hugues jn macklon ns faculty nardo l www ...€¦ · 17:30 hcg, progesterone and lh levels during...

FACULTY

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Presidents of the Congress

Professor Marco FilicoriChairmanGynePro Medical Group, Bologna, Italy

Professor Giuseppe De PlacidoHeadReproductive Medicine UnitDepartment of Obstetrics and GynecologyUniversity of Naples “Federico II”, Naples, Italy

International Scientific Committee

Fleming R (UK)Hugues JN (FR)Macklon NS (UK)Nardo L (UK)Pellicer A (ES)Ubaldi F (IT)

E.C.M. • Continued Medical EducationThe congress participates in the Continued MedicalEducation National Program instituited by the ItalianMinistry of Health. The credits assignment is being processed.

Italian Scientific Committee

Caserta D (IT)Chiantera A (IT)Cicinelli E (IT)Colacurci N (IT)Colao A (IT)Crosignani PG (IT)Dessole S (IT)Genazzani AR (IT)Locci M (IT)Moscarini M (IT)Nappi C (IT)Perino A (IT)Petraglia F (IT)Pivonello R (IT)Volpe A (IT)Zullo F (IT)

Local Organizing Committee

Clarizia R (IT)Mollo A (IT)Ranieri A (IT)Strina I (IT)

ENDORSEMENTS

REGIONE CAMPANIAwww.regione.campania.it

COMUNE DI NAPOLIwww.comune.napoli.it

ALMA MATER STUDIORUMUNIVERSITÀ DI BOLOGNAFACOLTÀ DI MEDICINA E CHIRURGIAwww.unibo.it

UNIVERSITÀ DEGLI STUDI DI NAPOLI FEDERICO IIFACOLTÀ DI MEDICINA E CHIRURGIAwww.unina.it

SECONDA UNIVERSITÀ DEGLI STUDI DI NAPOLIwww.unina2.it

MIDDLE EAST FERTILITY SOCIETYwww.mefs.org

AGUIASSOCIAZIONE GINECOLOGI UNIVERSITARI ITALIANIwww.aguionline.it

AOGOIASSOCIAZIONE OSTETRICI GINECOLOGI OSPEDALIERI ITALIANIwww.aogoi.it

SIGOSOCIETA' ITALIANA DI GINECOLOGIA E OSTETRICIAwww.sigo.it

ORDINE DEI MEDICI CHIRURGHI E ODONTOIATRIDI NAPOLI E PROVINCIAwww.ordinemedicinapoli.it

MINISTERO DELLA SALUTEwww.salute.gov.it

SOTTO L’ALTO PATRONATO DELPRESIDENTE DELLA REPUBBLICA ITALIANAwww.quirinale.it

WE THANK

Ferring InternationalCenter S.A.Chemin de la Vergognausaz, 50CH - 1162 Saint Prex - SWITZERLANDPhone +41 58 301 00 00Fax +41 58 301 00 10www.ferring.com

IBSA Institut Biochimique S.A.Via del Piano, 116915 Pambio Noranco - SWITZERLANDPhone +41 58 360 1000Fax +41 58 360 1677www.ibsa-international.com

Merck Serono S.p.A.Via Casilina, 12500176 Roma (RM) - ITALYPhone +39 06 703841Fax +39 06 70384643www.merckserono.it

Merck Serono S.A.9, chemin des Mines, Case postale 54CH-1211 Geneva 20 - SWITZERLANDPhone +41 22 414 3000Fax: +41 22 414 2179www.merckserono.com

Schering-Plough S.p.A.Via Fratelli Cervi snc20090 Segrate (MI) - ITALYPhone +39 02 210181Fax +39 02 21018602www.schering-plough.it

Takeda Italia Farmaceutici S.p.A.Via E. Vittorini, 12900144 Roma (RM) - ITALYPhone +39 06 50260Fax +39 06 5011709www.takeda.it

Scientific Secretariat

Dr. Graciela E. CognigniDr. Lodovico ParmegianiGynePro Medical CentersVia T. Cremona 8I-40137 Bologna, ItalyTel. +39 051 442094Fax +39 051 [email protected]

Dr. Carlo AlviggiReproductive MedicineDepartment of Obstetricsand GynecologyUniversity of Napoli "Federico II"Via Sergio Pansini 5I-80131 Napoli, ItalyTel. +39 081 746 2699Fax +39 081 746 [email protected]

Organizing Secretariat

GynePro EducationalVia Lame, 44I-40122 Bologna, ItalyTel. +39 051 223260Fax +39 051 [email protected]

www.gynepro.it

Congress Venue

Royal Continental HotelVia Partenope 38/44I 80121 Napoli, Italywww.royalcontinental.itTel. +39 081 245 2068Fax +39 081 764 [email protected]

GENERAL INFORMATION

PROGRAM AT A GLANCE

Thursday Friday Saturday30 September 2010 1 October 2010 2 October 2010

morning

8:30-10:45 8:30-11:00Session 1I Session IV

ANOVULATION AND FSH FORMULATIONSMILD STIMULATION AND REGIMENS

10:45-11:15 11:00-11:30coffee break coffee break

11:15-12:45 11:30-13:00IBSA MERCK SERONO

SYMPOSIUM SYMPOSIUM

13:00-14:00 13:00-14:00lunch lunch

afternoon13:15-13:45

14:00-16:15 14:00-16:10Opening CeremonySession III Session V

13:45-14:30 GnRH LH ACTIVITY:Keynote Lecture ANALOGS FORMULATIONS

AND REGIMENS14:30-17:00Session 1

BASIC ASPECTS

17:00-17:30 coffee break

17:30-19:00FERRING

SYMPOSIUM

19:00Welcome Reception

at Castel dell’Ovo

14:00-16:00 14:00-15:45 FREE ORAL COMMUNICATIONS FREE ORAL COMMUNICATIONS

Session I Session II 16:15-17:00 16:10-16:40 coffee break coffee break

17:00-18:30 16:40-18:45

SCHERING-PLOUGH Session VI SPECIAL REGIMENS

SYMPOSIUM

18:45 Close of the Congress

Thursday • 30 September

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OPENING CEREMONY

13:15 Welcome and Opening of the CongressFilicori M (IT)De Placido G (IT)

13:45 KEYNOTE LECTUREThe ovarian lifecycle: from genesis to preservation (AB-00)Oktay K (US)

SESSION I BASIC ASPECTS

Sponsored by IBSA PHARMACEUTICALS

Chairpersons: Fleming R (UK), Di Carlo C (IT)

14:30 Genetics of folliculogenesis (AB-01)Raijkovic A (US)

14:55 Follicle and oocyte aging (AB-02)Artini PG (IT)

15:20 Reproductive hormone receptor dynamics in developing follicles(AB-03)Mason H (UK)

15:45 Androgens, follicle development, and oocyte quality (AB-04)Andersen CY (DK)

16:10 AMH, antral follicle count, and other parameters to predictCOS and ART outcome (AB-05)Broekmans FJ (NL)

16:35 The endometrium in ovarian stimulation (AB-06)Macklon NS (UK)

17:00 COFFEE BREAK

17:30 FERRING PHARMACEUTICALS SYMPOSIUM

GONADOTROPINS, BACK TO THE FUTURE?

Chairperson: Ubaldi F (IT)

17:30 hCG, Progesterone and LH levels during ovarianstimulation: the GOOD, the BAD an the UGLYPlatteau P (BE)

17:55 Progesterone and hCG driven LH activity in GnRHantagonist cyclesBosch E (ES)

18:20 Use of AMH to decide between GnRH agonists orantagonists in controlled ovarian stimulationFleming R (UK)

18:45 Discussion

19:00 WELCOME RECEPTIONat Castel dell’Ovo

AUDITORIUM

AUDITORIUM

AUDITORIUM

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SESSION II ANOVULATION AND MILD STIMULATION

Sponsored by MERCK SERONO PHARMACEUTICALS

Chairpersons: Nardo L (UK), De Leo V (IT)

08:30 Aromatase inhibitors (AB-07)Requena A (ES)

08:55 Metformin update (AB-08)Palomba S (IT)

09:20 Low-dose gonadotropins in anovulation (AB-09)Hugues JN (FR)

09:45 Controlled ovarian stimulation for intrauterine insemination(AB-10)Somigliana E (IT)

10:10 Minimal ovarian stimulation for ART (AB-11)Fauser B (NL)

10:45 COFFEE BREAK

11:15 IBSA PHARMACEUTICALS SYMPOSIUM

MANAGING PCOS PATIENTS IN ASSISTED REPRODUCTION

Chairpersons: Loutradis D (GR), Requena A (ES)

11:15 Choice of GnRH analog regimens for controlled ovarianstimulation in PCOS (SS-01)Orvieto R (IL)

11:40 Use of highly purified FSH in PCOS (SS-02)Grudzinskas G (UK)

12:05 Effects of in vivo and in vitro gonadotropinsupplementation in PCOS patients undergoing oocytein vitro-maturation (SS-03)Fadini R (IT)

12:30 Discussion

13:00 LUNCH

AUDITORIUM

AUDITORIUM

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AUDITORIUM

AUDITORIUM

SESSION III GnRH ANALOGS

Chairpersons: Hugues JN (FR), Colao A (IT)

14:00 Improving efficacy of GnRH antagonists (AB-12)Lambalk C (NL)

14:25 Pituitary suppression regimens in poor responders (AB-13)Sunkara SK, Braude P (UK)

14:50 Oral contraceptive pre-treatment in GnRH antagonist cycles(AB-14)Griesinger G (DE)

15:15 Final follicle maturation trigger with GnRH agonists (AB-15)Humaidan P (DK)

15:40 Luteal phase support of GnRH antagonist cycles (AB-16)Nardo L (UK)

14:00 FREE ORAL16:00 COMMUNICATIONS

SESSION I

16:15 COFFEE BREAK

17:00 SCHERING-PLOUGH PHARMACEUTICALS SYMPOSIUM

NEW PERSPECTIVES IN CONTROLLED OVARIAN STIMULATION

Chairpersons: Nappi C (IT), Perino A (IT)

17:00 Optimisation of rFSH protocols:a patient focused approach (SS-04)Alviggi C (IT)

17:25 Evolution and future of antagonist protocols (SS-05)Palermo R (IT)

17:50 Corifollitropin a novel long acting rFSH:clinical features and clinician’s perspective (SS-06)Devroey P (BE)

18:15 Discussion

RoomARAGONESECATALANA

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SESSION IV FSH FORMULATIONS AND REGIMENS

Chairpersons: Pal L (US), Benagiano G (IT)

08:30 FSH structure-function relationships (AB-17)Wide L (SE)

08:55 Design and action of hyperglycosilated recombinant FSHmolecules (AB-18)Lustbader JW (US)

09:20 Androgen priming before FSH ovarian stimulation (AB-19)Fleming R (UK)

09:45 Effects of high-dose FSH regimens (AB-20)Pal L (US)

10:10 Gonadotropin stimulation and follicular phase progesteronelevels: mechanisms and outcome (AB-21)Bosch E (ES)

10:35 Tridimensional automated ultrasound for monitoring ovarianstimulation (AB-22)Raine-Fenning N (UK)

11:00 COFFEE BREAK

11:30 MERCK SERONO PHARMACEUTICALS SYMPOSIUM

THE FUTURE OF CONTROLLED OVARIAN STIMULATION: FROM REPRODUCTIVE PHYSIOLOGY AND GENOMICS TO INDIVIDUALIZED THERAPY

Chairpersons: Lenzi A (IT), Levi-Setti P (IT)

11:30 Welcome and introduction

11:40 Role and responsibilities of LH during early folliculargrowth: gonadal and extra gonadal effects (SS-07)Andersen CY (DK)

12:00 Customized COS to patients needs (SS-08)Humaidan P (DK)

12:20 From Subjectivity to Objectivity in gametes/embryoselection (SS-09)Montag M (DE)

12:40 LH vs hCG in endometrial gene expression profile(SS-10)Horcajadas JA (ES)

13:00 LUNCH

AUDITORIUM

AUDITORIUM

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Saturday • 02 October

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AUDITORIUM

RoomARAGONESECATALANA

AUDITORIUM

SESSION V LH ACTIVITY: FORMULATIONS AND REGIMENS

Sponsored by FERRING PHARMACEUTICALS

Chairpersons: Pellicer A (ES), Aboulghar M (EG)

14:00 LH activity-containing gonadotropin preparations (AB-23)Ubaldi F (IT)

14:25 Orally active LH agonists (AB-24)Van de Lagemaat R (NL)

14:50 Clinical use of recombinant LH (AB-25)Alviggi C (IT)

15:15 hCG in ovarian stimulation (AB-26)Filicori M (IT)

15:40 LH activity supplementation during GnRH antagonist cycles(AB-27)Devroey P (BE)

14:00 FREE ORAL15:45 COMMUNICATIONS

SESSION II

16:10 COFFEE BREAK

SESSION VI SPECIAL REGIMENS

Chairpersons: Alviggi G (IT), Bosch E (ES)

16:40 Controlled ovarian stimulation in oocyte donors, fertilitypreservation, and oocyte banking (AB-28)Remohi J (ES)

17:05 Strategies to prevent ovarian hyperstimulation (AB-29)Pellicer A (ES)

17:30 Controlled ovarian stimulation in endometriosis (AB-30)Arici A (US)

17:55 Therapeutic strategies in poor responders (AB-31)Loutradis D (GR)

18:20 Controlled ovarian stimulation in PCOS (AB-32)Aboulghar M (EG)

CLOSING CEREMONY

18:45 Close of the CongressFilicori M (IT)De Placido G (IT)

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FREE ORAL COMMUNICATIONS

OC-01 Fixed versus flexible GnRH antagonist administration protocol in patients with polycysticovaries: effect on total gonadotropin dose and pregnancy ratesRuiz Flores F, GarcÌa-Velasco JA, Requena A

Effectiveness of pioglitazone for preventing severe ovarian hyperstimulation syndromeCastellotti D, Cambiaghi A

Flexible antagonists protocol for FIV-ICSILorente González J, De Andrés CM, RÌos Castillo JE, Martín Sances L, Povedano CanizaresB, Velasco Sánchez E, Ramírez Montilla F, Arjona Berral JE

The LH receptor insLQ variant is a negative factor in ARTFilippini G, Spalvieri S, Lucchi S, Suter T, Jemec M

Assessment of the value of adding LH activity during the late stages of follicular maturationin ICSI cyclesSharkawy S, Azab H, Elshahed M, Amer M, Al-Inany H

Mild ovarian stimulation with hp-hMG in IVF: results from a perspective controlled studyBecattini C, Rizzello F, Cammilli C, Fantaccini I, Quitadamo L, Zambelli V

Neurotrophine follicular fluids concentration in infertility womenPalumbo M, Teodoro MC, Gulino F, Rubbino G, Ciotta L, Leonardi E, Cantarella G, Bernardini R

Differential approach to controlled ovarian stimulation in poor responders undergoingassisted reproduction cyclesStrelko G

Metformin treatment before and during IVF or ICSI in PCOS women with BMI < 28 kg/m3:A prospective, multicenter, randomised, double-blind studyKjøtrød SB, Rasmussen PE, Holst-Larsen T, Mellembakken J, Thurin KA, Kouru K,Humaidan P, Morin PL

Effects of inositol on oocytes' quality in patients with polycystic ovary syndromePalumbo M, Ciotta L, Stracquadanio M, Pagano I, Carbonaro A, Gulino F

Are all human-derived FSH products the same? A systematic review and meta-analysisusing direct and adjusted indirect analyses to determine if Fostimon® is more efficientthan Metrodin-HP®

Al-Inany H, Abou-Setta A

A prospective randomized non-inferiority study comparing recombinant FSH (RFSH) andhighly purified menotrophin (HP-HMG) in intrauterine insemination (IUI) cycles in coupleswith unexplained infertility and/or mild-moderate male factorSagnella F, Moro F, Lanzone A, Tropea A, Capalbo A, Gangale MF, Morciano A, Apa R

New strategies for human oocytes selection in ICSI technique: standard protocol definitionthrough bioinformatics softwareCariati F, Alviggi C, Vaccina A, Pivonello R, Fiorentino A, Leo P, De Placido G, Colao A

Higher LH levels are associated to increased pregnancy rates when leuprolide acetate isused for ovulation inductionChehin M , Haddad J, Bertolla R, Motta E, Fraietta R

Follicular volume increases by 35 to 40 % from HCG administration to oocyte retrievalPalumbo A, Hernandez J, Garcia R, Taraborrelli S, Filicori M

OC-02

OC-03

OC-04

OC-05

OC-06

OC-07

OC-08

OC-09

OC-10

OC-11

OC-12

OC-13

OC-14

SESSION I • Friday • 01 October

SESSION II • Saturday • 02 October

14:00

14:15

14:30

14:45

15:00

15:15

15:30

15:45

14:00

14:15

14:30

14:45

15:00

15:15

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Chairpersons: Ciampaglia W (IT), Iaccarino S (IT)

Chairpersons: Fiorentino A (IT), Locci M (IT)

OC-1515:30

ABSTRACTS

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6th World Congress on Ovulation Induction - Naples, Italy • 30 September - 2 October 2010

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THE OVARIAN LIFECYCLE: FROM GENESIS TO PRESERVATIONOktay KNew York Medical CollegeDepartment of Obstetrics & Gynecology, Westchester Medical Center, Valhalla, NY, USAe-mail: [email protected]

Ovarian reserve, made up of presumably non-replenishable primordial follicles is subject to many environmental andiatrogenic insults such as oxygen radicals and chemotherapy. Furthermore, aging results in not only depletion of thisreserve but is also associated with diminished oocyte quality which is associated with meiotic errors, alteredembryogenesis, increased incidence of pregnancy failures, and birth defects. The mechanism underlying thediminishment of oocyte quality is also unknown. Fertility Preservation field has evolved to circumvent the aforementionedfactors impacting future reproductive potential of young individuals, who wish to preserve and defer their fertility.In the last decade, we witnessed a rapid proliferation and evolution of fertility preservation techniques. Add to theevolution of cryopreservation technologies, a new controversy arrived regarding the presence or absence of germ-stem cells and whether the human ovarian reserve can be replenished. While several rodent studies have shownevidence for the existence of germ stem cells in the ovary, human data are lacking. We have on the other handobserved some evidence from human ovarian transplantation cases, which may give support to oocyte regenerationin adult life. This lecture will update the audience on the most recent developments from germ cell generation tooocyte aging and fertility preservation.

AB-01

GENETICS OF FOLLICULOGENESISRajkovic ADepartment of Obstetrics, Gynecology and Reproductive ScienceGenetics Division, University of Pittsburgh, Pittsburgh, PA, USAe-mail: [email protected]

Premature ovarian failure (POF) is defined as cessation of menstruation and associated elevation of gonadotropinlevels (FSH 1 40 IU/l) as a result of decreased ovarian function prior to the age of 40. An estimated 1% of thepopulation is affected before age 40, with 0.1% affected prior to age 30.Although the causes for POF are many, the majority of POF cases have idiopathic etiologies. Mechanisms long invokedin the pathogenesis of POF include abnormalities of the X chromosome or autosomes and autoimmune, infectious,and environmental causes. For some cases, a genetic basis has been shown, and, for genes such as FMR1, FSHR,FOXL2, BMP15, NOBOX and FIGLA functional data support causation.Because human ovaries are not easily accessible, our knowledge of ovarian development is largely derived fromanimal models. Mouse knockouts and naturally occurring mutations in sheep have been useful in identifying candidategenes for ovarian failure. New genetic technologies, such as array CGH, genomewide association studies, and massiveparallel sequencing will give us further insight into the genetic etiology of POF.

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FOLLICLE AND OOCYTE AGINGArtini PG1, Papini F1, Tatone C2, Monteleone P1, Genazzani R11 University of Pisa, Pisa, Italy2 University of Aquila, Aquila, Italye-mail: [email protected]

Numerous evidences support the idea that the chief regulator of female reproductive aging is the ovary. Femaleageing is the most significant determinant of success in IVF. Ovarian functional decline with aging has been so farstudied in depth in terms of accelerated depletion of the ovarian follicle pool and reduced ability to produce oocytescompetent for fertilization and development. Few studies have addressed the question about potential causal factorsof ovarian aging. According to the most relevant concept of ageing, age-associated malfunction results fromphysiological accumulation of irreparable damage to biomolecules as an unavoidable side effect of normal metabolism.Moreover, it was suggested that an important environmental factor responsible for oocyte senescence might berepresented by a reduced oxygen supply to the leading follicle, a condition dependent on a compromised perifollicularvascularisation. Future investigation of age-related molecular damage in the different ovarian components isimperative in order to develop strategies to possibly save or rescue the developmental potential of aged oocytes.

AB-03

REPRODUCTIVE HORMONE RECEPTOR DYNAMICS IN DEVELOPING FOLLICLESMason HD, Pellatt L, Laing K and Rice SDivision of Basic Medical Sciences, St. George's, University of London, London, UKe-mail: [email protected]

Folliculogenesis is a remarkable process involving the unprecedented growth and selection of one ovulatory folliclefrom many which begin the journey. At the earliest stage at which follicles join this process the follicle consists onlyof an immature oocyte and its surrounding sphere of flattened granulosa cells and yet even at this stage, endocrinereceptors are expressed. Our knowledge of the control of the initiation and growth of primordial follicles has beenhampered by the technical limitations of working with such small amounts of material. Our new ability to amplifythese small amounts of follicular mRNA and to apply this to microarrays has the potential to reveal the mechanismscontrolling the slow release of follicles and their subsequent progression. We have shown that multilaminar andtransitional follicles have greater similarity in their transcriptional profile than primary follicles. The number of genesaltered at the primary stage was very much lower than at the other stages and all were reduced, suggestion a‘quiescent’ phase in the follicle’s path. Although traditionally considered to be gonadotrophin-independent, FSHreceptors have been described on early follicles and data from patients with inactivating receptor mutations havealso revealed the extent of the involvement of gonadotrophins in early folliculogenesis. Using nested RT-PCR wefound very few follicles in the early stages of development to be FSH receptor-positive and in fact androgen receptor-positive follicles were more common. In addition to the endocrine gonadotrophins, the involvement of paracrinefactors permits the fine-tuning of growth and selection. Two of the most important groups of factors with extensiveroles are the TGFβ family and the IGFs. The importance of this process is further highlighted by the recent demonstrationof other receptors which are able to determine environmental nutritional status such as those of adiponectin andleptin.


