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Searching for the optimal GnRH antagonist regimen to compare with GnRH agonists in IVF Judith Anna Francisca Huirne

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Page 1: Searching for the optimal GnRH antagonist regimen to ... Huirne.pdf · agonists and antagonist we will evaluate the current place of GnRH antagonists in assisted reproductive therapies

Searching for the optimal GnRH antagonist regimen

to compare with GnRH agonists in IVF

Judith Anna Francisca Huirne

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The studies described in this thesis were performed at the Division of Reproductive Medicine, Department of Obstetrics and Gynaecology, VU Medical Center, Amsterdam, The Netherlands. Financial support for the printing of this thesis was kindly provided by Organon Nederland BV, Serono Nederland BV, Ferring BV, Johnson en Johnson Medical BV, Skills Meducation BV, Schering Nederland BV, Kapper new Look Amsterdam, and the Posthumus Meyjes Fonds, Kennemer Gasthuis.

ISBN-10: 90-8659-068-3ISBN-13: 978-90-8659-068-1

Thesis VU University Medical Center, Amsterdam – with summary in Dutch.

Cover- Chapterpagepictures: (more) happiness (www.kunstvoor jou.nl)Lay out: Nicole Nijhuis, Gildeprint Drukkerijen B.V., EnschedePrinting: Gildeprint Drukkerijen B.V., Enschede

© Judith A.F. Huirne, Amsterdam, The Netherlands, 2006No part of this thesis may be reproduced in any form or by any means, by print, photocopy, microfi lm or any other means without permission of the author.

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VRIJE UNIVERSITEIT

Searching for the optimal GnRH antagonist regimen

to compare with GnRH agonists in IVF

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad Doctor aan

de Vrije Universiteit Amsterdam,

op gezag van de rector magnifi cus

prof.dr. L.M. Bouter,

in het openbaar te verdedigen

ten overstaan van de promotiecommissie

van de faculteit der Geneeskunde

op vrijdag 16 februari 2007 om 13.45 uur

in de aula van de universiteit,

De Boelelaan 1105

door

Judith Anna Francisca Huirne

geboren te Eibergen

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promotor: prof.dr. R. Homburg

copromotor: dr. C.B. Lambalk

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Voor mijn ouders

Voor Joost

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ContentsPart 1 Considerations in selecting the optimal GnRH antagonist regimen 9

1 General introduction and outline 11

2 Gonadotrophin releasing hormone receptor antagonists 20 The Lancet 2001: 358; 1793-1803

3 Contemporary pharmacological manipulation in assisted reproduction 45 Drugs 2004: 64; 297-322

4 Dose-fi nding study of daily GnRH antagonist for the prevention of premature LH surges in IVF/ICSI patients: antide and hormone levels 79 Human Reproduction 2004: 19; 2206-2215

5 Dose-fi nding study of daily GnRH antagonist for the prevention of premature LH surges in IVF/ICSI patients: optimal changes in LH and progesterone for clinical pregnancy 97 Human Reproduction 2005: 20; 2359-67

6 Effect of an OC pill on follicular development in IVF/ ICSI patients receiving a GnRH antagonist: a randomized study 115 RBM-online 2006: 13; 235-245

7 Effect of timing gonadotrophins administration after OC withdrawal and rLH addition on follicular development and hormonal concentrations in GnRH antagonist cycles: a pilot study 133 Submitted

Part 2 Searching for the optimal comparison of GnRH antagonists and GnRH agonists 151

8 Cetrorelix in an OC-pretreated stimulation cycle comparing with buserelin in IVF/ICSI patients treated with rhFSH: a randomized, multicentre, phase IIIb study 153 Human Reproduction 2006: 21; 1408-1415

9 General discussion. Are GnRH antagonists comparable to GnRH agonists in IVF?: a debate 169 Submitted

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10 Epilogue 189

11 Summary 193

Appendices

Nederlandse samenvatting 199

Authors’ affi liations 205

Dankwoord 207

Curriculum Vitae 211

List of publications 213

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Considerations in selecting the optimalGnRH antagonist regimen

Part 1

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General introduction and outline

1

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General Introduction

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Searching for the optimal GnRH antagonist regimen for application in assisted reproduction.

After the discovery of the amino acid sequence of GnRH in 1967, several synthetic gonadotrophin releasing agonists and antagonists were developed to control the pituitary follicle stimulating hormone (FSH) and luteinizing hormone (LH) secretion. Most of the agents induced FSH and LH release after binding and were therefore called GnRH agonists. However, under continuous administration GnRH agonists induce pituitary desensitization by a mechanism still far from understood, leading to strong suppression of LH and a lesser extent FSH. In the 1980’s the fi rst reports were published using GnRH agonists in IVF to control the pituitary gonadotrophin release. The ability of GnRH agonists to suppress LH and to prevent premature LH surges allowed optimal timing of the hCG administration and ovum pick-up which improved IVF outcome in terms of pregnancy rates (Hughes et al., 1992). Placebo controlled studies revealed that in about 20% of women there was a premature LH increase that led to cancellation of the IVF cycle (Edwards et al., 1996, Janssens et al., 2000). It took nearly 10 more years to develop agents which were able to suppress gonadotrophin release directly by competitive receptor binding. But the introduction of the GnRH antagonists for clinical application was hampered by their histamine releasing properties and hence the induction of allergic reactions. Additional problems were their low solubility. Finally, after almost 30 years of trial and error these problems were solved resulting in the third generation GnRH antagonists with a safe profi le and low histamine releasing properties. (Abarelix/Praecis; Antarelix/Asta Medica; Antide/Serono; Cetrorelix/Serono) and Ganirelix/Organon). The latter two were offi cially registered for use in IVF therapy to prevent premature luteinization. Due to the ability of GnRH antagonists to inhibit gonadotrophin secretion rapidly without exerting any stimulation, less injections are required in comparison to the long GnRH agonist protocol which improves patients’ convenience (Huirne and Lambalk, 2001). After several dose-fi nding studies (Albano et al., 1997, Ganirelix dose-fi nding group 1998, Olivennes et al., 1998) initially two GnRH antagonist regimens emerged. The single fi xed protocol, in which a large dose is given on day 8 of gonadotrophin stimulation (Olivennes et al., 1998) and a fi xed multiple dose protocol started with daily subcutaneous GnRH antagonist injections from stimulation day 6 onwards, up to and including the day of hCG administration. These regimens turned out to be effective for the prevention of premature LH surges, with a shorter stimulation period, less gonadotrophins needed and resulted in lower number of reported side effects compared with the long GnRH agonist protocol (European Middle East Orgulatran Study group, 2001; Fluker et al., 2001). Although this antagonist regimen seems to be an attractive alternative to the long agonist protocol, several drawbacks became clear using this antagonist-only regimen in comparison to the long agonist in the fi rst comparative phase III studies (European Middle East Orgulatran Study group, 2001; Al-Inany and Aboulghar, 2002; Albano et al., 2000; Borm and Mannaerts, 2000; Fluker et al., 2001). The pregnancy rates were not different in the various individual studies. But a meta-analysis including the fi rst 5 comparative studies of GnRH antagonist only protocols compared with long agonist protocols, indicated that the number of clinical pregnancies might possibly be lower in the antagonist only protocols (Al-Inany and Aboulghar, 2002). In most individual studies the number of oocytes was lower with antagonist treatment (European Middle East Orgulatran Study group, 2001; Al-Inany and Aboulghar, 2002; Albano et al., 2000; Borm and Mannaerts, 2000; Fluker et al., 2001). The lower number of

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14

oocytes retrieved in the antagonist groups may play a role in the possibly lower pregnancy rates. Number of oocyts retrieved may be related to improved pregnancy rates, possibly via better embryo selection (Templeton and Morris, 1998). Furthermore, the initiation of FSH administration in a GnRH antagonist regimen is cycle dependent. It is mostly started on day 3 of the natural cycle which makes the treatment planning and scheduling very diffi cult (Huirne and Lambalk, 2001), and increases the clinical site workload. From 15 years of experience with the GnRH agonists in IVF we have learned that the long protocol starting in the midluteal phase of the preceeding cycle is the optimal protocol with regard to highest yield of oocytes, good quality embryos and the best pregnancy results (Huirne et al., 2004). The optimal treatment regimen for the use of GnRH antagonist for IVF to achieve the optimal number of oocytes and pregnancy rates is not yet established.

The studies reported in this thesis aim to search for factors which may improve GnRH antagonist regimen in terms of follicular development, oocyte yield and scheduling ability. What is the optimal GnRH antagonist dose, what is the effect of OC pre-treatment, LH addition and the timing of starting with rhFSH administration after OC withdrawal in GnRH antagonist cycles. Finally, after critical appraisal of currently available comparative studies between GnRH agonists and antagonist we will evaluate the current place of GnRH antagonists in assisted reproductive therapies.

REFERENCES

Albano C, Felberbaum RE, Smitz J, Riethmuller-Winzen H, Engel J, Diedrich K, and Devroey P (2000)

Ovarian stimulation with HMG: results of a prospective randomized phase III European study

comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and the

LHRH-agonist buserelin. European Cetrorelix Study Group. Hum Reprod, 15, 526-531.

Albano C, Smitz J, Camus M, Riethmuller-Winzen H, Van SA, and Devroey P (1997) Comparison of

different doses of gonadotropin-releasing hormone antagonist Cetrorelix during controlled

ovarian hyperstimulation. Fertil Steril, 67, 917-922.

Al-Inany H and Aboulghar M (2002) GnRH antagonist in assisted reproduction: a Cochrane review.

Hum Reprod, 17, 874-885.

Borm G and Mannaerts B (2000) Treatment with the gonadotrophin-releasing hormone antagonist

ganirelix in women undergoing ovarian stimulation with recombinant follicle stimulating

hormone is effective, safe and convenient: results of a controlled, randomized, multicentre

trial. The European Orgalutran Study Group. Hum Reprod, 15, 1490-1498.

Edwards RG, Lobo R, Bouchard P (1996) Time to revolutionize ovarian stimulation. Hum Reprod,

11, 917-19.

European Middle East Orgalutran study group (2001) Comparable clinical outcome using the

GnRH antagonist ganirelix or a long protocol of the GnRH agonist triptorelin for the prevention

of premature LH surges in women undergoing ovarian stimulation. Hum Reprod, 16, 644-651.

Fluker M, Grifo J, Leader A, Levy M, Meldrum D, Muasher SJ, Rinehart J, Rosenwaks Z, Scott RT, Jr.,

Schoolcraft W et al (2001) Effi cacy and safety of ganirelix acetate versus leuprolide acetate in

women undergoing controlled ovarian hyperstimulation. Fertil Steril, 75, 38-45.

Ganirelix dose-fi nding study group (1998) A double-blind, randomized, dose-fi nding study to

assess the effi cacy of the gonadotrophin-releasing hormone antagonist ganirelix (Org 37462)

to prevent premature luteinizing hormone surges in women undergoing ovarian stimulation

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Chapter 1

15

with recombinant follicle stimulating hormone (Puregon). The ganirelix dose-fi nding study

group. Hum Reprod, 13, 3023-3031.

Hughes EG, Fedorkow DM, Daya S, Sagle MA, Van de Koppel P, Collins JA (1992) The routine

use of gonadotropin-releasing hormone agonists prior to in vitro fertilization and gamete

intrafallopian transfer: a meta-analysis of randomized controlled trials. Fertil Steril, 58, 888-

896.

Huirne JA and Lambalk CB (2001) Gonadotropin-releasing-hormone-receptor antagonists. Lancet,

358, 1793-1803.

Huirne JA, Lambalk CB, van Loenen AC, Schats R, Hompes PG, Fauser BC, and Macklon NS (2004a)

Contemporary pharmacological manipulation in assisted reproduction. Drugs, 64, 297-322.

Janssens RM, Lambalk CB, Vermeiden JP, Schats R, Bernards JM, Rekers-Mombarg LT, Schoemaker J

(2000) Dose-fi nding study of triptorelin acetate for prevention of a premature LH surge in IVF: a

prospective, randomized, double-blind, placebo- controlled study. Hum Reprod, 15, 2333-2340.

Olivennes F, Alvarez S, Bouchard P, Fanchin R, Salat-Baroux J, and Frydman R (1998) The use of a

GnRH antagonist (Cetrorelix) in a single dose protocol in IVF-embryo transfer: a dose fi nding

study of 3 versus 2 mg. Hum Reprod, 13, 2411-2414.

Templeton A and Morris JK (1998) Reducing the risk of multiple births by transfer of two embryos

after in vitro fertilization. N Engl J Med, 339, 573-577.

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General Introduction

16

OBJECTIVES OF THIS THESIS

1. To review the existing literature on the development of GnRH antagonists, their mechanism of action and their possible clinical applications, especially in the fi eld of reproduction and IVF.

2. To provide an overview of the literature with respect to various drugs and therapeutic regimens (i.e. GnRH agonists and antagonists, urinary and recombinant gonadotrophins) used for ovarian stimulation in IVF.

3. To optimize the GnRH antagonist protocol by a. assessing the minimal effective dose of a new GnRH antagonist Antide to suppress LH levels and to prevent premature LH surges in IVF patients. b. identifying the relation between various induced LH levels by different GnRH antagonist dosages on implantation ability and pregnancy. c. studying the effect of OC pre-treatment in GnRH antagonist regimens, given to schedule IVF treatment cycles, on hormone levels and follicular development. d. studying the effect of timing stimulation with gonadotrophins and the addition of LH on hormone levels and follicular development.

4. To compare the oocyte yield and IVF outcome in patients treated with a long GnRH agonist regimen and an OC pre-treated fi xed day 6 GnRH antagonist protocol in which stimulation was started on day 5 after the last oral contraceptive pill.

5. To review the current literature to identify the optimal GnRH antagonist regimen in terms of follicular development, oocyte yield, pregnancy outcome and scheduling ability. Finally, after critical apraisal of currently available comparative studies between GnRH agonists and antagonist, we will evaluate the current place of GnRH antagonists in assisted reproductive therapies.

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OUTLINE OF THE THESIS

Considerations with respect to selecting the optimal GnRH antagonist regimen are discussed in part 1.

Chapter 2 provides an overview of the development of GnRH antagonists, their mechanism of action and possible indications in human reproduction and beyond. Chapter 3 addresses the broad spectrum of various drugs and therapeutic regimens (i.e. GnRH agonists and antagonists, urinary and recombinant gonadotrophins) used for ovarian stimulation in IVF. Chapter 4 reports a prospective randomized parallel trial to identify the minimal dose of a novel GnRH antagonist antide for the prevention of premature lutienizing hormone surges in IVF. Chapter 5 addresses the possible relation between various induced endogeneous LH and progesterone levels during GnRH antagonist therapy in IVF and the likelihood to achieve clinical pregnancies. Chapter 6 studies the effect of oral contraceptive pre-treatment on hormonal levels and follicular development in GnRH antagonist treated IVF patients. Chapter 7 addresses the effect of timing gonadotrophins administration after oral contraceptive withdrawal and rLH addition on follicular development and hormonal concentrations in GnRH antagonist cycles.

Considerations with respect to optimal comparison of GnRH antagonists and GnRH agonists for use in IVF are addressed in part 2 of this thesis.

Chapter 8 reports the results of randomized controlled trial comparing oral contraceptive pre-treated fi xed GnRH antagonist regimen with a long GnRH agonist protocol in IVF. Chapter 9 presents the search for the optimal GnRH antagonist regimen to compare with GnRH agonists in IVF. Chapter 10 addresses clinical relevance of this thesis and future perspectives of GnRH antagonists. Chapter 11 provides a brief summary.

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2Gonadotrophin releasing hormone receptor antagonists

JAF Huirne, CB Lambalk

The Lancet 2001: 358; 1793-1803

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ABSTRACT

Pulsatile gonadotrophin-releasing hormone (GnRH) stimulates the pituitary secretion of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and thus controls the hormonal and reproductive function of the gonads. Blockade of GnRH effects may be wanted for a variety of reasons—eg, to prevent untimely luteinization during assisted reproduction or in the treatment of sex-hormone-dependent disorders. Selective blockade of LH/FSH secretion and subsequent chemical castration have previously been achieved by desensitizing the pituitary to continuously administered GnRH or by giving long-acting GnRH agonists. Only recently have GnRH-receptor antagonists, that immediately block GnRH’s effects, been developed for clinical use with acceptable pharmacokinetic, safety, and commercial profi les. In assisted reproduction, these compounds seem to be as effective as established therapy but with shorter treatment times, less use of gonadotropic hormones, improved patient acceptance, and fewer follicles and oocytes. All current indications for GnRH-agonist desensitization may prove to be indications for a GnRH antagonist, including endometriosis, leiomyoma, and breast cancer in women, benign prostatic hypertrophy and prostatic carcinoma in men, and central precocious puberty in children. However, the best clinical evidence so far has been in assisted reproduction and prostate cancer.

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INTRODUCTION

The hormone gonadorelin, also known as gonadotrophin-releasing hormone or luteinizing-hormone-releasing hormone (GnRH, LHRH), induces both follicle-stimulating hormone and LH secretion by the pituitary gonadotrope cells in an orderly way which is crucial for the control of gonadal function and normal ovarian cyclicity. One logical consequence of the discovery of the aminoacid sequence of GnRH in 1967 was the development of synthetic agonists and antagonists. It was noted that sustained stimulation of the pituitary with GnRH itself or with a GnRH agonist caused desensitization, by post-receptor mechanisms that are still not well understood (Conn and Crowley, 1994). The result, not immediately but after some time, was a chemical hypophysectomy which was thought to be the indication for GnRH antagonists. At fi rst it seemed that the development of clinically safe agents would be simple to achieve by changing just one or two aminoacids but it was to take almost 30 years of trial and error with three or more replacements, including sometimes the use of unnatural aminoacids, before antagonists with acceptable pharmacokinetic, safety, and commercial profi les were developed. Today these GnRH-antagonists are at an advanced stage of clinical development.

PHYSIOLOGICAL PRINCIPLES

GnRHGnRH is a decapeptide (table 1) that was isolated and characterised by the groups of AV Schally and RCL Guillemin, the 1977 Nobel laureates. GnRH type I is the classic reproductive neuroendocrine factor (Carolsfeld et al., 2000). It is synthesised in the cytoplasm of the diencephalon of the brain and packaged into granules in the Golgi apparatus and then transported by the axons to the neuronal terminal, where it is released in a pulsatile fashion into the capillaries of the pituitary-portal circulation. GnRH mRNA has been found in the pituitary (Krsmanovic et al., 2000) and in extrapituitary tissue, including the placenta, ovary, myometrium, endometrium, and prostate and blood mononuclear cells, indicating an autocrine/paracrine role (Kang et al., 2000; Chegini et al., 1996; Cheng et al., 2000). The genes for human GnRH types I and II lie on chromosome 8 (8p11.2-p21) and chromosome 20 (20p13), respectively (Yang-Feng et al., 1996; White et al., 1998).

GnRH receptorThe GnRH receptor (GnRH-r) (fi gure 1) is a member of a family of receptors known as the rhodopsin-like G-protein-coupled receptor family, which also includes the thyrotropin-releasing hormone receptor (Sealfon et al., 1997; Probst et al., 1992). Most vertebrates have at least two types of GnRH receptor (Sealfon et al., 1997). The gene for GnRH type I receptor lies on chromosome 4 (4q21.2) (Leung et al., 1995), and the type II receptor is thought to be on chromosome lq (Millar et al., 2001). Cloning of a type II GnRH receptor in the marmoset showed that only 41 % is identical to the type I receptor and that, unlike the type I receptor, it has a carboxyl-terminal tail, which is important for rapid desensitisation (Millar et al., 2001). In human beings, controversial data have been obtained for extrapituitary expression of mRNA for GnRH-r in ovary, breast, endometrium, myometrium, placenta, prostate, and testis and in the various cancers of these tissues and their associated cell lines. Variable presence of the different GnRH-r transcripts (Kottler et al., 1995) and different distribution of type I and type II receptors could have a role. The type II receptor in mammals is more

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widely distributed than the type I receptor. It is expressed throughout the brain, including the pituitary and areas associated with sexual arousal, and also in diverse non-neural and reproductive tissues, suggesting various functions (Millar et al., 2001).

Aminoacid found at position:

1 2 3 4 5 6 7 8 9 10

GnRH-I pGlu His Trp Ser Tyr Gly Leu Arg Pro

GnRH-II 1 2 3 4 His 6 Trp Tyr 9 10

Abarelix D-Ala D-Phe D-Ala 4 5 D-Asp 7 Lys(iPr) 9 D-Ala

Antarelix D-Nal D-Phe D-Pal 4 Phe D-Hcit 7 Lys(iPr) 9 D-Ala

Cetrorelix D-Nal D-Phe D-Pal 4 5 D-Cit 7 8 9 D-Ala

Ganirelix D-Nal D-Phe D-Pal 4 5 D-hArg 7 hArg 9 D-Ala

Antide D-Nal D-Phe D-Pal 4 NicLys D-NicLys 7 Lys(iPr) 9 D-Ala

Nal-Glu D-Nal D-Phe D-Pal 4 D-Glu D-Glu 7 8 9 D-AlaTable 1. Structural formulae of native GnRH and third-generation GnRH-antagonists

GnRH signal transduction

GnRH binds to specifi c receptors on the gonadotrophe cells in the anterior pituitary. In gonadotrophes, GnRH receptor activation, after coupling to Gqs11 protein, leads to the generation of several second messengers, among which are diacylglycerol and inositol-4,5-triphosphate. The former leads to activation of protein kinase C and the latter to the production of cyclic AMP and release of calcium ions from intracellular pools (Stojikovic et al., 1994; Kaiser et al., 1997). Both events result in secretion and synthesis of LH and FSH. GnRH II selectively binds to the type II receptor and signalling is distinctly different from that in the type I receptor (Millar et al., 2001). The FSH and LH releasing potency of mammalian GnRH I and GnRH II, tested in Soay rams, showed that the FSH to LH ratio was higher after treatment with GnRH II than with GnRH I (Millar et al., 2001). These two GnRHs and GnRH receptor systems, along with different signalling pathways, provide the potential for differential FSH and LH secretion (Millar et al., 2001). The natural GnRH signal from the hypothalamus to the pituitary is episodic (every 1–4 h) and this ensures LH and FSH secretion. However, upon continuous stimulation with GnRH, via a long-acting agonist, there is fi rst a period of hypersecretion quickly followed by pituitary desensitisation and arrest of gonadotrophin secretion. This mechanism is still not clear except that post-receptor signalling is involved, true receptor loss (down-regulation) having only an initial role (Conn et al., 1994). A clear dichotomy of GnRH and competitive antagonist (in which antagonist blocks agonist-induced effects and vice versa) has not been shown in any human extrapituitary tissue that expresses the receptor (Emons et al., 1998). Why agonists and antagonists sometimes exert similar effects is not clear. If the effects seen with an antagonist result from direct blockade of the receptor while a similar effect with the agonist is achieved via desensitisation, one would expect antagonist effects to be mitigated by an agonist, but this

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does not usually happen. On the other hand, more recent data show that GnRH antagonist abolishes a dose-dependent antiproliferative effect of a GnRH agonist on human ovarian epithelial cells and in human granulosa-luteal cells (Kang et al., 2000). The antagonist reverses GnRH induced activation of mitogen activated protein (MAP) kinase which is known to regulate cell growth and differentiation (Kang et al., 2000; Kang et al., 2001). In in-vitro studies with cell lines of various tumour types, antiproliferative activity was shown by both types of analogue. In a cell line of a human kidney embryo antiproliferative effects of agonist and antagonist occurred only when expression of a high affi nity GnRH-r was induced, which strongly suggests direct, specifi c, receptor-mediated effects (Emons et al., 1998). GnRH-r stimulation of MAP kinases (MAPKs) in granulosa-luteal cells of human beings confi rmed the receptor mediation; additional studies showed that cellular responses (proliferation, hormone or peptide secretion) to MAPK stimulation are tissue specifi c and duration dependent (Kang et al., 2001). GnRH analogues may interfere with the signal transduction of growth factor receptors and related oncogene products associated with tyrosine-kinase activity Kaiser et al., 1997). So far, most studies have been done on the assumption that there was only the GnRH type I receptor. Cloning of the type II GnRH receptor provides the opportunity to study the different affi nity and potency of analogues to the different types of receptor and their signalling pathways and cellular reactions. Differential expression of the GnRH type I and type II receptors could have a role in the paradox of similar effects of both GnRH agonists and antagonists on proliferation in specifi c tumour cell lines. Certain mammalian GnRH type I antagonists behave as agonists on GnRH type II receptors (Millar et al., 2001). The antiproliferative effects of GnRH analogues on cell lines of these tumours are consistent with the activation of MAPK p38α by the type II receptor, which is known to be antiproliferative (Neil et al., 2001).

Figure 1. Schematic representation of human GnRH-r. Receptor composed of single polypeptide chain, with seven hydrophobic transmembrane domains, extracellular amino terminus, and intracellular carboxy terminus. Known glycosylation site is marked, and certain key functional residues are numbered.

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GnRH antagonistsUnlike the agonists, GnRH antagonists do not induce an initial stimulation of gonadotrophin release, but cause an immediate and rapid, reversible suppression of gonadotrophin secretion. The principal mechanism of action of GnRH antagonists is competitive receptor occupancy of GnRH-r (fi gure 2).

Figure 2. Mechanism of action of GnRH antagonists

Structural modifi cationsThe effects of a single aminoacid substitution may alter affi nity and agonist activity via modifi cation of a side-chain that interacts with the binding pocket and/or by altering the conformation of the peptide. Whether a ligand functions as agonist or antagonist is species dependent and is determined by the structure of the second extracellular loop of the GnRH-r (Sun et al., 2001). The structural features of GnRH antagonists are reviewed elsewhere (Haviv et al., 1998; Schally, 1999). The weak fi rst-generation drugs were hydrophilic and contained replacements for His at position 2 and for Trp at position 3. Inhibitory activity increased after incorporation of a D-aminoacid at position 6 but increased histamine-releasing activity resulted in anaphylactic reactions to the second-generation antagonists such as detirelix (Karten, 1992). In the third generation the undesirable risk of oedema was eliminated by replacing the D-Arg at position 6 by neutral D-ureidoalkyl aminoacids, to produce compounds such as cetrorelix, antide, azaline B, ganirelix, abarelix, and antarelix (Bajusz et al., 1988; Ljungqvist et al., 1988; Rivier et al., 1992; Nestor et al., 1992; Garnick and Campion, 2000; Cook and Sheridan, 2000; Deghenghi et al., 1993).

GnRH

FSH

LHNo intrinsic activity

Competative receptor blocking

GnRH neuron

GnRH antagonist

FSHLH

GnRH

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Name Manufacturer Route Status

Abarelix Praecis (Amgen)/ IM Phase III, prostate cancer

Sanofi -Synthelabo Phase II, endometriosis

Antarelix Asta Medica SC Phase I, prostate cancer

Cetrorelix (Cetrotide) Asta Medica/Serono SC Approved IVF. Phase II, benign prostatic hyperplasia, uterine myoma, prostate and ovarian cancer

Ganirelix acetate (Orgalutran, Antagon)

Organon SC Approved, female infertility

Antide Serono SC Phase II, IVF

Table 2. GnRH antagonists launched or under investigation

PharmacokineticsThe pharmacokinetic properties with regard to maximum antagonist administration (Cmax) are linear following single or multiple doses (Reissmann et al., 1995; Duijkers et al., 1998; Oberye et al., 1999). The predominant clearance is metabolic and hepatic (Sommer et al., 1994). Absorption is the time-limiting step for elimination. Plasma protein binding averages 80% and the plasma half-life of a non-depot antagonist varies from 5 to 30 h after single subcutaneous administration of clinical doses and is increased after multiple dose administration (table 3). Abarelix for injectable suspension allows single intramuscular administration every 28 days in patients with prostatic cancer (Garnick et al., 1999b; Garnick and Campion, 2000; Tomera et al., 2001.

Pharmacodynamics6 h (range 4–24 h) after administration of a GnRH-antagonist plasma LH concentrations will have fallen by 70% (range 52–91%) and plasma FSH by 30% (range 23–61%). The size and duration of this suppression are dose dependent (Sharpless et al., 1999; Rabinovici et al., 1992). Complete reversal is achieved in 24–72 h. Comparable suppression is seen after subcutaneous and intramuscular administration (Gonzalez-Barcena et al., 1994) but the intranasal route requires higher dosages (Fujimoto et al., 1997). In women with regular menstrual cycles, the LH suppressive effect is greatest at the time of ovulation (Hall et al., 1994). Any residual gonadotrophin in the serum after administration of an antagonist in maximally suppressive doses suggests either incomplete blockade of pituitary receptors or, and more probably, non-GnRH-dependent gonadotrophin secretion. The percentage decline in LH secretion achieved by GnRH-antagonists is consistently higher than the decline in FSH secretion (Sommer et al., 1994; Leroy et al., 1994; Kettel et al., 1991; Fluker et al., 1991; Couzinet et al., 1991), indicating the existence of some additional, non-GnRH-dependent control mechanism.

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Compound M/F Dose (mg) Tmax (h) Cmax (ng/mL) T1/2 (h) Refs

Single dose

Abarelix-depot Both 30–120 NA 7.5–38 >240 Wong 2000

Cetrorelix M 10 NA NA 20 Behre 1994

F 0.25 1·0 5.0 5.0 Duijkers 1998

F 1.0 1·0 21.2 9.4 Duijkers 1998

F 3.0 1·5 28.5 62.8 Erb 2001

Ganirelix F 0.25 1·1 14.8 12.8 Oberye 1999

Multiple dose†

Cetrorelix F 0.25 1·0 6.4 20.6 Duijkers 1998

F 1.0 1·0 21.1 77.4 Duijkers 1998

Ganirelix F 0.25 1·1 11.2 16.2 Oberye 1999

Table 3. Pharmacokinetic indices following single and multiple dose administrations of GnRH antagonists in (pre)clinical studies; phase I studies, which studied recommended dose as applied in phase III trials, and published in peer-reviewed journals. † After multiple dosing (daily): Tmax = time of maximum plasma concentration at steady state, Cmax = maximum plasma concentration at steady state, T1/2 = terminal half-life.

SAFETY AND TOLERABILITY

The third-generation antagonists have low histamine-releasing potency, and antide and azaline-B have lower histamine-releasing potencies than GnRH itself though neither agent is currently under clinical development (Bajusz et al., 1988; Nestor et al.,1992; Campen et al., 1995). Toxicological studies have confi rmed the safety of the third-generation drugs. There is no evidence for irreversible drug-related atrophy of the target organs or for mutagenic/clastogenic potential. Infl uence on the early embryonic development of rats could not be demonstrated (Reissmann et al., 1995; Cetrorelix report, 1994; Ganirelix report, 1995). Over 2000 patients have now been treated with ganirelix, cetrorelix, or abarelix, without evidence of anaphylactic reactions (Felberbaum and Diedrich, 1999; Albano et al., 2000; Brom and Mannaerts, 2000; The European and Middle East Orgalutran Study Group, 2001; Fluker et al., 2001). Adverse effects of antagonists are largely the same as those with agonists, and are attributable to hypo-oestrogenism in women and hypoandrogenism in males, including hot fl ushes, mood changes, headache, and decreased libido. Prolonged exposure could lead to loss of bone-mineral density to the same extent as the 2–8% after 6 months seen with agonists. One common side-effect is a local reaction at the injection site (redness and pain), which usually resolves within an hour (The European and Middle East Orgalutran Study Group, 2001; Fluker et al., 2001). Local skin reactions seem to be less common with antagonists than they are with agonists. No signifi cant changes in serum chemistry or haematological indices have been noted (Ganirelix report, 2000; Giuliani et al., 1998). Clinical experience to date indicates that GnRH antagonists are safe and well tolerated.

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Extrapituitary effectsThe safety of GnRH analogues in respect of structures such as the ovary, oocyte, and granulosa cell and the embryo has become a matter of debate since the discovery of extrapituitary human GnRH-receptors. The many paradoxical results in studies of ovarian steroidogenesis may be caused by factors such as the type, dose, and regimen of the analogues used, the patient’s ovarian status, the ovarian cell types used and the hormonal pretreatment of those cells, and the type of in-vitro stimulation, besides methodological differences in the experiments and physiological variation in receptor abundance and type (Millar et al., 2001; Brus et al., 1997; Janssens et al., 2000a; Hernandez et al., 2000. So far, intrinsic direct effects of GnRH antagonists on human ovarian steroidogenesis or cAMP production in vitro have not been demonstrated (Mannaerts and Gordon, 2000; Demirel et al., 2000; Ortmann et al. 2001). An important fi nding is the expression of GnRH and GnRH-r in the mouse embryo (Raga et al., 1999). Incubation of the murine embryos with a GnRH agonist enhanced preimplantation embryonic development whereas an GnRH antagonist completely blocked this development (Raga et al., 1999). The blockade could be reversed by a GnRH agonist. Hernandez has hypothesised that GnRH antagonists interact with the mitotic programming of cells involved in folliculogenesis, blastomere formation, and endometrium development (Hernandez, 2000). Moreover, there is evidence that GnRH (and agonists) increase the cleavage rate of bovine oocytes, an effect that is abolished by addition of a GnRH antagonist (Funston and Seidel, 1995). Although inhibition of sperm-binding to the zona pellucida was inhibited by GnRH antagonists in vitro (Morales et al., 1999), lower fertilization rates were not found in phase III studies of antagonists (Albano et al., 2000; Borm and Mannaerts, 2000; The European and Middle East Orgalutran Study Group, 2000; Fluker et al., 2001; Olivennes et al., 2000). The lower implantation rates seen with higher doses of antagonists in in-vitro fertilization (IVF) (The Ganirelix dose-Finding Study Group, 1998) are probably related to impaired endometrial receptiveness rather than a direct embryonic effect because cryopreserved embryos from these cycles yielded normal pregnancy rates (Kol et al., 1999). Follicular growth was also not infl uenced by the daily dose of GnRH antagonist (De Jong et al., 2001). Despite the reassurance to date, the safety GnRH analogues in respect of human embryo development remains a priority. So far no increased risk of birth defects or pregnancy wastage in pregnancies exposed to daily low-dose GnRH antagonist therapy in the fi rst weeks of gestation have been recorded (Ludwig et al., 2001).

THERAPEUTIC APPLICATIONS

On pharmacological grounds the primary indications for GnRH antagonists will be in any situation in which chemical gonadotropic hypophysectomy is required (fi gure 3).

