of group B (P = 0.0184), while VEGF-A,Angiogenins, and their different receptors did not show such any differencein normalised mRNA content between the two groups.

Conclusions: This in vivo study demonstrates for the first time that PLGF inthe endometrium biopsy together with the hysteroscopic appearance(vascularisation) of the endometrium might be an important prognosticfactor for the evaluation of the success rate in a therapy for infertility. Ourresults need a larger number of patients in order to confirm this hypothesis.

310.1 – EFFICIENCY OF IVM EGG COLLECTION

Bülent Gülekli, Obstetrics & Gynecology and Division of ReproductiveEndocrinology & IVF Unit, Dokuz Eylul University School of Medicine, Izmir,Turkey.

In vitro maturation (IVM) of immature oocytes retrieved from womenwithout any ovarian stimulation is a promising new treatment especially forwomen with polycystic ovary syndrome (PCOS), with many successfulpregnancies reported worldwide. Although Cha et al reported the first birthusing immature oocyte donation from oopherectomy specimens in 1991, in1994 it was Trounson and colleagues who put IVM in the clinical practicewhen they reported the first pregnancy using oocytes collected by

transvaginally ultrasound guided follicle aspiration from a patient withpolycyctic ovaries (1,2).

Before the first successful birth with in vitro fertilization (IVF) and embryotransfer, the first culture and maturation of human oocytes in vitro werecarried out on oocytes that were obtained by laparatomy. Similarexperiments were done by Steptoe and Edwards and they introducedlaparoscopic method for aspirating oocytes from the Graff follicles (3).Laparoscopic oocyte retrieval procedure may be performed either 2 or 3puncture technique depending on the type of the laparoscope and theexperience of the surgeon. Each follicle is punctured and aspirated at anavascular site. The needle may be left in the follicle to permit flushing andreaspiration in case that an oocyte is not identified in the initial aspirate.Following those first few births via laparoscopilly aspirated oocytes,ultrasound guided oocyte collection techniques has been described. Whilelaparoscopic oocyte retrieval is generally performed under generalanesthesia with endotracheal intubation the major advantages for the TVStechnique include decreased exposure to general anesthesia, lower chancefor operative complications, and feasibility of performing on an outpatientbasis. On the other hand laparoscopy identifies visible follicles on theovarian surface, whereas TVS also identifies intra-ovarian follicles.

There are many different ultrasound guided oocyte collection techniques.The first follicular puncture under ultrasound guidance was described in1981 and the same group was introduced transabdominal transvesicalultrasound directed oocyte recovery for the oocytes from the ovarianfollicles a year later (4,5). Oocyte retrieval for IVF by ultrasonically guidedneedle aspiration via the urethra was proposed by Parsons and coworkers in1985 (6). The above techniques have in routine use during the days ofconventional external transducers. Transvaginal sonography guidedtransvaginal follicular aspiration was initially reported in 1983 by Gleicher etal. and become dominant method over laparoscopy (7). In this techniquethe follicles are punctured with a single or double lumen needle connectedto the vaginal transducer with a guide. Aspiration with a negative pressure <20 Kpa (150 mm HG) is performed gently and there is no need to withdrawthe aspirating needle from the ovary to move the next follicle. This TVSguided follicular aspiration technique have become the method of choiceduring the years because of the refinements in instrumentation andincreased experience of the clinicians. Within the years the automaticaspiration and washing systems has been introduced and the follicles can beaspirated and, if necessary, washed with various systems. It has beenshown that oocyte retrieval rates may be improved by using this flushingsystems in IVF cycles, especially in patients who have low number offollicles (e.g. natural or minimal stimulation IVF cycles or in poorresponders).

Even transvaginal sonography (TVS) guided follicular aspiration has nowbecome the preferred procedure of choice for oocyte retrieval in IVM cyclescomparing with the conventional IVF oocyte retrieval it requires certainmodifications(8). In a typical IVM cycle oocyte retrieval was performedunder ultrasound control between 10 and 14 days after withdrawal bleedingusing a specially designed aspiration needle and an aspiration pressure of7.5 kPa which is half of conventional aspiration pressure. Althoughpregnancy rates have in general been relatively low, recent reports suggestimproving IVM success by hCG priming (9). All the follicles had to be < 10mm in diameter because data suggest that the presence of dominant follicleat the time of immature oocyte retrieval is deleterious to outcome in IVM(10,11). The maximum diameter of the follicle on the day of oocyte recoveryprovides one of the main differences between IVF and IVM cycles. Aspecially designed 17-gauge single lumen aspiration needle was introducedinto the follicle with the bevel facing downward to prevent loss of follicularfluid. To prevent blood clotting heparinized saline was used as the aspirationmedium. An IVM oocyte collection involves multiple ovarian punctures sincethe single channel aspiration needle tends to block frequently when passingthrough the dense ovarian stroma and follicular flushing is not performed.The collection also takes, on average, longer than IVF oocyte retrievalbecause of the repeated flushing of the needle and the tubing in order toprevent the blockage of the needle (12,13). Because of the multiple ovarianpunctures the IVM oocyte retrieval is likely to be more painful than a routineIVF oocyte collection. Consequently, the method of anesthesia used duringan IVM oocyte collection, especially in some patients who have previouspoor response with IV anesthetic, may become a general anesthesia.

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References

Cha KY, Koo JJ, Ko JJ, et al. Pregnancy after in vitro fertilisation of humanfollicular oocytes collected from nonstimulated cycles, their culture in vitroand their transfer in a donor oocyte program Fertil Steril 1991; 55: 109

Trounson A, Wood C and Kausche A. In vitro maturation and the fertilisationand developmental competence of oocytes recovered from untreatedpolycystic ovarian patients Fertil Steril 1994: 62; 353

Steptoe PC and Edwards RG. Birth after reimplantation of human embryo.Lancet 1978; 2: 366

Lenz S, Lauritsen G, Kejlow M. Collection of oocytes for IVF by ultrasonicallyguided follicular puncture Lancet 1981; 1: 1163

Lenz S, Lauritsen JG. Utrasonically guided percutaneous aspiration ofhuman follicles under local anesthesia: A new method of collecting oocytesfor in vitro fertilization. Fertil Sterl 1982; 36:673

Parsons J Riddle A, Booker M. Oocyte retrieval for in vitro fertilization byultrasonically guided needle aspiration via uretra Lancet 1985; i: 1076

Gleicher N, Friberg N, Fullan N et al. Egg retrival for in vitro fertilization bysonographically controlled vaginal culdocentesis Lancet 1983; 2: 508

Gülekli B,Demirtas E, Buckett WB. Immature oocyte collection. In In-vitroMaturation of Human Occytes Basic sciences to clinical application Edited byTan SL, Chian R, Buckett WB,pp 253-263 , 2007 InformaUK Ltd

Chian RC, Gülekli B, Buckett WM, Tan SL. Priming with human chorionicgonadotropin before retrieval of immature oocytes in women with infertilitydue to the polycystic ovary syndrome N Engl J Med 1999; 341: 1624

Russell JB. Immature oocyte retrieval combined with in-vitro oocytematuration. Human Reprod. 1998;13:63

Cobo AC, Requena A Neuspiller F et al. Maturation in vitro human oocytesfrom unstimulated cycles: selection of the optimal day for ovum retrievalbased on follicular size. Hum Reprod 1999:14;1864

Child TJ., Abdül-Jalil AK., Gülekli B., Tan SL., In vitro maturation andfertilisation of oocytes from unstimulated normal ovaries, polycystic ovariesand women with polycystic ovary syndrome. Fertil Steril 2001; 76:936

Child TJ, Phillips SJ, Abdul-Jalil AK, Gülekli B, Tan SL. A comparison of invitro maturation and in vitro fertilization for women with polycystic ovariesObstet Gynecol 2002; 100: 665

310.3 – HCG PRIMING FOR IVM

Weon-Young Son, Royal Victoria Hospital, Montreal, Canada.

A major side-effect of controlled ovarian hyperstimulation (COH) in patientswith polycystic ovary or polycystic ovarian syndrome (PCOS) is the risk ofovarian hyperstimulation syndrome (OHSS). In-vitro maturation (IVM) ofimmature oocytes represents a potential alternative for the fertility treatmentof these patients. Recently, applications of FSH (hMG) or hCG priming havebeen used before immature oocyte retrieval to improve the success rate ofIVM procedures.

Although it is still controversial, hCG priming before oocyte retrieval seemsbeneficial in terms of easier oocyte retrieval, easier oocyte identificationunder stereomicroscope, maturation competence, and may increase theharvest in vivo mature oocytes. Recently, new approaches attempted toimprove IVM program such as extending hCG stimulation or optimal hCGtiming before immature oocytes retrieval. Therefore, as a first option, hCG-priming IVM treatment can be offered to women with PCO(S) instead ofconventional IVF treatment with ovarian stimulation. In this session, I wouldlike you to introduce on embryological factors affecting the clinical outcomeof IVM cycles after hCG priming.

311.1 – PREIMPLANTATION GENETIC DIAGNOSIS (PGD) FOR CANCERPREDISPOSITION GENES

Yuval Yaron, Tel Aviv, Israel.

Inherited germ-line mutations in cancer predisposition genes areresponsible for about 10% of cancers and may explain aggregation ofcancer cases in some families. Such mutation may involve oncogenes,tumor-suppressor genes, mismatch-repair genes, and others. Once apathogenic mutation has been identified, it is possible to perform pre-symptomatic testing, as well as offer prenatal diagnosis and termination ofaffected pregnancies. This however poses significant psychological, ethical,moral and legal issues. Alternatively, it is possible to performpreimplantation genetic diagnosis (PGD) in single cells from embryosobtained by in vitro fertilization (IVF). Transfer of embryos free of themutation will ensure birth of unaffected offspring. While circumventingsome of these issues, PGD for cancer predisposition raises new dilemmas,and according to some opinions, presents the "slippery slope" towards"designer babies". In this talk I will discuss these issues vis-à-vis our clinicalexperience.

311.2 – PGD IMPROVED PGD TESTS AND CLINICAL APPLICATIONS

René Frydman, Service de Gynécologie-Obstétrique et Médecine de laReproduction, Hôpital Antoine Béclère; Univ Paris-Sud ; INSERM U782;Clamart, France.

Preimplantation genetic diagnosis (PGD) is used to analyze embryos geneticallybefore their transfer into the uterus. It was developed first in England in 1990, aspart of progress in reproductive medicine, genetic and molecular biology. PGDoffers couples at risk the chance to have an unaffected child, without facingtermination of pregnancy. Embryos are obtained by in vitro fertilization withintracytoplasmic sperm injection (ICSI), and are biopsied mostly on day 3;blastocyst biopsy is mentioned as a possible alternative. The genetic analysis isperformed, on one or two blastomeres, by Fluorescent In Situ Hybridization (FISH)for cytogenetic diagnosis, or Polymerase Chain Reaction (PCR) for moleculardiagnosis. Genetic analysis of the first or second polar body can be used to studymaternal genetic contribution. Only unaffected embryos are transferred into theuterus. To improve the accuracy of the diagnosis, new technologies are emerging,with Comparative Genomic Hybridization (CGH) and microarrays.

In Europe, depending on national regulations, PGD is either prohibited, or allowed,or practiced in the absence of recommendations. The indications arechromosomal abnormalities, X-linked diseases or single gene disorders. Thenumber of disorders being tested increases. In Europe, data collection from theyear 2004 reports that globally 69.6% of cycles lead to embryo transfer andimplantation rate is 17%. European results from the year 2004 show a clinicalpregnancy rate of 18% per oocyte retrieval and 25% per embryo transfer, leadingto 528 babies born. The cohort studies concerning the paediatric follow-up ofPGD babies show developmental outcomes similar to children conceived afterIVF-ICSI.

Recent advances include Human Leucocyte Antigen (HLA) typing for PGDembryos, when an elder sibling is affected with a genetic disorder and need stemcell transplantation. The HLA-matched offspring resulting can give cord blood atbirth. Preimplantation genetic screening (PGS) consists in euploïd embryoselection; it could be used for advanced maternal age, repeated implantationfailure, single embryo transfer or idiopathic recurrent pregnancy loss. Theseapplications are controversial. PGD for inherited cancer predispositions isdiscussed and social sexing remains prohibited in Europe.

Conclusion: PGD requires a close collaboration between obstetricians, fertilityspecialists, IVF laboratory and human geneticists. It needs intensive effort,expensive techniques and is demanding for the patients, but it offers tremendousopportunity for couples whose previous child has exhibited genetic abnormalities.The debate on certain indications is ongoing.

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312.1 – ULTRASOUND GUIDED VERSUS BLIND TOUCH EMBRYO TRANSFER:THE EGYPTIAN EXPERIENCE

O. Azmy, T. Taha, M Bibars, M. Refaat. Reproductive Medicine Department,National Research Centre, Cairo, Egypt.

Objective: To compare the success rate of ultrasound-guided (USG) embryotransfers versus clinical touch (CT) embryo transfers among patientsundergoing ICSI cycles.

Design: A case-control study

Setting: analysis of women records undergoing fresh embryo transfer at theNational Research Centre in the Department of Reproductive Medicine, overthree year period between 2004 and 2007.

Patients: Eight hundred and fifty three cycles of ICSI were analyzed asregards the technique of embryo transfer, whether by USG or the CTtechnique.

Methodology: Comparisons were made as regards the chemical and clinicalpregnancy rates as well as the ectopic pregnancy and miscarriage ratesbetween the two groups. Corrections were made for the patient’s age, causeof infertility, body weight, induction protocol, number and quality ofembryos transferred and the difficulty or blood stained procedure.

Results: The chemical pregnancy was significantly higher among the USGversus the CT embryo transfer techniques (47.6%, 25.5% respectively,P<0.001). Furthermore, the clinical pregnancy was higher among the groupusing the transvaginal USG during embryo transfer compared with those inthe group using the clinical CT method (32.3% and 12.3% respectively,P<0.0001). In-spite of the increased inconvenience a woman mayexperience during the abdominal ultrasound with embryo transfer, however,the woman has more than double the chances of being pregnant if theembryos were transferred under vision rather than blindly (Relative risk1.86; 95%CI 1.53 to 2.25 , P<0.0001). Nevertheless, there were nosignificant differences in ectopic pregnancy or miscarriage rates betweenthe two groups.

Conclusion: In our experience, although patient’s discomfort is more whileusing the USG technique ET, we believe that this maneuver significantlyimproves the chances of achieving a successful pregnancy.

312.2 – A MICROSYSTEM WITH WHITE LIGHT OPTICAL SENSOR TO QUALIFYTHE CYTOPLASM MATURITY OF HUMAN OOCYTES

Florie Vidberg1, Clotilde Amiot1, Christian Pieralli 2, Bruno Wacogne2, AlphéeBAILLY1, Christiane Joanne1, Germain Agnani3, Christophe Roux1. 1Servicede Génétique Histologie Biologie du Développement et de la ReproductionCHU; 2Institut FEMTO-ST; 3Service de Gynécologie Obstétrique CHU;Besançon, France.

Introduction: During ICSI attempts oocytes reaching metaphase II aremicroinjected. A morphological examination under a microscope providesthe usual means for determining oocyte maturity based on the meioticstatus. On the other hand, cytoplasmic maturity is difficult to assess usingthis method. In this study the cytoplasm maturity was analysed using amicrosystem recording of white light transmission spectra of oocytes.

Materials & Methods: The microsystem consists of an optical sensorintegrated onto a silicon anodic bonded lab-on-chip (LOC). This LOC wasinitially devoted to micro fluidic application for moving and trapping a singleoocyte. In the present study the oocyte is held with a micropipette.

Two aligned optical fibres were connected to the LOC surface. A white lightsource is launched into the illumination fibre (50 µm core diameter). Thelight propagates through the oocyte and is collected with another fibre (100µm core diameter). The transmission spectrum of the oocyte is recordedand divided by the transmission spectrum of the IVF medium only.

The reference transmission spectrum I1(λ) is given by I1(λ) = I0(λ)C1Rwhere I0(λ) is the illumination spectrum, C1 the coupling coefficientbetween fibres and R(λ) the spectra response of spectrophotometer. Thecollected spectrum is given by I2(λ)=I0(�)T(λ)C2R(λ) with T(λ) oocytetransmission spectrum and C2 the new coupling coefficient between fibresdue to light focalisation by oocyte. The global spectrum is:I2(λ)/I1(λ)=(C2/C1)T(λ).

Oocytes excluded from ICSI attempts were tested after denudation: 57

Prophase I (PI) germinal vesicle present, 33 metaphase I (MI) no first polarbody and no germinal vesicle, 10 metaphase II (MII) first polar bodypresent. Immature oocyte (PI) and (MI) were cultured to capable ofundergoing final in vitro maturation. 15 primordial follicles isolated fromovarian tissue were also analysed.

For each group the minimum of the transmission (arbitrary unit, a.u.) andcorresponding wavelength position (nm) of the spectra were computerizedand compared with T test of Student and ANOVA.

Results: The 4 oocyte groups presented different transmission spectrumprofiles. Prophase I, Metaphase I and Metaphase II have significantminimum of transmission (0.34, 0.44, 0.49 a.u. respectively - p<0.05) andare characterized by a progression of their transmission spectrum with thedegree of maturity. Primordial follicles presented with a different white lighttransmission profile (0.21 a.u.).

Conclusions: Transmission spectrum analysis of oocyte by using of thismicrosystem, a non deleterious technology allows the assessment ofcytoplasm maturity degree during ICSI attempts. This technology could bean alternative to microscope observation. Moreover, it could play a roleduring follicle culture or oocyte in vitro maturation protocols.

312.3 – HYSTEROSCOPIC ENDOMETRIAL EMBRYO DELIVERY (HEED):TRANSFER OF DAY 2-5 EMBRYOS

Michael Kamrava1, Asha Bhargava2, Jerry Hall3; 1West Coast IVF Clinic,Beverly Hills; 2LA Center for Embryo Implantation, Beverly Hills; 3UCLAGeffen Medical School, Los Angeles; CA, USA.

Introduction: Since the inception of in vitro fertilization (IVF), the procedurehas seen many advances that have significantly improved pregnancy ratesas well as a reduction in complication rates. Embryo transfer at the cleavagestage began as the standard of practice both because of limitations in theability to culture later stage embryos at that time and because of the abilityof human embryos at that time to develop even at an early stage whenplaced into the uterus. However, improved understanding of embryometabolism led to the development of a culture sequence that optimizesgrowth and implantation at various stages of development, includingblastocysts.

The benefits of blastocyst stage transfer have been established for theroutine blind transfer technique of catheter introduction into the uterinecavity when a patient produces a large number of oocytes or has provencapacity to produce blastocysts. Higher pregnancy and live birth rates areobserved for other than cleavage stage embryos, especially in patients withhigh numbers of eight-cell embryos present 72 hours after fertilization. Thisalso implies improved success with the transfer of fewer embryos, whichmay translate into fewer multiple gestations and the ability to implant themost viable embryo(s) available. However, recent recommendations in usinglower medication dosages for the controlled ovarian hyperstimulation, andin patients with lower response or advanced age, the number of developingembryos are limited and cleavage stage embryo transfers may be moreadvantageous. Also, since a significant number of patients embryos developpoorly beyond day three, early transfer is clinically prudent.

In this study, we use a flexible mini-hysteroscope with a flexible catheter fordirect delivery of embryos onto the endometrium under direct visualization.We have previously shown that our technique for blastocyst stageimplantation resulting in a high pregnancy outcome. Now we seek to explorethe outcomes of embryo transfer at the cleavage stage versus the blastocyststage, using our novel approach. We hope to further optimize pregnancyand birth rate outcomes and thereby minimize the risk of multiple births andcomplications.

Objectives: To compare pregnancy and birth outcomes of embryo transfersperformed on day two or three or five performed under direct visualization.

Materials and Methods: 32 consecutive patients with Infertility of variousorigins underwent Hysteroscopic Endometrial Embryo Delivery (HEED) onday two or three or day five after fertilization. Controlled ovarianhyperstimulation was done using standard protocols. Transvaginal oocyteretrieval was performed under local anesthesia with mild sedation. Allwomen received some type of luteal support, be it progesterone or hCG.Oocytes were fertilized and cultured in early cleavage medium (IrvineScientific) at 37 degrees C and 5% CO2 in air. Embryos were transferred at48-120 hours post fertilization.

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Results: Table 1. Pregnancy Outcomes by Day of Transfer

Day 2 Day 3 Day 5

Average Age (Years) 39 35 37

Total number of patients 16 13 3

Number of cancellations 0 0 0

Number of patients with retrieval 16 13 3

% Total pregnancies per retrieval 40.0 61.5 66.7

% Spontaneous abortions of total pregnancies 0 25.0 0

% Multiple pregnancies of total pregnancies 16.7 0 50.0

% Ectopic pregnancies 0 0 0

% Ongoing live births per transfer 33.3 30.8 66.7

Our results show that the percent of total pregnancies per retrieval washighest on day of transfer five, though this is not significantly higher thanthe percent on day of transfer three or two due to the limited number ofpatients transferred on day five. The percent of total ongoing live births pertransfer is not different between transfer on day two and three, and percentof ongoing pregnancies occurring with transfers on day five is higher thantransfers on day three or two. (Chi square value for day two vs. five = 1.36,0.5<P<0.1; chi square value for day three vs. five = 1.34, 0.5<P<0.1).

Of pregnancies that occurred with transfer on day three, fifty percentresulted in miscarriage or biochemical pregnancy, while only sixteen percenthad this result when transfer occurred on day two. Only one multiplepregnancy occurred with day of transfer two and five, while none of thetransfers on day three produced a multiple pregnancy. Finally, no ectopicpregnancies were observed in either group.

Conclusions: These results using Hysteroscopic Endometrial Embry Delivery(HEED) show for the first time that the technique is very effective with bothcleavage stage embryos and blastocysts, including day two embryos. Theseresults are even more remarkable due to our clinical bias to replace embryoson day 2 when presented with poor quality or low numbers of embryos. Ourfindings could offer new hope of success for a large number of poorprognosis IVF patients.

References:

Blake DA, Farquhar CM, Johnson N, Proctor M. Cleavage stage versusblastocyst stage embryo transfer in assisted conception. Cochrane Databaseof Systematic Reviews 2007, Issue 4. Art. No.: CD002118. DOI: 10.1002/14651858.CD002118.pub3.

Practice Committee of the American Society for Reproductive Medicine.Blastocyst production and transfer in clinical assisted reproduction. FertilSteril. 2004 Sep;82 Suppl 1:S149-50.

Schoolcraft WB, Gardner DK. Blastocyst versus day 2 or 3 transfer. Seminarsin Reproductive Medicine 2001; 19:259-268.

312.4 – NOVELHAND FREE ULTRASOUND GUIDED EMBRYO TRANSFERMETHOD USING NEW STABILIZING DEVICE

A. Rahim Hallob, Basildon University Hospital and Brentwood FertilityCentre, Essex, UK.

Embryo Transfer is a major and most important step of Invitro Fertilisationand Intracytoplasmic Sperm Insemination Treatment Cycles. The newMethod will shorten the time needed for ultrasound guided ET, preventunnecessary manipulation,and free the operator to carryout other neededtasks during ET such as second person check. The new stabilizing and fixingdevice allows the outer catheter to be fixed in place on to the speculum,therefore, minimising intrauterine movements of the tip of the outer andinner catheters during ET and reduce truama to the endometrium. Itsbenefits come at a relatively cheap cost, as well as being user friendly.

312.5 – CERVICAL MUCUS STATUS CAN BE ACCURATELY ESTIMATED BYTRANSVAGINAL ULTRASONOGRAPHY DURING FERTILITY EVALUATION

Igal Wolman, Tamar Birenbaum Gal, Ariel J Jaffa. Ultrasound Unit inObstetrics and Gynecology Lis Maternity Hospital, Tel-Aviv Medical Center,Tel-Aviv, Israel.

Objective: To correlate the diameter of the cervical canal determined bytransvaginal ultrasonography (TVS) to clinical assessment of the cervicalmucus.

Design and Setting: Prospective study in an academic ultrasound unit.

Patients: Women (n = 101) undergoing an infertility workup or treatment.

Interventions: Ovulatory cycle evluation.

Main Outcome Measure(s): Cervical diameter measurements made fromTVS images were correlated to a cervical mucus score which was assessedby a different observer.

Results: The cervical canal diameter values correlated well with thecalculated cervical mucus scores. The mean ± SE cervical canal diameterwas 0.9 ± 0.1 mm for women with a low (≤5) and 2.1 ± 0.1 mm for womenwith a high (>5) cervical mucus score (P<0.001). A cervical canal diameterof 1 mm emerged as a single predictor of favorable versus hostile cervicalmucus, with a sensitivity and specificity of 83.7% and 80.8%, respectively.

Conclusion(s): TVS, which is routinely used as a tool for follicular growthmonitoring in patients undergoing ovulation induction, might alsosimultaneously be used for estimating cervical mucus measurements. Withno additional effort or expense, these data may help to optimize individualpatient management.

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312.6– ULTRASOUND MONITORING AND HORMONAL PROFILES IN SERUMAND FOLLICULAR FLUID FOR UNSTIMULATED IVF-CYCLES INRELATION TO “EMPTY FOLLICLE SYNDROME”

Mikael Tang-Pedersen, Lars Westergaard. Fertility Clinic, Odense UniversityHospital, Faculty of Health - Institute of Clinical Research, University of SouthernDenmark, Odense, Denmark.

Introduction: Over the years one observation remains constant inunstimulated / natural cycle IVF. That is the approx. 20 % of so called“empty follicles”. Empty follicle syndrome (EFS) has been debated, but sinceit seems a constant finding a deeper understanding of the follicularmaturation process is desirable. In this study we investigate hormonal levelsin follicular fluid (FF) and serum for unstimulated IVF-treatment of ±oestradiol-primed patients from start of the cycle till day of oocyte pick-up(OPU), along with ultrasonic measurements of follicle size and number andendometrial thickness.

The aim is to identify possible correlations between endocrinology,ultrasound monitoring and failed cycles due to “EFS”.

Materials And Methods: This retrospective analysis includes 68 womenunder the age of 37, referred to IVF-treatment due to tubal, male orunexplained factor, and with regular cycles from 26-34 days and FSH <15U/L. Ultrasound (US) examination and blood samples were undertakenduring the early, mid and late follicular phases until the appearance of adominant follicle > 16 mm, with a corresponding endometrium > 8 mm,when an hCG-injection of 6500 IU was administered 34 hours prior to OPU.The aspiration of the mature follicle provides FF for measurements oflevelsof AMH, progesterone, oestradiol, androstendione and testosterone. Themature oocyte is fertilized and transferred 2 days post-OPU.

Results: A total of 68 patients underwent 70 cycles of unstimulated IVF. OPUwas cancelled in13 of these due to premature ovulation. Of the remaining 55patients who went through OPU, 12 had an “empty” large follicle and from45 women a mature oocyte was retrieved. Of these 30 were fertilized and 26transferred at 4-cell stage. FF-concentration of progesterone in the12 cycleswith EFS were significantly lower than in the 45 cycles with retrieval of amature oocyte (9.5 ± 2.2 vs. 13.5 ± 1.1µg/ml; p = 0,042), despite the factthat US- criteria were met. Ratio of E2/P4 was significantly higher in EFS.Compared to OPU cycles with retrieval of a mature oocyte, serum-LH levelson day of OPU was significantly lower for EFS ( p=0,0413) whereas se-FSHand se-E2 were similar in the two groups. Ratio s-(E2/LH) is alsosignificantly lower (p=0,0363) confirming a trend towards a slightly highers-E2 in EFS.

Follicular mean diameter and endometrial thickness on last day ofmonitoring were similar in EFS and cycles with successful OPU.

Conclusions: Cycles with EFS exhibit different hormonal profiles than cycleswith mature oocytes retrieved. This indicates some degree of immaturityand/or a less capacitated oocyte-cumulus complex. However this is notvisualized by standard 2-D ultrasound monitoring.

312.7 – A STUDY OF EPIGENETIC ALTERATIONS ASSOCIATED WITHPREMATURE OVARIAN FAILURE

Manda Ghahremani1, Courtney Hanna2, Luana Avila2, Maria Penaherrera2,Karla L Bretherick2, Margo R Fluker1-2, Wendy P Robinson2. 1Department ofObstetrics & Gynecology, 2Department of Medical Genetics, University ofBritish Columbia, 3Genesis Fertility Centre, Vancouver, BC, Canada.

Introduction: Premature ovarian failure (POF) affects 1% of women with alargely idiopathic and poorly understood etiology. The objective of this studywas to identify specific epigenetic alterations by measuring DNA methylationof gene regulatory regions in women with POF vs. controls.

Materials and Methods: Blood samples were collected from idiopathic POFpatients (amenorrhea for at least 3 months and 2 serum FSH levels of >40mIU/ml obtained > 1 month apart prior to age 40) and control women (CW)(healthy pregnancy after age 37 with out a pregnancy loss). Genomic DNAwas extracted from EDTA anticoagulated blood and bisulfite converted foranalysis using the Illumina Golden Gate Methylation Panel which measuresDNA methylation at 1506 CpG sites in the promoter regions of 807 genes in18 POF and 21 CW. Candidate genes with altered epigenetic marks betweenPOF and CW were identified based on 3 selection criteria: a nominal p-value<0.05, absolute methylation difference of >5% and False Discovery Rate(FDR) of <50% using the “Significance of Analysis of Microarrays” (SAM)version 3.01. Genes of interest were further analyzed for quantitativemethylation at specific CpG sites using pyrosequencing in 30 POF and 30CW.

Results: After comparison of DNA methylation profiles of POF and CW usingthe above mentioned criteria, 16 genes met our criteria as candidate geneswith altered epigenetic marks in the POF group. All of these genes werehypermethylated in the POF group as compared to CW. In addition, theAndrogen Receptor (AR) gene, which also was hypermethylated, met thelast two selection criteria and was followed up due to its functional role inthe ovary. To further validate these results, DNA methylation of the AR genewas quantified by pryosequencing in a larger group of POF and CW.Pyrosequencing showed significantly higher DNA methylation of the ARpromoter region in POF vs. CW (p=0.007).

Conclusions: This is a novel human study identifying epigenetic alterationsin POF. 16 genes were found to be hypermethylated in POF compared to CWand several of them have a functional relevance in follicular development orautoimmune processes. The further validate and quantify methylation ofthese genes, pyrosequencing experiments will be carried out, as was donefor the AR. The hypermethylation of the AR gene in POF patients may causedecreased level of the AR in these women. This is especially interestinggiven a recent report of induced POF in AR deficient mice. Specificepigenetic markers, as identified by DNA methylation array profiling inblood, may serve as useful biomarkers for POF and other fertility disorders.However, it will need to be determined if these methylation changes arepresent prior to diagnosis, or are a consequence of menopause itself.

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ORAL ABSTRACTS – WEDNESDAY

400.1 – DERIVATION AND CHARACTERIZATION OF HUMAN EMBRYONIC STEMCELLS

Outi Hovatta, Karolinska Institutet, Karolinska University Hospital Huddinge,Sweden and Geneve University Hospital, Switzerland.

Since the first outgrowths of cells from the inner cell mass of humanembryos (Fishel et al. Science 1986, Bongso et al. Hum Reprod 1998), andthe first permanent hESC lines (Thomson et al. Science 1998, Reubinoff etal, Nature Biotechnol 2000), an intensive research period of derivation,characterization, culture and differentiation of these cells has evolved.Because of the potential of hESC in cell therapy, differentiation protocols tomany cell types, such as several cell types of nervous tissue,cardiomyocytes, hepatocytes, hematopoietic stem cells, bone, and manyother cell types have been developed. It has been difficult in human toobtain clean populations of certain single cell types. Thinking of celltransplantation this is a problem since non-differentiated cells among thepopulations are tumorigenic. Even a single pluripotent cell can give rise to ateratoma. Before clinical trials this problem needs to be solved. Comparativegenomic hybridization and single nucleotide polymorphism profiling haveshown potentially malignant changes in these cells in culture.

Establishing clinical grade hESC has been another goal. There are strictrequirements for products which will be accepted for clinical use. Goodmanufacturing practice (GMP) quality system is required by both EuropeanMedicines Agency (EMEA) and Food and Drug Administration (FDA).Optimal clinical grade cells have not been in contact with any animal derivedsubstances, because they are immunogenic and may contain microbes. Thelines have to be derived and cultured from the beginning according to goodto the quality system. Our recent defined feeder cell free culture systemusing recombinant human laminin 511 (Rodin et al. 2009) and chemicallydefined culture medium has facilitated achieving this goal. Theimmunogenicity of hESC is another question which has to be solved.

Establishment of human induced pluripotent cells (Takahasi and Yamanaka2007), further development of such cells questioned the need of hESC anymore. However, too little is known about these cells to say that really can beused in human cell replacement. Possible genetic and epigenetic problemscaused by the reprogramming are to be solved, even though non-viraltransduction will be likely to be achieved. All the knowledge accumulatedregarding the characterization of hESC and their differentiation is alreadynow applicable to human iPS cells, even though there appears to be alsodifferences. For the time being, both cell types need to be studied.

401.3 – MINIMUM STIMULATION

Osamu Kato, Kato Ladies Clinic, Tokyo, Japan.

Shift from conventional IVF to minimal stimulation IVF

Our clinic has become the largest IVF center in the world. In 2008, thenumber of the IVF cycles carried out by us reached 20,000. For the last 14years, we have gradually shifted our method of IVF from conventional IVF tominimal stimulation IVF. Recently, we have also been performing natural IVFcycles, and the results are very encouraging.

In the past, like most IVF centers in the world, we mainly performedconventional IVF using large dose of gonadotropin. This caused many sideeffects and complications such as Ovarian Hyperstimulation Syndrome(OHSS) and multiple gestations. These have been and still are both medicaland social problems.

For the last 10 years, we have mainly used the Clomiphene, with or withoutHMG protocol, which was developed in our center. This has significantlyreduced the side effects of medication, and also reduced the patients’financial burden.

Our standard protocol for the minimal stimulation IVF using Gn-RH agonistas an oocyte maturation trigger

In the controlled ovarian hyper-stimulation method, which is now popular allover the world, intra-muscular injection of hCG is used as the maturationtrigger.

In our standard protocol for the natural and the minimal stimulation IVF, ourunique GnRH agonist nasal spray is used for triggering oocyte maturation.

In the minimal stimulation cycle, we administer 50mg of clomiphene citrate

from day 3 of the period to the day when the maturation trigger isadministered. Instead of an hCG injection, we utilize Gn-RhH agonist sprayas a maturation trigger to induce the LH flare up. Then, we aspirate oocytes33 hours later.

Single embryo transfer based on the Intra-tubal Embryo Recurrence Theory

Most importantly, we have been carrying out single embryo transfer for allIVF cycles for the last 5 years. As a result, our multiple pregnancy rate hasdropped to around 1 %. Along with this, the incidence of ectopicpregnancies has decreased in our clinic, due to the fact that we providedifferent treatment for women with tubal infertility to that we give thosewithout the tubal infertility factor. Specifically, we treat women with tubalinfertility with blastocyst transfer, and women without the tubal factor withday 2 ET. Based on this result, we believe the zygote which has beenreturned to the uterus moves to the Fallopian tube and develops in the tubeup to the blastocysts, then returns back to the uterus where it implantsitself. This has been shown in our animal studies. These animal studies,along with the human experience, suggest that blastocyst transfer may be asolution for implantation failures in women with tubal factor infertility.

402.2 – EMBRYONIC STEM CELLS DERIVED FROM PGD-AFFECTED EMBRYOSFOR MODELING HUMAN GENETIC DISORDERS

Dalit Ben-Yosef, Racine IVF Lab, Scientific and Managerial Director, LisMaternity Hospital, Tel-Aviv Sourasky Medical Center, Tel Aviv University, TelAviv, Israel.

Human embryonic stem cells (HESC) carrying specific mutations can beused as a valuable tool for studying genetic disorders in human. Onefavorable approach to obtain such mutant HESC lines is their derivationfrom affected preimplantation genetic diagnosed (PGD) embryos. Theseunique cells are especially important for modeling human genetic disorderfor which no good research models currently exist. They can be further usedto gain new insights on developmentally regulated events that occur duringhuman embryo development and that are responsible for the manifestationof genetically inherited disorders. In addition they have a great value for theexploration of new therapeutic protocols, including gene therapy-basedtreatments and disease-oriented drug screening and discovery.

The lecture will focuse on the importance of deriving HESC lines fromgenetically abnormal embryos. It will describe the first report on using PGD-derived HESC for modeling a developmentally regulated genetic disease –Fragile X.

403.1 – LAPAROSCOPIC TREATMENT OF UTERUS BICORNIS

Bruno J. van Herendael, Endoscopic Training Centre Antwerp (ETCA),Gyntech BVBA, Antwerp, Belgium.

The topic concerns the laparoscopic technique of the classical Strassmannoperation for bicornate uteri. The second look hysteroscopy andlaparoscopy prove that the technique is feasible and yields good results witha minimal discomfort for the patients.

403.3 – ROLE OF HYSTEROSCOPY IN INFERTILITY AND IVF

Luca Mencaglia, Geneva University Hospitals, Florence, Italy.

In the “”office hysteroscopy” the introduction of mini-endoscopes makeshysteroscopy even more simple and painless. The technique is an officeprocedure. We consider the “office hysteroscopy” a well tolerated innocuousprocedure which could be considered as a routine procedure in all the casesof infertility. A high level of expertise is not a prerequisite to performinghysteroscopy on an outpatient basis.

Hysteroscopy is able to detect important unsuspected endouterineabnormalities in 18% of patient with two failed IVF-ET and in whom HSG didnot show any abnormality.

Indication for “office hysteroscopy” in ART

Evaluation of the cervix

Evaluation of the uterine cavity

Evaluation of the endometrial phases

Evaluation of the tubal ostia

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The hysteroscopic surgery of pathologies like a submucous fibroids, uterineseptum and endometrial polyps, before IVF/ET treatment, improves results

Prospective studies demonstrates that at present is hysteroscopic surgery isthe method of choice to improve the cumulative pregnancy rate as well asthe live birth rate in selected women with intracavitary pathologies and ahistory of reproductive failure.

Surgical procedures through hysteroscopy must be reserved to experiencedhysteroscopists.

404.2 – OOCYTE SHARING AS TREATMENT OF FERTILITY

Kamal Ahuja, The London Women’s Clinic, London, UK.

The introduction of egg sharing in the UK in 1992 was greeted with deepsuspicion. Despite its practical appeal to patients, the concept wasportrayed as exploitative and unethical and it deeply polarised the opinionsof key stakeholders and also the media. The HFEA came under tremendouspressure to ban the practice and seemingly came very close to doing justthat. However, to their credit they resorted to a careful examination of thearguments through public debates and a consultation exercise before, in1998, accepting egg sharing as a licensed treatment. In 2007, some tenyears after the original decision, the HFEA reaffirmed their support of theoriginal decision by approving egg sharing as a suitable practice to obtainhuman eggs for stem cell research.

It is probably fair to say that apart from IVF itself no assisted conceptionprocedure has undergone such an in-depth scrutiny as egg sharing beforebecoming accepted. The key objectives of this presentation are: 1.Toexamine how the HFEA argued the merits of egg sharing and what wasinitially perceived as the shortcomings of egg sharing, 2. The promise eggsharing may hold for clinical research and treatment in future, and, 3. Thevigilance and research required to ensure that the practice does not deviatefrom its objective of providing a safe and affordable form of IVF treatmentfor those who otherwise face difficulties in procuring IVF treatment.

405.1 – OOCYTE MATURATION AND ANEUPLOIDY: THE MOLECULARPROTAGONIST IS APC

Keith T. Jones, The University of Newcastle Australia, Newcastle, Callaghan,Australia.

Oocyte quality is a major factor governing a woman’s fertility. Quality is poorwhen chromosome segregation errors occur during oocyte maturation.Such errors, which include trisomy 21, affect between 20-40% of all humanoocytes, and go on to produce mostly non-viable, aneuploid embryos;making chromosome segregation errors the leading cause of early embryoloss. The aetiology of aneuploidy in maturing oocytes has been heavilyinvestigated but remains obscure, with female age being the only well-characterized correlate.

The Spindle Assembly Checkpoint (SAC) is a universal, error-detectingmechanism, employed by all dividing cells to stall cell division untilchromosomes are ready to segregate. Although it was first thought that theSAC would be defective or absent in maturing oocytes, this is now knownnot to be the case. Instead, we have recently found that the maturingoocyte’s susceptibility to chromosome mis-segregation is due to its peculiarand unique regulation of the SAC’s target: the Anaphase-Promoting Complex(APC) during oocyte maturation.

406.3 – HOW TO STUDY AND SELECT THE BEST SPERM FOR ICSI

Branko Zorn, Andrology Centre, Department of Obstetrics and Gynaecology,University Medical Centre Ljubljana, Ljubljana, Slovenia.