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ANDROGENS, FOLLICLE DEVELOPMENT, AND OOCYTE QUALITYAndersen CYLaboratory of Reproductive Biology, University Hospital of Copenhagen, University of Copenhagen,Copenhagen, Denmarke-mail: [email protected]

The two gonadotropins FSH and LH exert several diverse and different functions on their target cells in the ovary.Whereas FSH receptors (FSHr) are exclusively located on the surface of the granulosa cells, LH receptors (LHr) areconstitutively expressed on the surface of theca cells and become expressed on the granulosa cells as the folliclematures. FSH has an important function in inducing LH-receptor expression on the granulosa cells and thereby changegranulosa cells sensitivity to gonadotropins.Activation of the gonadotropin receptors stimulates a number of well-characterized intracellular signal transductionpathways, like the cAMP induced pathway. However, other signal transduction pathways are also involved and anumber of biological actions and their regulation are not fully understood, including initiation and regulation ofreceptor expression.Our activities in connection with cryopreservation of human ovarian tissue for fertility purposes has allowed thecollection of both follicular fluid and granulosa cells from a number of small antral human follicles with a diameterof 3 to 9 millimetres. The mRNA expression of FSHr, LHr, AMH-r2, Androgen-receptor (AR) and CYP19 has beendetermined in these granulosa cells by quantitative RT-PCR. These expression data will be correlated to the hormoneprofiles in the corresponding follicular fluid, including measurements of AMH, inhibin-B, oestradiol, progesterone,androstendedione and testosterone.In these naïve human small antral follicles we found a highly significant correlation between AR and FSHR expression.In addition, there was a highly significant correlation between follicular fluid concentrations of androgens and FSHRexpression. Collective these data confirm earlier studies on primates and suggests that stimulating the AR prior tocontrolled ovarian hyperstimulation with exogenous gonadotropins may augment follicular FSH responsiveness.Thus, the presentation will provide new data on the interactions between the expressions of gonadotropins receptorsin immature granulosa cells from women in their natural menstrual cycle in relation to expression of AR, AMH-r2and CYP19 and discuss possible interactions with follicular steroid regulation and production.

AB-05

AMH, ANTRAL FOLLICLE COUNT, AND OTHER PARAMETERS TO PREDICT COSAND ART OUTCOMEBroekmans FDivision Female and Baby, University Medical Centre Utrecht, Utrecht, The Netherlandse-mail: [email protected]

Ovarian hyperstimulation for IVF aims at producing multiple embryos of high quality in order to make the chancesof an ongoing pregnancy occurring as high as possible. Two distinct problems may arise when performinghyperstimulation: too low response with inherent reduction in pregnancy rates and too high ovarian response withthe threats of ovarian hyperstimulation syndrome developing. Both outcomes of the stimulation also bear the riskof cycle cancellation. Therefore that prediction of the outcome response in terms of number of oocytes is of greatimportance. In addition, predicting the outcome of the ART cycle or sequence of cycles in terms of ongoing pregnancyis another feature that allows clinicians to select patient for whom ART with own oocytes may not be the properchoice.In prediction of the outcome ovarian response two issues are keynote. One is that the relation between FSH doselevel and the response of the ovaries in terms of number of dominant follicles growing, has not been clearlyestablished. So far, evidence has shown that the dose response curve is very steep, indicating that with only smallincreases in FSH level single follicle growth is turned into maximal ovarian response. This implies that in the vastmajority of patients the dosage administered (150 IU and over) will provide maximal stimulation of the ovaries. Onlyfactors like high BMI or the presence of specific FSH receptor variants may contribute to the creation of FSH levelsthat lead to submaximal stimulation of the ovaries.Second, the use of maximal stimulation dosages as a rule implies that the ovarian response is not principally dependentupon the dose of FSH applied, but on the size of the cohort of the FSH sensitive antral follicles present in the ovariesat the time of stimulation. The antral follicle cohort size is fully determined by the ovarian reserve status of theindividual woman and expressed by female age and possibly by ovarian reserve tests. These two factors are thereforethe most important tools for response prediction.Predicting outcome ongoing pregnancy necessitates a marker that somehow depicts the increasing decline of follicleswith a competent oocyte with advancing ovarian ageing. As most markers relate to the quantity of follicles and therelation between quantity and quality of oocytes is not fully understood, tools that correctly identify very poorprognosis cases in ART have been difficult to identify.In poor response prediction several ovarian reserve tests (FSH, AFC, AMH) have shown to be accurate predictors.However, the added value to female age can be considered doubtful. Moreover, if a poor response is predicted, noclear options for altered management other than counselling are available. Especially, the application of higher FSHdosages in predicted poor responders is not evidence based. Applying ovarian reserve tests in actual poor respondersin the first IVF cycle may however help to classify the nature of the poor response and estimate the remaining chancesfor ongoing pregnancy. Especially if no other signs of reduced reserve are present prognosis is fairly good andcontinuation of IVF treatment justified.Hyperresponse to ovarian stimulation is more and more considered as a condition in which low quality or immatureoocyes are added to a basal number of best quality oocytes. Prevention of hyperresponse has been mainly basedon patient profiles, like very young age and PCOS, as well as the general use of modest dosing schemes not exceeding200 IU in first cycles. Recent review studies have shown that the correct prediction of hyperresponse is possible byusing tests like the AFC or AMH. Still, whether effective strategies to manage predicted hyperresponders can bedeveloped is currently not known.Identification of poor prognosis cases regarding chances of ongoing pregnancy has been notoriously difficult usingquantity markers like Basal FSH, AMH and the AFC, especially if female age is firstly used as baseline test. The addedvalue to knowing female age in individual data analysis (IMPORT (Individual Meta-analysis of Patient data for OvarianReserve Testing) study group) has shown to be absent. This implies that routine screening of ART patients prior tostarting treatment should be limited to response profiling.


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THE ENDOMETRIUM IN OVARIAN STIMULATIONMacklon NSUniversity of Southampton - Complete Fertility Centre Southampton, Princess Anne Hospital, Southampton, UKe-mail: [email protected]

As IVF success rates begin to plateau despite ongoing development in embryo culture and selection technologies,more attention is being given to understanding the role of the other participant in human implantation: theendometrium. Since the early days of IVF it has been understood that ovarian stimulation disrupts the luteal phase,and current regimens include hormonal support to sustain the early conceptus, and support implantation.However, only in recent years has the impact of ovarian stimulation on the integrity and function of the endometriumbecome understood.Initial reports of advanced development of the endometrium according to Noyes histological criteria indicated thatthere may be a problem. Clinical studies, showing higher implantation rates when embryos were transferred tooocyte donation recipients, in whom the endometrium has not been exposed to supraphysiological levels of sexsteroids, supported these observations. Elegant studies in rodents demonstrated that the duration of the windowof implantation is dependent on levels of estrogen administered in the luteal phase (Ma et al 2003). At a molecularlevel, ovarian stimulation has been shown to impact on integrin and e-selectin expression which are key factorssupporting apposition of the embryos prior to implantation. More recently, the focus has switched to the impactof ovarian stimulation on endometrial gene expression, and it has become clear that extensive gene expressiondysregulation occurs during the crucial window of implantation (Horecajas et al, J 2009).In addition to studies looking at the impact of ovarian stimulation on gene expression, our group has developed anon-invasive means of analyzing the protein content of endometrial secretions at the time of embryo transfer inIVF cycles. Using this technique, we have identified significant effect of ovarian stimulation on the expression of anumber of cytokines, chemokines, growth factors and signaling factors in the secretions of the endometrium , withwhich the embryo begins the molecular dialogue which will ultimately determine whether the embryo will implantsuccessfully or not. (Boomsma et al, 2010)The evidence for a detrimental effect of ovarian stimulation on endometrial receptivity is now clear, and raisesquestions as how best to ameliorate this effect. The move towards milder stimulation regimens, increasing use ofcryopreservation of embryos and transfer in a natural cycle are addressing this issue. However, adjunctive therapiesaimed at improving endometrial receptivity in IVF cycles have as yet not been shown to be beneficial.Ma et al. PNAS 2003 100:2963Horcajadas JA et al. J Clin Endocrinol Metab. 2008 Nov;93(11):4500-10Boomsma CM, et al, Fertil Steril 2010 (e-pub ahead of print)

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AROMATASE INHIBITORSRequena A1, García-Velasco JA1,21IVI-MADRID, Madrid, Spain2 Rey Juan Carlos University, Madrid, Spaine-mail: [email protected]

Aromatase is a microsomal member of the cytochrome P450. Aromatase converts androstenedione to estrone andtestosterone to estradiol. Therefore, aromatase inhibitors (AI) are able to inhibit the conversion of androgens intoestrogens by blocking the estradiol synthetase or human aromatase in a potent, specific, and reversible way.Although its capacity to reduce serum estradiol levels makes letrozole a great drug for postmenopausal women withbreast cancer, over the last years AI have been widely used in assisted reproduction due to the low estradiol levelsthat induce and it causes modifications in the hypothalamic–pituitary–ovary axis, including: (i) release of thehypothalamic–pituitary axis from estrogenic negative feedback and FSH secretion is increased, with the resultantstimulating effect on the growth of ovarian follicles.(ii) increase of intraovarian androgens secondary to aromatase inhibition.The third-generation AI includes two non-steroidal inhibitors, anastrozole and letrozole, and a steroidal agent,exemestane. Letrozole has been the most widely used in human reproduction. The dose used has been between 2,5to 5 mg per day during 5 days starting usually on day 2. At present, there is enough evidence to support that letrozolecontributes to follicular recruitment and development, and is as effective as other methods of ovulation induction.It has been indicated in PCOS women with good results and in ovulation induction for IUI.Other indications of AI have been:(i) In ART it has been recommended for low responder patients that would take advantage of the endogen secretionof gonadotrophins together with an increase of intraovarian androstenedione and testosterone concentrations thatcould improve the outcome results in this group of patients.(ii) AI has been also proposed to use during the luteal phase after an ART cycle. The administration of 2.5 mg ofletrozole during the luteal phase drastically reduces serum estradiol levels and restores LH secretion sooner. This maybe of interest for egg donors but also in patients at high risk of OHSS who freeze all their oocytes/embryos, or whocancel hCG administration to reduce the potential risk that high E2 levels.(iii) As coadjuvant treatment in oncological patients who are undergoing an ART cycle for fertility preservation. Inpatients with estrogen-dependent breast cancer, the addition of letrozole 5 mg/day to gonadotrophins in ovarianstimulation protocols decreased significantly the levels of estradiol without affecting oocyte quality, fecundationrate and number of embryos obtained. Therefore it is highly recommended the use of letrozole in these cases.Finally in the past there has been a controversy regarding the security of these drugs due to the publication of onestudy that found an increase of fetal anomalies when these drugs were used for ovulation induction. But cohortstudies do not show an increase of congenital malformations among offspring of mothers who conceived withletrozole treatment for infertility. Because of the short half-life of AI, the biological plausibility of the teratogeniceffects when these drugs are used in the early follicular phase can be discarded.


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METFORMIN UPDATEPalomba S, Falbo ADepartment of Obstetrics & Gynecology, University “Magna Graecia” of Catanzaro, Catanzaro, Italye-mail: [email protected]

Introduction: Metformin is an insulin sensitizer widely used for the treatment of patients affected by type-2 diabetesmellitus. More recently, since many women with polycystic ovary syndrome (PCOS) are insulin resistant, metforminwas introduced in clinical practice for the treatment of these patients. Other than its insulin-sensitizing action,metformin has other targets. In particular, data available in literature investigated and showed any beneficial effectof metformin not only for the treatment of all PCOS-related disturbances such as menstrual disorders, anovulatoryinfertility, increased abortion or complicated pregnancy risk, hyperandrogenism, endometrial, metabolic andcardiovascular abnormalities, but also for the prevention of the syndrome.Methods: A systematic review of the literature.Conclusions: In PCOS patients with anovulatory infertility and not previously treated, the administration of metforminplus CC is not better than monotherapy, thus the combined approach should be avoided as initial treatment ofanovulatory infertility. No specific recommendation can be given regarding the use of CC or metformin since thebest first-step option to induce ovulation in infertile anovulatory PCOS patients remains unclear and, probably,depends on the wishes of the patient. On the other hand, PCOS patients who have failed to ovulate with CC maybenefit from the addition of metformin. It is possible to hypothesize that metformin therapy would augment theinduction of ovulation in CC-resistant women because of its favourable change in androgens, gonadotropins, andinsulin, through mechanisms distinct from those of CC. It is plausible to assume that women with CC-resistancereceiving metformin have an increased response to CC secondary to an improved follicle steroidogenesis caused bythe effect of metformin administration.Certainly, metformin monotherapy seems to be more effective and cheaper than LOD for treating anovulatoryinfertile CC-resistant PCOS patients, and LOD should be reserved for inducing ovulation only in anovulatory PCOSpatients with other suspected and/or diagnosed factors of subfertility. At the moment, data on the efficacy ofmetformin pre-treatment before CC in CC-resistant patients are controversial and inconclusive. In patients whoreceived gonadotropins as treatment for anovulation, metformin addition increases the rate of mono-ovulationsreducing the risk of cancelled cycles, whereas in infertile PCOS patients scheduled for IVF cycles metformin co-treatment reduces the OHSS risk and, thus, its use should be planned for patients at high-risk for OHSS.

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LOW DOSE GONADOTROPHINS FOR ANOVULATIONHugues JNReproductive Medicine - Jean Verdier Hospital - University Paris XIII, Paris, Francee-mail: [email protected]

From experience gained in the monitoring of ovarian response to gonadotrophin therapy by Brown3 in 1978, it hasbeen possible to develop concepts regarding the mechanisms which operate during the normal menstrual cycle.Firstly, the concept of FSH “threshold” reflects that the ovary has a threshold requirement for FSH below whichfollicular development does not occur. Moreover, due to different sensitivity to FSH, developing follicles are notrecruited simultaneously during the FSH rise of the early follicular phase. Secondly, the concept of FSH “window”described by Baird1 results from the evidence that follicular growth lasts as long as the FSH level is above the individualFSH threshold. Consequently, the decrease in FSH plasma levels during the mid follicular phase ensures the closureof the window and restricts the number of developing follicles to a single preovulatory one which is preservedbecause of an increased sensitivity to FSH (Zeleznik12). These two concepts are critical to explain the process thatleads to ovulation of a single dominant follicle in humans.Additionally, it has been shown that administration of low doses of exogenous FSH to normo-ovulatory women candisrupt these mechanisms for single follicle selection and dominance by preventing a decrease in FSH and wideningthe FSH window. Administration of FSH in the early stage of follicular phase stimulates follicular recruitment butsustained FSH administration is required to maintain follicular growth (Lolis8; Schipper10).Therefore, the durationrather than the magnitude of FSH administration affects follicular development.These data provided the rationale for the development of the so-called “step protocols” in women with chronicanovulation.The step-up protocols have been firstly proposed for ovulation induction in normogonadotrophic anovulatory women,including polycystic ovary syndrome. It was postulated that a stepwise increment of exogenous FSH plays a key roleto select the most FSH-sensitive follicle, detected on ultrasound. The regimen of FSH increase is actually critical toprevent multifollicular recruitment. Indeed, the low dose protocol proved to be safer than the conventional protocol.Furthermore, as the duration of the initial FSH administration (14 versus 7 days) seems to be essential to preventfrom the risk of multifollicular development, the chronic low dose step-up protocol was eventually considered asthe safest and the most effective regimen of FSH administration in women with chronic anovulation (Homburg5).The reason to maintain the same dose for 14 days came from clinical trials who reported that the mean durationof FSH administration before triggering ovulation is about 2 weeks. Presumably, FSH receptor is up-regulated duringa long term FSH administration. However, despite such a wise FSH administration regimen, the rate of monofollicularovulation is only about 50 % and the risk of multiple pregnancy cannot be fully excluded.Furthermore, the physiological relevance of keeping constant the FSH dose once follicular selection is achieved hasbeen questioned because it is not in accordance with other evidence that FSH requirement is actually reduced asfollicular development proceeds to final maturation.


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In order to mimic physiology more closely, the principle of step-down protocol has been developed. After an initialloading dose of FSH to induce follicular recruitment, a decremental regimen of FSH administration is applied to leadto atresia most developing follicles. This protocol proved to be effective to simultaneously decrease plasma FSH levelsduring the late stimulation period and the number of medium-size follicles as well. However, it requires experienceand skill to adjust the FSH doses and to prevent atresia of the whole cohort.In line with this observation, we suggested to combine an initial stepwise administration of FSH and a subsequentdecremental dose regimen to better control the risk of multifollicular development. This “sequential” FSH administrationregimen confirmed the ability of the step down regimen to reduce the number of medium-size follicles and thepotential risk of overstimulation (Hugues6).Comparison of efficacy and safety between the step-up and step-down FSH protocols has been performed in fewclinical trials. In a prospective randomised study (Van Santbrink11), both protocols gave comparable clinical outcomeswhile a reduced stimulation period was observed when using the step-down regimen. In contrast, in a largermulticenter study (Christin Maitre4), the low dose step-up protocol proved to be safer than the step-down one.Reasons for these discrepancies may be related to different patients‘characteristics, such as the presence ofhyperandrogenaemia and overweight which greatly affect the ovulation induction results.Collectively, step protocols proved to be quite effective to reduce the incidence of over-response. However, there isstill room for improvement and 2 major issues need to be addressed.The first deals with the issue of the optimal FSH starting dose. The measurement of circulating plasma FSH levels isnot able to adequately determine the FSH threshold. Therefore, a prediction model has been developed, based onclinical, endocrine and ultrasound characteristics of patients. It was shown that BMI, cycle history, ovarian responseto clomiphene citrate as well as basal FSH levels are significantly correlated with the individual FSH response (Imani7).Additionally, women with a high number of follicles and high plasma androstenedione levels upon initial screeningare at higher risk of multifollicular development (Mulders9). Therefore, an individual effective starting dose mustbe determined to better ensure ovulation of a single follicle. Nowadays, the antral follicular count should beintegrated in the predictive model for the starting dose because it proved to be effective in women stimulatedbefore insemination.The second issue is related to the potential benefit of LH supplementation. This issue has been addressed inhypogonadotrophic hypogonadism women known to be also at risk of multifollicular development. While it hasbeen established that the minimal daily effective dose of LH is about 75 IU to sustain steroidogenesis, the synergisticeffect of LH and FSH on folliculogenesis was still uncertain. Balasch2 has recently shown that a pre-treatment withr.hLH alone during 7 days actually increases the ovarian sensitivity to FSH as demonstrated by the reduction in themean daily effective FSH dose. These results attest for the potential “priming” role of LH in the determination ofFSH threshold.In conclusion, the optimal protocol for ovulation induction in women with chronic anovulation has been partlyestablished. However, a key issue remains the determination of the individual starting dose. While predictive modelshave been recently developed, it seems safer to recommend a “dose finding” low dose step-up protocol as the firstline therapy in order to determine the individual FSH dose response. In a subsequent cycle, a step-down regimenmay be applied with a starting dose slightly above the effective response dose. In every case, strict compliance tocancellation criteria is recommended to reduce the risk of multiple pregnancy.

Key references:1. Baird D. A model for follicular selection and ovulation: lessons from superovulation. J Steroid Biochem. 1987,

27: 15-23.2. Balasch J., Fabregues F, Carmona F et al. Ovarian Luteinizing Hormone priming preceding Follicle-Stimulating

Hormone stimulation: clinical and endocrine effects in women with long-term hypogonadotroic hypogonadism.J. Clin. Endocrinol. Metab. 2009, 94: 2367-2373.

3. Brown JB. Pituitary control of ovarian function - Concepts derived from gonadotrophin therapy. Aust. N.Z.J.Obstet. Gynaec. 1978, 18: 47-54.

4. Christin-Maitre S., Hugues JN. , on behalf of the recombinant FSH study group. A comparative randomizedmulticenter study comparing the step-up versus step-down protocol in polycystic ovary syndrome. Hum Reprod.2003, 18: 1626-1631.

5. Homburg R., Howles C.M. Low-dose FSH therapy for anovulatory infertility associated with polycystic ovarysyndrome: rationale, results, reflections and refinements. Hum. Reprod. Update 1999, 5: 493-499.

6. Hugues JN., Cedrin-Durnerin I., Avril C. et al. Sequential step-up and step-down dose regimen: an alternativemethod for ovulation induction with follicle-stimulating hormone in polycystic ovary syndrome. Hum. Reprod.1996, 11: 2581-2584.

7. Imani B., Eijkemans MJC, Faessen GH et al. Prediction of the individual follicle-stimulating hormone thresholdfor gonadotropin induction of ovulation in normogonadotropic anovulatory infertility: an approach to increasesafety and efficiency. Fertil. Steril. 2002, 77: 83-90.

8. Lolis DE., Tsolas O., Messinis IE. The follicle-stimulating hormone threshold level for follicle maturation insuperovulated cycles. Fertil. Steril. 1995, 63: 1272- 1277.

9. Mulders AM. Prediction of chances for success or complications in gonadotrophin ovulation induction innormogonadotrophic anovulatory infertility. Reprod. BioMed. Online. 2003, 7:170-178

10. Schipper I., Hop WC., Fauser BCJM. The follicle-stimulating hormone threshold/window concept examinedby different interventions with exogenous FSH during the follicular phase of the normal menstrual cycle: durationrather than magnitude of FSH increase affects follicle development. J.Clin. Endocrinol. Metab. 1998, 83: 1292-1298.

11. Van Santbrink EJP., Fauser BCJM. Urinary follicle-stimulating hormone for normogonadotropic clomiphene-resistant anovulatory infertility: prospective, randomized comparison between low-dose step-up and step-downdose regimens. J. Clin. Endocrinol. Metab. 1997, 82: 3597-3602.

12. Zeleznik AJ, Kubik CJ. Ovarian responses in macaques to pulsatile infusion of follicle-stimulating hormone andluteinizing hormone: increased sensitivity of the maturing follicle to FSH. Endocrinology, 1986, 119: 2025-2032.

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CONTROLLED OVARIAN STIMULATION FOR INTRAUTERINE INSEMINATIONSomigliana ECentro di Fertilità, Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italye-mail: [email protected]

The association of controlled ovarian hyperstimulation (COH) to intrauterine insemination (IUI) cycles appears toenhance the pregnancy rate. According to the two available Cochrane metanalyses on this point, the OR for pregnancyin unexplained infertility and male factor subfertility is 2.1 (95%CI: 1.2, 3.5) and 1.5 (95%CI: 0.9-2.4), respectively.The best protocol of treatment remains however to be determined. A major difficulty in this area is that the chancesof pregnancy of IUI is generally low (10-15%). The sample size of randomized controlled trials aimed at investigatenew protocols is consequently extremely high (more than 1000 couples) so that pivotal studies are in fact unavailable.Despite these difficulties, there is some evidence showing higher pregnancy rate with the use of gonadotropinsrather than antiestrogens. Moreover, a recent metanalysis also supports the benefits of adding GnRH antagonistsin gonadotropins-stimulated cycles to prevent spontaneous ovulation and better temporize the insemination.A major concern in this field is the risk of multiple pregnancies and in particular high-order multiple pregnancies.This new consciousness have lead to markedly reduce the dosages of drugs administered for COH (mild ovarianstimulation). Although this attitude is considered effective in preventing multiple pregnancies, it remains a matterof debate whether mild stimulation can maintain overall pregnancy rates.