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Figure 3. Effects of GnRH antagonists. Schematic representation of effects on different levels of hypothalamic-pituitary-gonadal axis.

(1) Immediate blockade of the effect of gonadotropic hormones -eg, in IVF to prevent the normal midcycle rise in LH.(2) Indirectly, to block gonadal sex-hormone secretion via blockade of pituitary gonadotrophin secretion. An antagonist can, theoretically, be given to patients with a sex-hormone-dependent disease such as benign prostatic hypertrophy and prostate carcinoma, leiomyoma and endometriosis, or precocious puberty.(3) The treatment of cancer, based on evidence that GnRH antagonists exert direct negative effects on growth of certain malignant tumours.The only two registered GnRH antagonists for IVF are cetrorelix (Cetrotide; Serono International) and ganirelix (Orgalutran, Antagon; Organon). Both are approved only for use during IVF.

Prevention of premature LH surge in IVFPlacebo-controlled studies of GnRH agonists revealed that in about 20% of women there was a premature LH increase that led to cancellation of the IVF cycle (Edwards et al., 1996; Janssens et al., 2000b). Preventing this untimely physiological change would clearly be benefi cial. Moreover, the IVF harvest is bigger with more embryos, allowing better selection so that, on average, the outcome in terms of pregnancy rates is better (Templeton and Morris, 1998). It takes time for a state of desensitization to be reached and, to start with, LH secretion actually increases (fl are-up). In an IVF setting, treatment with a GnRH agonist usually begins in the midluteal phase of the menstrual cycle preceding the IVF treatment cycle; this is the so-called long protocol (fi gure 4). With an antagonist, immediate blockade of pituitary gonadotrophin secretion when premature luteinization during IVF stimulation is imminent seemed an obvious approach. Several studies of dose and treatment schedules have been done (The Ganirelix Dose-Finding Study Group, 1998; Olivennes et al., 1998; Albano et al., 1997), and two general approaches have emerged. The fi rst is a single subcutaneous injection of a large dose on about the eighth day of stimulation with gonadotrophins

Pituitary level

Hypothalamic level

Gonadal level

GnRH neuron

Extrapituitarydirect effects

Chemical

castration

Selective chemical

hypophysectomy

LH & FSH

EFFECTS

Estragens Androgens

testisovary

GnRH GnRH antagonist

LH & FSH

Pituitary

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(fi gure 4). The alternative is fi ve or six daily injections of a small dose from about day 6 of stimulation until the day that hCG for fi nal oocyte maturation is given (fi gure 4, table 4).

Compound Dose LH-surge Refs

Cetrorelix 3 mg single dose 0/34 Olivennes 1998

Cetrorelix 0.25 mg/day 0/30 Albano 1997

Ganirelix 0.25 mg/day 1/69 Ganirelix group 1998

Antide 0.5mg/day 0/32 Huirne 2004

Table 4. Prevention of premature LH surge; minimal effective dose of GnRH antagonists in patients undergoing in-vitro fertilization. Single subcutaneous injection on stimulation day (SD) 8 or until oestradiol level ≥400 pg/mL (Olivennes et al., 1998). Multiple injections at one per day subcutaneous, from stimulation day 6 or 7 up to hCG administration day (The Ganirelix Dose-Finding Study Group, 1998; Albano et al., 1997; Huirne et al., 2004). LH-surge defi ned as LH >10 IU/L after fi rst antagonist injection.

Table 4 summarises suggested daily dosages based on extensive dose-fi nding reports (The Ganirelix Dose-Finding Study Group, 1998; Olivennes et al., 1998; Albano et al., 1997; Huirne et al., 2004). The next step was to establish whether a GnRH antagonist is at least as effective as a GnRH agonist as the established reference medication. So far, four such studies of repeated antagonist injections have been reported (Albano et al., 2000; Borm and Mannaerts, 2000; The European and Middle East Orgulatran Study Group, 2001; Fluker et al., 2001) and one with a single-dose regimen (Olivennes et al., 2000) (table 5).

Figure 4. GnRH analogue treatment regimens in in-vitro fertilization. Schematic representation of most commonly used regimen in phase III trials.

hCG or GnRH

GnRH agonist

FSH

FSH

GnRH antagonist

day 6of FSH

day 2 or 3 of menses

desensitization

hCGAGONIST

multiple dose

single dose

FSH

day 2 or 3 of menses hCG or GnRH

day 7or 8of FSH

ANTAGONIST

21-24 of the preceeding cycle

GnRH antagonist

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Antagonist group Agonist group

Drug dose (mg)

FSH dose (IU/day)

Drug Dose (mg)

FSH dose (IU/day)

No Refs

Multiple dosing regimen

Cetrorelix 0.25 150 HMG Buserelin 0.6 150 HMG 293 Albano 2000

Ganirelix 0.25 150 rFSH Buserelin 0.6–1.2 150 rFSH 701 Borm 2000

Ganirelix 0.25 150 rFSH Triptorelin 0.1 150 rFSH 337 EME Orgal. Study 2001

Ganirelix 0.25 225 rFSH Leuprolide 1–0.5 225 rFSH 297 Fluker 2001

Single dose regimen

Cetrorelix 3.0 150 HMG Triptorelin 3.75 225 HMG 154 Olivennes 2000b

Table 5. GnRH antagonist versus GnRH agonists: treatment schedules of phase III trials in in-vitro fertilization. Multiple antagonist dosing one subcutaneous injection daily from stimulation day 6,7 or 8 up to hCG day; single dosing one subcutaneous injection on stimulation day 7 or if oestradiol 500 pg/mL or follicle >14 mm. For agonists multiple dosing with daily subcutaneous or intranasal (buserelin) administration, starting midluteal phase of menstrual cycle preceding ovarian stimulation cycle (long protocol). Single dosing regimen is one triptorelin depot injection during preceding luteal phase.

With repeated injections (table 6), a consistent fi nding is that duration of treatment with gonadotrophins is shortened by 1–2 days. With an antagonist slightly fewer follicles are seen at the time of hCG injection so the number of recovered oocytes tends to be lower. A likely explanation is that an agonist suppresses the natural cycle follicular recruitment initiated by the intercycle FSH rise (Albano et al., 2000; Borm and Mannaerts, 2000; The European and Middle East Orgalutran Study Group, 2001; Fluker et al., 2001) so that longer treatment with gonadotrophins is required, which allows more follicles to enter the growing phase. No signifi cant difference was found with respect to percentages of metaphase II oocytes, fertilization rates, and number of good quality embryos (The European and Middle East Orgalutran Study Group, 2001; Fluker et al., 2001). Pregnancy rates were high in both groups in all four studies but in every one the rate was lower in the antagonist group. A meta-analysis of the fi ve randomized trials (table 5), shows an overall signifi cantly lower rate of pregnancy of 5%. This meta-analysis unfortunately included the study that compared a single-dose analogue regimen with different gonadotrophin starting dose as an additional variable (Al-Inany et al., 2001). There is some concern that this may be a consequence of the currently advised treatment regimen. It has been suggested that the larger numbers of oocytes and embryos with agonists allow better selection (Templeton and Morris, 1998), although the numbers of good quality embryos do not seem to be different. A direct adverse effect on the embryo cannot yet be ruled out but is not likely. There are many reports of pregnancies inadvertently exposed to a GnRH agonist without any adverse effect (Janssens et al., 2000a). In IVF treatment the risk of embryonic exposure to an antagonist is minimal. Cetrorelix plasma and follicular fl uid levels fell signifi cantly after hCG administration, and at minimally effective doses cetrorelix was not detectable during ovum retrieval and embryo transfer (Ludwig et al., 1999). Nevertheless, only long-term follow-up of the children born after IVF procedures in which these new drugs have

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been used can be conclusive. Follow-up of neonatal outcome after pregnancies established in large phase II/III trials (474 pregnancies after ganirelix, 134 after a long agonist protocol (Bonduelle, 2001), and 227 children after centrorelix (Ludwig et al., 2001) did not show any negative effect. No major side-effects have been reported. Minor side-effects were limited to pain and redness at the injection site. This happened with both types of analogue but was less common with the antagonist (The European and Middle East Orgalutran Study Group, 2001; Fluker et al., 2001). The smaller number of injections and the reduction in the duration of IVF treatment are welcomed by patients and this could be the most important reason why GnRH antagonists will fi nd a therapeutic role in IVF clinics. So far, however, no large and moreover no blinded comparisons of GnRH agonists and antagonists with pregnancy outcome as the endpoint have been done. A further advantage of antagonists is that less FSH is needed.Antagonist blockade allows immediate reversal of pituitary gonadotrophin secretion. This means that in IVF ovulatory ripening can be triggered via endogenous gonadotrophins by using a GnRH agonist (Felberbaum et al., 1995; Christin-Maitre et al., 2000). One advantage could be prevention of ovarian hyperstimulation syndrome, which is thought to result at least in part from the prolonged LH effect of hCG (Itskovitz-Eldor et al., 2000). Finally, the use of GnRH antagonist blockade of premature luteinization can be used in IVF with very low or without any hormonal stimulation (De Jong et al., 2000), lower risk of overstimulation, and simplifi cation of the procedure (Olivennes et al., 2000a). A possible disadvantage of an antagonist in IVF is its narrow therapeutic range with the currently advised doses for repeated injections. Patient compliance needs to be high because there is a risk of premature LH secretion if an injection is missed.Another disadvantage is the unpredictable timing of the start of the IVF procedure, which begins with the administration of FSH on day 3 of the woman’s cycle and so depends on how regular her menstruation is. This problem may be solved by pretreatment with an oral contraceptive (Van Loenen et al., 2001).

OTHER INDICATIONS: FEMALE REPRODUCTIVE SYSTEM

On theoretical grounds, many future indications are feasible for GnRH antagonistic compounds (table 7).

Ovarian hyperstimulation syndromeCurrently, ovarian hyperstimulation syndrome is managed by electrolyte and fl uid administration, rest, paracentesis, and, in severe forms, respiratory support. The patient may benefi t from immediate blockade of endogenous LH secretion. High doses of a GnRH antagonist may be used for this purpose (De Jong et al., 1998).

Polycystic ovary syndromePolycystic ovary syndrome (PCOS) is characterised by oligomenorrhoea, hyperandrogenism, and cystic appearance of the ovaries. Raised LH levels are thought to be responsible for the high androgen levels that adversely affect the development of follicles. Theoretically, blockade of endogenous LH secretion by an antagonist combined with ovulation induction could result in improved follicular development. The abnormal LH secretion is thought to result from an abnormal endogenous GnRH signal from the hypothalamus (Hayes et al., 1998).

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Female reproductive systemOvarian hyperstimulation syndromePolycystic ovary syndromeEndometriosisLeiomyomaOvarian cancerEndometrial cancerBreast cancerMammographyMale reproductive systemBenign prostate hypertrophyProstate cancerMale contraceptionPaediatricsPrecocious pubertyDelay of puberty in children with small statureMiscellaneousPituitary adenomaGonadal protection during radiation or chemotherapyParaphiliasDirect antitumour effectsDrug targeting

Table 7. Other indications (being explored or future) for GnRH antagonists

Studies in monkeys showed that blockade of the endogenous GnRH signal can be competed with by exogenously administered pulsatile GnRH which restored gonadotrophin secretion and ovulation (Gordon et al., 1992). So far the same combination in PCOS patients normalised gonadotrophin secretion but failed to induce ovulation (Dubourdieu et al., 1993).

EndometriosisWith endometriosis, an oestrogen-dependent disorder, treatment with a GnRH antagonist is likely to be just as effective as treatment with a GnRH agonist. Phase III trials comparing abarelix with leuprolide are underway (Martha et al., 1999).

LeiomyomaTreatment of leiomyoma with GnRH antagonists, by daily injections or by depot injections, results in shrinkage of the leiomyoma by 30–50% within 4–8 weeks (Felberbaum et al., 1999; Gonzalez-Barcena et al., 1997). The avoidance of the fl are-up in gonadotrophin secretion seen with the GnRH agonist may explain the rapid reduction in fi broid size. Autocrine/paracrine effects mediated via myometrial GnRH receptors may also be involved.

Endometrial ablationAmong other future benign gynaecological indications are prevention of endometrial proliferation - eg, with diffi cult dysfunctional bleeding and as pretreatment before hysteroscopic surgery (Fauser et al., 2000).

Gynaecological cancersThe original rationale for a GnRH agonist in the treatment of ovarian cancer was to block the endogenous LH and FSH secretion which were thought to stimulate tumour growth. However,

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a role for gonadotrophins in ovarian epithelial cancer remains controversial. Clinical studies have recorded objective responses (9% to 26%) with GnRH agonists, and there is evidence for a direct antiproliferative effects of GnRH analogues (Emons and Schulz, 2000). A role for GnRH antagonists has yet to be established but studies are underway (Emons et al., 2000). Favourable effects of GnRH agonists in patients with recurrent endometrial cancer have been reported (Emons and Weiss, 2000). These favourable effects could be related to reduction in oestrogen output but direct inhibitory effects are likely also. GnRH agonists and antagonists inhibit proliferation in endometrial cancer cell lines (Noci et al., 2000), but no clinical data on the effects of antagonists are available.Treatment with GnRH agonists of patients with advanced metastatic breast cancer has been shown to be effective and safe though in clinical practice antioestrogenic therapy by tamoxifen prevails. Human breast cancer does express the GnRH receptor with moderately high-affi nity binding, inducing inhibition of cell proliferation in cultured cell lines (Kakar et al., 1994; Eidne et al., 1987). Although some studies with agonists show inhibition, others found no effect (Miller et al., 1985). Unfortunately, at the moment there are no reports or studies underway with regard to the clinical application of GnRH antagonists in breast cancer.

MammographyInjection of a GnRH antagonist before screening mammography, to achieve an antioestrogenic effect on the breast, could permit better resolution (Fauser et al., 2000).

OTHER INDICATIONS: MALE REPRODUCTIVE SYSTEM

Benign prostatic hypertrophyIn one clinical study injections of cetrorelix (5 mg twice daily for 2 days and then 1 mg twice daily for 2 months) resulted in a rapid 27% reduction in prostate volume and a 53% reduction of symptoms with improvement in quality of life (Comaru-Schally et al., 1998). During follow-up urinary symptoms usually improved and sexual function was enhanced. However, phase III comparative studies have yet to be done.

Prostate cancerGnRH agonists have advantages over orchidectomy and oestrogens and were fi rst-choice drugs, mostly initially combined with antiandrogens, in the hormonal treatment of prostate cancer (Tomera et al., 2001). However, the initial androgen surge as result of the LH fl are may cause signifi cant complications such as ureteric obstruction and severe pain from bone metastasis. This effect does not occur with GnRH antagonists (Tomera et al., 2001; Gonzalez-Barcena et al., 1996; Mcleod et al., 2000; Garnick et al, 1999; Stricker, 2001; Trachtenberg et al., 2001). With cetrorelix 1–2 mg per day after an initial loading of 10 mg over 2–5 days, castrate testosterone values are sustained with reductions in prostate specifi c antigen (PSA), regression of metastases, and rapid improvement of disseminated prostate cancer (Gonzalez-Barcena et al., 1996). Immediate and sustained suppression of tumour is achieved, with a rapid decline in prostate volume compared with conventional treatments (Mcleod et al., 2000). Abarelix-depot 100 mg by intramuscular injection every 28 days with an additional injection on day 15 causes rapid and profound reduction in androgen, gonadotrophin and PSA in men with various stages of prostate cancer, superior by comparison with the GnRH agonists up to 29 days (Tomera et al., 2001; Gonzalez-Barcena et al., 1996; Mcleod et al., 2000; Garnick et al,

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1999; Stricker, 2001; Trachtenberg et al., 2001; Gittelman et al., 2001) and comparable during 85 to 169 days (Trachtenberg et al., 2001; Gittelman et al., 2001). An additional advantage of GnRH antagonists by comparison with GnRH agonists is the long-term (up to 169 days) suppression of FSH (Garnick and Campion, 2000; Trachtenberg et al., 2001), since FSH has been implicated as a potential growth factor for prostate cancer (Ben-Josef et al., 1999). At 12 weeks the overall response to abarelix-depot is 74% (9% complete response, 22% partial, 43% stable disease) (Garnick et al., 1999a), median PSA and testosterone levels declined with 90% of baseline, cancer pain was reduced and narcotic use declined with improvement of urinary complaints (Koch et al., 2001). A New Drug Application has been submitted to the US Food and Drug Administration, and has been fi led for approval in Europe for its use in prostatic cancer.

Male contraceptionWeekly 200 mg testosterone injections cause reversible oligozoospermia in 98% of men, and resulting pregnancy rates are lower than those for the oral contraceptive for women (Swerdloff et al., 1998). However, reported side-effects are weight gain, oily skin, and acne and if the dose is lowered contraceptive effi cacy declines. Azoospermia can be achieved with lower dosages (25 mg weekly) if combined with a GnRH antagonist (Pavlou et al., 1991). Low sperm counts can be achieved with an initial combination of a daily subcutaneous antagonist (Nal Glu) and weekly low-dose testosterone followed by low-dose testosterone alone (Swerdloff et al., 1998). The newly developed long-acting GnRH antagonist preparations could bring us one step closer to the goal of a satisfactory male hormonal contraceptive.

OTHER INDICATIONS

Puberty disorders

GnRH agonists are the established treatment of central precocious puberty. They act via desensitization of the pituitary to prematurely released endogenous GnRH. There are no clinical data on GnRH antagonists in this context, though animal experiments are encouraging (Roth et al., 2000). Another future indication may be the delay of normal puberty in children with short stature but so far this has only been tested with a GnRH agonist (Kamp et al., 2001).

MiscellaneousAmong other possible indications are the use of GnRH antagonists in the hormonal treatment of pituitary gonadotrophin producing tumours (Daneshdoost et al., 1990; Chanson et al., 1994), and the protection of the gonads in a similar way as agonists may act during radiation or chemotherapy (Ataya et al., 1995a and b; Levitsky and Owens, 1999). Finally, GnRH antagonists may be useful as pharmacological agents in treatment of hypersexuality and paraphilias (Levitsky and Owens, 1999).

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FUTURE PROSPECTS

Current GnRH antagonists seem to be safe and effective additions to the hormonal armamentarium for several conditions though published clinical evidence so far is mainly in IVF and prostate cancer. The hope remains that all current indications for GnRH agonist therapy will also become applications for GnRH antagonists. With respect to direct anti-tumour effects we do not yet know whether agonists or antagonists will prevail. More studies, looking at tissue-specifi c distribution of GnRH receptor types, their analogue affi nity, potency, and specifi c signalling pathway activation are needed. In all situations sound comparative studies, and publication of them, are essential. Expected long-term developments include non-peptide orally active GnRH antagonists (Haviv et al., 1998; Ashton et al., 1999) and targeted drug administration for some cancers (Schally, 1999). Finally, licensed GnRH antagonists are ideal research tools for further exploration of the pathophysiology of the human reproductive system.

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Contemporary pharmacologicalmanipulation in assisted reproduction

JAF Huirne, CB Lambalk, ACD van Loenen,

R Schats, PGA Hompes, BCJM Fauser, NS Macklon

Drugs 2004, 64, 297-322

3

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ABSTRACT

Follicle-stimulating hormone (FSH) treatment to induce follicular development in anovulating women and multiple follicular development for assisted conception has been incorporated in almost all reproductive treatment cycles in the form of either urinary, purifi ed urinary or recombinant preparations. Besides improved tolerance and theoretically lower chances of infection by prions, the latter may be more effective in terms of clinical pregnancy rates, FSH requirement and cost effectiveness. The low-dose, step-up protocol to induce monofollicular development, which is applied worldwide, has to compete with the equally effective but health economically benefi cial step-down protocol. The long protocol using recombinant FSH (rFSH) 150 IU/day is advocated when using gonadotrophin-releasing hormone (GnRH) agonists in in vitro fertilization (IVF) or intracytoplasmatic sperm injection treatment. However, the current paradigmatic hyperstimulation came under scrutiny after the introduction of the GnRH antagonists, which allow milder and more convenient approaches with acceptable cancellation and pregnancy rates but lower requirements for FSH. Risk of ovarian hyperstimulation syndrome (OHSS) can be further eliminated if recombinant luteinizing hormone (rLH) or GnRH agonists are used to trigger oocyte maturation and ovulation, the latter require pituitary responsiveness and are therefore excluded in agonist protocols. FSH and LH are both required for appropriate folliculo- and steroidogenesis. In hypogonadotropic women, the addition of LH (human menopausal gonadotrophin, human chorionic gonadotrophin (hCG) or rLH) is therefore obligate to achieve appropriate follicular growth and pregnancy. The role of LH in ovulation induction is still a matter of debate, although in GnRH agonistic protocols there seems to be a ‘therapeutic window’, levels that are too high or too low have detrimental effects on IVF outcome. To broaden the pharmaceutical armoury, recent efforts have been directed towards the development of novel GnRH antagonists and FSH preparations with optimal pharmacokinetic, pharmacodynamic and safety profi les. Alternative strategies with fewer adverse effects and higher benefi t/cost ratios are under development. However, before the GnRH agonist is abandoned for the antagonist as standard therapy, the cause of the observed possible lower pregnancy rates with the latter need to be clarifi ed. In addition, prospective studies investigating possible direct effects of GnRH analogues, optimal dose-fi nding studies and treatment regimens under different conditions, with or without pharmacological coadministration and for different indications, should be performed to optimize the effi cacy and tailor treatment strategies to individual needs. Since the discovery and isolation of gonadotrophins (i.e. FSH, LH, hCG and GnRH, an extensive variety of pharmacotherapeutic preparations have been developed in order to be used in ovulation induction and assisted reproductive therapy (ART). This paper focuses on the use of gonadotrophins, GnRH analogues, and the new developments in this fi eld. We provide insight into the different types of drugs used, their mechanism of action and how they manipulate the hypothalamic-pituitary-ovarian axis. We discuss the current situation in the use of pharmacological manipulation in assisted reproduction based on the development of various therapeutic strategies and comparative studies.

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GONADOTROPHINS

In the 65 years since the gonadotrophin hormones were discovered (Zondek and Ascheim,1927), FSH has attained a central role in contemporary infertility therapies. Clinical applications include the treatment of anovulatory infertility and controlled ovarian stimulation in women being treated with in vitro fertilization (IVF). In addition, FSH is used to treat male infertility caused by gonadotrophin defi ciency (hypo-gonadotrophic hypogonadism). The extraction and purifi cation of gonadotrophins from postmenopausal urine was developed in Italy in the late 1940s. Although the fi rst human menopausal gonadotrophin (hMG) was developed in 1949, clinical interest in these compounds did not grow until the 1960s, when the fi rst hMG-assisted live human birth was reported (Lunenfeld et al., 1962). The fi rst clinically available gonadotrophin preparations contained a mixture of FSH and LH in a ratio of activity of 1:1, and a large number of urinary proteins. Early production techniques were crude and labour intensive, requiring up to 30L of urine to produce enough hMG for one treatment cycle. Improvements in purifi cation techniques led to increasing relative amounts of the active ingredients and the fi rst urine-derived preparation containing only urinary FSH (uFSH) became available in 1983. The development and application of production techniques based on immunoaffi nity chromatography with monoclonal antibodies enabled the production of highly purifi ed uFSH (Metrodin®). Although improvements in purity and specifi c activity were thus achieved, the large quantities of urine required and a massive increase in worldwide demand for FSH compounds for infertility treatment put increasing pressure on production and availability. In the 1990s, recombinant DNA technology led to the development and clinical introduction of human recombinant FSH (rFSH) by stable transfection of the common α- and the β-FSH subunit into Chinese hamster ovary cells. This development promised not only unlimited availability, but also improved purity and batch-to-batch consistency in comparison with urine-derived products. Since 1996, rFSH has been clinically available, in the form of follitropin-α (Gonal-F®) and follitropin-β (Puregon®). More recently, recombinant hCG (rhCG) and recombinant LH (rLH) have been added to the clinical armoury for ovarian stimulation and assisted conception. Both LH and hCG are heterodimer glycoproteins, composed of two non-covalently linked subunits (α and β). For the production of rLH and rhCG, the human genes are also transfected into Chinese hamster ovary cells, encoding for the subsequent α and β subunits, producing lutropin-α (Luveris®) and choriogonadotrophin-α (Ovidrel®), by repeated chromatographic steps (Fonjallaz and Loumaye, 2000, Hull et al., 1994). Recombinant technology has also opened the way towards the development of novel molecules with shorter- and longer-acting properties.In this article, the role of native FSH, LH and hCG are reviewed, and the clinical applications of current gonadotrophins preparations are discussed (see table I). Finally, some novel preparations soon to be clinically available are described.

What Does Native Follicle-Stimulating Hormone (FSH) Do? FSH is a complex heterodimeric glycoprotein consisting of two subunits produced in the anterior lobe of the pituitary gland. While the role of FSH in early follicle development is unclear, late follicular development is FSH dependent. The great majority of human oocytes are destined to undergo atresia (Baker, 1963, Schwartzman et al. 1993). Only those follicles able to respond to stimulation by FSH and which gain increased FSH sensitivity will enter the fi nal stage of development and ovulate (Gougeon, 1993, Macklon and Fauser, 1998).

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Compound generic name

Active substrate FSH , LH ratio Route Tradename Europe/USA

Follicle-stimulating hormone (FSH)

Menotrophin SC uFSH and uLH 1 : 1 SC HMG-Ferring, Humegon, Menogon,Pergonal, Merional/Reponex

3 : 1 SC Normegon

Purifi ed menotrophin uFSH and uLH 1 : 1 SC Menopur

Urofollitrophin uFSH and uLH 1 : <0.01 SC Metrodin, Follegon

Purifi ed urofollitrophin uFSH and uLH 1 : <0.001 SC, IM Metrodin HP/ Fertinex, Bravelle

Follitrophin-α rFSH 100% FSH SC Gonal-F

Follitrophin-β rFSH 100% FSH SC Puregon/Follistim

Luteinizing hormone (LH)

Lutrophin-α rLH 100% rLH SC Lhadi, Luveris

Human chorionic gonadotrophin (hCG)

Choriogonadotrophin uhCG hCG IM Pregnyl, Profasi, Novarel

Choriogonadotrophin-α rhCG 100% hCG SC Ovidrel/Ovitrelle

Table I. Gonadotrophins commonly used in assisted reproductive therapy. IM = intramuscular; rFSH = human recombinant FSH ; rhCG = recombinant hCG ;SC = subcutaneous; uFSH = urinary FSH; uhCG = urinary hCG; uLH = urinary luteinizing hormone.

As a result of the demise of the corpus luteum and a subsequent decrease in progesterone and oestradiol production, FSH levels increase at the end of the luteal phase of the menstrual cycle (Roseff et al., 1989, Le Nestour et al., 1993). The high FSH levels, which occur during the luteofollicular transition, give rise to continued growth of a limited number (or cohort) of follicles (Hall et al., 1992). Subsequent development of this cohort during the follicular phase becomes dependent on continued stimulation by gonadotrophins (Macklon and Fauser, 2001b). Each growing follicle possesses a threshold requirement for stimulation by circulating FSH (Brown, 1978, Van der Meer et al., 1994). This threshold level needs to be passed to ensure ongoing preovulatory development. In the normo-ovulatory cycle only one follicle will become responsive to FSH above this threshold and become capable of converting the theca cell-derived substrate androstenedione to oestradiol by induction of the aromatase enzyme. In response to negative feedback from rising oestradiol and inhibin levels, the FSH level falls in the late follicular phase. The dominant follicle has increased sensitivity to the falling FSH level and continues growing. Those follicles that commence the latter stages of development after FSH levels start to fall undergo atresia (fi gure 1). The duration of this ‘FSH window’, during which FSH levels are above the threshold required to stimulate ongoing development, determines the number of follicles which can develop to the preovulatory stage (Baird et al., 1987, Fauser and van Heusden, 1997). These advanced stages of follicular development are open to therapeutic intervention with exogenous FSH.

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Figure 1. FSH rise during a natural ovulatory cycleIn the spontaneous normo-ovulatory cycle, the intercycle rise in serum follicle stimulating hormone (FSH) levels exceeds the threshold for recruitment of a cohort of follicles for further development. The number of follicles recruited is determined by the duration (‘window’) for which the serum FSH is above the threshold at which recruitment occurs (adapted from Macklon and Fauser 1997, with permission from Cambridge University Press).

FSH in Clinical Practice FSH in Anovulatory Infertility The aim of ovulation induction in anovulatory women is the formation and ovulation of a single dominant follicle. In order to achieve this, specifi c treatment and monitoring protocols are needed. While several approaches to ovulation induction with gonadotrophins have been described, the two most frequently used in clinical practice are the low-dose step-up and the step-down protocols. The initially described ‘standard’ step-up protocol had a starting dose of FSH 150 IU/day. However, this regimen was associated with a high complication rate. Multiple pregnancy rates of up to 36% were reported, while ovarian hyperstimulation occurred in up to 14% of treatment cycles (Fauser and van Heusden, 1997). As a result, this protocol has been largely abandoned. A low-dose, step-up protocol (Franks et al., 1988) designed to allow the FSH threshold to be reached gradually has now become the most widely used regimen, reducing the risk of excessive stimulation and development of multiple preovulatory follicles. In this protocol, the initial subcutaneous or intramuscular dose of FSH is 50-75 IU/day and the dose is increased if, after 14 days, no response is observed on ultrasonography (and serum oestradiol monitoring). Increments of 37.5IU are then given at weekly intervals up to a maximum of 225 IU/day. The detection of an ovarian response is an indication to continue the current dose until hCG can be given to trigger ovulation. In a series describing outcomes using the low-dose, step-up regimen in 225 women with polycystic ovary syndrome (PCOS) treated over a 10-year period, ovulation rates of 72% per cycle were reported, 45% of these women conceived as a result of ovulation induction (White et al., 1996).

threshold

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The low-dose, step-up protocol is associated with a lower incidence of multiple folliculogenesis and hyperstimulation than the standard protocol (Buvat et al., 1989) and pregnancy rates appear similar (Shoham et al., 1991). However, the results of the low-dose, step-up protocol are negatively infl uenced by obesity (White et al., 1996), age (McClure et al., 1992) and insulin resistance (Dale et al., 1998). More recent studies focusing on further reducing the starting dose have reported the feasibility of commencing with 50IU (Hayden et al., 1999) and 37.5IU (Balasch et al., 2000). However, while ovulation can be achieved with this approach, the stimulation period may be further extended (Balasch et al., 2000). Step-down protocols are aimed at rapidly achieving the FSH threshold for stimulating follicle development. Current regimens normally begin therapy with 150 IU/day started shortly after a spontaneous or progesterone-induced bleeding. This dose is continued until a dominant follicle (≥10mm) is seen on transvaginal ultrasonography. The dose is then decreased to 112.5 IU/day followed by a further decrease to 75IU 3 days later, which is continued until hCG is administered to induce ovulation (Fauser et al., 1993). Should no ovarian response be observed after 3-5 days, the FSH dose can be further increased. For some patients an initial dose of 150 IU/day may be too high, refl ecting major individual differences in the FSH threshold. The appropriate starting dose may be determined by using the low-dose step-up regimen for the fi rst treatment cycle in order to assess the individual FSH response dose or by applying a formula for the calculation of the individual response dose (van Santbrink et al., 2002, Imani et al., 2002). Experience with the step-down protocol in a series of 82 women suggested that the duration of treatment and total gonadotrophin dosage were reduced compared with the low-dose, step-up protocol. Moreover, monofollicular growth was more frequently achieved (Van Santbrink et al., 1995). These fi ndings were subsequently confi rmed by a prospective, randomized comparison of low-dose, step-up and step-down regimens (van Santbrink et al., 1997). The clinical benefi ts of a more physiological means of stimulating follicle development were refl ected in an incidence of monofollicular cycles of 88% compared with 56% observed in women treated with the step-up regimen, presumably reducing the risk of multiple pregnancy and hyperstimulation. Potential health-economic benefi ts were also apparent since those treated with the step-down regimen required a mean duration of treatment of just 9 days, as opposed to 18 days in women treated with the low-dose, step-up regimen. A recent multicentre, randomized study comparing the step-up versus step-down protocol using rFSH reported a shorter duration of stimulation when the step-down protocol was used (Christin-Maitre et al., 2003). The cumulative rate of clinical gestations did not differ between the two groups but, in contrast with the fi ndings of an earlier single-centre study (Van Santbrink et al., 1997), the step-up protocol was associated with a higher rate of monofollicular development and a lower rate of ovarian hyperstimulation. These differences may refl ect the necessity for increased skill and care in monitoring step-down stimulation cycles, which is easier to ensure in a single-centre setting.The balance between success and complications resulting from ovulation induction is dependent on many factors, including patient characteristics, gonadotrophin preparations and dose regimens used, the intensity of monitoring ovarian response to stimulation and willingness to cancel the cycle in case of hyper-response. Cumulative success rates of ovulation induction are reported to be around 75% (Eijkemans et al., 2003), with a coinciding incidence of multiple pregnancies of <10% and of ovarian hyperstimulation syndrome (OHSS) of <2% .

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The degree to which the type of FSH compound employed may infl uence outcomes in ovulation induction remains the subject of controversy. A meta-analysis comparing the effectiveness of daily uFSH with daily hMG in a step-up regimen for inducing ovulation in women with polycystic ovary syndrome (PCOS) who had not responded to clomifene (clomifene citrate) demonstrated no difference in pregnancy rates per treatment cycle (Hughes et al., 1996). However, women given FSH were less likely to have moderately severe or severe OHSS. Subsequent studies remained consistent with these conclusions but have suggested a less marked difference in the risk of OHSS (Yarali et al., 1999). With respect to rFSH, a multicentre, prospective trial found that the cumulative ovulation rates were comparable with those achieved with purifi ed uFSH (95% after three cycles) (Coelingh Bennink et al., 1998). The total dose of rFSH needed and duration of treatment was less, and the complication rates were similar. In a meta-analysis of randomized, controlled trials comparing rFSH with uFSH for ovulation induction in women with clomifene-resistant PCOS, no signifi cant differences were demonstrated for the ovulation rate (odds ratio [OR], 1.19, 95% CI 0.78-1.80). Moreover, the ORs for pregnancy rate (0.95, 95% CI 0.64-1.41), miscarriage rate (1.26, 95% CI 0.59-2.70), multiple pregnancy rate (0.44, 95% CI 0.16-1.21) and OHSS (1.55, 95% CI 0.50-4.84) showed no signifi cant difference between rFSH and uFSH (Bayram et al., 2002). Purifi ed uFSH has some LH activity but rFSH does not. Experience with rFSH in hypogonadotrophic hypogonadal women (WHO class 1) indicates that those women who have very low serum LH levels (<0.5 IU/L) need exogenous LH activity (derived from LH or hCG) to maintain adequate oestradiol biosynthesis and follicle development (Koustra et al., 1996).