Since the introduction of intracytoplasmic sperm injection (ICSI) muchprogress has been made in resolving male infertility due to sperm-oocytefusion defects. In order to improve the results of ICSI in all causes of maleinfertility, selection of the best sperm becomes a priority. This has beenmade possible by the development of various methods for sperm evaluation.

In infertile men, numerous candidate genes and transcripts involved insperm cell development and fertilization are being studied using microarrayanalysis; they allow the recognition of genetic and epigenetic factors of maleinfertility. Sperm aneuploidy screening in sperm from the ejaculate or testisis recommended for prediction of implantation.

Proteomics technology enables the evaluation of protein function at themolecular level. Testicular spermatogonial stem cells will permit the use ofgene-therapy in severest forms of spermatogenesis defects.

The use of high magnification light microscopy imaging method iscomplementing classical light microscopy in evaluating sperm morphology.

Fertilization potential is assessed by sperm staining assays using antibodiesagainst acrosomal proteins and sperm penetration assay.

Determination of sperm apoptosis markers, i.e. mitochondrial membranepotential integrity, plasma membrane translocation of phosphatidylserine,caspase activation and DNA denaturation/fragmentation providesinformation on sperm function, which assists the clinician with therapydecision.

The detection of reactive oxygen species (ROS) deserves to be routinelyperformed. Besides classical luminescence, flow cytometry technologypermits accurate intracellular ROS determination.

Before ICSI, sperm is selected by swim-up, glass wool filtration or densitygradients. The hyaluronic acid binding identifies mature sperm with lowfrequency of aneuploidy.

Magnetic-activated cell sorting using annexin V-conjugated microbeadseliminates apoptotic sperm. Sperm selection could also be based onmembrane electrostatic charge.

Sperm selection at high magnification using Nomarski interference contrastis useful to identify more precisely the size and the number of nuclearvacuoles.

After testicular or epididymal extraction, sperm motility-vitality is enhancedby antioxidants.

Excellent post-thaw motility is being observed after testicular spermfreezing.

Sperm selection tests will be presented and comments based on theauthor’s experience provided.

407.1 – STUDY ON EFFECTS OF MALE REPRODUCTIVE TRACT INFECTIONSWITH AEROBIC BACTERIA ON SPERM QUALITY

A. Kaluarachchi, S.Wijeratne, P.K.B.Mahesh, H.R. Seneviratne. Departmentof Obstetrics & Gynecology, Faculty of Medicine, Colombo, Sri Lanka.

Introduction: This study was performed to evaluate the association betweenmale reproductive tract infections with aerobic bacteria and semen qualityamong men who are seeking infertility treatment at a tertiary care setting.

Methodology: Male partners of infertile couples who presented for treatmentto the professorial unit at the De Soyza Hospital for Women were recruitedfor the study (n=200). Seminal fluid analysis and seminal fluid culture wereperformed according to the WHO criteria. Mid-stream urine culture wasperformed on the same day to exclude urinary tract infections. Results wereanalyzed using Statistical Package for Social Sciences (SPSS) 15.0.

Results: Normal sperm concentration was seen in 69.5% (n=139/193);normal sperm motility was seen in 39.5 %( n=79/193);normal spermmorphology was seen in 38% (n=76/179) and normal sperm vitality wasseen in 36.5 %( n=73/179). Pathogenic organisms were isolated in seminalfluid in 8% of males (n=16/191) with a colony count of more than 103,out ofwhich two also had concomitant urinary tract infections with the samepathogen. Another 7.5%(n=15/191) showed a mixed growth of organisms.Organisms isolated in positive cultures were, Staphylococcus aureus in37.5%(n=6), beta(β) hemolytic Streptococci in 18.8%, (n=3), alpha(α)hemolytic Streptococci in 12.5% (n=2), non-hemolytic Streptococci in 6.3%(n=1) and Escherichia coli in 5.9% (n=1). Analysis did not show a significantassociation between the seminal fluid infections with aerobic organisms andseminal fluid parameters. P values for individual parameters were,concentration (0.465), motility (0.608), morphology (0.869) and viability(0.137).

Conclusion: Infections with aerobic organisms have not shown an effect onseminal fluid parameters.

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407.2 – SEMINAL MORPHOLOGY AND THE OUTCOME OF ASSISTEDREPRODUCTION TECHNIQUES

A. Kaluarachchi1; S. Wijeratne1, P.K.B.Mahesh1, C. Nelson2, H.R. Seneviratne1. 1Department of Obstetrics & Gynecology, Faculty ofMedicine; 2 Vindana Reproductive Health Centre; Colombo, Sri Lanka.

Introduction: This study was performed to assess the value of assessingsperm morphology and its relationship with the outcome of In VitroFertilization (IVF) and Intra Cytoplasmic Sperm Injection (ICSI) .

Material and Methods- A descriptive correlation study was done using therecords of 306 couples who have sought In Vitro Fertilization (IVF) and IntraCytoplasmic Sperm Injection (ICSI) treatment for sub fertility during 2003 to2008 at a tertiary care centre. Data was analyzed using Statistical Packagefor Social Sciences (SPSS) 15.0. The male partner’s sperm morphology wascompared with Fertilization Rate, pregnancy and pregnancy outcome.

Results - Out of the 306 couples (mean age-Female-35.91 Male-38.65),238(77.8%) had undergone IVF alone and 49(16.0%) had ICSI, while19(6.2%) was treated with both techniques. In 300 (98.03%) of themhusband was the sperm-provider and a donor provided sperms in6(1.97%).The mean value of normal morphology was 34.39%( SD-13.45%).According to the World Health Organization criteria, 193(63.71%) of thesperm providers/donors had normal morphology. The mean FertilizationRate was 67.45% (SD-25.6%). The correlation between the morphology andFertilization Rate was significant (R=0.030, P=0.004). Mean values of Head,Mid Piece and Tail abnormalities were 59.86%, 12.91% and 11.59%respectively. Head and Tail abnormalities correlated negatively with theFertilization Rate, but it was not statistically significant. (Head-R=0.037/P=0.088, Mid Piece-R=0.001/P=0.788, Tail-R=0.24/P=0.171).Outof the 76(24.84%) who became pregnant with IVF and ICSI 20(26.3%) ofthem miscarried the pregnancy. There was no significant associationbetween successful pregnancy and morphology (P=0.178) or having amiscarriage and the morphology (P=0.976).

Conclusion- Sperm morphology has a significant impact on fertilizationwhile the relationship between successful pregnancy and miscarriage wasnot significant. Detail analysis of the type of morphological abnormality isuseful since it is related to the success of IVF and ICSI..

407.3 – UTERINE SEPTUM DECREASES THE IMPLANTATION RATE IN IVF /ICSI CYCLES

Tomaz Tomazevic, Helena Ban-Frangez, Irma,Virant-Klun, Ivan Verdenik.Slovenia, Ljubljana, Slovenia.

Objective: To evaluate the effect of hysteroscopic resection of a large uterineseptum (Class V according to the American Fertility Society (AFS)classification) and of a small partial uterine septum (Class VI according toAFS classification or arcuate uterus) on implantation after ET in stimulatedIVF and ICSI cycles.

Study design: The retrospective matched control study included 339 ETs instimulated IVF or ICSI cycles before hysteroscopic resection of a large orsmall partial uterine septum (113 and 176 ETs respectively) and 538 ETs instimulated IVF or ICSI following hysteroscopic resection of a large or smallpartial uterine septum ( 226 and 248 ETs respectively). For each ET in thestudy group, we found two consecutive ETs s from the IVF/ICSI registrywho had a normal uterus and were matched for age, BMI, stimulationprotocol, quality of embryos, the use of IVF or ICSI and for variousinfertility causes. The clinical pregnancy rate was the main outcomemeasure. Data on the septum length were obtained during hysteroscopicresection by comparing the length of the 1.3 cm long yellow tip of theelectric knife to the length of the resected septum.

Results: The implantation rate before hysteroscopic metroplasty wassignificantly lower , both in women with a small partial septum ( 13,6%before resection vs. 25,6% in the normal controls, OR 2,9; p<0,001) and alarge septum ( 12,4 % before resection vs. 29,2% in normal controls, OR2,1; p<0,002) compared to women with a normal uterus. After the surgery,the pregnancy rate was comparable to the pregnancy rate in women withnormal uterus: in both women with a small partial ( 23,2 % vs. 25,4%) andin women with a larger septum ( 21,8% vs. 20,4%) respectively.

Conclusion(s) Similar to a large uterine septum, a small partial uterineseptum or arcuate uterus negatively influences the implantation instimulated IVF and ICSI cycles .

407.4 – A NOVEL PROCEDURE FOR SEVERE CASES OF ADENOMYOSIS —SURGICAL TREATMENT BY THE TRIPLE-FLAP METHOD FORRECONSTRUCTION OF THE UTERINE WALL- OSADA PROCEDURE FORMASSIVE ADENOMYOSIS

Hisao Osada1, Toshiyuki Kakinuma1, Tom Kiyosi Fujii1, Sherman J Silber2,Keiichi Kato3, Osamu Kato3. 1Department of Obstetrics and Gynecology,Nihon University School of Medicine, Tokyo, Japan 2 Infertility Center of St.Louis, St. Luke’s Hospital, St. Louis, U.S.A. 3 Kato Ladies Clinic, Tokyo,Japan.

Introduction: A multiple flap reconstruction of the uterine wall followingwide excision of adenomyosis tissue was developed for women with severedysmenorrhea and infertility. Consecuctive series of operations in womenwith severe adenomyosis.

Materials and Methods: Severe adenomyosis causes infertility, severedysmenorrhea, and hypermenorrhea. Surgical reconstruction is problematicbecause of absence of any surgical plane or capsule, and fear of uterinerupture with pregnancy. In fact, the usual treatment is hysterectomy. Ourtreatment for even the most severe cases of adenomyosis involves widecomplete excision of affected tissues to assure adequate removal, followedby a multiple flap reconstruction of the uterine wall without overlappingsuture lines to prevent ruptures in subsequent pregnancies.

Results: The results were as follows: 1) Wider and more thorough excisionof the affected tissues were possible compared to the conventionalconservative surgery using a wedge resection. 2) The massive tissuedefects created by the wide excision of the lesion could be reconstructedwith an adequate thickness of uterine wall by overlapping the thin remainingmyometrial musculature with non-overlapping suture lines. 3) There wereno complications such as interstitial haematoma, suture diastasis,adhesions, etc. observed. 4) There was remarkable improvement indysmenorrheal and hypermenorrhea. 5) Twenty-six women wished toconceive following the surgical removal of the adenomyosis. Sixteen ofthem (61.5%) subsequently conceived. 13 cases went to term and all weredelivered by elective Caesarean section. There were no cases of uterinerupture. 6) There was only a 3.84% recurrence (4 cases) of theadenomyosis.

Conclusions: Wide complete excision of adenomyosis with multiflapreconstruction of uterine wall results in a dramatic reduction in bothmenstrual cramping and menstrual flow volume post-surgically, and inwomen who desired to conceive, allowed them to go to term without uterinerupture.

407.5 – PREVALENCE OF MARKERS FOR THROMBOPHILIA ANDIMMUNOLOGICAL DISORDERS IN PATIENTS HAVING IN VITROFERTILISATION AND EMBRYO TRANSFER

L. Mettler, A. Salmassi, A. G Schmutzler. Department of Obstetrics andGynaecology, University of Kiel, Kiel, Germany

Background: Birth rates after IVF remain low at 30%, causes arediscussable. Investigators reported an increased prevalence of singlethrombophilic or immunologic disorders.

Methods: In a first uncontrolled pilot study we studied 123 patientsundergoing IVF. Prior to initiation of IVF, patients were interviewed forhistorical and clinical evidence of hypercoagulatory state or immunologicaldisease. Blood samples were determined using a pattern of hereditary(protein-S, -C-deficiency, factor V Leiden), acquired thrombophilia(antiphospholipid antibodies) and immunological parameters (ANA,complement factor 4).

Results: No patient displayed clinical evidence of active immunological orhypercoagulative disease. Prevalence of protein S deficiency (11.2%),positive antiphospholipid antibodies (32.5%) and ANA (30%) weresignificantly higher than in the normal population (< 1%; p < 0.005; ~2%; p< 0.005; ~5%; p < 0.005). Overall 82/123 (66.7%) of the patients werepositive for any pathological marker, 31/123 (25.2%) of the patients had acombination of parameters of hereditary and/or acquired thrombophilia

April 19-22, 2009 68


and/or autoantibodies. Women with multiple abnormal tests were found tohave a strong tendency towards a lower pregnancy rate after IVF althoughthe difference did not reach statistical significance (38.7% vs. 51.2%respectively; p > 0.005).

Conclusions: In an unselected population of patients undergoing IVF, asubstantial share (66.7%) demonstrates pathological assays forthrombophilia or immunological disorder without symptoms of clinicallyactive disease. Every fourth patient (25.2%) displays combinations of theselaboratory parameters. These patients show a strong tendency towardsdecreased pregnancy rates after IVF. Therefore a risk-adapted strategy intreatment with low dose aspirin, heparin and/or prednisolone to decreasethe IVF failure rate seems appropiate.

407.6 – ASSESSMENT OF THE OVARIAN RESERVE FOLLOWINGCHEMOTHERAPY AMONG A COHORT OF CANCER PATIENTS WHOUNDERWENT UNILATERAL OOPHORECTOMY VERSUS PARTIALOOPHORECTOMY FOR OVARIAN TISSUE CRYOPRESERVATION

Foad Azem, Abir Massarwa, Ami Amit, Dalit Ben-Yosef, Tanya Cohen, TamarShwartz, Nava Mieraz, Rona Limor. Racine IVF Unit, Lis Maternity Hospital,Sourasky Medical Center, Tel-Aviv, Israel.

Background: The number of patients surviving cancer is increasing, hencereproductive potential becoming an important issue. Many reports havesuggested some parameters i.e.: FSH, anti Mulerian hormone and numberof follicles as for the assessment of ovarian reserve following chemo-radiotherapy.

Methods: The study included 24 cancer patients. 12 patients underwentpartial oophorectomty (Group A) and 12 patients underwent unilateraloophorectomy (Group B). Prior to freezing, the ovarian cortex was dissectedinto 1- to 2-mm and cut into 10 X5-mm sections. Twelve months followingthe end of chemotherapy we assessed several parameters of ovarian reserveincluding: menstrual status, day 3 (basal): FSH, E2, AMH and antral folliclecount (AFC). In 21 patients all parameters were analyzed while in 3 patientsonly AMH was available.

Results: The two groups were comparable in regard to average age,indications for chemotherapy, levels of basal FSH, AMH and AFC. We foundpositive correlation between FSH, AMH and AFC (p = 0.001) and (p = 0.019)correspondingly . Five patients in each group had reduced ovarian reserve

Conclusions: Ovarian reserve as assessed by the measurement of AMH,basal FSH and antral follicles count, showed no statistical differencebetween the study groups. The lack of difference is attributed to the fact thatthe two groups were comparable in the average age, indications forcryopreservation and mode of chemo/radiotherapy. In both groups ovarieswere exposed to chemotherapeutic agents which affected similarly theovarian reserve. In cases in which the damage was severe the ovarianreserve decreased below a threshold independent of the original ovarianreserve. While in cases in which the chemotherapeutic protocol mildlyaffected the ovary, the ovaries reserve stay above a threshold independent ofthe original mass.

In patients in whom the ovarian reserve could severely be affected followingchemotherapy; one should consider application of oophorectmy andcryopreservation whole ovary rather than partial cryopreservation.

407.7 – VASCULAR COMPLICATIONS OF OVARIAN HYPERSTIMULATION(OHS)

J-M. Dreyfus, A.Watrelot, A.Khebbe.; CRES, Lyon, France.

Treatment of infertility and A.R.T are now routinely performed. Efficiency ofdrugs and specially rFSH, agonists, antagonists in specific protocolsincreases success rates.

However, OHS is difficult to control and may conduct to an Ovarian HyperStimulation Syndrome (OHSS).Frequency of complications is probablyunderestimated.

The goal of this communication is to review vascular (arterial and venous)complications.Some of them can be particularly grave and possibly fatal.

We describe the type, the delay of occurrence, and the issue of the reportedaccidents in the literature. We report two arterial complications recentlyhappened in Lyon

We summarise ethiopathogeny, characteristic, supervision and treatment ofthese complications

We remind measures to prevent medical and legal complications of OHSS

408.1 – INCIDENCE OF ANEUPLOIDIES IN OVARIAN STIMULATIONTECHNIQUES

J. Remohí, Institut Universitari-Instituto Valenciano de Infertilidad. Valencia.España.

Several factors affecting gametes and embryos have been found to berelated to an increase in chromosome abnormalities: changes intemperature during oocyte culture and handling (Pickering et al., 1990;Almeida and Bolton, 1995), ageing of gametes (Badenas et al., 1989; Munnéand Estop, 1993), use of a 20% oxygen tension instead of 5% (Pabon et al.,1989; McKiernan and Bavister, 1990), hormonal stimulation in some mousestrains (Maudlin and Fraser, 1977), sub-optimal stimulation in humans(Hammitt et al., 1993). Furthermore, in patients with high response togonadotrophins there was an increased incidence of diploid oocytes (Tarinet al., 1990) and “in vitro” maturation (IVM) experiments with higherconcentrations of FSH in the culture media showed increased aneuploidyrates in MII oocytes (Roberts et al., 2005). All these data suggested thatsuperovulation protocols in IVF might increased the risk of aneuploidy in theresulting embryos.

Previous results from our group (Reis Soares, 2003) showed that, in oocytedonation cycles, those donors with higher response to standard doses ofgonadotrophins produced significantly higher number of chromosomallyabnormal embryos. Theses results are in concordance with other authorsthat showed higher incidence of aneuploidy with higher number of oocytesretrieved (Munné et al., 2006, Baart et al., 2007).

As the results were unexpected, we decide to run another study. We selectedyoung donors that were classified as “high responders” using aconventional stimulation protocol. These donors had a first cycle with morethan 20 oocytes and/or serum estradiol levels >3,000 pg/mL, on hCG day,without developing ovarian hyperstimulation syndrome We then performedtwo consecutive stimulation treatments on these donors. In treatment 1,donors received similar stimulation dose than in the initial cycle: 225 IU/dayof recombinant follicle-stimulating hormone (r-FSH, Gonal-F ®; Serono,Madrid, Spain or Puregon; Organon, Madrid, Spain) for the first 5 days,when serum E2 was assessed and gonadotrophin dose adjusted ifnecessary. In treatment 2, decreased daily doses of gonadotrophins wereadministered: 150 IU/day of recombinant follicle-stimulating hormone (r-FSH, Gonal-F ®; Serono, Madrid, Spain or Puregon; Organon, Madrid,Spain). Preimplantation Genetic Screening (PGS) was performed onembryos resulting from the two stimulation regimens. Chromosomes 13,15, 16, 17, 18, 21, 22, X and Y (Vysis Inc. Downers Grove, IL, USA) wereanalyzed and chromosomally normal embryos were transferred to therecipients on day-5. Exclusion criteria for oocyte recipients were: previoushistory of recurrent miscarriages or repetitive implantation failures; severemale factor (<5x106sperm/mL or <5% normal morphology sperm); otherindications for PGS. Our results showed a significant decrease in the meannumber of retrieved oocytes (22.4 vs. 14.9; p=0.0004) as well as in serumE2 levels (3030.4 IU vs. 2085.6; p=0.0025) with the mild-stimulationprotocol. The percentage of abnormal embryos was similar in bothprotocols (55.9% vs. 50.4%), as well as blastocyst rates (68.4%% vs.74.8%) in the conventional stimulation compared to mild-stimulationprotocol. There was no difference between the two stimulation protocols inthe average number of chromosomally normal blastocyst obtained perdonor (2.8±2.2 vs. 2.4±1.7). The clinical efficiency of the donation cycleswas similar in the conventional and mild-stimulation protocols, in terms ofthe percentage of pregnancies achieved after donation (63.0% vs. 57.9%),implantation rates (30.2 vs. 31.6) and number of live-births (10 after eachstimulation protocol). A moderate reduction on oocyte number and E2 levelswas observed in a subgroup of 12 donors. In this subgroup, significantlyhigher blastocyst rates were observed in the mild-stimulation protocolcompared to conventional stimulation (78.9%% vs. 61.1%; p=0.0493) andongoing pregnancy rates with the mild-stimulation protocol doubled thoseachieved with the conventional protocol (66.7% vs. 33.3%). These differences were translated into 9 live-births after mild stimulation and 5live-births after conventional stimulation. We concluded that in highresponder donors, both stimulation regimens (conventional vs. mild-

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stimulation) had a similar efficiency in producing chromosomally normalblastocysts and live-births. Therefore, a moderate decrease in dailygonadotrophins doses would not compromise the number ofchromosomally normal embryos available for transfer and the take homebaby rates. Despite inter-donor differences could be expected to thisdecrease in gonadotrophins doses.

In parallel, we run another study in normosresponse donors to compareembryo aneuploidies in natural vs. stimulated cycles in the same egg donor.Firstly, the donor underwent a non stimulated cycle. When the folliclereached 18 mm in diameter, rCG was administered and oocyte retrieval wasscheduled 36 h later. PGS was performed in the resulting embryos followinga similar laboratory protocol. Secondly, donors followed ovarian stimulationunder a GnRH agonist long protocol and a starting fixed dose of 150 UI ofrFSH and 75 UI of hp-hMG. Recovered oocytes were inseminated with thesame sperm donor. All the embryos from the stimulated cycle were alsoanalyzed and a maximum of 2 euploid embryos were transferred. Similaraneuploidy rate was observed in the natural vs. the stimulated cycle (44.8%vs. 48.6%) and therefore, we concluded that ovarian stimulation would notincrease the incidence of chromosomally abnormal embryos in this group ofpatients. Pregnancy rate was significantly higher in stimulated cyclescompared to natural cycles (50.0% vs. 13.3%; p=0.0006).

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- Badenas J, Santalo J, Calafell JM, Estop AM, Egozcue J. Effect of thedegree of maturation of mouse oocytes at fertilization: a source ofchromosome imbalance. Gamete Res. 1989; 24 (2):205-18.

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- Munne S, Estop AM. Chromosome analysis of human spermatozoa storedin vitro. Hum Reprod. 1993; 8 (4): 581-6.

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- Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient coolingto room temperature can cause irreversible disruption of the meioticspindle in the human oocyte.Fertil Steril. 1990; 54 (1): 102-8.

- Reis Soares S, Rubio C, Rodrigo L, Simon C, Remohi J, Pellicer A. Highfrequency of chromosomal abnormalities in embryos obtained from oocytedonation cycles. Fertil Steril. 2003; 80 (3): 656-7.

- Roberts R, Iatropoulou A, Ciantar D, Stark J, Becker DL, Franks S, Hardy K.Follicle-stimulating hormone affects metaphase I chromosome alignmentand increases aneuploidy in mouse oocytes matured in vitro. Biol Reprod.2005; 72(1): 107-18.

- Tarin JJ and Pellicer A. Consequences of high ovarian response togonadotropins: a cytogenetic analysis of unfertilized human oocytes. FertilSteril. 1990; 54 (4): 665-70.

- Verberg MF, Macklon NS, Nargund G, Frydman R, Devroey P, BroekmansFJ, Fauser BC. Mild ovarian stimulation for IVF. Hum Reprod Update. 2009Jan-Feb;15(1):13-29.

- Verpoest W, Fauser BC, Papanikolaou E, Staessen C, Van Landuyt L,Donoso P, Tournaye H, Liebaers I, Devroey P. Chromosomal aneuploidy inembryos conceived with unstimulated cycle IVF. Hum Reprod. 2008Oct;23(10):2369-71.

408.2 – BIOLOGY AND BIOCHEMISTRY OF AMH/MIS

Nathalie Josso, Nathalie di Clemente, Rodolfo Rey, J.Y. Picard. INSERM Unit782, Clamart, France.

The existence of a fetal testicular product responsible for the regression ofMüllerian ducts in male fetuses was discovered by Alfred Jost in the earlyfifties. The “Müllerian inhibitor” as he called it is now known as anti-Müllerian hormone (AMH) or Müllerian inhibiting substance (MIS). Amember of the TGF-ß superfamily, it is produced by Sertoli cells, mainlybefore puberty, and by postnatal granulosa cells. The level of circulatingAMH, which varies according to sex and developmental stage, providesvaluable information on the functional activity of gonadal somatic cells. AxelThemmen and Bart Fauser, in Rotterdam, have shown that serum AMH isalso an excellent marker of ovarian reserve (1), hence its increasing value inthe field of assisted reproduction. In contrast, apart from its key role ininducing the regression of Müllerian ducts in male fetuses at 8 fetal weeks,its biological action does not appear to significantly affect human gonadalphysiology, in either males or females.

To induce regression of Müllerian ducts in the human fetus, AMH must beexpressed before 8 fetal weeks. As shown by Zhan et al (2), coelomicepithelial cells expressing AMH receptors under the control of Wnt7 (3)undergo epithelio-mesenchymal transformation and migrate into the peri-Müllerian epithelium. AMH action also leads to a wave of apoptosis and toan accumulation of cytoplasmic ß-catenin in the peri-Müllerian mesenchyme(4). In the absence of AMH or its receptors, the Müllerian ducts develop intothe Fallopian tubes, uterus and upper vagina. Retention of Müllerian ductderivatives occurs in genetic males harboring mutations of AMH or its typeII receptor (5) leading to the persistent Müllerian duct syndrome. In malerodents, AMH also negatively affects Leydig cell differentiation and function(6).

In female mice, AMH controls the recruitment of primordial follicles into thegrowing follicle (see (7) for review). The higher rate of primordialrecruitment in AMH–deficient transgenic mice leads to prematureexhaustion of the follicle pool causing an early cessation of ovulation inageing animals. Mice heterozygous for the AMH-null allele have anintermediate phenotype. Thus mothers of PMDS patients would be expectedto undergo early menopause but data are not yet available. AMH alsodecreases sensitivity to FSH and may inhibit FSH-dependent selection offollicles for dominance.

AMH biochemistry is relatively straightforward. A glycoprotein homodimerlinked by disulfide bonds, it undergoes proteolytic processing to generate anactive 25 kDa N-terminal dimer. Processing is required for biological activity,as the latter is destroyed by mutations that block cleavage (8). AMH signalsthrough a specific type 2 receptor and through type I receptors shared withthe BMP family, ALK-2 and ALK-3. Smads 1, 5 and 8 act as its cytoplasmiceffectors (9).

Reference List

1. vanRooij, I. A. J., Broekmans, F. J. M., teVelde, E. R., Fauser, B. C. J. M.,Bancsi, L. F. J. M., de Jong, F. H., and Themmen, A. P. N. (2002) HumReprod 17, 3065-3071

2. Zhan, Y., Fujino, A., MacLaughlin, D. T., Manganaro, T. F., Szotek, P. P.,Arango, N. A., Teixeira, J., and Donahoe, P. K. (2006) Development 133,2359-2369

3. Parr, B. A. and McMahon, A. P. (1998) Nature 395, 707-710

4. Allard, S., Adin, P., Gouédard, L., di Clemente, N., Josso, N., Orgebin-Crist, M. C., Picard, J. Y., and Xavier, F. (2000) Development 127,3349-3360

5. Josso, N., Picard, J. Y., Rey, R., and di Clemente, N. (2006) PediatricEndocrinology Reviews 3, 347-358

6. Racine, C., Rey, R., Forest, M. G., Louis, F., Ferre, A., Huhtaniemi, I.,Josso, N., and di Clemente, N. (1998) Proc. Natl. Acad. Sci. (USA) 95, 594-599

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7. Visser, J. A. and Themmen, A. P. N. (2005) Mol Cell Endocrinol 234, 81-86

8. Belville, C., Van Vlijmen, H., Ehrenfels, C., Pepinsky, R. B., Rezaie, A. R.,Picard, J. Y., Josso, N., di Clemente, N., and Cate, R. L. (2004) MolEndocrinol 18, 708-721

9. Orvis, G. D., Jamin, S. P., Kwan, K. M., Mishina, Y., Kaartinen, V. M.,Huang, S., Roberts, A. B., Umans, L., Huylebroeck, D., Zwijsen, A., Wang,D., Martin, J. F., and Behringer, R. R. (2008) Biol Reprod 78, 994-1001

409.3 – FUNDING IVF TREATMENT - THE CANADIAN EXPERIENCE

Beverly Hanck, Infertility Awareness Association of Canada (IAAC),Montreal, QC, Canada.

This presentation sets out the rationale for government funding of in vitrofertilization (IVF) and other assisted reproductive technologies (ART) inCanada, and includes an economic analysis to support the recommendationthat Canada’s public health care system should provide safe and effectiveinfertility treatment for all Canadians who need it.

In Canada, the need for IVF far exceeds its accessibility, and the treatmentremains financially out of reach for many infertile Canadian couples.Because there has been virtually no funding, infertile Canadian couples oftenresort to cheaper but less effective alternatives such as ovarian stimulationand/or hormone injections. These regimens have a major downside in thatthere is a significant risk of multiple pregnancy. With ovarian stimulation,poor control over the number of mature eggs produced may result in thebirth of triplets, quadruplets and even higher-order multiples. Statisticsshow multiple-pregnancy rates of 30% through ovarian stimulation.

In the last ten years, Canada’s birthrate has dropped 25%, while the numberof multiple births has increased by 25% over the same period. Available datafrom Europe and North America suggest that infertility treatment accountsfor 30% to 50% of all twin births and for up to 80% of all higher-ordermultiple births. Of these higher-order multiple births about 50% areattributable to fertility pills and hormone injections.

Multiple pregnancies have very broad repercussions: they severely affectfamilies psychologically, medically and financially, while the cost toCanadian provinces is far greater than for singleton pregnancies, due toincreased needs for medical and social support. Multiple pregnancies alsolead to elevated health risks for mothers and infants, increased perinatal andneonatal costs and, in extreme cases, lifelong costs because of thedisabilities that occur more frequently in multiples and multiple related pre-term births.

Accordingly the authors recommend the following strategies: (1) educationof the medical and allied professions, as well as prospective parents; (2)monitoring of women during ovarian stimulation treatments, with the optionto switch to IVF; (3) reduction of the number of embryos transferred in IVFtreatment and encouragement of elective single embryo transfer (eSET),with couples seeking treatment at younger ages; (4) optimal availability ofIVF treatment, based on a 100% refundable tax credit, the cost of whichwould be easily offset by significant savings associated with reducedincidence of multiple pregnancies.

These strategies should result in at least a 50% decrease in the rate ofmultiple births in every province that adopts funded IVF.

409.4 – PSYCHOLOGICAL COMPLEXITIES SURROUNDING ENDING IVFTREATMENT

Janet Takefman, McGill Reproductive Centre, McGill University HealthCentre, Montreal, QC, Canada.

A question frequently posed by infertility specialists is when to stop IVFtreatment or when to advise patients that “enough is enough”. The answerto this question is challenging for a number of reasons. First, there is alwaysa theoretical probability of success with further attempts. Second, there isno continuum of benefits with each successive attempt. Finally, thephysician and patient likely base their decision to end IVF on differentcriteria. The physician will consider medical and risk factors, the patient,financial, personal and emotional costs. Telling a couple that IVF is no longerrecommended usually implies their dream of having a genetic child will beunrealized. Research has documented this transition process for couples asbeing long, difficult and complex. It is important that the physician

understand the emotional processes that a patient navigates in acceptingending IVF, as well as being cognizant of factors that predict adaptation sothe needs of the patient are better met at this endpoint. Effective andcompassionate doctor-patient communication has been shown to lessen theemotional sting of ending IVF for couples and contributes to patients bemore open to alternatives such as egg donation.

410.1 – EFFECTS OF L-GLUTAMINE AND STRAW SIZE, FREEZING RATE ANDTHAWING RATE UPON POST-THAW QUALITY OF HUMANSPERMATOZOA

Hussian Asherkaci, L.M. Aboshala, M.A. Danfour, O.A. Elsraite, M.S.Elmahaishi. Faculty of Science, Faculty of Medicine, 7th October Universityand Misurata Infertility Centre, Misurata, Libya.

The objective of this study was to investigate cryoprotective effect of L-glutamine, straw size, freezing rate and thawing rate in preserving motility ofhuman spermatozoa during the freezing-thawing process. MATERIALS ANDMETHODS: Normal Semen sample were collected by ejaculation, accordingto criteria of the World Health Organization (WHO), as well as spermaticmorphology according to the strict Kruger criterion. Swim up technique wascarried out to obtain 5-6 X 106 progressive motile sperms with a goodmorphology. In Experiment 1, three straw sizes (1mm, 2mm and 5mm),three freezing rates (straws suspended 2 , 7 and 9 cm above liquid nitrogen)and three thawing rates (in water at 20, 30 and 37 degrees C) upon post-thaw quality of sperm, and to determine the best treatment combination.Quality was expressed in terms of the percentage progressively motilesperm 5 min after thawing. In Experiment 2, the best straw size, freezingrate and thawing rate in preserving motility of human spermatozoa wereused to investigate the effects of L-glutamine at different concentrations(20mM and 50mM) on post-thaw sperm motility. Data were analyzed bymeans of a repeated measures factorial analysis of variance and meanscompared. Results: There were significant effects by straw size, freezingrate, thawing rate on human sperm cryopreservation procedure (P<0.05).The straw size 2mm, straws suspended above liquid nitrogen 7cm andthawing rates 37 degrees C were the optimal conditions for preservation ofhuman spermatozoa. Using these conditions we found that addition of L-glutamine with 50mM improve the survival rate of human sperm. Inconclusion: These new preservation protocol permit extended conservationof viable spermatozoa that may capable of supporting normal embryonicdevelopment and the live birth of healthy baby after ICSI.

410.2 – IN VITRO FERTILIZATION IN NATURAL CYCLE FOR CONCURRENTTRANSFER OF FRESH AND FROZEN/THAWED EMBRYOS

M. Anshina, A. Smirnova, N. Shamugia, E. Abliaeva, T. Troshina, I Kalinina,К.Ilyin. IVF & Genetics Centre “FertiMed”, Moscow, Russia.

Introduction: In women with normal ovulation we usually perform thetransfer of cryopreserved embryo(s) in natural cycle. The key point isappropriate timing of ovulation with consequent blastocyst thawing andtransfer on day 5 after ovulation. Pregnancy rate is high enough – 31% perembryo transfer, but we suggested that simultaneous transfer ofcryopreserved embryo(s) and fresh embryo from the natural cycle mayimprove the results.

Objective: to identify and compare the clinical pregnancy rate and multiplepregnancy rate after the transfer of cryopreserved embryos and afterconcurrent transfer of fresh embryo obtained after IVF in natural cycle andcryopreserved embryos.

Material and Methods: Two groups of women who had not more than 3cryopreserved embryos at blastocyste after previous IVF or IVF/ICSI cycles:group I – 47 women who underwent 55 transfers of cryopreserved embryosin natural cycle; group II – 29 women who underwent 31 natural cyclefollicle aspirations and concurrent fresh/frozen embryo transfers. In allpatients follicle aspiration was performed 26-32 hours after hCG injection(5000 IU). All embryos were transferred on day 5 after ovulation (group I) orfollicle aspiration (group II).

Results: In group I, 55 embryo transfers resulted in 17 pregnancies: 14singleton and 3 multiple. Two pregnancies stopped to develop at 6 and 8weeks of gestation. In group II, 31 embryo transfers resulted in 19pregnancies: 11 singleton and 8 twins. Totally 79 embryos were transferredin group II: 31 fresh embryos (9 at 6-8-cells stage and 22 at blastocyste

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stage) and 48 frozen/thawed blastocystes. There were 3 twins after thetransfer of one fresh and one frozen embryos. Spontaneous reduction ofone fetus at 6-8 weeks of gestation occurred in three of eight twinpregnancies and in one singleton pregnancy.

There was no difference in women’s age, mean number of transferredembryos and endometrium thickness on the day of transfer between groups.The clinical pregnancy rates per transfer was higher in group II in compareto group I (61% vs 31%, P=0,002). The implantation rate (34% vs 17%,P=0,04) and multiple pregnancy rate (42% vs 14%, P=0,01) also werehigher in group II.

Conclusions: Concurrent fresh/frozen embryo transfer is preferableespecially in patients with small number of frozen embryos. It may improveclinical pregnancy rate and reduce the rate of transfer cancellation whenembryos failed to survive after thawing. Only in three patients we areconfident that both fresh and frozen/thawed embryos were implanted. Buthigh multiple pregnancy rate possibly indicates high implantation potency ofboth types of transferred embryos.

410.3 – N-ACETYL-CYSTEIN IMPROVES RESULTS OF LONG-TERM CULTUREOF FROZEN / THAWED HUMAN OVARIAN TISSUE

R Fabbri1, G. Pasquinelli2, L. Montanaro3, V. Magnani1, F. Tamburini1, D.Keane4, Y. Cabello Vives5, S. Venturoli1. 1Human Reproductive Medicine Unit,S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy; 2ClinicalPathology Section, Department of Radiological and HistocytopathologicalSciences, S.Orsola-Malpighi Hospital and 3Department of ExperimentalPathology, University of Bologna, Bologna, Italy; 4Department of Obstetricsand Gynaecology, Royal College of Surgeons in Ireland, Rotunda Hospital,Dublin, Ireland; 5Human Assisted Reproduction Laboratory, FIVRECOLETOS, Policlinico Nuestra Senora de America, Madrid, Spain.

Introduction:Women treated for malignant disease have a risk of prematureovarian failure (POF). Their fertility may be preserved by ovarian tissue culture invitro. With in-vitro culture, ovarian tissue is usually kept under normoxicconditions; the oxygen concentration being considerably higher than in-vivo.Thus, the oxidative stress, the excessive production of reactive oxygen specimen(ROS) may contribute to the atresia observed in in-vitro cultures. The aim of ourstudy was to investigate the effect of N-Acetyl-Cystein (NAC), a radical-scavenger, supplemented to culture media on follicular and stromal preservation.

Material and Methods: The ovarian cortex of 3 patients was collected andimmediately cryopreserved, as previously described, Fabbri et al, 2003. Afterthawing, samples of ovarian cortex from each patient were collected formolecular and histological analysis (control t0). Additional samples were culturedat 37°C in an atmosphere of 6% CO2 for 16 weeks, in minimum essentialmedium (α-MEM) supplemented with antibiotics, Follicle Stimulating Hormone(FSH), Insulin-Transferrin-Selenium (ITS), Human Serum (HS), with NAC(MediumA) or without NAC (Medium B), or ITS, HS, FSH and Luteinizing Hormone (LH) witha peak level every 28 days, with NAC (Medium C) or without NAC (Medium D).

The medium was changed every second day. Sample collection for morphologicaland molecular analyses was carried out after 8 (t8) and 16 (t16) weeks of culture.The number and developmental stage of follicles was determined using LightMicroscopy (LM) on semithin sections stained with toluidine blue; follicular andstromal cell integrity was evaluated using Transmission Electron Microscopy(TEM) and assessment of the expression of GDF-9, Bcl-2 and Bax mRNAs byReal Time RT-PCR.

Results: LM and TEM showed an improved follicular and stromal structuralpreservation in Medium A with respect to Medium B. Molecular evaluationdemonstrated Medium A had increased expression of GDF-9 transcripts and ahigh Bcl-2/Bax ratio; these results were consistent with the good follicular andstromal preservation observed by morphological analyses in medium A withrespect to medium B.

The beneficial NAC effect was also apparent when comparing Medium C with D.However in this experimental condition the Bcl2/Bax ratio was apparently notaffected.

Conclusions: These results suggest that NAC is necessary to perform long-termcultures of human ovarian cortical tissue kept under normoxic conditions.Thebeneficial NAC effect is most likely related to its intrinsic radical-scavengeractivity which minimizes the excessive production of ROS, one of the majordeterminants of human ovarian cortical tissue degeneration in long-termcultures. Reducing ROS tissue damage with NAC is necessary to maximize the

synergic effect of FSH and / or LH with other nutrients required for folliclulardevelopment in long - term culture.

410.4 – VITRIFICATION OF DAY 3 EMBRYOS IMPROVES THE POST THAWPREGNANCY RATES

Hrishikesh Pai, Nandita Palshetkar, Rishma Pai. Lilavati Hospital IVF Centre,Lilavati Hospital, Bandra, Mumbai, India.

Introduction: Vitrification improves the oocyte, pronuclear stage andblastocyst freezing survival. However there are very few studies which havereported application of vitrification to 6-8 cell human embryos on day 3 ofculture. . In this study we describe the technique as well as demonstrate thesignificantly favorable outcome of day 3 vitrified embryos.