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MILD OVARIAN STIMULATION FOR ARTFauser BDepartment of Reproductive Medicine and Gynecology, University Medical Center, Utrecht, The Netherlandse-mail: [email protected]

The history of IVF has been characterized by profound ovarian stimulation protocols, in an attempt to optimisepregnancy rates per cycle. These approaches, aiming at generating as many oocytes as possible, were meant tocounterbalance shortcomings in in vitro oocyte fertilisation, embryo culture conditions, as well as embryo selectionfor transfer. Another reason often put forward to justify profound stimulation is the cryostorage of surplus embryos,providing additional pregnancy chances in subsequent unstimulated cycles. Over the years ovarian stimulationprotocols have become extremely complex and time consuming, associated with much patient discomfort andconsiderable complication rates. Moreover, costs of applied medication may outweigh the cost of IVF treatmentitself.Mild IVF may involve mild ovarian stimulation, mild transfer policies (i.e. single embryo transfer in selected patients),or both. Both strategies result in a reduction in the pregnancy rate per cycle. Only when cryopreserved embryotransfer cycles are also included overall pregnancy rates become comparable in IVF units with good laboratoryperformance. The aim of milder forms of ovarian stimulation is to render stimulation less complex, less time consumingand less costly, while improving patient acceptability by reducing side effects and chances for complications. Reportedpregnancy rates per started cycle are also reduced following mild stimulation. However, mild stimulation improvethe cost effectiveness of IVF (hopefully resulting in augmented access to IVF) and may also reduce drop-out rates.Therefore, overall cumulative pregnancy rates of a given treatment strategy are likely to be similar as shown by somerecent studies. Certainly, the recent introduction of GnRH antagonist helped to reduce overall drug consumptionand considerably reduced the duration of stimulation. Therefore, more IVF cycles can be performed in a given periodof time when viewed from a per treatment rather then a per cycle paradigm.Further improvement of embryo selection and cryostorage is warranted for the wider acceptance of mild ovarianstimulation and single embryo transfer. Moreover, more individualized approaches may reduce both hyporesponse(and cycle cancellation) and hyperresponse and therefore further improve safety and efficacy of treatment. Hence,the focus of further development in ovarian stimulation should shift from mild stimulation towards mild ovarianresponse.


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IMPROVING EFFICACY OF GnRH ANTAGONISTSLambalk CBDivision of Reproductive Medicine, VU University Medical Center, Amsterdam, The Netherlandse-mail: [email protected]

Available systematic reviews comparing studies between GnRH agonists and antagonists for prevention of thepremature LH surge in IVF hormonal stimulation protocols performed so far, include likely less favourable GnRHantagonist regimens. After critical appraisal of the various studied GnRH antagonist regimens in terms of folliculardevelopment and IVF outcome, it is suggested that early suppression of endogenous FSH helps to optimize folliculardevelopment in terms of synchronous growth and quantity. A stable and early suppression of LH levels during theentire period of stimulation may be an advantage for implantation and pregnancy outcome. In this respect, singledose and particularly flexible protocols seem to be less advantageous. Early FSH and LH suppression can be achievedby early GnRH antagonist administration (from stimulation day 1 onwards) or by oral contraceptive (OC) pretreatment.Indeed a systematic review of reports comparing long protocol GnRH agonist versus fixed GnRH antagonist startingon stimulation day 6 shows equivalence of efficacy in terms of ongoing pregnancy. An adequately powered studycomparing standard GnRH agonist protocols with 'long' GnRH antagonist protocol is underway.

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PITUITARY SUPPRESSION REGIMENS IN POOR RESPONDERSSunkara SKAssisted Conception Unit, Guy's and St. Thomas' Hospitals NHS Foundation Trust, London, UKe-mail: [email protected] ; [email protected]

Background: Despite several years of research and several studies there is yet no clear consensus on the best pituitarysuppression regimen for poor responders undergoing IVF treatment, with the available studies yielding conflictingresults.Methods: We conducted a systematic review and meta-analysis of randomized controlled trials to compare IVFtreatment outcomes in previous poor responders undergoing a subsequent IVF treatment cycle. Searches wereconducted on MEDLINE, EMBASE, Cochrane Library, Web of Science and National Research Register. Study selectionand data extraction were conducted independently by two reviewers.Results: We identified 21 randomised controlled trials comprising 1142 women with a previous poor responseundergoing a subsequent IVF treatment cycle. The regimens used in the comparisons were the GnRH agonist longregimen, the GnRH agonist short regimen, the GnRH antagonist regimen, the GnRH agonist long stop regimen andother modifications of the GnRH agonist long regimen. Meta-analysis of these studies showed a significantly higherclinical pregnancy rate with the GnRH agonist long compared to the GnRH agonist short regimen (RR: 4.66; 95%CI: 1.43, 15.17; P = 0.01), no significant difference between the GnRH agonist short compared to the GnRH antagonistregimen (RR: 0.94; 95% CI: 0.63; 1.39; P = 0.75), the GnRH agonist long compared to the GnRH antagonist regimen(RR: 0.85; 95% CI: 0.53, 1.37; P = 0.51) and the GnRH agonist stop regimen compared to the GnRH agonist longregimen (RR: 1.74; 95% CI: 0.67, 4.47; P = 0.25),. Meta-analysis of these studies for the outcome of number of oocytesretrieved showed the GnRH agonist long stop regimen to be significantly better than the GnRH agonist long regimen(WMD: 2.42; 95% CI: 2.06, 2.77; P < 0.00001). The GnRH agonist long regimen was significantly better than the GnRHagonist short regimen (WMD: 1.36; 95% CI: 0.30, 2.43; P = 0.01) and the GnRH agonist short regimen was significantlybetter than the GnRH antagonist regimen (WMD: 0.55; 95% CI: 0.14, 0.96; P = 0.008). However, meta-analysis ofstudies comparing the GnRH agonist long with the GnRH antagonist regimen did not show a significant differencein the number of oocytes retrieved between the two regimens (WMD: - 0.15; 95% CI -1.17, 0.88; P = 0.78). Meta-analysis for outcomes of cycle cancellation rate, and ongoing pregnancy rates also showed either no significantdifference or inconsistent findings.Conclusion: Currently available evidence does not favour any particular pituitary suppression regimen for poorresponders undergoing IVF treatment with the results showing inconsistent findings.


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ORAL CONTRACEPTIVE PILL PRE-TREATMENT IN GnRH ANTAGONIST CYCLESGriesinger GDepartment of Obstetrics and Gynecology, University Clinic of Schleswig-Holstein, Campus Luebeck,Luebeck, Germanye-mail: [email protected]

Introduction: In the long GnRH-agonist protocol, ovarian stimulation can be arbitrarily initiated after pituitarydesensitization has been achieved, while the initiation of stimulation in GnRH-antagonist protocols relies on theoccurrence of menstruation. This hampers scheduling IVF treatment to meet the organizational needs of both patientsand IVF centers, which has important economical and practical implications. Thus, oral contraceptive pill (OCP) pre-treatment in GnRH-antagonist cycles has been advocated to schedule oocyte retrieval to exclude weekends.Methods: A literature search was performed using the bibliographic databases MEDLINE, CENTRAL and EMBASEcovering the period until November 2009. In summary, six RCTs (encompassing 1,343 randomized patients) on OCPpre-treatment in GnRH-antagonist cycles are available (Griesinger et al., Fertil Steril in press). One of the trials wasconducted in an expected ‘poor response’ patient population, whereas the other five trials were conducted in anaverage, normo-ovulatory IVF patient population. All trials used combined OCPs with 30μg ethinyl estradiol and150μg gestogen (either desogestrel or levonorgestrel), and the duration of pill pre-treatment ranged from 14-28days. The pill-free interval between cessation of OCP treatment and initiation of stimulation was 2-3 days in twoof the studies, while in the remaining 4 studies a 5 day interval was used. Ovarian stimulation was performed withrecombinant FSH in all studies. Ongoing pregnancy rate per randomized patient was expressed as an odds ratio (OR)with 95% confidence intervals (CI) for each study and these data were combined for meta-analysis with the softwareRevMan using the Mantel/Haenszel method. When the outcome of interest was of a continuous nature, the differenceswere pooled across the studies resulting in a weighted mean difference (WMD) with 95% CI.Conclusions: The probability of an ongoing pregnancy per randomized woman was found to be statisticallysignificantly lower in patients with oral contraceptive pill pre-treatment (fixed effects model; RR 0.80, 95% CI: 0.66to 0.97; p=0.02; rate difference: -5%, 95% CI: -10% to -1%; p=0.02). Six studies provided data for this outcome. Therewas no statistical significant heterogeneity between individual studies. Duration of stimulation (random effectsmodel; WMD: +1.35 days, 95% CI: +0.62 to +2.07; p<0.01) and gonadotropin consumption (random effects model;WMD: +360 IUs, 95% CI: +158 to +563; p<0.01) were significantly increased after OCP pre-treatment, however, therewas statistical significant heterogeneity (p<0.01) between individual studies for both outcomes. The number ofcumulus-oocyte-complexes was not significantly different between the patient groups (fixed effects model; WMD:+0.6 oocytes, 95% CI: -0.08 to +1.25; p=0.09), with no statistical significant heterogeneity between studies. Conclusively,the negative impact of routine use of OCP pre-treatment in an IVF program needs to be carefully balanced with thebenefit of treatment scheduling (spreading workload for the IVF center).

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FINAL FOLLICLE MATURATION TRIGGER WITH GnRH AGONISTSHumaidan PThe Fertility Clinic, Skive Regional Hospital, Skive, Denmarke-mail: [email protected]

Introduction: Human chorionic gonadotrophin (hCG) has been used as a surrogate for the mid-cycle luteinizinghormone (LH) surge for several decades. Due to structural and biological similarities with LH, hCG binds to andactivates the same receptor as LH, the LH/hCG receptor. This receptor is among others constitutively located on thecacells, but also on granulosa cells from a follicle size of 8-12 mm.Despite the fact that hCG effectively secures final oocyte maturation and ovulation, its use as a surrogate for LH hasgot several drawbacks - first and foremost a sustained luteotropic effect, facilitating ovarian hyperstimulationsyndrome (OHSS).Methods: Recently GnRH antagonist protocols for the prevention of a premature LH surge were introduced, allowingfinal oocyte maturation to be triggered with a single bolus of a GnRH agonist (GnRHa). GnRHa is as effective as hCGfor the induction of ovulation, and apart from the LH surge a FSH surge is also induced. Until recently, prospectiverandomized studies reported a poor clinical outcome when GnRHa was used to trigger final oocyte maturation inIVF/ICSI antagonist protocols, presumably due to a luteal phase deficiency, despite standard luteal phase supplementationwith progesterone and oestradiol.As GnRHa triggering of final oocyte maturation could possess advantages over hCG triggering in terms of a reducedif not eliminated risk of OHSS, the retrieval of more mature oocytes, and a higher patient convenience, the challengehas been to rescue the luteal phase, which could be differentiated depending on patient category: the OHSS riskpatient, the normo-responder patient and the oocyte donor. In the literature now several studies report a lutealphase rescue with a reproductive outcome comparable to that seen after hCG induced final oocyte maturation. Theparamount aim should be the improvement of pregnancy rates without increasing the risk of OHSS.Conclusions: Although more research is needed to further explore the optimal luteal phase support after GnRHatriggering, this mode of triggering final oocyte maturation is now a valid alternative with potential benefits.


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LUTEAL PHASE SUPPORT OF GnRH ANTAGONIST CYCLESNardo L, Mohiyiddeen LSt Mary's Hospital, Department of Reproductive Medicine, Manchester, UKNorth West Fertility, Liverpool & Manchester, University of Manchester, Manchester, UKe-mail: [email protected]

Luteal phase deficiency is a common problem in assisted reproduction techniques and has been described in cyclesusing pituitary down-regulation with GnRH antagonists used to prevent premature LH surge. Conventional regimensof luteal phase support involve different application forms and doses of therapeutic agent like HCG, progesteroneand estradiol. A recent study has shown that the route of progesterone administration for luteal phase support canbe an important prognostic factor in the antagonist cycles. A beneficial effect of GnRH agonist administered in theluteal phase has also been demonstrated. The administration of a single dose of GnRH agonist in the luteal phasewas shown to increase pregnancy, implantation and birth rates. GnRH antagonist administration in the luteal phasein patients with OHSS, combined with embryo transfer and exogenous oestradiol and progesterone supplementationresulted in successful clinical outcome. The administration of aromatase inhibitor (letrozole) during the luteal phasereduces serum E2 levels and has been shown to be helpful in patients at risk of OHSS. Appropriate luteal supportnot only assures good clinical outcome but can also help prevent risk of OHSS.

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FSH STRUCTURE-FUNCTION RELATIONSHIPSWide LDepartment of Medical Sciences, Clinical Chemistry, University Hospital, Uppsala, Swedene-mail: [email protected]

Human FSH is a glycoprotein hormone produced in a very large number of different FSH isoforms in each pituitary.FSH is heterodimeric and composed of an alpha- and a beta-subunit. Each subunit is decorated with two asparagine-linked oligosaccharide chains and it is the variations in the structure of these four N-linked glycans that give rise tothe large number of FSH isoforms. The spectra of FSH isoforms may show large differences between pituitaries asthe synthesis of the isoforms is related to the sex and age of the individuals.When the isoforms reach the blood circulation there is a selective survival in the circulation of the different FSHisoforms. A consequence of this is that the spectrum of isoforms reaching the target organs, the ovaries and thetestis, is different from that secreted from the pituitary. Furthermore, a consequence of the pulsatile secretion ofFSH from the pituitary, combined with this selective survival in the circulation, is a continuous change in the spectrumof FSH isoforms circulating and reaching the target organs.At least 14 different anionic N-linked glycan chains have been isolated from human pituitary FSH. They usuallyterminate with sialic acid but there are also some with sulfonated β1-4-linked N-acetylgalactosamine (SO3--GalNAc).Enzymes regulating the sulfonation and the sialylation pathways during synthesis compete for the same substrate,a subterminal GlcNAc residue on the biantennary glycan chains. Both these terminal groups are negatively chargedand determine the anionic character of the oligosaccharides and of the entire FSH molecule.We have developed a method to estimate the average number of sialic acid and sulfonated GalNAc residues per FSHmolecule in serum and the distributions of molecules with zero to four sulfonated residues. The composition of FSHisoforms in serum varies during the menstrual cycle, changes after menopause and is different in women withpolycystic ovarian syndrome and in men compared with healthy women. The content of sialic acid residues on FSHduring the menstrual cycle is highest in the latter part of the follicular phase and in early luteal phase and has anadir at midcycle and in late luteal phase. The content of sulfonated GalNAc residues on FSH is lowest in the latefollicular phase and then gradually increases to a maximum in the late luteal phase. At menopause, there is aconsiderable increase in the number of sialic acid and a small decrease in sulfonated residues per FSH molecule.The primary effect of oestradiol (E2) in E2 treated post-menopausal women was a decrease in the sialylation of FSHand, due to competition for the same substrate, secondarily and consequentially a minor increase in the sulfonationof FSH. When a progestogen, norethisterone acetate, was added the effect was primarily an activation of thesulfonation pathway during the synthesis of FSH. Our studies indicate that androgens primarily inhibit the sulfonationof FSH.The FSH isoforms circulating soon after a GnRH challenge contain less number of sialic acid and more of sulfonatedGalNAc residues per molecule. These FSH forms are more similar to those extracted from pituitaries. Our studies withGnRH receptor blockade show that sulfonation of FSH is associated with a more rapid disappearance of these isoformsfrom the blood circulation while sialylation prolongs its survival in the circulation. Less sialylated isoforms of FSHexhibit higher receptor affinity and in-vitro biological activity than their more sialylated counterparts.Exogenous FSH preparations used for ovulation induction contain several FSH isoforms. Twelve hours after subcutaneousadministration the more sialylated of these FSH isoforms dominate in the circulation.Recent references: Wide et al: J Clin Endocrinol Metab, 92: 4410-4417, 2007; 94: 958-964, 2009; 95: 383-389, 2010;Upsala J Med Sci, 115: Early Online, 1-10, 2010.


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DESIGN AND ACTION OF HYPERGLYCOSYLATED RECOMBINANT FSH MOLECULESLustbader JW, Pollak SV, Weenen C, Yu B, Klein J, Trousdale RK , Lustbader EG, Lobel LDepartment of Obstetrics & Gynecology, Division of Reproductive Sciences, College of Physicians &Surgeons, Columbia University, New Yorke-mail: [email protected]

Introduction: Due to its relatively short half-life, current gonadotropin formulations must be administered as dailyintramuscular or subcutaneous injections. Long-acting hFSH formulations may decrease the number of requiredinjections as well as minimize pain and discomfort associated with therapy. Fusion of the carboxyterminal peptide(CTP) of hCG to the N-terminus of FSHβ results in a follitropin agonist with an extended half-life (hFSH-CTP), attributedto the presence of four additional O-linked glycosylation sites on the CTP moiety. Alternatively, we have studiedthe pharmacokinetics and pharmacodynamics of novel hFSH analogues containing additional N-linked carbohydrates.Material and Methods: DNA sequences encoding amino acid polypeptides containing alternating ser-gly residueswith 0, 1, 2 or 4 N-linked glycosylation signal sequences (Asn-Ala-Thr) were synthesized. These sequences were ligatedin-frame into SV40 expression vectors containing hFSHβ and α-subunit encoding cDNAs. The vectors were transfectedinto CHO-K1 cells, and high-secreting clones were grown in spinner bottles. The expressed proteins, single-chainfusions consisting of hFSHβ-N (0,1,2 or 4) - α(hFSH-N0, 1, 2 or 4 respectively), were purified by immunoaffinitychromatography using an α-subunit specific antibody, and the proteins were quantified using an hFSHβ−specific RIA.In vitro bioactivity was assessed in CHOK-1 cell cultures expressing the hFSH receptor (hFSHR). Pharmacokineticparameter estimates were obtained from serial serum assay of product at .5, 1.0, 3.0, 6.0 and 12 hours in immaturefemale SD rats (21 days old) following a single IV injection (2800 ng) of hFSH-N0,1,2,4 or control material (saline,native hFSH, hFSH-CTP, n=3 for each group). In vivo bioactivity was determined by administering a single subcutaneousinjection of hFSH-N(X) analogues or control (saline, hFSH or hFSH-CTP) at a dose of 2800 ng to immature 21-day oldrats (n=3 for each group) and assessing ovarian weight gain 3 days post-injection.Results: Receptor binding and signal transduction was confirmed by the induction of comparable levels of cAMPin CHOK-1 cells expressing hFSH receptor exposed to hFSH-N(X) and native hFSH. Serum concentration-time curvesfollowing IV injection in rats could be explained by a two-compartment model; half-life of elimination for hFSH,hFSH-CTP, hFSH-N2 and hFSH-N4 was 3.7h, 6.9h, 7.9h and 7.8h respectively. Mean ovarian weight (mg, standarddeviation) 3 days post-SC injection of hFSH, hFSH-CTP, hFSH-N2 and FSH-N4 in rats was 16.7 ± 1.5, 25 ± 3.5, 22 ± 3.6and 29.5 ±1.3, respectively.Conclusions: The addition of polypeptide stretches containing 0, 1, 2 or 4 N-linked glycosylation signal sequencesto hFSH does not interfere with proper folding, receptor binding or signal transduction of the fusion protein. TheFSH-N2 and -N4 analogues have a half-life of elimination comparable to an hFSH mutant containing 4 added O-linked carbohydrates (FSH-CTP), and approximately double that of native hFSH. In vivo biopotency of the hFSHchimera containing 2 or 4 additional N-linked sugars was increased in comparison to native hFSH and comparableto hFSH-CTP. In rodents, the use of the hyperglycosylated FSH analogues rhFSH-CTP and rhFSH-N4 stimulated a largernumber of eggs after ovulation and a higher number of 8-cell embryos with in vitro culturing comparable to rhFSH.Thus, the use of hyperglycosylated FSH analogues potentially provides an additional novel therapy for ovarianstimulation in infertile women. One concern that arose from our studies was that the delivery rate associated withrhFSH-CTP was lower than expected compared with the delivery rates for rhFSH, rhFSH-N4 and PMSG (gold standardfor mouse ovarian stimulation). Further studies are necessary to elucidate the source of the discrepancy betweenthe antral follicle, egg production, and in vitro embryo data relative to the delivery and litter size data observedwith rhFSH-CTP therapy. Interestingly, use of the N-linked hyperglycosylated rhFSH-N4 was not associated with areduction of live birth rates or smaller litter sizes and was statistically equivalent to PMSG for all fertility parametersevaluated. HFSH-N4 may have important clinical applications for patients requiring gonadotropin therapy and meritsfurther investigation. By defining the role of differential glycosylation, therapeutic derivatives developed throughcarbohydrate manipulations could be expanded to a wider breath of recombinant proteins not just restricted to theglycoprotein hormone family.