FSH in Assisted Conception In contrast with ovulation induction regimens, where the aim is to induce monofollicular development in an anovulatory woman, FSH is applied in assisted conception to obtain large numbers of oocytes for IVF and subsequent selection of embryos for intrauterine transfer. Higher doses of gonadotrophins are therefore administered. The most commonly applied regimen is the ‘long protocol’ where gonadotrophins are administered following down-regulation with a GnRH agonist for the prevention of premature luteinization. The question of what is the optimal dose of FSH required to stimulate the development of adequate numbers of follicles without causing the potentially dangerous OHSS has been the subject of a number of recent studies. The most frequently applied starting doses in normo-ovulatory women undergoing ovarian hyperstimulation for IVF is 150 or 225 IU/day. In one study comparing these two starting doses of rFSH in combination with the GnRH antagonist cetrorelix in women undergoing ovarian hyperstimulation for IVF or intracytoplasmatic sperm injection (ICSI), the number of oocytes retrieved was signifi cantly higher in the 225IU group (11.0 ± 4.6 vs 9.1 ± 4.4, p = 0.024), but the ongoing pregnancy rates per started cycle and per embryo transfer did not differ signifi cantly between the groups (Wikland et al., 2001). Similar results were reported in another study in which 138 patients received rFSH at a dose of 150 or 250 IU/day. The mean number of oocytes retrieved was 9.1 in the 150IU group compared with 10.6 in the 250IU group (Out et al., 2000). In women between 30 and 39 years of age a decline in oocytes obtained was observed, but this was not overcome by an augmented daily dose of rFSH from 150 to 250IU. Moreover, ongoing pregnancy rates did not differ signifi cantly between the two study groups. Starting doses <150 IU/day have also been examined. In a study of 179 women randomized to receive rFSH at a dose of either 100 or 200 IU/day, fewer oocytes were obtained after the lower dose (5.7 vs 12.0, p < 0.001) (Out et al., 2001). However, while no differences

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in vital pregnancy rates per started cycle (16.9% vs 19.2%) or per embryo transfer (19.3% vs 26.2%) were observed, the number of cancelled cycles was higher in the 100 IU group. Thus, the collective evidence to date would suggest that 150 IU/day is an appropriate starting dose for most women undergoing ovarian hyperstimulation for IVF with rFSH as part of a long GnRH agonist or GnRH antagonist protocol. With the clinical introduction of GnRH antagonists, the opportunity has arisen to look again at current paradigms for ovarian hyperstimulation. Contemporary protocols aim at achieving maximal numbers of oocytes. However, the disadvantages of this approach, which include the risks of ovarian hyperstimulation, expense, inconvenience and adverse effects for the patient, are becoming increasingly recognised and alternative less aggressive approaches are now being investigated (Fauser et al., 1999, Macklon and Fauser., 2000). The availability of GnRH antagonists has opened the way to the development of novel regimens, in which the endogenous production of FSH can simply be supplemented by lower doses of FSH, thus extending the FSH ‘window’ and enabling a number of oocytes to achieve dominance (fi gure 2) (Macklon and Fauser, 2001a, Schipper et al., 1998, Hohmann et al., 2001). Following the publication of a pilot study indicating the feasibility of this approach in clinical IVF (De Jong et al., 2000), a prospective, randomized study comparing this approach with the long protocol indicated that while fewer oocytes may be obtained, the pregnancy rate per started cycle is not reduced (Hohmann et al., 2003). Ongoing studies are assessing the cost/benefi t aspects of milder approaches to ovarian stimulation for FSH.

Figure 2. FSH window after exogenous FSH administration. When the duration of the follicle-stimulating hormone (FSH) ‘window’ is extended by the administration of exogenous FSH in the mid-late follicular phase, more follicles may achieve dominance, leading to the multiple dominant follicle selection that is appropriate for in vitro fertilization treatment (adapted from Macklon and Fauser 2001, with permission from Cambridge University Press).

Since the fi rst pregnancies resulting from the use of rFSH were reported in the early 1990s, several controlled trials comparing rFSH with uFSH have been published. One study of 123 women found that the two preparations were equally effective (Recombinant Human FSH Study

10

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Group, 1995). However, in three larger studies of 981 (Out et al., 1995), 235 (Bergh et al., 1997) and 496 women Schats et al., 2000), the dose of a different rFSH preparation and the duration of rFSH treatment needed to stimulate follicle development were less and more oocytes were recovered, implying greater effi cacy for rFSH. More recently, a total of 18 studies comparing rFSH with uFSH preparations were subject to a meta-analysis in which the clinical pregnancy per cycle started was the primary focus. The overall OR was 1.21 (95% CI 1.04-1.42) in favour of rFSH. This equates with a 3.7% (95% CI 0.8-6.7) absolute increase in clinical pregnancy rate for patients stimulated with rFSH compared with those stimulated with uFSH. Moreover, the total FSH dose was lower by 406 IU (95% CI 185-627) with rFSH (Daya and Gunby, 2001, Daya, 2002). In contrast, a recent meta-analysis of fi ve studies comparing the effectiveness of hMG and rFSH in IVF following down-regulation indicated higher clinical pregnancy rates with hMG (relative risk [RR] 1.22, 95% CI 1.03-1.44] (Van Wely et al., 2003). No differences in ongoing clinical pregnancy rates were observed. Inconsistency in the results of randomized, controlled trials and meta-analyses in this commercially sensitive area may be due to chance and are to be expected given differences in methodology, participants and clinical settings (Collins, 2003). The debate as to whether rFSH is more effi cacious then uFSH in IVF treatment is unlikely to be resolved in the near future. In practice, the choice of preparation is based increasingly on other factors, such as relative cost, purity, batch variance, side effects and possible long-term risks (Daya, 2003). OHSS is the most commonly reported serious adverse event. No statistically signifi cant differences in the incidence of OHSS have been found between uFSH and rFSH preparations. Pooled data from 14 comparative trials (including four that reported no cases of OHSS) in a meta-analysis (Daya and Gunby, 2001), indicated that the incidence of OHSS was 2.0% in the rFSH group and 1.4% in the uFSH group (RR 1.50, 95% CI 0.88-2.58). In terms of cost effectiveness, recent studies have indicated benefi ts with rFSH over uFSH. The increased yield of mature oocytes, requiring a lower total FSH dose and a shorter period of treatment, combined with a higher clinical pregnancy rate per started cycle would certainly indicate that rFSH is likely to be a more cost-effective option than uFSH. However, rFSH remains considerably more expensive than uFSH. In a recent study addressing this issue, Markov decision analysis was used to model the outcomes of a large number of ART cycles using rFSH and uFSH (Daya et al., 2001. Analysing 5000 stimulated cycles on a Markov cohort of 100 000 patients, the number of pregnancies achieved was predicted to be signifi cantly higher with rFSH than with uFSH (40 575 vs 37 358, p < 0.0001). The cost per successful pregnancy calculated within the context of the UK National Health Service was € 5906 ± 455 in the rFSH group and € 6060 ± 547 in the uFSH group (p < 0.0001). Fewer cycles of treatment were required to achieve an ongoing pregnancy with rFSH (3.83 vs 4.29). Applying this approach to the context of the US health system, the same group showed that rFSH was more cost effective than uFSH. A recent analysis of the mean cost of achieving an ongoing pregnancy was carried out in the context of a randomized trial of highly purifi ed hMG versus rFSH (Lloyd et al., 2003). While the two preparations appeared to be equally effective, hMG was associated with a signifi cantly lower cost per pregnancy of £10 781 (95% CI 9056-12 919) compared with £11 883 (95%CI 11 883-17 891) with rFSH. The two clinically available rFSH preparations, follitropin-α and follitropin−β have been compared in a recent prospective study in 492 women (Harlin et al., 2002). No statistically signifi cant differences were found for numbers of follicles on the day of hCG administration, numbers of oocytes retrieved and clinical pregnancy rate per started cycle.

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What Does Native Luteinizing Hormone (LH) Do? LH is essential for timing of ovulation, the regulation of target tissue responses and the production of steroid hormones. In a natural cycle, a sudden and profound midcycle peak of LH and FSH levels (LH and FSH surge, lasting for about 48 hours) is observed, which is regulated by a complex mechanism that integrates multiple neurotransmitters and sex steroids (Shoham et al., 1995). Oestrogens play important functional roles in the reproductive system by, among other functions, stimulating cervical mucous production and endometrial proliferation. Persistently elevated oestrogen levels in combination with a small, but distinct, rise in progesterone, which results in increased pituitary sensitivity for GnRH and a substantial rise of GnRH (Caraty et al., 1995), induce the LH surge. Gonadotrophin surge attenuating factor has also been implicated in the regulation of the LH surge timing and amplitude (De Koning, 1995). The LH surge is obligate for the resumption of meiotic maturation of the oocyte, rupture of the dominant follicle resulting in the release of the oocyte, and luteinization of thecal and granulose cells resulting in the formation of the corpus luteum, providing support for the early stages of pregnancy. During follicular development, LH has a synergistic action with FSH. Theca cells are stimulated by LH to convert cholesterol into androstenedione and testosterone by cytochrome P450 side chain cleavage oxidases and 3β-hydroxy-steroid dehydrogenase. Aromatase activity in the granulosa cells is induced by FSH, and converts androstenedione and testosterone into estrone and oestradiol. The involvement of two cell types (granulosa and theca cells) and two hormones (LH and FSH) to produce oestrogens from cholesterol has led to the concept of the 'two cell, two gonadotrophin' theory (Fevold, 1941). In vivo evidence for this concept has grown in recent years. In 1992, the effect of rFSH administration was described on a woman with isolated gonadotrophin defi ciency as a result of a previous hypophysectomy (Schoot et al., 1992). For the fi rst time, the effects of FSH alone could be studied in the absence of endogenous and exogenous LH. While multiple follicular growth was induced, oestradiol levels remained low and no pregnancy ensued. Subsequent case reports confi rmed these fi ndings Balasch et al., 1995, Battaglia et al., 2000). Recent evidence points to a central role for LH in monofollicular selection and dominance in the normal ovulatory cycle. Despite falling FSH levels in the late follicular phase, the dominant follicle continues to develop. One of the actions of FSH is the induction of LH receptors on granulosa cells. While granulosa cells from early antral follicles respond only to FSH, those from mature follicles, possessing receptors to both gonadotrophins, are responsive to both FSH and LH. The maturing dominant follicle may become less dependent on FSH because of the ability to respond to LH levels, which remain relatively constant during the follicular phase (Zeleznik et al., 1984). Elegant studies employing rLH in down-regulated women have indicated that although follicles of ≥14mm require gonadotropic stimulation for continued oestradiol production, they are responsive to either FSH or LH (Sullivan et al., 1999). In contrast, less mature follicles which do not express LH receptors undergo atresia as a consequence of falling FSH levels.

LH and Human Chorionic Gonadotrophin (hCG) in Clinical Practice Both LH and hCG are heterodimeric glycoproteins with identical α subunits. The major differences between the two hormones include the sequence of the β subunit, the regulation of their secretion, their receptor affi nity and their clearance (Weissman et al., 1996, Yen et al., 1968). Until recently, hMG, a urinary extract containing a fi xed combination of LH and FSH, was the only source of exogenous LH. Therefore, exogenous hCG (which activates the LH receptor as a result of structural and biological similarities to LH, and which is easy to

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extract from urine of pregnant women) has been used for decades to replace endogenous LH during FSH stimulation protocols but is associated with a number of disadvantages. Currently, human gene products like rLH and rhCG are available for pharmacological application. The success in clinical studies of pure FSH preparations, increasingly devoid of LH, has served to enhance the impression that excess LH is detrimental to oocyte development and chances of pregnancy following therapeutic intervention. However, a number of recent clinical studies, together with an increasing understanding of the function played by LH in oocyte maturation, have begun to redefi ne the role of LH as a therapeutic agent in anovulatory fertility and IVF.

LH in Anovulatory Infertility The treatment of hypogonadotrophic women with FSH alone leads to follicular development but not pregnancy (Schoot et al., 1992, Balasch et al., 1995). Exogenous LH is therefore required to treat this form of anovulatory infertility. Until recently, hMG was the only source of exogenous LH for this group of patients. rLH now offers the possibility for a more sophisticated and individualized approach to treatment. Recent studies have demonstrated the safety and appropriate dose required to effect follicle development and subsequent pregnancy (European Recombinant Human LH Study Group, 1998). It has been established that resting levels of 1-10 IU/L should be suffi cient to provide maximal stimulation to thecal cells (Chappel and Howles, 1991). In a recent study of hypogonadotrophic women undergoing treatment with rFSH and rLH, a dose of 75 IU/day of rLH was observed to result in follicular development and pregnancy. However, further increases in LH levels above the ‘threshold’ level needed to gain a response did not appear to induce a greater degree of ovarian stimulation (Burgues, 2001).In normogonadotrophic anovulation, endogenous LH does not normally require supplementation. Indeed, the focus on LH in this group of patients has been primarily directed at reducing the potential detrimental effects associated with excessive LH (Shoham et al., 1993). More recently, however, the demonstration of the importance of LH in optimizing oocyte quality has reopened the debate as to the role of LH in ovulation induction (Filicori and Cognigni, 2001). Supplementation of LH activity may offer advantages in some patients by hastening large follicle development and therefore shortening the duration of treatment (Filicori et al., 1999). Moreover, the work of Sullivan et al. referred to a potential therapeutic role for LH in affecting monofollicular stimulation, as part of a sequential ovarian stimulation protocol following initiation with rFSH (Sullivan et al., 1999). As the availability of recombinant gonadotrophins leads to increasing knowledge of the processes of follicular development and selection, further improvements in the effi cacy and safety of ovulation induction may follow.LH/hCG in Assisted Conception Although there is clear evidence for the crucial role played by LH in normal follicular development, the necessity for LH supplementation in ovarian hyperstimulation for IVF remains a contentious issue. Excessive suppression of LH levels following down-regulation with GnRH agonists has been associated with a detrimental effect on outcome of IVF (Fleming et al., 1998, Westergaard et al., 2000). On the other hand, a number of studies comparing uFSH with hMG which measured LH levels suggested that resting levels of LH following down-regulation are suffi cient to support development and maturation of follicles and oocytes in normogonadotrophic women (Daya et al., 1995,

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Loumaye et al., 1997). In one study, no signifi cant differences in either performance or clinical outcome among groups of patients with varying degrees of LH suppression were observed (Balasch 2001). However, another recent study using GnRH agonists suggested that LH levels that are too low or too high on stimulation day 8 for IVF can have a negative impact on fertilization and clinical pregnancy rates (Humaidan et al., 2002). Taken together, recent studies suggest that midfollicular LH levels <0.5 IU/L are likely to be detrimental to IVF outcome. Therefore, there may be a role for LH supplementation in some patients undergoing ovarian stimulation for IVF. Moreover, supplemental LH may offer a new strategy for dealing with poor response to ovarian stimulation with rFSH.Recent studies in IVF patients have confi rmed the earlier observed ability of LH activity alone to complete follicle and oocyte maturation. In a study using hCG to provide LH activity, Filicori et al. showed that low-dose hCG can stimulate the growth and support the maturation of larger follicles in the absence of FSH administration (Filicori et al., 2002). In addition, LH activity without FSH appeared to cause a selective reduction in the number small preovulatory follicles. This work has opened new possibilities for further refi ning ovarian stimulation regimens for IVF, allowing suffi cient stimulation for successful IVF treatment while reducing the risks associated with excessive multifollicular development (Filicori et al., 2002).

LH/hCG to Trigger Ovulation or Resumption of Meiotic Division The most used substitute for the midcycle LH surge in ovulation induction and ART is hCG. However, hCG may be an additional promoting factor for the often observed dangerous complication of OHSS in ovarian hyperstimulated cycles, which may be explained by the longer half-life of hCG compared with LH (Weissman et al., 1996, Yen et al., 1968). To avoid this serious event, alternative ways for triggering ovulation are under development, using rLH, native GnRH or a GnRH agonist (see section 2.2.3). Three prospective, randomized, controlled trials (with 84, 190 and 297 patients, respectively), between rhCG and uhCG implicated rhCG to be as effi cacious as uhCG in terms of IVF outcome, with the benefi t of improved local tolerance (Driscoll et al., 2000, European rhCG Study Group, 2000, Chang et al., 2001). In a prospective, randomized trial, 259 infertile women were treated with either uhCG or rLH in doses of 5000, 15000, 30000 or 15000 IU plus 10 000 IU 3 days later (European r-LH Study Group, 2001). A single dose of rLH was as effective in inducing fi nal follicular maturation and early luteinization in IVF as uhCG 5000 IU, but with a signifi cant reduction in OHSS. The dose with the highest effi cacy to safety ratio was between rLH 15 000 and 30 000IU (European r-LH Study Group, 2001). Moreover, less intense circulatory changes were observed after rLH 5000IU than after uhCG 5000IU (Maau et al., 2002). Altogether, rLH seems to be an attractive alternative for hCG with equal effi cacy, but reduced risk for OHSS and improved local tolerance.

Safety Aspects of Gonadotrophins An advantage offered by rFSH preparations over urinary products is improved tolerability. The absence of contaminating urinary, nonhormonal proteins reduces the (rare) complication of local reactions to exogenous gonadotrophins at the injection site (Albano et al., 1996). Furthermore, rFSH used in clinical trials was reported to be well tolerated by both patients undergoing ART and anovulatory patients treated for ovulation induction, the most common adverse event was associated with treatment is reaction at the injection sites.

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In terms of long-term risks, urinary preparations are associated with a theoretical risk of transmission of prion proteins, which have been identifi ed in human urine (Shaked et al., 2001). Although infections by urine prions in humans and animals has not been reported, and in 40 years of use no such infections have been identifi ed, the risk of prion disease such as new variant Creutzfeldt-Jakob disease has been deemed by some to be suffi cient to advise against the use of uFSH, uLH or uhCG (Matorras et al., 2002). However, others consider the risk to be minimal and not in itself a reason to prescribe recombinant preparations over urinary ones (Balen, 2002). Moreover, studies of follitropin-α and follitropin−β suggest that the incidence of OHSS is similar with both products, while a number of small studies have suggested that the incidence of local reactions following injection of follitropin-α may be lower than with follitropin-β (Harlin et al., 2002).

New Developments With Gonadotrophins The degree of purity and batch-to-batch consistency in activity achieved in the production of rFSH allows improved consistency in dose from day-to-day and cycle-to-cycle that was diffi cult to achieve with urine-derived products. However, the currently applied system of determining activity means that in the clinical setting variations in activity may be observed. The bioactivity of uFSH and rFSH compounds is determined by an in vivo bioassay which is cumbersome and subject to such variation that an ampoule labelled to contain 75IU may range in true activity from FSH 50 to 120IU. The constant relationship between FSH mass (¼ g) and biological activity (IU) has allowed the development of a fi lled by mass rFSH product which promises greater control and consistency in optimising the dose for an individual patient. In a double-blind study, in which 131 patients undergoing ART were randomized in rFSH batches fi lled by IU (FbIU) or fi lled by mass (FbM), improved consistency in clinical outcome was observed in the FbM group (Hughes et al., 2003). Longer-acting rhFSH preparations are also undergoing clinical studies. The half-life of rhFSH, around 34 hours, is not markedly different from that of urine-derived products. As a result, daily injections are required in order to cause ovarian stimulation. Manipulation of the rhFSH molecule may alter its pharmacokinetic properties. FSH is a member of the gonadotrophin/ thyrotrophin hormone family, which is characterised by a heterodimeric structure consisting of a common α-subunit and a hormone-specifi c β-subunit (Pierce and Parsons, 1981). The β-subunit of the pregnancy hormone hCG is distinctly different from the other hormones in this family owing to an extension at the carboxyl end, that is, a C-terminal peptide (CTP) with four O-linked oligosaccharides. Analysis of the β-hCG coding sequence suggests that this extension results from the loss of the termination codon of the ancestral LH (Fiddes and Goodman, 1980). Both hCG and LH bind to the same receptor and exhibit comparable bioactivity in vitro. The CTP extension of the β-subunit of hCG has been shown to be responsible for the reduced clearance and resulting major enhancement of in vivo bioactivity. In an attempt to create a long-acting FSH agonist preparation, chimeric genes containing the sequence encoding the CTP of β-hCG fused with rFSH were constructed (Fares et al., 1992). The fi rst human exposure in hypogonadal males showed that rFSH-CTP could be administered safely and showed an extended half-life of 95 hours (Bouloux et al., 2001). Recently, the half-life of rFSH-CTP in women has been shown to be dose dependent. The elimination half-life was around twice that of rFSH (Duijkers et al., 2002). Clinical studies are now ongoing in both IVF and ovulation induction patients, and the fi rst ongoing pregnancy using rFSH-CTP in IVF treatment has recently been reported (Beckers et al.,2003).

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GONADOTROPHIN-RELEASING HORMONE (GNRH) AND ITS ANALOGUES

Thirty years after the discovery of the amino acid sequence of GnRH, also known as gonadorelin or LH-releasing hormone (LHRH), agonistic and antagonistic analogues were introduced in assisted reproduction. After an initial short period of gonadotrophin hypersecretion, continuous administration of GnRH or its agonists cause desensitization, resulting in a state of chemical hypophysectomy by a mechanism still far from understood (Conn and Crowley, 1994). GnRH antagonists cause an immediate and rapid, reversible suppression of gonadotrophin secretion, by competitive occupancy of the GnRH receptor. The development of agonists with good clinical safety was relatively simple by just changing one or two amino acids. The potency was increased by the replacement of glycine at position 6 by D amino acids and the replacement Gly-NH2 at the C-terminus by NH2-ethylamide binding to the proline at position 9, resulting in nonapeptides (Karten and Rivier, 1986). GnRH agonists have the same effect on gonadotrophin release after binding the type I receptor as native GnRH. The main difference of GnRH agonists used in clinical practice compared with native GnRH is that the half-life and bioavailability are prolonged as a result of increased lipophilicity. It took almost 30 years to obtain a GnRH antagonist with an acceptable pharmacokinetic, safety and commercial profi le. The fi rst-generation antagonists, containing replacements for His and Trp at position 2 and 3, respectively, had low suppressive activities. The potency in the second generation was increased after the incorporation of a D-amino acid at position 6, but resulted in increased anaphylactic reactions due to the increased histamine-releasing activity. These problems were resolved in the third generation by the replacement of D-Arg at position 6 by D-ureidoalkyl amino acids (Schally, 1999). The reversible state of chemical hypophysectomy, which can be achieved by both types of analogues, offers an important pharmacological tool which can be used to manipulate hormone secretion in a broad variety of disorders. The most promising indication for GnRH analogues was the use in controlled ovarian hyperstimulation (COH) protocols for the prevention of a premature LH surge. In both the natural and stimulated cycle, IVF treatment was hampered by the occurrence of premature luteinization and ovulation. Placebo-controlled studies of GnRH agonists revealed that, without an agonist, there was a premature LH increase that lead to the cancellation of the IVF cycle in about 20% of women (Edwards et al., 1996, Janssens et al., 2000b). Preventing the LH surge is clearly benefi cial. Moreover, the oocyte yield is higher with more embryos, allowing better selection leading to an increase in pregnancy rate (Templeton and Morris, 1998). In 1984, GnRH agonists were introduced in IVF (Porter et al., 1984), thereafter, many treatment regimens have been developed. In most centres the long agonist protocol has become the standard procedure. The immediate and dose-dependent suppressive action of antagonists provides a convenient alternative to agonists. Their administration results in the suppression of LH (about 70%) and FSH (about 30%) serum levels after approximately 6 hours. GnRH agonists may be preferred in indications in which long-term, profound suppression of the endogenous gonadotrophin release is desired. Conversely, GnRH antagonists are preferred if the initial gonadotrophin surge caused by GnRH agonists is undesirable.

What Does Native GnRH Do? Since the isolation and characterisation of the ten amino acids of GnRH type I by the groups of Schally and Guillemin (Baba et al., 1971, Burgus et al., 1971), many GnRH types have been

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isolated in different species (Sealfon et al., 1997). In humans, so far three types of GnRH have been isolated. Compared with GnRH type I, types II and III have three and two different amino acids, respectively. GnRH type II has been found to be identical to chicken GnRH and GnRH type III is equal to salmon GnRH (Yahalom et al., 1999). The genes for human GnRH type I and II are identifi ed on chromosome 8 (8p11.2-p21) and 20 (20p13), respectively (Yang-Feng et al., 1986, White et al., 1998). The gene locus of the third isoform has not been identifi ed yet. The physiological meaning of the multiple isoforms in the human brain has not yet been elucidated, nor has the distribution of different types of GnRH receptors in the different tissues. GnRH type I is the classical hypothalamic reproductive neuroendocrine factor, which is synthesised in the diencephalon and then transported by the axons to the neuronal terminal. There it is released in a pulsatile fashion into the capillaries of the pituitary-portal circulation. Thereafter, it binds to the GnRH receptors in the pituitary gonadotrophic cells after which it stimulates the release of LH and FSH in an orderly way, which is crucial for the control of normal gonadal function.

GnRH Agonists in Clinical Practice GnRH agonists may be delivered intranasally or subcutaneously. Subcutaneous formulations induce more stable serum levels, and the inter- and intra-individual variability is less than with intranasal formulations. In addition, short- and long-acting or slow-releasing subcutaneous formulations are available (Devreker et al., 1996). Although depot preparations seem to be more convenient for the patient since this requires fewer injections, some concern has been proposed about the possible interference with the luteal phase and embryo development (Devreker et al., 1996). In terms of IVF outcome, no clear benefi cial effect can be advocated for long-acting products. Therefore, in general, short-term agonists will be prescribed to minimise any compromising effect on an emerging pregnancy. GnRH agonist dosages used in IVF are derived from treatment schedules used in disseminated prostate cancer (Tolis et al., 1982), in which complete gonadal suppression is necessary. In ART, GnRH analogues are used to prevent a premature LH surge, for which it may be enough to suppress gonadotrophin secretion only partially. Using lower dosages could have some advantages, such as avoidance of a direct effect of agonists on the ovary, oocyte, embryo and endometrium. There are some comparative studies, which indicate that the daily dose of agonists used in IVF may be decreased without compromising the results (Simon et al., 1994, Eldendy et al., 1998). Only one prospective, randomized, double-blind, placebo-controlled, single-centre, dose-fi nding study has been performed in IVF for a GnRH agonist (triptorelin), 240 patients were included. The dosage necessary for suppressing the spontaneous LH surge was only 15-50% (15-50μg) of the dose needed for the treatment of prostate cancer (100μg), which is usually used in IVF (Janssens et al., 2000b). Halving the dose of a daily administered GnRH agonist at the beginning of the stimulation has been successfully performed in normal (Elgendy et al., 1998) and poor responders (Feldberg et al., 1994, Olivennes et al., 1996b), without adverse effects on the quality of ovarian response to stimulation. In conclusion, it is very likely that the dosage of the commonly used GnRH agonists is far too high. In fact, dose-fi nding studies should be conducted before proper comparative studies between various GnRH agonists and between various GnRH agonists and antagonists in IVF can be performed. The GnRH analogues commonly used in assisted reproduction are described in table II.

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Generic drug name

Type Minimal effective dose to prevent a LH surgea

Route Brand name Europe/USA

References

GnRH agonists

Leuprolide Nonapeptide NA SC Lucrin/Lupron

Buserelin Nonapeptide NA SC, IN Buserelin/Suprefact

Nafarelin Decapeptide NA SC, IN Synarel

Triptorelin Decapeptide 25 μg/day SC Decapeptyl/Trelstar depot

Janssens 2000

GnRH antagonists

Cetrorelix 3rd generation

0.25 mg/day or 1x 3mg

SC Cetrotide Albano 1997Olivennes 1998

Ganirelix 3rd generation

0.25 mg/day SC Orgulatran/Antagon

Ganirelix dose-fi nding study Group 1998

Table II. Gonadotrophin releasing hormone (GnRH) analogues commonly used in assisted reproductive therapy (ART). a If coadministered only with follicle-stimulating hormone and/or luteinizing hormone for in controlled ovarian hyperstimulation in ART. GnRH = gonadotrophin-releasing hormone; IN = intranasal; LH = luteinizing hormone; NA = not assessed; SC = subcutaneous.

GnRH Agonists in Assisted Conception The use of GnRH agonists results in lower cancellation rates as a result of prevention of the premature LH surge, improved follicular recruitment with a larger number of oocytes recovered and improvement in routine organization of assisted reproduction (Zorn et al., 1987). Many treatment schedules with the use of GnRH agonists in ART, particularly in ovarian hyperstimulation IVF/ICSI treatments, have been designed. The duration and initiation of agonist administration before the start of the actual ovarian stimulation varies widely. Initiation of the agonist treatment may either be in the early follicular or midluteal phase of the preceding cycle. This cycle may be a spontaneous cycle or an artifi cial one under the infl uence of the administration of progestogens and/or oestrogens. In the long or desensitization protocol the agonist starts in the follicular phase or in the early, mid- or late-luteal phase in the preceding cycle until hCG administration. Stimulation with gonadotrophins is started when pituitary and ovarian suppression has been achieved. A meta-analysis comparing ultra-short, short and long IVF protocols showed a higher number of oocytes retrieved and higher pregnancy rates with the long protocol, although more ampoules of gonadotrophins were needed (Daya et al., 2000). In terms of gonadotrophin suppression and number of retrieved oocytes, the midluteal phase of the preceding cycle is the optimal moment for the initiation of the GnRH agonist, compared with the follicular, early or late luteal phase (Pellicer et al., 1989, Kondaveeti-Gordon et al., 1996, San Roman et al., 1992). A major advantage of the long protocol of GnRH agonist administration is the contribution to the planning of the ovum pick-up (OPU), since both the initiation of exogenous gonadotrophins after pituitary

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desensitization and the administration of hCG can be delayed without any detrimental effect on IVF outcome (Chang et al.., 1993, Dimitry et al., 1991). The short or fl are-up protocol combines GnRH agonist therapy, started at cycle day 2, with gonadotrophins initiated 1 day later. The immediate stimulatory action of the GnRH agonist serves as the initial stimulus for follicular recruitment. Adequate follicular maturation is on average reached in 12 days, which should allow enough time for suffi cient pituitary desensitization in order to prevent any premature LH surges (Smitz et al., 1990). The use of a long protocol in poor responders has been found to result in reduced ovarian responses to hormonal stimulation (Muasher, 1993). The short GnRH agonist protocol has been proposed as a better stimulation protocol for poor responders. The initial stimulatory effect of GnRH agonist on pituitary hormone levels may improve the ovarian response (Padilla et al., 1996). On the other hand, this short protocol might increase gonadotrophins in the early phase, which induces enhanced ovarian androgen release and that is associated with declined oocyte quality and reduced ongoing pregnancy rates compared with the long protocol (Loumaye et al., 1989). Nevertheless, experience to date shows that the short protocol has an important role in the treatment of poor responders (Fasouliotis et al., 2000). In the ultra-short protocol, the agonist is given during a period of 3 days in the early follicular phase. At the second day of agonist administration stimulation with gonadotrophin is started (Acharya et al., 1992).In the ‘long-short’, ‘early cessation’ or ‘discontinuation-long’ protocol, several investigators have tried to shorten the duration of GnRH agonist administration by early cessation. The agonist is started mid-luteal in the preceding cycle and discontinued during, or even before, the FSH treatment is started. Several prospective, randomized, controlled studies have been performed comparing the ‘early cessation’ and the ‘long’ protocols (Fujii et al.,1997, Cedrin-Durnerin et al., 2000, Dirnfeld et al., 1999, Garcia-Velasco et al., 2000). Increased hMG/FSH requirement and cancellation rates were reported after early cessation in 137 normo-ovulating IVF patients (Fujii et al.,1997), but the opposite was found in a study which included 230 normo-ovulating IVF patients (Cedrin-Durnerin et al., 2000), although pregnancy rates were the same in both studies (Fujii et al.,1997, Cedrin-Durnerin et al., 2000). The paradoxical dip in serum LH levels after early cessation, leading to signifi cantly lower oestradiol levels on the day of hCG, may have a deleterious effect on oocyte quality (Fujii et al.,1997, Cedrin-Durnerin et al., 2000). The early discontinuation protocol may improve ovarian responsiveness based on a hypothetical effect on the ovary and was, therefore, tested in poor responders. However, this approach reported no further advantages compared with the long protocol to these patients in terms of pregnancy and implantation rates (Dirnfeld et al., 1999, Garcia-Velasco et al., 2000), although the number of retrieved oocytes were signifi cantly higher and the amount of required gonadotrophins were reduced after early cessation (Garcia-Velasco et al., 2000). In conclusion, the currently available data are in general not in favour of an ‘early cessation’ protocol but it may have some benefi cial effects in poor responders. ‘Coasting’ may be defi ned as a treatment regimen whereby gonadotrophin therapy is discontinued while continuing the GnRH agonist. This delay by a variable number of days in administering hCG injection is to trigger oocyte maturation prior to oocyte retrieval until lower oestradiol levels are attained. It has been suggested that this approach prevents severe OHSS by reducing FSH-stimulated granulosa cell proliferation and consequently reduction of available granulasa cells for luteinization (Sher et al., 1995). This allows continued follicular growth and maturation while reducing the risk of OHSS (Fluker et al., 1999). However, in a recent systematic review it was concluded that there is a lack of randomized, controlled trials

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comparing ‘coasting’ with no ‘coasting’ (D’Angelo and Amso, 2002). Only one prospective, comparative trial in 60 IVF patients showed a similar incidence of moderate and severe OHSS whether ‘coasting’ was applied or not (Egbase et al., 1999).GnRH agonists are not frequently used during intrauterine insemination (IUI) since a longer pre-treatment period is required and the risk of multiple pregnancies is increased because of increased follicular growth. Two prospective, randomized studies on this subject reported similar effects on cycle fecundity or live-birth rates between patients treated with hMG alone or in conjunction with GnRH agonists, although hMG requirement was increased in the agonist group (Sengoku et al., 1994, Nuojua-Huttunen et al., 1997).