Material and Methods :Since January 2007 , vitrification of day 3 embryoswas carried out with the open technique of Cryotop (kitazato,japan ) usingtwo step protocol with ethylene glycol, DMSO and sucrose ascryoprotectants.After equilibration in vitrification media, 1 to 2 embryoswere loaded on to the tip of the Cryotop straw in a minimum volume of <0.1microlitre. . The straw was then plunged into liquid nitrogen. The tip wascovered with cover straw and stored. Patients were prepared for frozen thawembryo transfer using depot Gnrh agonist in conjunction with hormonereplacement therapy. An average of 2 to 3 Embryos was thawed using a fourstep protocol. The embryo survival in the form of > 50 % blastomeres intactand 100 % blastomere intact was calculated. Assisted laser hatching in theform of partial zona thinning was carried out on all embryos .After 2 hoursof culture in the incubator, the embryo transfer was carried out.

Results: 220 patients were subjected to vitrification. 70 frozen thawvitrification cycles were done from January 2007 till date. 95 % of embryosthawed had 100% intact blastomeres. The B HCG positive rate was nearly68.5 % (48/70) and the clinical pregnancy rate was 61.4(43/70) % perembryo transfer.

Conclusion: This is one of the few studies on day 3 vitrification of 6 to 8 cellembryos. The method is significantly successful. However the largestdrawback in the open method is the potential risk of contaminating thestraws .Ongoing trials comparing the outcome of vitrified embryos using aclosed system and an open system are being carried out in our unit.

410.5 – HIGH SURVIVAL AND PREGNANCY RATE AFTER SINGLE EMBRYOTRANSFER USING CRYOTOP BLASTOCYST VITRIFICATION METHOD

Tsuyoshi Okubo, Shoukichi Teramoto, Masashige,Kuwayama. ShinbashiYume Clinic, Shinbashi Minato-ku, Tokyo, Japan.

Objective: Multiple pregnancy is one of the most serious problem in IVF-ET,but single embryo transfer (SET) can solve the problem. Various badinfluences considered, SET is highly recommended to pregnant patient incase of IVF-ET. Since the opening of our clinic, we have cryopreserved all ofembryos by the cryotop vitrification method in substitute for the slowfreezing method. That survival rate was almost 95%(n=1089), thissurprisingly good result shows that almost of all vitrified blastocysts can betransferred with very little damage. We have performed SET by usingcryotop blastocyst vitrification method. Then we report our clinical outcomethat is high survival and pregnancy rate but no multiple pregnancies.

Design: The study in our clinic was conducted from May 2007 to August2008 for 695 patients in as the minimal stimulation IVF cycles. A total of695 patients were transferred that one cryopreserved blastocyst vitrified bythe cryotop vitrification method.

Materials and Methods: All of the patients were treated by the minimalstimulation IVF cycles using clomiphene citrate and recombinant FSH.Administration of 50 mg clomiphene citrate was initiated on cycle day 3 andthat of 150 IU recombinant FSH was injected every other day from day 8.When the size of the dominant follicle and the estradiol concentrationreached the predefined values, gonadotropin-releasing hormone agonist wasadministered to induce follicular maturation. Oocytes were then retrieved ataspiration 3035 h later since dosed nasal GnRHa spray. Then maturedoocytes were inseminated by conventional IVF or ICSI procedure. When theblastocyst developed over 170μm diameter, they were vitrified by thecryotop vitrification method (Kuwayama, 2006) on day 5 to 7. All blastocystwere underwent laser assisted hatching after thawing, then it was culturedfor before ET at least for 2 hours retrieval. Blastocysts were transferred to

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the patient visibility of the catheter under ultrasound guide.

Results: The mean number of oocytes retrieved was 2.8 per treatment cycle. The number of patients cryopreserved blastocysts by cryotop vitrificationmethod was 695 and mean age of patient was 37.0±4.1 years old. Of all1029 IVF-ET cycles initiated, the rate for blastocyst survival on day 5, 6 and7 were 99.0%(489/494), 91.2%(486/533) and 85.5%(53/62), respectively.The rates for clinical pregnancy, ongoing pregnancy and multiple pregnancywere 50.9%(524/1029), 43.8%(451/1029) and 0%(0/1029) , respectively.

Conclusions: The Cryotop vitrification method enabled us to obtain excellentblastocyst survival rate on day 5 to 7. Furthermore the SET protocol usingin this study resulted in highly pregnancy rate without multiple pregnancies.In short, the Cryotop vitrification blastocysts method is very valuable forSET, it can be possible for patients both maintaining high pregnancy rateand preventing from multiple pregnancies under an appropriate endometrialcondition.

410.6 – COMPARISON OF VITRIFICATION VERSUS SLOW COOLING FORBLASTOCYST CRYOPRESERVATION IN AN IVF PROGRAM

Andrew Dorfmann, Micheal Geltinger, Michael,Sisson, Simone Yap, IwaszkoMelissa. Genetics & IVF Institute, Fairfax, VA, USA.

Introduction: In recent years vitrification has been utilized as an alternativeto conventional slow cooling methods for the cryopreservation of embryosand oocytes. In the present study either slow cooling or closed vitrification(Cryotip™) was used to cryopreserve blastocysts. The purpose of this trialwas to determine if we could improve our results using a closed vitrificationsystem for cryopreservation of blastocysts. Results were compared betweena standard slow cooling method and vitrification.

Materials and Methods: Embryo culture was done by conventionalmethods using Vitrolife G series sequential media. All embryos werecultured in 5% O2 and 6% CO2. All embryos were cultured to the blastocyststage prior to cryopreservation on day 5 or 6. Embryos were only selected ifthey had a well developed inner cell mass, a clear and healthytrophectoderm layer, and a blastocoel cavity comprising at least 50% of themass of the embryo. Vitrification was carried out using Cryotips® fromIrvine scientific using previously published methodology. Slow cooling wasperformed using 0.25 cc straws and subsequently thawed with thaw kitsfrom Vitrolife. Statistical comparisons were performed using chi-squaredanalysis.

Results: During the study period, June 2007 through December 2008:167patient thaws were attempted; 82 patient thaws using their own oocytes ofwhich 29 were slow cooled and 53 were vitrified and 85 patients usingdonor oocytes of which 33 were slow cooled and 52 were vitrified. 464 totalBlastocysts were thawed or warmed; 280 were vitrified, and 184 were frozenby slow cooling; 225 from patients own oocytes and 239 from donoroocytes. The overall survival rate for slow cooled blastocysts was 83/184(45%) vs 206/280 (73.5 %). For patients using their own eggs the survivalrate was 34/88 (39%) in the slow cool group and 102/138 from thevitrification group (74%). From patients using donor oocytes the survivalrate was 49/97 (50%) and 104/142 (73%) respectively. Differences betweenall groups were statistically significant; p<0.001. The clinical ongoing ordelivered pregnancy rates were as follows: patients using their own oocytes;slow cooling: 7/29 (24%) versus vitrification 22/52 (42%) (Not statisticallysignificant), patients using donor oocytes; slow cooling 9/33 (27%) vsvitrification 26/52 (51%) (p < 0.05).

Conclusions: Vitrification is a highly efficient and successful method ofcryopreservation when used for blastocyst stage embryos. In our hands ityielded significantly better results than that achieved with standard slowcooling methodologies. Embryo survival, viability, and quality were excellentand produced good pregnancy rates in this initial series.

410.7 – FROZEN EMBRYO TRANSFER: THE EFFECT OF NUMBERTRANSFERRED, STAGE TRANSFERRED AND SYNCHRONISATION ONTHE CLINICAL OUTCOME

Tarique Salman, Ariel Zosmer, Amanda,Tozer, Luca Sabatini, Talha Al-Shawaf. Barts and the London Centre of Reproductive Medicine, Rochdale,Lancs, UK.

Introduction: Elective single embryo transfer in fresh and in frozen embryotransfer (FET) cycles has been advocated to reduce multiple pregnancies. Inthis study we analysed the effect of transferring 1 vs. 2 good qualityembryos, the number of embryos thawed, the number of blastomeres atfreezing and the synchronisation of the embryos and the endometrialdevelopment at transfer on the outcome of FETs.

Material and Methods: We retrospectively analysed 818 FET cycles. 48(5.6%) cycles resulted in failed thaw, leaving 770 consecutive non donoroocytes FET cycles. Good quality embryo was defined as <15%fragmentation post thaw. The transfer of day 2 (D2) embryos on day 3 (D3)post ovulation (in natural cycle FETs) or after the start of progesteronesupplementation (in HRT cycle FETs) or D3 embryos transferred on D2respectively was defined as an asynchronous FET cycle.

Results: The overall clinical pregnancy (CPR) and live birth (LBR) rates were18.3% and 14.3% per transfer respectively. One embryo was transferred in123 (15.9%), 2 embryos were transferred in 632 (82.1%) and 3 embryoswere transferred in 8 (1%) cycles. In 413 (53.6%) of the FET cyclesembryos of good quality were only transferred, in the remainder theembryos quality were mixed.

Analysis of outcome by the number of good embryos transferred: One goodquality embryo (1xFET) was transferred in 74 cycles, resulting in 6 (8.1 %)CPR and LBR. Two good quality embryos (2xFET) were transferred in 339cycles resulting in 83 (24.7%) CPR and 68 confirmed LBR (20.1%) (8pregnancies final outcome is unknown). The CPR and LBR were significantlyhigher in the 2xFET vs. 1xFET (p=0.015).

Twin sacs were noticed in 21 cases and triplets in 2. Twelve (10.3% perLBR) sets of twins, and 2 (1.7% per LBR) set of triplets were delivered. Theoverall multiple LBR was 12%.

Analysis of outcome by the number of embryos thawed: Cycles where only1 embryo was transferred the LBR was 1.9% when only 1 embryo wasthawed, 9% when 2 embryos were thawed and 14.2% when more than 2embryos were thawed. In cycles where 2 embryos were transferred CPR andLBR were 20.5% and 17.6% when only 2 embryos were thawed, 17.1% and12.8% when 3 embryos were thawed and 22.4% and 19.4% when morethan 3 embryos were thawed respectively.

Analysis of outcome by the number of cells at cryopreservation: When 2embryos were transferred and both were frozen at the 2 cells stage the LBRwas 5.1% but if both embryos had 3 or more cells when frozen the LBR wassignificantly higher at 20.2% (p =0.0014 ).

Effect of synchronising freezing day to transfer day: Out of 770 cycles, 94(12.5%) were asynchronous by +/- 1 day. There was no statistical differencein the CPR (18.5% vs. 17.0%) or LBR (14.9% vs. 15.9%) between thesynchronous and asynchronous groups respectively.

Conclusions: Transferring 2 good quality embryos in FET cycles resulted insignificantly higher CP and LB than single FET. The transfer of embryosfrozen at 3 or more blastomeres stage is more successful than that ofembryos frozen at 2 blastomeres stage. One day asynchronisation betweenembryos’ and endometrial development does not affect LBR.

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ABSTRACTS – POSTERS

P.001 – MISOPROSTOL FOR RIPENING OF CERVIX IN NULLIGRAVIDASHYSTEROSCOPY

Seddigheh Abdollahi-Fard, Abolfazl Bohlouli. Tabriz University of MedicalSciences, Tabriz, Eastern Azarbaijan, Iran.

Hysteroscopy is an operation in which the gynecologist examines the uterinecavity using a small telescope inserted via the vagina and the cervix and thisinstrument is to diagnostic and operative purpose in intrauterine pathology.In the non pregnant state the cervical canal is very narrow and the cervixresists mechanical force to open it and there is much complication inherentto mechanical dilatation including cervical tearing, uterine perforation, andbleeding and intra abdominal organs damage. these complications is moreprofound in nulliparous patients.

Material and Methods: To determine the effect of self administered oralmisoprostol 200 mg (a synthetic prostaglandin E1 analogue) vaginally thenight before procedure to all infertile or sub fertile patients withhysteroscopy indication and compared with non used cases.108 patientswith indication for hysteroscopy including vaginal bleeding, septate uterus,endometrial thickness or mass (polyp,submucosal myoma) were includedand half of patients offered self administered misoprostol 200 mg the nightbefore admission and the other half did not get any medication beforeprocedure for cervix ripening .the procedure time and complications andsurgeon satisfaction was compared in two groups.

Results: the effect of misoprostol in ripening of the cervix was significanteven in nulliparous post menopause patients and the hegar no-7dilatatorwas replaced without any resistance.

Conclusion: We would recommend this inexpensive and easy to use regimento infertile or sub fertile women prior to undergoing operative hysteroscopyto reduce the risk of complications and facilitate cervical dilatation.

P.002 – RESPIRATORY ACTIVITY AND ULTRASTRUCTURAL FEATURES OFBOVINE EMBRYOS DEVELOPED IN DIFFERENT CULTURE SYSTEMSUSING SERUM-FREE OR SERUM-CONTAINING MEDIA

Hiroyuki Abe1, Shoko Yamashita2, Hiroyoshi Hoshi2. 1Graduate School ofScience and Engineering, Yamagata University, Yonezawa; 2ResearchInstitute for the Functional Peptides, Yamagata; Japan.

Introduction: Scanning electrochemical microscopy (SECM) measuringsystem has been employed to quantify the respiratory activity of embryos inseveral animal species including humans. Respiration is a useful parameterfor evaluating embryo quality as it provides important information aboutmetabolic activity. Recently, we have found that there is correlation betweenembryo quality and respiratory activity in bovine embryos. It has beenreported that culture condition affects embryo quality and serum may be akey factor. The aims of this study were: (1) to assess the oxygenconsumption; (2) to examine the ultrastructural features of bovine embryosat different developmental stages cultured in serum-free and serum-supplemented media.

Materials and Methods: Bovine oocytes were matured in IVMD101 medium(Research Institute for the Functional Peptides: IFP, Japan) and inseminatedin BO-based medium. For serum-free culture, inseminated ooocytes werecultured to blastocyst stage in IVD101 medium (IFP, Japan) in anatmosphere of a low oxygen condition (5% CO2/5% O2/90% N2) at 38.5C˚.For serum-supplemented culture, inseminated oocytes were cultured inHPM199 medium (IFP, Japan) supplemented with 5% calf serum(HPM199+CS) in the presence of bovine cumulus/granulose cells in ahumidified atmosphere of 5% CO2 in air. Oxygen consumption by individualbovine embryos was non-invasively quantified by SECM measuring system.Some embryos were prepared for transmission electron microscopy.

Results: Oxygen consumption has been monitored at various developmentalstages of bovine embryos cultured in IVD101 and HPM199+CS media (Table1). Oxygen consumption rates of the single embryos were low from 2-cell to8-cell stages (0.48-0.52×1014/mol•s-1). In serum-free culture, an increase inoxygen consumption rate was found at the morula (1.03×1014/mol•s-1) stageand blastocysts showed an even higher oxygen consumption rate(1.86×1014/mol•s-1). On the other hand, the oxygen consumption of morulaeand blastocysts produced in serum-supplemented medium were lower thanthose of embryos cultured in serum-free medium. Ultrastructural analysis

revealed that many of the mitochondria of morulae and blastocysts culturedin serum-supplemented medium were immature, consistent with acorrelation between respiration activity and development of mitochondria.

Table 1: Oxygen consumption rates of the bovine embryos cultured inserum-free medium (IVD101) and serum-supplemented medium(HPM199+CS)

Embryonic stage Oxygen consumption rate (×1014/mol•s-1)

IVD101 HPM199+CS

2 cell 0.46±0.05 (17) 0.52±0.04 (6)

4 cell 0.45±0.03 (17) 0.47±0.04 (6)

8 cell 0.46±0.02 (10) 0.52±0.04 (10)

Morula 1.10±0.05 (23) 0.70±0.05 (12) *

Blastocyst 1.99±0.07 (35) 1.33±0.10 (12) *

*Significant differences (P<0.05, compared with embryos cultured inIVD101 at same embryonic stages). The numbers in parentheses representthe numbers of embryos examined.

Conclusion: These results demonstrated that the culture conditions (serumsupplemented or not) affect the respiratory activity, mitochondrialmorphology, and embryo quality. Measuring oxygen consumption withSECM may have broad applications for determining suitable cultureconditions for embryos.

P.003 – ENDOMETRIOSIS SEVERITY AND IVF OUTCOME

Germain Agnani, Arnaud Collin, Xavier Dellis, Pascale Lagré, ChristineSouquet, Christophe Roux, Christianne Joanne, Didier Riethmuller. Servicede Gynécologie Obstétrique, CHU Besançon, Besançon, France.

Objective: The aim of this study was to evaluate the impact of endometriosison In Vitro Fertilization (IVF) outcome, focusing on the initial surgicalapproach, surgical difficulties, and ovarian reserve.

Retrospective review of 200 consecutive first oocyte retrievals in patientswith endometriosis.

Materials and methods: Severity of endometriosis was evaluated during thefirst laparoscopy according to the American Fertility Society (AFS)classification and a simplified classification

( 3 subgroups).

Subgroup A : superficial peritoneal and ovarian implants.

Subgroup B : endometrioma (diameter ≥ 5mm) without deeply infiltratingendometriosis.

Subgroup C : deeply infiltrating endometriosis involving the bladder,sigmoid, uterosacral ligaments or vagina.

Male infertility and anovulation were excluded. Surgical treatment wasalways performed before IVF.

Follicular count was estimated just before the first IVF attempt.

Data were analysed using the chi square test for qualitative variables and theKruskal Wallis one-way analysis of variance for quantitative variables,statistical significance was set at p<0.05.

Results: Clinical pregnancy rate was significantly lower in group C ( group A: 42%, group B : 50%, group C : 5.9%, p<0.001 ).

Subgroups A, B, and C were comparable when we considered female age,BMI, duration of infertility, sperm concentration, basal FSH level,endometrioma diameter before laparoscopy (groups B & C), follicular count,oocyte number, embryo number, and classical parameters of stimulation.

The percentage of laparotomies and ovarian cystectomies was similar insubgroups B and C.

Total top quality embryo number and top quality transferred embryo numberwere significantly lower in group C (p<0.05).

Early complications after IVF gradually increased with the severity ofendometriosis ( group A : 5%, group B : 20%, group C : 38%, p<0.001 ).

The surgical approach including a second look offered the best results(p<0.01).

Clinical pregnancy rate was also significantly influenced by total embryo

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number (p<0.01), top quality embryo number, top quality transferredembryo number (p<0.001), detection of endometrioma before stimulation(p<0.02), and BMI (p<0.02).

In a multivariate logistic regression including top quality transferred embryonumber, endometriosis subgroups, BMI, and also age, basal FSH level, andfollicular count, three variables were selected : top quality transferredembryo number, endometriosis subgroups, and detection of anendometrioma. Fewer differences were observed when the AFS classificationwas considered.

Conclusions: As opposed to recent studies which minimize the impact ofendometriosis on IVF results, our study suggested that the persistence ofactive lesions is more deleterious than mild follicular destruction andadhesions.

P.004 – HOW DO HYPOGONADOTROPHIC HYPOGONADISM PATIENTS DO INASSISTED REPRODUCTION CYCLES?

Gülnaz Şahin, Cüneyt Barut, Ayşin Akdoğan, Halil Abdülrezzak, Rafael Levi,Ege Tavmergen Göker, Erol Tavmergen. Ege University Family Planning andInfertility Center, Izmir, Turkey.

Introduction: Hypogonadotrophic hypogonadism (HH) is a rare condition offemale infertility. The purpose of this study was to evaluate the assistedreproduction treatment (ART) cycle characteristics and outcomes of womenwith HH and compare them with a group of normal responders’ cycleoutcomes.

Design: Retrospective analysis

Materials and Methods: Analysis was carried out on 26 HH female patientswith 40 ART/ICSI cycles during 1996-2008 performed at Ege UniversityFamily Planning and Infertility Center. Age matched 66 healthy women wereselected as a control group with a history of regular menses, normal basalendocrine hormone levels and undergone ART with long protocol cycles formale factor infertility from the same center. Controlled ovarian stimulationwas performed with human menopausal gonadotrophin (HMG) in HH groupand recombinant FSH combined with gonadotrophin releasing hormoneagonist for control group.

All IVF cycle outcomes were evaluated. Their age, infertility duration, basalserum hormone levels, the mean diameters of both ovaries onultrasonographic examination, total and daily dosage of gonadotrophins,serum E2 levels and LH levels on the day of human chorionic gonadotrophin(HCG) injection, number of total oocytes, metaphase II (MII) oocytes,transferred embryos, implantation rates and pregnancy rates were analyzed.Statistical analysis was performed with SPSS version 15.0. Mann-Whitney -U test, Chi-square tests were used to compare groups. p < 0.05 wasconsidered statistically significant.

Results: Totally 36 cycles in HH group and 59 cycles in control groupreached to the embryo transfer. The mean ages in the HH group were33±4.5 (range 26-42) and 31.1±2.6 (range 28-37) of the control group. Themean infertility durations were 9±3.9 years and 8.4±4.0 years, respectively.The mean diameters of both ovaries on ultrasonographic examination weresmaller than control group (p<0.01). Basal serum FSH, LH levels weresignificantly lower than control group (p<0.05). Although HMG was used inthe HH group, the control used only FSH containing preparates. Dailydosage of gonadotrophin requirement and total dosage of gonadotrophinswere significantly higher in HH patients than controls, 5.6±1.1 vs. 3.7±0.5and 60.7±18.5 vs.31.8±8.6 ampoules, respectively (p<0.01). Duration ofstimulation was significantly longer in HH group (p<0.01). Serum E2 levelson the day of HCG injection was not different between groups but meanserum LH levels on the same day was significantly higher in control groupthan HH patients, 2.3±1.5 vs. 0.76± 0.73, respectively (p<0.05). Meannumber of oocytes, MII oocytes were the same. Although more embryoswere transferred in HH group, their implantation rates (15.7±22.8vs.25.4±30.5), pregnancy rates per embryo transfer (44.4% vs.55.9%) andclinical pregnancy rates per embryo transfer cycle (38.9% vs. 50.8%)seemed to be lower than controls, even though not statistically significant.

Conclusions: Although higher consumption of gonadotrophin dosages andlonger duration of ovarian stimulation is needed in HH patients, theirpregnancy rates in IVF cycles were not different than normal responderpatients.

P.005 – IMPACT OF SURGICAL TREATMENT OF VARICOCELE ON THEBIOLOGICAL ASSESSEMENT OF IN VITRO FERTILIZATION (IVF)

Mounir Ajina, Loussaief Wafa, Ibala Samira, Ben Regaya Lassad, HidarSamir, Khairi Hedi, Saad Ali. Unit of Medicine of Reproduction, Sousse,Tunisia.

Introduction: Surgical treatment of varicocele makes it possible to restorethe adequate temperature of spermatogenesis, but the re-establishment ofthe fertilizing capacity remains doubtful. The aim of this work is to evaluatethe impact of varicocele and of its surgical repair on the biologicalassessment of in vitro fertilization.

Materials and Methods: We studied 97 patients carrying varicocele, 46% ofwhich was operated. Among these patients, 16% had a grade III and 55%had the grade II. Varicocele was on the left in 79% of case. All the couplesprofited at least from a cycle of in vitro fertilization and for each couple weanalyzed: semen parameters, fertilization rate, segmentation rate andembryonic quality. Statistical study was made by software SPSS11.0.

Results: Average age of patients was of 38±5, 08 years with a primaryinfertility duration average of 6, 81±3, 95 years. Infertility was of male originin 55% of case and mixed origin in 40%. The analysis of semen finds: amean spermatic concentration of 23, 05±35,71millions/ml, a mean mobility(a+b) of 15±10%, % of abnormal forms of 65±30% and 30±28 % of deadspermatozoa. 12% of our patients were azoospermic with an average rate ofFSH of 7, 28±4, 78 UI/ml. assisted fertilization was indicated in 68% ofcouples and traditional fertilization in 11%of couples. During the first cycle,7, 77±4, 93 follicles were aspirated of which only 66, 48±33, 09% reachedthe stage of zygote after insemination. The fertilization and segmentationrates were respectively of 50, 96±35, 81% and 81, 02±103, 49%. Theaverage number of type I embryo transferred was of 1, 82±1, 25.

Conclusion: The biological assessment of in vitro fertilization was fadedamong patients carrying varicocele, especially the fertilization rate. On thehand segmentation rate and embryonic quality were both preserved. In ourstudy the surgical clean of varicocele does not improve the oocytefertilization rate. This same rate does not vary also to a significant degreeaccording to the grade of varicocele.

P.006 – NONENZYMATIC ANTIOXYDANT SEMINAL ANALYSIS IN INFERTILEMEN AND ITS EFFECT ON THE BIOLOGICAL ASSESSEMENT OF INVITRO FERTILIZATION (IVF)

Mounir Ajina1, Atig Fatma1, Ibala Samira1, Loussaef Wafa1, Hidar Samir1,Khairi Hedi1, Kerkeni Abdelhamid2, Saad Ali1. 1Unit of Medicine of theReproduction, Hospital F. Hached; 2Laboratory of Biophysics, Faculty ofMedicine, H Bourguiba Monastir; Sousse, Tunisia.

Introduction: The aim of this study consists on evaluating the nonenzymaticantioxidant statute of infertile patients programmed for in vitro fertilizationand the repercussions of this statute on the biological assessment of in vitrofertilization.

Material and Methods: Our work concerns 57 couples including sixWitness patients profited from a spermatic nonenzymatic exploration: Zinc,Malondialdéhyde and Glutathion. The proportioning of seminal zinc wascarried out by the spectrophotometry of atomic absorption to flame, the acidMalondialdéhyde (MDA) was evaluated by the thiobarbituric method of theacid (TBA) and the proportioning of the various forms of glutathion (GSH)(total, oxidized and reduced) was based on the coupling of the groupingsthiols of the GSH with the dithiobisnitrobenzoate (DTNB) which istransformed into a compound coloured in yellow and detectable with 412nm.

Results: Our results showed that the fertilization rate increased moderatelyamong patients group. Therefore, oxidative parameters (Zinc, GSH andMDA) were also raised in patients group than in witness one. Inasthenozoospermic group of patients, the MDA rate was converselycorrelated with mobility (p=0, 04). However, total GSH was positivelycorrelated with fertilization (p=0, 03) and with embryonic segmentation rate(p=0, 02).

Conclusion : The increase in the seminal concentrations in MDA amonginfertile patients can partly explain the bad spermatic quality observed aswell as the weak fertilisation rate. But, the positive correlation of

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antioxydants with the rate fertilization confirm the paternal nonenzymaticantioxydant contribution in the early embryonic development.

P.007 – OVARIAN HYPERSTIMULATION USING GONADOTROPIN UPREGULATECFTR EXPRESSION IN VIVO: IMPLICATIONS FOR HYDROSALPINXENLARGEMENT AND UTERINE FLUID ACCUMULATION DURINGOVULATION INDUCTION

Louis Chukwuemeka Ajonuma1, 2, Lai Ling Tsang1, Sun Yee Lam1, HsiaoChang Chan1. 1Epithelial Cell Biology Research Center, Department ofPhysiology, Faculty of Medicine, The Chinese University of Hong Kong,Shatin, NT, Hong Kong; 2Faculty of Dentistry, The University of Hong Kong,Hong Kong SAR.

Introduction: Controlled ovarian hyperstimulation (COH) for ovulationinduction is associated with formation and/or sudden increase inhydrosalpinx fluid (HF) as well as reflux into the uterine cavity leading topoor in vitro fertilization (IVF) treatment outcome. However, the mechanismsunderlying fluid formation during gonadotropin administration for ovarianstimulation have not been thoroughly investigated. Since cystic fibrosistransmembrane conductance regulator (CFTR), a cAMP-dependent ionchannel is known to modulate fluid secretion in the female reproductivetract, the present study investigated whether ovarian hyperstimulationincreases CFTR expression in vivo after gonadotropin administration.

Materials and Methods: We examined CFTR expression after gonadotropin(HMG, PMSG and HCG) administration in comparison with controls usingreverse transcriptase-polymerase chain reaction (RT-PCR),immunohistochemistry and immunofluorescence staining in a rat ovulationinduction model.

Results: RT-PCR revealed that CFTR expression in the uteri of gonadotropintreated rats was significantly higher than controls and no difference wasnoted in the ovriectomized and control rats. Immunostaining showedenhanced CFTR immunoreactivity in the uterine epithelium of rats with intactovaries when compared to ovriectomized rats.

Conclusions: These results suggest that ovulation induction upregulate theexpression of CFTR via ovarian hormones. Upregulation of CFTR leading toincreased transepithelial fluid transport results in the enlargement ofhydrosalpinx and HF reflux into the uterine cavity during ovarianhyperstimulation. These findings may provide grounds for a better treatmentstrategy for infertile patients undergoing ART.

P.008 – BEGIN OF HUMAN LIFE AND RELIGIOUS IDEAS ABOUT RESEARCH ONHUMAN EMBRYO

Mahzad Akbarpour2, Reza Omani Samani1, Seyed Taha Merghati1.1Department of Epidemiology and Reproductive Health, ReproductiveMedicine and Cell Science Research Center; 2Department of Embryology,Reproductive Medicine and Cell Science Research Center; Royan Institute,Tehran, Iran.

Doing research on human embryo is a big controversy in around the worldand according to different religions, there are different ideas about it. Beginof In-vitro fertilization and entrance of embryo donation made a complexsituation about using human embryos in research. Embryonic stem cellsand hope of treatment of incurable disease made it even more complex. Iranis the only Islamic country that practices donation programs and also hashuman embryonic stem cell lines. Here in this paper, we present the Islamicidea about the begin of human life, permission to do the research on humanembryo and therapeutic abortion. The basic presentable conclusions are asfollows:

1. Pre implantation embryo is not considered as human or potential humanand can be used in research with the permission of the parents.

2. After implantation, although the embryo is not considered human, butnobody can touch it. Any manipulation of embryo in the uterus isconsidered try to abort the child.

3. After 120 days (for Shiaa Muslims) and 50 days (for Sunni Muslims), it isbelieved that soul goes inside the fetus so it is considered a human, andno abortion is allowed.

4. Before this date, it is OK to do the therapeutic abortion if there is anabsolute medical reason.

P.009 – PREDICTORS OF SUCCESS IN GNRH ANTAGONISTS CYCLES FOR IVF-ICSI

Aysin Akdogan, Gülnaz Sahin, Cuneyt Barut, Rafael Levi, Ege Goker, ErolTavmergen. Family Planning & Infertility Research & Treatment Center, EgeUniversity, Izmir, Turkey.

Introduction: Controlled ovarian stimulation with GnRH antagonists forprevention of LH surge is largely used in mild ovarian stimulation protocols.The purpose of the study was to evaluate prognostic factors for achievingpregnancy in antagonist cycles.

Material & Methods: A total of 1396 IVF patients with their first cycles weretreated with rec FSH and GnRH antagonist stimulation for IVF –ICSI between2005-2007 at the Ege University Family Planning and Infertility Researchand Treatment Center were evaluated. Patients age, duration of infertility,fertility cause, menstruel regularity, basal endometrium thickness, basalendocrine parameters (basal FSH, LH, E2, prolactine),total FSH doses anddose regulation, total antagonist doses, duration of stimulation, maximumE2, maximum endometrial thickness, number of aspirated oocytes, MIIoocytes and transferred embryos were recorded for analysis to predictpregnancy rates. Multivariable logistic regresion analysis was performedwith a backward LR elimination procedure, a p-value<0,1 was used as acriterion for exclusion.

Results: Of the 1396 initiated cycles, 179(12,5%) were cancelled due to:insufficient responce, empty follicule, fertilisation defect and bad qualtyembryos. The pregnancy rate was 40.8%. Over all patients’ mean age was33,6±5,0 and duration of infertility was 8,7±5,4 years. Infertility causes weremale factor (52,4%), idiopathic (27,5%), tuboperitoneal factor (10,1%),ovulatuar factor (8,6%) and others (1,3%). In a multivariable logisticregression analysis, age, basal endometrial thickness, basal prolactinelevels, maximum endometrial thickness and number of transferred embryoswere found to be associated with pregnancy. The P-values and odds ratios(95%CIs) were respectively 0.042: 0.966(0.934,0.999) for age, 0.013:0.899(0.826,0.978) for basal endometrium, 0.043: 0.986(0.972, 1.000) forbasal prolactine, 0.001: 0.1.153(1.068,1.244) for maximum endometrialthickness, 0.001:1.751(1.394,2.200) for number of transferred embryos.

Conclusions: In this analysis the results revealed that while an increase inwomen age, basal endometrial thickness and basal prolactine levels cause adecrease in pregnancy rates. An increase in maximum endometrial thicknessand number of transferred embryos have a positive effect in pregnancyrates.

P.010 – IMPLANTATION AND CLINICAL PREGNANCY RATES IN DAY 2-3TRANSFER VERSUS BLASTOCYST TRANSFER-A RETROSPECTIVECOMPARATIVE STUDY

H. Tijani, S. Patwardhan, S. Keay, S. Montgomery, R. Kennedy, Rubina Ali.Centre of Reproductive Medicine, University Hospitals Coventry andWarwickshire, Coventry, UK.

Introduction: Recent advances in cell culture media have led to a shift in Invitro fertilisation (IVF) practice from early cleavage embryo transfer toblastocyst stage transfer. Initially, there were contradictory reports thatblastocyst transfer did not improve pregnancy rate. However, more recentevidence has shown significant difference in pregnancy and live birth ratesin favour of blastocyst transfer. The purpose of this study was to comparethe reproductive outcome of early cleavage embryo transfer and blastocyststage transfer.

Material and Methods: A retrospective analysis of implantation and Clinicalpregnancy rates was carried out for women who underwent In vitrofertilization and Intracytoplasmic sperm injection (ICSI) at the Department ofReproductive Medicine, UHCW, from June 2006 to June 2008. A total of1016 women were involved in the analysis. Of these 524 had IVF while 491had ICSI. Eight hundred sixty seven had early cleavage embryo transferwhile 149 had blastocyst transfers.

Results: Evidence of a significant difference in number of positivepregnancies between the two treatment groups was detected in favour ofblastocyst transfers: 32.5% in day 2-3 embryo and 38.9% in blastocystgroup with unadjusted success rate RR of 1.22, 95% CI of 0.99 to 1.52 andadjusted success rate RR of 1.35, 95% CI of 1.07 to 1.70. A similar trend

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was observed in the successful clinical pregnancies. A total of 28.3% clinicalpregnancies in day 2-3 embryo group and 34.2% clinical pregnancies in theblastocyst group with unadjusted success rate RR of 1.21, 95% CI of 0.95to 1.55 and adjusted success rate RR of 1.34, 95% CI of 1.03 to 1.75.Adjustment was made for age, FSH, gravidity, number of embryos for andnumber of attempts at transfer as well as treatment type, that is IVF or ICSI.

Conclusions: This study provides evidence that there is a significantdifference in number of positive pregnancies and clinical pregnancies infavour of blastocyst transfer. This trend in the difference persists even afteradjusting for confounding factors. This further corroborates recent evidencein favour of blastocyst transfer in selected patients.

P.011 – DEBATE IN EMBRYO DONATION: SOURCE OF EMBRYOS, FROZENFROM INFERTILE OR FRESH FROM FERTILE?

Leila Alizadeh, Mohammad Reza Rezania Moalem, Seyed Taha Merghati,Reza Omani Samani. Department of Ethics of Royan Institute, Tehran, Iran.

For many infertile couples there is no treatment except joining a third partyto the family. Iran is the only Islamic country in which, donation andsurrogacy is practiced and in other Islamic countries they are consideredforbidden or “Haram”. Recently, embryo donation law has been approved byIran’s parliament and now it is legal in Iran. But there is a misunderstandingin the source of embryos: they can be obtained from surplus frozenembryos of infertile couples or embryos can be made from donated spermsand eggs from fertile married couples. Here in this paper we discussedethical, religious and legal aspects of these two procedures and present theadvantages and disadvantages of them. Meanwhile, the new term “bothgamete donation” has been defined for the procedure that is practiced hereinstead of “embryo donation”. In conclusion we can say: 1) Iranian lawmeans both “embryo donation” so covers surplus embryos from otherinfertile couples and “both gamete donation” so covers making embryosfrom a fertile couple. 2) As gamete donation is practiced in Iran upondecrees of clergy leaders, we have no law or legislation against “bothgamete donation”. 3) There are so many ethical, legal and religiousquestions about “both gamete donation” to be answered. 4) Ethical andreligious questions are very fewer about “embryo donation” comparing to“both gamete donation” program, and 5) Embryo sharing is a good way fordonation of fresh embryos.

P.012 – MULTIDISCIPLINARY TASK FORCE FOR MANAGEMENT OF WOMENWITH CANCER AND FERTILITY PRESERVING ISSUES INSWITZERLAND

Alexandra Ambrosetti, Marina Bellavia, Victoria Ibecheole, Khalil Zamman,Jean-Bernard Dubuisson, Dorothea Wunder-Galié, D. de Ziegler. Onco-gynécologie, HUG, Geneva, Suisse.

Introduction: The desire for future pregnancy expressed by cancer patientsof reproductive age needs to be addressed with a degree of urgency that isdictated by the cancer treatment.

The inherent complexity of this emerging field mandates having amultidisciplinary structure capable of offering (a-remove) better and safermanagement than individual initiatives might do. We are reporting (Wereport) the activity of such a multidisciplinary regional task force that existsin Switzerland since 2006, “Réseau Romand de Cancer et Fertilité” (RRCF).

Material and Methods: Between 07/2006 to 01/2009, a total of 32 women(20 breast cancers and 12 other cancers or autoimmune diseases) wereprospectively evaluated for fertility preservation measures before adjuvantchemotherapy. Of these, 16 underwent controlled ovarian hyperstimulation(COH) for cryopreservation of 2-PN embryos or oocytes (11 receivedletrozole- gonadotropin-GnRH antagonist protocols, 2 long GnRH-aprotocols, 2 GnRH antagonist protocols and 1 a GnRH agonist microflare-upprotocol). Only 1 woman underwent ovarian tissue cryopreservation. Onewoman with squamous cervical cancers (FIGO stages Ia1) underwenttrachelectomy. The remaining 14 patients declined any fertility-preservingprocedure after thorough counselling.

Results: In women with breast cancer (mean age 31.81, range 25-39), (anaverage of) 14 ± 8.73 oocytes were retrieved and 11.66 ± 7.37 oocytes or7.25 ± 5.80 pronucleids were cryopreserved per patient. In women withother cancers or autoimmune diseases (mean age 30, range 21-39), 11.8 ±

9.06 oocytes were retrieved and 10.5 ± 6.36 oocytes or 5.25 ± 4.57pronucleids were cryopreserved per patient.

Time between surgery and chemotherapy was 34.5 ± 9.25 days for patientswith breast cancer undergoing IVF(Proposition: Chemotherapy was notdelayed in breast cancer patients undergoing fertility perservationtechniques with an interval between surgery and adjuvant chemo of 34.5 ±9.25 days.) Peak E2 levels at the time of ovulation was 1210 pmol/L (range540-2190 pmol /L) in patients with breast cancer using the letrozole-gonatrophin- GnRh antagonist protocol and 5550 pmol/L(range 1830-10430pmol/L) in patients with other cancers or autoimmune diseases.

Conclusions: Cancer and Fertility is an emerging new field of reproductivemedicine. The approach to patient-care should be multidisciplinary toensure that the best options are offered to women based on currentknowledge whilst ensuring that optimally timed curative oncologicaltreatment is not compromised. which needs to be approached in the contextof a multidisciplinary strategy for assuring that the best options are offeredto cancer patients at the right time. In doing this, utmost care should bedeployed for not disturbing the cancer treatment and notably, delaying thestart of adjuvant chemotherapy.

P.013 – THE EFFECTS OF LIF AND EGF ON MOUSE OOCYTE MATURATION ANDPREIMPLANTATION EMBRYO DEVELOPMENT IN VITRO

Iraj Amiri1, Ali Amini2, Maryam Parvini2, Narges Mirahadi. 1Dept of Anatomyand Embroyology, Medical School, Hamadan University of Medical Sciences,Hamadan; 2Dept. of Biology, Faculty of Science, Kermanshah University,Kermanshah, Iran.

Introduction: Recent studies have demonstrated that mammalianpreimplantation embryos are exposed to a mixture of many different growthfactors and cytokines, expressed by the follicles, oviducts and endometrium.Receptors for many of these growth factors have also been shown to beexpressed by preimplantation embryos. In vitro culture of human andanimal’s embryos in conventional media lacking growth factors can result insuboptimal growth and a variety of short-term and long-term developmentalabnormalities. One of these factors is Leukemia inhibitory factor (LIF).Theaim of this study was to evaluate the effects of LIF on the mousepreimplantation embryo development.

Materials and Methods: Six to eight weeks old NMRI mice weresuperovulated by injection of 10IU PMSG and 10IU HCG 48h later. Themated mice were killed 48 hours after hCG injection, oviducts were flushedand two-cell embryos collected and divided randomly to two groups(Control and treatment).Control medium was HTF and treatment mediumwas HTF+1000u/ml LIF. In each group the embryos were cultured in anincubator at 37°C with 5% CO2 for 72h.The state of embryo developmentwas evaluated in 24 hours interval using inverted microscope.