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ANDROGEN PRIMING BEFORE FSH OVULATION INDUCTIONFleming RGCRM Ltd., Glasgow University, Glasgow, UKe-mail: [email protected]

IntroductionThe roles of androgens in folliculogenesis and in antral follicle function are simultaneously indisputable, but unclear.There are generally two areas for consideration through the life of a follicle, and the importance and influence ofandrogens requires a great deal more elucidation in both. Androgens appear to play an important role infolliculogenesis and follicular survival through the many weeks of development prior to the stages of gonadotrophinsensitivity – the stages of paracrine control. Any clinical applications of androgens aimed at addressing these stageswould require a protracted strategy.On the other hand, the role of androgens within the functions of antral follicles, growing under the influence ofFSH and LH, represent a relatively short treatment approach. In this latter context therapeutic approaches wouldaddress improved function rather than increased follicular numbers, as the number of antral follicles present hasbeen determined by other, preceding, factors.There have been a number of clinical explorations to explore the potential benefits of deployment of androgensto influence both circumstances.Folliculogenesis, survival and androgensThere is good scientific evidence indicating that the androgen receptor is critically important in folliculogenesis ina number of species. The activity of oocytes or the growth factor GDF9, a product of developing oocytes whichpromotes follicular development, is much enhanced by androgen treatment in terms of granulosa cell division andsteroidogenesis (Hickey et al, 2005). Studies such as these indicate that occupation of the androgen receptor ongranulosa cells is important for follicle survival.There is a rationale linking androgens and the degree of folliculogenesis seen in adult women because of the commonpathological complications of polycystic ovarian syndrome (PCOS). PCOS is a condition of excessive folliculardevelopment to the latter stages of development and also of hyperandrogenaemia, and the degree of each is stronglylinked. Furthermore, in primate models, androgen treatment during pregnancy can lead to the development ofovaries reminiscent of PCOS in the offspring (Abbot et al, 2008). Corresponding evidence in the human is less clear-cut, but family studies with hyperandrogenic women do suggest that similar circumstances may apply.At the other end of the spectrum, in the adult woman with PCOS, suppression of gonadotropins, and suppressionof androgens may reduce the symptoms of PCOS, but it is not clear that there is an impact upon the degree offolliculogenesis, as the number of antral follicles seen in the ovary appears to remain excessive despite a numberof therapeutic approaches reducing androgen activity.Three recent studies have explored the effect of androgen suppression, induced by metformin treatment, uponfolliculogenesis in women with PCOS. The consensus is that circulating androgens do not have a causative role inthe pathological hyper-folliculogenesis found in PCOS. Correspondingly, this evidence does not support the use ofprotracted androgen treatment in women with a diminished ovarian reserve.Nevertheless, there have been a number of small reports that protracted treatment with DHEA, a weak androgen,may lead to an improvement in the ovarian reserve of women with a poor response to FSH stimulation for IVF. Sofar, there is no published study with a controlled approach to examining this potentially interesting strategy. Themost encouraging evidence so far comes from the group of Norbert Gleicher in New York. The most recent of these(Goyal, 2009) was carried out in women with a low ovarian reserve determined by circulating AMH concentrations. After a minimum of 1 month treatment with DHEA (25mg tid), a significant (though modest) increase in AMH wasachieved. Subsequent IVF treatment suggested that those with an increase in the AMH during treatment had ahigher chance of pregnancy.It is proposed that DHEA supplementation may influences ovarian growth factor activity. This in turn may increasefollicular survival through to the later developmental stages, and there may also be greater benefits in terms of eggquality and reductions in aneuploidy leading to reduced miscarriage and increased live-birth outcomes.Much further work is required in this sizeable cohort of women attending IVF clinics world-wide, and AMH can nowbe use as a marker for identifying patients and exploring benefit. However, although many women are now treatingthemselves with DHEA, there is currently very little evidence to support its use.Follicular paracrinology and androgensThe two main cell types of antral follicles, granulosa and theca, although separated by a basement membrane interactin many ways. There is a constant dialogue exchanged between them for both steroidogenesis and growth.Androgens, which derive from theca cells, promote granulosa cell sensitivity to FSH (Hillier and Tetsuka, 1997) andit is entirely logical to explore this function for therapeutic reasons.However, it is inconceivable that any modification to peripheral concentrations androgens will influence those atthe follicular level, which are approximately 1000 fold higher. Correspondingly, any effect of peripheral androgentreatment could only influence follicular function through indirect mechanisms. Two well controlled studies havebeen reported using short term transdermal administration of androgens. There was negligible evidence to supportits use more widely, although one of the studies reported an increased proportion of patients achieving egg pick-up.In contrast to the direct use of androgens, it is possible to influence local follicular concentrations of androgens byeither driving more theca cell activity (with LH or hCG), or by blocking the primary androgen catabolic step – thearomatization to estrogens (using aromatase inhibitors). Both approaches have been explored in normal womenundergoing IVF with profound gonadotropin suppression. In neither case was there any indication of a quantitativeeffect, but there was limited evidence suggesting possible qualitative benefit. Further work is required to providemore comprehensive evidence.In summary, numerous strategies have been explored to determine the effects of androgens, and factors promotingandrogens, to influence quantity and quality of oocytes for assisted reproduction. So far, none has provided convincingevidence of benefit to advise wider use outside of research applications.ReferencesHickey et al (2005). Androgens augment the mitogenic effects of oocyte-secreted factors and growth differentiationfactor 9 on porcine granulosa cells. Biol Reprod., 73, 825-32.Abbott et al (2008) Fetal programming of adrenal androgen excess: lessons from a nonhuman primate model ofpolycystic ovary syndrome. Endocr Dev. 13, 145-58.Hillier SG, Tetsuka M. (1997) Role of androgens in follicle maturation and atresia. Baillieres Clin Obstet Gynaecol.11, 249-60.


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EFFECTS OF HIGH DOSE FSH REGIMENS – “LESS IS MORE!”Pal LYale University School of Medicine, Dept.of Obstetrics, Gynecology and Reproductive Sciences, Div.ofReprod. Endocrin. & Infertility, New Haven, CT, USAe-mail: [email protected]

Ovarian events in a spontaneous menstrual cycle entail recruitment and progressive growth of a single ovarian folliclethat eventually attains dominance, at the expense of the remainder of a committed follicular cohort. In the contextof assisted reproductive techniques (ART), the aim of controlled ovarian hyperstimulation (COH) is to optimizerecruitment and growth of as many of the available follicular cohort, as can be achieved with due regard to avoidingrisk for ovarian hyperstimulation syndrome. The quantitative ovarian response during COH is determined not onlyby age and the underlying contributor to infertility (young women, and those with polycystic ovary syndrome orhypothalamic amenorrhea are recognized as ‘‘high responders’’) but also by the dose of gonadotropins employedduring COH. Suboptimal ovarian responses to exogenous gonadotropins are known to reflect compromised ovarianreserve, and such occurrence is commonly managed by increasing gonadotropin dose in a subsequent attempt atART. While improved quantitative ovarian responses and lower cycle cancellation rates are shown to relate to useof higher dose of gonadotropins, these benefits have not translated to improved treatment success, i.e. live birthfollowing ART. On the contrary, higher dose gonadotropin use is even suggested to hold potential for detriment,i.e. lower cycle related success rates, leading to suggestions that a more conservative approach to COH be encouraged. Critical concepts relating to folliculogenesis are reviewed and data relating gonadotropin regimens for COH withthe goals of ART are summarized. Evidence relating adverse implications of excess gonadotropin dose for treatmentsuccess following reproductive assistance strategies will be discussed and a case made that gentler COH strategiesmaximize the risk: benefit ratio by minimizing risks for ovarian hyperstimulation syndrome and maximizing treatmentrelated reproductive success.

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GONADOTROPIN STIMULATION AND FOLLICULAR PHASE PROGESTERONELEVELS: MECHANISMS AND OUTCOMEBosch EInstituto Valenciano de Infertilidad. Universidad de Valencia, Valencia, Spaine-mail: [email protected]

Ovarian stimulation is performed for IVF with the aim of obtaining a proper number of oocytes to ensure the selectionof good quality embryos for transfer. However, it causes a dramatic variation of the normal hormonal profile on themenstrual cycle, that include a 5 to 10 fold increase on Estradiol (E2) serum levels in the follicular phase, due tomultiple follicular development, and a subtle raise in serum progesterone (P) levels, before the human chorionicgonadotrophin (hCG) administration.The influence of elevated serum P levels during IVF cycles on pregnancy rates is a matter of continued debate amongstfertility clinicians. Efforts to resolve this question have been impeded by the various assays used to measure P andthe different, arbitrary threshold values for defining ‘high’ P levels. Although there is not a clear consensus to definea detrimental P level, most authors use a value between 1.0 and 1.5 ng/mL for cut-off level. This elevation has beenshown to occur in a considerable number of IVF cycles, and for both kind of GnRH analogues used for preventinga premature LH surge. Thus, in GnRH agonist cycles, it has been described in 5 to 35% of cycles and in 20 to 38%of GnRH antagonist cycles.In a recent study, the ongoing pregnancy rate has been related to serum P level the day of hCG administration inan unselected population of more than 4,000 cycles. In it, it is clearly shown that success rate is significantly decreasedwhen P was > 1.5 ng/mL, regardless the type of GnRH analog used. Whether this detrimental effect on cycle outcomeis due to a negative impact on the oocyte-embryo quality or on endometrium repectivity remains unclear. Whiledata from studies performed in oocyte donation cycles suggest that the oocyte and embryo qualities are unaffectedby high P levels, a very recent study shows that the endometrium gene expression profile at the time of implantationis significantly altered on samples of women with P > 1.5 ng/mL.


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TRIDIMENSIONAL AUTOMATED ULTRASOUND FOR MONITORING OVARIANSTIMULATIONRaine-Fenning NNottingham University Research and Treatment Unit in Reproduction (NURTURE), Division of HumanDevelopment, School of Clinical Sciences, University of Nottingham, Nottingham, UKe-mail: [email protected]

Serial assessment of follicle number and size is routinely employed to assess the response to ovarian stimulation(‘follicle tracking’) and to time oocyte retrieval during assisted reproduction treatment. Two-dimensional (2D)ultrasound is used to identify and scroll through each ovary whilst the observer qualifies the number of folliclespresent. An objective assessment of the size of the larger follicles, commonly those measuring more than 10 mm,is then made through a series of two-dimensional measurements of their perceived mean diameter. There is noconsensus as to how this should be performed or how many measures of each follicle should be taken. Such assessmentbecomes increasingly difficult and less reliable when there are numerous follicles of different sizes as occurs withmultifollicular recruitment during controlled ovarian stimulation.Accurate assessment of the size of follicles is important as the timing of oocyte maturation and subsequent eggcollection is based on the principle that a follicle is more likely to contain a mature oocyte when it measures between12 and 24 mm in diameter (1, 2). Whilst follicles measuring more than 18 mm in diameter are pre-ovulatory andinvariably contain metaphase II oocytes, many smaller follicles contain mature oocytes capable of fertilisation albeitat a reduced rate . The proportion of mature oocytes derived from follicles measuring more than 15 mm in diameterappears to be relatively constant (5, 6). Significantly less mature oocytes are recovered from follicles with a meandiameter of less than 15 mm and only 30% of the oocytes retrieved from follicles measuring 12–14 mm will bemature. Follicles measuring less than 9 mm in diameter are not routinely measured or aspirated as whilst they containdevelopmentally competent oocytes these often require in vitro maturation (7) and fertilization rates are higher infollicles measuring more than 10 mm although it is unclear whether rates increase progressively after this stage 8,9). Regardless of the absolute size of the follicle, its final maturation must be induced prior to oocyte retrieval. Thisis achieved through the administration of specific doses of human chorionic gonadotrophin (hCG) or recombinantluteinising hormone (LH) and is usually based on the total number of follicles measuring more than 10 mm andspecifically those measuring more than 17-18 mm (10, 11, 12). Oocyte maturation is essential prior to egg collectionas it induces the final oocyte nuclear meiotic maturation, germinal vesicle breakdown, and cumulus expansion inthe follicle (13, 14). The timing of the administration of these drugs is based on the size of the leading follicles. Anaccurate assessment of follicle size, and appropriate timing of oocyte maturation and egg collection, could resultin the retrieval of a higher number of mature ooctes and, theoretically, improved fertilisation rates and a higherchance of pregnancy.Ultimately two-dimensional measurements of diameter are relatively arbitrary as no non-uniform three-dimensionalobject actually has a true diameter. Two-dimensional measurements are used in clinical practice as they are easy toperform and three-dimensional data acquisition is a relative new technique. Volume is a more appropriate measurementof a three-dimensional structure however and is quantifiable with both two- and three-dimensional ultrasound. 3Doffers a more direct measure of volume as estimations from 2D information are based on certain geometric assumptionsand have obvious imitations therefore. 3D volume calculations can be performed manually and, more recently,automatically. Sonography-based Automated Volume Count (SonoAVC: GE Medical Systems, Zipf, Austria) is a newsoftware programme, which can be applied to datasets acquired using three-dimensional (3D) ultrasound (15). Itindividually identifies and quantifies the size of any hypoechoic region within these three-dimensional datasetsproviding an automatic estimation of their absolute dimensions and volume. Whilst SonoAVC may be used todelineate and quantify the volume and relative dimensions of any fluid-filled area, it is particularly useful whenthere are two or more adjacent regions of interest. SonoAVC can quantify an unlimited number of volumes, whichmakes it an ideal tool for the assessment of the ovary in women undergoing controlled ovarian stimulation as partof assisted reproduction treatment. SonoAVC is an ideal and, potentially, clinically important tool for the assessmentand measurement of follicles during controlled ovarian stimulation therefore and the fact that each hypoehoeicarea is individually colour coded also eliminates the possibility of measuring the same follicle more than once.To use SonoAVC the user needs to acquire a three-dimensional dataset. This is then displayed using the multiplanarview and a region of interest selected by manually moving a box to fit the maximal proportions of the ovary. Oncethe dataset has been correctly positioned SonoAVC is implemented. The automated analysis takes around six secondsand the individual follicles are then displayed together with their dimensions and relative sizes (Figure). There arelimitations to the technique however and it is important to recognise these. Some follicles are not identified followingthe initial automatic assessment and some follicles are incorrectly identified and in most cases there is some extra-ovarian artifact. There are several options for post-processing that require manual or automatic modification of theinitial output. Manual techniques may be used for single follicles but the automated options are applied to thedataset as a whole. The most simplistic tool involves clicking on individual follicles that have not been identified aswell as those that have been incorrectly included. These appear and disappear respectively. The next step involvesdrawing around follicles to identify them as separate or single entities although this may also be achieved by usingthe ‘separation’ and ‘growth’ functions that modify how each follicle is colour-filled. The latter option is good whenthere are aberrances across the whole ovary but in most cases editing is required for a few follicles and the manualtechnique is preferable. It is important to accept these limitations and not to simply expect to acquire a 3D volume,press a button and have a perfectly defined folliculogram: some additional work is likely to be needed in all cases.Once the post-processing completed SonoAVC provides three measurements for each colour-coded area: the meandiameter (relaxed sphere diameter), the maximum dimensions (x, y, z diameters), and the volume of each colour-coded region. The ‘relaxed sphere diameter’ is the diameter the follicle would achieve if it formed a perfect sphere.This is infrequently seen physiologically especially during controlled ovarian stimulation where multiple follicles liein close proximity and their overall expansion is limited by the ovarian cortex. Many follicles are ellipsoid but severalobtain a more obtuse and irregular structure and in some cases have a more cuboidal appearance than a sphericalone. Follicles, whilst being three-dimensional, are rarely spherical therefore and yet current practice is based uponthe two-dimensional estimation of a mean diameter, a measurement that has only developed through necessitybecause of a lack of three-dimensional technology and due to time constraints placed on the majority of assistedconception units. The two-dimensional estimate of a presumed diameter may actually be sufficient in practice asthe validity of any measurement technique is less important than the biological accuracy needed. Unfortunatelywhilst the former is readily quantifiable the latter is much more difficult to define requiring extensive prospectivemulticentre trials in units with varying practices. SonoAVC will allow for this however and addresses many if not allof the current issues surrounding follicle tracking as it provides highly valid measures of both follicle diameter andvolume that are more reliable than those made using conventional two-dimensional ultrasound and in a shortertime frame thereby improving the clinical work flow (15, 16). In a study comparing the automated measurementsto those derived from measuring the follicular aspirate we found SonoAVC estimated the volume of a follicle towithin ± 0.5 cm3 . This was significantly more accurate than volume measurements derived using algorithms (thesphere and ellipsoid formulae) based on the mean follicle diameter calculated by both 2D and 3D techniques.6th World Congress on Ovulation Induction - Naples, Italy • 30 September - 2 October 2010

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Follicular diameter measurements were also more reliable when made with SonoAVC which provides two differentmean diameters . Other groups have confirmed the validity of the software both in vitro and in vivo using phantomsof known volume (19) and follicular aspirates (20) respectively and its reliability for follicle assessment (21, 22).We have also conducted a randomized controlled trial to assess the clinical impact of these improved automatedmeasures of follicle diameter on the outcome of assisted reproduction treatment (23). The aim of the study was toevaluate the effect of timing final follicular maturation and egg collection on the basis of follicular measurementsmade automatically with SonoAVC against those made with conventional 2D ultrasound. Our primary outcomemeasures included the number of oocytes collected and their maturity whilst fertilization rates, embryo cleavage,and pregnancy were secondary outcome measures. We hypothesized that these outcome measures would besignificantly higher in subjects whose egg collection was timed according to follicular measurements made withSonoAVC as a result of the increased reliability and accuracy this software offered. Final follicular maturation wasinduced by giving 250 mcg of recombinant coriogonadotrophin alfa (Ovitrelle, Merck Serono) when there were threeor more lead follicles measuring 18 mm or more and oocyte retrieval was performed 36 hours later using a singlechannel needle according to a local standard operating protocol (SOP). Complete data were available on 68 subjectsand there were no differences between the groups in terms of the cause of subfertility, age, body mass index or inthe ovarian stimulation protocols as measured by the number of days of ovarian stimulation or in the total dose ornumber of ampoules of gonadotrophins used. Direct comparison between SonoAVC and real-time 2D ultrasoundrevealed no significant differences in any of the outcome variables (Table). The study was not designed or poweredto evaluate differences in pregnancy rates but the lack of difference between the two techniques and the embryologydata suggest this is unlikely to be improved by the automated measurement technique but larger prospective studiesare required to address this. The only other group to apply the software prospectively reported a higher fertilisationrate in 20 patients whose egg collection was timed on the basis of measurements made with SonoAVC although thestudy was too small to evaluate the effect on pregnancy (24).It is unclear at present whether SonoAVC improves the outcome of assisted reproduction treatment when it is usedto determine the timing of final follicular maturation and egg collection. There may be a ‘window of opportunity’for oocyte retrieval or that the measures obtainable using conventional 2D ultrasound are sufficient to determinethe timing of events. We know that a mature oocyte can be obtained from a follicle measuring anywhere between12 and 24 mm and that these oocytes are capable of fertilisation. There is a degree of flexibility in the timing ofoocyte retrieval therefore and it is unclear what degree of measurement accuracy is required for follicular assessmentwhen multifollicular recruitment is desired. The need for precise timing may be more for natural cycles, mild IVF andin ovulation induction and intrauterine stimulation where a monofollicular response is needed.Ultimately measurements of diameter are relatively arbitrary as no non-uniform three-dimensional object actuallyhas a true diameter. Two-dimensional measurements are only used in clinical practice as they are easy to performand three-dimensional data acquisition and automated analysis are relatively new techniques. Volume is a moreappropriate measurement of a three-dimensional structure however and despite the current lack of an apparentclinical benefit we still feel SonoAVC has great potential as it is quicker than conventional 2D imaging and theresultant measurements are more reliable and valid which gives the user, and our patients, more confidence. Furtherstudies are required as always and it will be interesting to see how the technology is accepted into clinical practiceand how it affects a units workflow and success rates.ACKNOWLEDGEMENTSSonoAVC was developed by GE Medical Systems in association with K plus Competence Center (Advanced ComputerVision) and part funded by the K plus Program.


Figure: A three-dimensional multiplanar view of a stimulated ovary following the application of SonoAVC.Each follicle has been individually colour-coded and its dimensions, which have been automatically calculated, areshown on the right of the image as follows; relaxed sphere diameter; d(V), its maximum dimensions (dx, dy, dz) andtheir mean (m-d), and its volume (V).

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SREFERENCES1. Bergh C, Broden H, Lundin K, Hamberger L. Comparison of fertilization, cleavage and pregnancy rates of oocytes

from large and small follicles. Hum Reprod 1998;13:1912-5.2. Wittmaack FM, Kreger DO, Blasco L, Tureck RW, Mastroianni L, Jr., Lessey BA. Effect of follicular size on oocyte

retrieval, fertilization, cleavage, and embryo quality in in vitro fertilization cycles: a 6-year data collection. FertilSteril 1994;62:1205-10.

3. Trounson A, Anderiesz C, Jones G. Maturation of human oocytes in vitro and their developmental competence.Reproduction 2001;121:51-75.

4. Tsuji K, Sowa M, Nakano R. Relationship between human oocyte maturation and different follicular sizes. BiolReprod 1985;32:413-7.

5. Rosen MP, Shen S, Dobson AT, Rinaudo PF, McCulloch CE, Cedars MI. A quantitative assessment of follicle size onoocyte developmental competence. Fertil Steril 2008.

6. Scott RT, Toner JP, Muasher SJ, Oehninger S, Robinson S, Rosenwaks Z. Follicle-stimulating hormone levels oncycle day 3 are predictive of in vitro fertilization outcome. Fertil Steril 1989;51:651-4.

7. Haines CJ, Emes AL. The relationship between follicle diameter, fertilization rate, and microscopic embryo quality.Fertil Steril 1991;55:205-7.

8. Penzias AS, Emmi AM, Dubey AK, Layman LC, DeCherney AH, Reindollar RH. Ultrasound prediction of folliclevolume: is the mean diameter reflective? Fertil Steril 1994;62:1274-6.

9. Salha O, Nugent D, Dada T, Kaufmann S, Levett S, Jenner L et al. The relationship between follicular fluid aspiratevolume and oocyte maturity in in-vitro fertilization cycles. Hum Reprod 1998;13:1901-6.

10. Ghosh C, Buck G, Priore R, Wacktawski-Wende J, Severino M. Follicular response and pregnancy among infertilewomen undergoing ovulation induction and intrauterine insemination. Fertil Steril 2003;80:328-35.

11. Kolibianakis E, Bourgain C, Albano C, Osmanagaoglu K, Smitz J, Van Steirteghem A et al. Effect of ovarianstimulation with recombinant follicle-stimulating hormone, gonadotropin releasing hormone antagonists, andhuman chorionic gonadotropin on endometrial maturation on the day of oocyte pick-up. Fertil Steril 2002;78:1025-9.

12. MacDougall MJ, Tan SL, Jacobs HS. In-vitro fertilization and the ovarian hyperstimulation syndrome. Hum Reprod1992;7:597-600.

13. Wiesak T. Role of LH in controlled ovarian stimulation. Reprod Biol 2002;2:215-27.14. Eppig JJ. Intercommunication between mammalian oocytes and companion somatic cells. Bioessays 1991;13:569-

74.15. Raine-Fenning N, Jayaprakasan K, Clewes J. Automated follicle tracking facilitates standardization and may

improve work flow. Ultrasound Obstet Gynecol 2007;30:1015-8.16. Raine-Fenning N, Jayaprakasan K, Deb S, Clewes J, Joergner I, Dehghani Bonaki S, et al. Automated follicle

tracking improves measurement reliability in patients undergoing ovarian stimulation. Reprod Biomed Online.2009 May;18(5):658-63.

17. Raine-Fenning N, Jayaprakasan K, Clewes J, Joergner I, Bonaki SD, Chamberlain S et al. SonoAVC: a novel methodof automatic volume calculation. Ultrasound Obstet Gynecol 2008;31:691-6.

18. Raine-Fenning N, Jayaprakasan K, Chamberlain S, Devlin L, Priddle H, Johnson I. Automated measurements offollicle diameter: a chance to standardize? Fertil Steril. 2009 Apr;91(4 Suppl):1469-72.

19. Deutch TD, Joergner I, Matson DO, Oehninger S, Bocca S, Hoenigmann D, et al. Automated assessment of ovarianfollicles using a novel three-dimensional ultrasound software. Fertil Steril. 2009 Nov;92(5):1562-8.

20. Lamazou F, Arbo E, Salama S, Grynberg M, Frydman R, Fanchin R. Reliability of automated volumetric measurementof multiple growing follicles in controlled ovarian hyperstimulation. Fertil Steril. 2010 Mar 11.

21. Salama S, Arbo E, Lamazou F, Levailllant JM, Frydman R, Fanchin R. Reproducibility and reliability of automatedvolumetric measurement of single preovulatory follicles using SonoAVC. Fertil Steril. 2009 Apr;93(6):2069-73.

22. Rodriguez-Fuentes A, Hernandez J, Garcia-Guzman R, Chinea E, Iaconianni L, Palumbo A. Prospective evaluationof automated follicle monitoring in 58 in vitro fertilization cycles: follicular volume as a new indicator of oocytematurity. Fertil Steril. 2009, 93(2):616-20.

23. Raine-Fenning N, Deb S, Jayaprakasan K, Clewes J, Hopkisson J, Campbell B. Timing of oocyte maturation andegg collection during controlled ovarian stimulation: a randomized controlled trial evaluating manual andautomated measurements of follicle diameter. Fertil Steril. 2009 Jun;94(1):184-8.