GnRH Agonists in Anovulatory Infertility In the early 1980s, the fi rst studies using GnRH agonist in combination with hMG for anovulatory patients with PCOS showed optimistic results, seven of eight patients conceived (Fleming et al., 1985). Nevertheless, subsequent prospective, randomized studies indicated that GnRH agonists provide no benefi t over hMG therapy alone and did not reduce the tendency of the polycystic ovary to multifollicular development, cyst formation or OHSS (Scheele et al., 1993). It has even been shown that the addition of GnRH agonists in low-dose, step-up protocols may cause multiple follicular growth, probably by interfering in the integrity of the hypothalamic-pituitary-ovarian axis (van der Meer et al., 1996). GnRH agonists have also been employed to enhance responsiveness of patients with PCOS to other ovulation-induction drugs such as pulsatile GnRH. However, rates of ovulation were disappointing and miscarriage rates were as high as 45% (Filicori et al., 1994, Shoham et al., 1990). In a systematic review, it was assessed that an ovulation induction protocol with a GnRH agonist as an adjunct to FSH/hMG compared with FSH/hMG alone did not improve pregnancy and OHSS rates, and should therefore not be recommended as a standard treatment for this patient group (Hughes et al., 2000).

GnRH Agonists to Trigger Ovulation or Resumption of Meiotic Division In accordance to the assumed midcyle endogenous GnRH rise in the natural cycle (Caraty et al., 1995), GnRH analogues may be used as an alternative way for hCG to trigger the endogenous LH and FSH surges, and subsequent fi nal maturation of the oocytes and ovulation (Emperaire and Ruffi e, 1991, Lanzone et al., 1994). Since hCG is believed to contribute to the occurrence of the OHSS, because of its prolonged circulating half-life compared with native LH, this strategy seems to be an attractive alternative to prevent OHSS. In the early 1990s, it was shown that single-dose GnRH agonists administrated in IVF patients undergoing COH were able to induce an endogenous rise in both LH and FSH levels, leading to follicular maturation and pregnancy (Gonen et al., 1990, Itskovitz et al., 1991). Mean serum LH and FSH levels rose over 4-12 hours and were elevated for 24-34 hours after administration of a GnRH agonist compared with approximately 6 days of elevated LH levels after hCG 5000IU administration. The capacity of a single administration of a GnRH analogue to trigger follicular rupture in anovulatory women or in preparation for IUI has been well established and seems to induce lower OHSS rates with comparable or even improved results, despite short luteal phases, than with hCG cycles (Emperaire and Ruffi e, 1991, Lanzone et al., 1994, Romeu et al., 1997). Interest for this approach was lost during the 1990s because GnRH agonists were introduced in ovarian hyperstimulation protocols to prevent premature luteinization by pituitary desensitization, precluding stimulation of the endogenous LH surge. However, interest returned after the recent introduction of GnRH antagonist protocols in which

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pituitary responsiveness is preserved.This new concept of triggering fi nal oocyte maturation after GnRH antagonist treatment by a single GnRH agonist injection was successfully tested in patients undergoing COH for IUI (Olivennes et al., 1996a) and in high responders for IVF (Itskovitz-Eldor et al., 2000). None of these patients developed OHSS. The effi cacy and success of this new treatment regimen was established in a prospective, multicentre trial in which 47 patients were randomized to receive either triptorelin 0.2mg, leuprorelin 0.5mg or hCG 10 000IU (Fauser et al., 2002). The LH surges peaked at 4 hours after agonist administration and returned to baseline after 24 hours, the luteal phase steroid levels were also closer to the physiological range compared with the hCG groups. In terms of triggering the fi nal stages of oocyte maturation, similar outcomes were observed in all groups as demonstrated by the similar fertilization rates and oocyte quality (Fauser et al., 2002). A prospective, randomized study in 105 stimulated IUI cycles with a GnRH antagonist in patients with clomifene-resistant PCOS, showed statistically signifi cantly more clinical pregnancies after ovulation triggering by a GnRH agonist than with hCG (28.2% vs 17% per completed cycle, respectively) (Egbase et al., 2002). Thus, this new approach of ovulation triggering seems to be an attractive alternative to hCG in ART if administered in GnRH antagonist-treated cycles, with lower OHSS and similar or improved IVF outcome.

GnRH Antagonists in Clinical Practice GnRH Antagonists in Assisted Conception GnRH antagonists have been recently introduced in clinical practice for COH in ART cycles to prevent premature luteinization. So far, cetrorelix (Cetrotide®) and ganirelix (Orgulatran® or Antagon®) are the two registered antagonists for this indication. Because of the acute gonadotrophin suppressive activity of these agents, GnRH antagonists may be administered at any time during the follicular phase of a treatment cycle to prevent a premature LH surge. Several studies have been performed to determine the minimally effective or optimal dose, and treatment schedule in IVF patients (Albano et al., 1997, Olivennes et al., 1998, Ganirelix Dose-fi nding Study Group, 1998). Two general approaches have emerged. In the single dose protocol, one injection of cetrorelix 3mg (ganirelix is not provided in a depot formulation) is administered in the late follicular phase on stimulation day 8 or 9 is suffi cient to prevent a LH surge, although in slow responders a repeat injection may be needed (Olivennes et al., 1998). In the multiple dose antagonist protocol cetrorelix or ganirelix 0.25mg is given daily from the sixth or seventh day of gonadotrophin stimulation onward, including the day of hCG administration (Albano et al., 1997, Ganirelix Dose-fi nding Study Group, 1998, Felberbaum et al., 2000). Four large industry-sponsored prospective, multicentre clinical trials comparing daily antagonist injections with long agonist protocols in IVF patients undergoing COH were reported (Albano et al., 2000, Borm and Mannaerts, 2000, European and Middle East Orgulatran Study Group, 2001, Fluker et al., 2001). With an antagonist, the duration of gonadotrophin treatment is shortened by 1-2 days and slightly fewer follicles are seen at the time of hCG injection compared with an agonist. Therefore, the number of recovered oocytes tends to be lower. A likely explanation is that long agonist protocols extend the duration of the ‘FSH window’ by suppressing the intercycle FSH rise. In these studies, no signifi cant difference was found with respect to percentages of metaphase II oocytes, fertilization rates and number of good quality embryos. Pregnancy rates were high in both groups in all four studies but in every one the rate was lower in the antagonist group.A meta-analysis of fi ve large randomized trials shows an overall signifi cantly lower rate of

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pregnancy of 5% (OR, 0.75, 95% CI 0.62-0.97). This meta-analysis also included the study that compared a single-dose analogue regimen with different gonadotrophin starting doses as an additional variable (Al-Inany and Aboulghar, 2002). It has been hypothesized that the lower pregnancy rates may be a consequence of the currently advised treatment regimen. It has also been suggested that the larger numbers of oocytes and embryos with agonists allow better selection, although the numbers of good quality embryos do not seem to be different. The antagonist was started on a fi xed day of stimulation (day 6) in these studies, which may be too early for some patients, and may lead to a diminished number and quality of oocytes (Albano et al., 2000, Borm and Mannaerts, 2000, European and Middle East Orgulatran Study Group, 2001, Fluker et al., 2001, Al-Inany and Aboulghar, 2002). More fl exible alternative regimens are currently under development. Prospective studies comparing the fi xed antagonist protocol with a fl exible protocol, in which the daily antagonist administration is started when at least one follicle has reached a size of 14mm, showed no differences in IVF outcome except that the dose of GnRH antagonist was reduced in the fl exible protocol (Crespo et al., 2002, Mansour et al., 2002). Moreover, one study even reported more oocytes per OPU despite lower FSH requirements in the fl exible protocol than with the fi xed protocol (Ludwig et al., 2002). Concerning OHSS, the results of the four comparative studies are inconclusive. Although three studies show decreased OHSS incidence with the antagonist (Albano et al., 2000, Borm and Mannaerts, 2000, European and Middle East Orgulatran Study Group, 2001), one study showed a higher incidence of OHSS (Fluker et al., 2001). The meta-analyses, which compared the fi ve large comparative studies, showed signifi cantly lower OHSS after GnRH antagonists than after GnRH agonists (RR 0.36, 95% CI 0.16-0.80) (Al-Inany and Aboulghar, 2002). Recently, in a prospective, randomized study, higher vascular endothelial growth factor mRNA and protein levels were expressed in IVF patients undergoing COH treated with GnRH agonists versus antagonists (Raga et al., 2002). This provides a hypothetical biological explanation for the clinical experiences of the lower observed OHSS rates in antagonist cycles. Retrospective comparison of pregnancy rates after transfer of frozen-thawed two-pronucleate oocytes obtained either in long agonistic protocols or in antagonist protocols, showed no differences in implantation, pregnancy and abortion rates (Seelig et al., 2002, Nikolettos et al., 2000). On a theoretical basis, the use of GnRH antagonists may be preferred above GnRH agonists in poor responders. First, GnRH antagonists are given in the late follicular phase of COH and are not involved in the early period of folliculogenesis, which may be critical for poor responders with a limited cohort of follicles. Secondly, FSH levels are less ‘over-suppressed’ by GnRH antagonists than with GnRH agonists in long protocols, probably leading to less exogenous gonadotrophin being required. Finally, some investigators assume that GnRH agonists may have a direct deleterious effect on the ovary, which could be especially important for patients who are poor responders (Craft et al., 1999). In a small prospective, comparative study, Akman et al. evaluated the effect of a GnRH antagonist in IVF cycles versus IVF cycles without any GnRH analogue addition in 40 poor responders (Akman et al., 2000). There were no statistical differences with regard to cancellation rate, IVF outcome and FSH requirement. In an additional small, prospective, randomized trial comparing an agonistic fl are-up protocol and antagonistic multiple-dose protocol in 48 poor responders in IVF, no differences could be found either (Akman et al., 2001). A small, matched, case-control study indicated that slightly fewer ampoules of FSH were needed in antagonist- versus agonist-treated poor responders in IVF (Nikolettos et al., 2001). To establish

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these results and to advocate a particular treatment regimen in poor responders, further randomized studies with larger sample sizes are required. Accurate prospective trials comparing GnRH antagonists with agonists in patients with PCOS, undergoing IVF, have not yet been published.On theoretical grounds, prevention of excess LH secretion in gonadotrophin-stimulated IUI might be benefi cial. One inconclusive study has been published with regard to the use of a GnRH antagonist in IUI (Ragni et al., 2001). This indication obviously requires further study before general introduction.

GnRH Antagonist in Ovulation Induction Triggering ovulation with GnRH agonists in GnRH antagonist-treated cycles is discussed previously. The coadministration of pulsatile GnRH to induce ovulation in GnRH antagonist-treated cycles provides a new treatment option but clinical data on its use are scanty. Studies in monkeys showed that blockade of the endogenous GnRH with an antagonist can be reversed by exogenously administered pulsatile GnRH, which restored gonadotrophin secretion and ovulation (Gordon et al., 1992). In a very small pilot study, the same combination in PCOS patients normalised gonadotrophin secretion but failed to induce ovulation (Dubourdieu et al., 1993). In a small, noncontrolled study in 20 patients with PCO, it was indicated that daily ganirelix 0.25mg coadministered with rFSH 75 or 150IU might be an effective protocol for ovulation induction in these patients but prospective, randomized trials are warranted to confi rm these preliminary fi ndings (Elkind-Hirsch et al., 2003).

Safety Aspects of GnRH Analogues Although GnRH analogues have acquired an important place in ART, caution remains with regard to the use of these drugs. The safety aspects of GnRH analogues, related to direct effects on extrapituitary structures such as the ovary, the oocyte, granulosa cells and the embryo, are a matter of debate, since the discovery of extrapituitary GnRH receptors in humans (Hernandez, 2000, Janssens et al., 2000a). GnRH and GnRH receptors are expressed in developing mouse embryos at the mRNA and protein levels. The incubation of the embryos with a GnRH agonist enhanced the preimplantation embryonic development in a dose-dependent way, whereas a GnRH antagonist could completely block this development (Raga et al., 1999). Recently, it has been demonstrated that GnRH mRNA and GnRH proteins are produced in the human fallopian tube during the luteal phase of the menstrual cycle at the same time spermatozoa and oocytes are deposited in the oviduct to promote their union and nurture the resultant zygotes and early embryos (Casan et al., 2000). These results underline the high priority for the safety aspects of GnRH analogues and stress the need for prospective, comparative studies concerning in vivo effects of the GnRH analogues on fertilization, early embryonic development and implantation in humans, before the agonist should be abandoned for antagonists in general practice (Blumenfeld, 2001). On the basis of the inverse association between implantation rates and ganirelix dose in the higher dosage groups in the large dose-fi nding study (Ganirelix Dose-Finding Study Group, 1998), the possibility of direct effects of antagonists on human embryos is of concern. However, this adverse effect was not observed on the freeze-thaw embryos of these cycles, suggesting that there is no direct negative effect of the GnRH antagonist on the quality of oocytes and embryos, but perhaps on the endometrium (Kol et al., 1999). In accordance with the reported association between low LH levels (<0.5 IU/L) and lower ongoing pregnancy rates in IVF cycles (Westergaard et al., 2000), as discussed earlier, LH levels are thought to have a role in the lower pregnancy rates in GnRH antagonist-treated cycles,

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since these cycles often lead to extensive suppression of endogenous LH activity if combined with rFSH administration. However, in a large retrospective study in which patients with the latter regimen were divided in two groups based on the LH levels on the day of hCG administration, with a cut-off level of 0.5 IU/L, no differences were found in pregnancy rates and outcome (Merviel et al., 2002). Although, it has been recently proposed that it might be more appropriate to look at an LH ‘window’ instead of a single LH cut-off level, since there seems to be a ‘threshold’ LH level, below which oestradiol production is not adequate, and a ‘ceiling’ level, above which LH may be detrimental to follicular development (Shoham, 2002). There does not appear to be an increased risk of birth defects or pregnancy wastage in human pregnancies exposed to daily low-dose GnRH agonist therapy in the fi rst weeks of gestation. Obviously, there are no controlled trials on this subject. Although several authors claim normal outcome of pregnancy after inadvertent administration of a GnRH agonist during early pregnancy, Lahat et al. reported a high incidence of attention defi cit hyperactivity disorder in long-term follow-up of children inadvertently exposed to GnRH agonists early in pregnancy (Lahat et al., 1999). The follow-up data on pregnancy, birth and neonatal outcome of 227 children born after IVF or ICSI cycles in which cetrorelix was used, showed no abnormal results compared with outcome after commonly used long agonist protocols (Ludwig et al., 2001). The safety of the novel antagonistic protocol was established on the hand of the similar follow-up results, collected during clinical development trials, of 340 ongoing pregnancies after ganirelix and 134 after a long agonist protocol (Boerrigter et al., 2002).

New Developments With GnRH Analogues The availability of GnRH antagonists with good clinical safety on the commercial market provides an important alternative for the extensively used GnRH agonists for a variety of indications. As experience with these drugs increases, new protocols can be developed. Additional prospective, comparative studies should be performed to establish the optimal treatment regimen for the different indications. The application of the different regimens should be fi ne-tuned and adapted to the individual needs and properties of specifi c patient groups. For example, the minimal stimulation protocols which regained interest after the introduction of the GnRH antagonists might be specifi cally attractive for high responders or in patients in which the ovarian reserve is not thought to be diminished (e.g. if IVF is indicated because of tubal dysfunction). The same can be said for the application of ‘natural’ cycle IVF with late follicular phase GnRH antagonist administration, which is a convenient and inexpensive procedure (Pelinck et al., 2002, Rongieres-Bertrand et al., 1999). It is important to realise that the use of GnRH analogues for different indications or in different treatment schedules requires adjusted dose-fi nding studies. The pituitary responsiveness depends on the timing of administered GnRH analogues, but also on the coadministered medication. In soft-stimulation IVF cycles with clomifene, 21.5% premature LH surges occurred despite daily cetrorelix 0.25mg administration (Engel et al., 2002) and elevated LH levels during the follicular and luteal phase (Tavaniotou et al., 2002) were observed, indicating that the required antagonist dose in this protocol is above 0.25mg/day. Alternate-day administration of 0.25mg antagonist in IVF/ICSI patients undergoing COH resulted in 5.8% premature ovulations, which seems to be an acceptable alternative (Al-Habib et al., 2002).To facilitate planning the starting moment of an antagonist cycle, independent of the menstrual period, oral contraceptive (OC) pre-treatment has been evaluated in a few small

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prospective studies, in which the antagonist was started 3 days after OC withdrawal. OC pre-treatment induced a longer stimulation period with more oocytes, lower LH levels and lower pregnancy rates than the antagonist cycles (van Loenen et al., 2001). The cause for longer simulation period and more oocytes recovered might be the oestrogen-induced suppression of the intercycle FSH rise and, subsequently, the prevention of some sensitive follicles being selected, resulting in a more synchronised and larger cohort, allowing an increased ‘FSH window’ (Fanchin et al., 2003). Therefore, this strategy is hypothesized to be an advantage for poor responders. An uncontrolled, prospective study showed a mean number of six oocytes in 68 poor responders and a high clinical pregnancy rate (31% per cycle) (Stanovici et al., 2002). However, large prospective, randomized controlled studies are warranted to draw solid conclusions on this topic. New antagonists are under development. Abarelix and degarelix are long-acting formulations which have been recently developed to be used for the management of sex steroid-dependent pathologies, in which the avoidance of the initial fl are effect might be preferred over the currently used GnRH agonists (Tomera et al., 2001, Broqua et al., 2002). Expected long-term developments are nonpeptide orally active GnRH analogues (Ashton et al., 1999). The debate on the possible cause of the lower implantation rates after GnRH antagonist cycles compared with GnRH agonist cycles triggered a new fi eld of research which focuses on the possible direct effects of GnRH analogues on fertilization, early embryonic development and endometrium. The existence of the recently discovered additional GnRH receptor (type II) in mammals (Millar et al., 2001) could have a role in the confusing and inconclusive results of many studies on direct extrapituitary effects of GnRH agonists and antagonists as reviewed by Janssens et al. (Janssens et al., 2000a), since most studies assumed to deal with only one GnRH receptor. The type II receptor is equal to the chicken GnRH receptor, displays different affi nity for the different analogues, and has different effects on gonadotrophin release after binding by a GnRH agonist or antagonist (Ashton et al., 1999). The effect on gonadotrophin release of GnRH antagonists after binding to the type I receptor or the type II receptor is also different (Kang et al., 2000). Additional studies are under development to gain insight in the functional properties of the GnRH receptors and their impact on GnRH analogue binding (Hovelmann et al., 2002).The different effect of analogues on the different GnRH receptors and their tissue specifi c distribution should be clarifi ed before rational choices for clinical application of specifi c analogues in specifi c indications can be made, and to gain insight in possible extrapituitary side effects.

CONCLUSION

To broaden the pharmaceutical armoury, recent efforts have been directed towards the development of novel GnRH antagonists, FSH and LH preparations with optimal pharmacokinetic, pharmacodynamic and safety profi les in addition to the available GnRH agonists and urinary gonadotrophins. Alternative strategies with fewer adverse effects and higher benefi t to cost ratios are under development. Prospective studies investigating possible direct effects of GnRH analogues, optimal dose-fi nding studies and treatment regimens under different conditions, in different types of patients, with or without pharmacological coadministration and for different indications, should be performed to optimise the effi cacy and tailor treatment strategies to individual needs.

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Acknowledgements No sources of funding were used to assist in the preparation of this manuscript. The authors have no confl icts of interest that are directly relevant to the content of this manuscript.

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6.

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Dose-fi ndings study of dailyGnRH antagonist for the preventionof premature LH surges in IVF/ICSI patients: antide and hormone levels

JAF Huirne, AC van Loenen, R Schats, J McDonnell,

PG Hompes, J Schoemaker, R Homburg, CB Lambalk

Human Reproduction 2004: 19; 2206-2215

4

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Antide dose-fi nding study: antide and hormonal levels

78

ABSTRACT

BackgroundThe aim of this study was to defi ne the minimal effective dose of antide (Iturelix) to prevent premature luteinizing hormone (LH) surges in in vitro fertilization (IVF) patients.

Materials and methodsIn a prospective, single centre study, 144 IVF/ICSI patients were stimulated with rhFSH from cycle day 2 and from cycle day 6 onwards, cotreated with daily 2 mg/2 ml (n=30), 1 mg/ml (n=30), 0.5 mg/ml (n=31), 0.5 mg/0.5 ml (n=23) and 0.25 mg/ml (n=30) GnRH antagonist (antide). Serum samples were taken three times daily during antide administration to assess antide

and hormone levels. The minimal effective dose was defi ned as the lowest dose group with <2 LH surges (LH >12.4 IU/l and progesterone >2 ng/ml). ResultsSerum antide levels, mean LH and E2 levels per day and their area under the curves were dose-related to antide. The bioavailability of antide almost doubled after dilution in larger volumes. Pre-injection LH levels gradually increased during GnRH antagonist treatment. LH surges occurred in the lowest dose groups 0.5 mg/ml (3.2%), 0.5 mg/0.5 ml (6.7%) and 0.25 mg/ml (13.3%). Hence, 0.5 mg/ml is considered to be the minimal effective dose. Antide was overall well tolerated and safe. Conclusions 0.5 mg/ml antide is the minimal effective dose to prevent an untimely LH surge in IVF patients stimulated with rhFSH.

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INTRODUCTION

GnRH agonists have been used in IVF cycles for many years to prevent premature LH surges. Placebo-controlled studies of GnRH agonists reveal that LH surges occur in ∼20% of IVF patients, leading to cancellation of the IVF cycles (Edwards et al., 1996; Janssens et al., 2000). The long protocol became the standard in most centers and proved to be benefi cial for the quality and number of retrieved oocytes, resulting in more embryos (Daya, 2000). In general, more embryos allow better selection so that the outcome, in terms of pregnancy rates, is improved (Templeton and Morris, 1998). It takes time to induce a state of desensitization after GnRH agonist administration. Unlike the agonists, GnRH antagonists cause an immediate and rapid, reversible suppression of gonadotrophin secretion (Huirne and Lambalk, 2001). These compounds can be administered when premature luteinization during IVF stimulation is imminent, reducing the number of GnRH antagonist injections required. Antagonist therapy has proved to be a very convenient alternative to the long agonist strategy. The effi cacy of this strategy, using single or multiple dosing regimen, has been demonstrated in several studies with ganirelix and cetrorelix (Ganirelix dose-fi nding Study Group, 1998; Albano et al., 1997; Olivennes et al., 1998). Both compounds belong to the so called third generation GnRH antagonists which exhibit low histamine releasing properties and can therefore be safely administered with low risks for allergic reactions (Bajusz et al., 1988; Rivier et al., 1992). Another compound belonging to this generation of GnRH antagonists is antide (Iturelix, Serono, Geneva, Switzerland) (Ljungqvist et al., 1988). Low histamine releasing properties in comparison to Ganirelix or Cetrorelix were reported, even lower than native GnRH (Bajusz et al., 1988; Ljungqvist et al., 1988; Nestor et al., 1992). The current study was designed to select the minimal effective dose of antide for the prevention of premature LH surges during ovarian stimulation when administered daily by subcutaneous (s.c.) injections. Effi cacy was assessed in terms of hormone suppression, IVF outcome and safety aspects. This report will focus on the hormone and antide levels in the

various dose groups. The effect of various induced LH levels on IVF outcome will be reported elsewhere (Huirne et al., 2005).

MATERIALS AND METHODS

PatientsAll patients scheduled for IVF or ICSI treatment in our department during a period of 7 months were assessed for eligibility. A total of 144 patients participated in the study. All patients were aged between 21 and 39 years, had spontaneous regular menstrual cycles between 25 and 35 days, two ovaries, a normal uterine cavity, a body mass index ≤30 and had at least two spontaneous menstruations after the last clomiphene citrate or gonadotrophin

treatment. Women with elevated hormone levels (FSH ≥10 IU/l or LH ≥8 IU/l or prolactin levels (≥800 mIU/l) on cycle day 2 or 3 were excluded from participation. Patients with polycystic ovaries syndrome (defi ned as oligomenorrhoe and elevated LH levels or signs of hyperandrogenism) were also excluded. Also excluded were patients with abnormal hematological or biochemical parameters, patients with any previous ART cycle with fewer

than 3 oocytes, known allergy or hypersensitivity to human gonadotrophin preparations or GnRH analogues. The protocol was approved by the Committee on Ethics of Research

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80

involving Human Subjects of the Vrije Universiteit medical center (VUmc), Amsterdam, The Netherlands. All participants signed informed consent.

Study designA phase II, single center study, conducted in two phases: a double-blind phase with two parallel treatment groups was followed by an open phase (see Figure 1). In the double-blind phase 60 patients were randomized to two different treatment groups (A: 2 mg/2 ml, B: 1 mg/ml). To improve patients’ convenience, the 2 mg in group A was given as two injections of 1 mg/ml antide, one injection in the morning and one in the evening, since we expected that one injection with a volume of 2 ml would be too painful. Patients in group B received placebo in the morning and 1 mg/ml antide in the evening. Since none of the two groups turned out to be a failure group (i.e.with ≥2 LH surges) we decided to add an open phase in which three additional treatment groups with lower antide dosages were studied (0.5 mg/0.5 ml, 0.5 mg/ml, 0.25 mg/ml), administered once daily. The additional arms were added in a consecutive order and patients were enrolled in a chronological way. New evidence which became available after the start of this study suggested that the bio-availability of antide increases after dilution in larger volumes of glucose 5% (Data on fi le, Serono, Geneva). Therefore in two groups (0.5 mg/ml and 0.25 mg/ml) antide was diluted in larger volumes of glucose 5% solution; 0.5 mg and 0.25 mg in 1 ml, respectively. This means that in two arms 0.5 mg antide was administered but in group C it was diluted in 1.0 ml of glucose 5% solution and in group D it was diluted in 0.5 ml of glucose 5% solution.

Each group was intended to contain 30 patients unless more than 1 LH surge occurred, which according to the protocol lead to the discontinuation of that particular dose group and was considered to be a failure dose. More than 1 LH surge per 30 patients was considered to be unacceptable for clinical use in IVF patients.

MaskingTreatment packs for the double-blind phase of the study were prepared according to the randomization list by Serono International (Geneva, Switzerland). Patient packs, containing antide/placebo or antide/antide vials, were labeled with unique study identifi cation numbers, provided by Serono International (Geneva, Switzerland); placebo vials contained a sterile isotonic aqueous solution. When eligible, patients were enrolled to the study by one of the two responsible trained researchers and received a unique study number in a chronological order at the start of the fi rst stimulation day. The code was not known to the executors of

the study. Assignment to group A or B was therefore double blind, assignment to group C, D or E was dependent on the chronological entry of the study.

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Figure 1. Flow diagram, showing the fl ow of the participants through each stage of the trial. RFSh, recombinant FSH; antide, GnRH antagonist; rhCG, recombinant hCG; OPU, ovum pick-up; ET, embryo transfer. All patients included had follow-up of serum samples throughout the entire treatment period (three times daily) and during ET, no serum values were missing.

Treatment protocolThe treatment protocol is illustrated in Figure 2. On day 2 or 3 of a spontaneous menstruation, rhFSH (Gonal-F®, Serono, Aubonne, Switzerland) was given as a single daily s.c. injection. The starting dose varied between 150–300 IU, depending on previous ovarian response, but was fi xed for the fi rst 5 days. After this period, depending on ovarian response as assessed by daily ultrasound, the rhFSH dose could be adjusted. All antide, placebo and rhFSH injections from stimulation day 6 (S6) onwards, were given subcutaneously, by a trained research

professional. From stimulation day 6 onward, up to and including the day of recombinant human chronic gonadotrophin (rhCG; Ovitrelle®, Serono) administration, daily antide was given. Recombinant hCG was administered as soon as one follicle ≥18 and two follicles were ≥16 mm. Thirty-six hours after rhCG administration, ovum pick-up (OPU) was performed transvaginally and ultrasound guided. The OPU was followed by IVF with or without ICSI, a maximum of three embryos were replaced 2–3 days thereafter. Luteal support (200 mg progesterone vaginally, three times daily) was started 1 day after OPU until the third week of pregnancy or a negative pregnancy test.

Eligible patients asked to participate (n=153)

Exclusion just before allocation (n=9) • Abnormal uterine cavity by ultrasound (n=1) • BMI > 30 kg/l2 at moment of inclusion (n=5) • Abnormal biochemical parameters (n=1) • Refused participation (n=2)

Randomized (n=60) Additional arm is added in chronological order (n=84)

GroupA (2mg/2ml)

(n=30)

GroupB(1mg/ml)

(n=30)

GroupC(0.5mg/ml)

(n=31)

GroupD(0.5mg/0.5ml)

(n=23)

GroupE(0.25mg/ml)

(n=30)

had rFSH (n=30) had Antide (n=30)

OPU (n=30) ≥ 1oocyte (n=30) ET (n=30)

had rFSH(n=30) had Antide (n=30)

OPU (n=30) ≥ 1oocyte (n=30) ET (n=29)

had rFSH(n=31) had Antide (n=31)

OPU (n=31) ≥ 1oocyte (n=31) ET (n=30)

had rFSH(n=23) had Antide (n=23)

OPU (n=21) ≥ 1oocyte (n=21) ET (n=20)

had rFSH(n=30) had Antide (n=30)

OPU (n=30) ≥ 1oocyte (n=30) ET (n=28)

Analysed (n=30) Excluded (n=0)

Analysed (n=30) Excluded (n=0)

Analysed (n=31) Excluded (n=0)

Analysed (n=23) Excluded (n=0)

Analysed (n=30) Excluded (n=0)

Number of patients assessed for eligibility: not recorded

had rhCG (n=30) had rhCG (n=30) had rhCG (n=31) had rhCG (n=22) had rhCG (n=30)

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82

Figure 2. Schematic overview of the treatment schedule. Schematic overview of the treatment regimen with a GnRH antagonist (antide) in patients undergoing ovarian hyperstimulation with rhFSH (Gonal-f®). S1, stimulation day 1; S6, stimulation day 6; OPU, ovum pick-up; ET, embryo transfer

AssessmentsOne to three months before randomization, serum samples were taken to assess haematology (haemoglobin, haematocrite, white cell count, red cell count and platelets), biochemistry (sodium, potassium, creatinine, total bilirubin, calcium, total cholesterol,

alkaline phosphatase, SGPT, LDH, total protein) and hormone levels (FSH, LH, oestradiol, progesterone and prolactin), taken on cycle day 2 or 3. Urine was checked for glucose, ketones, haematuria and proteinuria. On S1 (stimulation day 1), before any study drug was administered, a blood sample was taken to perform a pregnancy test and to assess FSH, LH, oestradiol (E2) and progesterone (P4) levels and a transvaginal ultrasound was

performed to measure follicular activity, endometrial thickness and to exclude the presence of cysts. During antide administration, three samples per day were taken (in the morning before any injection, in the evening prior to Gonal-f or antide injection,

and 20–84 min later) to assess serum levels (FSH, LH, E2, P4 and antide). The potential variation in timing of the evening post-injection bloodsampling was intended to allow pharmacokinetic and pharmcodynamic modeling. The mean sample time was 34.6 (SD

3.9) min after antide injection (range of the mean per patient varied from 30 to 53 min). The transvaginal ultrasound was performed daily to assess follicular development and endometrial thickness. On the day of embryo transfer (ET), serum samples were taken

to measure the antide level, and 7–11 days after OPU to measure the levels of progesterone and antide. The prestudy haematological and biochemical parameters were reassessed after

the treatment cycle to evaluate potential changes. Finally, 23–25 days after ovum pick-up, serum hCG levels were measured. If positive, a vaginal ultrasound was performed 35 and 42 days following rhCG administration, to record the number of fetal sacs and fetal heart activity. Ultrasound was repeated at a gestational age of 12 weeks. The pregnancy results will be reported elsewhere (Huirne et al., 2005).

Local tolerance after the different injections was assessed 60 min and 12 h after each antide injection. Pain, itching, tenderness, redness, swelling and bruising were recorded on

If ≥ 1 follicle ≥1 mm 2 follicles ≥ 1 mm

rhFSH

GnRH antagonist

day of rhFSH

day 2 or 3 of menses

( S1)

rhCG OPU ET

Cycle day 1

( S ) 6

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83

a 4 point scale (none, mild, moderate and severe) of a diary card. All other side effects were reported daily on this diary card as well.

Serum assessmentBlood samples were processed to serum immediately after collection and stored at –20°C. Routine haematology, biochemistry and urine assessment were performed by the local laboratory (The Central Laboratory of the VUmc) using commercially available

immunometric assays. To detect LH surges, all morning samples were assessed daily by this local laboratory to measure the LH levels, using immunometric assay kits from Amerlite (Amerlite, Amersham, Bucks, UK). An LH surge was defi ned as LH >10 IU/l and P4 >2 ng/ml using this assay. Halfway through the study we were forced to change the assay, since Amerlite assays were not available anymore. We decided to use Delphia (Dephia, Wallac, Finland) assays. During the transition period of the assays, we assessed LH levels using both assays in 89 patients. Excellent correlation was observed between the two assays (R=0.98). A regression analyses revealed that the coeffi cient of LH was 1.24 using Delphia assay in comparison to Amerlite assay, thus the LH cut-off level 10 IU/l assessed by Amerlite was equivalent to 12.4 IU/l if assessed by Delphia assay.

For defi nitive analyses of all hormone and antide levels, as presented in this report, all serum samples (taken three times daily) were assessed retrospectively by LCG Bioscience Services LTD. E2 was measured using Sorin Radioimmunoassay, P4 using DPC Coat-a-count, RIA solid phase coated tube separation, FSH and LH using Serono MAIAclone IRMA. The lower limit of quantifi cation for LH was 1 IU/l. For the retrospective analyses we defi ned an LH surge as LH >12.4 IU/l and P4 >2 ng/ml in one or more samples, taking all samples (three times daily) into account from S6 until hCG administration day, equivalent to the cut-off

levels using the Delphia assays. The retrospective centralized analysis of serum antide levels were performed by Woods Assay (RIA), all samples were analyzed in triplicate, 1 μg/l is the Limit of Quantifi cation.

Outcome measuresThe primary endpoint of this study was to determine the minimal effective dose, defi ned as the lowest dose group in which fewer than two LH surges occurred. Secondary endpoints were drug requirements, serum hormone and antide levels and safety aspects.