Results: There was not any significant difference in the rate of morollaformation after 48 hours. In comparing blastocyst formation and hatchingrates, 60 and 72 hours after culture, there was significant differencebetween control and treatment groups (p< 0.008).

Conclusion: LIF doesn’t provide obvious stimulation in the early mouseembryo development until morolla stage; however, it has positive effects onblastocyst formation and hatching.

Key Words: LIF, Preimplantation embryo, Mouse

P.014 – CHROSOMAL ANALYSIS OF THE ABORTUSES IN INFERTILE PATIENTSAFTER TREATMENT

Tomoka Aniya1, Yoshiharu Nakaoka1, Sachiyo Tarui1, Aya Ohgaki1, KengoSugihara1, Mamoru Ida1, Aisaku Fukuda1, Yoshiharu Morimoto2. 1IVF OsakaClinic; 2IVF Namba Clinic; Osaka, Japan.

Objectives: Cytogenetic abnormalities of the conceptus are well recognizedcauses of pregnancy loss. Most of these abnormalities are the result ofchromosomal accidents at the time of parental meiosis, fertilization andearly cell division in the zygote. On the other hand, it is well known thatspontaneous miscarriage rate in infertile patients is approximately 20-25%and higher than that in natural pregnancy.

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The first objective of the present study was to assess the chromosomalabnormality rate by analyzing karyotype of the abortuses in the pregnanciesafter infertility treatments. The second objective was to clarify therelationship between chromosomal abnormality rate and maternal age orearly fetal development during pregnancy.

Methods: Chromosomal analysis was performed on 289 abortuses from theinfertile patients. Eighty five cases were conceived by classical infertilitytreatment such as timed intercourse or intrauterine insemination, 40 casesby conventional IVF (IVF) and 164 cases by intracytoplasmic sperm injection(ICSI). The average maternal age in these 3 groups were not different.Chorionic villi obtained from dilatation and curettage were cultured andkaryotyped by G-banding techniques. All procedures were performed afterobtaining informed consent from the patients.

Results: Two hundreds seventeen cases (75.0%) had abnormalchromosome constitutions, in which 152 cases (70.0%) were autosomaltrisomy, 32 cases (14.7%) were structural abnormalities, 8 cases (3.7%)were triploidy, 4 cases (1.8%) were tetraploidy and 10 cases (4.6%) were Xmonosomy. Chromosome 16 and chromosome 22 were the most frequentautosomal trisomies. There are no difference of incidence of chromosomalabnormalities in the groups of classical infertility treatment (81.0%), IVF(74.3%) and ICSI (70.8%). But polyploidy rate in either IVF (0%) or ICSI(1.7%) was lower than that in classical infertility treatment group (14.0%).Chromosomal abnormality rate (81.7%) in the advanced maternal age (�35years) was significantly higher than that (63.9%) in younger age (<35years). Aneuploidy rate of chromosomal abnormalities was increased whenmaternal age advanced,

Conclusions: The present study had that abortuses in infertile patients afterany infertility treatment showed high incidence of abnormal karyotype,approximately 70�. Assisted reproductive technologies does not onlyincrease the rate of chromosomal abnormalities in abortuses, but alsoprevent the polyploidy by observation of number of pronucleus in thelaboratory. The incidence of chromosomal abnormality is strongly related tomaternal age.

P.015 – DEVELOPMENT OF THE SAFETY CYTOPLASMIC EXCHANGE METHODIN PRONUCLEAR STAGE OOCYTES; PREVENTION OF MITOCHONDRIACOEXISTENCE IN CYTOPLASMIC THAT USED THE PRONUCLEARMICRO INJECTION

Fumihito Aono1, Kojiro Kawano1, Masashige Kuwayama1, Yuji Takehara2,Osamu Kato2; 1Advanced Medical Research Institute of Fertility, Kato LadiesClinic, Shinjuku; 2 Kato Ladies Clinic, Shinjuku; Tokyo, Japan.

Introduction: The effectiveness of oocyte cytoplasmic exchange has alreadybeen demonstrated for the treatment of the infertility cause of defectivecytoplasm and of the prevention of heredity of the mitochondrial diseases(Kuwayama, 58th ASRM meeting, 2002�. However, mitochondria is broughtin with the nuclear when the cell membrane fusion method (McGrath andSolter, Science 1983) is used, and two kinds of mitochondria, derivationfrom both of donor and recipient mitochondria, exist togetherconsequentially in the donor cytoplasm. Because mitochondria coexistencedoes not exist in nature, and the safety in the situation is uncertain, thesetwo types of mitochondria coexistence is an obstruction in a clinical use ofthis technology application. To prevent mitochondria coexistence, we triedthe development of the pronuclear microinjection method.

Materials and Methods: The pronuclear stage oocytes with informedconsent were used for the experiment. A pronuclear was removed from thecell membrane crushed oocyte that was crushed by the pulse of the Piezomicromanipulator (the cell membrane crushing method) in modified P1medium containing 60% of serum substitute supplement (SSS, IrvineScientific). To remove the cytoplasm containing mitochondria, the removedpronuclear was washed by the repetitive pipetting with the small glasspipette assembled in the micromanipulator. The cytoplasm-free pronuclearwas injected directly to the donor cytoplasm with the glass pipette of theinside diameter 15 or 25 μm. The pronuclear injected oocytes were culturedwith Cleavage Medium (SAGE) containing 10% of SSS for 30 minutes 5%CO2 5% O2 and 90% N2 at 37C. The oocyte that the cell membrane hadbeen maintained intact after 30 minutes from the pronuclear microinjectionwas judged survival and the oocyte that was able to confirm the pronuclearintact was judged the pronuclear microinjection success.

Results: The survival rate in the pronuclear micro injected oocyte using apipette inner diameter 15 or 25 μm were 93% (37 / 40) and 50% (20 / 40)(P<0.01), and the pronuclear microinjection success rate were 78% (29 /37) and 70% (14 / 20), respectively.

Conclusion: The pronuclear that was removed from the oocyte withoutcytoplasm is able to inject into the enucleated cytoplasm directly. The resultshows the possibility that this pronuclear microinjection method becomesthe effective treatment of the infertility cause of defective cytoplasm, and ofthe prevention of heredity of the mitochondrial diseases.

P.016 – SWITCHING FROM PN SLOW FREEZING TO BLASTOCYSTVITRIFICATION: IMPACT ON IVF OUTCOME

Marina Argyrou, Massia Moschopoulou, Chris Karamalegos, Sofia Doriza,Tania Karagianni, Christina Mentorou, Stephen Davies, Minas Mastrominas;Embryogenesis Assisted Conception Unit, Marousi, Athens, Greece.

Introduction: For many years our policy was to routinely slow-freezeembryos at 2PN stage. In our experience, survival & pregnancy rates afterthawing were better than cleavage stages. We experimented with severalprotocols of vitrification, but were not satisfied with survival quality until wewere introduced to the Cryotop method. Vitrification as a proceduretheoretically reduces intracellular damage due to ice crystallization inducedby other freezing protocols.

We changed our policy of freezing during July 2008, where patientsundergoing treatment cycles were advised to culture all embryos untilproposed transfer dates and spare blastocyst(s) of good quality vitrified forsubsequent use as opposed to blindly freezing a proportion of embryos at2PN stage and allowing the remainder to progress for fresh transfer.

We assess here the impact of this change in policy had on the outcome ofIVF cycles during this period of transition.

Materials & Methods: In this study we included all patients from period ofFebruary-June 2008 who underwent slow freezing at 2PN stages (Group A,n=163 patients). They all followed our standard procedure where if 7 ormore fertilized oocytes are available they are advised to have at least 3embryos frozen, & allow the remaining to proceed for transfer either on day3 or at the blastocyst stage, depending upon doctors recommendation.

The vitrification protocol included in this study were performed betweenJuly- December 2008 (Group B, n=108 patients). The protocol required thatall embryos are cultured to the blastocyst stage. Either one or twoblastocysts were transferred, and the remaining blastocysts immediatelyvitrified using Croptop method.

Both groups A & B consisted of patients undergoing 1st cycle at our center,ICSI procedure used, & >6 embryos available. Cycle outcome was thenassessed as clinical pregnancy & implantation rates.

Results: Groups A & B did not differ in terms of mean age (Group A:34.0±4.2, & Group B: 33.6±3.9), causes of infertility, number of oocytesretrieved (Group A: 14.9±5.5 & Group B: 15.2±5.2 respectively), & numberof oocytes fertilized (Group A: 8.8±3.4, & Group B: 9.1±3.2). There werestatistical differences in the numbers of embryos transferred (Group A:2.8±0.2, & Group B: 1.4±0.2), clinical pregnancy rates (Group A:72/163:44.2%, & Group B: 59/108:54.6%) & implantation rates (Group A:104/508:20.5%, & Group B: 71/158:44.9%)

Conclusions: The switch from routinely freezing at 2PN to Blastocyst had afundamental benefit on our IVF programme by improving clinical pregnancyrates in 1st cycle patients after transferring fewer embryos. These increasedrates are attributed to the patients with blastocyst vitrification having all oftheir cohort of embryos cultured, and greater numbers from which tochoose the best embryo(s) for ET. Routinely in our programme, we do notsee significant differences in pregnancy rates between day3 & Blastocyst ET.

We were allowed to make this change in protocol because of the efficiencyand effectiveness of the cryotop vitrification technique which allows freezing& warming blastocysts with virtually no loss of embryos & potential.

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P.017 – EFFECT OF HYDROSTATIC PRESSURE ON CUMULUS AND OOCYTECOMPLEX (COCS) DERIVED FROM IN VITRO CULTURED PREANTRALFOLLICLES

Zahra Rashidi, Mehri Azadbakht, Ali Amini. Razi University, Kermanshah,Iran.

Introduction: In vitro maturation of oocytes is a safe and effective treatmentoffered in some fertility centers for assisted reproduction. Hydrostaticpressure as physical force is effective in reproduction system and there is anincrease in intrafollicular pressure between 15-20 mmHg in the ovulatingfollicle during the late stage of the ovulatory process. Cumulus cells play acritical role in oocyte maturation and fertilization. Whether the degree of celldeath in the cumulus–oocyte complex (COCs) has an impact on oocytedevelopment potential is unclear. Physical forces may to

assure the incidence of cell death in (COCs). In this study, we examined theeffect of hydrostatic pressure on the viability of COCs derived from in vitrocultured preantral follicles.

Materials & Methods: Preantral follicles were isolated from 12-day-oldfemale NMRI mice, each follicle cultured individually in microdrops of MEM-� culture medium under oil during 12 days. On day 12, follicles withdiameter nearly 500 μm and good quality were induced using 7.5 mIU/mlhuman chorionic gonadotropin (HCG) for in vitro maturation. Then follicleswere subjected to 20 mmHg hydrostatic pressure for 30 min and culturedfor 24 h. Viability of cumulus cells and oocyte were assessed withdifferential staining (propidium iodide & bisbenzimide) on 0 and 24 h afterculture.

Results: indicate that, hydrostatic pressure had not changed viability ofoocyte in 0 and 24 h after culture (p<0.05).At the 0 h, viability of thecumulus cells were reduced in hydrostatic pressure treated follicles (93%)compared to control (97%); (p<0.05). After 24 h viability of the cumuluscells were reduced in hydrostatic treated follicles (93%) compared to control(85%); (p<0.05).

Conclusion: hydrostatic pressure had the mild effect on cell death incidencein cumulus cells without any effect on oocyte viability. Hydrostatic pressuremay play a role in oocyte maturation and fertilization by improving inreleasing and mediating signals to oocyte.

P.018 – EFFECT OF HYDROSTATIC PRESSURE ON IN VITRO MATURATION OFOOCYTES DERIVED FROM IN VITRO CULTURED PREANTRAL FOLLICLES

Zahra Rashidi, Mehri Azadbakht, Ali Amini. Razi University, Kermanshah,Iran.

Introduction: Oocyte in vitro maturation (IVM) is an important reproductivetechnology that involves artificial removal of cumulus-oocyte complex(COCs) from antral follicles significant progress has been made in thedevelopment for the IVM, but there is limited efficiency. Hydrostaticpressure as physical force is effective in reproduction system and there is anincrease in intrafollicular pressure (15-20 mmHg) in the ovulating follicleduring the late stage of the ovulatory process. In this study, we examinedthe effect of hydrostatic pressure on in vitro maturation of oocyte derivedfrom in vitro cultured preantral follicles.

Materials and Methods: Preantral follicles were isolated from 12-day-oldfemale NMRI mice, each follicle cultured individually in microdrops of MEM-α culture medium under oil during 12 days. On day 12, follicles withdiameter nearly 500 μm and good quality was induced using 7.5 mIU/mlhuman chorionic gonadotropin (HCG) for in vitro maturation. Then folliclesdivided in control and experiment groups. In experiment group follicles weresubjected to 20 mmHg hydrostatic pressure for 30 min and then folliclesfrom two groups were cultured for 24-48 h.

Results: After 24 h percent of metaphase II (MII) oocyte were increased inhydrostatic pressure treated follicles(16%) compared to control (9%);(p<0.05). After 48 h percent of metaphase II (MII) oocyte were increased inhydrostatic pressure treated follicles (33.3%) compared to control (20.2%);(p<0.05).

Conclusion: Hydrostatic pressure may play a critical role in oocytematuration. It can be a useful tool for the improvement of oocyte in vitromaturation.

P.019 – COMPARATIVELY EVALUATION OF INFERTILE (RIF) AND FERTILEWOMEN ENDOMETRIAL BIOPSIES AT THE ULTRASTRUCTURAL LEVELBY TEM

L. Bahar1, Z.N. Candan2, S. Kahraman2, T. Baykal1. 1Mersin University,Medicine Faculty, Histology and Embryology Department, Mersin; 2IstanbulMemorial Hospital, ART & Reproductive Genetics Center, Istanbul; Turkey.

Introduction: Implantation of embryo in the uterus wall, which involvescomplex series of interactions and events, is the first requirement forembryo to develop beyond the blastocyst stage. Failure in this regard is stillwidely considered as an obstacle staying ahead of the improvement of thesuccess in assisted reproduction (IVF) and could be mostly attributed to thepoor endometrial receptivity. During menstrual cycle, endometrium, inrelation to the establishment of endometrial receptivity, undergoes severalmorphological changes at the physiological, structural, biochemical andmolecular levels to prepare supportive environment for blastocystacceptance and implantation. Understanding of these changes wouldfacilitate to discover the still unknown biological mechanisms ofimplantation. Since problems in pregnancy are frequently associated withpre and peri-implantation period, considerable advances following theextensive research will contribute to the enhancement in both implantationand pregnancy rates. In this study, we aimed to comparatively investigatethe endometrium tissues of infertile patients with at least 3 previous IVFfailure and fertile patients on the implantation window by transmissionelectron microscope (TEM) in order to evaluate the differences at the cellularlevel.

Methods: Two groups were defined. In a study group, 21 infertile women,having history of repeated implantation failure (RIF) in ≥ 3 IVF trails, wereinvolved. In a control group, 9 fertile women, who applied to clinic forgynecologic problems, were recruited after having informed consents.Endometrial sampling of each patient in both groups was performed on the19-21 day of the menstrual cycle, which was detected by ultrasonographicexamination of existence of corpus luteum and measurement ofprogesterone level. Subsequently, endometrial samples were prepared forTEM analysis and ultra thin sections of randomly selected 3 patients fromeach group were comparatively analyzed.

Results: By TEM analysis, all fertile group endometrial biopsies showeduniformly distributed higher number of pinopodes presenting epithelial cells.Moreover, less number of ciliated cells among the pinopodes was observedas well as cell displaying microvilli at the apical surface were well organizedwith well defined cellular junctions. On the other hand, comparison betweenendometrial biopsies of infertile group and fertile group demonstratedmarked differences, which could affect the functionality of endometrium andimplantation. Less number of pinopodes and rather unmature pinopodeswere obvious in RIF cases endometrial biopsies. Additionally, more ciliatedcells, which were unexpected in the mid luteal phase were still present ininfertile group endometrium and apical surface was less organized withloosen interactions between cell displayed microvilli as well as less numberof stromal cells with inadequate extracellular cell matrix were observed.Decidualisation of stromal cells was not frequent and epithelial cells, makingup endocrine gland were less in number, inadequately vacuolated andcontained prominently heterochromatin nucleus.

Discussion: Recurrent implantation failure in IVF is still one of the majorproblems remains to be unsolved. In general, underlying causes for RIF areattributed to the problems related with embryos, the endometrial factors orimmunity. However, endometrial based problems during the implantationwindow are widely proposed as a main reason for RIF. Despite themysterious complex interactions between embryo and endometrium duringimplantation, comparison of the ultrastructural features of endometrium atthe implantation window between infertile and fertile patients can deducenew explanations for RIF. TEM analysis of endometrial samples from RIFgroup reveals that dramatic changes at the ultrasructural level could beunderlying cause for infertility of these patients. As our best knowledge, thisstudy was the first comparatively evaluating endometrium of RIF and fertilegroup by TEM.

Geneva79


P.020 – EMBRYO QUALITY IN POLYCYSTIC OVARY SYNDROME

M. Balawi1,2, M. D. Cuquerilla2. 1Department of obstetrics and Gynecology.General Hospital; 2Clinica Rubal, Gynecology and Reproduction; CiuadadReal, Spain.

Introduction: polycystic ovary syndrome (PCOS) is the most common causeof anovulatory infertility in women. Folliculogenesis in the PCOS ovary isoften disrupted, leading to suboptimal oocyte competence for fertilization.This alteration in oocyte development is likely due to intrinsic moleculardefects in oocyte along with the state of androgen excess in PCOS patients.Many therapeutic options are available to infertile couples with PCOS,including controlled ovarian hyperstimulation and in vitro fertilization. Theaim of this study is to compare the embryo quality in women with PCOS andwomen with normal ovaries undergoing in vitro fertilization (IVF/ICSI).

Material and Methods: is a retrospective study conducted in Clinica Rubalduring the period between 1st September 2007 and 30th December 2008. Thenumber of embryos included in the study was 497 embryos, 249 comingfrom 31 cycles in women with PCOS, and 248 coming from 50 cycles inwomen with normal ovaries. To define patients with PCOS we used thecriteria of the consensus of Rotterdam 2003. We also used the criteria ofASEBIR (Spanish Association for the Biological study in Reproduction) toclassify the embryo quality.

Results: The average age of women was 32.09 years for the PCOS groupand 33.8 years for the control group. Both groups were comparable. Thenumber of embryos, very good or good (A + B) in the PCOS group washigher, 177 (71%) compared with the control group 156 (62.9%) with astatistically significant P 0.02. However, the pregnancy rate was comparablein both groups, 52% in the PCOS group vs. 51% in the control group, withno significant P.

Conclusions: embryos coming from polycystic ovaries are not of inferiorquality compared with those from normal ovaries, and the pregnancy rate isthe same in women with or without polycystic ovaries undergoing IVF/ICSI

P.021 – EXPRESSION OF NEUROMEDIATORS/EFFECTORS IN CYTOPLASMICMATURATION OF HUMAN OOCYTES

Elisabetta Tosti, Yves Menezo, Moncef Benkhalifa. UNILABS, Laboratoired'Eylau, Paris, France.

Introduction: In the last years more and more attention has been paid to theeffectors of oocyte maturation usually rather described as involved in thedevelopment of nervous system. Neuregulins have been shown to havediverse functions in the development of the nervous system. Brain-derivedneurotrophic factor also known as BDNF is member of the "neurotrophin"family of Nerve growth factors, found in the brain. For the neurotransmitterswe have looked at the mainly at GABA and serotonin, due to theirinvolvement in early embryonic development in invertebrates.

Material and Methods: According to the bioethical law, all the experimentswere performed on GV oocytes retrieved for ICSI, after conrolled ovarianstimulation. There is no transcription during the final stages of oocytematuration and the first embryonic divisions are under maternal controlbefore maternal to zygotic transition. So analysis of the oocyte gives aninteresting pictures of early embryonic cleavages.The protocol used for thehuman oocytes has been already published. The analyses were performed,on six pools of 10 oocytes, with microarrays, using the Affymetrix HG U-133plus 2.0 chips. Amplification was performed via double IVT (in vitrotranscription, two cycles).

Results: Growth factors First of all, the neuregulins (NRG) 1 and 4 areexpressed, but the intensity is 10 times higher for NRG1. NGFI-A bindingprotein 1 (EGR1 binding protein 1 and nerve growth factor receptor(TNFRSF16) associated protein 1 are expressed in all the samples.

Neurotransmitters: Serotonin 5-hydroxytryptamine (serotonin) receptor 7(adenylate cyclase-coupled) and 5-hydroxytryptamine (serotonin) receptor3, involved in depolarization of the membrane, are expressed.

GABA GABA(A) receptor-associated protein-like 1, 2, and 3 are expressed.The solute carrier family 6 (neurotransmitter transporter, GABA), member11 and the diazepam binding inhibitor (GABA receptor modulator, acyl-Coenzyme A binding protein) are very significantly expressed . GABAAreceptors are ligand-gated ion channels We did not find any expression of

the receptor for melatonin, however the N-Acetylserotonin O-methyltransferase catalyzing the final reaction in melatonin biosynthesis isactively expressed.

Conclusion: Modulators and growth factors generally described aseffecteurs of nervous system are present in the oocyte. Some of them couldbe considered like some members of the EGF network, as oocyte secretedfactors involved in cytoplasmic maturation. Receptors of neuromediatorsare expressed: they are involved either in ion channel physiology and/or inactivation of cAMP dependant pathways. Based on their effect on earlyembryo development, in invertebrate, the implications for human assistedreproductive technology should not be overlooked.

P.023 – EFFECT OF SERUM PROTEIN SUBSTITUTE (SPS) ON THE MATURATIONOF HUMAN GV- OR MI-OOCYTES IN VITRO

Hwang-Yun Cho1, Jung-Lim Choi1, Seok-Yoon Lee1, Yong-Soo Heo1, Won-Don Lee1, San-Huyn Yoon2, and Jin-Ho Lim2. 1Maria Fertility Hospital,2Fertility Research Center, Maria Medical Foundation, Seoul, Korea.

Introduction In vitro maturation (IVM) of immature oocytes for infertilepatients is an attractive treatment, because it can reduce side effects ofovarian stimulation with gonadotropins. However, there has been littleinformation about the suitable conditions for human IVM.

Human follicular fluid (HFF) has been used in human IVF program as aprotein source, but it is an undefined supplement. Because HFF has a risk ofcontamination and batch-to-batch variation, it should be replaced bysynthetic serum substitute. This study was carried out to investigatewhether serum protein substitute (SPS) is used as protein supplement forhuman oocyte maturation.

Materials and Methods Immature oocytes were obtained from 60 patientsunderwent controlled ovarian hyperstimulation (from February to August2008). A total of 280 immature oocytes, 183 of germinal vesicle (GV) and 97of metaphase I (MI), were cultured in IVM medium (YS medium containing7 IU/ml rFSH, 1 IU/ml rLH, and 10 ng/ml EGF) supplemented with either40% (v/v) HFF or 40% (v/v) SPS (Sage/Cooper Surgical co.). Maturationrates were compared between the two groups after 24 hrs.

Results The maturation rate of SPS group (64.1%; 59/92) from GV oocyteswas similar to HFF group (59.3%; 54/91). No difference was also thematuration rate of MI oocytes between HFF (84.3%; 43/51) and SPS(89.1%; 41/46).

Conclusion These results suggest that SPS should be use as a proteinsupplement for human oocyte maturation like HFF.

P.024 – DEVELOPMENTAL POTENTIAL ON DAY 3 FAST-CLEAVING EMBRYOSAND PREGNANCY OUTCOME OF FAST-CLEAVING EMBRYOSTRANSFER CYCLES

Hye Won Choi1, Hee Jung Kang1, Mi Ra Shin1, Myo Kyung Kim1, Sun-HeeLee1, Mi Kyoung Koong2, In Ok Song2, Chun Kyu Lim1; 1Laboratory ofReproductive Biology and Infertility, 2Department of Obstetrics andGynecology, Cheil General Hospital & Women’s Healthcare Center,Kwandong University College of Medicine, Seoul, Korea.

Introduction: It has been considered that 7~9 blastomeres are the optimalcell number of embryo for normal development on day 3. An association hasbeen demonstrated between day 3 fast-cleaving embryos (≥ 10 cells) andblastocyst formation rate. We set out to determine the pregnancy potentialof fast-cleaving embryos transferred on day 3.

Materials & Methods: Day 3 embryos were divided into three groupsdepending on their cell number: slow-cleaving embryos (4 ~ 6 cells),normal-cleaving embryos (7 ~ 9 cells) and fast-cleaving embryos (≥ 10cells). The blastocyst formation rate of each group was assessed in cyclesthat the embryos were transferred on day 5 after oocytes retrieval. Thepregnancy outcome of each group was evaluated in cycles that embryoswere transferred on day 3 after oocytes retrieval. Only embryos of same kindwere transferred in each cycle and the pregnancy outcome was evaluated.

Results: Blastocyst formation rate of day 3 fast-cleaving embryos (30.0%)was significantly higher (P<0.01) than that of the slow-cleaving embryos(16.9%). However, there was no significant difference between fast-cleaving

April 19-22, 2009 80


embryos and normal-cleaving embryos (34.2%). A total of 50 embryos(2.2±1.0) were transferred in 23 patients on day 3 and all of them were fast-cleaving embryos. The clinical pregnancy rate (12/23, 52.2%) andimplantation rate (19/50, 38%) were significantly higher (P<0.05) than them(13.8% and 4.3%, respectively) of cycles that slow-cleaving embryos weretransferred. And the clinical pregnancy rate and implantation rate werehigher than them (43.5% and 27.9%, respectively) of cycles that normal-cleaving embryos were transferred. However, there was no significantdifference. Four patients have delivered six healthy babies and sixpregnancies are ongoing. Two pregnancies have been aborted during firsttrimester.

Conclusions: According to our data, the developmental potential of day 3fast-cleaving embryos is similar to normal-cleaving embryos. The day 3fast-cleaving embryo transfer cycles showed similar clinical pregnancy rateand implantation rate to them of cycles that normal-cleaving embryos weretransferred. Our findings demonstrate that fast-cleaving embryos on day 3have a comparable potential to normal-cleaving embryos in achievingpregnancies.

P.025 – LUTEAL ESTRADIOL/GNRH ANTAGONIST SUPPRESSION VERSUS ORALCONTRACEPTIVE PRETREATMENT IN YOUNG POOR RESPONDERS

Nur Dokuzeylul, MeteGurol Ugur, Guvenc Karlikaya, Hale Karagozoglu, AynurErsahin, Mustafa Acet, Mehmet Karaca, Semra Kahraman. IstanbulMemorial Hospital IVF and Reproductive Genetics Unit, Istanbul, Turkey.

Introduction: The purpose of this study is to compare cycle outcomes foryoung poor responders(pr) treated with luteal estradiol patch (lep)suppression versus an oral contraceptive (oc) pretreatment.

Materials and Methods: We reviewed the medical records of all cyclesperformed in Istanbul Memorial Hospital IVF Unit from January 1, 2007 to,November 30, 2008. In a review of our clinical data, we found 37 patientswho were treated with lep and 79 patients who were treated with oc.Patients were included if they were considered pr, as defined by one of thefollowing criteria (1)≤4 oocytes retrieved in previous stimulation;(2) basalFSH levels >10 mIU/mL (3)low estradiol(E2) level on the day of hCGadministration (<800pg/mL) in previous stimulation. For lep protocols,patches containing 7.8mg estradiol/25cm2 was started on luteal day 21 andit was replaced a new one two days later. Patients then remained on the lastE2 patch until the patch fell off or until day of hCG administration. On the 2ndday of the transdermal E2 patch, the patients started taking antagonist dailyganirelix acetate 0.25 mg subcutaneously for 3 days. For oc protocol ,the pillcontaining gestodene 0.075mg and ethinyl estradiol 0.003 mg was startedat 3. day of previous cycle and continued for 21 days. Ultrasound andestradiol measurements were used to monitor follicular development.Oocytes were harvested by transvaginal ultrasound guided follicularpuncture 35-36 hours after 10000IU hCG administration. Results arepresented as mean±standard error. P <0.05 was considered statisticallysignificant.

Results: Both groups were comparable with regards to mean age(30.84±0.48 versus 30.99±0.33years; P= 0.82), mean duration of infertility(7.34±0.73 vs. 7.37±0.51 years; P=0.34), mean number of previous trials(2.30±0.20 vs. 2.14±0.11 P=0.37 ), mean body mass index (24.82±0.54 vs.24.79±0.47 kg/m²; P=0.47) and mean value of day 3 FSH levels(10.70±0.56vs. 10.37±0.40 IU; P=0.64) respectively. Although there is no significantdifference between two groups, the mean total amount of gonadotropinsrequired for COH in lep group is higher than the oc group (4428.36±234.16versus 4009.34±168.30 IU; P=0.27). There was also no remarkabledifference between the mean duration of stimulation period in each group(9.32±0.28 versus 9.37±0.28; P=0.85). There were also no significantdifferences in the mean thickness of endometrium (10.98±0.41 versus10.68±0.18 mm; P=0.85) and the mean serum estradiol (E2) levels, on theday of trigger of oocyte maturation between two groups. (1173.29±101.47versus 1230.63±65.036 pg/ml; P=0.11). There were also no significantdifferences in the mean number of retrieved oocytes (5.30±0.43 versus5.63±0.28; P=0.61), M ΙΙ oocytes (4.63 ± 0.38 versus 4.67±0.24; P=0.94)the mean number of PN (3.91±0.33 versus 4.03±0.21; P=0.62). and themean number of transferred embryos (2.32±0.14 versus 2.28±0.08;P=0.62). Although the implantation rates and clinical pregnancy rates werehigher in the oc group,there were no significant difference in implantationrates (22.2% versus 25.4%; P=0.11) and clinical pregnancy rates between

two groups (35.1% versus 41.7%; P=0.11). The cancellation rates werealso similar between two groups. (8.1% versus 6.3%; P=0.51).

Conclusions: Young poor responders in the oc group showed a trend towardimproved implantation and clinical pregnancy rates which did not achievestatistical significance. Today both protocols accepted as viable options forpoor responder patiens ≤37years.

P.026 – OUTCOME OF MILD STIMULATION CYCLES IN POOR RESPONDERSWITH SHORT-TERM APPLICATION OF GNRH ANTAGONIST AND LOWDOSE GONADOTROPINS USING CLOMIPHENE CITRATE

Nur Dokuzeylul, MeteGurol U ur, Guvenc,Karlıkaya, Hale Karagozoglu, AynurEr ahin, M. Kavrut, Mustafa,Acet, Semra Kahraman; Istanbul MemorialHospital IVF and Genetics Center, Istanbul, Turkey.

Introduction: The objective of this study is to evaluate the efficacy ofminimal stimulation IVF with GnRH antagonist and rFSH or/and HMG incycle using clomiphene citrate in young (37< years old ) and 38-42 yearsold poor responders.

Materials and Methods: We reviewed the medical records of all cyclesperformed in Istanbul Memorial Hospital ART and Genetics Departmentfrom January 1, 2006 to, December 30, 2008. In a review of our clinicaldata, we found 146 patients who were treated with clomiphene citrate (cc)and gonadotropins in young poor responders(Group A) and 150 patients in38-42 years old poor responders (Group B).Patients were included if theywere considered poor responders,as defined by one or more of thefollowing criteria (1)≤4 oocytes retrieved in previous stimulation;(2) basalFSH levels >10 mIU/mL (3)low estradiol(E2) level on the day of hCGadministration (<800pg/mL) in previous stimulation. For both groups ccwas started on cycle day 3 and it has been continued for 5 days.On the 5thday of cc, the patients started taking gonadotropins and daily ganirelixacetate was started 0.25 mg subcutaneously when follicles reached 13-14mm in diameter. Ultrasound and estradiol measurements were used tomonitor follicular development. Oocytes were harvested 35-36 hours after10000IU hCG administration. Clinical pregnancy was defined as thepresence of a gestational sac on ultrasound.The primary end point was thepregnancy rate. Data are compared by using student-t test and chi-squaretest.Results are presented as mean±standard error. P <0.05 was consideredstatistically significant.

Results: The mean age (33.08±3.30 vs. 40.10±1,36 years; P=0.0001) andthe mean duration of infertility (8.00±4.85 vs. 11.13±7.10 years; P=0.0001)were significantly different between two groups. Both groups werecomparable with regards to mean number of previous trials (3.07±2.04 vs.3.20±2.19 P=0.591), mean body mass index (24.78±4.64 vs. 26.32±4.44kg/m²; P=0.91) and mean value of day 3 FSH levels(12.16±6.05 vs.12.39±10.51 IU; P=0.213) respectively. Although there is no significantdifference between two groups, the mean total amount of gonadotropinsrequired for controlled ovarian hyperstimulation in group B is higher thanthe group A (1434±999.83 vs 1426±1072.36 IU; P=0.37). There was also noremarkable difference between the mean duration of stimulation period ineach group (4.47±2.32 vs 4.56±2.31; P=0.79). There were also nosignificant differences in the mean thickness of endometrium (8.21±2.13 vs8.27±2.31 mm; P=0.313) and the mean serum estradiol (E2) levels, on theday of trigger of oocyte maturation between two groups (836±453.02 versus731±585.88 pg/ml; P=0.696). There were also no significant differences inthe mean number of retrieved oocytes (3.47±2.43 vs 2.79±2.60; P=0.622),metaphase ΙΙ oocytes (2.76 ± 1.80 vs 2.38±1.97; P=0.32), the mean numberof fertilized oocytes(2.33±1.53 vs 2.00±1.32; P=0.08). and the meannumber of transferred embryos (1.91±0.94 vs 1.73±0.83; P=0.377). Wealso found that there was no significant difference in the cancellation rates(21.4% vs 24.6%) and in the implantation rates between two groups (13.5%vs 7.6%; P=0.095).The clinical pregnancy rates per ET(21.7% vs 10.6%;P=0.03) and ongoing pregnancy per ET(16.5% versus 6.1%; P=0.02) aresignificantly different between two groups.

Conclusions: Young poor responders showed a trend toward improvedclinical and ongoing pregnancy rates by cc protocol.Thus it can be acceptedas a viable option for young poor responder patients.

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P.027 – SOLUBLE FORMS OF FAS AND FAS LIGAND CONCENTRATIONS IN THESEMINAL PLASMA OF SUBFERTILE MEN WITH OR WITHOUTVARICOCELE

Yehia El-Garem1, Mohamed El-Sawi2, Adel El-Shafei1. 1 Department ofDermatology, Andrology & STD; 2Department of Clinical Pathology; Facultyof Medicine, Alexandria University, Alexandria, Egypt.

Introduction: The presence of a varicocele was often cited as the mostcommon cause of reduced male fertility and decrease sperm quality.

The exact association between varicocele and male infertility is not wellestablished. It is suggested, however, that varicocele is related to reducesemen quality, decline in spermatogenesis.

An altered apoptotic process has been found to be closely associated withmale infertility. The Fas system has been implicated as a key regulator ofgerm cell apoptosis in the mammalian testis.

Up to 10% of sperm cells in the ejaculate of men with a varicocele wereapoptotic, as compared with 0.1% in fertile controls. That report concludedthat varicocele induces apoptosis, which is initiated in the testicular tissueand is then expressed in the semen.

Soluble Fas (SFas) was found to inhibit Fas mediated apoptosis. The activityof sFasL in inducing apoptosis has been extensively debated in the literature.There is evidence both in favor of and opposing its apoptosis inducingactivity. Recent reports suggest that human sFasL inhibits FasL-mediatedapoptosis, indicating that the shedding of FasL from the membrane to formSFasL is a mechanism for down-regulating of its killing activity.

Objective: The aim was to measure the soluble forms of Fas and Fas ligandconcentrations in seminal plasma of subfertile men with or withoutvaricocele, to study the effect of varicocele on Fas pathway andspermatogenesis and correlate levels of sFas and sFasL with semenparameters.

Patients: Twenty subfertile male with varicocele (group A) and twentysubfertile male without varicocele (group B) and ten fertile controls (groupC).

Methods: All patients were subjected to semen analysis, hormonal assay(FSH, testosterone) with chemiluminescence assay, scrotal duplex anddetection of seminal plasma levels of sFas and sFasL by double antibodyenzyme linked immunoassay.

Results: The levels of sFas ranged between 30-110 pg/ml in group A,between 90-202 pg/ml in group B, and between120-130 pg/ml in group C.The levels of seminal plasma sFasL ranged between 0.8-2.6 pg/ml in groupA, between 0.9-1-6 pg/ml in group B and between 1.0-2.6 pg/ml in group C.Significant correlation between sFas levels and sperm count in the group A(r=0.33, p=0.002)., There was positive significant correlation between sFaslevels and percent of rapid and slowly progressive sperms motility in groupA (p=0.03) and group B(p=0.01),while there was Positive significantcorrelation between sFas levels and motile sperms in group A (r=0.44,p=0.007) while there was insignificant correlation between sFas levels andpercentage of motile sperms in the other 2 groups (p>0.05). There wasnegative significant correlation between sFasL levels and percent of motilesperms in group A (r=-0.3, p=0.05), group B (r=-0.3, p=0.03), group c (r=-0.4, p=0.05). No correlation between sFas, Fasl and sperm morphology andwith hormonal profile.

Conclusion: Apoptosis play an important role in pathophysiology of impairedspermatogenesis in subfertile patients with varicocele. SFas concentration inthe seminal plasma is a better marker for apoptotic changes than the sFasL.The sFasL concentration in the seminal plasma implies the fine regulation ofSFasL in the function of the Fas system and consequently, of the apoptoticprocess in the human genital tract.

P.028 – EFFECT OF CAFFEINE ON HUMAN SPERM MOTILITY IN VITRO

Omar Elsraite, Mohamed Danfour, Bashir Awin, Mohamed Awin, YousefAwin, Ahmed Fakron, Mohamed Elmahaishi. Faculty of Medicine, 7thOctober University - Misurata IVF Centre, Misurata, Misurata, Libya.

The effect of caffeine on the sperm motility was investigated after Incubation30min In In Vitro Fertilization Medium.

Materials and Methods: Normal Semen sample were collected byejaculation, according to criteria of the World Health Organization (WHO), aswell as spermatic morphology according to the strict Kruger criterion. Swimup technique was carried out to obtain 20 X 106 progressive motile spermswith a good morphology. The motility speed of the preincubatedspermatozoa introduced into the fertilization medium containing 0 mM or 5mM and 10 mM caffeine were examined in capillary tube (25cm) ascm/30minutes.

Results: The results demonstrated that addition of caffeine withconcentration 5mM to the fertilization medium show significant increase(P<0.05) in sperm speed motility from 7cm/30min to 11cm/30min. Howeverhigher concentration of caffeine (10mM) show no significant effect.

Conclusion: Caffeine stimulates human sperm hyperactivated motility. Theseresults demonstrated that Caffeine enhanced several motion spermparameters and suggest a potential use of the Caffeine in infertile patientswith motility defects undergoing artificial insemination.

P.029 – COMPARISON OF TEST-YOLK BUFFER FREE SPERMCRYOPRESERVATION MEDIA TO A TEST-YOLK BASED SPERMFREEZING MEDIUM

Samira Es-slami1, Susan Tarshala2, Richard Rawlins2, Rebecca Gilbert1.1R&D Department, Irvine Scientific, Santa Ana, CA; 2Rush Center forAdvanced Reproductive Care, Chicago, IL; USA.

Introduction: Sperm Maintenance Medium (SMM, Irvine Scientific) is an eggyolk free sperm cryopreservation medium containing glycerol and sucroseas cryoprotectants and Human Serum Albumin (HSA). CryoSpermTM

(Medicult) is a protein free sperm cryopreservation medium containingglycerol and raffinose as cryoprotectants. This study was conducted tocompare the performance of these two Test-yolk buffer free media to testyolk buffer (TYB) containing medium: Sperm Freezing Medium-TYB (SFM-TYB, Irvine Scientific).

Material and Methods: Semen samples were obtained from 25 patients. Allsamples had normal concentrations and motility according to WHOreference values (≥20 X 106 spermatozoa/ml and ≥50% Motilespermatozoa). Routine semen analysis was performed under lightmicroscopy to determine the motility and forward progressive motility (FP)of the samples. The semen from each patient was divided into equalaliquots and cryopreserved in each of the 3 sperm cryopreservation mediaaccording to the manufacturer’s instructions. After thawing, the % motilityand % FP were determined for each sample, the % recovery post thaw wasdetermined by comparing the pre-freeze and post thaw values. After removalof the cryoprotectants using a separation gradient, % motility and % FP postwash were determined. All assessments were made by the same observer.Comparisons between groups were performed by using One Way ANOVAfollowed by Student t test. A P value of <0.05 was considered statisticallysignificant. The results are presented as mean ± SEM.