24. Murtinger M, Aburumieh A, Rubner P, Eichel V, Zech MH, Zech NH. Improved monitoring of ovarian stimulationusing 3D transvaginal ultrasound plus automated volume count. Reprod Biomed Online. 2009 Nov;19(5):695-9.


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LH ACTIVITY-CONTAINING GONADOTROPIN PREPARATIONSGiuliani M, Rienzi L, Sapienza F, Romano S, Colamaria S, Magiulli R, Ubaldi FGENERA, Centre for Reproductive Medicine, Valle Giulia Clinic, Rome, Italye-mail: [email protected]

In the natural cycle, follicle growth is driven by a delicate interplay of FSH and LH that affects theca and granulosacells, leading to the selection of a single dominant follicle through a series of feedback mechanisms. The synergisticrelationships of FSH and LH are essential for oocyte development and maturation, but the need for exogenous LHadministration during controlled ovarian hyperstimulation (COH) has, nevertheless, remained controversial. Thosein favor of LH supplementation argue that pituitary desensitization with GnRH agonists or antagonists results insuch profound LH suppression that proper oocyte maturation does not take place. Moreover its most significantfunction is probably in the resumption of meiosis. Faultless completion of meiosis I and II is crucial for chromosomalintegrity and, hence, oocyte diploidy. Opponents of that view, however, claim that even marginal concentrationsof endogenous LH are sufficient for appropriate oocyte development. The impact of different gonadotrophinpreparations used in ovarian stimulation, such as urinary hMG (which contains FSH and LH activity) and recombinantFSH (which contains just FSH) preparations, on treatment outcome in women undergoing controlled ovarianhyperstimulation for assisted reproduction technologies (ARTs) has been widely debated.Several data of the literature indicate that LH activity plays a role in ovarian stimulation and show that there aremajor pharmacodynamic differences between these preparations in follicular development, endocrine response,embryo quality and endometrial status and show a beneficial effect of LH-containing stimulation on diploidy ratesin preimplantation embryos.In a systematic review of randomized trials, representing the current best evidence on the comparison hMG versusrFSH for controlled ovarian hyperstimulation following an agonist long down-regulation protocol in IVF or ICSItreatment, Coomarasamy et al. showed a significant 4% increase in live birth rate with hMG for the study populations.Although this difference is statistically significant, the clinical significance of this difference is a matter of judgementfor couples undergoing IVF or ICSI treatment, and the clinicians who look after them. None of the seven trialsindividually showed a statistically significant benefit towards hMG, although five of the seven trials including thethree largest trials, showed a trend favouring hMG.Despite the clinical effectiveness and the reduced cost of hMG, a few more issues deserve addressing. First, cliniciansand couples may still prefer to use rFSH for reasons such as elimination of the risk of viral or prion infections, althoughthese infections have never been demonstrated, and therefore remain only as a theoretical risk. Secondly, rFSHrepresents a potentially unlimited source of FSH. Thirdly, further research into rFSH holds the promise of longer-acting forms (with the result of reduced numbers of injections) or even orally active versions of FSH. Finally, if theclinical superiority of hMG is due to the LH it contains, then it may be possible to add recombinant LH or even hCGto rFSH to achieve the same results.However, these possibilities need to be confirmed in trials before such a step is considered for clinical practice.

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ORALLY ACTIVE LH AGONISTSvan de Lagemaat RDepartment of Women’s Health, Merck Research Laboratories, Oss, The Netherlandse-mail: [email protected]

Introduction: In assisted reproductive technology, the pre-ovulatory LH surge is mimicked by human ChorionicGonadotropin (hCG) which is administered subcutaneously for triggering final oocyte maturation and induction ofovulation. In order to allow oral administration, improve homogeneity and reduce elimination half-life, researchhas concentrated on Low Molecular Weight (LMW) LH compounds. Our efforts to develop orally bioavailableLuteinizing Hormone (LH) receptor agonists have led to the discovery of Org 43553.Results: Org 43553 is a potent stimulator of the human LH receptor in vitro (EC50 3.7nM), and binds to the endodomainof the LH receptor by allosteric interaction. It has 30 times less activity at the FSH receptor and does not activate theTSH receptor.Ovulation induction by Org 43553 was demonstrated in a mouse in vitro ovulation model and in an in vivo cyclicrat model after a single oral intake. The ovulated oocytes were of good quality as demonstrated by successfulfertilization and implantation of normal embryos. In male rats testosterone production was substantially inducedafter oral administration.Pharmacokinetic analyses showed high oral bioavailability in rats (79%) and dogs (44%) with a shorter half-lifecompared to hCG (3.4 vs 5.6 hrs in the rat).In a rat model for Ovarian Hyperstimulation Syndrome (OHSS), Org 43553 did not induce vascular leakage (sign ofOHSS) and showed reduced VEGF levels.First human exposure studies are ongoing and indicate that LMW LH agonist Org 43553 is orally active, with arelatively shorter half-life compared to hCG and is well tolerated.Conclusions: Org 43553 is the first LMW LH-R mimetic with demonstrated in vivo efficacy upon oral administrationwhich could replace subcutaneously administered hCG. The elimination half-life of Org 43553 is substantially shorterthan hCG, which could potentially represent a clinical benefit in reducing the risk for OHSS. Thus oral active LHagonists are anticipated to introduce a safer and more convenient therapy for fertility treatment.

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CLINICAL USE OF RECOMBINANT LHAlviggi CDipartimento di Scienze Ostetriche Ginecologiche Urologiche e Medicina della Riproduzione, Universitàdegli Studi di Napoli “Federico II”, Naples, Italye-mail: [email protected]

An increasing body of evidence clearly supports the idea that personalising controlled ovarian stimulation (COS)regimens optimises the outcome of in vitro fertilisation (IVF) procedures. Availability of recombinant products allowsindependent use of the two gonadotrophins, giving the opportunity for evaluating the impact of LH and differentFSH:LH ratios on ovarian response. On these bases, LH supplementation has been tested in different subgroups ofpatients. Available data indicate that recombinant LH (r-hLH) is able to improve the ovarian response and the outcomeof IVF in women identified as “hyporesponders” to r-hFSH monotherapy. In particular, the concept of “hypo-response”to COS has been proposed to identify normogonadotrophic women who have normal estimated ovarian reserve but,when stimulated with standard GnRH-a long protocol require high amounts of FSH to obtain an adequate numberof oocytes retrieved (De Placido et al., 2001, 2004, 2005; Ferraretti et al., 2004; Mochtar et al., 2007; Devroey et al.,2009). These women seem to be distinct from classical poor responders because they have normal ovarian reserve,but unexpectedly show sub-optimal response when stimulated with standard regimens. Conversely, specific adjustmentsof classical protocols, including the use of r-hLH supplementation seem to optimise their ovarian response.On the basis of the current literature, it is possible to argue that hypo-response is related to genetic characteristics.More specifically, it has been found that this condition is associated with an increased frequency of a common andless bioactive LH polymorphism (v-LH).Recent data clearly demonstrated that normogonadotrophic women displaying higher-endogenous LH levels duringclassical GnRH-a long protocol benefit from r-hLH supplementation, suggesting that less performing LH receptorsmay be involved. Taken together, these lines of evidence support the idea that testing women for specific geneticcharacteristics may allow tailored gonadotrophins administration. A pharmacogenomic approach to COS may leadto develop new tailored and cost-effective treatments.

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HUMAN CHORIONIC GONADOTROPIN IN OVARIAN STIMULATIONFilicori M, Cognigni GE, Ciampaglia W, Pocognoli P, Taraborrelli S, Tabarelli C, Infante FE,Parmegiani L, Bernardi SReproductive Medicine Unit, GynePro Medical Centers, Bologna, Italye-mail: [email protected]

Chorionic gonadotropin (CG) has been clinically employed since the outset of ovulation induction in anovulatoryand infertile women. It was long recognized that luteinizing hormone (LH) and CG shared the same receptor (hencenamed LH/CG receptor) and that their main mechanism of action outside of pregnancy was to stimulate ovariantheca cells to produce the androgen substrate needed for aromatase system conversion into estrogens in granulosacells (GC). Furthermore, it became soon evident that an additional key physiologic mechanism of LH was GC interactionat the completion of the follicular phase to provide the signal for final follicle and oocyte maturation through themid-cycle LH surge. As the amount of LH needed to pharmacologically reproduce the mid-cycle LH surge was huge,while CG was plentiful (through extraction from pregnant women urine) and highly potent, this latter hormone waschosen instead of LH to complete ovarian stimulation and trigger ovulation.Because of its higher sialic acid content, CG is 6-8 times more potent than LH and has a much longer serum half-life.Thus, less CG than LH (in unit values) can be used but this is also associated with a greater risk of excessive ovarianstimulation. Recombinant (r) LH has been found to be effective for final follicle maturation trigger, but a dose of25-30,000 IU of rLH is needed to this end. Both LH and CG are present in human menopausal gonadotropin (hMG)preparations and the CG content of this class of medications has increased in more recent highly purified (HP) hMG.CG content has been linked to improved clinical efficacy of HP hMG.LH activity can be supplemented during follicle-stimulating hormone (FSH) stimulation under the form of rLH or CG.Though FSH supplementation with rLH has been largely disappointing (possibly because of inadequate dosing), wedemonstrated that addition of moderate amounts of hCG (50 IU/day, corresponding to 300-400 IU of LH) can enhanceestrogen secretion, selectively stimulate large follicle growth (>14 mm diameter), and reduce FSH dose requirementsand treatment duration. We also found that LH activity can replace FSH in the last days of ovarian stimulation, hencesignificantly reducing FSH consumption. In addition, low-dose hCG appears to be capable of selectively stimulatinglarge follicle growth, limit the occurrence of small preovulatory follicles (<10 mm) and potentially reduce theoccurrence of the ovarian hyperstimulation syndrome (OHSS). It was shown that these regimes are effective whenhCG, rCG and rLH are used, and in both GnRH agonist and antagonist cycles.Thus, LH activity supplementation (in adequate dosages) and late replacement of FSH appear to be effective andefficient manners to improve COS efficacy in OI and ART.

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LH ACTIVITY SUPPLEMENTATION DURING GnRH ANTAGONIST CYCLESDevroey PCentre for Reproductive Medicine, UZ Brussels, Brussels, Belgiume-mail: [email protected]

The question remains if the LH activity during GnRH antagonist cycles has an influence on the follicular stimulationand especially the follicular recruitment. Surprisingly enough the concentration of LH during the early follicularphase before GnRH antagonist administration did not influence the follicular outcome neither the pregnancy rates.This observation has been confirmed by studies where daily administration of recombinant FSH was administeredbut also in cycles where a 7 days one shot sustained release of recombinant FSH has been given.From these observations it is transparent that the median LH concentrations in GnRH antagonist cycles don’t influencepregnancy outcomes. One could argue that supplementary administration of LH could influence the follicularrecruitment. In prospective randomized trials it has been clearly demonstrated that adding LH does not improvepregnancy rates.A crucial question remains if recombinant FSH in GnRH antagonist cycles can be replaced by low dose hCG (200 units).In a randomized controlled trial it has been clearly demonstrated that from a diameter of 12 mm onwards recombinantFSH can be replaced by low dose hCG. Health economics of low dose hCG administration worldwide is of paramountimportance.In conclusion: median LH concentration during the follicular phase does not influence pregnancy outcome in GnRHantagonist cycles stimulated with recombinant FSH. In several studies, it has been clearly demonstrated that addingLH does not improve follicular recruitment, neither follicular response. Recombinant FSH in the late follicular phasecan be easily replaced by low dose hCG.

References:Among patients treated with FSH and GnRH analogues for in vitro fertilization, is the addition of recombinant LHassociated with the probability of live birth? A systematic review and meta-analysis. Kolibianakis EM, KalogeropoulouL, Griesinger G, Papanikolaou EG, Papadimas J, Bontis J, Tarlatzis BC. Hum Reprod Update. 2007 Sep-Oct;13(5):445-52.Are endogenous LH levels during ovarian stimulation for IVF using GnRH analogues associated with the probabilityof ongoing pregnancy? A systematic review. Kolibianakis EM, Collins J, Tarlatzis B, Papanikolaou E, Devroey P. HumReprod Update. 2006 Jan-Feb;12(1):3-12.Profound LH suppression after GnRH antagonist administration is associated with a significantly higher ongoingpregnancy rate in IVF. Kolibianakis EM, Zikopoulos K, Schiettecatte J, Smitz J, Tournaye H, Camus M, Van SteirteghemAC, Devroey P. Hum Reprod. 2004 Nov;19(11):2490-6.LH concentrations do not correlate with pregnancy in rFSH/GnRH antagonist cycles. Doody K, Devroey P, GordonK, Witjes H, Mannaerts B. Reprod Biomed Online. 2010 Apr;20(4):565-567Can 200 IU of hCG replace recombinant FSH in the late follicular phase in a GnRH-antagonist cycle? A pilot study.Blockeel C, De Vos M, Verpoest W, Stoop D, Haentjens P, Devroey P. Hum Reprod. 2009 Nov;24(11):2910-6.

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CONTROLLED OVARIAN STIMULATION IN OOCYTE DONORS, FERTILITYPRESERVATION, AND OOCYTE BANKINGRemohi JEquipo IVI, Instituto Valenciano de Infertilidad, Valencia, Spaine-mail: [email protected]

IntroductionOocyte cryopreservation has been one of the most fascinating challenges of assisted reproduction technology andits introduction into the clinical practice has been a goal since many years. One of the most interesting advantagesof oocyte cryo-storage is the greater flexibility granted to ovum donation programs, since no synchronization wouldbe needed, becoming safer and easier procedures since a quarantine period could be attained prior to the donationof the oocytes. Recently vitrification procedures are achieving satisfactory results particularly Cryotop method isattaining great improvements in both post-thaw survival and clinical outcome.MethodsOocyte vitrification by using Cryotop method. In brief oocytes are gradually equilibrated in a solution containing7.5% EG plus 7.5% DMSO during 12 -15 minutes. Vitrification step was carried out in a solution containing 15% EG;15% DMSO plus 0.1M sucrose in 1 minute. Vitrified oocytes were banked during a minimum of six months. Afterthis quarantine period were donated to appropriate recipients. Data on survival, fertilization, embryo quality andclinical outcome has been recorded and compared with the outcome achieved from fresh donated oocytes.ResultsThe average time of estradiol replacement for recipients of our egg-bank ovum donation program is 15.5 ± 4.6 days,being significantly lower than the spent for patients waiting for the reception of fresh oocytes (22.4 ± 5.4; P<0.05).Survival rate after warming is over 90%. The potential of vitrified oocytes is consistently similar to the one attainedfor fresh ones: fertilization rate (74.2% vs. 73.3%); cleavage rate on day (95.3% vs. 96.0%); clinical pregnancy rate(55.4% vs. 55.6%); ongoing pregnancy rate (49.1% vs. 48.3%) and implantation rate (39.9% vs. 40.9%) for vitrifiedand fresh oocytes respectively (NS).ConclusionsThese evidences indicate that nowadays oocyte cryopreservation by means of vitrification is providing a highlyeffective tool within ART, attaining similar outcomes than those obtained with fresh oocytes, thus allowing itsapplication into clinical practice.

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STRATEGIES TO PREVENT OVARIAN HYPERSTIMULATIONPellicer AInstituto Valenciano de Infertilidad (IVI), University of Valencia, Valencia, Spaine-mail: [email protected]

OHSS has been prevented and treated empirically over the years because its pathophysiology remained unknown.Employing animal models, it has been shown that increased vascular permeability (VP), a landmark of OHSS, isassociated with a hCG-induced ovarian expression of vascular endothelial growth factor (VEGF) and its receptor 2(VEGFR2). Binding of VEGF to VEGFR2 disrupts cellular junctions and increases VP. Dopamine and its agonists reverseincreased VP in animals by inhibition of VEGFR2 phosphorilation. To prevent OHSS, two steps are mandatory: toreduce or eliminate the effects of hCG, and/or to block VEGF binding to VEGFR2. Therefore, the first single measureto prevent OHSS is to cancel those patients who are at serious risk of developing OHSS, i.e. serum E2>4000 pg/mland/or >30 follicles developed. The second relevant measure is to avoid a pregnancy, which means vitrification ofall embryos, provided the high survival rates achieved with this technique. Once hCG has been withdrawn, a differentstrategy is recommended to mature the oocytes and two main measures should be taken in the luteal phase to avoidearly symptoms of OHSS. Final oocyte maturation is achieved employing a GnRH agonist bolus to trigger endogenousLH in GnRH antagonist-drive cycles. Moreover, GnRH antagonists can be employed successfully along the luteal phase.In addition, dopamine agonists can be added also in the luteal phase. We showed in a randomized and double blindstudy on oocyte donors at risk of developing OHSS that 0.5 mg/day cabergoline (Cb2) significantly reducedhemoconcentration (p<0.01) and ascites (p=0.005) as compared to placebo. The incidence of moderate OHSS was20.0% and 43.8%, respectively (p=0.04).In a second study, another dopamine agonist Quinagolide (QN) was employed in a prospective, randomized, placebocontrolled cycle in IVF patients. QN was used in three different doses (50 μg/day, 100 μg/day, 200 μg/day) in womenat risk of developing OHSS and compared to placebo. QN overall and the 200 μg/day group were associated with asignificant (p=0.019 and p=0.046) reduction in the frequency of moderate/severe early OHSS. QN significantly reduced(p=0.033) the incidence of ultrasound evidence of ascites in patients who did not achieve pregnancy, while therewas no significant difference for those who did become pregnant. QN did not have a detrimental effect on clinicalpregnancy. An increase in the incidence of adverse events was seen with increasing doses. Finally, in those patientswho develop ascites causing discomfort, therapeutical paracentesis has proved to be effective.

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CONTROLLED OVARIAN STIMULATION IN ENDOMETRIOSISArici ADepartment of Obstetrics, Gynecology & Reproductive Sciences, Yale University School of Medicine. NewHaven, CT, USAe-mail: [email protected]

Although infertility and endometriosis are clearly connected, uncertainty persists over the causal relation betweenthe two. Believed to be one of the leading causes of infertility, endometriosis has a prevalence of 2–5% in fertileand 25–40% in infertile women. The optimal choice of management for endometriosis-associated infertility remainsobscure. Removal or suppression of endometrial deposits by medical or surgical means constitutes the basis ofendometriosis management. Current evidence indicates that suppressive medical treatment of endometriosis doesnot benefit fertility and should not be used for this indication alone. Surgery is probably efficacious for all stagesof the disease. Controlled ovarian hyperstimulation with intrauterine insemination is recommended in early-stageand surgically corrected endometriosis when pelvic anatomy is normal. In advanced cases, in vitro fertilization (IVF)is the treatment of choice. In choosing a stimulatory regimen in women with endometriosis, there seems to be nodifference among various gonadotropins. On the other hand, there is evidence to suggest that the use of prolongedGnRH-agonist downregulation is beneficial in women with endometriosis.IVF outcomes in patients with endometriosis remain controversial. Several studies have shown that women withendometriosis have a lower ovarian response to gonadotropins. One reason for the reduced ovarian response maybe the presence of endometriomas per se, which may negatively influence ovarian function and may impose difficultiesand risks during oocyte retrieval. On the other hand, many studies have shown that the presence of an ovarianendometrioma does not have a deleterious effect on oocyte fertilization, embryo quality, implantation, or pregnancyoutcome. Another reason for the reduced ovarian response may be due to previous excision of these cysts.Overall, there is insufficient evidence to support a strategy of systematic surgical treatment of endometriomas beforeIVF. Furthermore, there are now several lines of evidence that suggests that laparoscopic cystectomy for endometriomasbefore commencing an IVF cycle does not improve fertility outcomes and may be hazardous on the ovarian reserve.There is also a concern that repeated ovarian hyperstimulation during IVF may stimulate the growth of endometrioticlesions and lead to recurrence of endometriosis. The data suggest that temporary exposure to high estrogen levelsin women during ovarian hyperstimulation for IVF is not a major risk factor for endometriosis recurrence in womentreated with assisted reproductive technology.In summary, expectant management may be a reasonable approach in younger patients with early stage disease anda shorter duration of infertility. Controlled ovarian hyperstimulation with intrauterine insemination is a good optionin mild and surgically corrected disease. In patients with advanced stage endometriosis, IVF is the treatment of choice,and prolonged Gn-RH-agonist downregulation may be useful.

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THERAPEUTIC STRATEGIES IN POOR RESPONDERSLoutradis D1st Department of OB/GYN, Athens Medical School, Division of Human Reproduction, Athens, Greecee-mail: [email protected]

The standard goal of all fertility treatments is the improvement in pregnancy rates in patients with infertility problems.Within the past years, ovulation induction has contributed to the success of assisted reproduction techniques, in vitrofertilization (IVF) and embryo-transfer (ET). The efficacy of these techniques depends on a personalized protocol ofcontrolled ovarian hyperstimulation (COH) and an adequate oocytes recruitment . The response of several patientsto ovarian stimulation protocols used as a routine is not always as expected. A failure to respond adequately tostandard protocols and to recruit an adequate number of follicles is called ‘poor response’. The lack of clear, uniformdefinition concerning the poor responders and the lack of large-scale randomized studies make data interpretationvery difficult for precise conclusions.Optimistic data have been presented with the use of high doses of gonadotropins rFSH or HMG , the flare up GnRH-aprotocol (standard or microdose), the stop protocols, the luteal onset of GnRH-a and the short protocol. The use ofGnRH antagonist may be associated with simpler stimulation protocols, lower gonadotropin requirements, reducedpatient costs, and shorter downtimes between consecutive cycles. Recent data suggest a potential beneficial effectof aromatase inhibition by the administration of letrozole prior to gonadotropin stimulation .The use of LH inovarian stimulation in IVF-ET cycles has a beneficial effect in the quality of oocytes, as well as in the pregnancy rate. Natural cycle or a modified natural cycle seems to be an appropriate strategy for poor responders. Growth hormoneand pyridostgmine in poor responders has been found to show a significant improvement in live birth. There isevidence that sort term pretreatment with transderm testosterone or long term treatment with DHEA has beneficialeffect in poor responders. Also low dose of aspirine , adjunctive use of L-arginine ,and glucocortioids administration are alternative therapieutics approaches but further trials are needed to support the beneficial effect in patientsconfirmed as poor responders.Available data seem to indicate that COCs given before treatment could be effective in patients who are resistantto clomifene citrate and in poor responders. The use of ICSI in poor responders is not justified unless there areconcomitant indications such as Male factor of infertility and maybe in cases of unexplained infertility, where ICSIhas been shown to decrease fertilization failure, however there is no difference in clinical pregnancy rates.Molecular biology tools such as the single nucleotide polymorphisms (SNPs), have also been considered to assist themanagement of this group of patients. The clinical implications of SNPs (FSHR, ESR1, ESR2) are highly important andthe ultimate goal is the application of genetic markers as routine diagnostic tests before ovarian stimulation, inorder to predict the ovarian response. The frequency distribution of the Ser680Asn polymorphism of the FSHR, inpatients with ovarian dysfunction (OD defined as FSH>10 mIU/mL) and in ‘poor responders’ (PR) demonstrates thatin OD patients the FSHR Ser/Ser variant was more prevalent (45.5%), while the Asn/Ser variant is correlated withmore follicles and oocytes. We also examined the frequency distribution of the Pvu II and Rsa I polymorphisms ofthe estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2) genes respectively, in patients undergoing IVF. Thisstudy shows that CC allelic variant (ESR1) is associated with higher pregnancy rates compared to TC,TT allelic variant(31.3% vs 23.7% ,21%). The frequency distribution of ESR1 allelic variants in relation to FSH levels revealed a largerpresence of the CC genotype in women with FSH>10 mIU/mL (30.8%) compared to women with FSH<9 mIU/mL(20%).Many studies have evaluated the use of various ovarian stimulation regimens to improve the outcome of poorresponders undergoing IVF treatment. There is no sufficient evidence to support the routine use of any particularintervention either for pituitary downregulation, ovarian stimulation or adjuvant therapy in the management ofpoor responders. More data from good quality controlled trials are needed. However, a trend for an overallimprovement in ovarian response has been shown with some of these protocols giving hope for improving pregnancyrates.REFERENCES1. Poor responder protocols for in-vitro fertilization: options and results.