Statistical analysisThe treatment groups were compared depending on the nature of the variables, [i.e the analysis of variance (ANOVA) or the analysis of covariance (ANCOVA), Chi-square tests, Fisher’s

exact test or non-parametric ranking methods like Kruskall–Wallis and Mann–Whitney U tests]. Results are reported as mean±SD. Correlations were calculated using Pearson’s correlation coeffi cient. P<0.05 was considered to be statistically signifi cant. Analyses were conducted on an intention to treat base. An overall dose-response test for trend with the treatment groups were performed on all effi cacy data. The statistical hypothesis of no treatment effect was tested against the ordered alternative hypothesis that at least one antide dose is superior to the lowest dose group and that the response is decreased, or at least equal to those of the previous group when decreasing the dose. The statistical methods used to test this hypothesis were directly related to the nature of the variable, i.e. ANOVA with linear contrast, Jonckheere–Terpstra test or a Cochran–Armitage test. To identify different covariates on pharmacokinetic parameters, univariate analysis within NONMEM was used.

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Antide dose-fi nding study: antide and hormonal levels

84

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Total exposure to antide and hormone levels was expressed as area under the curves (AUC) during antagonist administration (i.e. S6-hCG day). For this calculation, the sum of the mean

daily levels of all days during antide treatment were taken.

To calculate the induced change in serum levels in comparison to the basal level on stimulation day 6 (the moment on which the antagonist was started), AUC was calculated after subtraction of the basal level on S6 of all samples, defi ned as AUC–S6.

The sample size was not calculated; we based our groups size on clinically relevant arguments. More than one LH surge per 30 patients was considered to be unacceptable for clinical use in IVF patients. Therefore the number of patients was intended to be 30 per group, unless more than one LH surge occurred.

RESULTS

Patients’ characteristicsBaseline characteristics were similar between the fi ve groups, except for type of fertility disorder (see Table I). Baseline biochemistry and hematology parameters in serum and urine were comparable between the fi ve groups (data not shown), as were baseline FSH, LH and progesterone levels. The baseline oestradiol levels were signifi cantly higher in group C (0.5 mg/ml) and E (0.25 mg/ml) in comparison to groups A (2 mg/2 ml) and B (1 mg/ml).

Number of patients per treatment stageSixty patients (30 in each group) were enrolled in the double blind phase. In the open phase, 31 patients were successfully enrolled in group C (0.5 mg/ml). Group D failed after the inclusion of 23 patients, since two LH surges occurred. In the last group (E: 0.25 mg/ml) a total of four LH surges occurred. At the time of the second LH surge, which according to the protocol leads to the discontinuation of the treatment arm, the last fi ve patients

of this treatment group had already started with antide and were allowed to fi nish the started treatment. Two additional LH surges occurred in the last fi ve patients of this arm. All patients received antide and rhFSH. One patient (group D: 0.5 mg/0.5 ml) did not receive rhCG and had no ovum pickup, because of premature ovulation. Six additional patients did not undergo an embryo transfer because of total fertilization failure (one in 1 mg/ml, one in 0.5 mg/ml, two in 0.5/0.5 mg and two in 0.25 mg/ml group).

Stimulation phaseThe mean duration of antide and rhFSH administration was 5 (range 2–9) and 9 days (range 6–13), respectively. This was comparable between the groups (P=0.89) (see Table II). The mean Gonal-F® starting dose was similar in the various dose groups and varied between 223 and 238 IU (P=0.78). Additionally, the mean dose Gonal-F® per day and the mean total dose of

Gonal-F® were also similar in the various dose groups (P=0.98 and 0.91, respectively). Because of insuffi cient response, the Gonal-F® dose was more often increased in the higher antide

dose groups (P=0.04).

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Antide treatment group (daily dose) P value

A(2mg/2ml)

B(1mg/ml)

C(0.5mg/ml)

D(0.5mg/0.5ml)

E(0.25mg/ml)

No of patients (ITT) 30 30 31 23 30

Duration antide (days) 5.0 (1.7) 4.9 (1.1) 4.8 (1.6) 5.1 (1.4) 5.2 (1.6) 0.89

Total antide dose (mg) 10.0 (3.3) 4.9 (1.1) 2.4 (0.8) 2.5 (0.7) 1.3 (0.4) <0.01*

Duration rhFSH (days) 9.0 (1.7) 8.9 (1.1) 8.8 (1.6) 9.1 (1.4) 9.2 (1.6) 0.89

Gonal-F starting dose (IU) 223 (62) 229 (59) 224 (48) 228 (51) 238 (52) 0.78

Mean rhFSH/day (IU) 246 (79) 246 (74) 232 (48) 242 (51) 243 (59) 0.98

Total rhFSH (IU) 2241(914) 2196(718) 2079(669) 2209(576) 2280(814) 0.91

Patients with rhFSH ↑a 13 (43%) 10 (33%) 7 (23%) 9 (39%) 3 (10%) 0.04*

Table II. Drugs requirements. Values are means (± SD). Analyses were performed on intention to treat base (ITT). For statistical analyses, one-way ANOVA tests were used if normal distribution and equal variance criteria were met, otherwise the Kruskal-Wallis tests was used. aX2-square test was used. ∗P < 0.05.

Serum antide levelsThe daily antide levels, the mean antide levels per day and the total AUC of antide during treatment were different between the various treatment groups (P<0.001) (see Table III and

Figure 3). A two-compartment disposition model with fi rst order absorption and fi rst order elimination best described the pharmacokinetics of antide. The residual error model was a combination of an additive and a proportional part. The absorption half-life was ∼10 min. The calculated bioavailability was different between the treatment groups: the higher the concentration the lower the relative bioavailability data (data not shown). The dilution of 0.5 mg antide in 1.0 ml (group C) versus 0.5 ml (group D) increased the mean antide levels/day from 0.56 to 0.87 μg/l, respectively, (P=0.01) (see Figure 3). The antide serum levels were below the limit of detection on the day of embryo transfer and during the post treatment visit. Using univariate analysis within NONMEM, body weight was found to linearly affect the apparent clearance, the apparent central volume and the absorption rate constant (data not shown). BMI also affected the pharmacokinetic parameters of antide but at a lower degree of signifi cance compared to body weight. After the full covariate selection was performed,

only the effect of body weight on apparent clearance and the apparent central volume remained in the fi nal, model. Mean weight was 66.2 kg (range 45–94 kg), and mean height 167.7 cm (range 150–186), no differences were found between the various dose groups.

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Antide treatment group (daily dose) P value

A(2mg/2ml)

B(1mg/ml)

C(0.5mg/ml)

D(0.5mg/0.5ml)

E(0.25mg/ml)

No of patients (ITT) 30 30 31 23 30

Antide S7 2.5 (0.8) 1.5 (0.7) 0.8 (0.4) 0.5 (0.3) 0.3 (0.1) <0.001*

Antide S8 2.6 (0.6) 1.8 (0.8) 1.0 (0.4) 0.6 (0.3) 0.4 (0.2) <0.001*

Antide hCG day 3.1 (0.8) 2.0 (0.4) 1.0 (0.4) 0.8 (0.4) 0.3 (0.2) <0.001*

Antide-AUCa 30.4(10.6) 23.0 (8.3) 21.6 (7.4) 12.9 (3.4) 16.1 (5.8) <0.001*

Antide-AUC/dayb 6.2 (1.4) 4.6 (0.8) 4.5 (1.0) 2.6 (0.7) 3.1 (0.7) <0.001*

Table III. Serum antide levels. Serum levels (ug/l) are pretreatment morning samples. Values are mean (± SD). Analyses were performed on intention to treat (ITT) base, using both Kruskal Wallis and Jonckheer Terpstra tests for each parameter. S7= stimulation day 7 etc. a Antide-AUC is total sum of the mean daily antide levels (mean of the 3 samples taken daily). bAntide-AUC per day, is calculated out antide-AUC/total days of antide administration. ∗P < 0.001.

Figure 3. Mean antide serum levels per day. Box and wisker plot of the mean antide levels per day in the various treatment groups; A (2 mg/2 ml, B (1 mg/ml), C (0.5 mg/ml), D (0.5 mg/0.5 ml), E (0.25 mg/ml). The median levels were different between all treatment groups, P<0.001 (Mann–Whitney U). The trend for linear association with the dose is highly signifi cant P<0.001 (Jonckheere–Terpstra). Dots above the box plots are the outliers.

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LH levels during stimulationLH levels immediately decreased after the fi rst antide injection. LH values in the various treatment groups are presented in Table IV. The LH levels on S8 and the LH-AUC were different between the antide dose groups (P=0.03 and <0.001, respectively); Kruskall–Wallis and were dose related (both P<0.001). Thus high LH levels were associated with low antide levels. The differences in LH levels induced by antide, expressed by LH-AUC–S6 were also dose-related to the treatment groups (P<0.001).

Antide treatment group (daily dose) P value

A(2mg/2ml)

B(1mg/ml)

C(0.5mg/ml)

D(0.5mg/0.5ml)

E(0.25mg/ml)

No of patients (ITT) 30 30 31 23 30

LH S6 (IU/l) 2.0 (1.3) 1.6 (0.9) 2.0 (1.4) 2.0 (1.6) 2.0 (1.2) 0.65

LH S8 (IU/l) 1.5 (0.8) 1.8 (1.0) 2.1 (1.3) 2.0 (1.2) 2.5 (1.4) 0.03

LH hCG (IU/l) 2.1 (1.5) 2.1 (1.2) 2.6 (1.4) 2.2 (1.9) 3.2 (2.8) 0.19

LH AUC S6-hCG 8.7 (5.7) 9.9 (5.5) 12.9 (7.7) 11.1 (6.3) 16.2 (8.8) <0.01*

LH AUC–S6 -1.7 (3.8) 2.0 (5.0) 4.4 (6.4) 2.6 (10.1) 7.2 (9.8) <0.01*

FSH S6 (IU/l) 15.8 (6.7) 14.9 (6.0) 12.1 (4.1) 12.9 (4.5) 13.0 (4.3) 0.12

FSH S8 (IU/l) 16.7 (7.2) 16.9 (7.0) 13.4 (4.3) 14.9 (4.7) 14.0 (4.3) 0.36

FSH hCG (IU/l) 17.8 (7.1) 17.5 (7.3) 13.3 (3.7) 15.9 (5.5) 14.0 (4.5) 0.05

FSH AUC S6-hCG 76.0 (40.6) 73.1 (32) 58.0 (28) 65.3 (22.9) 64.5 (33) 0.25

Table IV. Serum gonadotrophin levels. Serum levels are pretreatment morning samples. Values are mean ± SD. Analyses were performed on intention to treate (ITT) base, using Kruskal Wallis test. S6=stimulation day 6 etc.; hCG = hCG day; E2 = oestradiol; P4=progesterone; ET = embryo transfer day. LH AUC–S6 = LH area under the curve from S6 tot hCG day, substracted by the baseline levels of LH on S6. ∗P < 0.05.

A gradual increase of the mean pre-injection LH levels were observed from S7 to the day of rhCG (Figure 4). Overall, the mean LH serum levels increased with 0.76 IU/l (95%CI: 0.45–1.07).

This increase was clearest in the lowest dose group (P=0.04), but was also present in some individual plots in the higher dose groups (see Figure 5).

LH surgesBefore antide administration (on S6) one patient (in the 0.5 mg/ml group) had a LH of 15.1 IU/l with a progesterone of 1 ng/ml; after antide administration the LH levels decreased to 3.8 IU/l. During antide treatment, eight patients had a LH >12.4 IU/l and seven had a concomitant P4 > 2 ng/ml before the criteria of hCG administration were met; one (LH 13.1 IU/l and P4 2.0

ng/ml) in the 1 mg/ml group, two in the 0.5 mg/0.5 ml group (LH 15.2 IU/l and P4 2.6 ng/ml, LH 19.4 IU/l and P4 4.4 ng/ml), none in the 0.5 mg/ml group (one patient had a LH of 14.1 but

a P4 of 1.8 ng/ml) and four in the 0.25 mg/ml group (LH 13.9 IU/l and P4 3.6 ng/ml, LH 21.0 IU/l and P4 3.9 ng/ml, LH 32.0 and P4 2.0 ng/ml, LH 12.3 IU/l and P4 30 ng/ml). The incidence

of LH surges was signifi cantly different between the dose groups (P=0.04).

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Figure 4. Mean serum levels: FSH, LH, oestradiol (E2) and progesterone (P4). Mean serum LH, FSH, E2 and P4 levels: pretreatment levels (on cycle day 2 or 3 before the study = PRE), on stimulation day 1 (CD2 or 3 during the study, before any medication was given) and daily from stimulation day 6 (S6) up to and including stimulation day 9 (S9) in the morning (am) and evening (pm).

FSH, oestradiol and progesterone levels during stimulationOn the day of hCG, the FSH levels were higher in group A (2.0 mg/ml) and group B (1.0 mg/ml) in comparison to the lower dose groups (P=0.05). The total AUC of the FSH levels were not signifi cantly different between the individual treatment groups (P=0.25) (see Table IV).

The steroid hormone levels are presented in Table V. E2 levels increased in all groups during antide administration (see Figure 4). The mean E2 levels/day and the induced E2 AUC, adjusted for baseline levels on S6 (E2-AUC–S6) were proportionally dose-related to the treatment group (P=0.009 and <0.001, respectively), with the highest E2 levels in the lowest antide dose groups. The same was so for the calculated E2 levels per follicles >10 mm on the day of hCG administration, with the highest E2 levels in the lowest dose group (0.25 mg) and the lowest E2 levels in the highest antide dose group (2.0 mg), 501 (228) and 388 (168) pmol/l/follicle, respectively (P=0.05).

FSH (IU/L)

0,02,04,06,08,0

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S6

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S7

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S7

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S8

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2mg1mg0.5/0.50.5/ml0.25mg

n≥23 n≥23 n≥21 n≥14

LH (IU/L)

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S6

am

S6

pm

S7

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S8

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E2 (pmol/L)

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1000

2000

3000

4000

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Figure 5. Plot of LH and antide levels in an individual patient. LH and antide levels from S6 to rhCG day of a patient receiving 1 mg/ml antide.

Antide treatment group (daily dose) P value

A(2mg/2ml)

B(1mg/ml)

C(0.5mg/ml)

D(0.5mg/0.5ml)

E(0.25mg/ml)

No of patients (ITT) 30 30 31 23 30

E2 S6 (pmol/l) 1138 (705) 1236 (542) 1283 (642) 1289 (886) 1540 (718) 0.15

E2 hCG (pmol/l) 3941 (2126) 4920 (2290) 4705 (3130) 4435 (2559) 5034 (2701) 0.45

Mean E2/day (pmol/l)a 2197 (1134) 2701 (1014) 2654 (1460) 2518 (1346) 3193 (1220) 0.02*

E2 AUC–S6 5766 (4367) 8165 (5347) 7767 (7489) 6968 (4851) 9723 (5746) 0.06

P4 S6 (ng/ml) 0.7 (0.3) 0.7 (0.3) 0.9 (0.4) 0.8 (0.4) 0.8 (0.3) 0.50

P4 hCG (ng/ml) 1.5 (0.7) 1.5 (0.7) 1.6 (0.6) 1.4 (0.8) 1.7 (0.8) 0.40

Mean P4/day (ng/ml)a 1.0 (0.3) 1.0 (0.4) 1.2 (0.4) 1.0 (0.5) 1.1 (0.3) 0.46

P4 AUC–S6 (ng/ml) 0.5 (2.1) 1.2 (1.8) 0.9 (1.7) 0.5 (1.7) 1.6 (3.0) 0.33

Table V. Serum steroid hormone levels. Serum levels are pretreatment morning samples. Values are mean (± SD). Analyses were performed on intention to treat ( ITT) base, using Kruskal Wallis tests. S6 = stimulation day 6. E2 = estradiol; P4 = progesterone; ET = embryo transfer day. E2 AUC–S6 = E2 area under the curve from S6 tot hCG day, substracted by the baseline levels of E2 on S6. P4 AUC–S6 = P4 area under the curve from S6 tot hCG day, substracted by the baseline levels of P4 on S6. aAUC/day = the mean area under the curve per day, calculated out of all daily morning samples from S6-HCG day divided by the stimulation days. ∗P < 0.05.

On all days, the mean P4 levels were comparable in the various dose groups. Progesterone levels showed large fl uctuations during the day (see Figure 4). All calculated AUC or induced differences in P4 levels (P4-AUC, P4-AUC–S6) did not differ between the various treatment groups and the P4 levels were not signifi cantly correlated to the actual antide levels. But linear regression analyses of the P4-AUC (P<0.001, R2=0.36) revealed that, if adjusted for the duration of the treatment, the absolute P4 levels were dependent on LH-AUC (β1=0.61, P<0.001) and the number of follicles at hCG day (β2=0.20, P=0.004).

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Safety and toleranceOverall, antide was well tolerated, with none of the patients experiencing a serious adverse event related to medication. The most frequently recorded side effects were general disorders

(especially fatigue and headache) and gastro-intestinal disorders (abdominal pain and nausea). 83.1% of all patients recorded at least one local skin reaction (redness, tenderness, itching, bruising or swelling) with transient redness (25%) and tenderness at the injection site (22.2%) being most frequent. Most patients considered the local reactions to be mild. The local reactions mostly resolved spontaneously within 1 h.8.8% of patients had at least one moderate local reaction occurring 1 h after antide injection and were mostly found in treatment groups A and B. No severe local reactions were observed 1 h after antide injection.

In the minimal effective dosegroup (D: 0.5 mg/ml), a moderate local reaction observed 1 h after the injections was observed in only one patient. The biochemical and hematological parameters measured in all patients were similar before and after the treatment cycle (data not shown).

DISCUSSION

In this study we established that a daily subcutaneous dose of 0.5 mg/ml antide, when given from the 6th day of stimulation with rhFSH, is suffi cient to prevent signifi cant rises in LH

and progesterone in IVF/ICSI. We confi rm that the approach of a daily small dose of a GnRH antagonist is useful in IVF/ICSI stimulation protocols to avoid cancellation of the procedure

due to premature lutinization that otherwise would occur in ∼20% of all started cycles (Janssens et al., 2000).

We used the same standard defi nition for premature luteinization as others in GnRH antagonist dose-fi nding studies, namely a rise of LH above a predetermined threshold plus a signifi cant increase in serum progesterone values (Albano et al., 1997; Ganirelix dose-fi nding Study Group, 1998). According to our defi nition, groups D (0.5 mg/0.5 ml) and E (0.25 mg/ml) were considered to be failure dosages, and group C (0.5 mg/ml) appeared to be the minimal effective dose, in which no rises occurred. It should be noted that sampling blood three times per day increases the detection of any signifi cant change in LH. The design of the study was chosen on ethical grounds, with the aim of minimal exposure to possible premature luteinization. We were aware of the risk of a possible imbalance in baseline characteristics.

Retrospectively it appeared this was present as regards type of fertility disorder and pretreatment oestradiol levels between the various groups. Nevertheless, these factors had no effect on pharmacokinetics and hormonal dynamics.

As expected there was a clear dose dependency of LH secretion throughout the stimulation period with lowest levels with the highest antide dosages. Similar dose dependency was seen in the other GnRH antagonist dose-fi nding studies (Albano et al., 1997; Ganirelix dose-fi nding Study Group, 1998). This study clearly demonstrated that the dilution of 0.5 mg antide in a larger volume of glucose 5% increased the bioavailability of antide and resulted in a lower incidence of LH surges. The antide serum levels were almost doubled if the same dose of antide (0.5 mg) was diluted in 1 ml instead of 0.5 ml.

A clear and intriguing observation in our study was the remarkable rise of preinjection LH levels throughout the antide treatment period. It occurred with all dosages, but was particularly clear in the lowest antide dose groups. This gradual increase of basal

LH secretion while daily injections of a GnRH antagonist are given seems a common feature with this treatment strategy (Ganirelix dose-fi nding Study Group, 1998). It can also

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be seen in healthy unstimulated women (Oberye et al., 1999). Apparently some escape of pituitary LH secretion takes place, which even occurs while serum concentrations of antide remain constant. One explanation is that the pituitary develops an increased sensitivity to endogenous hypothalamic GnRH under these circumstances. The mechanism by which this takes place remains obscure. Possibly changes in hormonal milieu play a role. Increasing oestradiol levels during stimulation may modify pituitary sensitivity. The gradual escape

was indeed most prominent in the lowest antide dose groups, which was associated with the highest oestradiol levels, both in our study and also in the Ganirelix dose-fi nding study (Ganirelix dose-fi nding Study Group, 1998). Alternatively, pituitary GnRH receptors may have been up-regulated by the GnRH antagonist itself as suggested by several authors (Gordon et al., 1994). A fi nal explanation for the LH increase may be that the GnRH antagonists directly intervene in the ultrashort-loop feedback mechanisms comprising GnRH autoregulation (Krsmanovic et al., 1999).

The FSH levels on the day of hCG were higher in the highest antide dose groups, while there was a trend of a dose dependency in mean daily FSH levels. The adjustments of daily FSH dose, based on insuffi cient follicular response, may have been responsible for this. Although similar total FSH quantities were used in all antide dose groups, signifi cantly more patients had rhFSH dose increases in the highest antide dose groups.

The mean oestradiol levels per day and the induced changes in oestradiol levels during antide administration in comparison to the baseline levels, were inversely related to the antide dose. This is in agreement with an earlier report (Ganirelix dose-fi nding Study Group, 1998). Similarly, the highest levels of oestradiol production per follicle ≥11 mm were found in the lowest antide dose group. The dose-related oestradiol response to the GnRH antagonist, independent from the number of follicles, indicates a decrease of its production by granulosa cells, probably as a result of reduced availability of androstendione substrate.

It is often suggested that late follicular progesterone secretion is LH dependent. However, a straightforward LH dependency of progesterone secretion was not obvious in our study. Nevertheless, this relation became clear after correction for numbers of follicles on the day of hCG. The total AUC of progesterone increased by 0.6 ng/ml when LH-AUC increased by 1 IU/L. Apparently, the absolute level of progesterone production is also dependent on the total number of follicles. This means that high progesterone levels, independent of high LH levels, may also be the result of profound follicular growth in the ovary.

Overall, antide was well tolerated and safe in terms of side effects and infl uence on biochemical and haematological parameters. The reported reactions were all minor and most resolved spontaneously within 1 h. Only 9% of the patients had a moderate or severe

local skin reaction 1 h after antide administration during the entire treatment period. This was lower than with other antagonist studies employing the same assessment methods, scales and criteria for these side effects (Ganirelix dose-fi nding Study Group, 1998; Middle East Orgalutran Study Group, 2001). This can be explained by the much lower histamine releasing property of antide compared to the clinically available GnRH antagonists (Bajusz et al., 1988; Rivier et al., 1992).

In conclusion, 0.5 mg/ml antide is the minimal effective dose to prevent LH surges in hyperstimulated cycles for IVF or ICSI. LH levels increase gradually during GnRH antagonist treatment. The bioavailability increases if antide is diluted in a larger volume. Antide was well tolerated and safe.

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Acknowledgements The authors would like to thank Ted Korsen and Agnes Wierink-Wanders, research nurses for their very active support to this study; Carien van de Brand and Floris holzheimer, Paxel Mirai, for the monitoring of this study. This study was sponsored by Serono International, Geneva, Switzerland.

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Ljungqvist A, Feng DM, Hook W, Shen ZX, Bowers C and Folkers K (1988) Antide and related antagonists of

luteinizing hormone release with long action and oral activity. Proc Natl Acad Sci USA 85, 8236–8240.

Nestor JJ, Jr, Tahilramani R, Ho TL, Goodpasture JC, Vickery BH and Ferrandon P (1992) Potent gonadotropin

releasing hormone antagonists with low histamine-releasing activity. J Med Chem 35, 3942–3948.

Oberye JJ, Mannaerts BM, Huisman JA and Timmer CJ (1999) Pharmacokinetic and pharmacodynamic

characteristics of ganirelix (Antagon/Orgalutran). Part II. Dose-proportionality and gonadotropin suppression

after multiple doses of ganirelix in healthy female volunteers. Fertil Steril 72, 1006–1012.

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Olivennes F, Alvarez S, Bouchard P, Fanchin R, Salat-Baroux J and Frydman R (1998) The use of a GnRH antagonist

(Cetrorelix) in a single dose protocol in IVF-embryo transfer: a dose fi nding study of 3 versus 2 mg. Hum

Reprod 13, 2411–2414.

Rivier J, Porter J, Hoeger C, Theobald P, Craig AG, Dykert J, Corrigan A, Perrin M, Hook WA and Siraganian

RP (1992) Gonadotropin-releasing hormone antagonists with N omega-triazolylornithine, -lysine, or -p-

aminophenylalanine residues at positions 5 and 6. J Med Chem 35, 4270–4278.

Templeton A and Morris JK (1998) Reducing the risk of multiple births by transfer of two embryos after in vitro

fertilization. N Engl J Med 339, 573–577.

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5Dose-fi ndings study of dailyGnRH antagonist for the preventionof premature LH surges in IVF/ICSI patients: optimal changes in LH and progesterone for clinical pregnancy

JAF Huirne, AC van Loenen, R Schats, J McDonnell,

PG Hompes, J Schoemaker, R Homburg, CB Lambalk

Human Reproduction 2005: 20; 359-67

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ABSTRACT

BackgroundAn optimal range of LH concentrations for achieving pregnancy has not been established. The aim of this study was to investigate the effect of various LH levels induced by different

GnRH antagonist doses on IVF outcome. Material and methodsThis was a prospective, single centre study including 144 IVF patients, stimulated with

recombinant FSH from cycle day 2, and co-treated with daily GnRH antagonist (antide/Iturelix) (2 mg/2 ml, 1 mg/ml, 0.5 mg/ml, 0.5 mg/0.5 ml or 0.25 mg/ml) from cycle day 7 onwards. Serum samples were taken three times daily. ResultsClinical pregnancies were only observed within a particular range of change in LH levels. The upper and lower thresholds for the mean LH area under the curve (AUC), adjusted for the baseline LH level before the antagonist was started (LH AUC–S6; S6=stimulation day 6) were –2.2 and 12.4 (IU/l) respectively (a negative value=below baseline levels). There were no clinical pregnancies outside these threshold values. Similar results were found for progesterone, the threshold levels of progesterone AUC–S6 were 3.98 and –1.21 ng/ml. Moreover, there were no pregnancies with progesterone levels >0.26 ng/ml/follicle on the day of hCG. ConclusionsExcessive or insuffi cient suppression of LH and progesterone levels during GnRH antagonist administration and high progesterone/follicle on hCG day seems to be associated with impaired clinical pregnancy rates.

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INTRODUCTION

GnRH antagonist can be used safely to suppress LH levels and to prevent premature LH surges in IVF-stimulated cycles (Huirne and Lambalk, 2001). For many years, long agonist protocols have been the standard treatment regimen for this indication. Recently GnRH antagonists have been introduced in many IVF centres since their use improved patients’ convenience in comparison to the long agonist protocol. Three GnRH antagonists have been studied in proper dose-fi nding studies for their daily use in IVF patients to prevent premature LH surges; ganirelix (Ganirelix dose-fi nding Study Group 1998), cetrorelix (Albano et al., 1997; Olivennes et al., 1998) and antide (Huirne et al., 2004). In a ganirelix dose-fi nding study, the implantation rate was inversely associated with the antagonist dose. The highest pregnancy rates were found in the minimal effective dose group (0.25 mg) and low pregnancy rates were especially found in the high dosage groups (2 and 1 mg) (Ganirelix dose-fi nding Study Group, 1998). The infl uence

of GnRH antagonist on implantation became a matter of debate after the publication of fi ve major comparative studies between GnRH agonists and antagonists in IVF, which reported consequent lower pregnancy rates in the antagonist groups (Albano et al., 2000; Borm and Mannaerts, 2000; European Middle East Orgulatran Study Group, 2001; Fluker et al., 2001). These differences were not statistically signifi cant in the individual studies, but a meta-analysis of these studies showed a signifi cantly lower pregnancy rate of 5% (Al-Inany and Aboulghar, 2002). The mode of LH suppression and the pattern of circulating LH levels vary

between GnRH antagonist and GnRH agonist cycles. After an initial period of gonadotrophin hypersecretion, GnRH agonists induce desensitization, mostly resulting in profound and stable LH levels during the entire stimulation period (Janssens et al., 2000; Westergaard et al., 2001). In contrast, the GnRH antagonist treatment regimens allow higher LH levels in the early stimulation period, since antagonist is usually started from stimulation day 6 onwards. The LH levels rapidly decline after the start of the antagonist treatment, often followed by a gradual increase later in the cycle (Ganirelix dose-fi nding Study Group, 1998; Oberye et al., 1999; Huirne et al., 2004). Differences in the pattern and the level of LH suppression between agonist and antagonist regimens may play a role in the observed differences in pregnancy rates.

The role of LH on implantation is still not fully elucidated. It has been established that severe suppression of LH using GnRH agonists is associated with impaired IVF outcome (Fleming et al., 1998; Howles, 2000; Balasch et al., 2001; Filicori, 2002) and increased pregnancy loss (Westergaard et al., 2000). A certain LH threshold needs to be achieved for adequate folliculogenesis and steroidogenesis, which is required to provide an appropriate milieu for successful fertilization and implantation (Hillier, 1994; European Recombinant human LH Study Group, 1998; Howles, 2000; Filicori, 2002; Shoham, 2002). On the other hand, there are convincing data suggesting that elevated LH levels are associated with impaired fertilization and pregnancy rates and with higher miscarriage rates (Stanger and Yovich, 1985; Howles et al., 1986; Homburg et al., 1988; Regan et al., 1990; Chappel and Howles, 1991; Shoham, 2002; Tesarik and Mendoza, 2002; Loumaye et al., 2003), the so called ‘ceiling’ effect (Hillier, 1994). The optimal LH levels to provide an endocrine milieu which results in the highest number of clinical pregnancies in patients undergoing IVF, in protocols with FSH stimulation, are still a matter of debate and have rarely been studied in GnRH antagonist-treated cycles. One recent study demonstrated that exposure to high LH levels in the early follicular phase of GnRH antagonist-treated cycles is associated with a reduced chance of pregnancy (Kolibianakis et al., 2003a). An additional randomized controlled trial in which 257 women were randomized

to receive either 150 or 200 IU rhFSH per day showed a trend towards higher pregnancy rates,

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despite lower number of oocytes retrieved, in the lower recombinant (r)FSH dose group in which higher LH levels were found (Out et al., 2004). However, lower early follicular phase LH and oestradiol levels after early administration of the GnRH antagonist, stimulation day 1 versus day 6 in 60 patients, did not alter the IVF outcome (Kolibianakis et al., 2003b).

In a previous study we demonstrated that various endogenous LH levels can be induced by different doses of repeated GnRH antagonist injections in IVF/ICSI patients undergoing ovarian stimulation with rhFSH (Huirne et al., 2004). The aim of the present study was to examine the effect of various LH concentrations, induced by different GnRH antagonist doses, on the outcome of IVF.

MATERIALS AND METHODS

PatientsAll patients scheduled for IVF or ICSI treatment in our department during a period of 7 months were assessed for eligibility. In this period, 660 IVF cycles were performed in 290 patients; of these, 158 patients were eligible and 153 were asked to participate. A total of 144 patients was included. All patients were aged between 21 and 39 years, had spontaneous regular menstrual cycles between 25 and 35 days, two ovaries, a normal uterine cavity, a body mass index ≤30 kg/m2 and had had at least two spontaneous menstruations since the last clomiphene citrate or gonadotrophin treatment. Women with elevated hormone levels (FSH ≥10 IU/l or LH ≥8 IU/l or prolactin levels (≥800 mIU/l) on cycle day 2 or 3 were excluded from participation. Patients with polycystic ovarian syndrome (defi ned as oligomenorrhoea and elevated LH levels or signs of hyperandrogenism) were also excluded. Also excluded were patients with abnormal haematological or biochemical parameters, patients with any previous assisted reproduction cycle with fewer than three oocytes, known allergy or hypersensitivity to human gonadotrophin preparations or GnRH analogues. The protocol was approved by the Committee on Ethics of Research involving Human Subjects of the VUMC, Amsterdam, The Netherlands. All participants signed informed consent forms.

Study designA phase II, single centre study, conducted in two phases—a double-blind phase with two parallel treatment groups—was followed by an open phase. In the double-blind phase, 60 patients were randomized to two different treatment groups (A: 2 mg/2 ml; B: 1 mg/ml). To improve patients’ convenience, the 2 mg in group A was given as two injections of 1 mg/ml antide, one injection in the morning and one in the evening, since we expected that one injection with a volume of 2 ml would be too painful. Patients in group B received placebo in the morning and 1 mg/ml

antide in the evening. Since none of the two groups turned out to be a failure group (i.e. with two or more LH surges) we decided to add an open phase in which three additional treatment groups with lower antide dosages were studied (0.5 mg/0.5 ml, 0.5 mg/ml, 0.25 mg/ml). The additional arms were added in a consecutive order and patients were enrolled in a chronological fashion. New evidence which became available after the start of this

study suggested that the bioavailability of antide increases after dilution in larger volumes of glucose 5% (data on fi le: Serono International, Geneva). Therefore in two groups (0.5 and 0.25 mg/ml) antide was diluted in larger volumes of glucose 5% solution: 0.5 and 0.25 mg in 1 ml respectively. This means that in two arms, 0.5 mg antide was administered but in group

C it was diluted in 1.0 ml glucose 5% solution and in group D it was diluted in 0.5 ml glucose 5% solution.

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Each group was intended to contain 30 patients unless more than one LH surge occurred, which according to the protocol led to the discontinuation of that particular dose group and was considered to be a failure dose. More than one LH surge per 30 patients was considered to be unacceptable for clinical use in IVF patients. Additional observations were performed, to search for optimal GnRH antagonist and/or LH levels. We made scatter plots to see whether an optimal range could be found for clinical pregnancy.

MaskingTreatment packs for the double-blind phase of the study were prepared according to the randomization list by Serono International (Geneva, Switzerland). Patient packs, containing antide/placebo or antide/antide vials, were labelled with unique study identifi cation numbers, provided by Serono International (Geneva, Switzerland),

placebo vials contained a sterile isotonic aqueous solution. When eligible, patients were enrolled into the study by one of the two responsible trained researchers and received a unique study number in a chronological order at the start of the fi rst stimulation day. The code was not known to the executors of the study. Assignment to group A or B was therefore double-blind, assignment to group C, D or E depending on the chronological

entry of the study.