Results: There was no significant difference in post thaw motility recoveryand FP recovery between samples cryopreserved in SMM and SFM-TYB (%motility recovery: 77±2.7 vs. 79±2.7; % FP recovery: 81±2.2 vs. 83±2.2).Motility (%) and FP (%) post wash were similar as well (% motility: 87±1.1vs. 87±1.1; % FP: 86±1.3 vs. 87±1.3). Samples cryopreserved inCryoSperm had significantly lower % motility recovery (62±3.6) whencompared to SMM and SFM-TYB. Percent FP recovery post thaw was lower(75±2.5) than other media but did not reach significance. Post wash %motility (78±3.2) and % FP (81±2.2) were both significantly lower whencompared to SFM-TYB and SMM.

Conclusions: TYB based media have long been considered a first choicecryopreservation media for human sperm. In this study we showed that theperformance of SMM, containing both glycerol and HSA without TYB, wasequivalent to TYB based medium SFM-TYB. The performance CryoSpermwas lower than SFM-TYB and SMM’s. The absence of Albumin inCryoSperm could explain its lower cryoprotective ability.

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P.030 – USE OF HUMAN-DERIVED FSH VERSUS RECOMBINANT FSH RESULTSIN MORE EMBRYOS SUITABLE FOR CRYOPRESERVATION

P. Fancsovits, GZs. Tóthné, Á. Murber, E. Hauzman, Z. Papp, J. Rigó Jr., J.Urbancsek. First Department of Obstetrics and Gynaecology, SemmelweisUniversity, Faculty of Medicine, Budapest, Hungary.

Introduction: The outcomes of IVF may change according to the type ofhormonal stimulation. However, there are very few studies analysing theeffects of different gonadotrophin preparations on oocyte and embryoquality. Our prospective randomised study analyses the effect of highlypurified human-derived FSH (hFSH) and recombinant FSH (r-FSH) on oocyteand embryo quality in ICSI cycles.

Materials and Methods: Seventy IVF cycles were randomly allocated tohuman-derived gonadotrophin (Fostimon-HP®; IBSA, Lugano, Switzerland)or recombinant gonadotrophin (Gonal-F®; Serono, Rome, Italy) for ovarianstimulation. Women with the following criteria were included in the study:infertility due to severe andrological factor, maternal age between 18 and 40years, BMI 19–30 kg/m2, <3 previous completed cycles and basal FSH<10IU/l within 3 months prior the study. Oocytes were fertilised by ICSItreatment. Oocyte morphology, fertilisation rate and embryo quality werecompared between the two groups. Assessment of oocyte morphologyincluded the most frequent oocyte dismorphisms, i.e., first polar bodyabnormalities (fragmented, enlarged or immature), enlarged perivitellinespace, considerable cytoplasmic vacuolisation or granularisation. Embryoswere graded according to their morphology observed at 42–48 hours and at66–72 hours after ICSI. Morphological grading was based on the numberand size of blastomeres and the amount of fragmentation. In one r-FSHcycle, where oocyte collection was performed, the male partner did notdeliver a semen sample for fertilization and the oocytes of this cycle werenot injected. Thus, this cycle was excluded from further analysis of oocyteand embryo morphology and pregnancy rate calculations. The Mann-Whitney U-test and the χ2 test were used for statistical analysis.

Results: A total of 389 oocytes in 35 cycles were collected in the h-FSHgroup and 415 oocytes in 35 cycles were collected in the r-FSH group.Patients’ age, length of stimulation and number of gonadotrophin ampoulesused for stimulation were similar in the two groups. The number of oocytes[11.1±3.9 vs. 11.9±4.1] and mature (MII) oocytes [9.9±4.1 vs. 10.6±4.3]were not significantly different between the h-FSH and r-FSH group. Thefrequency of morphologically abnormal oocytes (including abnormal firstpolar body [53.8% vs. 55.5%], enlarged perivitelline space [32.1% vs36.7%], cytoplasmic vacuolisation [7.1% vs. 4.1%] or granularisation[28.0% vs. 26.0%] were similar. The fertilisation rate was significantlyhigher in the h-FSH than in r-FSH group [239/347; 68.9% vs. 217/362;59.9%; P=0.01]. Morphological characteristics of embryos on day 2 and day3 were similar between the two groups. However, significantly moreembryos were cryopreserved in the h-FSH group [91/239, 38.0% vs.62/217, 28.6%; P=0.03]. Positive βhCG-tests [16/35, 45.7% vs. 14/34,41.2%], clinical pregnancy rates [13/35, 37.1% vs. 11/34, 32.4%] andimplantation rates [17/109; 15.6% vs. 20/111; 18.0%] were similar betweenthe two groups.

Conclusions: Stimulation with recombinant gonadotrophin for ICSItreatment seems to result in a similar number of oocytes and similar oocytemorphology as stimulation with highly purified human-derivedgonadotrophin. Oocytes obtained with h-FSH stimulation showed higherfertilisability, whereas embryo morphology, pregnancy and implantationrates did not differ between the groups. However, patients might benefitfrom h-FSH stimulation because of a higher proportion of embryos suitablefor cryopreservation.

P.031 – PSYCHOLOGICAL INFLUENCE OF MALE FACTOR AS THE CAUSE OFTHE COUPLE’S INFERTILITY ON MEN WHO ARE UNDERGOINGASSISTED REPRODUCTIVE TREATMENTS: A PRELIMINARY STUDY INA TURKISH POPULATION

Banu Kumbak Aygun, Irem Atak, Rukset Attar, Gazi Yildirim, NarterYesildaglar, Ates Karateke, Cem Ficicioglu. Department of Obstetrics andGynecology, Yeditepe University Hospital, Istanbul, Turkey.

Objective: To investigate the psychological influence of male factor infertilityas the cause of the infertility problem on men in couples undergoing

assisted reproductive treatments (ART) in Turkey.

Materials and Methods: A prospective study was carried out in a total of 105men, of whom 43 with male factor infertility, 31 with female factor infertilityand 31 with unexplained infertility diagnosis. The men answeredquestionnaires; the State Trait Anxiety Inventory(STAI), State Trait AngerScale(STAS) and the Beck Depression Inventory (BDI) during the treatmentcycle.

Results: No significant differences between groups were found on measuresof anxiety, depression or anger.

Conclusion: In Turkey, male factor infertility as the cause of the couple’sinfertility problem does not have an adverse effect on the psychologicalstatus of men undergoing ART. This study suggests that men’spsychological adjustment to their own infertility diagnosis is not consideredto be psychologically ill.

P.032 – DOES CYTOKINE PROFILE IN SERUM AND FOLLICULAR FLUID DIFFERAFTER OVARIAN STIMULATION WITH GNRH AGONIST LONG ORANTAGONIST PROTOCOL IN WOMEN UNDERGOING ASSISTEDREPRODUCTIVE TREATMENT?

Banu Kumbak1, Oya Akcin1, Rukset Attar1, Gazi Yildirim1, Nihan Tecellioglu1,Serdar Oztezcan2, Cem Ficicioglu1;. 1ART Unit, Department of Obstetric andGynecology, Yeditepe University Hospital, Istanbul, Turkey 2Department ofBiochemistry, Yeditepe University Hospital, Istanbul, Turkey.

Objective: To assess whether follicular fluid (FF) and serum (S) levels ofcytokines in women undergoing assisted reproductive treatments (ART)differ between GnRH agonist long and antagonist cycles.

Materials and methods: Eighty-five women who underwent ART either withGnRH agonist long (n=34) or antagonist multiple-dose flexible protocol(n=51) were studied. FF samples were collected at the time of oocyteretrieval and measured either by ezyme-linked immunosorbent assaytechnique using commercially available kits [interleukin (IL)-1β, IL-6, IL-8,IL-12, tumor necrosis factor (TNF)-α] or Griess method [nitric oxide (NO)].Serum levels of those cytokines and NO were also assessed on the day ofoocyte retrieval.

Results: No significant differences were found in the FF concentrations of IL-1β, IL-6, IL-8, IL-12, TNF-α and NO between the agonist long andantagonist cycles (p>0.05). The serum concentrations of those cytokineswere also similar in the two groups except NO (1.4±1.1 vs. 2.2±1.9 μM;p=0.038) and IL-6 (14.3±4.8 vs. 20.5±12.2 pg/ml; p=0.008) levels whichwere found to be significantly lower in antagonist cycles.

Conclusion: It has been demonstrated that there is no significant differencein follicular microenvironment in terms of IL-1β, IL-6, IL-8, IL-12, TNF-αand NO levels between GnRH agonist long and antagonist protocols.However, serum IL-6 and NO levels which were found to be significantlylower in women from antagonist group might be of clinical value whichshould be evaluated with future investigations.

P.033 – MEIOTIC AND REDOX CONSEQUENCES OF GLYCOLYSIS INHIBITION INBOVINE CUMULUS-OOCYTE COMPLEXES DURING IN VITROMATURATION

Cynthia Gutnisky1, Gabriel Dalvit1, Jeremy Thompson2, Pablo Cetica1.1Institute of Research and Technology in Animal Reproduction (INITRA),Area of Biochemistry, School of Veterinary Sciences, University of BuenosAires, Argentina; 2The Robinson Institute, School of Paediatrics andReproductive Health, The University of Adelaide, Australia.

Glucose is necessary for cumulus-oocyte complex in vitro maturation.Glucose catabolism by glycolysis within cumulus cells produces ATP andsubstrates that can be subsequently oxidized by the oocyte. In other somaticcells, the glycolytic pathway is negatively regulated by ATP by inhibition ofphosphofructokinase. Sodium fluoride (NaF) can also inactivate the pathwayby inhibiting the glycolytic enzyme, enolase. The intermediary metabolism ofglucose also produces NADH, which, besides being used for metabolism,also helps regulate the oocyte REDOX potential. This work studied the effectof the inhibition of glycolysis during in vitro maturation on glucose uptakeand lactate production of COCs and the nuclear maturation rate of theoocyte. Because of the results obtained, a further aim was to determine theeffect of inhibition of glycolysis on oocyte REDOX state and rate of meiotic

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maturation. COCs were recovered by aspiration of antral follicles (2-5 mm indiameter) and only oocytes completely surrounded by a compact andmultilayered cumulus oophorus were used. Glucose uptake and lactateproduction from spent media was measured by spectrophotometric assaysfollowing culture of individual COCs in 20 µl drops of maturation media.Maturation was carried out in medium 199 supplemented with 5 % fetalbovine serum, FSH, LH and gentamycin (control) and ATP (1, 10, 20 and 40mM) or NaF (2, 3, 4, 5 mM). After 22 h of maturation, COCs were removedfrom culture to evaluate the rate of meiotic completion to the metaphase IIchromosome configuration. In order to study the redox potential of theoocyte, groups of 50 COCs were cultured in maturation media describedabove (control) supplemented with 10 mM ATP or 3 mM NaF. The redoxlevel of the oocyte and the maturation stage was measured at 4 differenttime points (0 – 9 – 15 – 22 h). Oocytes were stained with the fluorescentprobes, Redox Sensor Red CC-1 and MitoTracker Green and observed bylaser scanning confocal microscopy. The images were analysed usingPhotoshop 9.0 software. To assess the maturation stage, oocytes werestained with 10 µg/ml Hoechst and evaluated under fluorescencemicroscopy. COCs matured in the presence of ATP and NaF showed a dosedependant inhibition in glucose uptake and lactate production (p<0.05), andin their progression to metaphase II (p<0.05). Oocytes showed changes intheir REDOX level throughout maturation (p<0.05). However, these changeswere not observed either with ATP or with NaF (p<0.05), and an inhibition inthe migration of mitochondria was observed in the presence of thesecompounds (p<0,05). High positive correlations between the REDOX leveland mitochondrial activity were observed (r>0.82; p<0.05). Both, ATP andNaF blocked nuclear maturation at the germinal vesicle stage (p<0.05).These results demonstrate that glycolytic pathway activity is essential forbovine oocyte in vitro maturation. The oocyte REDOX levels vary during thematuration process and they are related to mitochondrial activity. Theinhibition of nuclear maturation by ATP and NaF may be linked to changes inthe REDOX potential and the inhibition of the migration of mitochondria.

P.034 – PREGNANCY RATE OF FROZEN-THAWED PRONUCLEUS STAGESEMBRYOS FROM IVF AND ICSI

M.E. Hammadeh, M. Deryal, A.S. Amer, C. Fischer-Hammadeh. Departmentof Obstetrics and Gynaecology, University of Saarland, Homburg/Saar,Germany.

Introduction: The purpose of this study was to determine and compare thesurvival and pregnancy rate of frozen-thawed pronucleate stage embryos ofIVF and ICSI patients.

Material and Methods: 431 women underwent either IVF (n=191) or ICSI(n=240). All women received GnRHa for down regulation and rec FSH(Gonal-F) for controlled ovarian hyperstimulation. After 18-24 hours ofoocyte insemination (IVF) or sperm injection (ICSI) the pronucleate stageembryos were randomly assigned to be cryopreserved or to continuedevelopment for potential embryo transfer (≤3Embryos) according to thefemale age (cut-off point =35 year). Pronucleate stage embryos were frozenin phosphate–buffer saline (PBS) medium containing 1, 2 propanediol(PROH), sucrose and 20% patients’ serum in three steps (0.5 mol PROH,1.0 mol PROH, 1.5 mol PROH plus 0.1 mol sucrose) at room temperaturefor 10 min. each. The freezing process was initiated at 24 hour afterinsemination or oocytes injection. The mini straws were cooled slowly from22 °C until -7°C (2 °C /min.), after holding them at this degree for 10 min.,they were cooled further until -30°C at a rate of -0.3 °C/min. followed bycooling until -170 °C/min. at 20°C /min. They are then plunged into liquidnitrogen for storage. Seeding was done at -7°C. After thawing of pronucleatestage embryos by removing the straw from liquid nitrogen into water bath30 °C. The cryoprotectants were removed in four steps dilution using PBSwith PROH and sucrose. The pronuclear stage oocytes are inspected forsurvival before being incubated for 24 hours. Embryo transfer wasperformed a 24 h culture. Up to three pronucleate embryos were transferredonly if cleavage takes place.

Results: The mean number of obtained oocytes, fertilized, fertilization rate ofIVF patients was (12.9±5.7; [n=2458]; 8.9±4.2, [n=1701], 69.2%). However,1210 pronucleate stage embryos were frozen, 850 were thawed. Besides,558 were survived the freeze-thaw process (65.6%. survival rate). Thissurvived Pronucleate stage embryos were cultured until the first or seconddivision was completed (24 hours), Embryo transfer was performed at 24hours after thawing. 34 women became pregnant (17.8%). In ICSI program

the mean number of obtained oocytes, fertilized, fertilization rate was(12.7±5.3, [n= 3038]); 7.3±2.8; [n=1755]; 57.8%). A total of 1210pronucleate oocytes embryos were frozen and 864 were thawed. However,only 592 were survived (survival rate 68.5%). 22 pregnancies were achieved(9.2%; p=0.068). In both groups The pronucleate stage embryos survivalrate after freeze thawing procedure was 67.1% and the pregnancy rate was13.6%.

Conclusion: Despite the indications and techniques of becoming pronucleusstage embryos were different, the survival and pregnancy rate was similarand added more than 10% pregnancy rate for ART program.

P.035 – URINARY HMG FOLLOWING RECOMBINANT FSH FOR CONTROLLEDOVARIAN HYPERSTIMULATION YIELDS BETTER CLINICAL OUTCOMESTHAN RECOMBINANT FSH ALONE IN GNRH ANTAGONIST IVF /ICSICYCLES

Atsushi Haruki, Tomoko Inoue, Fujio,Migishima, Keijiro Ito, Hirotsugu Oku,Yoshiharu Morimoto. IVF Namba Clinic, Osaka City, Osaka province, Japan.

Introduction: Recombinant FSH has an important role for controlled ovarianhyperstimulation in IVF/ICSI cycles and GnRH antagonists have beenintroduced in many IVF centers. But it has not yet become evident whetherrecombinant FSH alone or urinary hMG for controlled ovarianhyperstimulation is more effective in GnRH antagonist IVF/ICSI cycles. Thisstudy was aimed at comparing the two preparations for controlled ovarianhyperstimulation in patients with a GnRH antagonist protocol.

Material and Methods: In the retrospective study, we analyzed a total of 154patients following a GnRH antagonist IVF/ICSI cycle from June 2006 untilJune 2008. The exclusion criteria were age > 40 years and WHO Group Ianovulatory women. Oral contraceptives were administered to all womenduring the prior period of treatment cycles. The patients were divided intotwo groups by means of gonadotropic preparations for ovarian stimulation.Ovarian stimulation was initiated with recombinant FSH from day 3 of thecycle for both groups. For one group, recombinant FSH was givencontinuously until the day of inducing ovulation (rFSH group). For the othergroup, recombinant FSH was followed by urinary hMG at least from the day5 of cycles until the day of inducing ovulation (hMG group). Cetrorelix in thedose of 0.25mg was started to be administered daily when the leadingfollicle reached 14 mm. When at least two follicles reached a mean diameterof >17mm, 5000IU of HCG was given and oocytes were retrieved 36-38hours after HCG injection. A good-quality embryo was defined by theirmorphologic features and appropriate cleavage rate. A good morphology ofblastocyst was defined as having a well-expanded blastocoele cavity, a well-defined inner cell mass and a single layer of trophectoderm cellssurrounding the cavity. After conception, we followed the patients until 8thweeks of pregnancy. Clinical pregnancy was defined as presence of agestational sac.

Results: In both groups, no significant difference was observed in thebackground factors of the patients as an age, the past number of IVF/ICSItreatment cycles, basal FSH level. There was no significant difference in theserum estradiol and progesterone levels measured on the day of hCGinjection, a number of mature oocytes and the fertilization rate. Asignificantly longer duration of stimulation (9.6 versus 8.4 days) and thus asignificantly increased requirement for gonadotropic dose was present inthe hMG group. A rate of good morphology blastocyst was similar amongthe two groups, but a rate of good-quality embryos in the rFSH group washigher than that in the hMG group (31.5% versus 15.0%; P=0.06). Clinicalpregnancy rate per embryo transfer (20.0% versus 39.4%; P=0.06) and therate of early pregnancy loss (28% versus 8.3%) for the rFSH and hMGgroups respectively differed in favor of the hMG groups.

Conclusions: A quality of the embryos in the rFSH group was higher than inthe hMG group. However, a lower ongoing pregnancy rate was seen in therFSH group, most probably due to a worsened environment of implantationincluding endometrial receptivity.

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P.036 – LEUKOCYTOSPERMIA AND ITS CORRELATION WITH SPERMPARAMETERS IN PATIENTS REFERRED TO KASHAN INFERTILITYCENTER DURING 2007-2008

Hassan Hassani Bafrani1, Razieh Afrugh2, Fatemeh Fruzanfard3. 1Departmentof Anatomy, 2School of Medicine, 3Department of Obestetric & Gynecology,School of Medicine, Kashan University of Medical Sciences, Kashan, Iran.

Introduction: The reactive oxygen species (ROS) derived from abnormalsperm or from WBC or both of them. ROS in semen is responsible for thelipid peroxidation, sperm motility defect, and decreases the spermfertilization ability. The aim of this study was to determine sperm quality(count, motility, morphology) in infertile men with or withoutluckocytospermia and abnormal patterns of ROS production in.

Materials and Methods: We retrospectively reviewed semen analysis of 110infertile men referred to Infertility Center during 2007-2008.

Semen was centrifuged at 1200 ×g for 5 min to separate seminal plasma.The aliquots were stored at −80 °C until analyzed. Seminal plasma and serum were resuspended in phosphate buffer saline forMDA levels in seminal plasma by spectophotometric assays.

Results: After semen analysis in 110 patients, the semen samples were putin luckocytospermia group (n=30), and nonluckocytospermia group (n=35).In nonluckocytospermia leukocyte number was <0.25 × 106

Sperm motility had significantly different between leukocytospermia andnonluckocytospermy patients, and also in healthy men (P<0 .05 and P<0.001 respectively).

ROS level also had significantly difference between leukocytospermia andhealthy men, and between nonluckocytospermy patients with healthy men(P<0 .001 and P< 0.0001 respectively).

Conclusions: With respect to correlation of leukocytospermia with spermmotility, and important role of sperm motility in fertilization, we canconclude that detection and treatment of leukocytospermia, after excludinggenitourinary infection, may have positive influence on fertility andpregnancy rates in infertile couples. In respect of relationship betweensperm motility and leukocytospermia, and beneficial effect of motility,leukocytospermia can affect fertility capability patners. However, applicationof antioxidant in leukocytospermia treatment, but final effect of theantioxidant is still controversial.

P.037 – INVESTIGATING OF SPERM PARAMETERS AND PREGNANCYOUTCOME AFTER INTRAUTERINE INSEMINATION

Hassan Hassani Bafrani1, Maasoomeh Abedzadeh2, Fatemeh Fruzanfard3.1Department of Anatomy, 2School of Medicine, 3Department of Obestetric &Gynecology, School of Medicine, Kashan University of Medical Sciences,Kashan, Iran.

Introduction: Infertility prevalence is about 10-15 percent. IUI is one of thetreatment methods in infertility. The IUI success rate is Influenced bydifferent factors. Different studies indicated that Induction of ovulation andIUI increased the chance of pregnancy in the infertile couple special withspecially male factor infertility. The aim of this study was to investigate thesuccess rate of IUI with related factor in the kashan fertility and sterilitycenter.

Material and Methods: 84 infertile couple that underwent 102 IUI cycles withwashed husband semen were included in this study. All patients’ chartswere reviewed for age, IUI number, semen characteristics and pregnancyrate.

Findings: Total pregnancy rate were 11.8 percent per cycle and 14.3% perinfertile couple. The women age mean in pregnant group was 25.8 ± 26years old and in non pregnant group was 29 ± 5.3 years old. The t-studenttest in both groups were significant. The majority of pregnancies wereAchieved in women less than 35 years old (13.3%). Induction ovulationprotocol in 66/7% women was HMG. All of pregnancies were achieved incouples with first IUI cycle.

Post wash semen parameter including : Sperm count ≥ 10 × 10 6 , motility ≥50%, normal morphology ≤50% were observed in 91.7%, 100% and83.3% of pregnancy rate respectively but difference statistically were notsignificant in two groups.

Conclusion: This study indicated that pregnancy rate per cycle was 11.8%.IUI success rate decreased when the woman age was more than 35 yearsold or sperm count < 10 × 10 6 . 10 million sperm must be InseminatedWhen the normal morphology was less than 50%.

P.038 – OBSTETRIC OUTCOMES OF PREGNANCIES FROM IN-VITROMATURATION OF OOCYTES TREATMENT

H.M. Tuong, V.T.N. Lan, D.Q. Vinh, P.H. Tuan. IVF Van Hanh, Ho Chi Minh,Vietnam.

Introduction: In-vitro maturation (IVM) of oocytes has been developedrecently and has become more popular at IVF centers worldwide. There havebeen few data on the pregnancy and obstetric outcomes of babies born fromthis technique. This study is to report the obstetric outcomes of the firstcohort of babies following IVM treatment in Vietnam.

Material and methods: This was an observational study conducted onwomen conceived and then gave births from IVM treatment due to theindication of PCOS at IVF Van Hanh, Ho Chi Minh City, Vietnam fromSeptember 2007 to September 2008. Pregnant women were examined forweight gain; complications occurred during pregnancy such as pre-eclampsia and diabetes mellitus. Ultrasonography was used to investigatenumber of fetus, nuchal translucency, and 3-D morphology of the fetus. Thebabies born from IVM treatment were recorded with regards to mode ofdelivery, gestation at birth, number of babies, birth weight, gender, apgarscore, and complications at birth such as respiratory distress syndrome,intracranial hemorrhage, and major birth defects.

Results: There were 31 women conceived and gave births, amongst them,there were 20 singleton and 11 twin-pregnancies. The mean female age was29.8 ± 2.8 years. The mean weight gain was 15.0 ± 4.7 kg. No case of pre-eclampsia and diabetes mellitus was recorded. There was no case ofabnormal nuchal translucency and 3-D morphological ultrasonography. Atotal of 42 babies were born from the IVM treatment, in which, there were18 male (42.9%) and 24 female (57.1%). Two cases had vaginalspontaneous delivery, others had cesarean sections. There were 6 pretermbirths; the mean gestation at birth was 37.6 ± 1.9 weeks (30 – 40 weeks).The mean birth weight was 2602 ± 480 g (1700 – 3450 g). Two babies hadapgar score at 1 minute of 6, and 40 babies had score of >/= 7. No severecomplications and major congenital defects were recorded at births.

Conclusion: Pregnancies from IVM treatment has good obstetric outcomes.Data from the first cohort of babies born from IVM program in Vietnam arereassuring for women undergoing IVM treatment.

P.039 – THE IMPACT OF SERUM AND FOLLICULAR FLUID ANTI-MULLERIANHORMONE LEVELS ON IN VITRO FERTILIZATION OUTCOMES INNORMAL AND POLYCYSTIC OVARY SYNDROME WOMEN

S. Arabzadeh1, G. Hossei1, B. Rashidi2, M. Agha Hosseini3. 1School ofBiology, University College of Science, University of Tehran; 2ImamKhomeini Hospital, Vali-e-Asr Reproductive Health Research Center, Schoolof Medicine, Tehran University of Medical Science, 3Shariati UniversityHospital IVF center, School of medicine, Tehran University of MedicalScience; Tehran, Iran.

Introduction: Anti-mulleian hormone (AMH) is a biomarker that predicts thenumber of antral follicles and is involved in follicle arrest for women withpolycystic ovary syndrome (PCOS). This study investigates the relationshipbetween serum or follicular fluid (FF) AMH concentrations and In VitroFertilization (IVF) outcomes in normal and PCOS women.

Material and Methods: Women with PCOS, (20-47) years, (n=33), andnormal ovulatory control, (21-42) years, (n=56), body mass index 21-33kg/m2 in PCOS vs. 19-35 kg/m2 in normal controls were recruited from Vali-e-Asr Reproductive Health Research center and Shariati University HospitalIVF center during this retrospective study. On the cycle day 3, serumsamples and on the day of oocyte retrieval FF from more than onepreovulatory follicle (16-20 mm in diameter) was collected for AMHmeasurements by using Elisa method. Association between AMH levels andoocyte number, oocyte maturation, fertilization rate, implantation rate,embryo grade, biochemical and clinical pregnancy were assessed with one-sample Kolmogorov-smirnov, spearman, Mann Whitney and binary logisticregression statistical tests.

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Results: Median (range) serum AMH level was markedly increased in thePCOS group [15.06 (0.10-50.70) vs. 3.38 (0.42-9.91) in normal controls;P<0.0001]. Similarly, median (range) FF AMH level was higher in PCOSgroup (8.21 (0.39-127.59) vs. 2.13 (0.62-13.69) in normal controls;P<0.05). In both groups, serum AMH levels showed a significant positivecorrelation with oocyte number (r = 0.40; P<0.01 vs. r = 0.49; P<0.01 inPCOS) and oocyte maturation (r=0.46; P<0.0001 vs. r = 0.59; P<0.0001 inPCOS). Embryo grade remained irrespective of AMH concentrations inserum or in FF in both normal control and PCOS groups. There was aninverse relation between fertilization rate and serum AMH levels (r = -0.29;P<0.05); a positive relation between FF AMH levels and implantation (r =0.32, P<0.05) and biochemical pregnancy (P<0.05) rates in normal controlgroup but not in PCOS group. However, no significant correlation wasobserved between serum or FF AMH levels and clinical pregnancy in bothgroups.

Conclusion: Concentration of AMH in serum, but not in FF, may constitute auseful marker of oocyte number and oocyte maturation in PCOS and normalcontrol group. While, AMH levels in FF could be a predictive factor ofimplantation and biochemical pregnancy rates in normal control but not inPCOS women.

P.040 – SERUM ESTRADIOL VALUE AND ESTRADIOL / METAPHASEIIOOCYTERATIO ON THE OUTCOME OF IN VITRO FERTILIZATION WITHCONTROLLED OVARIAN HYPERSTIMULATION

Mamoru Ida1, Fumie Nagata1, Kengo Sugihara1, Yoshiharu Nakaoka1, AisakuFukuda1, Yoshiharu Morimoto2. 1IVF Osaka Clinic; 2IVF Namba Clinic; Osaka,Japan.

Introduction: The aim of the present study was to evaluate the peak estradiol(E2) value on the day of human chorionic gonadotropin(HCG) administrationand the ratio of E2 per number of metaphaseIIoocytes retrieved (E2/MIIratio) during the controlled ovarian hyperstimulation (COH) withgonadotropin-releasing hormon(GnRH)-agonist or GnRH-antagonist, on theoutcome of in vitro fertilization-intracytoplasmic sperm injection (IVF-ICSI)cycles.

Materials and Methods: Retrospective analysis was performed on thepatients who completed IVF-ICSI cycles from January 2006 to July 2008.The elimination of bias in this selection was achieved by excluding eitherpoor responders or high responders: women who achieved E2 levels<500pg/ml or >5000pg/ml on the day of HCG. Other exclusion criteria werefrozen-thawed embryo transfer cycles. Two COH protocols were used: 537patients were treated with the midluteal long GnRH-agonist protocol(agonist group) and 162 patients with flexible multidose GnRH-antagonistprotocol (antagonist group).The selection of types of analog was largelydependent on clinical characteristics of the patients. Each group was dividedinto four subgroups according to their peak E2 value on the day of HCG:≤1000pg/ml; 1001-2000pg/ml; 2001-3000pg/ml; >3000pg/ml.Additionallyeach group was divided into another four subgroups according to theirE2/MII ratio: ≤100pg/ml;101-200pg/ml;201-300pg/ml; >300pg/ml. Numberof MIIoocytes retrieved, number of embryos transferred and pregnancyrates were assessed .

Results: Pregnancy rate per transfer cycles was significantly higher inagonist group (45.4%) than in antagonist group (33.3%)(p,<0.01).Pregnancy rates in E2 value of ≤1000pg/ml, 2001-3000pg/ml, and>3000pg/ml were comparable between agonist and antagonist groups, whilepregnancy rate in E2 value of 1001-2000pg/ml was significantly higher inagonist group than in antagonist group(p<0.05).In both agonist andantagonist groups, there was no difference in pregnancy rates betweendifferent E2 subgroups.Pregnancy rates in E2/ MII ratio of 101-200pg/mland 201-300pg/ml were comparable between agonist and antagonistgroups, while pregnancy rates in E2/MII ratio of ≤100 pg/ml and >300pg/mlwere significantly higher in agonist group (p<0.05 for both).Furthermore,there was no difference in pregnancy rates among the different E2/MII ratiosubgroups in agonist group. However, higher pregnancy rate was observedin E2/MII ratio of 101-200 pg/ml comparing to those in ≤100 pg/ml or>300pg/ml in antagonist group (p<0.05 for both).

Conclusions: The peak E2 value on the day of HCG cannot predict clinicaloutcome either in agonist group or antagonist group. While E2/MII ratiocannot predict clinical outcome in agonist group, only the patientsundergoing GnRH-antagonist protocol with E2/MII ratio of 101-200 pg/ml

achieved successful IVF outcome compared to other E2/MII ratiocategories.Consequently, this retrospective study implies that the decision ofappropriate HCG administration is not so critical in GnRH-agonist protocol,but is very critical in GnRH-antagonist protocol to obtain best IVF-EToutcome.

P.042 – COMPARISON OF CUMULATIVE PREGNANCY RATES IN IVF-ET CYCLESBETWEEN CULTURE OF ALL FERTILIZED EGGS AND SPLIT ZYGOTESAFTER FREEZING AT SIBLING PRONUCLEAR STAGES IN THE PATIENTSHAVING SMALL NUMBER OF ZYGOTES

Myo Kyung Kim1, Hee Jung Kang1, Dong-Wook Park1, Seung Bum Hong1, MiRa Shin1, Inn Soo Kang2, Jin Young Kim2, Chun Kyu Lim1. 1Laboratory ofReproductive Biology and Infertility; 2Department of Obstetrics andGynecology, Cheil General Hospital & Women’s Healthcare Center;Kwandong University College of Medicine, Seoul, Korea.

Introduction: It is well known that cryopreservation of supernumeraryembryos could increase the cumulative pregnancy rates. However, there isno consensus regarding cryopreservation at the PN stages to improvepregnancy outcomes in IVF cycles with a small number of zygotes. Thisstudy was carried out to see whether cryopreservation of split PN zygotescould increase the cumulative pregnancy rates in the patients having eight2PN-zygotes.

Materials and Methods: This retrospective study analyzed IVF or ICSI cyclescarried out between Jan. 2003 to Dec. 2007. We selected 138 embryotransfer (ET) cycles that were estimated eight zygotes (2PN) after 20-22hours insemination/ICSI and less than nine fertilized zygotes (included 1PNor delay developed embryos). Total ET cycles were divided into two groups:group I (n=86); total fertilized embryos were cultured to transfer on day 3without PN stage freezing. Group II (n=52); after some sibling zygotes werefrozen at the PN stages, the others were cultured to day 3 until to transfer.Clinical pregnancy outcomes were compared with between two groups infresh ET cycles and cumulative ongoing pregnancy rates were estimatedafter subsequent frozen-thawed ET.

Results: There were no significant differences in female mean age, numberof retrieved oocytes and total fertilized embryos between two groups.Number of cultured embryos was significantly lower in group II (5.2±0.5)than in group I (8.4 ± 0.7) (P<0.01). Also, number of transferred embryoswas significantly lower in group II (3.3 ± 0.6) compared with group I (3.6 ±0.6) (p<0.01). The number of beta-hCG positives and delivery rates (51.2 vs46.2 % and 47.7 vs 44.2%) after fresh ET were slightly higher in group Ithan in group II. The differences were not statistically significant. However,the cumulative ongoing pregnancy rates after frozen-thawed ET cycles wereslightly higher in group II (51.9%) than in group I (47.7%).

Conclusions: Cryopreservation of sibling zygotes could slightly increasecumulative pregnancy rates in the patients having less than ten fertilizedeggs compared with all zygotes cultured group. This IVF-ET policy couldoffer higher chances to cumulative pregnancies and the patients could havebenefit on cost effectiveness.

P.043 – COMPARISON OF CLINICAL OUTCOMES ACCORDING TO DAYS OFEMBRYO TRANSFER IN ART CYCLES

Sun-Hee Lee1, Hee Jung Kang1, Dong-Wook Park1, Hye Won Choi1, SeungBum Hong1, Hae Ok Kim2, Chan Woo Park2, Chun Kyu Lim1. 1Laboratory ofReproductive Biology and Infertility, 2Department of Obstetrics andGynecology, Cheil General Hospital & Women’s Healthcare Center,Kwandong University College of Medicine, Seoul, Korea.

Introduction: The majority of embryo transfers (ETs) to date have beenperformed on day-3 to reduce potential risks for developmental arrests of invitro cultured embryos before ETs. Development of sequential media hassignificantly improved culture conditions that could allow for blastocysttransfer on day-5. While day-5 ETs provide higher clinical pregnancyoutcomes with reduced risks of multiple pregnancies, they still havepotential risks of developmental arrests of IVF embryos. The aim of thisstudy was to evaluate clinical outcomes of day-4 ETs and compare theefficacy of day-4 ETs with other days of ETs in our center.

Material and Methods: From January 2006 to August 2007, total 1124cycles were analyzed – 897 day-3, 121 day-4, and 106 day-5 ETs. The cycles

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with number of retrieved oocytes < 5, female age >37 and/or any geneticfactors were excluded. The rates of matured oocyte, fertilization, goodembryo, and clinical pregnancy were examined among three groups. Chisquare test and ANOVA were used for statistic analysis. P values < 0.05were considered significant.

Results: There were no significant differences among three groups withrespect to mean age of female and rates of matured oocyte, fertilization andgood embryo. Clinical pregnancy rate of day-4 ETs (55.4%) was significantlyhigher than that of day-3 ETs (41.5%) (p=0.0051). There was no significantdifference in clinical pregnancy rates between day-4 and day-5 ETs.

Conclusions: Day-4 ETs had clinical pregnancy rates as high as day-5 ETsand it is significantly higher than that of day-3 ETs. Day-4 ETs may provide abetter option to achieve a high clinical pregnancy outcome with reducedrisks of multiple pregnancies and developmental arrests of embryos.

P.044 – OUTCOME AFTER HYSTEROSCOPIC CORRECTION OF UTERINEANOMALIES

Kay Jayakrishnan, RamAnupama. KJK Hospital, Trivandrum, Kerala, India.

Introduction: Mullerian anomalies are the frequent congenital malformationof the genital tract. It is associated with various types of reproductive failure,including recurrent miscarriage,late abortion and preterm delivery .

Methods: 123 women with mullerian anomalies were detected during a eightyear period from January 2001 to December 2008 in a prospective cohortstudy. The anomalies were detected by pelvic sonography including 3 Dscan, diagnostic hysteroscopy, hysterosalpingography and MRI scan. Of this52 patients (55% ) presented with primary infertility and 42 patients (44 %) with secondary infertility.In this study we have excluded male factorinfertility, unmarried patients and other causes of infertility. These patientswere subjected to laparohysteroscopy .Complete septum in 59patients(62.7%) and partial septum in 8 (8.5%)cases. 13 patients hadunicornuate uterus with rudimentary horn, 6 patients had arcuate uterus,3had bicornuate uterus,3 had mullerian agenesis and 2 presented withbicornis unicollis. Surgical intervention was in the form of septal resectionin 67 (71.2%) of cases, lateral metroplasty in 6 (6.3%) cases of unicornuateuterus, rudimentary horn excision in 3 (3.1%), right rudimentary horn withhematometra excision in 1case and vaginoplasty in a single case.Conception rate following surgical intervention, effort of cervical encerclageon pregnancy, surgery to conception interval, mode of conception and routeof delivery were followed up in our study group.

Results: 33conceptions (35.1%) occurred in our study group. 90.9% of ourconceptions within 1 year after surgery and rest within 4 years after surgery.17 patients (51.5%) conceived spontaneously, 9 (27.2%) followingcontrolled ovarian stimulation,6 (18.1%) patients following controlledovarian stimulation combined with IUI and 1 case following in vitrofertilization. We have done cervical encerclage in 8 (11.9%) cases of septumresection. All of them delivered at term ( 100 % live birth rate . Theseresults were statistically significant.About 19(57.5%) delivered by caesareansection and 7( 21.2%) by vaginal route. We had a follow up percentage of68.08%.

Conclusions: Endoscopic surgery restores normal uterine cavity, reducesmorbidity and post operative adhesions and thereby improves theconception rates.

P.045 – DO WE HAVE RESERVE TO RAISE EFFECTIVENESS OF IVF?

Leonid Kuzmichev, Elena Kalinina, Veronika Smolnikova, Marina Shakhova,Khatuna Surmava, Liya Kazaryan. Moscow, Russia.

Introduction: In our centre from May 2006 to may 2007 were performed1208 IVF cycles.

Materials and Methods: Among these female factor of infertility wasdiagnosed in 35% of cases, male – in 34% and both factors were present in21% of cycles. One attempt of IVF was performed in 27,4% of patients; twoattempts in - 30,2%; three attempts in - 28,8%; fore and more attempts - in13,3% of all cases. The protocols of ovarian stimulation we have used werethe following: antagonists in combination with human menopausalgonadotropins in 23,1% cases, antagonists in combination withrecombinant follicle stimulating hormone in – 24,7%; agonists incombination with human menopausal gonadotropins – in 25,8% cases;

agonists in combination with recombinant follicle stimulating hormone in –30%.

Results: After prompt analysis the following results were registered:pregnancy rate after IVF was 30,9% and after ICSI 31,8%; delivery rate afterIVF and ICSI were 26,24% and 27,9% accordingly; mean number ofembryos transferred in each cycle were 2,3 and 2,4 after IVF and ICSI (wewould like to mention the fact that after September 2007 maximal number ofembryos transferred were equal to 2). As to deal with embryologic stage wehad transfer of embryo only on day 3 in 21,7% of cases, on day 5 in –39,8%; and on day 3 and 5 in – 38,4%. In all our embryo transferprocedures we used COOK catheters. After embryo transfer procedure forluteal support were used utrogestane and crynone (8%) in 79% and 21% ofcases accordingly. From all attempts first attempt was effective in 64% ofwomen, second – in 17,6%; third – in 9,8%; effectiveness of four and moreattempts was less then 2% per cycle. We also tried to examine effectivenessof attempts in accordance to age of the patients. The data were following: inwomen younger then 27 years pregnancy rate was equal to 36,9%; in groupof 28-35 years – 30,5%; in 36-40 year group – 26,4%; women older then41 years – 15,8%. When analyzing the number of embryos transferred percycle we found out that after transfer of three embryos pregnancy rate was34,7%; delivery rate – 29,5%; number of multiple pregnancies – 15%. Incontrary after transfer of 2 embryos we had the further results: pregnancyrate was 30,9%; delivery rate – 26,2%; number of multiple pregnancies –9%. From all started cycles 317 (26,24%) cases ended with delivery ofbabies. Among all these were diagnosed 27 (8,5%) multiple pregnancies.