Loutradis D, Vomvolaki E, Drakakis P. Curr Opin Obstet Gynecol. 2008 Aug;20(4):374-8.2. Pharmacogenetics in ovarian stimulation--current concepts.

Loutradis D, Vlismas A, Drakakis P, Antsaklis A. Ann N Y Acad Sci. 2008 Apr;1127:10-9.3. Different ovarian stimulation protocols for women with diminished ovarian reserve.

Loutradis D, Drakakis P, Vomvolaki E, Antsaklis A. J Assist Reprod Genet. 2007 Dec;24(12):597-6114. FSH receptor gene polymorphisms have a role for different ovarian response to stimulation in patients entering

IVF/ICSI-ET programs.Loutradis D, Patsoula E, Minas V, Koussidis GA, Antsaklis A, Michalas S, Makrigiannakis A. J Assist Reprod Genet.2006 Apr;23(4):177-84

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CONTROLLED OVARIAN STIMULATION IN PCOSAboulghar MFaculty of Medicine, Cairo University, Cairo, EgyptThe Egyptian IVF center, Maadi, Cairo, Egypte-mail: [email protected]

Ovulation induction for polycystic ovarian disease (PCO) is associated with difficulties and complications as under-and over-stimulation are relatively common.For non IVF, minimal stimulation protocols (step up) which start with a small dose of FSH and increase gradually isthe commonly used protocol. Step down protocol is also used with lower risk of cancellation of the cycle.For IVF, ovarian stimulation is associated with a high risk of OHSS. That is why in the long GnRHa protocol, smalldose of FSH should be used if the response is very strong the dose should be reduced. Coasting is performed if E2level reaches over 3000 pg.ml by stopping FSH administration and monitoring E2 level. In later stages of inductionof ovulation, small doses of hCG could replace FSH.GnRH antagonist protocol is associated with a lower risk of OHSS and it allows the chance of triggering ovulationwith a bolus of GnRHa to reduce the risk of OHSS.Both urinary highly purified FSH and recombinant FSH have the same pregnancy rate after IVF.

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FERRING PharmaceuticalsSymposium

Thursday, 30 September 201017:30 - 19:00

Gonadotropins, back to the future?

Chairperson: Ubaldi F (IT)


IBSA PharmaceuticalsSymposium

Friday, 1 October 201011:15 - 12:45

Managing PCOS Patients in Assisted Reproduction

Chairpersons: Loutradis D (GR), Requena A (ES)

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CHOICE OF GnRH ANALOG REGIMENS FOR CONTROLLED OVARIAN STIMULATIONIN PCOSOrvieto RDepartment of Obstetrics and Gynecology, Barzilai Medical Center, Ashkelon, and Ben Gurion UniversitySchool of Medicine, Beer Sheva, Israele-mail: [email protected]

Background: In vitro fertilization (IVF) and embryo transfer (ET) is an effective therapy for PCOS patients, and resultsin pregnancy rates that are comparable with those for women with tubal factor infertility. Many controlled ovarianhyperstimulation (COH) strategies have been offered for the treatment of patients with PCOS undergoing IVG.Nevertheless, no compelling advantage for one stimulation protocol over another has been hitherto established andthe optimal stimulation protocol is still under debate. The ESHRE/ASRM-Sponsored PCOS Consensus Workshop Grouphas recently argued for the need to perform further randomized controlled trials (RCTs) comparing FSH stimulationprotocols with use of GnRH agonist versus GnRH antagonist. However, in an era where IVF success, individualization,and careful tailoring of the COH protocol and patient’s safety are interrelated and mandatory, such RCTs would raiseseveral ethical and legal issues.Aims: To present the available evidences that examine the influence of the type of GnRH-analogue used duringCOH on IVF outcome.Materials and Methods: A literature review was conducted for all relevant articles assessing the effect of theGnRH-analogue used during COH on PCOS patients’ IVF outcome.Results: In the vast majority of studies, and particularly the largest, PCOS women undergoing the midluteal longGnRH agonist suppressive protocol required longer stimulation and more gonadotropin ampoules, had an increaseincidence of severe ovarian hyperstimulation syndrome (OHSS), with a tendency toward a higher clinical pregnancyrate compared to patients undergoing the multidose GnRH antagonist protocol.Conclusions: In PCOS patients undergoing IVF-ET cycles, COH using the midluteal long GnRH agonist suppressiveprotocol is associated with a tendency toward a higher clinical pregnancy rate than the flexible multidose GnRHantagonist protocol. However, because POCS patients are at high risk to develop severe OHSS, it would be prudent,in the first IVF cycle attempt, to offer these patients the GnRH antagonist COH protocol, with its inherent lower riskof OHSS. Moreover, it enables the substitution of hCG with GnRH agonist to trigger ovulation, with the consequentelimination of severe OHSS.

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USE OF HIGHLY PURIFIED FSH IN PCOSGrudzinskas GConsultant Gynaecologist, Editor RBM Online, web site: www.grudzinskas.co.uke-mail: [email protected]

It is now accepted that PCOS affects about 1 in 10 women of reproductive age, characterised by multiple endocrinologicaland metabolic abnormalities, with clinical symptoms of hyperandrogenaemia and anovulation commonly seen. Asthe majority of women with PCOS have abnormal menstrual cycles, many have anovulation as the cause of theirinfertility.The medical treatment typically begins with anti-oestrogens such as clomiphene citrate strategies whereas FSHinjections, and/or ovarian drilling in some women, should be kept as a second option. The principles of FSH ovulationinduction/ augmentation/ superovulation, typically involving gradual step-up regimen of increasing doses of FSH atweekly intervals until the planned response is achieved, are considered to be the same regardless of the source ofFSH (recombinant/highly purified). Indeed, the clinical studies available so far did not show differences between thetwo types of FSH preparations, neither there was a rationale to sustain the idea that one of the two could be better.More recently, it has been shown that the FSH released from the pituitary in PCOS patients is more extensivelyglycosylated than that found in age-matched women (Wide et al. 2007) suggesting the hypothesis that the FSH typecould play a role in the dynamics of follicular development in PCO. Assuming that human-derived and recombinantFSH preparations are different in this respect, with low complexity (rodent-type) glycosylation in rFSH, some differencesin bio-activity could indeed occur.Fostimon is a human-derived FSH that is claimed by the manufacturer to be further enriched in highly glycosylatedFSH species, which justifies evaluation in PCOS patients. The first available randomised study (Aboulghar et al. 2010)of PCOS patients treated with Fostimon revealed 1) a higher fertilization rate 2) double the number of embryosfinally available for cryopreservation than that reported in the Gonal-F group. The pregnancy rates in the freshcycles were not different. In another study (retrospective) Palomba et al. (2010) found that in metformin-treatedPCOS patients, Fostimon usage had resulted in a lower drug administration and a trend for a lower cancellation rate(13.0% vs 7.1%, n.s.).In summary, the newly available data indicate that the treatment outcome in PCOS patients may be sensitive to thetype of FSH administered. These differences, although small, justify further investigations of suitable design and size.

References:Aboulghar, M., et al., Prospective, randomized study comparing highly purified urinary follicle-stimulating hormone(FSH) and recombinant FSH for in vitro fertilization/intracytoplasmic sperm injection in patients with polycystic ovarysyndrome. Fertil Steril, 2010. 2010: p. 24.

Palomba S. et al., Recombinant FSH versus highly purified FSH for controlled ovarian stimulation in women withpolycystic ovary syndrome under metformin. Gynecological Endocrinology, accepted for publication.

Wide, L., et al., Sulfonation and sialylation of gonadotropins in women during the menstrual cycle, after menopause,and with polycystic ovarian syndrome and in men. J Clin Endocrinol Metab., 2007. 92(11): p. 4410-7.

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EFFECTS OF IN VIVO AND IN VITRO GONADOTROPIN SUPPLEMENTATION INPCOS PATIENTS UNDERGOING OOCYTE IN VITRO-MATURATIONFadini R, Dal Canto M, Mignini Renzini M, Coticchio GBIOGENESI Reproductive Medicine Center, Istituti Clinici Zucchi, Monza, Italye-mail: [email protected]

Oocyte in vitro maturation (IVM) has been developed to prevent the risk of ovarian hyperstimulation syndrome(OHSS) and other drawbacks derived from conventional ovarian hyperstimulation with gonadotropins. In its originalscheme, IVM involves the retrieval of immature germinal vesicle (GV) stage oocytes enclosed in their vestment ofcumulus cells from small- and mid-sized antral follicles. These oocytes are placed in culture for 24-36 hours to permitthe resumption of meiosis and achievement of meiotic maturation, signified by the emission of the polar body I. Invitro maturation is routinely performed in the presence of follicle stimulating hormone (FSH) and human chorionicgonadotropin (HCG), but in fact experiments in animal models suggest that paracrine factors of the EGF-like familycould assist oocyte maturation in a more specific fashion.IVM is particularly suited for the treatment of infertile patients presenting with polycystic ovaries (PCO) or polycysticovary syndrome (PCOS), because of an elevated risk of OHSS and the high number of antral follicles occurringspontaneously in these subject. More recently, it has been proposed that regularly cycling women may benefit fromIVM, despite the reduced number of antral follicles available for recovery.Because collective experience has shown that the efficiency of IVM performed in total absence of gonadotropinsupplementation is reduced in comparison to conventional ovarian stimulation, it has been suggested that mild FSHpriming during the early follicular phase and/or induction of final maturation with HCG could improve quantity andquality of immature oocytes. In effect, after HCG administration a proportion of oocytes (15-20%) is found alreadymature at retrieval. This contributes to increase the overall fraction of mature oocytes available for treatment andcould improve the clinical outcome. However, current evidence is conflicting. In PCO patients, initially it was reportedby Mikkelsen and Lindenberg (2001) that FSH priming was able to improve the clinical outcome, but more recentstudies (Son et al., 2006) have not confirmed a beneficial effect of FSH. In the same category of patients, Chian etal. (2000) have found that HCG induction leads to a higher rate of mature oocytes and determines a trend towardshigher pregnancy rates. In recent years, our group has extended the experience of oocyte maturation to normo-ovulatory women. In a prospective randomized study (Fadini et al., 2009), we allocated 400 women to four differenttreatments: no stimulation, HCG priming, FSH priming, combined FSH and HCG priming. The maturation and overallrates of mature oocytes available for treatment were higher in the FSH/HGC group (77.4 and 82.1%, respectively).These percentages were around 60% after HGC priming alone and dropped to 48-50% in the absence of HCG,irrespective of FSH administration. The clinical pregnancy rate per transfer was significantly higher (29.9%) in theFSH/hGC group in comparison to all other regimens (15.3%, 17.3% and 7.6% in the no supplementation, FSH, andHCG categories, respectively). Therefore, it appears that a combined supplementation of FSH and HCG can improvethe IVM outcome while FSH or hGC alone do not seem to be beneficial. Studies are in progress in our centre toestablish whether these conclusions can be extended to PCO and PCOS patients.

ReferencesChian RC, Buckett WM, Tulandi T, Tan SL 2000 Prospective randomized study of human chorionic gonadotrophinpriming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. HumanReproduction 15, 165–170.

Fadini R, Dal Canto M, Mignini Renzini M., Brambillasca F, Comi R, Fumagalli D, Lain M, Merola M, Milani M, De PontiE 2009 Effect of different gonadotrophin priming on IVM of oocytes from women with normal ovaries: a prospectiverandomized study. Reproductive BioMedicine Online 19, 343–351.

Mikkelsen AL, Lindenberg S 2001 Benefit of FSH priming of women with PCOS to the in-vitro maturation procedureand the outcome: a randomized prospective study. Reproduction 122, 587–592.

Son WY, Yoon SH, Lim JH 2006 Effect of gonadotrophin priming on in-vitro maturation of oocytes collected fromwomen at risk of OHSS. Reproductive BioMedicine Online 13, 340–348.


Schering-Plough PharmaceuticalsSymposium

Friday, 1 October 201017:00 - 18:30

New perspectives in controlled ovarian stimulation

Chairpersons: Nappi C (IT), Perino A (IT)

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OPTIMISATION OF rFSH PROTOCOLS: A PATIENT FOCUSED APPROACHAlviggi CDipartimento di Scienze Ostetriche Ginecologiche Urologiche e Medicina della Riproduzione, Universitàdegli Studi di Napoli “Federico II”, Naples, Italye-mail: [email protected]

The use of recombinant FSH (r-hFSH) in association with GnRH-analogues represents a consolidated strategy forcontrolled ovarian stimulation (COS) in women undergoing IVF/ICSI. According to an increasing body of evidence,the starting dose of FSH represents a relevant aspect in optimising the outcome of both COS and IVF. In this context,different predicting factors should be taken into account. Available literature clearly demonstrates that algorithmswhich combine age, BMI and basal FSH with antral follicular count are effective in identifying the optimal r-hFSHstarting dose. These models show that, at least in “good prognosis” patients a starting dose of r-hFSH rangingbetween 112.5 and 150 IU is appropriate. Conversely, higher FSH doses increase costs without modifying pregnancyrates. The hypothesis that “predicting factors” are useful in choosing stimulation protocols has been further testedby other groups. In this context, the role of Anti-Müllerian Hormone (AMH) has been evaluated. Preliminary studiessuggest that the association of GnRH-agonist (GnRH-a) long protocol with low-intermediate doses of r-hFSH stillrepresents a reliable “first line” treatment in patients with normal AMH. Conversely, GnRH-antagonists (GnRH-ant)seem to be more efficient in women with “low” and “high” AMH. In this complex scenario, there are lines of researchindicating “mild stimulation” as first choice regimen in young, gonadotrophic women. More specifically, treatmentwith GnRH-ant in association with 150 IU of r-hFSH may reduce stimulation burdens maintaining optimal outcomeof IVF.Interestingly, in about 12-15% of young “good prognosis” patients, the ovarian response to standard GnRH-a longprotocol plus FSH is sub-optimal. More specifically, an initial low response is seen, leading to an increase in the dailydose of FSH, resulting in a higher total FSH consumption (e.g. >2500 IU). These observations lead to the developmentof the concept of “hypo-response” to COS to identify normogonadotrophic women who have normal estimatedovarian reserves but require high amounts of FSH to obtain an adequate number of oocytes retrieved. These womenseem to be distinct from classical poor responders because they have normal ovarian reserve, but unexpectedly showsub-optimal response when stimulated with standard regimens. Conversely, specific adjustments of classical protocolsseem to optimise ovarian response.On the basis of the current literature, it is possible to argue that hypo-response could be related to geneticcharacteristics, including specific polymorphisms of the genes of FSH receptor and LH. More specifically, it has beendemonstrated that carriers of a polymorphic variant of the FSH receptor (FSH-R) where aminoacid asparagine (Asn),at position 680, is replaced by Serine (Ser), benefit from higher FSH doses than those expected from age, BMI andbasal FSH. We have recently focused our research on a common polymorphism of LH (v-LH), due to two pointmutations in the β subunit gene, both altering the aminoacid sequence (Trp8Arg and Ile15Thr). Our results indicatethat the presence of v-LH is associated with ovarian resistance to FSH and higher r-hFSH consumption during COS.It should be underlined that both FSH ser/680 and v-LH are common worldwide. More specifically, homozygosis forser/680 is demonstrable in about 26% of female population, whereas v-LH carrier frequency varies from 0 to 52%in various ethnic groups.In conclusion, current literature indicates that predicting factors, including biomarkers and antral follicular countmay be useful in identifying appropriate COS strategies. These aspects, together with evaluation of geneticcharacteristics may be helpful in choosing both GnRH-analogues regimens and starting doses of r-hFSH. In this context,increasing compliance and reducing stimulation burdens and costs should be also considered as crucial aims intailoring the approach to IVF.

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EVOLUTION AND FUTURE OF ANTAGONIST PROTOCOLSPalermo RAMBRA (Associazione Medici e Biologi per la Riproduzione Assistita), Nuova Casa di Cure DEMMA,Palermo, Italye-mail: [email protected]

Induction multiple follicular development (MFD) by gonadotropin is the first and crucial step in all assisted reproductivetechnologies (ART) procedures. The evidence produced in almost three decades of experience and clinical studiesabout induction of MFD, support the idea (and the practice) that a stable and early inhibition of endogenousgonadotropins may be beneficial to achieve follicular synchronization and the highest clinical pregnancy rates. Thismay be achieved by combining the gonadotropin administration either with a GnRH-agonist or a GnRH-antagonist.GnRH-agonist-gonadotropin co-treatment was introduced in the mid eighties and for almost twenty years it hasbeen considered the standard for induction of MFD. GnRH-antagonists were introduced in recent years in ovarianstimulation for ART. Two GnRH antagonist protocols were developed involving either multiple or single administration.In the multiple dose protocol, the GnRH antagonist was administered continuously until the day of hCG administration,starting 5 days after stimulation with gonadotropins. The minimal dose shown to prevent the occurrence of apremature LH rise in the great majority of patients was shown to be 0.25 mg. In clinical trial data and meta-analyses,treatment with these GnRH-analogues is associated with similar live birth rates but reduced treatment burden,compared with GnRH-agonist long protocols. Moreover, GnRH-antagonist co-treatment results in shorter and morecost-effective ovarian stimulation protocols as compared with GnRH-agonist (long) protocols. In addition, there issome evidence showing a reduced incidence of early OHSS by the use of a GnRH-antagonist for inhibition of LHrelease and a GnRH-agonist to trigger final oocyte maturation.In recent years, increased understanding of the physiology of follicle development and a new thinking about criticalaspects of patients management has lead to more individualized stimulation approaches. Personalized managementstrategies, based on individual patient characteristics, have been proposed and the further development of strategiesmay represent real progress towards individually tailored fertility treatment. Evidence in favour of the so called mildovarian stimulation for IVF is accumulating in recent literature. Specifically, young, good responders and polycysticovary syndrome patients may benefit from mild stimulation. Mild stimulation protocols, comprising a GnRH-antagonistfor pituitary suppression and low-dose FSH stimulation in the mid-to-late follicular phase can reduce gonadotrophindosage, cutting costs, time commitments and the need for intense monitoring. Mild stimulation also diminishespatient distress and complications. When combined with the transfer of fewer embryos, multiple birth rates arereduced. Unquestionably, those are precious achievements in a patient-centred treatment perspective and shouldbe the goals for the patients present management and future research.


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CORIFOLLITROPIN A NOVEL LONG ACTING rFSH: CLINICAL FEATURES ANDCLINICIAN’S PERSPECTIVEDevroey PCentre for Reproductive Medicine, UZ Brussels, Brussels, Belgiume-mail: [email protected]

Corifollitropin alfa is a recombinant fertility hormone designed as a sustained follicle stimulant with the samepharmacodynamic profile as rFSH, but with a markedly prolonged duration of action. A single subcutaneous injectionreplaces the first seven injections of any daily rFSH preparation.A prospective randomized controlled trial (ENGAGE), statistically powered to demonstrate the non inferiority in theongoing pregnancy rate of a single injection of corifollitropin alfa 150 μg versus 7 daily injections of recombinantFSH when used in a GnRH antagonist protocol, has been performed in women with a body weight of > 60 kg. 1506patients were enrolled, 756 in the corifollitropin alfa group and 750 in the control group. The ongoing pregnancyrate was 39% per started cycle in the corifollitropin alfa arm and 38% per started cycle in the control group. Thedata indicate that a single injection of corifollitropin alfa is efficacious and non inferior to 7 daily injections ofrecombinant FSH. In one third of the patients stimulated with corifollitropin alfa and r-FSH the criterion for hCGadministration (at least 3 follicles of ≥ 17 mm) has been met on day 8 of treatment day, not requiring additional FSHinjections.The rate of OHSS following corifollitropin alfa was similar to that with daily r-FSH. Hospitalisation of OHSS wasrequired in 1.9% of patients treated with corifollitropin alfa and in 1.2% of patients treated with daily r-FSH.Additionally a second smaller randomized controlled trial has been performed with a single injection of 100μg inwomen with a body weight ≤ 60 kg (ENSURE trial). The trial evaluated the number of oocytes as primary endpoint.The mean number of oocytes retrieved per started cycle was significantly higher for corifollitropin alfa (13.3 ± 7.3)compared to daily rFSH (10.6 ± 5.9), but well within the prespecified equivalence range.In conclusion, corifollitropin alfa is a novel recombinant fertility hormone that can be administered as a singleinjection to initiate and sustain multiple follicular development for the first seven days of COS prior to IVF/ICSI.

References:

The Corifollitropin Alfa Dose-finding Study Group. A randomized dose-response trial of a single injection ofcorifollitropin alfa to sustain multifollicular growth during controlled ovarian stimulation. Hum. Reprod. 2008;23(11):2484-2492.

Kolibianakis EM, Albano C, Camus M, Tournaye H, Van Steirteghem AC, Devroey P. Prolongation of the follicularphase in in vitro fertilization results in a lower ongoing pregnancy rate in cycles stimulated with recombinant follicle-stimulating hormone and gonadotropin-releasing hormone antagonists. Fertil. Steril.2004;82(1):102-107.

Devroey P, Fauser BC, Platteau P, Beckers NG, Dhont M, Mannaerts BM. Induction of multiple follicular developmentby a single dose of long-acting recombinant follicle-stimulating hormone (FSH-CTP, corifollitropin alfa) for controlledovarian stimulation before in vitro fertilization. J. Clin Endocrinol Metab.2004;89(5):2062-2070.

Devroey P, Boostanfar R, Koper NP, Mannaerts BM, Ijzerman-Boon PC, Fauser BC. A double-blind, non-inferiority RCTcomparing corifollitropin alfa and recombinant FSH during the first seven days of ovarian stimulation using a GnRHantagonist protocol. Hum. Reprod. 2009;24(12):3063-3072.

Mannaerts B. Corifollitropin alfa ENSURE Study Group. Corifollitropin alfa for controlled ovarian stimulation in IVFtreatment: results from a randomized trial in lower body weight women. RBM Online 2010;21:66-76.