Treatment protocolOn day 2 or 3 of a spontaneous menstruation, rhFSH (Gonal-F®; Serono, Switzerland) was given as a single daily s.c. injection. The starting dose varied between 150 and 300 IU, depending on previous ovarian response, but was fi xed for the fi rst 5 days. After this period, depending on ovarian response as assessed by daily ultrasound, the rhFSH dose could be adjusted. All antide, placebo and rhFSH injections from stimulation day 6 (S6) onwards were given subcutaneously, by a trained research professional. From stimulation day 6 onward, up to and including the day of rhCG (rhCG; Ovitrelle®; Serono) administration, daily antide

was started. rhCG was administered as soon as one follicle was ≥18 mm and three follicles were ≥16 mm. Thirty-six hours after rhCG administration, oocyte retrieval was performed transvaginally and ultrasound-guided. The oocyte retrieval was followed by IVF with or without ICSI; a maximum of three embryos was replaced 2–3 days thereafter. Luteal support (200 mg progesterone vaginally, three times daily) was started 1 day after oocyte retrieval until the third week of pregnancy or a negative pregnancy test.

AssessmentsOne to three months before randomization, serum samples were taken to assess hormone levels (FSH, LH, oestradiol, progesterone and prolactin), taken on cycle day 2 or 3. On stimulation day 1 (S1), before any study drug was administered, a blood sample was taken to perform a pregnancy test and to assess FSH, LH, oestradiol (E2) and progesterone levels and a transvaginal ultrasound was performed to measure follicular activity, endometrium thickness and to exclude the presence of cysts. During antide administration, three samples per day were taken (in the morning before any injection, in the evening prior to Gonal-F or antide injection, and 20–84 min later), to assess serum levels (FSH, LH, E2, progesterone and antide). The potential variation in timing of the evening post-injection blood sampling was intended to allow pharmacokinetic and pharmcodynamic modelling (data not shown). The mean sample time was 34.6 (SD 3.9) min after antide injection (range of the mean per patient varied from 30 to 53 min). Transvaginal ultrasound was performed daily to assess follicular development and endometrium thickness. On the day of embryo transfer,

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serum samples were taken to measure the antide level, and 7–11 days after oocyte retrieval to measure the levels of progesterone and antide. Finally, 23–25 days after oocyte retrieval, serum hCG levels were measured. If positive, a vaginal ultrasound was performed 35 and 42 days following rhCG administration, to record the number of fetal sacs and fetal heart activity. Ultrasound was repeated at a gestational age of 12 weeks.

Serum assessmentBlood samples were processed to serum immediately after collection and stored at –20°C. Routine haematology, biochemistry and urine assessment were performed by the local laboratory (The Central Laboratory of the VUMC) using commercially available

immunometric assays. LH and FSH levels before inclusion, in morning samples, were assessed by the local laboratory using immunometric assay kits (Amerlite; Amersham, UK). Patients were excluded for high LH and FSH values on cycle day 3, respectively >8 and >10 IU/l, using immunometric assay kits (Amerlite). Half-way through the study we were forced to change the assay, since Amerlite assays were no longer available. We decided to use Delphia (Finland) assays. During the transition period of the assays, we assessed LH levels using both assays in 89 patients. Excellent correlation was observed between the two assays for the measurement of LH (r=0.981) and FSH (r=0.996) A regression analysis revealed that the coeffi cient of LH was 1.24 using Delphia compared with Amerlite, thus the LH threshold level of 8 IU/l assessed by Amerlite was equivalent to 9.9 IU/l if assessed by Delphia assay. In addition, the coeffi cient of FSH was 1.28, thus the FSH threshold level of 10 IU/l assessed by Amerlite was equivalent to 12.8 IU/l if assessed by Delphia assay.

For defi nitive analyses of all hormone and antide levels, as presented in this report, all serum samples (taken three times daily) were assessed retrospectively by LCG Bioscience Services Ltd. E2 was measured using Sorin Radioimmunoassay, progesterone using DPC Coat-a-Count radioimmunoassay solid phase coated tube separation, FSH and LH using Serono MAIAclone IRMA. The lower limit of quantifi cation for LH was 1 IU/l. For the retrospective

analyses, we defi ned an LH surge as LH > 12.4 IU/l and progesterone >2 ng/ml in one or more samples, taking all samples (three times daily) into account from S6 until hCG administration day, equivalent to the threshold levels using the Delphia assays. The retrospective centralized analysis of serum antide levels was performed by Woods Assay (radioimmunoassay); all samples were analysed in triplicate; 1 mg/l was the limit of quantifi cation.

Outcome measuresDrug requirements, stimulation results, IVF outcome and its relationship to serum hormone and antide levels.

Statistical analysesTreatment groups were compared depending on the nature of the variables, i.e. analysis of variance (ANOVA) or analysis of covariance (ANCOVA), χ2-test, Fisher’s exact test or non-parametric ranking methods such as Kruskal–Wallis and Mann–Whitney U-tests. Results are reported as mean ± SD. Correlations were calculated according to Pearson’s correlation coeffi cient. P<0.05 was considered to be statistically signifi cant. Analyses were performed on all subjects who were randomized or received Gonal-F (all patients who were included in the study) unless otherwise reported. The number of patients included in A, B, C, D and E were 30, 30, 31, 23 and 30 respectively. An overall dose–response test for linear trend with the treatment groups was performed on all effi cacy data. For continuous normally distributed data, a linear contrast of the treatment groups was tested in a one-way ANOVA; for ordinal

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(ordered categorical) data, a Jonckheere–Terpstra test and for binary data a Cochran–Armitage test were used.

This pilot study was not powered to calculate pregnancy rates, but group size was based on clinically relevant arguments. More than one LH surge per 30 patients was considered to be unacceptable for clinical use in IVF patients. Therfore the number of patients was intended to be 30 per group unless more than one LH surge occurred. This study employs patients included in a previous dose-fi nding study in which IVF outcome and hormones were examined (Huirne et al., 2004).

Total exposure to antide and hormone levels was expressed as area under the curves (AUC) during antagonist administration [i.e. from stimulation day 6 (S6) to hCG administration day]. For this calculation, the sum of the mean daily levels (= sum of three samples per day/3) of all days during antide treatment was taken. To calculate the induced change in serum levels in comparison to the basal level on S6 (the time-point at which the antagonist was started), AUC was calculated after subtraction of the basal level on S6 of all samples, defi ned as AUC–S6 (see Figure 1).

Figure 1. The method of area under the curve (AUC) calculation of the change in hormone levels. S1 = stimulation day 1; S6 = stimulation day 6.

RESULTS

The general results (i.e. basic characteristics and cancellation rates), pharmacokinetic and the induced hormone levels of this antide dose-fi nding study were reported elsewhere (Huirne et al., 2004). The ultrasound fi ndings during stimulation are presented in Table I and the IVF outcome in Table II. There were no differences in number of mature follicles (≥11 mm), mean follicular size and endometrium thickness on the day of hCG administration between the various dose groups (see Table I). Also no differences were found in number of oocytes retrieved or mean number of metaphase II oocytes per patient; the latter was only assessed and analysed in ICSI. Fertilization rate, total number of embryos, good quality

embryos (grade I and II) and number of embryos transferred were also not different. There was a tendency toward higher pregnancy and implantation rates in the middle dose groups

S6 hCGS1

GnRH-antagonist

levels

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(0.5 and 1.0 mg/ml; not signifi cant). None of the patients in this study had moderate or severe symptoms associated with OHSS necessitating hospitalization.

Antide treatment group (daily dose) P

A(2mg/2ml)

B(1mg/ml)

C(0.5mg/ml)

D(0.5mg/0.5ml)

E(0.25mg/ml)

No. of patients 30 30 31 23 30

n follicles ≥11mm S6 3.1 (3.0) 2.8 (1.6) 3.8 (2.5) 3.0 (2.6) 3.9 (3.2) 0.42

n follicles ≥11mm hCGa 10.5 (4.8) 10.6 (3.6) 9.8 (3.2) 9.7 (4.0) 10.8 (4.3) 0.92

n follicles ≥15mm hCGa 5.6 (2.5) 6.0 (2.5) 5.4 (1.6) 5.5 (2.3) 6.0 (2.3) 0.92

n follicles ≥17mm hCGa 3.5 (1.4) 4.0 (1.7) 3.1 (0.9) 3.5 (1.4) 4.2 (2.6) 0.13

Endometrial thickness hCGa

10.8 (2.3) 10.7 (2.0) 10.9 (1.8) 10.2 (1.6) 10.9 (2.1) 0.77

Table I. Late follicular phase ultrasound measurements. Values are means (± SD). Analyses were performed using Kruskal-Wallis tests, except for the endometrial thickness we used ANOVA. Analyses were performed on all patients included into the study, unless otherwise indicated. S6 = stimulation day 6; hCG = day of hCG. a analyses restricted to the patients who received hCG. n follicles = number of follicles

In contrast to the serum hormone levels, none of the IVF outcome parameters was linearly related to the dose groups or antide levels. The relationship between the AUC of antide and LH during antide administration and the occurrence of clinical pregnancies is plotted in Figure 2. No pregnancies were observed in relation to either very high or very low LH or antide levels, suggesting an optimal window for total exposure of these compounds for the occurrence of pregnancy. The upper and lower thresholds for the total LH AUC were 22.3 and 4.47 respectively. The total amount of LH secreted was the highest with the lowest antide

serum levels, while the lowest LH AUC values were found in association with the highest antide AUC values. Variation in the AUC values was higher in comparison to the absolute LH and antide levels per day, which showed better correlation (Huirne et al., 2004). The upper and lower levels for antide AUC were 10.2 and 36.3 (μg/l) respectively. Scatter plots of the LH and antide levels on S7 and S8 and the day of hCG did not show any relationship or threshold level for the occurrence of clinical pregnancies (data not shown).

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Antide treatment group (daily dose) P

A(2mg/2ml)

B(1mg/ml)

C(0..5mg/ml)

D(p.5mg/0.5ml)

E(0.25mg/ml)

No. of patients 30 30 31 23 30

No. oocytes 10.8 (6.8) 10.4 (5.2) 10.1 (5.1) 9.9 (4.2) 10.8(5.9) 0.99

No. metaphase II oocytesa 7.6 (5.3) 8.3 (5.3) 7.9 (4.3) 5.2 (2.8) 8.0 (3.5) 0.45

Fertilization rateb 0.6 (0.3) 0.6 (0.2) 0.7 (0.3) 0.5 (0.2) 0.7 (0.3) 0.20

No. embryos 5.5 (4.8) 5.9 (3.4) 5.4 (3.5) 4.5 (3.6) 6.3 (5.0) 0.60

No. good quality embryosc 3.5 (3.5) 4.2 (2.5) 3.3 (2.3) 2.8 (3.2) 4.5 (3.5) 0.05

No. embryos transferred/ET 1.9 (0.5) 2.1 (0.4) 2.0 (0.3) 2.0 (0.3) 1.9 (0.4) 0.29

Implantation rated (%) 6.7 (22) 13.8 (30) 16.1 (30) 7.9 (19) 8.9 (24) 0.50

Biochemical pregnanciese (%) 3 (10.0%) 10 (33.3%) 10 (32.3%) 3 (13.0%) 5 (16.7%) 0.08

Clinical pregnanciesf (%) 3 (10.0%) 6 (20.0%) 8 (25.8%) 3 (13.0%) 4 (13.3%) 0.48

Ongoing pregnanciesg (%) 3 (10.0%) 6 (20.0%) 7 (22.6%) 3 (13.0%) 4 (13.3%) 0.65

Table II. IVF outcome. Values are mean(± SD). Analyses were performed on all patients included into the study, unless otherwise indicated. Kruskal-Wallis tests were used, except for % IVF and pregnancy parameters: X2 or Fisher’s exact tests. aAnalyses only in ICSI patients. bFertilization rate = 2PN fertilized oocytes/number of oocytes inseminated. c number of grade I and II embryos. d The mean implantation rate = sum of all individual implantation rates/number of subjects, implantation rate = number of gestational sacs per subject/ number of embryos transferred per subject. e Biochemical pregnancy = hCG >50 IU/l. f Clinical pregnancy = intrauterine pregnancy with at least one fetus with heart activity. g Ongoing pregnancy = intrauterine pregnancy with proof of at least one vital fetus at 12-16 weeks after embryo transfer.

Figure 2. LH and Antide AUC levels in relation to clinical pregnancy

b AUC S6-hCG, corrected for baseline levels on S6

Antide-AUC (ug/L)

6050403020100

LH-A

UC

(IU

/L)

40

30

20

10

0

Vital Pregnancy

no

yes

LHwindow?

Antide window?

Clinical pregnancy

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To study the effect of the induced change in LH during antide administration, we calculated the LH AUC–S6. Clinical pregnancies could only be observed within a narrow range of induced change in LH levels (LH AUC–S6) (see Figure 3), indicating the existence of an optimal window of LH changes (either an increase or decrease in relation to endogenous baseline levels) to obtain clinical pregnancy. No clinical pregnancies were observed if the LH levels increased too much (LH AUC–S6 >12.4 IU/l), or if LH decreased too much (LH AUC–S6 <–2.2 IU/l) (a negative value means that the levels are decreased in comparison to the baseline levels).

Figure 3. Change in LH: expressed as LH-AUC–S6 in relation to clinical pregnancy

Based on these LH AUC–S6 threshold levels, outside which no pregnancies occurred, we

divided the patients retrospectively into three groups: a lower group with LH AUC–S6 <–2.2 (IU/l), a middle group with LH AUC–S6 ≥–2.2 and ≤12.4 (IU/l), and a higher group with LH AUC–

S6 >12.4 (IU/l). There were no differences in baseline characteristics and baseline hormone levels (see Table III). Also no differences were found with respect to duration of treatment, rhFSH requirements, E2 levels or FSH levels (data not shown). Additionally, the number and quality of oocytes, fertilization rate, number and quality of embryos and number of embryos replaced were also not different between the three groups (see Table III). On the basis of our selection criteria for the three groups, changes in LH and progesterone levels were different between the three groups (see Table III).

Antide-AUC (ug/L)

6050403020100

LH- A

UC, a

djus

ted

for S

6 (IU

/L)

40

30

20

10

0

-10

-20

-30

Vital Pregnancy

no

yes

Window for change in LH

Antide window?

Clinical pregnancy

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Change in LH: LH-AUC-S6 (IU/l) P

Lower group<-2.2

Middle group≥-2.2 and ≤12.4

Higher group>12.4

Number of patients 24 106 14

Age (years) 34.3 (3.3) 32.9 (3.5) 31.8 (3.2) 0.09

BMI (kg/m2) 22.6 (2.7) 23.5 (2.9) 25.1 (3.8) 0.11

Duration fertility (years) 4.4 (3.4) 4.3 (3.1) 3.3 (2.1) 0.63

No. of follicles <11mm S1 7.5 (4.9) 7.6 (4.0) 8.7 (5.5) 0.85

LH, CD2/3a 3.9 (1.6) 3.4 (1.2) 4.1 (1.1) 0.06

FSH, CD2/3a 9.0 (5.1) 7.9 (2.5) 7.7 (2.3) 0.72

E2, CD2/3a 323 (547) 211 (237) 239 (133) 0.25

P4 , CD2/3a 2.8 (1.3) 2.8 (2.0) 5.5 (10) 0.48

Changes in LH and progesterone

LH-AUC-S6 -6.7 (5.5) 2.9 (3.8) 19.3 (6.8) <0.01

Progesterone-AUC-S6 0.01 (2.3) 0.88 (1.8) 3.3 (2.8) <0.01

IVF outcome parameters

No. oocytes 10.5 (7.0) 10.5 (5.2) 9.8 (5.0) 0.78

No. of metaphase II oocytesb 8.4 (4.7) 7.1 (4.3) 8.6 (3.5) 0.32

No. of embryos 6.1 (5.1) 5.5 (3.9) 5.2 (3.8) 0.94

No. of good quality embryosc 4.0 (3.4) 3.7 (3.1) 3.4 (2.2 0.84

Implantation rated 0 12.6 % 0

No. of clinical pregnancies (%)e 0 (0%) 24 (29%) 0 (0%)

Table III. Baseline charachteristics of three groups with different changes in LH levels,expressed as LH-AUC-S6. Values are means (± SD). Analyses are performed on all patients included in this study, unless otherwise stated. LH-AUC-S6 = LH area under the curve, adjusted for baseline levels on stimulation day 6. The threshold values for change in LH levels (LH-AUC-S6) were based on the occurrence of clinical pregnancy (see Figure 3). P-values were calculated using Kruskal-Wallis or Fisher’s Exact tests. a CD2/3 = baseline value on cycle day2 or 3 before any medication was given. bAnalyses only in ICSI patients. c number of grade I and II embryos.dThe mean implantation rate = sum of all individual implantation rates/number of subjects, implantation rate = number of gestational sacs per subject/ number of embryos transferred per subject. e Clinical pregnancy = intrauterine pregnancy with at least one fetus with heart activity.

LH is related to oestradiol and progesterone production (Pearson’s correlation between LH-AUC and E2-AUC is 0.40; P<0.001; between LH AUC and progesterone-AUC is 0.55; P<0.001; between LH-AUC–S6 and progesterone-AUC–S6 is 0.55; P<0.001; and between LH AUC and progesterone/follicle and is 0.39; P<0.001). In an attempt to obtain information on individual association of these parameters with clinical pregnancy, we created scatter plots of these individual parameters in relation to the LH AUC–S6 (see Figure 4). We did not observe any optimal area for change in E2 levels, expressed as E2-AUC–S6 (see Figure 4a). In contrast, the change in progesterone, expressed as

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progesterone AUC–S6, showed clear upper and lower thresholds for the occurrence of clinical pregnancies, 3.98 and –1.21 ng/ml respectively (Figure 4b). To correct for the effect of the number of follicles, we calculated the E2 level per mature follicle on the day of hCG day (E2/follicle ≥11 mm) as plotted in Figure 5a. These E2 levels/mature follicle did not show any optimal level for the occurrence of clinical pregnancies, in agreement with the absolute E2 levels or change in E2 levels. To obtain a parameter for premature luteinization, adjusted for the total number of follicles, we calculated the progesterone levels per follicle ≥11 mm on the day of hCG (progesterone/follicle) (see Figure 5b). For the progesterone/follicle on hCG day, a clear upper threshold with respect to the occurrence of clinical pregnancy was seen. No pregnancies occurred if the progesterone level was >0.26 ng/ml/follicle.

Figure 4. Change in oestradiol and progesterone levels, expressed as E2-AUC–S6 and P4-AUC–S6 in relation to LH levels and clinical pregnancy

Figure 5. E2/follicle and P4/follicle on the day of hCG administration in relation to LH levels and clinical pregnancy

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DISCUSSION

The primary aim of this paper was to assess IVF outcome parameters and the optimal range for LH levels induced by different doses of daily GnRH antagonist administration. No signifi cant differences were found in IVF outcome parameters between the various treatment groups in which dosages varied between 2.0 mg/2 ml and 0.25 mg/ml antide. Number of follicles, mature follicles, oocytes, quality of oocytes, number of metaphase II oocytes, and number and quality of embryos were all similar between the various treatment groups. With respect to the clinical pregnancies, again no signifi cant differences could be found between the dose groups, although the middle dose groups (0.5 and 1.0 mg/ml) seemed to be optimal for clinical and ongoing pregnancy rates.

These results compare well with a ganirelix dose-fi nding study (Ganirelix dose-fi nding Study Group, 1998). In previous studies it was demonstrated that different endogenous LH levels can be induced by different GnRH antagonist dosages (Duijkers et al., 1998; Ganirelix dose-fi nding Study Group, 1998; Oberye et al., 1999; Huirne et al., 2004). Thus, different GnRH antagonist dosages can be used as an instrument to induce deliberately different LH levels. To test our hypothesis that the induced differences in LH levels may be responsible for the observed optimal GnRH antagonist range rather than the antagonist level itself, we analysed in detail the relationship between LH levels, LH AUC levels and pregnancy rates. In line with the LH window postulated by others (Hillier, 1994; Shoham, 2002), we found an optimal area in LH AUC for the occurrence of a clinical pregnancy. No pregnancies were observed when the total LH AUC (calculated out of three samples taken per day) was >22.3 or <4.47 IU/l. The absolute LH levels taken on a single sample day, either day 7, 8 or hCG day, did not show any clear relationship with pregnancy rate. These fi ndings are in line with an earlier report (Balasch et al., 2001). Apparently, the parameter LH AUC associates more strongly with pregnancy rates than the absolute LH level taken in one single sample. Possibly, the AUC during the entire antagonist treatment period is a better representative

of LH exposure. The association between LH AUC levels and pregnancy rates became clearer when the changes in LH levels (expressed as LH AUC, adjusted for the baseline LH levels) were taken into account. Apparently, too large changes—either increases or decreases in LH levels during the antagonist administration period—rather than absolute AUC levels, are associated with a decreased chance of clinical pregnancy. These fi ndings were independent of baseline characteristics and other parameters of IVF outcome (see Table III). The occurrence of a pregnancy is effected by many factors and variables other than LH and progesterone. The only signifi cant covariant to clinical pregnancy in a univariate analyses was change in LH levels (p=0.006).

Thus changes in the endocrine milieu seem to be important for the IVF outcome in terms of clinical pregnancy. Figure 6 gives our interpretation of these results. The physiological basis for the association of the increased change in LH levels and lower pregnancy rates is not clear. It may interfere with the correct sequence of maturational changes and right synchronization between nuclear and cytoplasmic maturation (Mattioli and Barboni, 2000; Luborsky et al., 2002) but it may also cause advanced endometrium maturation (Kolibianakis et al., 2002, 2003c).

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Figure 6. The interpretation of change in LH levels (LH-AUC–S6) in relation to clinical pregnancy. S1 = stimulation day 1; S6 = stimulation day 6.

Caution is needed to extrapolate our fi ndings in general to GnRH antagonist studies using the minimum effective dose for the prevention of untimely LH surges in IVF stimulated cycles, since we induced extreme (high and low) hormone levels. This probably explains in part why others do not fi nd an association between LH levels and IVF outcome, with a smaller range of the LH levels in these studies (Balasch et al., 2001; Balasch and Fabregues, 2002).

LH is required to produce progesterone by luteinizing the granulosa cells and oestradiol by producing androstendione as a substrate for oestradiol. In a previous study we demonstrated that LH levels in GnRH antagonist cycles were related to progesterone and oestradiol (Huirne et al., 2004). In an attempt to obtain information on the individual association of these parameters with clinical pregnancy, we performed scatter plots of these parameters with changes in LH levels and studied the occurrence of clinical pregnancy. Similar to the observed window for changes in LH,

changes in progesterone levels also seemed to be closely related to the occurrence of clinical pregnancy. Absolute progesterone levels on hCG day were not correlated with clinical pregnancy. This is consistent with the fi ndings of others (Urman et al., 1999). However, in an observational study, progesterone levels >1.2 ng/ml on the day of hCG as a parameter of premature luteinization were associated with lower pregnancy and implantation rates

(Bosch et al., 2003).

Absolute progesterone levels are related to the total amount of luteinized granulosa cells, thus total number of follicles may be a confounder for the absolute progesterone levels on the day of hCG. Therefore we corrected the absolute progesterone levels on the day of hCG for the total number of follicles (progesterone/follicle), as a parameter for premature luteinization. This parameter showed a clear ceiling effect for the occurrence of clinical pregnancy. No pregnancy occurred in our study when the progesterone levels/follicle on the day of hCG exceeded a certain threshold (0.26 ng/ml/follicle).

Since LH and progesterone were closely correlated in the current study, we could not discriminate which of the two should be held responsible for the observed differences in pregnancy rates. No association was found for changes in oestradiol levels nor for the absolute oestradiol levels on the day of hCG administration with or without correction

GnRH antagonist

S1 S6 hCG

Window for change

in LH

LH-AUC-S6/day

3.0

0.0

-0.4 •

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for the total number of mature follicles on that day. Similar results were found in other studies using GnRH antagonists (Ganirelix dose-fi nding Study Group, 1998). Altogether it seems to be that changes in LH and/or progesterone levels play a specifi c key role in the occurrence of clinical pregnancy rates in GnRH antagonist-treated IVF patients. Apparently a certain stability of these hormones associates with clinical pregnancy. These fi ndings are in agreement with the observed lower pregnancy rates in the group with higher LH levels, in

which hCG was administered 2 days later than a control group in GnRH antagonist-treated patients (Kolibianakis et al., 2003d). We speculate that our fi ndings may have implications particularly for specifi c patient groups which are sensitive for high fl uctuations in LH levels. For example, in patients with extreme low or high body mass indexes, the minimum effective dose may result in respectively too strong or insuffi cient suppression of LH levels. In addition, patients with high baseline LH levels (polycystic ovarian syndrome or patients with diminished ovarian reserve) may be more prone to larger changes (decreases) in LH levels. Based on our results, it is expected that depending on the level of LH and progesterone suppression during antagonist suppression, some patients will benefi t from adjustment of the GnRH antagonist dose or addition of LH. These fi ndings are in line with a study, using GnRH agonists, demonstrating that addition of LH was benefi cial for patients who had a very low endogenous LH level and detrimental for those with high endogenous LH levels (Loumaye et al., 2003). The possibility of improving pregnancy rates in GnRH antagonist-treated cycles by adaptation of the GnRH antagonist dose or the addition of LH where the LH levels are inappropriate, or using a treatment regimen resulting in stable LH levels, should be studied in a prospective manner.

In conclusion, this is the fi rst study demonstrating that the dynamics of LH and progesterone play a critical role in implantation when GnRH antagonist is used. No pregnancies occurred when the LH and progesterone changed too much (either increase or decrease) during GnRH antagonist administration due to insuffi cient or too high dosages of the GnRH antagonist. Moreover, a clear ceiling effect of high (absolute) progesterone levels per follicle could be observed. Further studies are required to investigate whether pregnancy rates can be improved by changing the treatment strategies, which result in stable and appropriate LH levels during the IVF cycle.

AcknowledgementsThis study was sponsored by Serono International, Geneva, Switzerland.

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blind, multicentre clinical trial comparing starting doses of 150 and 200 IU of recombinant FSH in women

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Westergaard LG, Laursen SB and Andersen CY (2000) Increased risk of early pregnancy loss by profound suppression

of luteinizing hormone during ovarian stimulation in normogonadotrophic women undergoing assisted

reproduction. Hum Reprod 15, 1003–1008.

Westergaard LG, Erb K, Laursen SB, Rex S and Rasmussen PE (2001) Human menopausal gonadotropin

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Effect of an OC pill on follicular

development in IVF/ICSI patients

receiving a GnRH antagonist:

a randomized study

JAF Huirne, AC van Loenen, J Donnez, C Pirard

R Homburg, R Schats, J McDonnell, CB Lambalk

RBM-online 2006: 13; 235-245

6

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ABSTRACT

BackgroundThis prospective randomized study compared the effectiveness of a gonadotrophin releasing hormone (GnRH) antagonist protocol with or without oral contraceptive (OC) pretreatment on the number of oocytes retrieved in IVF/ICSI patients. Material and MethodsSixty-four patients were randomized to start recombinant human FSH (rhFSH) on day 2 or 3 after OC withdrawal (OC group) or on day 2 of a natural cycle (control group). From stimulation day 6 onwards, all patients were treated with daily (0.5 mg/ml) GnRH antagonist (Antide). ResultsOC pretreatment resulted in signifi cantly lower starting concentrations of FSH, LH and oestradiol (P < 0.001) and a thinner endometrium (P < 0.0001). In the early stimulation period, fewer large follicles were found after OC pretreatment, leading to a signifi cantly extended stimulation period (11.6 versus 8.7 days, P < 0.0001) with more follicles on the day of recombinant human chorionic gonadotrophin administration (15.4 versus 12.5, P = 0.02) and more oocytes retrieved (13.5 versus 10.2, P < 0.001) as compared with the control group. ConclusionGnRH antagonist regimen, pretreated with OC, prevented the early endogenous FSH rise and improved follicular homogeneity, resulting in more oocytes. As a consequence of the extended treatment period, more rhFSH was required.

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INTRODUCTION

Gonadotrophin-releasing hormone (GnRH) antagonists have recently been introduced in assisted reproduction cycles to prevent premature luteinization (Al Inany et al., 2005; Del Prato and Borini 2005; Kolibianakis et al., 2005). The third generation antagonists turned out to be safe, with low histamine releasing properties and lower number of side effects in comparison with the long agonist protocols (European and Middle East Orgulatran Study Group, 2001; Fluker et al., 2001). Antide belongs to this generation of antagonists, with very low histamine releasing activity (Bajusz et al., 1988; Rivier et al., 1992). The minimal effective dose of Antide to prevent premature LH surges in IVF treatment cycles is 0.5 mg/ml (Huirne et al., 2004), and this compares well with the two GnRH antagonists which are registered for the prevention of premature LH surges (cetrorelix and ganirelix) (Huirne and Lambalk, 2001). Due to the immediate and dose-dependent suppressive action, GnRH antagonists require only a short administration period and signifi cantly fewer injections in comparison with the long agonist protocol. Therefore, from a patient´s point of view, GnRH antagonist regimens seem to be an attractive alternative for the long agonist protocol, which is still the standard therapy to prevent a premature LH surge in many IVF centres. However, the initiation of FSH administration in a GnRH antagonist regimen is cycle dependent and mostly started on day 2 of the natural cycle (Albano et al., 1997; Ganirelix Dose-Finding Study Group, 1998; Olivennes et al., 1998; Huirne and Lambalk, 2001). As a consequence, the date of oocyte retrieval cannot be predicted prospectively. Some kind of cycle scheduling has signifi cant practical and economical benefi ts due to improved prediction of the egg retrieval and scheduling of the workload (Templeton et al., 1984; Frydman et al., 1986). Oral contraceptive pills (OC) can be used to induce a withdrawal bleeding and to adjust the cycle to the patients’ and centre’s time plan. Additionally, using OCs, the endogenous gonadotrophin release is suppressed and the endogenous FSH rise, which normally precedes the luteofollicular transition in a natural cycle, can be attenuated (De Ziegler et al., 1998; Van Heusden and Fauser, 1999). By using a GnRH antagonist, slightly fewer follicles are seen at the time of recombinant human chorionic gonadotrophin (rhCG) injection and the number of recovered oocytes tends to be lower in comparison with the standard long agonist protocols (Albano et al., 2000; Borm and Mannaerts, 2000; European Middle East Orgulatran Study Group, 2001; Fluker et al., 2001). A likely explanation is that the long agonist protocols extend the duration of the ‘FSH window’ by suppressing the intercycle FSH rise. It is postulated that OC pretreatment in GnRH antagonist cycles, due to the prevention of the endogenous FSH rise, will lead to improved follicular homogeneity and therefore more large follicles on the day of rhCG and more oocytes retrieved in comparison with the GnRH antagonist cycle without OC pretreatment. The aim of this study was to test this hypothesis and to assess the feasibility, in terms of effi cacy and convenience, of this new scheduling regimen.

MATERIAL AND METHODS

Study design and patient characteristicsThis study, an open label randomized controlled study, was conducted in two centres: VU Medical Centre, Amsterdam, The Netherlands and Universite Catholique de Louvain, Cliniques Universitaires Saint-Luc Bruxelles, Belgium. The protocol was approved in both centres by the local Ethics Committees. The study was performed according to

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the Helsinki declaration. Sixty-four patients were randomly allocated to undergo an oral contraceptive (OC group) treatment followed by a GnRH antagonist regimen or a GnRH antagonist regimen without OC (control group). The treatment assigned to each patient was determined according to a computer generated randomization list. Patients needed to have a regular IVF or intracytoplasmic sperm injection (ICSI) indication (i.e. idiopathic infertility after six unsuccessful intrauterine inseminations, infertility based on male or tubal factor), a spontaneous regular ovulatory menstrual cycle, two ovaries and a normal uterine cavity, age between 18 and 38, and to have given their informed consent. Patients with FSH >12 IU/l on cycle day 2–4, body mass index >30 kg/m2, abnormal gynaecological bleeding, an extrauterine pregnancy within the last 3 months, any previous assisted reproductive technique cycles with fewer than 3 oocytes or severe ovarian hyperstimulation syndrome (OHSS), or patients with any contraindication to receive gonadotrophins or oral contraceptives, were excluded from the study. Patients with polycystic ovarian syndrome (defi ned as patients with oligomenorrhoea and at least two of the following criteria: elevated LH concentrations, signs of hyperandrogenism, or polycystic ovaries by ultrasound) were also excluded.

Treatment protocolOn cycle day 2 or 3, patients were randomized to receive either OC pretreatment (OC group) or not (control group) (see Figure 1). The control group started with recombinant human FSH (rhFSH) (Gonal-f® .Serono, Geneva, Switzerland) on day 2 or 3 of a natural cycle. In the OC group, patients started with daily OC pills (Microgynon 30®; Schering, Madrid, Spain, containing 30 μg ethinyl oestradiol and 150 μg levenorgestrel) on cycle day 2–3 for a variable period of 14–28 days. The date of the last OC intake was to be decided by the investigator on administrative criteria to schedule the initiation of stimulation. Instead of taking a fi xed number of days of OC pretreatment, it was decided to vary this duration allowing analyses of its effect on IVF outcome. Gonal-f administration was started 2 or 3 days after OC withdrawal, independent of their bleeding pattern. In both groups, rhFSH was administered daily up to the day of r-hCG administration. 6

Figure 1. Schematic overview of the treatment regimen with a gonadotrophin-releasing hormone antagonist (Antide) in patients undergoing ovarian stimulation with recombinant human FSH (rhFSH) (Gonal-f®). Patients were randomized in the oral contraceptive pretreatment group (OC group) or in the control group. S6 is stimulation day 6; rhCG = human chorionic gonadotrophin; OPU = ovum pick-up; ET = embryo transfer.