Conclusion: Differential approach to IVF programme optimizes and leads toincrease of IVF results.

P.046 – THE EFFECTS OF SPERMPREPARATION ON THE INITIATION OFCAPACITATION OF HUMAN MALE GAMETES

Gudrun Keck, Nadja Gneist, Evelyn,Gouma, Katrin Hanseroth, BirgitLeuchten, Berit Thieme, Ina,Trinkaus, Wolfgang Distler. UniversitatsklinikumDresden IVF-Labor, Dresden, Sachsen, Germany.

Introduction: Glycodelin (GD) is a progesterone-regulated lipocalin proteinof the reproductive axis with diverse actions in cell recognition and celldifferentiation. It is a 28 to 30-kD glycoprotein synthesized in variousglands, notably in the male and in the female reproductive organs.Glycodelin appears in different glycoforms on sperm surface and in seminalplasma (GD-S), in amniotic fluid (GD-A), in follicular fluid (GD-F) and incumulus cells (GD-C).

GD-S bound to the surface of human spermatozoa is known to inhibit thecapacitation of sperms, which is a prerequisite for fertilization. Thus, itneeds to be removed from the surface to enable the capacitation of thesperms. In in-vivo-conception GD-S is naturally removed during the passagethrough the cervical mucus. In contrast; up today there is nothing knownabout the fate of GD-S during in vitro fertilization. Therefore the aim of thisstudy was to investigate whether or not GD-S is removed during the semenpreparation procedure prior in vitro fertilization.

Materials and Methods: Ejaculates were assessed of men attending our IVFprogram. The study was approved by the Ethics Committee of the UniversityHospital Dresden. All patients gave signed informed consent. Only ejaculatesfrom patients with normozoospermia were included in the study (nEJ = 32).Semen preparation was carried out via swim up procedure (SUP). Theamount of bound GD-S on sperm surface was investigated on each 200sperms prior and each 200 sperms after SUP per ejaculate. GD-S wasvisualized by immunocytochemistry staining with a monoclonal antibodyMf8 against the glycodelin protein core. The stainings were imaged and theGD-binding on spermatozoa was semi-quantified by scoring (nSP =12 800).The amounts of bound GD both on sperm surface in ejaculate and in spermsuspensions after SUP were correlated (SPSS 12.0).

Results: With the method established in our workgroup it was possible todetect and to semiquantify bound GD-S in both; the fresh ejaculates, as wellas on the surface of sperms SUP. The results of the scoring showed adecreased amount of bound GD-S after SUP in all investigated ejaculates.The decrease was statistically significant (p< 0.05). The findings indicatethat semen preparation via SUP significantly reduces the amount of boundGD-S on the surface of sperms, enabling capacitation of sperms even invitro.

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Conclusion: With these results we could prove for the very first time that animportant prerequisite for initiation of capacitation of human sperms, theremoval of GD-S, can be achieved invitro by the swimming up semenseparation method.

P.048 – IMMUNOCOMPETENT CELLS OF PLACENTA FROM WOMEN OF HIGHFERTILITY AGE IN CASE OF PATHOLOGICAL PREGNANCY

Natalia Linkova, Artem Kostylev, Irina Kostyuchek, Viktoria Polyakova,Natalia Palchenko. Ott Institute of Obstetrics and Gynecology, Saint-Petersburg, Russian Federation.

Introduction: Women older than 30 years (high fertility age) frequently havedifficulties in pregnancy such as: threat of an abortion, spontaneousabortions, gestosis, uterine inertia, fetal intrauterine hypoxia. Thus,pregnancy and labors in this age are often links with changes in the system“mother- placenta-fetus” and can be the cause of obstetric and perinatalpathology. The pathological processes are accompanied with changes ofimmune status, which, in turn, can be the important indicator of thoseviolations.

The aim was to study various immunocompetent cells in placentas ofwomen older than 30 years. Our task was detection of NК-cells (naturalkillers), Т-killers, Т-supressors, B-lymphocytes and plasma cells in aplacentas of women in chosen age category.

Materials and Methods: 10 placental biopsies from women with a variouspathology (6 had gestosis of light and middle degree and 4 - had otherpregnancy pathology). The average women’s age was 33,7 years (from 30till 38 years). Immune cells were identified by according markers: CD40(plasma cells), СD20 (B-lymphocytes), CD8 (Т-killers and Т-supressors),CD4 (Т- supressors) and CD16 (natural killer cell). Slides with tissuespecimens were photographed at augmentation 400x. Quantitative analysisof immunohistochemical expression was done by using computer imageanalisator «Video-Test, Morphology-5». Relative square (S, %) and intensityof a staining (I, relative units) were estimated.

Results: NK-cells were founded in placenta (SCD16=3.36±1.13 %,ICD16=0.42±0.10). The large value of intensity and low values of square canbe connected with high localization’s degree of NK-cells. Though NК-cellscompound only 10-15 % from a total number of lymphocytes, they realizedsuch important function as destruction of target-cells without direct contact,with the help of perforine protein, and, accordingly, without development ofimmune response which realized by Т- and B-lymphocytes. Small amount ofcells, expressing CD8 (SCD8=1.9±1.6%, ICD8=0.22±0.17) were alsodetected in placental tissue. We suppose that absence of CD4 in placentaindicates on absence of Т-supressors. Positive response at CD8 is cause ofpresence only Т-killers or low differentiated T-lymphocytes.

Conclusion: Pathological pregnancy for women of high fertility age connectswith immune responses by NK-cells and only insignificant part realize by Т-killers.

P.049 – COMPARISON OF FERTILIZATION AND PREGNANCY RATES AFTER ICSIWITH FRESH AND FROZEN-THAWED SURGICALLY RETRIEVED SPERM

Shaw Ni Amy Lee, M.N. Lim, C.F. To, S.L. Yu. Department of Obstetrics &Gynaecology, CARE, Singapore General Hospital, Singapore.

Aim: This retrospective study aimed at comparing the results ofintracytoplasmic sperm injection with fresh and frozen-thawed spermobtained after microsurgical epididymal sperm aspiration (MESA) andtesticular sperm extraction (TESE) in azoospermic patients.

Materials and Methods: A total of 56 fresh cycles of ICSI treatment werecarried out using fresh and frozen-thawed surgically retrieved spermbetween February 1994 to December 2008, 23 using fresh retrieved spermand 33 using frozen-thawed retrieved sperm. ICSI was performed usingthese sperm on the wives’ oocytes after undergoing controlled ovarianhyperstimulation and oocyte recovery with ultrasound guidance. Amaximum of 3 embryos were selected for transfer on either Day 2 or Day 3after oocyte recovery. Retrospectively, the fertilization and pregnancy rateswere compared. Results were analyzed statistically using chi-square test.

Results: No significant difference was observed in the fertilization rate usingfresh and frozen-thawed surgically retrieved sperm (60.3% versus 55.5%);p = 0.270. The pregnancy rate is similar using both fresh and frozen-thawed

surgically retrieved sperm with no significant difference (p = 0.944). Thepregnancy rate is 34.8% using fresh surgically retrieved sperm and 39.4%using frozen-thawed one.

Conclusions: The fertilization and pregnancy rates after ICSI using fresh andfrozen-thawed surgically retrieved sperm were comparable. Thus, it showsthe chance of achieving a clinical pregnancy following ICSI was notcorrelated to the status of the sperm whether it is fresh or frozen.Cryopreservation of surgically retrieved sperm did not alter pregnancyoutcome. Recovery of surgically retrieved sperm for cryopreservation fromthe husband is strongly recommended before ovarian stimulation asinstances have been found of failure to obtain sperm on day of oocytecollection, hence, the oocytes will not be wasted. Also, it is certainly a validoption in these group of patients as ICSI may be performed later or even inanother center using the frozen-thawed surgically retrieved sperm withoutjeopardizing the success rate.

P.050 – MATURITY OF OOCYTE MAY IMPACT OUTCOME OF IN VITROMATURATION DURING CONTROLLED OVARIAN HYPERSTIMULATIONFOR PREVENTING SEVERE OVARIAN HYPERSTIMULATION SYNDROME

Soo Jin Chae1, Chang Young Hur2, Kyung Sil Lim3, Jin Ho Lim4. MariaFertility Hospital, Seoul, Korea.

Introduction: In vitro maturation (IVM) during controlled ovarianhyperstimulation (COH) is effective method for prevention of ovarianhyperstimulation (OHSS). We investigated the factors which may influenceclinical pregnancy of IVM during COH for preventing OHSS.

Materials and Methods: We included the women who had undergone OHSSin the previous COH cycles or had susceptibility for OHSS between January,2006 and December, 2007 by review of medical record in Maria fertilityhospital. Women with only first autologous cycle of IVM during COH wereenrolled. After at least 5 days’ stimulation with gonadotropin, if the diameterof leading follicle 12-14mm with more than 20 growing follicles, 10,000IUhCG was injected. After 36-38hr, oocytes were aspirated using 19 gaugeneedle. Immature oocytes filtered using mesh were cultured with YSmedium with 1 IU/ml FSH, 10 IU/ml hCG and 10ng/ml recombinant humanepidermal growth factor. After denudation with hyaluronidase, ICSI wasperformed. Zygotes were co-cultured with cumulus cells in 10μl YS mediumwith 10% human follicular fluid and were transferred. We compared theclinical pregnancy group and non-pregnant group according to thedevelopmental and clinical variables.

Results: A total of 110 patients received IVM during COH cycles (103 forcleavage stage embryo and 7 for blastocyst). Overall ongoing pregnancyrate was 34.5% (38/110). Minimal to moderate OHSS was developed in4.5% (5/110) and severe OHSS was not shown. Clinical pregnancy group(n=44, 40%) showed similar clinical variables such as age (mean±SD;32.0±2.9 years vs. 32.1±3.2 years), FSH (5.7±1.8 mIU/ml vs. 4.5±1.4mIU/ml), number of previous IVF cycle (1.0±1.5 vs. 0.7±1.2), etiology ofinfertility, total dose of gonadotropin (16.2±6.7 ampoules vs. 18.1±7.5ampoules), duration of stimulation (6.9±1.2 days vs. 7.2±1.8 days),endometrial thickness at transfer (9.4±1.5mm vs. 9.9±1.5 mm), and numberof embryos transferred [cleavage; 4.6±0.9 (n=40) vs. 4.7±0.9 (n=63),blastocyst; 2.3±0.5 (n=4) vs. 2.3±0.6 (n=3)] compared with non-pregnancygroup (n=66). Number of MII oocytes to those of total oocytes retrievedratio is significantly higher in clinical pregnancy group [479/968 (49.5%)]than non-pregnancy group [408/980 (41.6%), P<0.001]. Day 1 fertilizationrate was significantly different between clinical pregnancy group [365/479(76.2%)] and non-pregnancy group [337/408 (82.6%), P=0.019]. Clinicallypregnant women showed no significant difference in total fertilization rate[603/798 (75.5%) vs. 646/823 (78.5%)], day 2 fertilization rate [208/282(73.8%) vs. 264/355 (74.4%)], and day 3 fertilization rate [30/37 (81.1%)vs. 47/70 (67.1%)] compared with non-pregnant women.

Conclusions: Women with more mature oocytes may show higherpregnancy rate of IVM during COH than those with less mature oocytesdespite lower initial fertilization rate. Maturity of oocytes may be a predictorfor clinical pregnancy of IVM during COH for preventing severe OHSS.

April 19-22, 2009 88


P.051 – USING HIGH MAGNIFICATION MICROSCOPY TO EVALUATE FREZZINGEFFECT ON SPERM HEAD INTEGRITY

Gemma López, Rafael Lafuente, Sergi Rovira, Olga Cairó, Mario Brassesco.CIRH. Clínica Corachan. Barcelona, Spain.

Introduction: Evidence in long IVF series suggests that sperm morphology,according to a strict criteria, predicts an important information about fertilityrates. The aim of this study is to evaluate the effect of freezing on spermhead integrity after capacitation by gradient method, analyzing the presenceof nuclear vacuoles.

Materials & Methods: Prospective study that analyses samples fromaccepted donors after a preliminary study in our clinic. We have analysedthe sample before 1 hour post-ejaculation and the same sample oncethawed, after be prepared with density gradients and frozen. The analysiswas made in real time through inverted microscope Leica AM 6000 with100x immersion objective and 1.6x multiplier (8000x). A total of 100spermatozoids were counted in every sample, and divided into the followingcategories:

Group I: without nuclear vacuolesGroup II: 1 or 2 small vacuolesGroup III: 1 large vacuoleGroup IV: many small vacuolesGroup V: 1 large vacuole and other vacuoles

Results: Average of all samples analysed

Fresh sample: 12.7% of group I; 25.5 % of group II; 30.3% of group III;17.16% of group IV; 14.33% of group V.

Prepared and Frozen samples: 6% of group I; 25.3% of group II; 27% ofgroup III; 23% of group IV and 18.7% of group V.

Comparing between the fresh samples and frozen ones, we observe areduction in the number of spermatozoa without vacuoles, and a lightincreasing of spermatozoa with large vacuoles and with numerous smallones, in those samples analysed after thawing.

Conclusions: By means of sperm magnification we have observed that aftercapacitation and freezing there is an increasing of spermatozoa presentingaltered morphology. We continue studying further and trying to evaluate itsrepercussion on fertility.

P.052 – IN VITRO MATURATION, FERTILIZATION AND EMBRYODEVELOPMENTAL CAPACITY OF MOUSE GERMINAL VESICLE OOCYTESFOLLOWING STEPWISE CRYOPRESERVATION

Reza Mahmoudi1, Mina Dehghani2, Hamdollah Delaviz1. 1Department ofAnatomy and Embryology, Medicine Faculty, Yasuj University of MedicalScience, Yasuj; 2Dept. of Surgery, Namazi Hospital of Shiraz University,Shiraz; Iran.

Backround: Cryobiology is a very important tool in reproductive biology.This procedure can benefit the cancer patient wishing to preserve fertilitybefore initiation of any destructive chemotherapy or radiation therapy. It is asubstitute for embryo cryopreservation and thereby avoids associatedethical issues. Oocyte cryopreservation technology can lead to theestablishment of “oocytes banks” and provides solutions to ovarian failurepatients.

Objective: The aim of this study was to evaluate viability, fertilization andsubsequent developmental to blastocyts of mouse germinal vesicle oocytesafter single and stepwise vitrification procedure.

Material and Methode: Oocytes were obtained from 3- 4 week old femalemice 48hr after i.p. injection of 7.5 IU pregnant mare serum gonadotropin(PMSG). Collected oocytes before vitrification were exposed tocryoprotectant, which was composed of 30% (v/v) ethylene glycol, 18%(w/v) Ficoll-70, and 0.3 M sucrose, either by single step or in a step-wiseway. After vitrification and storage in liquid nitrogen, the oocytes werethawed and washed two times in medium TCM199 and then subjected to invitro maturation, fertilization and culture for blastocysts.

Results: The oocytes survival rates after vitrifying-warming, maturation rate,the capacity of fertilization and embryonic development to blastocyst wereexamined in vitro. The oocytes surviving, maturing to MII, fertilization,cleavage and blastocyst rate in the step-wise exposure were significantly

higher (P<0.05) compared with corresponding rate in the single stepprocedure.

Conclusion: The results of present study indicated that germinal vesicleoocytes vitrified stepwise procedure had positive effect on survival,maturation, fertilization cleavage and blastocysts rate than single stepprocedure.

Key words: Germinal Vesicle Oocyte, Vitrification, mouse, Ethylene glycol,blastocyst.

P.053 – PROSPECTIVE RANDOMIZED STUDY TO COMPARE THE EFFICACY OFRECOMBINANT HUMAN CHORIONIC GONADOTROPIN (R-HCG) VERSUSURINARY HCG (U-HCG) IN FINAL FOLLICULAR MATURATION OF 365PATIENTS UNDERGOING ASSISTED REPRODUCTIVE TECHNOLOGIES(ART)

Zeng Yong1, Hu Xiaodong1, Mo Meilan1, Song Cheng1, Zhang Wei M. Meilan1

Ye Xingshen2, Diego Ezcurra3. 1Shenzhen Zhongshan Urology Hospital,China; 2 Merck Serono China; 3Merck Serono SA, Geneva, Switzerland.

Objective:To compare oocyte and embryological outcomes of 365 IVF cyclestriggered with r-hCG versus u-hCG

Design: Prospective randomized study

Materials/Method: Three hundred and sixty five infertile patients from theCenter of Assisted Reproductive Techniques, Shenzhen Zhongshan UrologyHospital - China, eligible for IVF/ICSI treatment, participated in this study. Allpatients were treated with long pituitary down-regulation with the injectionof 1.25 mg of GnRHa in the mid-luteal phase of the preceding cycle. Fivedays after menses, ultrasound scanning was performed to confirm theachievement of down-regulation (absence of an ovarian cyst andendometrial thickness of less than 6mm). Controlled ovarianhyperstimulation (COH) was initiated with a daily dose of 150-375 IU ofrecombinant human FSH (r-hFSH) or human menopausal gonadotrophin(hMG). The ovarian response was monitored by ultrasound scanning.Urinary LH level was tested when the leading follicle was ≥ 14 mm. Cycleswere cancelled if follicles remained with a diameter of ≤10 mm after 14 daysof stimulation. Patients with more than two follicles ≥18 mm, wererandomized to receive 250 μg of recombinant human chorionicgonadotrophin (r-hCG) by subcutaneous injection or 10,000 IU of urinaryhuman chorionic gonadotrophin (u-hCG) by intramuscular injection.Transvaginal ultrasound-guided oocyte retrieval was scheduled 36 hr afterhCG injection and all visible follicles were aspirated. Fertilization wasperformed by IVF or ICSI (patients with poor sperm quality). Embryos with≥6 blastomeres and ≤20% fragments were defined as good quality.Continuous variables were expressed as mean ± SD and were comparedbetween the two groups using Student’s t-test. Categorical variables werecompared using Chi-square test. Differences were considered significantwhen the p value <0.05.

Results: The characteristics of IVF cycles utilizing r-hCG and u-hCG werecompared (Table 1). The results expressed as mean ± SD or percentages,as appropriate, are summarized below.

Table 1

Variable uhCG 10.000 IU Ovidrel 250 mcg P value

Number of patients 181 184

Age (years) 32.2 ± 5.6 32.0 ± 5.8 0.739

Years of infertility 4.6 ± 3.2 4.8 ± 3.5 0.606

Mean gonadotropin utilized 43.2 ± 14.5 41.6 ± 15.9 0.328

(75 IU vials)

Days on treatment 11.9 ± 2.5 12.0 ± 2.1 0.840

Number of oocytes retrieved 13.3 ± 6.0 12.9 ± 6.5 0.521

MII ratio (MII/oocytes retrieved) 94.45% 95.07% 0.480

Fertilization ratio IVF 84.37% 82.68% 0.246

(oocytes fertilized/oocytes inseminated)

Cleavage rate 97.61% 97.96% 0.583

Embryo excellence ratio IVF 41.45% 44.70% 0.297

(good quality embryos/all embryos)

Embryo excellence ratio ICSI 38.02% 44.78% 0.010

Conclusions: The number of oocytes retrieved and the percentage of thosethat were mature (MII ratio) were not significantly different when triggeringfinal follicular maturation with recombinant or urinary hCG. Cleavage rate

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was not different for both treatment groups and embryo excellence rate(good quality embryos/total embryos produced), in ICSI patients, wassignificantly lower with urinary hCG than the comparator. The rest of thevariables analyzed were not significantly different between the two treatmentgroups. The results of this study suggest that r-hCG is effective forachieving final follicular maturation and the production of good qualityembryos in patients undergoing ART.

P.054 – WHEN SHOULD WE FREEZE ALL ZYGOTES TO PREVENT OHSS IN IVFCYCLES WITH OVARIAN STIMULATION, E2 VALUE OF HIGHER THAN5000PG/ML OR 3500PG/ML?

Yukiko Miyaki1, Asuka Ohtani1, Kengo Sugihara1, Mamoru Ida1, YoshiharuNakaoka1, Aisaku Fukuda1, Yoshiharu Morimoto2. 1IVF Osaka Clinic; 2IVFNamba Clinic; Osaka, Japan.

Introduction:Controlled ovarian hyperstimulation (COH) increases thenumber of oocytes retrieved and is making it possible to select better qualityembryos for embryo transfer (ET). On the other hand, ovarianhyperstimulation syndrome (OHSS) is one of the most serious side effects.Cryopreservation of all embryos is one of the alternatives to avoidaggravation of OHSS and also implantation failure due to extremely highestrogen. Our present indication for cryopreservation of all zygotes is E2value of higher than 5000pg/ml on the day of HCG administration. In thepresent study, we investigated the occurrence of OHSS and pregnancy ratein fresh cycles among three different ranges of E2:3500-4000pg/ml, 4000-4500pg/ml and 4500-5000pg/ml to reevaluate the appropriate E2 value forcryopreservation of all zygotes to prevent the occurrence of OHSS.

Material and Methods: Sixty eight fresh ET cycles (65 cases) with E2 valuesbetween 3500 and 5000pg/ml on the day of HCG administration fromJanuary, 2006 through December, 2007 were analyzed retrospectively. Allcases were stimulated by gonadotropins with GnRH analog. COH started onday 3 of menstrual cycles and ovarian follicles were monitored bytransvaginal ultrasonography from day 7. When more than two folliclesreached 18 mm in diameters, 5000 unit of HCG was administered. Oocyteswere retrieved 36 hours after HCG administration. Cleavage or blastocytstage embryos were transferred and occurrence and intensity of OHSS werefollowed for 4 weeks after transfer. All cases were divided into 3 groups bythe values of E2 on the day of HCG (Group A: 3500 < E2 <4000, Group B:4000 < E2 <4500 and Group C: 4500 < E2 <5000). The occurrence of OHSSwith it’s intensity and pregnancy rate in each group were compared.

Results: The mean age of the patients and the number of embryostransferred among these three groups were similar. There were nosignificant differences in pregnancy rates among Group A (57.5%: 23/40),Group B (59.1%: 13/22) and Group C (50.0%: 3/6). Occurrences of OHSSwere comparable among Group A (20.0%: 8/40), Group B (27.3%: 6/22)and Group C (33.3%: 2/6) and there were no significant differences of theirintensity.

Conclusions: There were no differences of pregnancy rates and alsooccurrences of OHSS among the three E2 values from 3500pg/ml to5000pg/ml on the day of HCG administration. The present study reaffirmedthat the present our criteria, E2 value higher than 5000pg/ml, for freezingtotal zygotes to prevent OHSS is appropriate. However, we should followvery carefully the IVF patients with peak E2 value of 3500pg/ml or higher onthe day of HCG in stimulation cycles.

P.055 – THE EVALUATION OF THE TIME EFFECT OF CO-CULTURE SYSTEM ONSPERMATOGONIAL STEM CELL COLONIZATION

M. Mohamadi1, M. Movahedin1, M. Koruji2. 1Department of Anatomy, TarbiatModares University. 2 Basic sciences, University of Social Welfare andRehabilition Sciences; Tehran, Iran.

Introduction: Spermatogonial stem cells are unique population of cells withself-renewing potential, and are the only stem cells in the body that transmitgenetic information to the next generation and can be cultured for extendedperiods in the presence of feeder cells such as sertoli and STO cells.

Objective: This study aimed to compare the effect of co-culture with sertoliand STO cells on the number and diameter of mouse spermatogonial stemcell colonies to improve an in vitro culture system capable of supportingspermatogonial stem cell colonization.

Materials and Methods: The testis of mouse minced into small piecesmechanically and enzymatic digestion was performed to separate the cellsfrom seminiferus tubules. Lectin-coated dishes used for sertoli cell isolationfrom spermatogonial stem cells. With the formation of a confluent layer ofsertoli cells, spermatogonial stem cells transferred on this feeder layer. Alsomitomycin C-treated STO fibroblast cell line was used. In control group,spermatogonial stem cell cultured on a feeder free culture dishes. Thenumber and diameter of mouse spermatogonial stem colonies was assessedafter 3, 7 and 10 days of culture by an inverted microscope.

Results: The results of one-way ANOVA and Tukey post hoc test showedsignificant differences between the mean number and diameter of thecolonies between sertoli, STO and control groups in 7th and 10th days(p<0.05), however there wasn’t significant difference between these threegroups in 3th day (p>0.05).

Conclusions: The present study demonstrated that Sertoli cell may havemore positive influence on the process of in vitro colonization.

P.056 – EVALUATION OF SERUM ANTI-MULLERIAN HORMONE(AMH) LEVEL INTHE IVF

Miyuki Nagaki, Kaori Goto, Yoko Kumasako, Eiko Otsu, TakafumiUtsunomiya. St-Luke Clinic, Oita, Japan.

Introduction: The age of infertility patient when start the treatment isincreasing older because an age at marriage is going to up in Japan. As anew marker to examine the ovarian reseve, Anti-Mullerian hormone(AMH)was reported. The AMH level in in-vitro fertilization was investigated.

Materials and Methods: A total of 172 cycles of patients who scheduled in-vitro fertilization from January 2008 to July 2008 were devoted to our study.

The blood sampling for the AMH measurement was performed at the ovarystimulation was started (on the fifth day of menstrual cycle).

AMH levels were measured duplicately and caliculated the mean AMH level.

AMH levels in the serum were assayed by a commercially available enzyme-linked immno sorbent assay(ELISA) kit, AMH used leader 680 modelmicroplates (BIO-RAD company), and absorbance was measured at 450nm.

Results: The mean age of 172 cycles of patients who measured the AMHlevel was 36.4±4.4 years old.

As the patient’s age became higher, the mean AMH level were decreasedsignificantly (The mean AMH level of patients who are less than 34 yearsold : 2.68ng/ml , 35 - 39 years old : 1.83ng/ml, more than 40 years old :1.03ng/ml).

When a high number of follicles were recognized at the time of the oocytepick up, AMH level were increased(Less than 3 follicle : AMH0.45ng/ml, 4-6follicle : AMH1.27ng/ml, 7-9 follicle : AMH2.13ng/ml, 10-15 follicle :AMH3.08ng/ml, More than 16 follicle: AMH4.1ng/ml).

In 159 stimulation cycles, the AMH level of the no-oocyte group(AMH :0.33ng/ml) of 24 cycles was compared with the normal group (AMH :2.10ng/ml) of 135 cycles. The AMH level tended to be lower in the no-oocyte group than the normal group.

The AMH level was compared in 76 cycles of embryo transfer (ET) groupwith 27 cycles of ET cancelled group(not including all 2PN stage freezebecause of avoidance from OHSS). The AMH level was significantly lower inET cancelled group(AMH: 0.33 ng/ml) than ET group (AMH: 1.73ng/ml).

Embryo growth on the third day after oocyte pick up showed better growthwhen AMH was high level, there was not a significant difference(Thepercentage of good embryos of the AMH level 1ng/ml group: 75.0%, theAMH level 1 - 2ng/ml group: 85.7%, the AMH level more than 2ng/ml group:95%).

The pregnancy rate per stimulation cycle with an AMH level less than 1ng/mlgroup was 9.8%, In contrast, the pregnancy rate with an AMH level morethan 1ng/ml group was 25%. It showed a significant difference.

Conclusion: In IVF treatment, the AMH level on the fifth day of a menstrualcycle(before HMG start), will be able to predict the IVF outcome.

April 19-22, 2009 90


P.057 – 3D POWER DOPPLER MEASUREMENT OF PERIFOLLICULARVASCULARIZATION IN NORMAL AND POLYCYSTIC OVARIES DURING INVITRO FERTILIZATION

A. Nazzaro1, A. Salerno1, M.D. Limongelli 1, G. Carlomagno2.1Physiopathology of Human Reproduction Unit, AORN “G. Rummo”Hospital, Benevento; 2Department of Obstetrics and Gynecology, Universityof Rome “La Sapienza”, Roma; Italy.

Objective: Ovarian angiogenesis and subsequent intrafollicular O2 content isa critical point in determining oocyte competence and IVF outcome. Wewanted to evaluate the differences, if any, in ovarian perifollicular blood flow(PFBF) in normal and polycystic ovaries (PCOS) during IVF by using three-dimensional power Doppler (3D pD).

Materials: 80 unexplained infertile women and 74 women with ultrasoundevidence of PCOS were enrolled. Controlled ovarian hyperstimulation (COH)was induced by long or short protocol + rFSH(�-follitropin). A GE730Voluson machine with a transvaginal volume transducer was used.3DpD was used to assess total follicle number per ovary (FNPO), ovarianvolume (OV) and ovarian vascularization indexes (OVI). Stored volumeswere analyzed by the VOCALTM program. Mean greyness-MG, vascularizationindex, flow index and vascularization flow index were serially calculatedthroughout the follicular phase of ovarian stimulation. Serum FSH, LH andinsulin were also checked. Ovulation was induced by hCG administration.

Results: We observed conflicting results in PCOS women as OVI appearedpositively related to FNPO rather than OV or FSH /LH levels. In the presenceof FNPO > 20 OVI were all significantly higher in PCOS women, than innormal ones, in every step of stimulated cycles independently from pituitarydown regulation, whereas in the presence of FNPO > 12 but < 20(consensus standard) OVI were lower in PCOS women following pituitarysuppression. MG values were similar in both groups but total stromal areawas significantly increased in PCOS. Normoandrogenic ovulatory womenneeded a higher amount of �-follitropin to get the same PFBF vascularizationindexes than the PCOS ones. Insulin resistance appeared positively relatedwith OVI increasing.

Discussion: PCOS is a heterogeneous disease. FNPO better relate withovarian vascularization and angiogenesis during COH than FSH/LH levels orOV. Women with a total FNPO > 20 are at higher risk of ovarianhyperstimulation. Insulin resistance may play a role in ovarian hyperresponse in PCOS.

P.060 – CRYOPRESERVATION OF SUPERNUMERARY OOCYTES. RESULTS

Isabel Margarita Carrasco1, Carolina Ortega1, Marina Ríos1, Verónica Sáez1,Patricio Donoso1, René Salinas1, Rodrigo Enríquez1, Patricio González1.1Reproductive Medicine Unit., Clínica Alemana de Santiago. Universidad delDesarrollo. Santiago, Chile.

Introduction: Oocyte cryopreservation is a new option for countries and/orcenters where embryo cryopreservation is not allowed. This techniquerepresents a tool to preserve supernumerary oocytes after ovarianhyperstimulation, avoiding the medical risks and costs of a new cycle. Theaim of this report is to communicate the first results in our country of anoocyte slow freezing cryopreservation protocol in IVF patients, freezing allthe mature supernumerary oocytes. Oocyte survival, fertilization, embryonicdevelopment, implantation and pregnancy rates are analyzed.

Material and Methods: Supernumerary oocytes from patients who did notget pregnant after a conventional IVF cycle and had at least 3 oocytesavailable for cryopreservation, from November 2006 to October 2008. Slowfreezing technology was performed. Oocytes were denudated and placed ina choline based medium with 1.5M PrOH for 10 minutes, followed by 15minutes on the same medium plus 0.2 M sucrose. Oocytes were loaded inplastic straws and transferred into an automated Cryo Bath Freeze Control.Temperature was decreased gradually from 22ºC to -6ºC and seeding wasperformed. After 10 minutes temperature was decreased in 3 steps (-32ºC,-40ºC and free fall). Straws were placed in liquid nitrogen at -196 ºC.

For oocyte thawing the cryoprotectant was removed at room temperature byserial dilutions using 0.2M, 0.1M and 0M sucrose solutions. Oocytes werecultured in Cook Fertilization Medium at 37ºC in an atmosphere of 6% CO2 inair. ICSI was performed 2-3 hours after thawing. The number ofmicroinjected oocytes per patient (1 to 5) did not deferred from fresh cycles

and was decided considering female age and cause of infertility, thus not allthe surviving oocytes were inseminated. Cook Cleavage Medium was usedfor embryo culture. Embryo transfer (ET) was done on day 2-3 afterinsemination. ßhCG determination was performed 15 days after ICSI andclinical pregnancy was assessed by transvaginal ultrasound 3 weeks later.

Results: A total number of 524 MII oocytes from 58 cycles were frozen andin 25 cycles 193 were thawed. Oocyte survival rate was 41% (79/193). Themean number of microinjected oocytes was 3.6 ± 1.4 and fertilization ratewas 69.1 %. A mean of 2.3 ± 1.2 embryos were transferred. Embryonicdevelopment rate was 93.6 % from which 34.1 % corresponded to embryosof good morphologic quality and appropriate cleavage. No significantdifferences on embryo quality was found between fresh cycle and frozen-thawed cycles (p<0.2). Nineteen cycles reached ET. The 6 failed cycles weredue to: no surviving oocytes (2), no fertilization (2) and arrested embryos atpronuclear stage (2). The mean age of patients who had ET was of 33.4 ±4.4 years. Implantation, clinical pregnancy and live birth rates per ET were14.0 %, 31.6 % and 21.1 %, respectively. One ongoing pregnancy and fourliveborns were achieved. No multiple pregnancies occurred. The cumulativepregnancy rate was 42.2 % (21.2 % from fresh cycles plus 21.1% fromthawed cycles).

Conclusions: In our experience oocyte cryopreservation is an option to avoidsupernumerary oocytes disposal in centers where embryo cryopreservationis not allowed. Although the survival rate is not high when all thesupernumerary oocytes are frozen, the one that do survive give rise toembryos with acceptable implantation rate.

P.061 – OOCYTE CRYOPRESERVATION. CHILEAN CENTER EXPERIENCE

Carolina Ortega, Isabel Carrasco, Marina,Ríos, Veronica Sáez, PatricioDonoso, René Salinas, Rodrigo,Enriquez, Patricio González. ReproductiveMedicine Unit.ClÌnica Alemana Stgo.U del Desarrollo.Stgo,Chile., Santiago,Region Metropolitana, Chile.

Introduction: Oocyte cryopreservation (OC) has become an interestingalternative in countries or institutions in which embryo freezing is forbidden.In addition, this technique can be offered to women at risk of prematureovarian failure (oncologic treatment), or those who have decided to delaymotherhood. Our OC program started in October 2006 under the frameworkof a limited number of oocytes to be inseminated since embryocryopeservation is not allowed. All surplus mature oocytes arecryopreserved using slow-freezing (SF) protocol. The aim of this study is toshow our results from frozen-thawed cycles including oocyte survival,fertilization, implantation, embryo quality and clinical pregnancy rates.

Materials and Methods: We included 23 patients (25 frozen-thawed cycles)who had cryopreserved supernumerary oocytes from a previousunsuccessful fresh cycle (November 2006 until October 2008). SF wasperformed in all cycles using 1,5 M propanediol and 0,2 M sucrose in aCholine based Medium. Oocytes were loaded into plastic straws andtransferred to a Cryo Bath Freeze Control. Decreasing concentrations ofpropanediol and sucrose made thawing procedure. During the thawingcycle, patients were followed up on a natural cycle until the leading folliclereached at least 17 mm. hCG (5000 IU) were administered to enable propertiming with the thawing process. Oocytes were thawed after 36 hours instraws of 5 oocytes. ICSI was done 2 hours after thawing. Embryo transferwas performed on days 2 or 3 of development. The number of oocytes to beinseminated was decided considering women’s age and diagnosis ofinfertility. A ßhCG test was performed on day 15 after ICSI and US 3 weeksafter. Micronised vaginal progesterone as luteal support was started on theday of ICSI.

Results: A total of 524 supernumerary mature oocytes from 56 cycles (54patients) were frozen with SF technique. Of these, 187 oocytes (25 cycles)were thawed and 19 cycles (17 patients) were transferred. The mean agewas 33.4 ± 4.4 years (24-42). The survival rate per thawed oocytes was41,2% (77/187). The injected oocytes were 3,6 ±1,4 and the transferredembryos were 2,3 ± 1,2. The fertilization, implantation and clinicalpregnancy rates per thawed oocytes were 61%, 14% and 24% respectively.The embryonic development was 93,6% with 34,1% good quality embryos.The number of live birth was 4 (16%), three healthy children, one pretermdelivery at 24 weeks who did not survived and three spontaneous abortions.No multiple pregnancies were recorded. The cumulative pregnancy rate pertransfer was 46,4%.

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Conclusions: The present study showed the results of thawing cycles thatdid not achieve pregnancy in fresh cycle, hence when patients who becomepregnant in the fresh cycle choose to thaw their oocytes, the cumulativepregnancy rate may rise.

The survival rate was 42,6%, and reflects the survival of all cryopreservedmature oocytes, no matter the oocyte quality before freezing, maternal ageor response to stimulation protocols.

Oocyte cryopreservation is an option to improve pregnancy rates in thosecentres in which embryo freezing is not allowed or for those couples whoreject embryo cryopreservation.

P.062 – CUMULUS OOCYTE COMPLEXES FROM SMALL ANTRAL FOLLICLESDURING THE EARLY FOLLICULAR PHASE OF SPONTANEOUS CYCLESIN RHESUS MONKEYS CAN EXPAND AND YIELD OOCYTES CAPABLEOF MATURATION IN VITRO

Marina C. Peluffo1, Richard L. Stouffer1,2, Jon .D. Hennebold 1,2, Mary B.Zelinski1. 1Division of Reproductive Sciences, Oregon National PrimateResearch Center; Beaverton; 2Department of Obstetrics and Gynecology,Oregon Health & Sciences University, Portland; OR, USA.

Introduction: Cryopreservation of the ovarian cortex is one experimentaloption for restoring fertility in cancer survivors. This strategy relies on thedevelopment of primordial/preantral follicles post-thaw in vivo or in vitro toyield mature oocytes for subsequent in vitro fertilization of intracytoplasmicsperm injection. However, there are many small antral follicles (SAF) in theovarian medulla that could be a potential source of oocytes for in vitromaturation (IVM) in tissue not used for cryopreservation. The aim of thisstudy was to determine the minimal diameter of an antral follicle fromspontaneous menstrual cycles in macaques that could yield a cumulus-oocyte complex (COC) capable of expansion and oocyte maturation in vitro.

Materials and Methods: Ovaries were removed from adult rhesus monkeys(n=6) during the early follicular phase (days 3-4) of spontaneous cycles.SAF were dissected from the ovarian medulla after collagenase treatment.The COCs from healthy SAF (devoid of dark oocytes or granulosa cells) wereextracted (n=87). SAF were divided into five groups according to theirdiameter; Group 1: < 0.5 mm; Group 2: 0.5-0.99 mm; Group 3: 1.0-1.49mm; Group 4: 1.5-1.99 mm; and Group 5: 2.0-2.5 mm. Individual COCsfrom each group were cultured for 48 h in TALP (Tyrode’s, albumin, lactate,pyruvate) alone, TALP + follicle stimulating hormone (FSH) + luteinizinghormone (LH) (n=46 COCs), SAGE (SAGE®, CooperSurgical, Inc.) alone, orSAGE + FSH + LH (n=41 COCs). Images of COCs were acquired at collection(0 h), 24 and 48 h post-treatment to assess cumulus cell expansion. At 48h, oocyte maturation and diameter were measured after treatment of COCswith hyaluronidase.

Results: Of the total COCs collected, the majority distributed into Group 3(69%), with fewer in Groups 1 (2%), 2 (14%), 4 (9%) and 5 (6%). In allgroups, cumulus-oocyte expansion was seen 24 and/or 48 h post-treatment.There were no differences in the percentage of oocytes that resumedmaturation to metaphase I (MI) or continued maturation to metaphase II(MII) at 48 h in vitro between media alone and media + gonadotropins, sodata were combined within media. Oocyte maturation was not observed inGroup 1 in either media. In TALP, the percentages of MI oocytes observed inGroups 2, 3 and 4 were 20% in each and were similar to those seen in SAGE(14% and 20% in Groups 2 and 3, respectively). A greater proportion ofoocytes achieved MII in both media. The percentage of MII oocytesobtained were not different (p >0.05) when cultured in either TALP or SAGEin Groups 2 (40 vs. 29%), 3 (31 vs. 16%) and 4 (40 vs. 33%). The diameterof MII oocytes was similar regardless of SAF diameter, and did not differbetween TALP (108 ± 2 μm, mean ±SEM) and SAGE (107 ± 6 µm).

Conclusions: These data indicate, for the first time in primates, thatcumulus-enclosed oocytes derived from healthy SAF at least 0.5 mm indiameter obtained during the early follicular phase can meiotically mature invitro. The competence of these oocytes to undergo fertilization after IVMwith subsequent embryonic development is under investigation. Thus, thecohort of SAF present in the ovaries of spontaneous menstrual cycles priorto selection of the dominant follicle can provide COCs as an additionalsource of oocytes for IVM and fertility preservation. Whether COCs obtainedfrom SAF outside of the early follicular phase are also suitable for IVMremains to be determined.