Merck Serono PharmaceuticalsSymposium

Saturday, 2 October 201011:30 - 13:00

The future of controlled ovarian stimulation:from reproductive physiology and genomics

to individualized therapy

Chairpersons: Lenzi A (IT), Levi-Setti P (IT)

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ROLE AND RESPONSIBILITIES OF LH DURING EARLY FOLLICULAR GROWTH:GONADAL AND EXTRA GONADAL EFFECTSAndersen CYLaboratory of Reproductive Biology, University Hospital of Copenhagen, University of Copenhagen,Copenhagen, DenmarkE-mail: [email protected]

The two gonadotropins FSH and LH exert through receptor stimulation several diverse and different functions ontheir target cells in the ovary. FSH receptors (FSHr) are exclusively located on the surface of the granulosa cells fromearly on in development, and LH receptors (LHr) expression occurs constitutively on the surface of theca cells. Inaddition, LHr becomes expressed on the granulosa cells as the follicle reaches the preovulatory stages. Expressionof LHr on the granulosa cells is orchestrated by FSH stimulation of the FSHR, which thereby play an important rolein changing granulosa cells sensitivity to gonadotropins during the final stages of follicular development.However, knowledge on how LHr expression develop on granulosa cells, and in the later stages of folliculogenesison cumulus cells, is not known in great detail. This is partly due to the scarce availability of human material andpartly due to the fact there are no good immunohistochemical methods for detection of LHr and a number of othermethods have shortcomings in connection with measuring LHr and the density of LHr that occur normally in granulosacells.We have used molecular biology methods to measure LHr mRNA expression in human granulosa and cumulus cellsfrom various sources. First, in connection with cryopreservation of human ovarian tissue for fertility purposes wehave collected granulosa cells from a number of small antral human follicles with a diameter of 3 to 9 millimetres.Second, we have obtained cumulus-oocyte complexes from women undergoing IVM treatment and lastly we haveobtained granulosa and cumulus cells from conventional IVF treatment.In mRNA isolated from these cells we have analysed the expression of FSHr, LHr, AMH, AMH-r2, Androgen-receptor(AR) and aromatase (CYP19) by quantitative RT-PCR. The presentation will focus on these data and where availablethey will be associated to the corresponding follicular fluid hormone profiles.


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CUSTOMIZED COS TO PATIENTS NEEDSHumaidan PThe Fertility Clinic, Skive Regional Hospital, Skive, Denmarke-mail: [email protected]

Introduction: With the recent development of recombinant gonadotropins (FSH and LH), it has become possibleto further adjust the stimulation protocol according to the expected needs of the patient. In this respect, the possiblebeneficial role of exogenous LH activity supplementation for stimulated ART cycles has received increasing attention.According to the two-cell, two- gonadotropin theory (Fevold, 1941), both FSH and LH are required for normalfolliculogenesis in humans. LH stimulates the production of androgens in the theca cells, which in turn are aromatisedto estradiol by the granulosa cells under the action of FSH. However, at a follicle size of 8-10 mm in normogonadotropicwomen, the granulosa cell also acquires LH receptors in addition to the FSH receptors, already present. Once LHreceptors are expressed in the granulosa cell, LH is able to regulate both steroidogenesis and growth of the follicle;thus, from this moment on FSH function can to a large extent be replaced by LH activity.Methods: During recent years an increasing body of scientific evidence has raised the question whether the LHhormonal environment achieved after down-regulation with either GnRHa or GnRH antagonist is really optimal forall patients, or whether sub-groups of patients exist who might benefit from exogenous LH supplementation.Several studies have until now addressed the effect of LH activity supplementation. The results of these studiesindicate that subgroups of normogonadotropic patients - patients > 35 years of age (Marrs et al. 2004, Humaidanet al., 2004, Bosch et al., 2008; Matorras et al., 2009) and patients with an initial sub-optimal response to FSH onlypreparations (Barrenatexea et al., 2000; De Placido et al., 2004; Ferraretti et al., 2004; Ruvolo et al., 2007) - indeedseem to benefit from modifications of the stimulation protocol in terms of exogenous LH activity supplementation.Moreover, additional exogenous FSH in patients with polymorphisms in the FSH receptor gene improves the ovarianresponse after COS (Mayorga et al., 2000).Possible biological reasons for a beneficial effect of modification in the stimulation of these sub-groups will bediscussed.Conclusions: Age, LH gene polymorphisms and FSH receptor polymorphisms are some of the factors, known untilnow to influence the ovarian response after COS. A customized stimulation in patients with these characteristicsimproves the ovarian response and the reproductive outcome. Ovarian response to stimulation with FSH is a polygenictrait and thus, the future scenario of ART will include pharmacogenetics in order to define the specific needs ofgonadotropins (FSH and LH) to secure the most optimal ovarian response.

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FROM SUBJECTIVITY TO OBJECTIVITY IN GAMETES/EMBRYO SELECTIONMontag MDept. Gyn. Endocrinol. & Reprod. Medicine, University of Bonn, Bonn, Germanye-mail: [email protected]

Quality of gametes and embryos is a key issue in assisted reproduction. Its main application is for selecting the bestgametes / embryos to achieve higher pregnancy and implantation rates. The ultimate aim is a higher take homebaby rate preferentially using a single embryo for transfer. Quality of gametes and embryos does partly depend onthe starting material but the laboratory aspect is equally important. This includes not only the actual work and howit is performed but also the quality of every item or product which is used throughout the process. Therefore,standardization of the laboratory is a prerequisite for the assessment of embryo quality. Otherwise differences inembryo quality will reflect quality fluctuations in lab work or consumables rather than the embryo’s real potentialto give rise to a viable pregnancy.Standardization is equally important for assessing gamete and embryo quality and the goal is to move from subjectiveto objective evaluation. A major criterion for quality assessment still is morphology. However, although morphologyis a good point to start with, it is not a reliable and objective tool. Morphology assessment rather gives a snapshotthan a true reflection of a complex process. Time-lapse imaging of embryo development has shown that morphologyof a momentum can be misleading.There are numerous technologies in use on the level of sperm, oocytes and embryos, but only few are fully objective.Imaging is applied in various ways and among the new concepts is polarized light microscopy for sperm and foroocytes. Spindle detection and automated zona imaging can help in identifying good quality oocytes. A step furtheris continuous image acquisition allowing time-lapse analysis of early embryo development and preliminary dataalready show that an adequate timing of early embryo development is important. Imaging is not an ultimatetechnology but it is a good add-on in combination with other emerging technologies which are already now in useto improve outcomes. A clear way to go to for the future is being non-invasive and physiological relevant and sometechnologies are already in line with that. Examples are respiration measurements to investigate oxygen consumptionin oocytes, metabolomic profiling using near infrared spectra of spent embryo culture medium and direct measurementof amino acid turnover in culture medium as well. Some of the results obtained with that technology are intriguing.Metabolomic studies have shown that the morphologically best embryo does not necessarily show the best metabolomicprofile – questioning again the value of morphology assessment. And in terms of amino acid turnover quiescenceseems to be a pretty successful strategy.It is generally accepted that measuring something is a way towards objectivity. The reliability of all the techniquesmentioned above is greatly dependent on how robust the results achieved are under standardized conditions. Thisdoes still require further work and presumably much more trials in order to establish the scientific and the clinicevidence that it does work.

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LH vs hCG IN ENDOMETRIAL GENE EXPRESSION PROFILEHorcajadas JAiGenomix, Valencia, SpainFundación IVI, Valencia, Spaine-mail: [email protected]

The endometrium is a highly dynamic tissue with the capacity to undergo physiological changes in response to steroidhormones with the ultimate aim of creating a receptive status in a synchronized manner with the arrival of theimplanting blastocyst during the window of implantation (WOI) between days 19 and 21. For more than 60 yearshistologic evaluation has been considered a standard for clinical diagnosis based on morphological observations.These classic articles have been widely quoted, and followed as ‘‘the diagnostic tool’’ for endometrial dating. However,their accuracy and functional relevance as a predictor of endometrial receptivity have been questioned in randomizedstudies. With the arrival of the Genomic Era and the development of sophisticated technical analysis and bioinformatictools, new perspectives of the analysis and classification of subclasses of the disease have emerged. Using microarraytechnology we have created the ERA (Endometrial Receptivity Array), a customized microarray for endometrialevaluation, With the help of the bioinformatics we have also developed a predictor for ERA analysis. This is the firsttime that a molecular tool based on microarray technology can be used clinically in reproductive medicine to evaluatethe endometrium. This genomic tool (ERA+predictor) is a unique innovative and objective procedure for clinicalendometrial evaluation that will probably improve endometrium-related evaluations and diagnoses. The ERA is alsoa new molecular research tool for endometrial research as it contains a finite number of genes involved in endometrialreceptivity, thus avoiding the use of whole genome microarrays, which cuts down on costs and simplifies the dataanalysis. Furthermore, the methodology presented herein could serve to inspire new molecular approaches fordiagnoses or evaluations, and to also switch from anatomical to molecular medicine.


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FIXED VERSUS FLEXIBLE GnRH ANTAGONIST ADMINISTRATION PROTOCOL INPATIENTS WITH POLYCYSTIC OVARIES: EFFECT ON TOTAL GONADOTROPINDOSE AND PREGNANCY RATESRuiz Flores F, García-Velasco JA, Requena AIVI Madrid (Instituto Valenciano de Infertilidad), Madrid, SpainE-mail: [email protected]

Background: There seems to be a trend towards a higher pregnancy rate using the fixed GnRH antagonistadministration protocol, according to a recent meta-analysis. However, there is a lack of information comparing thefixed and flexible protocols in women with polycystic ovaries (PCO). The flexible protocol has also been associatedwith a significant reduction both in number of antagonist ampoules and amount of gonadotropins used. Weconducted a retrospective study from our electronic database of patients in women with PCO attended during theyear 2009 and we compared those who were assigned to a fixed protocol against those assigned to a flexible protocol.The objective of the study was to investigate the effect of either protocol on the amount of administered medication,with special interest in determining number of oocytes retrieved and quality of the embryos, as well as the pregnancyrate.Methods: A total of 118 patients with PCO were included for the study from our University associated private clinic.All of the included patients went through controlled ovarian hyperstimulation cycles with a GnRH antagonist protocol.We found 26 (22%) patients assigned to the fixed protocol and 92 (78%) patients to the flexible protocol. Wecompared the number of retrieved oocytes, percentage of fertilized oocytes, quality of embryos obtained, totalgonadotropin dose, number of days of stimulation and miscarriage and pregnancy rates between the two groupsof patients.Results: There was no statistically significant difference in age between both groups (p=0.51). The average numberof retrieved oocytes was 11.8 in the fixed group and 12.3 in the flexible group (p=0.958). Furthermore, the percentageof fertilized oocytes in the fixed group was 82.9% compared to 82.7% in the flexible group (p=0.776). The percentageof high-quality embryos in the fixed group was 50.4% and in the flexible group was 54% (p=0.575). The totaladministered gonadotropin dose and number of days of stimulation in the fixed group was 1256 IU and 8.8 daysrespectively, compared to 1579 IU and 10 days in the flexible group (p=0.002 for both). The miscarriage rate was23.1% in the fixed protocol group versus 15.2% in the flexible protocol group (p=0.379). The pregnancy rate was53.8% (14/26) in the fixed protocol group and 43.5% (40/92) in the flexible protocol group (p=0.349).Conclusions: There was a trend towards a higher pregnancy and miscarriage rate in the fixed group, but this wasnot statistically significant. More importantly, the fixed group needed fewer days of stimulation and a lower totalgonadotropin dose, and these two differences were statistically significant.

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EFFECTIVENESS OF PIOGLITAZONE FOR PREVENTING SEVERE OVARIANHYPERSTIMULATION SYNDROMECastellotti D, Cambiaghi AIstituto Paulista de Ginecologia, Ostetricia e Reproducao Humana Sao Paulo – Sao Paulo, BrazilE-mail: [email protected]

Objective: Ovarian hyperstimulation syndrome (OHSS) is a serious and potentially life-threatening iatrogeniccomplication in assisted reproduction. Serum and follicular fluid vascular endothelial growth factor (VEGF) concentrationshave been shown to be significantly elevated in patients who developed severe OHSS compared with those who didnot, suggesting a role for VEGF in the pathogenesis of this condition. Thiazolidinedione derivatives (TZDs), such aspioglitazone are approved as oral antihyperglycemic agents as therapy for noninsulindependent diabetes mellitus.They significantly improved insulin sensitivity, hyperandrogenism and ovulation rate without any serious side effectsrecent data indicate that TZDs decrease VEGF production by cultured granulose cells, and treatment with pioglitazonedoes not adversely impact the morphologic development of cultured murine embryos in vivo. The aim of this studywas to assess the effectiveness of pioglitazone for preventing the symptoms of OHSS after the hCG in IVF cycles.Design: Prospective randomized study.Material and methods: Between September 2009 and May 2010 we selected 24 women undergoing IVF treatmentat Instituto Paulista de Ginecologia e Obstetrícia (São Paulo, Brazil) considered at high risk of developing moderateor severe OHSS. All of them were submitted to the same protocol for controlled ovarian hyperstimulation (COH)with GnRH agonist and hMG. In group I (12 patients) the patients received conventional treatments for OHSS (IValbumin on the day of oocyte retrieval, rest and orientation for increase water intake and oral whey protein). Ingroup II (12 patients), besides conventional treatment all of them received pioglitazone 30mg orally once a daybeginning on the day of hCG.Results: No difference was found between the two groups in terms of patient’s characteristics. We classified OHSSin mild, moderate and severe according Schenker & Weinstein criteria. In group I 4 patients didn’t developed thesyndrome, 5 patients developed mild OHSS, 3 patients had moderate OHSS. None of them required hospitalization(we have shown in previous data the benefits of whey protein). In Pioglitazone group (group II) 8 patients didn’thave any symptoms of the syndrome, 4 patients developed mild OHSS and no patients had the moderate or severeform. None of them required hospitalization. We realized that patients in group I had a dramatically drop on estradiollevels right after pioglitazone intake, so they required a supplement of this hormone after the oocyte retrieval (whatis normally contraindicated for patients with OHSS risk). Clinical pregnancy rates were similar in both groups.Conclusion: The use of pioglitazone for preventing OHSS in high risk patients may be an excellent alternative asthe safety of TZDs in early pregnancy has been suggested by the literature. Nevertheless, larger studies with longerfollow up times are needed to confirm the effectiveness and safety of this drug.


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FLEXIBLE ANTAGONISTS PROTOCOL FOR FIV-ICSILorente González J, De Andrés CM, Ríos Castillo JE, Martín Sances L, Povedano Cañizares B,Velasco Sánchez E, Ramírez Montilla F, Arjona Berral JEReproductión Unit, University Hospital "Reina Sofía", Córdoba, SpainE-mail: [email protected]

Objectives: To determine whether flexible antagonist protocol is at least as effective as agonist long protocol inovarian stimulation practice of our unit.Material and methods: A retrospective study of 410 first cycles of patients undergoing IVF-ICSI between 2008 and2009 in our Unit of Reproduction. The cases were divided into two groups of 205 patients each. There were nodifferences between groups in age, body mass index, level of FSH on day 3, diagnosis, or type of gonadotropin usedin stimulation. Stimulation protocol. Before stimulation, all patients were administered 0.03 mg ethinyl estradioland 0.15 mg of levonorgestrel (Microgynon®, Schering AG) for 18 to 30 days. The stimulation started the 2nd-3rdday of the cycle at a dose of follitropin variable, depending on age, body mass index, basal FSH, and antral folliclecount. The type of gonadotropin used was at the discretion of the physician who initiated the cycle. The patternmore used was the so-called "combo", consisting of recombinant FSH (Gonal 1050®, Merck-Serono) mixed withhuman menopausal gonadotropin (hMG) (Menopur®, Ferring) in 2:1 ratio. Follicular maturation was performed with250 mg of choriogonadotropin alfa (Ovitrelle®, Merck-Serono) when appreciated at least 3 follicles ≥ 17 mm. Flexibleantagonists protocol. When a follicle reached a diameter of 14 mm, 0.25 mg of GnRH-antagonist were administered.Cetrorelix (Cetrotide®, Merck-Serono) or ganirelix (Orgalutran®, Organon) were used interchangeably. Agonists longprotocol. Six to seven days before the end of the contraceptive pills, the patients began subcutaneous administrationof 0.2 mg of leuprolide (Procrin®, Abbott Laboratories). Once started the period, this dose was halved until the dayof hCG administration inclusive. Follicular puncture and embryo transfer. Follicular puncture was performed 34-36hours after administration of choriogonadotropin, and embryo transfer was performed 2-3 days after. Usually twoembryos were transferred. The luteal phase support was performed with 400 mg of intravaginal micronizedprogesterone (Progeffik 200 mg, Effik) since follicular puncture until the day of pregnancy test or until the 10thweek of pregnancy.Results: There were no differences in pregnancy rate per cycle (23.4 vs 23.9, p= 0.90749) or per transfer (35.3 vs 36.0,p= 0.89927). The antagonists group needed fewer days of stimulation (9.3 vs. 9.7, p = 0.02987) and a lower dose offollitropin (without statistic difference). Although this group has fewer oocytes (6.0 vs 7.3, p = 0.00397) and embryos(3.2 vs 4.4, p= 0.00007), the transfer ratio with at least one good quality embryo was similar (71.3 vs 69.1, p= 0.69071).A higher rate of abortions in the antagonists group was observed, without reaching statistical significance (20.8%vs 12.2%, p = 0.25233).Conclusions: The flexible antagonists protocol need fewer injections and days of treatment than the agonists longregimen, making it more comfortable for patients and economic. We think it may be a good treatment regimen inany patient, although more studies are needed to clarify the possible highest rate of abortions.

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THE LH RECEPTOR insLQ VARIANT IS A NEGATIVE FACTOR IN ARTFilippini G, Spalvieri S, Lucchi S, Suter T, Jemec MProcreaLab SA, Lugano, SwitzerlandE-mail: [email protected]

Objective: The pituitary gonadotrophin LH plays an important role in follicular maturation and ovulation and iscrucial in the reproductive process. Recently a functional variant of the LH Receptor, insLQ in exon 1, has beenreported to increase LHR activity and has been correlated with a shorter survival term in breast cancer patients.Design: We hypothesized an influence of insLQ (ins) on the response of women to hormonal stimulation prior tooocyte pick-up during ART, in terms of the number of recovered mature oocytes and of the outcome (pregnancy).Materials and methods: Informed consent was obtained from all 88 women undergoing ART included in this study.Only women younger than 38 years old were included. Leukocyte DNA was extracted by standard protocols (QIAamp,QIAGEN); patients were genotyped by PCR amplification of LHR exon 1, with one 6-FAM labelled fluorescent primerfollowed by capillary electrophoresis and fragment analysis (ABI Prism 310, Applied Biosystems). Patients were dividedinto 3 groups according to genotype: ins/ins; ins/N; N/N (N = non insLQ). The number of mature oocytes recoveredand the percentage of pregnancies (bHCG level ≥10 mIU/ml on day 14) were determined.Results: The distribution of the three genotypes was: ins/ins: 6 women; ins/N: 32; N/N: 50. The mean number ofrecovered mature oocytes per patient was: 6 (1-13); ins/N 9 (1-28) and N/N 9.5 (2-33). Pregnancy rates were: ins/ins0% (0/6); ins/N 34% (11/32); N/N 32% (16/50).Conclusion: To our knowledge this is the first study concerning the potential influence of the LHR insLQ varianton the outcome of ART. Our data suggest that LHR insLQ homozygotes have poor outcomes of hormonal stimulation(mean oocytes = 6; pregnancy rate 0%), compared to nonLQ individuals (mean oocytes = 9-9.5, p=0.89; pregnancyrate: 32-34%, p=0.06). Because of the small number of insLQ homozygotes in this preliminary study, the analysis ofa larger group of patients is underway in order to confirm this preliminary result.


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ASSESSMENT OF THE VALUE OF ADDING LH ACTIVITY DURING THE LATESTAGES OF FOLLICULAR MATURATION IN ICSI CYCLESSharkawy S, Azab H, Elshahed M, Amer M, Al-Inany HCairo University, Cairo, EgyptE-mail: [email protected]

Objective: In patients on GnRH agonist long protocol, the impact of suppressed endogenous LH levels, on theoutcome is still debatable. It was reported that lower estradiol synthesis, lower oocyte and embryo yield, lowerfertilization rate , and higher frequency of early pregnancy wastage were observed in women down regulated witha GnRH agonist and stimulated with pure FSH preparations. Conversely, some papers reported that LH had a negativeeffect on oocyte quality and IVF outcome. Others failed to detect any relationship between serum LH levels andstimulation outcome. In this study we are evaluating the value of providing LH activity during the late stages offollicular development. We are testing the effect of such LH supplementation on the outcome of ICSI cycles.Participants & Methods: Four hundred seventy three couples were recruited for this study. They were assignedinto 2 groups; group A, the control group, comprised 262 patients and group B, the experimental group, comprised211 patients. Long down regulation protocol of ovarian stimulation was used for all patients. Highly purified FSHwas used throughout the stimulation period in the control group. In the experimental group, we replaced highlypurified FSH with HP-HP-hMG when the leading follicle was 10-12 mm. Our primary aim was to compare the outcomein terms of clinical pregnancy rate. Secondary outcomes included the number of stimulation days, total amount ofFSH administered, total number of embryos, and number of cancelled cycles.Results: The clinical pregnancy rate in group B, the experimental group, was significantly higher than that of groupA, the control group (40% versus 29%, p < 0.05). The odds ratio was 1.59 (95% CI 1.06-2.37). The number of stimulationdays was significantly higher in the group B compared to group A (p < 0.01). The total amount of FSH administeredwas significantly higher in group B compared to group A (56.97 ± 15.60 Vs 41.63 ± 9.81, p < 0.01). There were nosignificant differences between the 2 groups regarding the total number of embryos, the number of good embryostransferred, number of fair embryos transferred, the number of freeze embryos, and the number of cancelled cycles.Conclusion: Adding LH activity in late stages of follicular maturation during ovarian stimulation improves the clinicalpregnancy rate.