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The starting dose of rhFSH (150–300 IU) was maintained for 5 days, after which it could be adjusted according to the ovarian response (increase if fewer than 3 oocytes were ≥11 mm and decrease if a patient was at risk for OHSS) up to a maximal dose of 450 IU/day. From stimulation day 6 up to and including hCG day, a GnRH antagonist (antide/Serono) (0.5mg/ml per day) was given. A previous study demonstrated that this is the minimal effective dose to prevent premature LH surges in FSH hyperstimulated patients (Huirne et al., 2004). Subcutaneous injections were performed by trained health care professionals. Recombinant-hCG 250 μg (=6500 IU) (r-hCG; Ovitrelle®/Serono) was injected as soon as the largest follicle reached a mean diameter ≥18 mm and at least two other follicles a mean diameter ≥16 mm. A transvaginal oocyte retrieval was performed 34–38 h after rhCG administration under ultrasound guidance, followed by a standard IVF or ICSI procedure. Up to three embryos were replaced 2–3 days after oocyte retrieval. Remaining embryos were cryopreserved. After oocyte retrieval, intravaginal progesterone (3 times daily 200 mg progestan®; Organon, Oss, The Netherlands) was started as luteal support. This was continued up to a negative pregnancy test or at least the fi rst 3 weeks of pregnancy.

AssessmentsAfter giving written informed consent, a transvaginal ultrasound was performed on cycle day 2 or 3 to measure the antral follicular count and to exclude the presence of ovarian cysts or other abnormalities. A blood sample was taken to perform a pregnancy test. On stimulation day 1 and daily from stimulation day 6 onwards up to and including the day of rhCG, blood samples were taken in the morning before any drugs were given, to assess FSH, LH, oestradiol and progesterone concentrations, and transvaginal ultrasound was performed to monitor the number and size of the follicles and the endometrial thickness. Additional to the central assessed blood samples, oestradiol concentrations were also assessed locally to detect patients at risk for OHSS. The embryo quality was assessed on morphological criteria according to symmetry and extent of fragmentation of the blastomeres (Rijnders and Lens, 1993). In the case of ICSI, the cumulus cells were removed to assess the nuclear maturity. Side effects or local skin reactions were recorded daily on a personal diary card.

Hormonal assaysBlood samples were centrifuged at 1328 g within 1 h after withdrawal and stored at −20°C until assayed. Serum LH, FSH, oestradiol and progesterone concentrations were measured by a central laboratory (LCG Bioscience Services Ltd), using Sorin radioimmunoassay (Sorin, Italy) and DPC Coat-a-Count direct radioimmunoassay for oestradiol and progesterone respectively (DPC, Los Angeles, CA, USA). FSH and LH concentrations were analysed using Serono MAIAclone immunoradiometric assay.

StatisticsThe primary effi cacy endpoint was the number of oocytes retrieved on the day of oocyte retrieval. Secondary endpoints were number and size of follicles, cumulative dose of Gonal-f, duration of FSH treatment, number of (viable) embryos (transferred and cryopreserved), pregnancy and implantation rates, serum hormone concentrations, endometrial thickness and bleeding pattern. To test a difference of three oocytes under the assumption of a standard deviation of 4.2, α = 0.05 and β = 0.20 (power 80%), 30 patients were to be included in each arm. The estimated standard deviation was based on the standard deviation in a previous study in which 0.5 mg/ml antide was used without OC pretreatment (Huirne et

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al., 2004) and on a study in which OC pretreatment was given (Obruca et al., 2000). Based on these calculations, it was intended to randomize 32 patients in each arm. One patient, randomized to receive OC, withdrew consent after she was randomized, as she did not want to take contraceptives. For all continuous data, non-parametric rank sum tests (Mann−Whitney U) were used, and proportions were analysed using Fisher’s exact tests. Discrete variables with a limited range (e.g. the number of follicles, oocytes and embryos) were analysed using Poisson regression analyses. To test the effect of differences between the two centres on the main results, multivariate regression and univariate analyses were used. Analyses were based on an intention-to-treat principle, and all randomized subjects who received at least one dose of the OC pill or rhFSH were included (n = 63). Since there were no signifi cant associations between duration of OC pill intake and baseline characteristics or IVF outcome parameters, the OC pill group was treated as one single group for statistical analyses. All statistical tests were performed two-sided at the signifi cance level of 5%.

Randomization Treatment allocation was decided by an independent person from an independent monitoring company according to a computer-generated, blocked randomization list. The randomization was stratifi ed by centre.

RESULTS

Baseline characteristics and cancellationsA total of 64 patients were randomized, 32 patients to each group. The two treatment groups were comparable for demographic characteristics (see Table 1). Tubal and male factor were the main causes of infertility. All patients who started the stimulation phase received rhCG and had at least one oocyte retrieved (n = 63). Five patients did not undergo an embryo transfer (OC: n = 3, control: n = 2) due to nonfertilization (n = 2), insuffi cient embryonal maturation (n =2), and in one case embryo transfer was not performed since the patient was hospitalized because of tubal infection after oocyte retrieval (see Figure 2). Nevertheless, these patients were included for the analyses of the primary endpoint, since all patients had oocyte retrieval. 237

Stimulation characteristicsThe mean duration of OC intake was 19.7 (±3.8) days. Drug requirements are presented in Table 2. The stimulation period was approximately 3 days longer in the OC group (P < 0.001). The total amount of Gonal-f administered was substantially higher (P < 0.001) in this group, partly due to the longer duration of the stimulation phase as described above. The starting dose was similar in both groups. Based on inadequate follicular response, in 20 patients (63.5%) in the OC group and 12 patients (39%) in the control group the rhFSH dose was increased on stimulation day 6. Antide administration ranged between 1 and 12 days in the entire study, the mean duration in the OC group and the control group was 6.6 and 3.7 days respectively (P < 0.001).

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Randomization group

OC group Control group P value

Number of patients (ITT) 32 32

Age (years) 32.3 (4.0) 33.3 (3.8) 0.35

Body mass index (kg/m2) 22.5 (3.2) 22.8 (4.0) 0.84

Cycle length (days) 28.7 (1.3) 29.5 (1.8) 0.14

Primary infertility 22 (68.8%) 22 (68.8%) 1.00

Smoking habits (% smokers) 7 (21.8%) 7 (21.8%) 1.00

Duration of infertility (years) 5.0 (2.8) 4.6 (2.5) 0.84

Type of infertilitya:

Tubal factor 4 (12.5%) 10 (31.3%) 0.13

Endometriosis 3 ( 9.4%) 0 ( 0 %) 0.24

Male factor 18 (56.3%) 17 (53.1%) 1.00

Unexplained 8 (25.0%) 7 (21.9%) 1.00

Other 1 ( 3.1%) 0 ( 0 %) 1.00

Antral follicle countb 10.1(3.8) 9.7 (3.9) 0.70

Table 1. Comparison of patients characteristics, between the oral contraceptive (OC) and control groups. Values are mean (± standard deviation). Analyses were performed per intention to treat (ITT) using Mann–Whitney U-test, except for proportions where Fisher’s exact test was used. aMore options are possible. bNumber of follicles ≤ 11mm on cycle day 2 or 3 in a natural cycle.

The mean numbers of follicles ≥11 and ≥14 mm on stimulation days 6, 7 and 8 were signifi cantly fewer in the OC group compared with the control group (Figure 3, P < 0.05). Although fewer follicles ≥11 and 14 mm were observed in the OC group during the early stimulation phase, this resulted in 2−3 more large (≥11 and 14 mm) follicles on the day of rhCG administration (see Figure 3).

Randomization group OC group Control group P value

Number of patients 31 32

Daily dose of rhFSH (IU) 250 (64) 224 (56) 0.17

rhFSH starting dose (IU) 224 (55) 216 (56) 0.58

Duration of rhFSH treatment (days) 11.6 (2.1) 8.7 (1.6) <0.001*

Cumulative dose of rhFSH (IU) 2958 (1162) 1950 (616) <0.001*

rhFSH dose increase (% of patients) 20/31 (63.5%) 12/32 (39%) 0.08

Duration of Antide treatment (days) 6.6 (2.1) 3.7 (1.6) <0.001*

Table 2. Comparison of drug requirements in the oral contraceptive (OC) and control groups.Values are mean (± standard deviation). Analyses were performed in all patients receiving at least one OC pill in the OC group or one rhFSH injection in the control group, using Mann-Whitney U-test, except for the rhFSH dose increase which was analyses using Fisher’s Exact test. S6 = stimulation day 6. ∗ p < 0.05.

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Figure 2. The fl ow of the participants though each stage of the trial. OC = oral contraceptive; rhFSH = recombinant FSH; Antide = gonadotrophin-releasing hormone antagonist; rhCG = recombinant human chorionic gonadotrophin; OPU = oocyte retrieval; ET = embryo transfer.

Figure 3. Follicular growth pattern. (A), number of follicles ≥11 mm on various stimulation days and the day of recombinant human chorionic gonadotrophin (rhCG) administration (hCGd). (B) Number of follicles ≥14 mm on these days. Values are mean, with SD (error bars). Analyses were performed in all patients receiving at least one OC pill in the OC group or one rhFSH injection in the control group using Mann-Whitney U-tests. S1, S6, S7, S8 and S9 = stimulation day 1, 6, 7, 8 and 9 respectively. rhCG = day of rhCG administration. OC = oral contraceptive group. *P < 0.05

Eligible patients who gave informed consentwere randomized (n=64)

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had rhFSH (n=32) had Antide (n=32) had rhCG (n=32) OPU (n=32) ≥ 1oocyte (n=32) ET (n=30) luteal support (n=30) pregnancy test (n=30)

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Hormonal concentrationsHormonal concentrations on stimulation day 1 (S1) are presented in Table 3. The serum FSH, LH, oestradiol and progesterone concentrations were signifi cantly lower on S1 in the OC group versus the control group (P < 0.001 for FSH, LH and oestradiol, P < 0.02 for progesterone). The hormonal concentrations during the period of GnRH antagonist administration are presented in Figure 4. From stimulation day 6 onwards, including the rhCG day, the FSH concentrations were comparable in the two groups. The LH concentrations were signifi cantly lower on stimulation days 7, 8 and 9 in the OC group compared with the control group (P <0.01) (Figure 4). The oestradiol concentrations remained lower in the OC group until stimulation day 9 (P<0.01), on the day of r-hCG administration these concentrations became comparable (see Table 3). The progesterone concentrations were comparable in the two groups; on stimulation day 8 they were slightly lower (P = 0.009) and on the day of rhCG they were higher in the OC group in comparison with the control group (P = 0.013) (see Table 3).

Randomization group OC group Control group P value

Hormonal concentrations on S1

Number of patients 31 32

FSH (IU/l) 3.3 (2.3) 7.7 (3.0) <0.001*

LH (IU/l) 2.2 (1.5) 3.6 (1.3) <0.001*

Oestradiol (pmol/l) 156 (121) 241 (77) <0.001*

Progesterone (nmol/l) 2.2 (1.1) 3.0 (1.7) 0.020*

Hormonal concentrations on rhCG day

Number of patients 30 31

FSH (IU/l) 15.6 (6.5) 13.7 (4.3) 0.303

LH (IU/l) 1.6 (0.7) 2.4 (1.3) 0.004*

Oestradiol (pmol/l) 6220 ± 3708 5744 ± 3400 0.670

Progesterone (nmol/l) 5.2 (2.0) 4.0 (1.5) 0.013*

Table 3. Comparison of hormonal concentrations on stimulation day 1 and day of recombinant human chorionic gonadotrophin (rhCG) administration between the oral contraceptive (OC) and the control groups. Values shown are mean (± standard deviation). Analyses, using Mann-Whitney U-tests, were performed in all patients receiving at least one OC pill in the OC group or one recombinant human FSH (rhFSH) injection in the control group. S1 = stimulation day 1. * p < 0.05.

IVF harvestAll patients had an oocyte retrieval performed and at least 2 oocytes were retrieved. In the OC group, 3 more oocytes were retrieved compared with the control group (P < 0.001) (see Table 4). The mean number of inseminated oocytes was also higher in the OC group versus the control group (P = 0.03) as were the mean number of mature oocytes (metaphase II oocytes) in ICSI patients (P = 0.02), but the percentages of metaphase II oocytes and mean fertilization rates were not different in both groups (see Table 4). On average, the numbers of embryos were comparable in both groups, but the number of good quality embryos (grade I and II) were higher in the OC group versus the control group, 5.3 versus 3.7, (P < 0.01). The number of embryos cryopreserved was slightly higher in the OC group (see Table 4).

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Randomization group

OC group Control group P value

Number of patients 31 32

Number of oocytes 13.5 (6.7) 10.2 ± 6.0 <0.01*

Number of oocytes inseminated 10.9 (6.5) 9.2 ± 6.0 0.03 *

Number of patient with IVF 9 (29%) 13 (41%) 0.33

Number of metaphase-II oocytesa 10.0 (5.8) 8.9 ± 6.3 0.02 *

Metaphase-II oocytesa (%) 69.9 69.7 0.91

Number of embryos (2PN) 6.3 (4.7) 5.8 (4.8) 0.44

Fertilization rateb (%) 56 62 0.41

Number of good quality embryos (grI,II) 5.3 (4.5) 3.7 (4.1) <0.01*

Number of embryos replaced/ETc 2.3 (0.5) 2.1 (0.6) 0.77

Number of embryos cryopreserved 2.4 (3.6) 0.9 (2.4) <0.01*

Positive pregnancy test (%) 8/31 (26%) 13/32 (41%) 0.29

Clinical pregnancy rate (%) 4/31 (13%) 12/32 (38%). 0.04*

Ongoing pregnancy rate (%) 4/31 (13%) 8/32 (25%) 0.34

Implantation rated 0.07 0.22 0.05*

Positive pregnancy test/ETc (%) 8/28 (29%) 13/30 (43%) 0.28

Clinical pregnancy rate/ETc (%) 4/28 (14%) 12/30 (40%) 0.04*

Ongoing pregnancy rate/ETc (%) 4/28(14%) 8/30 (27%) 0.34

Table 4. IVF outcome in the oral contraceptive (OC) and control groups. Values are mean (± standard deviation). Analyses were performed in all patients receiving at least one OC pill in the OC group or one rhFSH injection in the control group, unless otherwise stated. Poisson regression analyses were used, except for proportions where Fishers’s Exact tests was used. S1=stimulation day 1; ET=embryo transfer; Positive pregnancy test is hCG serum level >10 IU; Clinical pregnancy= ≥ 1 intra-uterine sac by ultrasound at gestational age of 6 weeks; Ongoing pregnancy is intra-uterine heart activity at a gestational age of 12 weeks. aAssessed if intracytoplasmic sperm injection was performed. bFertilization rate = nr of embryos/nr of oocytes inseminated. cAnalyses were performed per embryo transfer. dImplantation rate = number of foetal sacs/number of embryos replaced, mean number of implantation rate is the mean of the individual implantation rates. * p < 0.05.

Although the number of patients with a positive pregnancy test (hCG >10 IU/l) was similar in both study groups, fewer clinical pregnancies (defi ned as ≥1 intrauterine fetal sac) were seen in the OC group (Table 4). In the OC group, three patients had a biochemical pregnancy, one had an ectopic (tubal) pregnancy and in all patients with an intrauterine sac, heart activity could be detected. In the control group, one biochemical pregnancy was found and in two patients with intrauterine sacs, no heart activity could be detected. Two of the remaining pregnancies with initial positive heart activity ended as missed abortions when heart activity failed, leaving eight ongoing prregnancies. The mean implantation rate, defi ned as the mean of all individual implantation rates, was lower in the OC group in comparison to the control group (P = 0.05) (see Table 4). The differences in ongoing pregnancy rates did not reach statistical signifi cance (see Table 4). Two twin pregnancies occurred in the control group.

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Although, the number of OC pills taken varied between 14 and 27 days, the 95% confi dence interval was relatively small (18.4−21.1). Therefore, it is assumed that the risk for any introduction of variance in outcome parameters by the duration of OC pill intake will be small. This is underlined by the fact that no signifi cant correlation could be found between basic characteristics or IVF outcome parameters (number of oocytes, embryos, pregnancy rates) and the duration of OC use (data not shown). There was also no signifi cant correlation between duration and amount of GnRH antagonist administration and any of the IVF outcome parameters (number of oocytes, embryos, pregnancy rates), (data not shown).

Differences between the two centresBaseline characteristics were similar in the two centres, except for duration of infertility. The mean duration was 5.5 (±2.7) in centre one and 3.2 (±2.0) years in centre two (P = 0.02). Main outcome results, in terms of duration and total dose of rhFSH used, serum hormone concentrations, number of oocytes and embryos, were similar in both centres.

Endometrium and bleeding characteristicsThe bleeding pattern before antagonist administration was different in the two groups (see Figure 5). The onset of withdrawal bleeding started mostly around days 2 and 3 of the stimulation. Three patients in the OC group were still bleeding on day 8 of the stimulation. From day 10 onwards, none of the patients experienced further vaginal bleeding. Patients in the control group started earlier with bleeding, and all were bleeding at the start of stimulation (by defi nition). The mean duration of bleeding was 4.1 days in the OC group and 3.4 in the control group. The period between the last day of bleeding and the day of rhCG administration were comparable in the OC and control groups, 7.6 and 6.3 respectively. Using logistic regression analyses, no signifi cant relation was observed between any of the bleeding characteristics (duration of the bleeding-free period before r-FSH administration or before rhCG administration, duration of the bleeding period) and clinical pregnancy rate.

Figure 5. Bleeding pattern; percentage of patients with vaginal bleeding on various stimulation days and on the day of recombinant human chorionic gonadotrophin (rhCG) administration.S1=stimulation day 1; S2=stimulation day 2 etc. OC=Oral contraceptive group; Control=control group.

Endometrial thickness was similar in both groups on the pre-study visit, stimulation day 1 and the day of rhCG administration. This parameter was only different on stimulation day 6, the mean endometrial thickness was >3 mm thinner in the OC group (see Figure 6).

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Figure 6. Endometrial thickness was measured before treatment (cycle day 2 or 3 of a natural cycle), and on stimulation day 1 (S1), stimulation day 6 (S6) and on the day of recombinant human chorionic gonadotrophin administration (rhCG). Values are mean, the error bars represent SD, analyses were performed per intention to treat using Mann−Whitney U-test.OC=oral contraceptive group; Control=control group. *p < 0.0001.

Safety evaluationThe treatment was well tolerated. In total, 117 new adverse events were reported in 51 patients. The majority of the adverse events (98%) were reported as mild, fi ve were moderate and one was severe (tubal infection after oocyte retrieval).

Randomization group

OC group Control P value

Number of patients 31 32

Nr patients with at least one AE 25 (80.6 %) 26 (81.2 %) 0.60

Mean nr AE per patient (±SD)a 2.0 (±1.4) 1.7 (±1.4) 0.44

Total new reported AE 62 55

Adverse events reported b

Administration site conditions 14 (45.2 %) 10/32 (31.3 %) 0.19

Headache 14 (45.2 %) 12 (37.5 %) 0.36

Abdominal discomfort 7 (22.6 %) 8 (25.0 %) 0.53

Gastrointestinal disorders 7 (22.6 %) 7 (21.9 %) 0.59

General disorders (fatigue) 6 (19.4 %) 5 (15.6 %) 0.48

Ovarian cysts 2 ( 6.5 %) 4 (12.5 %) 0.35

Mood changes 2 ( 6.5 %) 2 ( 6.3 %) 0.68

Other 10 (32.3 %) 7 (21.9 %) 0.26

Table 5. Adverse events (AE) occurring in the oral contraceptive (OC) and control groups during the study. Analyses were performed in all patients receiving at least one OC pill in the OC group or one recombinant human FSH (rhFSH) injection in the control group. Fishers Exact tests were used, unless otherwise stated. SD=standard deviation. aMann-Whitney U-test was used. bMore than one option is possible per patient; values are number of patients who reported these AE (percentage of patients in which these events were reported).

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The most frequently reported adverse events were injection site reactions (20.5%), headache (22.2%), abdominal pain (12.8%), gastrointestinal discomfort such as nausea (12.0%), fatigue (9.4%), ovarian cyst (5.1%) and mood changes (3.4%). Ovarian hyperstimulation was observed twice, only in the OC group; both cases were considered to be mild, treatment or admission was not required, one of these patients turned out to be pregnant. The number and type of reported adverse events per patient were similar in both groups (see Table 5). Overall, Antide injections were well tolerated.

DISCUSSION

The aim of this study was to assess the effect of oral contraceptive pretreatment in GnRH antagonist treated IVF/ICSI patients on the coordination of follicular development and the number of oocytes retrieved, in comparison to a control group without OC use. In addition, it was demonstrated the feasibility in terms of effi cacy and convenience of this scheduling regimen which was well tolerated by the patients. OC pretreatement offers a potential solution to the cycle dependency of the initiation of GnRH antagonist treatment.This new regimen allows, due to suppression of the hypothalamic−pituitary−ovarian axis by OC for a predetermined number of days, planning of the initiation of stimulation and therefore to a greater extent prediction of the oocyte retrieval, allowing scheduling of the workload. As soon as the stimulation is started, the oocyte retrieval is expected 13.6 (±2.1) days later in the OC pretreated group and 10.7 (±1.6) days later in the control group. A recent comparative study indeed demonstrated the same scheduling ability of an OC pretreated GnRH antagonist regimen compared with a long GnRH agonist protocol, with only 5.9% oocyte retrieval in weekends or public holidays (Huirne et al., 2006). Scheduling the initiation of the treatment might be especially helpful in patients with an irregular cycle. The application of such an approach in polycystic ovarian syndrome patients resulted in similar IVF outcome parameters in the OC pretreated GnRH antagonist and long GnRH agonist protocol (Hwang et al., 2004).

It was clearly demonstrated that OC pretreatment in a fi xed GnRH antagonist protocol resulted in a signifi cant reduction (P < 0.05) of large follicles (≥14 mm) in the early stimulation period (stimulation day 6), compared with the control group, indicating that the development of potential dominant follicles had been suppressed. The follicle cohort was more uniformly developed after OC pretreatment with almost no large follicles before stimulation day 8. As a consequence, the criteria of rhCG administration were met later in the OC group, resulting in an increased duration of the treatment with an extended period of rhFSH administration and leading in the end to more follicles and oocytes. A relative disadvantage was the increase in the total dose of rhFSH required. This new approach leads to a comparable number of oocytes retrieved as in most standard long protocols using a GnRH agonist (Barmat et al., 2005; Huirne et al., 2006; Rombauts et al., 2006). This is in contrast to the GnRH antagonist regimens without OC pretreatment, in which lower numbers of oocytes were retrieved compared with GnRH agonist cycles in several phase III studies (Albano et al., 2000; Borm et al., 2000; European and Middle East Orgulatran Study Group, 2001; Fluker et al., 2001; Al-Inany and Albouhar et al., 2002; Rombauts et al., 2005). The use of oestradiol before GnRH antagonist administration is also able to induce improved follicular development (Franchin et al., 2003). These studies and the present work point to a more homogeneous oocyte development after steroid hormone administration

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prior to GnRH antagonists. Apparently, the attenuated FSH rise in the OC group prevented the development of larger follicles in the early stimulation period. It has been shown that a large FSH rise occurs 5−7 days after OC withdrawal (Van Heusden and Fauser, 1999). This endogenous FSH rise can also be attenuated by oestrogens only, an FSH rise consistently occurred 3 days after discontinuation of oestrogen medication (De Ziegler et al., 1998). It was decided to start rhFSH administration no later than 2 or 3 days after discontinuation of the OC pills, in order to completely circumvent this endogenous FSH rise. Besides the signifi cantly lower FSH concentrations (P < 0.001) during the early stimulation phase, LH and oestradiol concentrations increased at a slower rate in the OC group. This suggests that the hypothalamic−pituitary axis was indeed more profoundly suppressed after OC pretreatment, if stimulation was started on day 2 or 3 after the last OC pill taken. 243 Improved oocyte yield was not reported in two studies in which stimulation was started 5 days after the last OC intake in GnRH antagonist cycles (Obruca et al., 2000; Barmat et al., 2005; Kolibianakis et al., 2006). Follicular development may be affected by the starting time of gonadotrophin administration after OC withdrawal. Further studies are required to establish this. Sustained suppression of LH, oestradiol and progesterone concentrations in the OC group may contribute to the observed differences in endometrial thickness on stimulation day 6. Apparently, changes have already occurred during the early follicular phase. No differences in endometrial thickness were measured further more on the day of rhCG administration. The extended period of exogenous rhFSH administration with ultimately similar oestradiol concentrations may be responsible for this. Alternatively, the differences in bleeding pattern may also affect the measured endometrial thickness. Fewer bleedingfree days were observed on stimulation day 6 in the OC group compared with the control group. The results are in agreement with the lower endometrial thickness reported on day 6 only of stimulation in the OC pretreated group compared with a control group (Kolibianakis et al., 2006).

Besides the improved follicular development and quantative oocyte recruitment, the number of inseminated oocytes, the absolute number of metaphase II oocytes in ICSI patients and the number of good quality embryos were also increased after OC pretreatment. However, the increased number of good quality embryos with its improved ability for optimal embryo selection did not lead to higher clinical pregnancy rates. In contrast, despite similar biochemical and ongoing pregnancy rates, lower clinical pregnancy and implantation rates were observed in the OC group, in comparison with the control group. Similar results with respect to improved follicular development and a trend towards lower implantation rates were recently reported after OC pretreatment in a fl exible GnRH antagonist regimen, with gonadotrophin administration 2 days after OC withdrawal (Rombauts et al. 2006). Several explanations for the lower implantation rates of this regimen can be postulated. In the fi rst place, the initiation time of rhFSH administration may play a role. Another study that started rhFSH on day 5 after OC withdrawal showed no differences in the rather high pregnancy rates per embryo transfer between the OC and a control groups, 39.7 and 41.2% respectively (Obruca et al., 2000). Treatment was started on day 2 or 3 after OC withdrawal, independent of the bleeding pattern, in an attempt to optimally synchronize the follicular cohort. As a consequence, most patients started with bleeding during the early stimulation period and some were even bleeding some days before rhCG administration. This difference in bleeding pattern may have had a detrimental effect on endometrial development and its receptivity. On the other hand, no signifi cant relation was observed between any of the bleeding characteristics and clinical pregnancy rate. Additionally, a very recent study, comparing a fi xed GnRH antagonist regimen with or without OC pretreatment, in which gonadotrophin administration was started 5 days after OC withdrawal, reported a signifi cant increased

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pregnancy loss after OC pretreatment (Kolibianakis et al., 2006). A second explanation for the lower clinical pregnancy rate may lie in the persistently lower LH concentrations observed in the OC group. The role of low LH concentrations on implantation in GnRH antagonists is still a matter of debate (Kol, 2005). Several studies could not fi nd any effect on pregnancy outcome in GnRH antagonist cycles of the absolute LH value on a specifi c day (Bosch et al., 2003; Cédrin-Durnerin et al., 2004; Merviel et al., 2004). Others found even lower pregnancy rates when the LH concentrations were rather high (Kolibianakis et al., 2003a,b). In a previous study, using antide, it was shown that the level of change in LH and progesterone rather than the absolute values may be important. If the LH and progesterone concentrations increased or decreased too much, no pregnancies occurred (Huirne et al., 2005). The LH concentrations in the current study were signifi cantly lower in the OC groups versus the control group (see Table 3). Unfortunately, due to the assay that was used, it was not possible to detect LH concentrations below 1.0 IU/l. Therefore, the actual LH concentrations might even have been lower than the reported values. As a third explanation it could be that the GnRH antagonist itself played a role. This is unlikely, since the clinical pregnancy rates in the control group, with GnRH antagonist only, were rather high (38%). In addition, no relation was found between the duration and total amount of GnRH antagonist and pregnancy outcome. Finally, it cannot be ruled out that the observed differences in clinical pregnancy rates are based on chance, since this study was not powered to study pregnancy rates. Thus, by selecting number of oocytes as the primary endpoint, this study has only limited value with respect to pregnancy and implantation rates, but it can be of use in future meta-analyses.

In conclusion, this study has demonstrated that this new GnRH antagonist treatment regimen with OC pretreatment, starting on the 2nd or 3rd pill-free day, is feasible and well tolerated and it may offer a new treatment strategy using antagonists which could facilitate better planning of the initiation of the treatment. OC pretreatment in GnRH antagonist cycles leads to initially lower FSH concentrations and as such improves follicular homogeneity but also results in an extended duration of rhFSH stimulation with more rhFSH required, with fi nally more oocytes at retrieval and more good quality embryos. However, the improved quantative oocyte yield and scheduling needs to be weighed against the unexpected possible lower pegnancy rates, which needs further study.

AcknowledgementThe authors thank Serono Geneva for kindly supplying the Antide.

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rFSH in women with oral contraceptives before in vitro fertilization. Fertility and Sterility 83, 321–330.

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surges in women undergoing ovarian stimulation. Human Reproduction 16, 644–651.

Fluker M, Grifo J, Leader A et al. 2001 Effi cacy and safety of ganirelix acetate versus leuprolide acetate in women

undergoing controlled ovarian hyperstimulation. Fertility and Sterility 75, 38–45.

Franchin R, Salomon L, Caselo-Branco A et al. 2003 Luteal oestradiol pre-treatment coordinates follicular growth

during controlled ovarian hyperstimulation with GnRH antagonists. Human Reproduction 18, 2698–2703.

Frydman R, Forman R, Rainhorn JD et al. 1986 A new approach to follicular stimulation for in vitro fertilization–

programmed oocyte retrieval. Fertility and Sterility 46, 657–662.

Ganirelix Dose-Finding Study Group 1998 A double-blind, randomized, dose-fi nding study to assess the effi cacy

of the gonadotrophinreleasing hormone antagonist ganirelix (Org 37462) to prevent premature luteinizing

hormone surges in women undergoing ovarian stimulation with recombinant follicle stimulating hormone

(Puregon). Human Reproduction 13, 3023–3031.

Huirne JA, Lambalk CB 2001 Gonadotropin-releasing-hormone-receptor antagonists. Lancet 358, 1793–1803.

Huirne JA, Hugues JN, Pirard C et al. 2006 Cetrorelix in an OC pretreated stimulation cycle compared to buserelin

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Huirne JA, Van Loenen AC, Schats R et al. 2005 Dose-fi nding study of daily GnRH antagonist for the prevention

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(GnRH) antagonist for the prevention of premature luteinizing hormone surges in IVF/ICSI patients: Antide

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Kolibianakis EM, Papanikolaou EG, Camus M et al. 2006 Effect of oral contraceptive pill pretreatment on ongoing

pregnancy rates in patients stimulated with GnRH antagonists and recombinant FSH

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Effect of timing gonadotrophins administration after OC withdrawal and rLH addition on follicular development and hormonal concentrations in GnRH antagonist cycles; a pilot study

JAF Huirne, AC van Loenen, J Donnez, C Pirard,

J McDonnell, R Schats, R Homburg, CB Lambalk

Submitted

7

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ABSTRACT

BackgroundThis trial was designed to assess the effect of starting time (day 2 versus day 5) of gonadotrophin stimulation after pretreatment with oral contraceptives (OCs) and rhLH addition on follicular development and hormonal concentrations in a fi xed GnRH antagonist protocol. Material and methodsFourty eight IVF/ICSI patients treated with a fi xed day 6, GnRH antagonist protocol, were randomized to start with daily 150 IU rhFSH 2 or 5 days after last OC taken, with or without addition of 150 IU rhLH/day. ResultsStarting on day 2 versus day 5 leads to signifi cantly lower starting concentrations of FSH, LH and oestradiol, a thinner endometrium (p<0.001) and less large follicles on S6, an extended stimulation period (8.0 versus 4.8 days, p<0.001) and more rhFSH required (1981 and 1474 IU, respectively) (p<0.001). Hormonal concentrations and number of oocytes inseminated were similar on the day of hCG administration (hCGd). Addition of daily rhLH leads to higher LH concentrations (all >1.0 IU/L), less large follicles on hCGd and a lower number of oocytes available for insemination (p<0.001).ConclusionsAlthough the follicular cohort is more uniformly developed if gonadotrophins administration is started 2 versus 5 days after OC withdrawal, this does not lead to an improved oocyte yield. Starting with gonadotrophins 5 days after OC withdrawal is to be preferred in view of shorter treatment and less gonadotrophin requirements. Addition of 150 IU rhLH per day in OC pretreated GnRH antagonist regimen results in higher LH levels but may produce less follicles and oocytes.

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INTRODUCTION

GnRH antagonists were recently introduced in assisted reproduction cycles for prevention of premature luteinization. Due to the immediate and dose-dependent suppressive action, GnRH antagonists require only a short administration period in comparison to the long agonist protocol which improves patient convenience (Huirne & Lambalk 2001). Several large phase III studies have proven the effi cacy of GnRH antagonists to prevent LH surges during controlled ovarian hyperstimulation in IVF and ICSI therapies (Albano et al. 2000; Borm & Mannaerts 2000; Olivennes et al. 2000; Fluker et al. 2001). The fi rst large studies did not show a signifi cant difference in pregnancy outcome, but a meta-analysis suggested a difference of 5% in favour of the long GnRH agonist protocols (Al-Inany & Aboulghar 2002). Less oocytes were retrieved in the GnRH antagonist protocols, but the stimulation period was shorter with less rhFSH required. The GnRH antagonist was administered daily from stimulation-day 6 onwards until the day of hCG administration in the fi rst large phase III studies in the multiple dosing regimens (Albano et al. 2000; Borm & Mannaerts 2000; Fluker et al. 2001) or on day 7 or 8 in the depot regimens (Olivennes et al. 2000). From 15 years of experience with the GnRH agonists in IVF we have learned that the long protocol starting in the midluteal phase of the preceeding cycle is the optimal protocol with regard to highest yield of oocytes, good quality embryos and the best pregnancy results (Huirne et al. 2004). The optimal regimen for the use of GnRH antagonists in IVF to achieve optimal number of oocytes and pregnancy rates is not established yet.

Several studies used oral contraceptive (OC) pretreatment in GnRH antagonist cycles for programming purposis or to prevent ovarian cyst formation. In some studies gonadotrophin administration was started 2 or 3 days (Cedrin-Durnerin et al. 2004; Cheung et al. 2005; Huirne et al. 2006a; Rombauts et al. 2006) and others 4 or 5 days after OC withdrawal (Obruca et al. 2000; Hwang et al. 2004; Sauer et al. 2004; Barmat et al. 2005; Kolibianakis et al. 2005; Huirne et al. 2006b; Koichi et al. 2006) in either fl exible or fi xed GnRH antagonist protocols. Sofar no studies have been published examining the effect of timing gonadotrophin administration after oral contraceptive pill withdrawal on hormonal levels and follicular development.