Supported by Oncofertility Consortium NIH 1 UL1 RR024926 (R01-HD058294, PL1-EB008542), NCRR-RR00163 and U54 HD55744.

P.063 – CORRELATION BETWEEN BODY MASS INDEX AND EMBRYOLOGYOUTCOMES IN ART PATIENTS AFTER TRIGGERING FINAL FOLLICULARMATURATION WITH 250 MICROGRAMS OF RECOMBINANT HCG

Kathleen Peters1, James Catt2, Diego Ezcura3; 1Merck Serono, Sydney,Australia; 2Monash IVF, Melbourne, Australia; 3Merck Serono SA Geneva,Switzerland.

Introduction: The objective of this retrospective observational study was toevaluate if 250 mcg recombinant human chorionic gonadotropin (r-hCG)achieved similar embryology outcomes in women with different body massindexes (BMI).

Materials and Methods: Two hundred ninety two (292) assisted reproductivetechnology (ART) patients eligible for intracytoplasmic sperm injection(ICSI), treated at Monash IVF (Melbourne Australia) between September2007 and December 2007, were included in the analysis. Women weretreated with GnRH agonist for mid-luteal down-regulation. Controlledovarian stimulation was initiated with 150 - 450 IU recombinant human FSH(r-hFSH) depending upon patient etiology. Final follicular maturation wasinduced with 250 mcg r-hCG (Ovidrel®) when at least two follicles hadreached 18 mm diameter. Oocyte retrieval was scheduled 38 hours followingr-hCG administration. ICSI was performed on all metaphase II oocytes.Syngamy was assessed at 24 hours post-insemination (hpi), day 2 between42 – 44 hpi, day 3 at 64 – 68 hpi. Blastocyst transfer occurred on day 5post-OPU. Patients were stratified according to their BMI: 17-20 kg/m2

(Group 1), 21-25 kg/m2 (Group 2), 26-29 kg/m2 (Group 3), 30-48 kg/m2

(Group 4). Data was analyzed utilizing JMP 7.0 software (SAS Institute).Continuous variables were expressed as mean ± standard deviation andcategorical variables were expressed as percent. ANOVA was utilized toanalyze continuous variables and Chi Square for categorical data; statisticalsignificance was established at p<0.05.

Results: Of the four BMI groups (G1 – G4), 36 patients were assigned to G1,96 to G2, 32 to G3 and 34 to G4. There were no significant differencesamong the different age-groups, which ranged from a mean of 33.8 (±3.6)to 34.8 (±4.4). Likewise there were no significant differences among thefour groups in the mean daily dose (in IU) of FSH (G1=262±119,G2=258±110, G3=244±115, G4=290±108). No significant differences amongthe four groups were noticed in the total number of oocytes retrieved(G1=9.6±6.0, G2=10.8±5.5, G3=10.9±6.4, G4=9.1±6.5) as well as thenumber of metaphase II oocytes available for injection (G1=7.8±5.2,G2=8.8±5.0, G3=8.8 +-4.9, G4=6.7±4.5). In terms of embryo characteristics,there were no differences among the groups in the number of 2 pronuclear(PN) embryos (G1=5.6±4.2, G2=5.8±3.8, G3=5.3±3.3, G4=4.6±4.0); thenumber of embryos transferred (G1=1.1±0.3, G2=1.1±0.4, G3=1.2±0.4,G4=1.3±0.4) or in the number of embryos cryopreserved (G1=1.5±2.5,G2=1.3±1.6, G3±0.9±1.2, G4=1.0±1.2).

Conclusions: The observations of this study need to be further tested in aprospective randomized clinical study.

P.064 – OVARIAN HYPERSTIMULATION SYNDROME – DO ASIANS DIFFERFROM THE WESTERNERS?

Hemashree Rajesh1, Stephanie Fook-Chong2, Su Ling Yu1. 1Department ofObstetrics and Gynaecology Singapore General Hospital, 2Department ofClinical Research, Singapore General Hospital; Singapore.

Introduction –We aim to to identify the variables associated with ovarianhyperstimulation in the Asian patients profile and compare them withwestern standards. In addition characteristics between patients with apolycystic ovary( PCO) on ultrasound and those without one( NPCO) werecompared.

Materials and Methods - This was a retrospective case analysis of in vitrofertilisation(IVF) records of patients at the Centre for Assisted Reproductionin a tertiary restructured hospital at Singapore, who developed moderate orsevere ovarian hyperstimulation syndrome( OHSS) during IVF stimulation. Atotal of 79 patients identified over 5 years were subdivided into early onsetand late onset hyperstimulation. Patient characteristics, ultrasoundfeatures, gonadotropin doses, stimulation sequelae and response were

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analysed. We used the Rotterdam criteria in the ultrasound recognition ofpolycystic ovaries. Statistical analysis was done using SPSS.B L E 1

Results: 63% of the population were Chinese with the rest - a mixture ofother Asian nationalities. The incidence of moderate to severe early OHSSwith an intention to treat was 10.9%( actual incidence 9.1%) with 86.1%having early OHSS. 63.3% had a PCO picture on ultrasound.

The mean estradiol level at hyperstimulation in a long cycle was comparablebetween a patient with PCO and non PCO( Mean 6009.1 pg/ml, vs Mean5391pg/ml, p=0.25). The average gonadotropin dose associated withhyperstimulation was also comparable. Gonadotropin doses and maximumestradiol levels did not vary between the long cycle and antagonist cycle in awoman <35 years with PCO(Mean 204.2 iu vs 168 iu).

There was no difference in BMI or severity of hyperstimulation betweenpatients with or without PCO ( p=0.600). However hyperstimulationshowed a tendency to be more severe in a PCO patient with a low BMI.Hyperstimulation was attributed to a high starting dose in 72.1% of patientsand to a 50iu step up in 23.5%.

After stimulation, ultrasound on day 5- 7, showed a higher follicle count ina patient with PCO than in a non PCO (p=0.001, mean 28 versus 20). Thiscorrelated with a significantly high initial antral follicle count in the PCOgroup( median 22.5 vs 10.5, p=0.003). At peak stimulation, there weremore large follicles(> 14) (p=0.010) and a trend towards more intermediatefollicles(p=0.079) in the PCO group. There was no significant difference inoocyte retrieval between the two groups. The number of follicles did notrelate to the grade of OHSS.

Severe OHSS presented significantly later on day 4( p= 0.004) after oocyteretrieval than moderate ovarian hyperstimulation(day 2). Prophylacticalbumin helped to reduce severe hyperstimulation. 43 patients had anembryo transfer and there was a 25% incidence of pregnancy. 90% ofpatients who presented with late OHSS were pregnant with a 50% incidenceof twins.

Conclusions – Gonadotropin doses at stimulation should probably conformto the western standards of 150iu in women less than 35 years and minimalincreases of 37.5iu should be considered at step-up. A total follicle count ofmore than 20 on day 5- 7 scan may be predictive of a subsequenthyperstimulation. Prophylactic albumin should be considered to reduce theincidence of hyperstimulation. Transfer should be abandoned in thepresence of high estradiol levels ( > 5200pg/ml) or when the total numberof intermediate and large follicle count exceeds thirty on the day of oocyteretrieval or when the oocyte retrieval exceeds nineteen eggs.Hyperstimulation may be more severe as the BMI decreases and late ovarianhyperstimulation must prompt a look out for twins. Variables seem to besimilar in the Asian population as compared with the west.

P.065 – IVM EXPERIENCE IN MEXICO, INITIAL EXPERIENCE

Luis Arturo Ruvalcaba, Alejandro Chavez-Badiola, Rocio Martinez, JoseMedina, Martha Garcia. Instituto Mexicano de Infertilidad, Zapopan, Jalisco,Mexico.

Background: Results for in-vitro maturation (IVM) are improving andbecoming a reality in several countries. Its low cost and low risk profile isvery attractive and probably should be considered as a treatment option indeveloping countries. In this communication we present our experience withIVM cycles in a Mexican IVF unit.

Methods: Patients with polycystic ovaries were recruited to our IVMprogramme. Collected oocytes were matured as described by Chian.Reported results are given in percentages of maturation and live-birth perseries.

Results: We achieved an overall 82% maturation rate from MII and GVoocytes. Although some patients are waiting to complete their treatmentcycles, this far we have only achieved a live-birth. In this case the patienthad to undergo vitrification of IVM oocytes due to personal reasons. Asingle intrauterine pregnancy was identified at seven gestational weeks(25% implantation rate). A healthy baby girl was delivered at 38 gestationalweeks.

Conclusions: IVM is a promising tool in the field of ARTs and although weare still looking to improve our pregnancy rates, we support that IVMfollowed by oocyte cryopreservation is, according to our results, a viable

alternative to preserve fertility in those patients in whom conventionalcontrolled ovarian stimulation is contraindicated.

P.066 – PER-FOLLICLE ANTI-MULLERIAN HORMONE SECRETION ANDPERIFOLLICULAR POWER DOPPLER ANGIOGRAPHY MAY REFLECTQUANTITATIVELY AND QUALITATIVELY THE OVARIAN FOLLICULARSTATUS IN A HIGH-RISK POPULATION OF INFERTILE WOMEN

Annalisa Salerno, Alfredo Nazzaro. Physiopathology of Human ReproductionUnit, AORN “G. Rummo” Hospital, Benevento, Italy.

Objective: The determination of ovarian reserve continues to be a challengeand we are at the continuous research of affordable markers helpful topredict cycle cancellation and quantitatively/qualitatively poor response toovarian stimulation. There are evidence that antimüllerian hormone (AMH) isinvolved in both the control of primordial to primary follicle growth and theirresponsiveness to FSH administration and that the acquisition of oocytedevelopmental competence is secondary to perifollicular vascularization(PV) and intrafollicular O2 content. In this study we have investigated therelationship between per-follicle AMH levels, PV, ovarian follicular statusand oocyte quality during controlled ovarian hyperstimulation (COH) in ahigh risk population of infertile women.

Materials and Methods: a total of 162 ICSI patients at high risk of cyclecancellation due to raised FHS, previous poor response and/or age ≥ 40years were studied. All of them underwent a cycle day 3 basal measures ofFSH and AMH, ovarian blood flow was evaluated by three dimensionalpowerDoppler angiography (3DpDA) and a total antral follicles count (tAFC)was obtained from 3D ovarian volumes in both ovaries by using a GE730Voluson machine. Basal ovarian blood flow was evaluated by the VOCALprogram. tAFC was obtained by the NICHE program. PV was quantified byseriate 3DpDA measurements during ovarian stimulation and blood flowindexes were off line calculated by the VOCAL program set to colorrendering mode. On the day of oocyte retrieval serum samples and follicularfluids from three follicles < 12 mm and three follicles between 17 and 21mm in diameter were collected and tested for AMH, E2 and Progesterone(P4) contents. Oocyte morphology, fertilization rate and embryo quality wereevaluated.

Results: Intrafollicular AMH levels were more than double in the follicle ≤12mm respect to the largest ones. Per-follicle AMH levels appeared to berelated to day 3 tAFC, number of follicles ≥ 12 mm and number of retrievedoocytes. High intrafollicular levels of P4 were inversely related to the AMHones in both follicular classes. Per-follicle AMH levels appeared inverselyrelated with requirement of FSH units during ovarian stimulation. PV indexesappeared to be unrelated to follicle diameters and ovarian volume but therewas a positive correlation with AMH levels (p<0.05). tAFC did not interferewith blood flow indexes. Cycle cancellation rate was correlated either withAMH levels (p<0.05) either with stromal blood flow (p<0.002), whereas tAFCalone did not (p>0.05). Women with basal AMH levels <1.60 ng/ml showedlower quality oocytes (dark central granulation, increased viscosity,aggregation of smooth endoplasmic reticulum, increased vitelline space)compared with the ≥ 1.60 ng/ml ones. The number of competent oocytesdropped to zero in case of undetectable basal stromal blood flowindipendentely from AMH levels (p<0.001). tAFC did not seemed to affectoocyte quality p(>0.05) but the number (p<0.05). Undetectable stromalblood flow but not AMH levels affect fertilization rate and embryo grading(p<0.001). Conclusions: Per-follicle AMH levels and 3DpDA may be used as prognosticbiomarker(s) of oocyte competence. The routinary use of 3DpDA to detectbasal stromal blood flow combined with AMH levels seems to be superior toAMH alone to predict both oocyte quality and developmental competence.Cycle day 3 basal ovarian blood flow and anti-Mllerian hormone (AMH) levelare useful tools to predict diminished oocyte quality and developmentalincompetence in infertile women.

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P.067 – ASYNCHRONOUS FROZEN EMBRYO TRANSFERS – DOES IT AFFECTRESULTS?

Tarique Salman, Amanda Tozer, Talha,Al-Shawaf, Luca Sabatini, ArielZosmer. Centre for Reproductive Medicine, St. Bartholomew’s Hospital,Barts and the London NHS Trust, West Smithfield, London, UK.

Introduction: In frozen embryo transfer (FET) cycles the transfer is usuallyplanned so that embryos’ and the endometrial development will besynchronous [i.e. day 2 (D2) embryos are transferred on D2 of the lutealphase etc.). Little information is available as to whether asynchrony mayaffect the outcome.

Materials and Methods: Data for FET cycles (1.1.2005 and 31.12.2007) wereretrospectively collected from our computerised database and patients’notes. In natural cycle FET (NC-FET) ovulation (day 0) was consider to occurthe day after a positive urine LH test. In hormone replacement cycles (HRT-FET) day 0 was considered the day when progesterone was started.

Results: 760 cycles were included: 666 synchronous (control), 94asynchronous (study). The clinical pregnancy (CPR) and live birth (LBR)rates per transfer were 18.5% and 14.9% vs. 17.0% and 15.9% respectively[(not significant (NS)]. The LBR for NC-FET or HRT-FET were 17.7% and10.9% (control) vs. 16.8% and 12.5% (study) (NS). In the study group theLBR for D2 embryos transferred on D3 was 16.6% (NC-FET – 17.1%; HRT-FET – 14.2%) and that for D3 embryos transferred on D2 was 12.5%(NC-FET – 14.2%; HRT-FET – 11.1%). In the control group the LBR for D2transfers was 15.2% (NC-FET – 17.8%; HRT-FET – 11.5%) and that for D3transfers was 10.6% (NC-FET – 16.6%; HRT-FET – 4.3%). There was nosignificant difference between any of the LBRs in the control and studygroups.

Conclusions: A one day asynchrony does not seem to affect the treatmentoutcome. The effect of a longer asynchrony needs further assessment.

P.069 – ULTRASOUND MONITORING AND HORMONAL PROFILES IN SERUMAND FOLLICULAR FLUID FOR UNSTIMULATED IVF-CYCLES INRELATION TO “EMPTY FOLLICLE SYNDROME”

Mikael Tang-Pedersen, Lars Westergaard. Fertility Clinic, Odense UniversityHospital; Faculty of Health - Institute of Clinical Research, University ofSouthern Denmark, Odense, Denmark.

Introduction: Over the years one observation remains constant inunstimulated / natural cycle IVF. That is the approx. 20 % of so called“empty follicles”. Empty follicle syndrome (EFS) has been debated, but sinceit seems a constant finding a deeper understanding of the follicularmaturation process is desirable.

In this study we investigate hormonal levels in follicular fluid and serum forunstimulated IVF-treatment of ± oestradiol-primed patients from start of thecycle till day of oocyte pick-up (OPU), along with ultrasonic measurementsof follicle size and number and endometrial thickness.

The aim is to explore possible correlations between endocrinology,ultrasound monitoring and failed cycles due to “EFS”.

Materials And Methods: 70 women under the age of 37, referred to IVF-treatment due to tubal, male or unexplained factor and with regular cyclesfrom 26-34 days and FSH <15 U/L were included in this project as aretrospective analysis.

Ultrasound (US) examination and blood samples were scheduled beside dayof OPU to early, mid and late follicular phase until the appearance of adominant follicle > 16 mm, with a corresponding endometrium > 8 mm. AnhCG-injection of 6500 IU is administered 34 hours prior to OPU. Theaspiration of the mature follicle provides follicle fluid for measuring levels ofAMH, progesterone, oestradiol, androstendion and testosterone. The matureoocyte is fertilized and transferred 2 days post-OPU.

35 of the 70 women had oestradiol-priming, 2 mg twice daily (Femanest®,Sandoz), in 3-10 days in order to predict most likely time for LH-peak andhence avoid premature ovulation as described in earlier work by deZiegler etal.

Delaying intermenstrual FSH-rise until a pre-defined day of lowering serumoestradiol is the feed-back mechanism behind this option.

Results: 70 patients underwent aspiration of follicles. 14 of these hadunexpected ovulation. Another 11 had an “empty” large follicle and from 45women a mature oocyte was retrieved. In cycles with EFS the FF-concentration of progesterone were lower than in cycles with a matureoocyte retrieved (9.5 ± 2.2 vs. 13.5 ± 1.1) µg/ml, with p = 0,037 despite thefact that US- criteria were met.

Conclusion: Cycles with EFS exhibit different hormonal profiles than cycleswith mature oocytes retrieved.

P.070 – INCIDENCE OF OVARIAN HYPERSTIMULATION SYNDROME INPATIENTS AFFECTED BY POLYCYSTIC OVARY SYNDROMEUNDERGOING IN VITRO FERTILIZATION: A PRELIMINARYRANDOMIZED STUDY

Alessandra Tirelli, Simone Giulini, Antonio La Marca, Francesca Tortolani,Susanna Xella, Daniela Tagliasacchi, Tiziana,Marsella, Annibale Volpe.Department of Obstetrics and Gynecology, University of Modena, Modena,Italy.

Introduction: Several ovarian stimulation protocols have been proposed overthe years for patients affected by polycystic ovary syndrome (PCOS)undergoing in vitro fertilization (IVF); however the optimal stimulationprotocol is still under debate.

It is expected that a satisfactory number of oocytes can be recoveredminimizing the risks of ovarian hyperstimulation syndrome (OHSS). OHSSis an exaggerated response to ovulation induction therapy and suchcomplication should always be accounted for in PCOS patients.

In the present study we compare the incidence of OHSS in PCOS patientsundergoing IVF, treated with Gonadotrophin Releasing Hormone (GnRH)agonist protocol or with GnRH antagonist protocol.

Material and Methods: PCOS was diagnosed according to the RotterdamESHRE/ASRM consensus on diagnostic criteria (2004). Fifty-two patientsaffected by PCOS were enrolled and randomly assigned to two differentovarian stimulation protocols. GnRH-agonist group (Group 1; 29 cases)received Leuprorelin 3.75 mg (Enantone 3,75; Takeda) in the midlutealphase and stimulation with 150 IU rFSH/day (Gonal-F; Serono) at least 15days after agonist injection. GnRH-antagonist treated patients (Group 2; 23cases) received 150 IU of rFSH/day starting on day 2-5 of menses,followed by Cetrotide 250 μg/day (Orgalutran, Organon) when the leadfollicles were 13–14 mm in mean diameter.

OHSS occurrence was diagnosed and classified according to a standardizedclinical criteria. The incidence of OHSS was compared among the twogroups. Additionally, pregnancy rate, dose of gonadotropins admnistered,stimulation time, number of oocytes retrieved, number of embryostransferred and number of “good embryos” on day 3 were assessed andcompared among groups.

Results: Six patients from Group 1 developed a mild OHSS duringstimulation and the treatment was thus suspended. No treatment wassuspended in Group 2. Mild OHSS after embryo-transfer (ET) developed in 7patients from Group 1 (24,1%) and in 3 patients from Group 2 (13%).Moderate OHSS was observed in 3 patients (10,3%) in Group 1 and in 0patients in Group 2. No patient developed severe OHSS. Differencesbetween groups were statistiscally significant according to one-way analysisof variance (ANOVA) (P<.005).

Pregnancy was achieved in 8 patients in Group 1 (pregnancy rate percycle=27.6%; pregnancy rate per ET=34.8 %) and in 7 patients in Group 2(pregnancy rate per cycle=30.4%; pregnancy rate per ET=30.4%). Meantotal amount of gonadotropin units administered per patient in Group 1 was1949 ± 95 IU and 1480 ± 169 IU in Group 2, (statistically significant; P<.01).The number of oocytes retrieved were significantly higher in Group1 (12.8 ±1.1 vs. 7.8 ± 0.7, respectively; P<.003). Stimulation time; number ofembryos transferred and number of “good quality” embryos did notstatistically differ in the two groups.

Conclusions: GnRH antagonist protocol is associated to lower incidence ofOHSS in PCOS patients, without reducing pregnancy rate.

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P.071 – EFFECTS OF ESTRADIOL/OOCYTE RATIO ON THE OUTCOME OFCONTROLLED OVARIAN STIMULATION FOR ASSISTED REPRODUCTIVETECHNOLOGY CYCLES WITH GONADOTROPIN RELEASING HORMONEAGONIST

Esra Tonguc, Turgut Var, Muammer Dogan, Leyla Mollamahmutoglu.Department of Reproductive Endocrinology, Zekai Tahir Burak Women’sHealth Research and Education Hospital, Ankara,Turkey.

Introduction: The aim of this study was to evaluate the effect of estradiollevel on the day of HCG(peak E2)/oocyte ratio on outcome of IVF using agonadotropin releasing hormone agonist (GnRH-agonist) based controlledovarian stimulation (COH) for assisted reproductive technology (ART ) cycle.

Materials and Methods: Of the patients who underwent IVF-ET at theDepartment of Reproductive Endocrinology at our hospital between theyears 2005-2007, 600 normal and high responders to the first cycle of COHwith GnRH-agonist were included in the study. Data was obtained frompatient records. Patients were designated into 3 groups based on peakE2/oocyte ratio (Group A:<100 pg/ml, Group B:100-200 pg/ml, GroupC:>200 pg/ml). A comparison between groups was made regarding ovarianstimulation characteristics, number of oocytes obtained, number oftransferred embryos, fertlization and pregnancy rates.

Results: After the division based on E2/oocyte ratio, in Group C(E2/oocyte:>200), the number of obtained oocytes, 2 PN (fertilized oocyte),M2, number of total and transferred embryos was statistically significantlylower than in the other two groups (p=0.001, 0.001, 0.001, 0.039, 0.045).Patients in group C had a significantly higher level of E2 on HCG day(p=0.001). However, there was no statistically significant difference betweengroups regarding number of high quality embryos, fertilization andpregnancy rates

Conclusions: The E2/oocyte ratio has no predictive value in determiningpregnancy rates for normal and high responders to IVF cycles using COHand GnRH-agonist.

P.072 – INFERTILITY PATIENT’S MENTAL HEALTH CONDITION USING THECORNELL MEDICAL INDEX

Keiko Ueno, Michiyo Sashiyama, Takafumi Utsunomiya. St. Luke Clinic, Oita,Oita, Japan.

Purpose: In 1997, the mental health condition of infertility patients in ourclinic was evaluated using the Cornell Medical Index. “As a result, thepatients with a high neurosis tendency did not continue medical treatmentas well as attending clinic regularly. The period was shorter than the others.”

Based on the above result, infertility patient’s mental health condition wasresearched once more using the Cornell Medical Index when they visited ourclinic for the first time and when we introduced them to birth facilitiesduring their pregnancy.

Object and Method: From February 2001 to May 2003, Cornell MedicalIndices were distributed to 464 female patients when they visited our clinicfor the first time (the response rate was 93%; 433 patients) . From October2003 to October 2004, Cornell Medical Indices were distributed to 238female patients that became pregnant after treatment, at that time they wereintroduced to birth facilities (the response rate was 77%; 184 patients). Ourexaminer handed the Cornell medical Index to patients and they werecollected later.

Result: 60% of the patients, belonging to group 4 (high neurosis tendency)when they visited our clinic for the first time, discontinued treatment, and90% of these patients gave up treatment within 6 months. A significantdifference was seen between group 4 and the other groups. Through thisresearch, we can say that the patients in group 4 did not continue treatmentregularly for a long period of time (p< 0.05). Regardless of the groups, over50% of the patients that discontinued their treatment gave up within 6months. If they belong to group 4, and they continued treatment regularlyover 6 months, they would have become pregnant within 2 years. The resultof the Cornell Medical Index at the time of introduction to birth facilities, therate of group 1 (the healthiest group) was significantly increased incomparison with their first visit to our clinic (p < 0.01).

Conclusion: With reference to p<0.05, compared with the other normalgroups, high rate of group 4 patients gave up treatment before they got

pregnant. Regardless of the groups, over 50% patients that gave up on theirtreatment gave up within 6 months and the other hand, 90% of patients thatwere introduced to birth facilities have become pregnant within 2 years.Consequently, we found that it is very important to support infertilitypatients with continued treatment, especially during the early stage of theirtreatment.

P.073 – CLINICAL EFFICACY OF A NOVEL EVALUATION METHOD WITHMEASUREMENT OF EMBRYO RESPIRATION ACTIVITY USING ASCANNING ELECTROCHEMICAL MICROSCOPY

Takafumi Utsunomiya1, Yoko Kumasako1, Kaori Goto1, Megumi Koike1,Masaki Yokoo2, Hiroyuki Abe3. 1St-Luke Clinic, Oita, Japan, 2TohokuUniversity Biomedical Engineering Research Organization (TUBERO),Sendai, Japan, 3Gradeuate School of Science and Engineering, YamagataUniversity, Yonezawa, Japan.

Introduction: Respiration is a useful parameter for evaluating embryo qualityas it provides important information about metabolic activity. The scanningelectrochemical microscopy (SECM) measuring system provides non-invasive and accurate measurement of the oxygen consumption (respirationactivity) of single human embryos. We have shown that there is correlationbetween embryo quality and respiration activity in human embryos at theearly developmental stage. In this study, for practical purposes, weexamined the clinical efficacy for IVF-elected single embryo transfer (eSET)patients using a modified-SECM measuring system.

Material and Methods: A total of 121 IVF-eSET patients gave consent totheir cycles being evaluated using a randomized study. In the morning after2-3 days culture following the conventional IVF or ICSI procedure, weevaluated the embryo quality using a morphological method, and the oxygenconsumption of individual embryos was measured using a modified SECMsystem. All cycles had two or more embryos that appeared reasonably goodmorphologically on ET day (ex. better than 6cell grade 3 by Veeck’s methodon Day3). Each embryo was transferred into a plate filled with medium. APt-microdisk electrode was lowered into the solution, and its tip potentialwas held at -0.6V to monitor the local oxygen concentration.

A single embryo, based on morphological evaluation (ME) or combingmorphological evaluation and measuring respiration activity (ME+MR), waselected by either decision and transferred into the patient’s uterus.

Results: In this study, seventy nine patients’ cycles (reasonably goodmorphologically on ET day) were devoted (61 cycles of ME eSET and 18cycles of ME+MR eSET). Pregnancy rate following of eSET by ME(conventional election) and ME+MR (novel method) was 39.3% (24/61) and50.0% (9/18), respectively. This result suggests a clinical efficacy forembryo quality evaluation by measurement of respiration activity.

Subsequently, among all the cycles that had two or more embryosrepresenting exactly the same excellent morphology by Veeck’s method (ex.8cell grade 1), the pregnancy rate in cycles (n=21) that transferred a singleembryo which was elected based on only morphology by examination underthe microscope was 38.1%, (8/21). Intriguingly, cycles (n=20) thattransferred a single embryo which was elected based on morphology andrespiration activity had a higher pregnancy rate: It was 60.0% (12/20).

Until now, 6 patients of eSET based on ME+MR resulted in a singletonpregnancy and the birth of normal healthy babies (4 male and 2 female)weighing 2520-3142g after 37-40 weeks’ gestation.

Conclusions: It is very difficult to judge which embryo has the most viabilityof all in eSET. Selecting one viable embryo using only the morphologicalmethod seems less objective. The results of this study support thehypothesis that measuring embryonic respiration provides additional andvaluable information about the embryo quality. It is indicated that thehighest pregnancy rate will come to fruition by electing the best embryo bycombining morphology evaluation and respiration activity evaluation in IVF-eSET. It is hoped that this new, novel method will prove to be more effectivefor IVF treatment.

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P.074 – THE INFLUENCE OF AGE ON THE BLASTOCYST GROWTH

Yuji Fujino, Eiko Wakimoto, Naomi Iida, Mayuko Hori, Kouji Koike, MegumiHoki, Yuka Koma, Ayako Yokoyama. Fujino Ladies Clinic. Suita, Osaka,Japan.

Introduction: The aim of this study is to examine the results of blastocystculture and transfer depending on female age for establishing an efficacystrategy for overall reproductive outcome.

Materials and Methods: Clinical evaluation of growing rates to the blastocyststage and pregnancy rates of blastocyst transfer from February 1999 toNovember 2004.Total 2593 cycles were performed oocytes pick-up andblastocyst culture. Ovarian stimulation was performed with ClomipheneCitrate (CC) and Gonadotropin (hMG) protocol. They received 50mg of CCbeginning on day 3 of her cycle, followed by 150 IU of hMG on cycle day 8and day 10. When at least one follicles of diameter reached at >18mm,GnRH analogue was sprayed nasally. Oocyte retrieval was carried out 32 to33h later using the flare-up effect of GnRH analogue. Oocytes wereinseminated with sperm and cultured in various sequential media. Blastocystwas transferred under the ultrasound guidance. Clinical pregnancy wasdiagnosed by the detection of gestational sac with the ultrasound. Asstatistic analysis, Correlation regression and Chi square analysis were usedwith StatView program to compare clinical pregnancy rate (PR) and growingrate to the blastocyst (GR) between each age. P<0.05 was consideredstatistically significant.

Results: The mean age of 2593 cycles was 38.6 +/- 4.3 years old (rangedfrom 30 to 49 years old). The mean number of retrieved oocytes of all cycleswas 3.1 +/- 2.0 (ranged from 1 to 10 oocytes). Cycles having blastocystswere 1234 cycles; those were 52.5% of all treated cycles. The GR was72.3% (34 cycles / 47 cycles) in 30 years old, 62.7% (104 / 185) in 35 yearsold, 51.2% (111 / 217) in 40 years old, and 24.7% (22 / 89) in 40 years old,respectively. There was significant correlation between the growing rates tothe blastocyst stage and age (P<0.01). The average number of transferredblastocysts was 1.3 +/- 0.5 per replacement. One hundred twenty eightclinical pregnancies were established, which included 12 set of twin. The PRper transfer cycle was 35.0% in 30 years old, 28.3% in 35 years old, 11.3%in 40 years old, and 11.1% in 45 years, respectively. There were nosignificant differences in PR of over 35 years old (P<0.05).

Conclusions: Results from this study confirm the expected age-dependentdecline of the growing rate to blastocyst and IVF pregnancy rates even whenhighly selected blastocysts are transferred. The effective treatment for thisage related decline maybe a very difficult problem in the recent reproductionmedicine.

P.075 – THE INFLUENCE OF LEADING FOLLICLES IN NATURAL CYCLE IVF/MTREATMENT

Seong-Ho Yang, Ki- Chul Kim, Chang-Suk Suh; Maria Fertility Hospital,Seoul, South Korea.

Introduction: Recently, it has been reported that successful live birthsobtained from natural cycle IVF combined with IVM (natural cycle IVF/M) forpatients with regular menstrual cycles (Chian et al., 2004; Lim et al., 2008).However, it is not well documented that how the recruitment of leadingfollicles in the ovaries affects the clinical outcome of the treatment cycle.Therefore, this study was to examine the influence of the leading follicles onclinical outcome of natural cycle IVF/M.

Material and Method: A total of 209 patients with regular menstrual cyclesunderwent 233 completed treatment cycles. When the leading folliclesreached 12-14 mm in diameter, 10,000 IU of human chorionic gonadotropin(HCG) was administrated 36-38 hours before oocyte retrieval. The collectedmature oocytes were inseminated by ICSI using the husband’s sperm 2hours later and the immature oocytes were cultured in 1 mL of maturationmedium. After one day of culture, the cumulus cells of oocytes wereremoved with 0.03% hyaluronidase and mechanical pipetting. In vitromatured oocytes were also inseminated by ICSI. Fertilization was assessed17-19 hours after ICSI to detect the appearance of two distinct pronucleiand two polar bodies. Embryo transfer (ET) was performed on day 3 or 4after oocyte retrieval. Clinical outcome was analyzed by the transferredembryos derived from the oocytes retrieved from the leading follicles or not.Group 1 (n=158 cycles): Transferred embryos derived from the oocytes

from the leading follicles and the small follicles; Group 2 (n=75 cycles):Transferred embryos derived from the oocyte from the small follicles only.

Results: The rate of clinical pregnancy in Group 1 (43.0%) was higher thanthat in Group 2 (37.3 %) but there was no significant difference betweengroups. Implantation rate were not significantly different between Group 1(17.4%) and Group 2 (16.3 %).

Conclusion:These results indicate that the clinical outcome of embryosproduced from the oocytes derived from the small follicles is not affected bythe presence of the dominant follicle. These results also suggest that theoptimal HCG injection time is the size of leading follicles reached to 12-14mm in diameter.

P.076 – EFFECTS OF BLOOD AND CERVICAL MUCUS ZINC, COPPER, CADMIUMAND LEAD LEVELS ON INFERTILITY IN WOMEN

Halil Ilgin, Atilla Yildirim, Hikmet Hassa. Eskisehir Osmangazi University(ESOGU) School of Medicine, Dept. Ob&Gyn Reproductive Health Center,Eskisehir, Turkey.

Introduction: This study was conducted to find out the effects of blood andcervical mucus zinc (Zn), copper (Cu), cadmium (Cd) and lead (Pb) levelson infertility in women.

Materials and methods: Thirty-five infertile and 15 fertile women made upour study group. The infertile women were referred to Reproductive HealthCenter of Dept.Ob&Gyn in ESOGU School of Medicine with no male factoras their cause of infertility. Fifteen controls were women attending theGynecology Clinic with no fertility problems and using no contraception.Whole blood (WB), blood plasma (BP) and cervical mucus (CM) sampleswere collected between menstrual days 14-21 and kept at -20º C. To obtainCM samples Gynetics Medical 4502-B IUI cannulas were used. Zn, Cu, Cdand Pb levels were measured in all samples using Hitachi (180-70) PolarizedZeeman Atomic Absorption Spectrophotometer in the Dept. of Chemistry ofESOGU Faculty of Science and Letters. Statistical analysis was made usingthe SPSS 13.0 for Windows. Ethical approval was obtained from ESOGUSchool of Medicine Ethics Committee before initiating the study.

Results: 1. BP and CM Zn and Cu levels were lower in the infertile group ascompared to the controls (p<0.001).

2. WB Pb levels were higher in the infertile group (p:0.023), whereas CM Pblevels did not differ (p>0.05).

3. WB and CM Cd levels were higher in the infertile group (p>0.05 andp:0.008, respectively).

4. Cd levels were higher in WB samples and lower in CM in smokers (fourwomen in each group, eight in total) (p>0.05).

5. Measurements did not differ statistically in women with primary orsecondary infertility (p>0.05).

6. CM levels of Zn, Cu and Cd increased paralel to the increases of bloodlevels of these elements, whereas this relation could not be found for Pblevels.

7. Negative correlation was found between BP Zn and CM Cd, WB Pb andCM Zn, WB Pb and CM Cu (p:0.001, r:-5.50; p:0.008, r:-3.72 and p>0.05, r:-2.87, respectively).

8. There was negative correlation in CM Zn and Cd and CM Cu and Cd(p:0.09, r:-2.44 and p:0.056, r:-2.73, respectively).

9. We found in our study, mean levels of Zn for BP and CM in women withno infertility problems as 1560 µg/L and 32.4 mg/L, respectively and meanlevels of Cu in the same group as 1785 µg/L and 1.32 mg/L.

Conclusions: That BP and CM Zn and Cu levels are lower, Cd levels arehigher and WB Pb levels are higher in the infertile women in our study canbe contributing factors to their infertility, as both Pb and Cd are knownreproductive toxins. Blood levels of Zn, Cu and Cd reflect their CM levels.That CM Cd levels are high when CM levels of Zn and Cu are low suggestsZn and Cu levels should be optimal for normal reproductive function.

We thank Temir Ali Demir, Asiye Berber and Zerrin Kaynak from the Dept. ofChemistry of ESOGU Faculty of Science and Letters, and Ahmet Musmul from theDept. of Biostatistics of ESOGU School of Medicine.

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P.079 – EFFECT OF LH TREATED OVINE OVIDUCTAL EPITHELIAL CELL CO-CULTURE SYSTEM ON MURINE PRE-EMBRYO DEVELOPMENT

Hiva Alipour, Poopak Eftekhari-Yazdi, Abdolreza Rastgarnia, MohamadrezaBaghaban Eslaminejad. Embryology Department, Royan Institute, ACECR,Tehran, Iran.

Introduction: This study was designed to develop a new co-culture systemand assess the effect of Luteinizing hormone using sequential media topromote development and increase the quality of 2-cell Ovine embryosthrough the 8-16 cell stage to morula and blastocyst stages.

Material and Methods: Monolayers for co-culture were prepared from Ovineoviduct epithelial cells (OOEC) in DMEM/F12 medium and in vivo-fertilized2-cell embryos were collected by flushing from superovulated NMRI mice.Co-culture media was treated with hCG as a surrogate for LH because of itsstability and purity. Embryos were cultured in G1/G2™Ver.5 drops alone andcontaining LH, as the control groups and on OOEC monolayers inG1/G2™Ver.5 drops alone and containing LH as the experimental groups.Development and quality rates were determined for all embryos daily andstatistically compared. At the end of the cultivation period, differentiallystained trophectoderm (TE) and inner cell mass (ICM) of expandedblastocysts from each group were examined microscopically.

Results: The embryos cultured on an OOEC monolayer in G1/G2™Ver.5drops treated with LH had a significantly higher developmental rate thanthose of the group without LH and the control groups (P≤.05). Theblastocysts from the LH treated group, in comparison with the groupwithout LH and the control groups, also had a significantly higher mean cellnumber (P≤.05).

Conclusions: These findings suggest that elevated periovulatory LH levelsmay promote preimplantation embryo development. These results haveimportant implications for assisted reproductive technologies in which co-cultures are used to improve pregnancy rates. Ovine Oviduct Epithelial cellco-culture system treated by LH could improve in vitro preimplantationembryo development both in terms of quality (increasing blastocystcellularity) and developmental rate.

P.081 – CLINICAL OUTCOME OF IN VITRO MATURATION CYCLES: EXPERIENCEOF 717 CASES IN MARIA FERTILITY HOSPITAL

Jeong-Ho Cha1, Hye-Jin Yoon1, Seok-Yoon Lee1, Won-Don Lee1, Chang-WonKang2, San-Hyun Yoon3, Jin-Ho Lim3. 1Maria Fertility Hospital, Seoul, Korea,,2Chonbuk National University, Jeonju, Korea, 3Fertility Research Center, MariaMedical Foundation, Seoul, Korea.

Introduction: The major benefits of in vitro maturation (IVM) includeavoidance of the risk of ovarian hyperstimulation syndrome (OHSS), reducedcost, and less complicated treatment for patients. This study reports theclinical outcome for 8 years of our IVM-ET program.

Materials and Methods: This study was performed retrospectively. A total of717 IVM cycles were analyzed from May 2000 to December 2007. Oocyteswere collected between day 7 and 13 of the menstrual cycle. The ovum pick-up day was estimated from over-all cycle length and endometrial thickness.The patients were administered 10,000 IU HCG 36 h prior to oocytecollection. A transvaginal ultrasound machine with a 19-gauge aspirationneedle was used to aspirate follicles. Follicular aspirates were filtered using70-mm mesh, and then the oocytes were isolated under astereomicroscope. Immature oocytes were cultured in YS maturationmedium. Fertilization of matured oocytes was induced by ICSI. 2PN zygoteswere co-cultured with cumulus cells in 20� of YS culture medium. Goodquality embryos were transferred on day 4 or 6 after oocyte retrieval.

Results: A total of 11,665 oocytes were retrieved from 717cycles (mean age= 32.0 ± 3.5 years). The maturation rate was 70.9% (8,269/11,665) andfertilization rate was 78.3% (6,478/8,269). Following transfer of embryos,clinical pregnancy was achieved in 223 cycles (31.1%, 223/717) with animplantation rate of 10.0% (330/3,289).

Conclusions: An acceptable pregnancy rate can be achieved through an IVM-ET program. These results suggest that in vitro maturation is a clinicallyuseful method for polycystic ovarian syndrome patients who have risksassociated with ovarian stimulation.

P.082 – PCOD AS RISK FACTOR FOR PRE-ECLAMPSIA: STUDY OF PRE-ECLAMPSIA,S RISK FACTORS IN SINGLETON PATIENTS IN THEFATEMIYEH HOSPITAL OF HAMEDAN

M. Farimani, R. Niazpor, M. Gharakhani, S. Rabie. Hamedan University,Hamedan, Iran.

Background: Preeclampsia is a major cause of maternal and fetal mortalityand morbidity. The incidence of preeclampsia is 2-10% depending on thepopulation studied. This disease is priority of WHO but pathophysiology ofpreeclampsia is not absolutely known. With attention of complicationpreeclampsia,s complications in pregnancy recognize of risk factors isobligatory for reducing of MMR , NMR and increasing of civil healthy index.