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MILD OVARIAN STIMULATION WITH hp-hMG IN IVF: RESULTS FROM APERSPECTIVE CONTROLLED STUDYBecattini C, Rizzello F, Cammilli C, Fantaccini I, Quitadamo L, Zambelli VFutura Diagnostica Medica P.M.A., University of Florence, Florence, ItalyE-mail: [email protected]

Background: Ovarian stimulation protocols for in vitro fertilization (IVF) with decreased dose of gonadotropins arebeing developed in recent years to minimize adverse effects. Recent studies suggest that the decreased number ofoocytes at retrieval following mild stimulation is associated with better outcomes. Furthermore, the relative importanceof LH during the follicular phase and its role in the stimulation of the follicle is still subject to extensive debate.However, recent meta-analyses and reviews demonstrated that hp-hMG is superior to rFSH with regard to clinicalefficiency. The objective of this study was to evaluate the efficacy of a mild stimulation protocol with hp-hMG in aselected group of patients.Setting: Private outpatient infertility clinic.Materials and Method(s): We studied patients undergoing IVF cycles for the first time in our clinic. The inclusioncriteria were: age ≤38 years, eumenorrhoic cycles, basal FSH < 10 mIU/mL, and infertility attributable to tubal,idiopathic, or moderate male factor. The study group included women (n.= 25) treated with 150 IU of hp-hMG(Meropur®), prospectively enrolled from March 2010. The controls (n.= 25) were retrospectively selected among ICSIcycles previously performed and treated with 150 IU of r-FSH (Gonal-f®, Puregon®). In both groups pituitary suppressionwas acheived with antagonist in a flexible protocol.Main Outcome Measure(s): The main outcome measure was pregnancy rate. Secondary outcomes included totalgonadotrophin used, lenght of stimulation, serum estradiol levels and follicles number at hCG day, number of oocytesretrieved, fertilization rate, implantation rate and cost of stimulation.Result(s): No differences were observed for total gonadotropin requirements, days of stimulation, oocyte yield andfertilization rate. No cases of drop-outs and OHSS were observed in both groups. A significantly higher pregnancyrate per embryo transfer was observed in hMG group (60% in the study group versus 32% in the controls). Althoughfinal estradiol levels showed similar values in both groups, they seem rising more slowly in the study group.Conclusion(s): Stimulation protocols including hp-hMG seem to be advantageous in term of pregnancy rate. Ourpreliminary data suggest that good prognosis patients undergoing IVF can successfully be treated with 150 IU ofmenotropin at a considerable financial savings. However, further, sufficiently powered prospective randomizedstudies applying this novel mild treatment regimen are required.


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NEUROTROPHINE FOLLICULAR FLUIDS CONCENTRATION IN INFERTILITY WOMENPalumbo M, Teodoro MC, Gulino F, Rubbino G, Ciotta L, Leonardi E, Cantarella G, Bernardini RCenter of Physiopatology of Human Reproduction, Dpt. Of Microb. Sc. And Gynaecological Sc., Universityof Catania, Catania, ItalyE-mail: [email protected]

The Nerve Growth Factor is the most important member of the neurotrophines' family and it was found in the ovariesof rats and humans together with its receptor. It has many functions: involved in the precocious survival of germcells, in the steroidogenesis control, in the expulsion of polar body, supporting the ovulation. Particularly, manyresearches strongly suggest that it has an important role in the folliculogenesis and cytoplasmatic development ofthe oocyte. In our preliminary study we proposed to determinate NGF levels, in the follicular fluid and in the serumof a group of patients submitted to a IVF cycle, for many causes of infertility, and then we put a relationship betweenthese results and oocyte quality. The considered sample was composed of 78 patients, aging between 19 and 44years old (Middle age: 32,88), and 14 of these women were illness (masculine sterility), defined like control group(CTR), 8 affected by idiopathic sterility, 6 had an eterozigosis of the MTHFR gene, 4 patients affected by a thyroidpathology, 18 suffered from PCO, 18 with tubaric factor and 10 with endometriosis. All the patients were submittedto IVF cycles, Fivet (4 patients) and Icsi (74 patients), in the period between October 2006 and March 2010. Wecollected follicular fluids during oocytes pick-up (FF) and serum basal sample (A) and after ovarian stimulation (B).From the results of this study we found that NGF concentrations in the FF were much higher than serum levels bothA and B. The NGF medium frequency was 213,76 pg/ml in the FF, instead of the medium frequency was 46,47 pg/mlin the serum A and it was 60,75 pg/ml in the serum B. Then the NGF levels in FF were about 4-5 times higher thanserum levels; moreover we didn't find any significant differences between the two serum. Finally from the obtainedresults we can assert that there is a paracrine secretion of NGF in the FF, and the concentrations of this growth factorcould be important for the follicular and oocyte growth.

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DIFFERENTIAL APPROACH TO CONTROLLED OVARIAN STIMULATION IN POORRESPONDERS UNDERGOING ASSISTED REPRODUCTION CYCLESStrelko G"Victoria" Genetics and Reproduction Clinic, P.Shupik National Medical Aacademy for Postgraduates,Obstetrics and Gynecology Department, Kyiv, UkraineE-mail: [email protected]

Introduction: Decreased ovarian reserve is a condition that cannot be reversed by means of medical treatment.There is a strict correlation between the AMH level, AFC and the number of follicles during stimulation. Severalprotocols to induce ovarian superovulation have been introduced in the clinical practice for poor respond patients,but the results have not been promising. We propose the differential stimulation regimen for poor respond patientsaccording to the AMH level and AFC.Materials and methods: A retrospective study of patients, who failed to respond traditional long GnRH agonistsprotocol, short flare-up or GnRH antagonists protocols. Inclusive criteria were: previous stimulation failures and ageless than 39 years old. A population of 52 women (aged 23 - 39 years), attending our infertility clinic from September2007 to March 2010 was screened before the controlled ovarian stimulation initiation. Serum AMH, FSH, E2 levelswere measured on the 2 - 3 day of the cycle. AFC was measured during TV ultrasound examination. Patients weredivided into 3 groups according to the AMH and AFC: 1. AMH 0,7 - 1,0 ng/ml; AFC ≥ 5 (n = 11) - traditional shortflare-up protocol was applied with 0,1 GnRH agonist (Diferilin®) and 300 IU of HP-hMG (Menopur®) daily from the2nd or 3rd day of cycle with further adjusting the dose of HP-hMG. 2. AMH 0,4 - 0,7 ng/ml ; AFC - 2 - 4 (n = 25) -Clomiphene citrate: the 100mg daily from the 3rd to 7th day of the cycle with HP-hMG 300 IU daily on the 7 - 11days with further adjusting the dose. GnRH antagonist (Cetrotide®), was started when at least one follicle reached14 mm. 3. AMH < 0,4 ng/ml; AFC - 1 - 3 (n = 16) - natural cycle with GnRH antagonist, when at least one folliclereached 14 mm and HP-hMG 150 IU daily.Results: There was no significant difference regarding BMI, duration and the type of infertility in three clinicalgroups. The FSH (9,8±5,3 mlU/ml, 11,2 ± 4,7 mlU/ml, 14,7 ±7,5 mlU/ml) levels were slightly higher in the third group(not statistically important). In total we managed to obtain oocytes in 76,9% of all patients. Duration of stimulationwas 10,7 and 8,9 days in 1st and 2nd clinical groups. The total dose of gonadotrophins was 2887,5 ±555 IU; 1537,5±487,5 IU ; 712,5±525 IU respectively in 1st, 2nd and 3rd clinical groups. The cancellation rate was the highest in thethird group - 37,5 %; 16% in 2nd and 18,1% in 1st group. Pregnancy rates were 27,3%, 28,0% and 6,3 % respectively.Conclusion: This study shows the benefits of the differential approach to stimulation protocols in poor responder,undergoing IVF: lower cancellation rate, lower total dose of gonadotrophins, acceptable pregnancy rate. Low levelof AMH and AFC results in discorageous prognosis. Further prospective randomized trials are required to evaluatethe benefits of different stimulation regimen in poor respond patients.


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METFORMIN TREATMENT BEFORE AND DURING IVF OR ICSI IN PCOS WOMENWITH BMI < 28 KG/M3: A PROSPECTIVE, MULTICENTER, RANDOMISED, DOUBLE-BLIND STUDYKjøtrød SB, Rasmussen PE, Holst-Larsen T, Mellembakken J, Thurin KA, Kouru K, Humaidan P,Morin PL1 Trondheim University Hospital, Dept Ob/Gyn, Trondheim, Norway2 Odense University Hospital , Dept Ob/Gyn, Odense, Denmark3 Haugesund Fertility Senter, Haugesund, Norway4 Section of Assisted Reproduction, Rikshospitalet, Oslo, Norway5 Sahlgrenska University Hospital, Dept Ob/Gyn Gothenberg, SwedenE-mail: [email protected]

Introduction: It is debated whether metformin co-treatment in IVF / ICSI significantly improve live birth, pregnancyor fertilisation rates. (Cochrane review 2009)Materials and methods: 8 clinics recruited a total of 149 PCOS (polycystic ovarian syndrome) women scheduledfor IVF or ICSI treatment. The patients were randomised by the hospital pharmacy making identical capsules ofmetformin (74) and placebo (75). Codes were not broken until last ongoing pregnancy in the study was 32 weekspregnant. All patients fulfilled the Rotterdam criterias, and all had BMI < 28 kg/m3. All patients had general diet-and life style councelling at screening, and were treated with metformin / placebo (1000mg bid) 4-5 months untilthe day of positive/negative s-hCG (pregnancy test). Patients were downregulated with nafarelin at least 14 daysbefore stimulation with recombinant FSH (fixed dose 112.5 IE). From cycle day 8 dose adjustments were allowed.Mean age was 29.5 years, and mean BMI 23, 8 kg/m3.Results: The primary endpoint in this study was clinical pregnancy rate defined as an intrauterine gestational sacby ultrasound week 7. According to intention to treat analysis the clinical pregnancy rate was 0.50 in metformingroup vs. 0.33 in placebo group (p=0.04). Live birth rates were 0.49 vs. 0.32 (p=0.04). Prior to IVF 14 patients droppedout, and during pre-treatment 15 spontaneous pregnancies occurred in metformin group vs. 8 in placebo group(p=0.10). In per protocol analysis among patients starting gonadotrophin stimulation, respectively 56 in metformingroup vs. 56 in placebo group, the biochemical pregnancy rate was identical 0.43 vs. 0.43. There were a difference(although not significant) in clinical pregnancy rate (0.39 vs. 0.30), and in live birth rate (0.38 vs.0.29). There wereno significant differences in the total number of oocytes (11.6 vs. 13.2), fertilisation rate (0.53 vs. 0.54), number ofcleaved embryos day 2 (6.0 vs. 7.6), number of transferred embryos (1.2 vs. 1.1), number of frozen embryos (2.5 vs.2.7), and number of good quality embryos (3.5 vs. 3.6). Total dose of gonadotrophin (1553 IE vs. 1531 IE), and numberof stimulation days (12.4 vs. 12.1) were also similar.Conclusion: In this prospective randomised, double-blind study metformin treatment before and during IVF or ICSIincreases live birth rates in PCOS women with BMI < 28 kg/m3. The number of spontaneous pregnancies during pre-treatment contributes notably to this result.

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EFFECTS OF INOSITOL ON OOCYTES’ QUALITY IN PATIENTS WITH POLYCYSTICOVARY SYNDROMEPalumbo M, Ciotta L, Stracquadanio M, Pagano I, Carbonaro A, Gulino FDipartimento di Scienze Microbiologiche e Scienze Ginecologiche, Sezione di Ginecologia, Ospedale SantoBambino, Università degli Studi di Catania, Catania, ItalyE-mail: [email protected]

Polycystic ovary syndrome is the most common cause of chronic anovulation infertility in women in fertile period.The supplementation of inositol, due to its ability to increase insulin sensitivity, improves the oocytes' quality andincrease the number of oocytes collected after ovarian stimulation in patients undergoing IVF (In Vitro Fertilization).The aim of our study is to determine the effects of myo-inositol on oocyte's quality on a sample of women withpolycystic ovary syndrome. The patients were divided into two groups: patients of Group A intook 2 g of myo-inositol+ 400μg of folic acid 2 times a day, continuously for 3 months, while Group B only 400μg of folic acid. At the endof treatment, the number of follicles of diameter >15 mm, visible at ultrasound during stimulation, and the numberof oocytes recovered at the time of pick-ups were found to be significantly greater in the group treated with myo-inositol, so as the average number of embryos transferred and embryo Grade G1. Significantly reduced was theaverage number of immature oocytes (vesicles germ and degenerated oocytes) too.


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ARE ALL HUMAN-DERIVED FSH PRODUCTS THE SAME? A SYSTEMATIC REVIEWAND META-ANALYSIS USING DIRECT AND ADJUSTED INDIRECT ANALYSES TODETERMINE IF FOSTIMON® IS MORE EFFICIENT THAN METRODIN-HP®Al-Inany H, Abou-Setta ADepartment of OB/GYN, Faculty of Medicine, Cairo University, Cairo, EgyptAlberta Research Centre for Health Evidence (ARCHE), University of Alberta, Edmonton, Alberta, CanadaE-mail: [email protected]

Background: Recent randomized trials and systematic reviews have challenged the claim for superiority of recombinantFSH (recFSH) compared with human-derived FSH (hFSH) products. Even so, much of the evidence comes from productsthat have been removed from the market. If the currently available Fostimon® is superior to the off-market Metrodin-HP®, then data from the latter should not be used to gauge the efficacy of the former.Methods: Electronic/hand searches (e.g. MEDLINE, EMBASE, CENTRAL) till January 2010 were performed to identifyrandomized trials (RCTs) comparing Fostimon® vs. Metrodin-HP®, or either hFSH product with recFSH. The primaryoutcomes were live-birth (LBR), ongoing pregnancy (OPR) and ongoing pregnancy/ live-birth rates (OPR/LBR). Thesecondary outcomes were clinical pregnancy (CPR), multiple pregnancy (MPR), OHSS and abortion rates, in additionto the cycle demographics. Data was extracted to allow for an intention-to-treat analysis and meta-analyzed usingcombined direct and adjusted indirect methods.Results: Twenty-two RCTs that met the inclusion criteria were analyzed: Fostimon® vs. Metrodin-HP® (two trials);Fostimon® vs. recFSH (eight trials); Metrodin-HP® vs. recFSH (12 trials). The LBR (OR = 1.72; 95% CI = 1.05 to 2.80),OPR/ LBR, and CPR were all significantly higher favoring Fostimon®. The OPR, MPR, OHSS and miscarriage rates werenot significantly different and the cycle demographics were not pooled due to high heterogeneity between theincluded trials.Conclusions: Evidence from RCTs suggests that Fostimon® is superior to Metrodin-HP® in terms of clinical outcomes.Therefore, care should be taken not to assume that all hFSH products have the same efficacy.

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A PROSPECTIVE RANDOMIZED NON-INFERIORITY STUDY COMPARINGRECOMBINANT FSH (rFSH) AND HIGHLY PURIFIED MENOTROPHIN (HP-hMG) ININTRAUTERINE INSEMINATION (IUI) CYCLES IN COUPLES WITH UNEXPLAINEDINFERTILITY AND/OR MILD-MODERATE MALE FACTORSagnella F, Moro F, Lanzone A, Tropea A, Capalbo A, Gangale MF, Morciano A, Apa RDepartment of Obstetrics and Gynaecology, Università Cattolica del Sacro Cuore, Rome, ItalyE-mail: [email protected]

Objective: To demonstrate the non inferiority of HP-hMG respect to rFSH regarding clinical pregnancy rate (PR) inIUI cycles.Design: Prospective randomized non-inferiority trial.Setting: Unit of Physiopathology of Human Reproduction, University Hospital Patients: Five hundred twenty-threepatients with unexplained infertility or mild male infertility undergoing a COH for IUI.Intervention: Patients were randomized for treatment with rFSH (262 patients) or HP-hMG (261 patients). Inseminationwas performed 34-36 hours after hCG injection.Main outcome measures: The primary outcome was clinical pregnancy rate (PR). The secondary outcome was thenumber of interrupted cycles for high risk of ovarian hyperstimulation syndrome (OHSS) and multiple pregnancy.Results: The clinical PR was 19.7% (95%CI 15.3 to 25.1) in HP-hMG group and 21.4% (95%CI 16.9 to 26.8) in rFSHgroup [absolute difference -1.7% (-8.6 to 5.2)]; therefore the non inferiority was demonstrated. The number ofinterrupted cycles for OHSS risk and multiple pregnancy was significanty higher in rFSH group, 8.4 % (95% CI 5.6to 12.4), compared to HP-hMG group, 1.2 % (95% CI 0.4 to 3.3).Conclusions: HP-hMG is not inferior respect to rFSH regarding clinical PR.


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NEW STRATEGIES FOR HUMAN OOCYTES SELECTION IN ICSI TECHNIQUE:STANDARD PROTOCOL DEFINITION THROUGH BIOINFORMATICS SOFTWARECariati F1, Alviggi C2, Vaccina A3, Pivonello R1, Fiorentino A2, Leo P3, De Placido G2, Colao A1

1 Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica Università degli Studi di Napoli Federico II, Naples, Italy2 Dipartimento di Scienze Ostetrico-Urologiche e Medicina della Riproduzione, Università degli Studi di Napoli Federico II, Naples, Italy3 GBS Business Analytics and Optimization-IBM Italia S.p.A, ItalyE-mail: [email protected]

Nowadays assisted fertilization is one of the main debated topic in gynaecology. The number of couples recurringto assisted reproduction techniques (ART) is increasing mainly due to socio-economic aspects, including the trendto plan pregnancy in more advanced age. This aspect, together with genetic and environmental variables, stronglycontributes to limit pregnancy rates. During last years different strategies of gamete and/or embryo selection havebeen proposed in order to optimise success rates in ART. In particular, in Italy, research has been focused on oocyteselection due to legislative aspects (Low 40/2004). On these bases, several papers addressed the topic of oocytesselection for ICSI technique. Nevertheless, an objective selection method doesn't exist and the choice of oocytes forfertilization exclusively depends on embryologist experience and on a "at first sight" impression. Recently, we focusedour attention on the possibility to develop a decision support system able to classify oocytes according to a scorebased on morphological features and patients' clinical data. The aim was to create a system able to support biologistsin oocyte non-invasive selection and identifying the ones with the highest probability to develop until of grade 1embryos. In order to reach this goal, as a first step, it was necessary to closely work with doctors and biologists inorder to identify main morphological features influencing oocyte quality, assign them a weight and find the betterway to measure them from high quality images analysis. After that, it was developed a standard data format collectingin a organized way all oocytes morphological features, zigote and embryo ones an patients' clinical data. At thesame time, an image acquisition protocol was identified and a standard way of capturing them was defined. Thiswas done by detecting optimal standard microscope settings in order to maximize measure extraction efficiency.Finally oocyte images were collected for eight months and used as a first data set to test a preliminary version ofthe score alghoritm and to discover unknown co-relations among clinical patience data/oocyte morphologialfeatures/embryo quality. As a first result, more than 150 oocytes images, taken in standard and comparable conditions,from 35 women were collected. Starting from them it was possible to keep the morfological features extraction(manually or automatically) and organization according to the standard format previously developed. All the oocyteimages data were also enriched with clinical data and post fertilization zigote and embryo quality parameters. Asa next result, a preliminary version of the scoring alghoritm was tested on these data. At the moment, the scoreeffectiveness is under evaluation by medical experts. Anyway, it is possible to conlcude that the results of this activitiesare important for fertilization experts because now it's possible to acquire images in a more consistent and standardconditions, to quikly collect all data in a structured format, to have a clear picture of all medical data and a quantitativemetric that is actually supporting their experience in oocyte selection.

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HIGHER LH LEVELS ARE ASSOCIATED TO INCREASED PREGNANCY RATES WHENLEUPROLIDE ACETATE IS USED FOR OVULATION INDUCTIONChehin M, Haddad J, Bertolla R, Motta E, Fraietta RFederal University of São Paulo, São Paulo, BrazilE-mail: [email protected]

Objective: in order to avoid ovarian hyperstimulation syndrome (OHSS) during controlled ovarian stimulation forin vitro fertilization (IVF), leuprolide acetate may be used for induction of LH peak in cycles utilizing GnRH antagonistfor pituitary down-regulation. Because results are still unsatisfactory using this approach, we set out to verify potentialmarkers of outcome in patients receiving leuprolide acetate for LH peak induction.Design: Case-control study.Material and Methods: The study included 40 patients who underwent ovarian stimulation for IVF using a GnRHantagonist protocol and that presented hyperresponse extreme enough to be considered for cancellation (to avoidOHSS), grouped according to outcome in controls (pregnant) and study (non-pregnant). LH peak was achieved usingGnRH agonist (Lupron®). Serum LH and FSH were assessed previously to initiation of the cycle. For statistical analysis,a Student's T-test was applied. Also, logistic regression was performed using occurrence of pregnancy as the dependentvariable, and LH, FSH, FSH:LH ratio, and female age as the independent factors (results are presented as odds-ratios[OR]). Significance cutoff was set at 5%.Results: Overall pregnancy rate was 31.6%. LH values were higher in the pregnant than in the non-pregnant group(9.6±3.2 and 6±2.2 mIU/mL, p=0.0003). Increasing values of FSH:LH ratio led to decreased odds of pregnancy (OR=0.023,p=0.016), increasing female age led to decreased odds of pregnancy (OR=0.861, p=0.001), and higher LH values ledto increased odds of pregnancy (OR=1.624, p=0.005). The area under the curve in a ROC graph when using FSH:LHratio and age was 0.788 (p=0.006), and when using LH and age, 0.805 (p=0.004), demonstrating that higher LH levelsin these patients lead to a better prognosis for pregnancy.Conclusion: Increased LH levels in patients undergoing IVF and using leuprolide acetate for ovulation inductionlead to better prognosis for pregnancy. For patients with lower LH or higher FSH:LH ratios, other strategies shouldbe considered. Support: none


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FOLLICULAR VOLUME INCREASES BY 35 TO 40 % FROM HCG ADMINISTRATIONTO OOCYTE RETRIEVALPalumbo A, Hernandez J, Garcia R, Taraborrelli S, Filicori MCentro de Asistencia a la Reproducción Humana de Canarias, La Laguna, Tenerife, Spain and ReproductiveMedicine Unit, GynePro Medical Group, Bologna, ItalyE-mail: [email protected]

Introduction: Automated follicular monitoring by 3D US and SonoAVC (Automated Volume Calculation) has recentlybeen introduced in clinical practice (Fertil Steril 2010;93:616). The new software Sono AVC allows reliable measurementsof follicular volume, introducing a new parameter for ovulation induction monitoring in clinical practice. We haveshown that a follicular volume between 0.6 and 0.7 cc on the day of hCG administration corresponds to matureoocytes and we have proposed that decisions on hCG administration could be based on follicular volume rather thanon follicular diameters. The objective of this study was to determine the extent of follicular volume growth fromhCG administration to egg retrieval.Material and methods: 38 women undergoing IVF in a single private fertility clinic were prospectively recruited.All women underwent automated follicular monitoring with the Sono AVC software using a voluson E8 Expert ora Voluson i (GE Medical Systems, Zipf, Austria) and a RIC5-9 MHz transvaginal probe. Follicular volumes measuredon the day of hCG administration and on the day of egg retrieval, just prior to the procedure were compared. Oocyteretrieval was performed 36 hours after hCG administration. Statistical evaluation was performed using the SPSSprogram.Results: Follicular volume increased by 0.12 to 0.85 cc depending on the original follicular size, with greaterincrements for larger follicles. This numbers correspond to a mean percent growth of 38%, independent of follicularsize.Conclusions: This preliminary study shows that sonoAVC may be a useful tool not only in clinical practice but alsofor the study of ovarian physiology, specifically the dynamics of follicular growth in both natural and stimulatedcycles.Support: None


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