Methods: Completion of questionnaire throughout direct interview withpatients . Kind of study was case/control and each preeclamptic patient wasmatched for age with one control patient and then patients compared withcontrol group. According to the data number of cases was 142 and controlgroup was 142 too. Details of questionnaire collecting and analyzed withtenth edition of SPSS .

Results: PCOD(history of hirsutism + oligomenorrhea and high BMI beforepregnancy) ,UTI , Previous preeclampsia in multiparous women , familyhistory of preeclampsia and passive smoking in case group Significantlywere higher than when compared with control group (P<0.05).

Also high rate of C/S in patients and more consumption of Aspirin , Folicacid, vitamin C & E were seen in preeclamptic group.

Conclusion: Some risk factors such as PCOD , UTI , previous preeclampsia ,positive family history and passive smoking may lead to developpreeclampsia .

P.087 – LARGE HEADED MULTIFLAGELLAR SPERMATOZOA SYNDROME

A. Sellami1, N. Feki1, N. Abid 1, A. Bahloul2, P. Ray3 , T. Rebai1, L. Keskes1.1Reproductive Biology laboratory Medical School and 2Male infertilityResearch Unit, Sfax, Tunisia; 3Department Genetics and Procreation,Grenoble, France.

Introduction: The development of the assisted reproduction technologies,essentially the intracytoplasmic sperm injection (ICSI), has revolutionizedthe treatment of male infertility. However, the use of this technique in someparticular cases of spermatogenesis disorders may lead to geneticallydefective and no viable embryos. Among these types of severe impairmentof male fertility, the large-headed multiflagellar spermatozoa syndrome is acommon infertility phenotype in North Africa population.

Material and Methods: Case report -Our observation is about an infertile 35-year-old man unable to conceive despite 2 years of unprotected intercourse.This patient stemming from a consanguine marriage and presenting afamilial history that revealed multiple cases of infertility, perinatal or low agedeaths. Repeated semen analysis revealed a severe oligoasthenospermiaand necrospermia with sperm count < 1 Million spz /ml, total motility <5%,vitality< 20%. Cytomorphological analysis using shorr coloration revealed asevere monomorphic teratozoospermia with 100% of abnormalmacrocephalic sperm heads. Moreover, 70% of spermatozoa carriedmultiple flagella. The midpiece was enlarged with cytoplasmic droplets in52% of spermatozoa. The multiple anomalies index (MAI) was 3.9 (normalvalue = 1.6), reflecting the high incidence of spermatozoal morphologicalabnormalities in this patient. Chromosomal analysis revealed a normalkaryotype (46,XY). A genetic factor was strongly suspected and a moleculargenotyping was assessed by a genome-wide microsatellite scan.

Results: A homozygous mutation (c.144delC) in the Aurora Kinase C(AURKC) gene was identified with a single nucleotide deletion in the AURKCcoding sequence. This founder mutation results in premature termination oftranslation, yielding a truncated protein that lacks the kinase domain.

Conclusions: Recent studies demonstrated that a homozygous mutation(c.144delC) in the Aurora Kinase C (AURKC) gene led to a meiotic divisiondeficiency and the production of large-headed multi-flagellar spermatozoa, aprimary infertility phenotype mainly observed in the Maghreb. Aurora KinaseC is a protein expressed in testis and implicated in the meiotic segregationduring spermatogenesis. This molecular defect leads to the genesis ofpolyploid spermatozoa with abnormal cytogenetic content. The treatment ofthese cases of infertility using ICSI would be unsuccessful and have a highgenetic risk.

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P.090 – EVALUATION OF RIF PATIENTS WITH RESPECT TO TYPE OF SPERMSELECTED FOR IMSI

Semra Mılık, Zafer Atayurt, Sevil,Unal, Hakan Yelke, Sebnem Yazıcı, FerhatCengiz, Semra Kahraman. Memorial Hospital IVF Center, Istanbul, Turkey.

Objective: To evaluate the effect of the sperm grade, selected at highmagnification according to their morphology and existence of nuclearvacuoles, on subsequent embryo development and pregnancy rates.

Design: Comparative prospective study

Materials and Methods: 81 couples having at least two previous unsuccesfultrials were included in the study. Spermatozoa used for IMSI were classifiedaccording to morphology of the head and existence and size of the nuclearvacuoles as Grade I:normal head morphology and no vacuoles, Grade II::normal head morphology and one small vacuole (4% of head surface),Grade III:normal head morphology and more than one small vacuole, GradeIV:abnormal head morphology and large vacuoles, a system previouslydescribed by Vanderzwalmenn et al. Spermatozoa having defects onneck,mid-piece and tail were not selected for microinjection, regardless ofhead morphology. Sperm selection was made by Zeiss Axioobserver A1inverted light microscope, equipped with DIC optic system and Narishigemicromanipulators, at a total of 8050X magnification on monitor. Patientswere grouped in three according to the grade of the sperms used as, Grade Ifor 36 of the cases, Grade II for 31 and GradeIII or IV for 14. There were nodifferences between groups with respect to mean age of women (33,1±5,5,31,5±5,5 and 31,5±5,2, respectively)

Results: 200,208 and 61 oocytes were injected for Groups 1,2 and 3respectively. Fertilization rates were similar between groups 1 and 2 (79.5%vs. %80.8%) but lower for the third group (70,9%). We have found also asimilar relationship between groups for the top quality embryos (more than6 blastomers,evenly sized and fragmentation lower than 10%) at day 3(45,2%,46,4% and 34,5%) and clinical pregnancy rates (40,9%,41,9% and35,7) as Groups 1 and 2 are similar and Group 3 is lower.

Conclusion: Our results have shown and also contribute to the previousfindings of similar studies that the normalcy of sperm selected for IMSIaffects the outcome positively. So it is important to search for the bestsperm when it is possible. But since there is no significant differencebetween the results of Grade I and II sperms, spending too much time tofind the best sperm should be avoided, taking into account the possiblenegative effect of incubation in PVP.

P.091 – COMPARISON THE EFFECTS OF CHOLESTEROL AND METHYL-BETA-CYCLODEXTRIN ON CRYOSURVIVAL OF C57BL/6 MOUSESPERMATOZOA

Shabnam Movassaghi1, Ghasem Saki2, Fatemeh Javadnia2, Marzieh Panahi2

Mahmoud Mahmoudi3; 1Department of Anatomy, School of Medicine, Islamic AzadUniversity-Tehran Medical Branch, 2Department of Anatomy, physiology researchcenter, School of Medicine, Jondishapour University of Medical Sciences, Ahwaz,Iran, 3Department of Biostatistics, School of Medicine, Tehran University ofMedical Sciences; Tehran, Iran.

Introduction: Cryopreservation induces damage to mouse sperm especiallyC57BL/6 mouse strain and results in a loss of motile and viable cells. Part ofthis damage occurs due to membrane alterations induced by the membranechanging from the fluid to the gel-state as the temperature decreased.Methyl-beta-cyclodextrin (MBCD), which leads to the stimulation ofcholesterol efflux from the cell membrane, is capable to improvingcryosurvival of frozen/thawed sperm in some mammalian species. Butanother observations suggest that adding cholesterol to sperm plasmamembrane may increase the membrane fluidity during cryopreservation.

MBCD and cholesterol-loaded-cyclodextrin (CLC) were examined for theirabilities to increase the cryosurvival of C57BL/6 mouse sperm. Theintactness of acrosome, motility and fertilizing ability of frozen/thawedspermatozoa were used to monitor cryosurvival.

Methods: This experimental study was performed in Cell Culture Laboratoryof Ahwaz Jondishapour University of Medical Sciences during autumn andwinter 2007. male mice were randomly divided in six groups: Inexperimental groups spermatozoa were exposed to different concentrationsof CLC or MBCD and subsequently cryopreserved. Spermatozoa in control

1 group were frozen without any exposure to CLC or MBCD and in control 2(vehicle), sperms were incubated with 4mM MBCD.Then the post-thawsperms were evaluated.

Results: The values of the intact acrosome, motility and fertilizing abilityincreased significantly with concentration of CLC compared to controls andMBCD experimental groups (P<0.05).

Conclusion: These results indicate that cryosurvival of C57BL/6 mousespermatozoa is enhanced by exposure to MBCD which loaded withcholesterol (CLC) before freezing.

P.093 – THE PROTECTIVE EFFECTS OF CARROT SEEDS EXTRACT ONSPERMATOGENESIS AND CAUDA EPIDIDYMAL SPERM RESERVES INGENTAMICIN TREATED RATS

Mohammad Nouri, Arash Khaki, Fatemeh Fathi Azar, Mohammad-Reza Rashidi.Tabriz University of Medical Sciences, Tabriz, East Azarbaijan, Iran.

Background: Carrot (Daucus carota L.) is known to possess antifertilityproperties in female. However, according to Iranian traditional medicine, theeffect of carrot on fertility is gender dependent and this food can increasethe potency in men. The aim of this study was to investigate the influence ofcarrot seed extract (CSE) on spermatogenesis, number and motility ofsperms in cauda epididyme in male rats.

Materials and Methods: Forty adult male rats were randomly divided into 5groups: normal group, groups receiving low- and high doses of CSE,animals that received high-dose of CSE with gentamicin, and gentamicintreated group. After 4 weeks treatment, serum samples were obtained forthe sex hormone analysis. Under anesthesia, testis, cauda epididymides andsperm ducts were dissected and sperm count, motility and caudaepididymis sperm reserves were determined. Histopathological changes oftestis were also studied to assess spermatogenesis. Data analysis wasperformed using one-way ANOVA followed by Tukey HSD tests.

Results: Administration of CSE caused a significant increase in the caudaepididymal sperm reserves (CESR) compared with the control (28.2 ± 1.8vs. 45.1 ± 2.0, ↔106). The extract could also protect testis from thegentamicin-induced necrosis. The CSE administration caused about 3.5-times increase in the LH levels of the rats even in spite of receiving 5mg/kg/day gentamicin with no significant effect on FSH levels. Thetestosterone concentrations in the group received 400 mg/kg CSE were 30%and 83% higher than its levels in the control and the gentamicin treatedgroup, respectively.

Conclusion: CSE is able to overcome reproductive toxicity of gentamicin andinduces spermatogenesis probably mainly through the elevation oftestosterone levels. It appears that this extract has opposite effects on maleand female reproductive systems.

P.095 – THE HISTOPATHOLOGIC EFFECTS OF ELECTROMAGNETIC FIELD ONPREIMPLANTATION PHASE OF MOUSE OVARY

Farzad Rajaei, Fatemeh Sabbagh Ziarani. Qazvin University of MedicalSciences, Qazvin, Iran.

Introduction: Life on earth has evolved in a sea of natural electromagneticfields (EMFs). Human data reviewed concern the potential reproductiveeffects (mainly, spontaneous abortions, low birth weight, and congenitalmalformations) of exposure to various sources of EMFs.

Materials and methods: 80 female mice were divided in to 2 groups. Controlgroup was not exposed to EMF and case group was exposed to 4 hours perday, 6 days a week for 2 weeks to 50 Hz & 0.5 mT EMF. Female mice in bothgroups on 8th day of exposing were superovulated and mated over thenight. Next morning females with a vaginal plug were identified as pregnantmice; at the time of implantation, the pregnant mice were killed andblastocysts were subsequently obtained from these mice by flushing theuterus horns. The samples of ovaries in all groups were taken and wereprocessed for light microscopic studies. The data have been comparedusing statistical methods (SPSS, t test and P<0.05).

Results: Results showed that the mean number of pregnant mice decreasedin EMF group) 50%) compared to the control group) 67.5 %( but thedifference between them was not significant. The mean number of fetusesper pregnancy was (9±4.8) in control group and (5.5±5.7) in experimental

April 19-22, 2009 98


group and statistical analysis were showed significant decrease betweenmean of 2 groups (P<0.03). The analysis of the size of monolayer primaryfollicle in EMF exposed groups did not show significant decrease in compareto control group (107.22±13.39, 105.86± 15.63 and P>0.810). Although thetotal number of follicles, number of monolayer primary follicle & corpusluteum, increase in compare to control group respectively but there was nosignificant differences between them.

Conclusion: The findings indicated that the EMF in short period of exposurehas negative effects on female mice fertility but histological studies show nonegative effects on ovaries.

P.096 – RELATIONSHIP BETWEEN SERUM ESTRADIOL LEVEL AND OOCYTEAND EMBRYO QUALITY FROM ICSI TRIALS

S. Masadnah, M.A Danfour, O.A.,Elsrait, Mohamed S. Elmahaishi. MisurataInfertility Centre, Misurata, Libya.

Oocyte quality affects early embryonic survival, the establishment andmaintenance of pregnancy, fetal development, and even adult disease.Quality, or developmental competence, is acquired during folliculogenesis asthe oocyte grows, and during the period of oocyte maturation. Assistedreproductive technologies involving ovarian hyperstimulation, perturb thisprocess and result in oocytes with reduced quality. The aim of the studywas to investigate the influence of serum oestradiol level in women whounderwent intracytoplasmic Sperm Injection (ICSI) program oocyte andembryo production quality.

Study Design: Retrospective, comparative.

Material and Methods: 600 patients were included. There were divided inthree groups depending on the serum oestradiol level: Group A:(<300pg/mL, group B: 300-500 pg/mL and group C: > 500 pg/mL). Therapeuticprotocol. All women were ≤42 years of age (average age 33; range 25-42.Ovarian stimulation was performed with gonadotrophins (Puregon &Menogon or Minipure) (short protocol) following down regulation of thepituitary with a gonadotrophin releasing hormone agonist (Gonopeptyl).When the leading follicle reached a mean diameter of 18-20 mm and serumoestradiol appeared adequate, human chorionic gonadotrophin (hCG,10,000 IU) were injected.

Statistical analysis: (SPSS 11) with one way ANOVA and person correlationwas used to analyze the patients age, number of metaphase II oocytesretrieval, oocyte quality, oocyte maturation, oocytes fertilized, embryocleavage and number, quality of transferred embryos and and serum βhCGamong three groups.

Results: There were statistically significant correlation between oocytenumber, serum βhCG among three groups and estradiol level, however therewere no correlation between serum oestradiol level and oocyte quality,oocyte maturation, oocytes fertilized, embryo cleavage and number, qualityof transferred embryos among three groups.

Conclusion: These retrospective data demonstrated that oestradiol serumlevel do not affect the quality of oocyte, potential fertilization and embryodevelopmental competence, however it may affect implantation level.Therefore serum oestradiol level may predict ICSI outcome and consideredas prognostic value.

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EXHIBIT DIRECTORY

FLOOR PLAN

April 19-22, 2009 100


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Exhibit Hours

Sunday, April 19 18:00-20:00 Official Welcome ReceptionMonday, April 20 08:30-18:00Tuesday, April 21 08:30-18:00Wednesday, April 22 08:30-18:00

EXHIBIT DIRECTORY

List of Exhibitors in Alphabetical Order

CCoommppaannyy NNaammee BBoooott hh NNuummbbee rr

CCD Lab 19

Cryo Bio Systems 11

Eppendorf AG 17

European Sperm Bank 14

Ferring Pharmaceuticals 32-33

GE Healthcare 38

Gynemed GmbH 24

IBSA Laboratories 15-16-27-28

Irvine Scientific 10

IUL Instruments/Ruskinn Life Sciences 8-9

IVF Online 18

ISIVF & IVM Behind Registration

Jaypee Brothers Medical Publishing 12

Karl Storz GmbH & Co KG 36-37

McGill Symposium Behind Registration

MediCult 31

Merck-Serono 42-43-44

MTG - Medical Technology Vertriebs-GmbH 30

Nanopoint Imaging 26

Nikon 39

Olympus Life Science Europa GmbH 34

Research Instruments Ltd. 6

Schering-Plough 40-41

Smiths Medical 25

Sparmed APS 5

Unisense 35

Vitrolife 7

Wisepress

April 19-22, 2009 102


CCD INTERNATIONALBOOTH: 19 Mrs GIBAULT Catherine48, rue des Petites Ecuries75010 ParisFrance

Telephone: +33 1 34 44 15 15Fax: +33 1 30 72 22 08E-Mail: [email protected]: www.ccdlab.comC.C.D. International has a long-standing tradition of developing medicaldevices for IVF and general OB/GYN practice. Our core competences includeextensive experience in the design, development and manufacturing ofcatheters and needles with state of the art, medical grade materials. Ourscientists and engineers bring extensive skills and broad experience towarddelivering the utmost in quality, design performance, reliability and value.Many C.C.D. devices are worldwide gold-standards such as Frydman®catheters, Echogyn® Embryoview®, Pipelle® by Dr Cornier, Pipelle® MarkII, H Pipelle®, S.I.S. Rudigoz catheter. Our new release, the ART of CCD®line for oocyte retrieval, has been designed to meet the most demandingneeds of experts in assisted reproductive techniques.

CRYO BIO SYSTEM BOOTH: 11Mrs. Béatrice Ledos29 rue Tronchet75008 ParisFrance

Telephone: +33 (0)149 240 505Fax: +33 (0)149 240 501E-mail: [email protected]: www.cryobiosystem-imv.comCryo Bio System, the expert in cryopreservation with its CBS™ HighSecurity straw concept, is now worldwide recognized for its HSV HighSecurity Vitrification Kit, a reliable, fast and secure device for oocyte andembryo vitrification. Cryo Bio System also manufactures IUI/ET catheters andbrand-new equipment for the ART and Biobanking markets.

EPPENDORF AG BOOTH: 17Dr. Heide Niesalla Barkhausenweg 122339 Hamburg Germany

Telephone: +49 40 53801 - 0Fax: +49 40 53801 - 556E-mail: [email protected]: www.eppendorf.comEppendorf is a biotechnology company that develops, manufactures anddistributes systems comprising instruments, consumables and reagents foruse in laboratories worldwide. The product range includes systems for cellmanipulation and microinjection, liquid handling, centrifugation as well ascomplete equipment for DNA amplification and biochips.

EUROPEAN SPERM BANKBOOTH: 14Dr. Peter Bower, Ph.D.Falkoner Alle 63, 2DK-2000 Frederiksberg, CopenhagenDenmark

Telephone: +45 38343600Fax: +45 38343646 E-mail: [email protected] Website: www.europeanspermbank.comThe European Sperm Bank is among the leading sperm banks in Europe. TheEuropean Sperm Bank offers patients a choice of highly screened donorsperm and to have it shipped to their clinic. We offer both Anonymous andOpen Identity donors, and all of our donors have IUI-ready samples.

FERRING PHARMACEUTICALSBOOTH: 32-33Ms. Elisabeth WeisChemin de la Vergognausaz 50CH-1162 Saint-PrexSwitzerland

Telephone: +41 58 301 00 00Fax: +41 58 3010371E-mail: [email protected]: www.ferring.comFerring is a Swiss-based research driven, specialty biopharmaceutical groupactive in global markets. The company identifies, develops and marketsinnovative products in the areas of fertility, gynecology, endocrinology,gastroenterology and urology. In recent years Ferring has expanded beyondits traditional European base and has offices in over 40 countries. To learnmore about Ferring or its products, please visit www.ferring.com and cometo the FERRING booth.

EXHIBITOR DETAILS

Geneva103


GE HEALTHCAREBOOTH: 38Mrs. Martina EmdeBeethovenstrasse 239D-42665 SolingenGermany

Telephone: +49 212 2802258Fax: +49 212 2802439E-mail: [email protected]: www.med.ge.comGE is dedicated to helping you transform healthcare delivery by drivingcritical breakthroughs in biology and technology. Our expertise in medicalimaging, medical diagnostics, patient monitoring systems and drugdiscovery is enabling healthcare professionals around the world discovernew ways to predict, diagnose and treat disease earlier. For additionalinformation visit: www.gehealthcare.com

GYNEMED MEDIZINPRODUKTE GMBH & CO. KG BOOTH: 24 Mrs. Andrea AndresenRadeberstr. 21D-23738 LensahnGermany

Telephone: +49/4363/903290Fax: +49/4363/9032919E-Mail: [email protected]: www.gynemed.deGynemed presents the GM501 media line, developed for handling andculture of human germ cells and embryos with biggest success. The GM501line offers a complete range of media needed in the IVF-lab. The media meetthe latest scientific cognitions and are established as standard in many labsworldwide.

IBSA INSTITUT BIOCHIMIQUE SABOOTH: 15-16-27-28Mrs. Elisabetta Artioli, Pharm. D.Via del PianoCH- 6915 Pambio Noranco, LuganoSwitzerland

Telephone: +41-58-3601624Fax: +41-58-3601647E-mail: [email protected]: www.ibsa.chIBSA is an international pharmaceutical company headquartered in Lugano,Switzerland, delivering tailored therapeutic solutions for follicular stimulationand luteal support. IBSA’s whole in-house manufacturing cycle provideshighly purified and highly glycosylated, human-derived gonadotrophins suchas hFSH (Fostimon), hMG (Merional), hCG (Choriomon). Other company'sfranchises include osteoarthritis, pain-management, dermatology and thyroiddiseases.

IRVINE SCIENTIFICBOOTH: 10Ms. Kiersten Carlin2511 Daimler Street Santa Ana, CA, 92705USA

Telephone: +1 949 261-7800Fax: +1 949 261-6522Website: www.irvinesci.comIrvine Scientific is the world leader in providing a complete line of media andsupplies for Assisted Reproductive Technologies. The success of ourcustomers is a direct result of our commitment to quality, consistency andreliability. Irvine Scientific is the exclusive U.S. distributor for Wallaceneedles and catheters.

IUL INSTRUMENTS GMBHBOOTH: 9Königswinterer Str. 409aD-53639 KönigswinterGermany

Telephone: +49-02223-9192-26Website: www.iul-instruments.deIUL Instruments GmbH is a leading provider of high quality laboratoryequipment to life science customers in Germany, Switzerland and Austria.We pride ourselves on our scientific expertise in microbiology, cell biologyand biotechnology and on our excellent support of customers. Ruskinn andIUL Instruments will be happy to demonstrate the Ac-tive™ IVF System for

ART laboratories at booths 8 & 9.

RUSKINN LIFE SCIENCES LTD.(IN COOPERATION WITH IUL INSTRUMENTS GmbH)BOOTH: 8Website: www.ruskinn.comRuskinn is one of the world’s leading suppliers of modified atmosphereworkstations. In July 2008, Ruskinn introduced the Ac-tive™ IVF System, atotally enclosed ART Workstation where all manipulations, embryo cultureand microscopic examinations can take place. Ac-tive™ significantly reducesgamete and embryo stress, thus increasing pregnancy rates.

IVFONLINEBooth: 18

Telephone: +1 519-826-5800Fax: +1 519-826-6947 Website: www.IVFonline.comLifeGlobal®: Increased worldwide success of global media; the onlyscientifically-based and clinically-proven single culture medium. SunIVF®:New dishes for larger volume culture. GPS™, Corral®, Universal - the safestdishes for IVF Coda®: New ECO Tower; continued success of Coda®Inlines - good standard practice. Free Fertility Magazine.

EXHIBITOR DETAILS

April 19-22, 2009 104


JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD.BOOTH: 12Mr. J.P. Vij4838/24, Ansari Road, DaryaganjNew Delhi 110 002India

Telephone: +91-11-43574357Fax: +91-11-43574314E-mail: [email protected]: www.jaypeebrothers.comJaypee Brothers is a medical publisher who publishes maximum obgynbooks. So far they have published about 200 titles exclusively in Obstetrics& Gynecology. They are a very high quality book publisher and the pricesare within the reach of the doctors and that is the secret of the popularity ofJaypee’s Obgyn books in the world.

KARL STORZ GMBH & CO. KG BOOTH: 35-36Ms. Sigrid LanzillottiMittelstrasse 8 D-72538 TuttlingenGermany

Telephone: +49 7461 7080Fax: +49 7461 708 105 E-mail: [email protected]: www.karlstorz.deKARL STORZ is a renowned manufacturer that is well established in all fieldsof endoscopy. The still family held Company has grown to one with aworldwide presence and 4000 employees. KARL STORZ offers a range ofboth rigid and flexible endoscopes for a broad variety of applications.

McGILL SYMPOSIUM, IVM & EGG FREEZINGWORKSHOPS

Telephone: +1-514-843-1729 Fax: +1-514-843-1476 E-mail: [email protected] Website: www.mcgillsymposium.com Learn first-hand how to incorporate IVM within your practice. Discover thetechniques of immature oocyte retrieval and identification. Acquire the latestknowledge of Cryobiology techniques performing vitrification of mouseoocytes using the McGill Cryoleaf. Participants will be able to implement thelearned techniques into their programs upon completion of this course.

MEDICULT BOOTH: 31Monsieur Denis AZRA, MediCult France 1 rue des VergersF-69760 LimonestFrance

Telephone: + 33 472 564 800Fax: + 33 472 564 801E-mail: [email protected] is a leader in delivering leading innovative ART solutions to thebenefit of families. Through innovation and product advancements, MediCulthas acquired in 2007 Humagen and in 2008 MidAtlantic Diagnostics;MediCult aims to help the # 1 dream of every infertile couple come true.

MERCK SERONOBOOTH: 42-43-44Ms. Mel LewisTelephone: +41 22 414 4778Fax: +41 22 414 3003E-mail: [email protected]: www.merckserono.com or www.merck.de

Merck Serono is the division for innovative prescription pharmaceuticals ofMerck, a global pharmaceutical and chemical group. Headquartered inGeneva, Switzerland, Merck Serono discovers, develops, manufactures andmarkets innovative small molecules and biopharmaceuticals to help patientswith unmet medical needs. Its North American business operates in theUnited States and Canada as EMD Serono.

Merck Serono has leading brands serving patients with cancer (Erbitux®),multiple sclerosis (Rebif®), infertility (Gonal-f®), endocrine and metabolicdisorders (Saizen®, Serostim®, Glucophage®, Concor®, Euthyrox®,Kuvan®).

With an annual R&D expenditure of around € 1bn, Merck Serono iscommitted to growing its business in specialist-focused therapeutic areasincluding neurodegenerative diseases, oncology, fertility and endocrinology,as well as new areas potentially arising out of research and development inautoimmune and inflammatory diseases.

MTG MEDICAL TECHNOLOGY VERTRIEBS-GMBHBOOTH: 30Opalstrasse 32D-84032 AltdorfGermany

Telephone: +49 871 97519-0Fax: +49 871 97519-70Website: www.mtg-de.comMTG – Your expert in advanced A.R.T. technology. We will be displaying pHOnlineTM for continuous pH recording from the incubator as well as themajor components of the integrated OCTAX platform: OCTAX Laser Shot™,OCTAX polarAIDETM for comprehensive spindle and zona analysis, OCTAXcytoScreenTM for real-time zoomed sperm analysis (“dry IMSI”).

EXHIBITOR DETAILS

Geneva105


NANOPOINT, INC.BOOTH: 26Ms. Cathy Owen900 Fort Street Mall, Suite A20Honolulu, Hawaii, 96813USA

Telephone: +1-808-457-1145Fax: +1-808-537-4245E-mail: [email protected]: www.nanopointimaging.comNanopoint, Inc. is a privately-held biotechnology company that is advancingthe study and treatment of diseases with its live cell imaging solutions.Nanopoint's cellTRAY® Imaging Systems with integrated microfluidics havebroad applications to life science research, drug discovery, and assistedreproductive technology.

NIKON INSTRUMENTS AGBOOTH: 39Mr. Lukas JuferIm Hanselmaa 10CH-8132 Egg/ZHSwitzerland

Telephone: +41 43 277 28 67Fax: +41 43 277 28 61E-mail: [email protected]: www.nikon.chFrom being instrumental in the first IVF births, to developing latesttechnologies, two core values are at the heart of Nikon’s IVF offerings:commitment to optical excellence; and an ethos of developing embryo-friendly workflows. Please come and see how Nikon’s latest developmentscould revolutionise embryology and remove stress: from the embryo’s andfrom the embryologist!

OLYMPUS LIFE SCIENCE EUROPA GMBHBOOTH: 34 Dr. Friederike LehmannWendenstrasse 14-18D-20097 HamburgGermany

Telephone: +49 40 23773 5406Fax: +49 40 23773 4647E-mail: [email protected]: www.microscopy.olympus.eu/microscopes/

Total clarity; total comfort Olympus is displaying the advanced IX71 invertedmicroscope system for easy and comfortable cell imaging. With a uniquetwo-tiered multi-port design for maximum flexibility, the IX71 gives brighterimages and modes for fluorescence, DIC, phase contrast and Olympus ReliefContrast. Also on display: micromanipulators and microinjectors from

Eppendorf.

RESEARCH INSTRUMENTS LTD.BOOTH: 6Mr. Justin RetallackBickland Industrial ParkFalmouth, Cornwall, TR11 4TAUnited Kingdom

Telephone: +44 (0) 1326 372753Fax: +44 (0) 1326 378783E-mail: [email protected]: www.research-instruments.comResearch Instruments will be displaying our Saturn Laser system, Integra Timicromanipulation system, new EZ-Strip denudation system and a range ofpipettes. Also on show is a complete range of environmental monitors, aswell as the IVF Witness automated gamete tracking system and IVF Trackerfor tracking consumables.

SCHERING-PLOUGHBOOTH: 40-41Mr. Ralph ZürcherESSEX Chemie AGWeystrasse 20CH-6006 Lucerne SwitzerlandTelephone: +41 79 358 03 18Fax: +41 41 368 49 36E-Mail: [email protected]: www.schering-plough.com/Essex Chemie AG, based in Lucerne, is the Swiss country operation ofSchering-Plough Corporation (S-P), based in Kenilworth, New Jersey, USA.In Switzerland Essex Chemie AG markets a broad range of innovative humanprescription products with the emphasis on allergology, anesthesia, CNS,dermatology, fertility, gynecology, hepatology, cardiovascular, immunology,mycology and oncology. Through its own biopharmaceutical research andcollaborations with partners, S-P creates therapies that help save andimprove lives around the world. The vision of the company is to “Earn Trust,Every Day” with the doctors, patients, customers and other stakeholdersserved by its colleagues around the world.

EXHIBITOR DETAILS

April 19-22, 2009 106


SMITHS MEDICAL INTERNATIONAL LTD.BOOTH: 25Miss Sarah FryPortex House, Military RoadHythe, Kent, CT21 6JLUnited Kingdom

Telephone: + 44 1303 236936Fax: + 44 1303 236777E-Mail: [email protected]: www.smiths-medical.comSmiths Medical worked with the pioneers in IVF in the design andmanufacture of the Wallace Classic Embryo Replacement Catheter and isassociated with some of the highest pregnancy rates in the world. SmithsMedical also manufactures a Single and Dual Lumen Oocyte RecoveryNeedle range.

SPARMEDBOOTH: 5Mr. Onur OzturkFarum Gydevej 89 DK-3520 FarumDenmark

Telephone: +45 39 40 25 03Fax: +45 39 40 25 64E-Mail: [email protected]: www.sparmed.dkSparmed is a Danish company which is worldwide exclusive distributer forIVFtech’s Oosafe® brand products such as Oosafe® Filter and Oosafe® AirCleaner. We aim to bring new products to ivf market cooperating withIVFtech which is the manufacturer of ivf workstations, anti-vibration tables,mini incubators and heating systems for ivf laboratory use. Sparmed is aturnkey IVF laboratory projects and clean air solutions expert.

UNISENSE FERTILITECH A/SBOOTH: 35Ms. Francesca BahrBrendstrupgaardsvej 21FDK-8410 AarhusDenmark

Telephone: +45 89449525Fax: +45 89449549E-mail: [email protected]: www.unisense.comUnisense Fertilitech A/S presents the EmbryoScope™ Advanced EmbryoIncubator, which allows time-lapse image acquisition of up to 72 developingembryos while maintaining a safe and fully controlled incubatorenvironment. The EmbryoScope™ Incubator will facilitate selection of the

best viable embryo, while minimizing disruptions to embryo development.

VITROLIFE SWEDEN ABBOOTH: 7Ms. Sara AureliusP.O. Box 9080SE-400 92 GöteborgSweden

Telephone: +46 31 721 80 00Fax: +46 31 721 80 90 Website: http://www.vitrolife.com We have over 25 years of experience in developing high quality products forART covering all steps of an IVF-treatment. Welcome to our booth to learnmore about the latest news in our product portfolio. Get the latest data onthe G5 Series™ and take a closer look at - Swemed Sense™ - a new needledesigned to minimize tissue damage, bleeding and patient discomfort.

WISEPRESS ONLINE BOOKSHOPThe Old Lamp Works, 25 High Path, Merton AbbeyLondon, SW19 2JLUnited Kingdom

Telephone: +44 20 8715 1812Fax: +44 20 8715 1722E-mail: [email protected]: www.wisepress.comWisepress.com, Europe’s leading conference bookseller, has a completerange of books and journals relevant to the themes of the meeting. Bookscan be purchased at the stand or, if you would rather not carry them, postedto you – Wisepress will deliver worldwide. In addition to attending 250conferences per year, Wisepress has a comprehensive medical and scientificbookshop online with great offers, some up to 40% off the publisher listprices.

EXHIBITOR DETAILS

Geneva107


Abdallah, Michel Abou 401.4Abdollahi-Fard, Seddigheh P.001Abe, Hiroyuki P.002Abir, Ronit 306.2Aboulghar, Mohamed 404.4Ahuja, Kamal 404.2Ajina, Mounir P.006Akbarpou, Mahzad P.008Akdogan, Aysin P.009Albertini, David 208.3Alipour, Hiva P.079Alizadeh, Leila P.011Ambrosetti, Alexandra 206.6, P.012Amiri, Iraj P.013 Aniya, Tomoka P.014Aono, Fumihito P.015Arabzadeh, S. P.039Argyrou, Marina P.016Asherkaci, Hussian 410.1Azadbakht, Mehri P.017, P.018Azem, Foad 407.6Bahar, L. P.019Bahçeci, M. 210.6Balawi, M. P.020Barak, Yona 304.1Belaisch-Allart, Joelle 301.3Belloc, Stéphanie 309.3Ben-Yosef, Dalit 402.2Benkhalifa, Moncef 104.3, 207.2, 210.1, 406.1, P.021Bernard, Artur 303.1Bersinger, N. 206.5Bischof, Paul 201.3Boivin, Jacky 409.5Bouchard, Philippe 205.2Buckett, William 209.4Campbell, Keith 400.2Cha, Jeong-Ho P.081Chae, Soo Jin P.050Chian, Ri-Cheng 302.2Chillik, Claudio 404.3Cho, Hwang-Yun P.023Choi, Hye Won P.024Dal Canto, Mariabeatrice 405.3Danfour, Mohamed 211.1de Geyter, Christian 401.1Demirol, Aygül 204.5, 210.4, 302.3Dogan, Muammer P.071Dokuzeylul, Nur P.025, P.026Donnez, Jacques 100.1, 300.1, 305.3Dor, Jehoshua 302.1Dorfmann, Andrew 410.6Dreyfus, Jean-Michel 407.7Dubuisson, Jean-Bernard 100.2, 403.2El-Garem, Yehia P.027Elmahaishi, Mohamed S. P.096Elsraite, Omar P.028Es-slami, Samira P.029Fabbri, Raffaella 410.3Fadini, Rubens 208.4Fanchin, Renato 408.4

Fancsovits, P. P.030Farimani, M. P.082Feki, Anis 102.3, 402.1Feldman, Lisa 105.2Fenichel, Patrick 408.3Ficicioglu, Cem P.031, P.032Fındıklı, Necati 210.2Frydman, Nelly 101.3, 209.2Frydman, René 101.1, 205.4, 311.2Fulka Jr., Josef 208.1Genazzani, Andrea Riccardo 309.6Germond, Marc 301.4Ghahremani, Manda 312.7Gianaroli, Luca 311.3Gilbert, Lucy 305.1Gneist, Nadja 211.4Göker, Ege 210.3Gomel, Victor 100.3, 403.4Gougeon, Alain 300.2Gülekli, Bülent 310.1Gürgan, Timur 104.2, 204.2Gutnisky, Cynthia P.033Haloob, A. Rahim 312.4Hammadeh, M.E. 207.6, P.034Hanck, Beverly 409.3Handyside, Alan 101.2, 200.1, 203.2, 203.3Haruki, Atsushi P.035Hassani Bafrani, Hassan P.036, P.037Holzer, Hananel 405.2Hovatta, Outi 102.1, 105.4, 209.1, 400.1Ida, Mamoru P.040Imthurn, Bruno 209.3Ishizuka, Bunpei 205.1Jayakrishnan, Kay 211.6, P.044Jones, Keith T. 405.1Josso, Nathalie 408.2Kaluarachchi, Athula 407.1, 407.2Kamrava, Michael 312.3Kang, Hee Jung P.042, P.043Kato, Keiichi 303.2Kato, Osamu 401.3Kazaryan, Liya P.045Keck, Gudrun P.046Keskes, L. P.087Kossowska-Tomaszczuk, K. 206.3Kostyuchek, Irina 211.2, P.048Kovacs, Peter 307.1Kuwayama, Masashige 307.3Kyono, Koichi 308.3Ledger, William 405.4Lee, Shaw Ni Amy P.049Leong, Milton 208.5Lessey, Bruce 201.2Leung, Peter C.K. 205.3Lim, Jin Ho 200.2Lindenberg, Svend 310.4López, Gemma P.051Macas, M. 206.1Mahmoudi, Reza P.052Mansour, Ragaa 406.2

PRESENTER INDEX

April 19-22, 2009 108


Marinakis, Gerasimos 211.5Mencaglia, Luca 403.3Mengistu, Meserat 105.3Mettler, Liselotte 204.4, 407.5Mikkelsen, Ann Lis 310.2Miyaki, Yukiko P.054Mılık, Semra P.090Morimoto, Yoshiharu 304.3, 306.3Movahedin, M. P.055Movassaghi, Shabnam P.091Mueller, Michael 100.4Munk, Mette 309.2Murdoch, Alison 404.1Nagaki, Miyuki P.056Nardo, Luciano 204.3Naumova, Anna K. 309.1Nazzaro, A. P.057Nouri, Mohammad P.093Nozha, Chakroun Feki 207.1Okubo, Tsuyoshi 410.5Ortega, Carolina P.060, P.061Osada, Hisao 407.4Pai, Hrishikesh 410.4Palermo, Gianpiero 306.4, 406.4Peluffo, Marina C. P.062Peters, Kathleen P.063Petignat, Patrick 204.1Pouly, J. Luc 208.2Prasad, Sudha 301.5Qiao, Jie 202.4Raine-Fenning, Nicholas 103.1, 202.3Rajaei, Farzad P.095Rajesh, Hemashree P.064Remohí Gimenez, José 408.1Roux, Christophe 312.2Ruvalcaba, Luis Arturo P.065Salerno, Annalisa P.066Sallam, Hassan 401.2Salman, Tarique 410.7, P.067Santi, Alessandro 309.7Schenker, Joseph 301.1Sermon, Karen 101.4Shoham, Zeev 201.1Shozu, Makio 304.4Silber, Sherman 305.2Smirnova, Anna 410.2Smith, Gary 306.1Smith, Venessa 207.3Son, Weon-Young 310.3Stadtmauer, Laurel 309.5Steiner, Hans-Peter 207.4Streuli, I. 206.4Swain, Jason 207.5Taha, Tamer Fouad 312.1Takefman, Janet 409.4Tan, Seang Lin 104.1, 300.3, 305.4Tanaka, Atsushi 308.2Tang-Pedersen, Mikael 312.6, P.069Tavmergen, Erol 210.5

Tepekoy, Filiz 309.4Tijani, H. P.010Tirelli, Alessandra P.070Tomazevic, Tomaz 407.3Tuong, H.M. P.038Tur-Kaspa, Ilan 201.4, 301.2, 103.2Ueno, Keiko P.072Ulug, U. 210.7Urner, F. 206.2Utsunomiya, Takafumi 307.2, P.073van der Poel, Sheryl 105.1, 409.1van Herendael, Bruno J . 403.1Vassena, Rita 402.3Veiga, Anna 102.2, 400.3Velleman, Ramon 308.1Verhaak, Chris 409.2Wakimoto, Eiko P.074Watrelot, Antoine 202.1Wenger, Jean-Marie 100.5Wolman, Igal 312.5Woodruff, Teresa K. 200.3Yang, Seong-Ho P.075Yaron, Yuval 311.1Yelian, Frank 304.5Yildirim, Atilla P.076Yong, Zeng P.053Yoshida, Atsumi 307.4Yoshida, Hiroaki 304.2Younas, Kinza 211.3, 211.7Young, Lorraine 203.1Zalel, Yuron 103.3, 202.2Zech, Herbert 308.4Zhang, John 303.3Zorn, Branko 406.3

PRESENTER INDEX

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