GnRH antagonist regimen pretreated with OCs compared with no OCs, in which rhFSH is started 2 or 3 days after OC withdrawal, induces deep suppression of LH and FSH levels and a more homogeneous development of the follicular cohort compared with no OC pretreatment (Huirne et al. 2006a). However, despite more oocytes and embryos of good quality in the OC pretreated group, the observed clinical pregnancies and implantation rates seemed to be lower in this (small) study (Huirne et al. 2006a). Whether the deep suppression of LH levels in such a protocol may effect reproductive outcome is still a matter of debate. Although the concentration of residual endogenous LH levels during GnRH agonist pituitary desensitization are usually suffi cient to achieve adequate follicular maturation and optimal IVF results (Sullivan et al. 1999; Balasch et al. 2001; Penarrubia et al. 2003), some normogonadotrophic women developed deep LH suppression which has been associated with adverse reproductive outcomes (Fleming et al. 1998; Westergaard et al. 2000; Esposito et al. 2001). Today, the optimal LH levels required in GnRH antagonist IVF cycles still need to be defi ned. Several studies could not fi nd any effect of the absolute LH value on a specifi c day during a fi xed GnRH antagonist cycle (Bosch et al. 2003; Merviel et al. 2004). Others found improved pregnancy rates when the LH levels were more profoundly suppressed on stimulation day 8 in a fi xed (multiple dose,

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starting day 6) GnRH antagonist protocol (Kolibianakis et al. 2003). In an earlier study with a fi xed (multiple dose, starting day 6) GnRH antagonist protocol we showed that rather than the absolute LH level, the changes in LH were associated with pregnancy outcome. No pregnancies occurred if the LH levels increased or declined too much (Huirne et al. 2005). The daily addition of 75 IU rhLH to rhFSH from stimulation day 1 onwards in an OC pretreated fl exible single dose GnRH antagonist protocol (Griesinger et al. 2005) or in a fi xed (multiple dose, starting day 6) GnRH antagonist protocol does not improve pregnancy outcome in an unselected IVF population (Cedrin-Durnerin et al. 2004). The various effects of LH addition probably depend on specifi c conditions, studied population and the level of LH suppression. LH levels are very deeply suppressed if the GnRH antagonist is administered in a fi xed (multiple dose, starting day 6) protocol directly (2 or 3 days) after withdrawal of OCs. Such a protocol leads to mean LH levels ≤ 0.5 IU/L in the early follicular phase (Huirne et al. 2006a). In the current study we examine the additional effect of rhLH administration under these conditions, i.e. in OC pretreated GnRH antagonist regimens in which rhFSH is started on either day 2 or day 5 after the last OCs.

The aim of the current pilot study was to test the effect of starting with gonadotrophin administration on day 2 versus day 5 after the last OC and the effect of the addition of rhLH to GnRH antagonist cycles on hormonal levels and follicular development.

MATERIAL AND METHODS

Study design and patients characteristicsThis open label randomized controlled study, was conducted in two centres: VU medical centre, Amsterdam, The Netherlands and Universite Catholique de Louvain, Cliniques Universitaires Saint-Luc Bruxelles, Belgium. The protocol was approved in both centres by the respective Ethics Committees and performed according to the Helsinki declaration. Fourty-eight patients were randomly allocated to start with rhFSH (150 IU/day) stimulation with or without daily addition of 150 IU rhLH, 2 days (day 2 group) or 5 days (day 5 group) after the last OC taken. The treatment assigned to each patient was determined according to a computer generated concealed randomization list. Patients needed to have routine IVF/ICSI indication, regular ovulatory cycles, two ovaries and a normal uterine cavity, aged between 18 and 38 and having given their informed consent. Patients with FSH > 12 IU/l on cycle day 2-4, a body mass index > 30 kg/m2, abnormal gynaecological bleedings, an extrauterine pregnancy within the last 3 months, any previous assisted reproductive technique (ART) cycles with less than 3 oocytes or severe ovarian hyperstimulation syndrome (OHSS), or patients with any contraindication to receive gonadotrophins or oral contraceptives were excluded from the study. Patients with polycystic ovary syndrome (defi ned following the Rotterdam consensus defi nition) were also excluded.

Treatment ProtocolAfter giving written consent, patients were randomized on day 2 or 3 of a natural cycle to start with gonadotrophins on day 2 or day 5 after the last OC and to be stimulated with daily 150IU rhFSH (Gonal-f®/Serono International, Geneva, Switzerland) or 150IU rhFSH plus 150IU rhLH (Luveris/Serono International, Geneva, Switzerland) up to the day of hCG administration, resulting in four groups: day2-rhFSH group, day2-rhFSH+rhLH group, day5-rhFSH group and day5-rhFSH+rhLH group (see fi gure 1).

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Figure 1. Treatment schedule. OC is oral contraceptive. hCG is given as soon as 1 follicle reached a size of ≥ 18mm and 2 other follicles of ≥ 16mm. OPU is ovum pick-up; ET= embryo transfer; S6=stimulation day 6.

All patients started with daily OCs (Microgynon 30®/Schering, Berlin, Germany, containing 30μg ethinyl oestradiol and 150μg levenorgestrel) on cycle day 2-3 for 21 days. The gonadotrophin dose was fi xed during the entire study. From stimulation day 6 up to and including hCG day, GnRH antagonist (Cetrorelix/Serono International, Geneva, Switzerland) in a dose of 0.25mg/day was given. Subcutaneous injections were performed by trained health care professionals. HCG 10.000 (Pregnyl®/Organon, Oss, The Netherlands) was injected as soon as the largest follicle reached a mean diameter ≥ 18 mm and at least 2 other follicles with a mean diameter ≥ 16 mm. A transvaginal ovum pick-up was performed 34-38 hours after hCG administration under ultrasound guidance, followed by a standard IVF or ICSI procedure. Up to three embryos were replaced 2-3 days after oocyte retrieval. Remaining

Day 2rhFSHgroup

Day 2 rhFSH+rhLH

group

n=12

n=12

OC

GnRHantagonist

2 days after last OC S6

OPU EThCG

OC rhFSH

rhFSH + rhLHGnRH

antagonist

Day 5 rhFSH group

Day 5 rhFSH+rhLH

group

OC

OC

5 days after last OC S6

GnRHantagonist

rhFSH

rhFSH + rhLH

n=12

n=12 GnRHantagonist

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embryos were cryopreserved. After ovum pick-up, intravaginal natural progesterone (three times daily 200mg Progestan®/ Organon, Oss, The Netherlands) was started as luteal support. This was continued until a negative pregnancy test or during the fi rst 3 weeks of pregnancy.

AssessmentsAfter randomization, a transvaginal ultrasound was performed on natural cycle day 2 or 3 to exclude the presence of ovarian cysts or other abnormalities. A blood sample was taken to exclude pregnancy. On day 2 and 5 after last OC intake and from stimulation day 6 onwards up to and including the day of hCG, blood samples were taken in the morning before any drugs was given, to assess FSH, LH, oestradiol(E2) and progesterone(P4) levels. Transvaginal ultrasounds were performed on these days to monitor the number and size of the follicles and endometrial thickness. In the case of ICSI, the cumulus cells were removed to assess the nuclear maturity. The embryo quality assessment was based on morphological criteria. On day 15-20 after rhCG administration a blood sample was taken to assess hCG value, if > 10 IU/l the test was repeated 2 to 4 days later to confi rm pregnancy. Pregnancy was defi ned as continuing increase in serum hCG. In that case, 4 weeks and 10 weeks after embryo transfer, an ultrasound was performed to assess the number of foetal sacs and heart activity. Clinical pregnancy was defi ned as the presence of a foetal sac, with or without heart activity. Ongoing pregnancy as a positive heart activity at a gestational age of 12 weeks.

Laboratory assays

Blood samples were centrifuged within 1 hour after withdrawal and stored at -20°C until assayed. Serum LH, FSH, E2 and P4 levels were measured by the endocrine laboratory (clinical chemistry) of VUmc, using commercially available immunometric assays. For FSH we used Delfi a (Delfi a, Wallac, Turku, Finland), with an interassay coeffi cient of variation (CV) of 9% at 3.5 and 5% at 15.5 IU/L. LH values were measured using Delfi a (Delfi a, Wallac, Turku, Finland), with a lower limit of detection of 0.3 IU/L and an interassay coeffi cient of variation (CV) of 8% at 0.46, 7% at 3.0 and 6% at 17 IU/L. Oestradiol and progesterone levels were measured using competitive immunometric assays (Delfi a; Wallac, Turku, Finland). For the assessment of oestradiol values the interassay CV was 8.5% at 111, 10 at 180 and 7 at 3000 and 6645 pmol/L. The lower limit of detection was 90 pmol/L. The interassay CV’s for progesterone assessment were 13% at 5, 9% at 30 and 7% at 120 nmol/L.

Statistical analysisThe primary effi cacy endpoint was the number of mature follicles (≥11mm) on the day of hCG administration. Secondary endpoints were cumulative dose and duration of rhFSH, number of oocytes and embryos, pregnancy- and implantation rates, serum hormone levels and endometrial thickness. Since this was a pilot study, no formal power calculation was performed. Both univariate and multivariate analysis of differences between the groups were carried out. Univariate tests on continuous outcome variables (e.g. age, BMI, cycle length) were carried out using analysis of variance (ANOVA) or Mann-Whitney U-test where normality requirements could not be achieved. Multivariate linear regression analysis were also carried out on these outcome variables with centre, stimulation day (2 or 5) and rhLH addition (yes/no) as explanatory variables. Proportions (e.g. fertilization-, implantation- and pregnancy rates) were analysed

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using Fisher’s Exact tests. Discrete variables with a limited range (e.g. the number of follicles, oocytes and embryo’s) were analysed using Poisson regression analysis with centre, stimulation day (2 or 5) and rhLH addition (yes/no) as explanatory variables. Analysis were carried out on an intention-to-treat principle (n=48). All statistical tests were performed two-sided at the signifi cance level of 5%.

Randomization Treatment allocation was decided by an independent person of an independent monitoring company according to a computer-generated, concealed randomization list, stratifi ed by centre.

Figure 2. Flow diagram, showing the fl ow of the participants through each stage of the trial OCs=oral contraceptives; OPU=ovum pick-up; ET=embryo transfer; ITT=intention to treat.

Day2 rhFSHgroup

OPU (n=11)

analysed per ITT (n=12)

Number of patients assessed for eligibility: not assessed

rhFSH (n=12)

OCs (n=12)

cetrorelix (n=12)

hCG (n=12)

ET (n=11)

Day2 rhFSH + rhLH group

Day 2 group (n=24)

Eligible patients who give informed consentwere randomized (n=48)

Day5 rhFSHgroup

Day5 rhFSH + rhLH group

rhFSH+rLH (n=12)

hCG (n=10)

OPU (n=10)

ET (n=10)

analysed per ITT (n=12)

OCs (n=12)

Day 5 group (n=24)

cetrorelix (n=11)

rhFSH (n=12)

OCs (n=12)

rhFSH+rLH (n=11)

OCs (n=12)

cetrorelix (n=12) cetrorelix (n=11)

OPU (n=10) IUI (n=1)

hCG (n=11) hCG (n=11)

OPU (n=11)

analysed per ITT(n=12)

ET (n=10) ET (n=10)

analysed per ITT (n=12)

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RESULTS

Baseline characteristics and cancellationsA total of 48 patients were randomized, 12 patients to each group. The four treatment groups were comparable for demographic characteristics (see table I). The fl ow of the participants through each stage of the study is presented in fi gure 2. One patient, randomized to start with rhFSH and rhLH on day 5 after the last OC, stopped the treatment during OC intake for personal scheduling reasons. One patient in the day 2 rhFSH+ rhLH group did not start with cetrorelix because of her lack of confi dence in the treatment. Three patients did not reach the hCG criteria; one in day 2 rhFSH+ rhLH group and two in the day 5 rhFSH group of which one received hCG followed by intra-uterine insemination. In addition, one patient (in the day2 rhFSH group) canceled the ovum pick-up because of personal reasons. Of the patients who had an ovum pick-up, one patient (in the day 5 rhFSH+rhLH group) did not undergo an embryo transfer because of non-fertilization.

Randomization groupDay 2 group Day 5 group P valuerhFSH rhFSH+rhLH rhFSH rhFSH+rhLH

Number of patients (ITT) 12 12 12 12

Age (years) 33.0 (2.9) 32.1 (3.5) 31.9 (2.2) 32.3 (2.8) 0.80

Body mass index (kg/m2) 21.9 (2.6) 23.7 (2.2) 20.8 (4.0) 23.9 (3.5) 0.70

Cycle length (days) 29.8 (2.1) 29.4 (2.3) 30.8 (2.4) 28.7 (2.4) 0.19

Primary infertility 5/12 (42%) 8/12 (67%) 9/12 (75 %) 9/12 (75%) 0.31

Duration of infertility (years) 2 1 2 3

Type of infertilitya: 2 0 1 1

Tubal factor 2 1 2 3

Endometriosis 2 0 1 1

Male factor 7 7 8 7

Unexplained 2 4 2 2

Other 0 0 1 0

FSH CD2/3 5.8 (1.7) 6.1 (2.1) 5.8 (1.5) 5.8 (1.3) 0.79

Estradiol CD2/3 146 (51) 143 (33) 116 (36) 118 (46) 0.17Table 1. Baseline characteristics. Values are mean (± standard deviation). Analyses were performed per intention to treat (ITT), using ANOVA, Mann-Withney U- and Fisher’s Exact tests. None of these variables showed a signifi cant center effect. a more options are possible.

Drugs requirementsOverall, cetrorelix administration ranged between 2 and 16 days. Detailed drugs requirements are presented in table II. The mean duration of cetrorelix administration in the day 2 and day 5 groups was 8.0 (±2.3) and 4.8 (±2.3) days, respectively (p<0.001). The stimulation period was signifi cantly longer in the day 2 groups compared with the day 5 groups, with a mean duration of 13.0 (±2.8) and 9.8 (±2.3) days, respectively (p<0.001). The addition of rhLH did not infl uence the length of the stimulation period. The total amount of rhFSH administered was substantially higher in the day 2 groups versus the day 5 groups, 1981 (±540) and 1474 (±343) IU/L, respectively (p<0.001). rhLH addition had no infl uence on rhFSH requirement. The total administered amount of rhLH was signifi cantly higher in the day 2 rhFSH+rhLH group

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compared with the day 5 rhFSH+rhLH group, 2100 (±524) and 1445 (±271) IU/L ,respectively (p<0.001).

Randomization group

Day 2 group Day 5 group P value

rhFSH rhFSH+rhLH rhFSH rhFSH+rhLH Day 2 or 5 LH+or -

Number of patients (ITT) 12 12 12 12

Total number OC pills 18.6 (4.9) 17.9 (3.8) 17.5 (4.1) 16.8 (3.4) 0.37 0.58

Duration of cetrorelix (days) 7.0 (1.6) 9.0 (3.5) 5.0 (2.7) 4.6 (1.8) <0.01∗ 0.39

Duration of rhFSH (days) 12.0 (1.6) 14.0 (3.5) 10.0 (2.7) 9.6 (1.8) <0.01∗ 0.39

Cumulative rhFSH (IU) 1775 (255) 2188 (672) 1500 (410) 1446 (271) <0.01∗ 0.17

Cumulative rhLH (IU) - 2100 (524) - 1445 (271) <0.01∗ -

Table 2. Drug requirements. Values are mean (± standard deviation). Analyses were performed in all patients receiving at least one OC pill using Mann-Whitney U-tests. Starting with rhFSH on day 2 or day 5 (day2 and day5) after the last OC pill and the addition of rhLH to rhFSH (yes or no) were compared separately. Duration = duration of treatment ∗p < 0.05

Hormonal concentrations Hormonal concentrations on stimulation day 1 (S1), and from stimulation day 6 onwards up to and including stimulation day 11 are presented in fi gure 3. The serum FSH, LH and oestradiol concentrations were signifi cantly lower on S1 in the day 2 versus the day 5 groups. Oestradiol concentrations were signifi cantly lower on S6 and S8 in the day 2 compared to the day 5 groups. Progesterone concentrations were low in all groups on S1 without signifi cant differences between the groups. There were no differences in FSH, LH and progesterone concentrations between the day 2 and day 5 groups on the other stimulation days (fi gure 3).FSH, LH, oestradiol and progesterone levels on the day of hCG administration were not infl uenced by the starting day of gonadotrophin administration after the last OC taken (data not shown). The LH concentrations were signifi cantly higher in the rhLH added groups from S6 onwards, with the mean LH concentrations being approximately 1 IU higher (fi gure 3). The mean LH concentration on the day of hCG administration was 0.53 (±0.32) in the rhFSH groups and 1.59 (±0.33 ) IU/L in the rhLH added groups (p<0.001). From S6 onwards, FSH, progesterone and the absolute oestradiol concentrations were not affected by addition of rhLH (fi gure 3). The only exception to this was the oestradiol concentrations which were signifi cantly lower on the day of hCG administration in the rhFSH groups compared to the rhFSH plus rhLH groups, 4372 (±2566) and 7190 (±3871), respectively (p=0.006). The mean oestradiol concentrations per mature follicle on the day of hCG administration were higher in the rhLH added compared with rhFSH only groups; with respect to follicles ≥ 11mm the mean concentrations were 276 (±152) and 552 (±283) pmol/L/follicle, respectively (p<0.001) and per follicle ≥ 14mm the mean concentrations were 380 (±216) and 709 (±316) pmol/L/follicle, respectively (p<0.001). The duration of stimulation did not infl uence this parameter.

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Figure 3. Hormonal concentrations. Values shown are mean. Analyses were performed in all patients receiving at least one OC pill, using Mann-Whitney U-tests. Starting with rhFSH on day 2 or day 5 (day 2 and day 5) after the last OC pill and the addition of rhLH to rhFSH (yes or not) were compared separately. On stimulation day 1(S1), the only signifi cant factor on LH and FSH concentration was day of stimulation, *day 2 versus day 5 (p<0.01). From stimulation day 6 (S6) onwards up to and including stimulation day 11( S11), the only signifi cant factor on LH concentrations was # rhLH addition (p<0.001), FSH concentrations during stimulation were not infl uenced by either starting day of gonadotrophin stimulation or rhLH addition. Oestradiol concentrations were signifi cantly lower in the day 2 groups compared to the day 5 groups on S6 and S8 (p<0.01). Progesterone concentrations were not infl uenced by either starting day of gonadotrophin stimulation or rhLH addition.

Follicular development and endometrial thicknessThe mean number of follicles on S6 and the day of hCG in the various groups are given in fi gure 4. On S1 there were no large follicles. On S6 the mean number of follicles ≥11 mm and ≥14 mm were signifi cantly lower in the day 2 versus the day 5 groups. On the day of hCG administration the number of follicles ≥11mm and ≥14mm were similar in the day 2 and day 5 groups. Addition of rhLH did not effect the number of large follicles on S6, but resulted in signifi cantly less follicles ≥ 11mm on the day of hCG administration.

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Figure 4. Follicular development. Number of follicles are presented as mean values. Analyses were performed using Poisson regression analyses with center, day of starting with gonadotrophins (day 2 versus day 5) and rhLH addition to rhFSH (yes or not) as explanatory variables. * On S6 the number of follicles ≥11 and ≥ 14 mm were signifi cantly different in the day 2 versus day 5 group (p<0.001). # On the day of hCG the number of follicles ≥11mm was signifi cantly lower in the LH added groups (p=0.003). Differences with respect to follicles ≥14mm did not reach statistical signifi cance (p=0.12).

The endometrium was signifi cantly thinner on S6 in the day 2 versus the day 5 groups (fi gure 5). Addition of rhLH had no effect on this parameter. Endometrial thickness was similar in all groups on S1 and day of hCG administration.

Figure 5. Endometrial thickness. Endometrial thickness is presented as mean values. Analyses were performed using analysis of variance (ANOVA). * The only factor of infl uence on the endometrial thickness was day 2 versus day 5 after last OCs on S6 (p<0.001).

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142

IVF harvestAll patients with an OPU had at least 2 oocytes retrieved. Number of oocytes retrieved was not effected by gonadotrophins starting day (day 2 versus day 5) but was signifi cantly lower if rhLH was added in comparison to the control group with only rhFSH, 9.2 (±5.2) and 12.1 (±6.3), respectively (p<0.01) (table III). The percentages of metaphase II oocytes (in ICSI patients), the total number of oocytes retrieved and the fertilization rates were similar in all groups (table III). The total number of embryos was lower in the rhLH added groups (p=0.05), stimulation day had no infl uence (p=0.53). The number of good quality embryos and embryos replaced was not different. The percentage of patients with a positive pregnancy test (hCG>10 IU/l) was high in all groups. The clinical pregnancy rates, defi ned as ≥ 1 intra-uterine sac on ultrasound at a gestational age of 6 weeks, varied between 25 and 50%. The ongoing pregnancy rates (positive heart activity on ultrasound, intra-uterine, at a gestational age of 12 weeks) varied between 17 and 42%. In this small study, no signifi cant differences could be observed in pregnancy rates between the various groups. The mean implantation rate, defi ned as the mean of all individual implantation rates varied between 15 and 40% and was not infl uenced by the day of stimulation or rhLH addition.

Randomization group

Day 2 group Day 5 group P value

rhFSH rhFSH+rhLH

rhFSH rhFSH+rhLH

Day2or5 LH+or-

Number of patients (ITT) 12 12 12 12

Number of oocytes (inseminated) 11.1 (7.3) 9.1 (6.0) 13.1 (5.2) 9.4 (4.7) 0.18 <0.01∗

Number of patients with ICSI 5/12 (42%) 5/12 (42%) 6/12 (50%) 6/12 (50%) 0.77 1.00

Percentage metaphase-II oocytesa 82% 87% 84% 86% 0.57 0.68

Total number of embryos 7.3 (4.8) 6.5 (4.9) 7.0 (3.4) 5.3 (4.3) 0.53 0.05*

Fertilization rateb 65 % 67% 58 % 51% 1.00 0.32

Number of good quality embryos (grI,II)

5.1 (4.1) 4.8 (3.2) 5.1 (2.8) 4.8 (3.0) 0.85 0.59

Number of embryos replaced/ETc 2.0 (0) 1.9 (0.32) 2.0 (0) 1.8 (0.42) 0.92 0.73

HCG levels 2 times >10 IU/l 7/12 (58%) 7/12 (58%) 5/12 (42%) 3/12 (25%) 0.15 0.77

Clinical pregnancy rate 6/12 (50%) 5/12 (42%) 5/12 (42%) 3/12 (25%) 0.56 0.56

Ongoing pregnancy rate 5/12 (42%) 5/12 (42%) 5/12 (42%) 2/12 (17%) 0.55 0.55

Mean implantation rate 36% 40% 35% 15% 0.58 0.77

Table 3. IVF outcome. Values shown are mean (±standard deviation). Analyses were performed per intention to treat (ITT), unless otherwise stated. We used poisson regression analyses with center, day of starting with gonadotrophins (day 2 versus day 5) and rhLH addition to rhFSH (yes or not) as explanatory variables, except for proportions we used Fischer Exact tests. Implantation rate = number of foetal sacs/number of embryos replaced per patient, mean implantation rate is the mean of the individual implantation rates. aAssessed in case ICSI was performed. bFR is fertilization rate =nr of embryos/nr of oocytes inseminated per patient.cAnalyses were performed per embryo transfer (ET).* p < 0.05.

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DISCUSSION

We developed this pilot study to obtain more basic information which could lead to optimalisation of hormonal treatment for IVF/ICSI involving the use of the GnRH antagonist and oral contraceptive pretreatment. The aim of the study was to explore the effect of timing of the initiation of gonadotrophin administration (day 2 versus day 5 after the last OC) and addition of rhLH in an OC pretreated fi xed GnRH antagonist regimen on hormonal concentrations and follicular development. OCs have been used in GnRH antagonist cycles for scheduling purposes (Vlaisavljevic et al. 2003; Cedrin-Durnerin et al. 2004; Bahceci et al. 2004; Sauer et al. 2004; Barmat et al. 2005; Cheung et al. 2005; Shapiro et al. 2005; Hwang et al. 2004, Huirne et al. 2006; Koichi et al. 2006; Rombauts et al. 2006). Some studies started with gonadotrophins on day 2 and others on day 5 after OC withdrawal. The effect of the timing of the start of gonadotrophin stimulation after OC withdrawal on hormonal concentrations and follicular development has not yet been studied in a direct way. One very recent study demonstrated that timing of exogeneous gonadotrophin administration after OCP-pretreatment effects LH and FSH levels during the GnRH antagonist therapy which may effect follicular development (Cedrin-Durnerin et al. 2006). In line with these results, we observed less large follicles in the early stimulation period (stimulation day 6) if gonadotrophin administration was started 2 days rather than 5 days after the last OC intake. In earlier studies using GnRH antagonists, OC pretreatment with rhFSH commenced on day 2 or 3 after the last OC’s resulted in a decreased number of large follicles in the early stimulation phase in comparison to a control group (Huirne et al. 2006a; Rombauts et al. 2006). This effect was not reported in studies in which stimulation was started 5 days after the last OC intake in GnRH antagonist cycles (Obruca et al. 2000; Kolibianakis et al. 2006a). The simplest explanation for the observation that more larger follicles were present on the 6th day of rhFSH stimulation, if rhFSH was started 5 days after the last OC intake, is that the total period over which follicles develope on own FSH had been 3 days longer. Starting with rhFSH 2 days versus 5 days after OC withdrawal resulted in a more homogeneous follicular cohort, but despite increased rhFSH requirements, fi nal number of mature follicles and number of oocytes available for insemination were similar in both groups. These results suggest that the most economic use of GnRH antagonists and rhFSH is to start stimulation 5 days after OC withdrawal.

Starting with stimulation on day 2 versus day 5 after the last OCP was associated with signifi cant lower FSH, LH and oestradiol concentrations and a thinner endometrium on stimulation day 6, despite the fact that initial endometrial thickness on stimulation day 1 was the same. Apparently, the endometrium develops slower in particular during the fi rst days of rhFSH stimulation under these circumstances. Obviously some subtle alterations of the endometrium take place during the fi rst days of the OC withdrawal period, resulting in increased thickness in a later stage if stimulation with rhFSH is started later after OC withdrawal. It is likely that the larger number of bleeding free days during stimulation, when gonadotrophin stimulation is started 3 days later, attribute to this.

Additionally we evaluated in a random way the effect of the rhLH addition on hormonal concentrations and follicular development. This may be relevant in situations with very deep suppression of LH concentrations such as GnRH antagonist treatment after OC pretreatment (Huirne et al. 2006a; Kolibianakis et al. 2006a; Rombauts et al. 2006). The current study shows that daily administration of 150 IU rhLH is suffi cient in this treatment regimen to increase the LH concentrations with aproximately 1 IU to mean serum LH concentrations above 1.2 IU/L during the entire stimulation period, which is supposed to be

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Timing stimulation and rLH addition in OC/GnRH antagonist regimen

the minimal concentration in hypogonadotrophic patients to achieve an optimal situation for ovulation and conceivement (Loumaye 2002). LH addition was not of infl uence on the observed follicular development in the early stimulation phase, but we found a signifi cant reduction in large follicles on the day of hCG administration. This can not be explained by any potential confounding effect of duration of stimulation or total amount of rhFSH used since these were similar in the rhFSH-only and rhLH-added groups. A number of studies indicate a benefi cial effect of LH or hCG administration in the late follicular phase during ovulation induction regimens using GnRH agonists. Stimulation periods are shorter, less drugs are required and there is a reduced number of smaller ovarian follicles in the late follicular phase (Filicori et al. 2002a and 2002b). However, studies in WHO I and II patients show that ovulation induction with LH containing, compared with FSH only regimen induce more follicular arrest, resulting in lower number of small follicles in the late follicular phase and a lower number of mature follicles on the day of hCG (Loumaye et al. 2003). These data indicate a LH ceiling effect for stimulation, and its physiolocical relevance for the proces of selection of dominant follicles either through direct infl uence or via increased androgen concentrations (Hillier 2001; Shoham et al. 2002). In a retrospective study containing 141 poor responders in IVF treated with a GnRH antagonist, a ceiling effect of LH could only be observed in patients with an age above 40 years, signifi cant less oocytes were retrieved in those patients treated with HMG and rhFSH compared with rhFSH alone (Chung et al. 2005). On the other hand, absolute LH concentrations during fi xed GnRH antagonist regimens on specifi c days were not related to ovarian response (Bosch et al. 2003, Merviel et al. 2004, Penarrubia et al. 2004). A very recent systematic review of current literature with respect to LH levels in IVF patients treated with GnRH agonists or antagonists, could not identify a relation between low endogeneous LH levels and a decreased probability of ongoing pregnany beyond 12 weeks (Kolibianakis et al. 2006b). Based on these results it is postulated that low endogenous LH levels during ovarian stimulation for IVF using GnRH analogues are not a rationale for LH supplementation to improve the probability of ongoing pregnancy beyond 12 weeks. On the contrary, it appears that the higher the level of midfollicular endogenous LH during IVF cycles, the lower the probability of ongoing pregnancy in GnRH antagonist cycles (Kolibianakis et al. 2004). The addition of 75 IU rhLH per day to rhFSH from stimulation day 1 onwards in a fi xed (multiple dose, starting day 6) GnRH antagonist protocol (Griesinger et al. 2005) or in a OC pretreated fl exible single dose GnRH antagonist protocol did not effect ovarian response or pregnancy outcome (Cedrin-Durnerin et al. 2004). In these studies possibly, LH dosages may have been too low or the groups were to heterogeneous to observe any detrimental effect on folliculogenesis or pregnancy rates.

The overall mean oestradiol concentrations during stimulation were not infl uenced by rhLH addition but the oestradiol concentrations per large follicle on the day of hCG administration were higher, indicating an increased availability of androgen substrate. The lower number of large follicles in the rhLH added groups, may have accounted for the overall equivalence of oestradiol concentrations compared to treatment with rhFSH alone. The similar oestradiol concentrations during the majority of the stimulation period are in agreement with the absence of any effect of rhLH addition on endometrial thickness on the day of hCG administration.

Our study was designed to evaluate endocrine and folliculodynamic features and it was underpowered to study the effect of any intervention on pregnancy and implantation rate. At this moment it is too early to draw any solid conclusion with respect to the optimal GnRH antagonist regimen to achieve optimal IVF outcome. But this pilot study does indicate

144

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that starting moment and rhLH addition both effect the hormonal concentrations and follicular development. These fi ndings underline the need for further basic studies assessing this topic to develop the optimal GnRH antagonist regimen, which remains important given the ongoing debate about the place of GnRH antagonists in ART.

In conclusion, starting with gonadotrophins 2 days versus 5 days after OC withdrawal results in lower FSH, LH and oestradiol concentrations, less larger follicles during the early stimulation phase with an increased duration of stimulation and more GnRH antagonist and rhFSH required but this does not lead to an increased number of large follicles on the day of hCG administration or ooyce yield. Daily addition of 150 IU rhLH to rhFSH results in increased LH concentrations and it reduces the number of mature follicles and oocytes.

AcknowledgementThe authors thank Serono for unconditionally supplying Cetrotide and Luveris.

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contraceptive pill on follicular development in IVF/ICSI patients receiving a GnRH antagonist: a randomized

study. Reproductive BioMedicine Online 13 235-245.

Huirne JA, Loenen ACD, Schats R, Mcdonnell J, Hompes PGA Schoemaker J, Homburg R and Lambalk CB 2005

Dose-fi nding study of daily gonadotropin-releasing hormone (GnRH) antagonist for the prevention of

premature luteinizing hormone surges in IVF/ICSI patients: optimal changes in LH and progesterone for

clinical pregnancy. Human Reproduction 20 359-367.

Hwang JL, Seow KM, Lin YH, Huang LW, Hsieh BC, Tsai YL, Wu GJ, Huang SC, Chen CY, Chen PH and Tzeng CR

2004 Ovarian stimulation by concomitant administration of cetrorelix acetate and HMG following Diane-

35 pre-treatment for patients with polycystic ovary syndrome: a prospective randomised study. Human

Reproduction 19 1993-2004.

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Koichi K, Yukiko N, Shima K, and Sachiko S 2006 Effi cacy of low-dose human chorionic gonadotropin (hCG) in a

GnRH antagonist protocol. Journal of Assisted Reproduction and Genetics 23 223-228.

Kolibianakis EM, Albano C, Kahn J, Camus M, Tournaye H, Van Steirteghem AC, Devroey P 2003 Exposure to high

levels of luteinizing hormone and estradiol in the early follicular phase of gonadotropin-releasing hormone

antagonist cycles is associated with a reduced chance of pregnancy. Fertility and Sterility 79 873-880.

Kolibianakis EM, Collins J, Tarlatzis B, Papanikolaou E, and Devroey P 2006b Are endogenous LH levels during

ovarian stimulation for IVF using GnRH analogues associated with the probability of ongoing pregnancy? A

systematic review. Human Reproduction Update 12 3-12.

Kolibianakis EM, Papanikolaou EG, Camus M, Tournaye H, Van Steirteghem AC, Devroey P 2006a Effect of oral

contraceptive pill pretreatment on ongoing pregnancy rates in patients stimulated with GnRH antagonists

and recombinant FSH for IVF. A randomized controlled trial. Human Reproduction 21 352-357.

Kolibianakis EM, Zikopoulos K, Schiettecatte J, Smitz J, Tournaye H, Camus M, Van Steirteghem AC, and Devroey P

2004 Profound LH suppression after GnRH antagonist administration is associated with a signifi cantly higher

ongoing pregnancy rate in IVF. Human Reproduction 19 2490-2496.

Loumaye E 2002 Ovarian stimulation: is exogenous LH necessary in all patients? Gynécologie, obstétrique &

fertilité 30 890-895.

Loumaye E, Engrand P, Shoham Z, Hillier SG, and Baird DT 2003 Clinical evidence for an LH ‘ceiling’ effect

induced by administration of recombinant human LH during the late follicular phase of stimulated cycles in

World Health Organization type I and type II anovulation. Human Reproduction 18 314-322.

Merviel P, Antoine JM, Mathieu E, Millot F, Mandelbaum J and Uzan S 2004 Luteinizing hormone concentrations

after gonadotropin-releasing hormone antagonist administration do not infl uence pregnancy rates in in

vitro fertilization-embryo transfer. Fertility and Sterility 82 119-125.

Obruca A, Fischl F, Huber JC 2000 Scheduling OPU in GnRH antagonist cycles. IVF Journal für Fertilität und

Reproduction 4 37.

Penarrubia J, Fabregues F, Creus M, Manau D, Casamitjana R, Guimera M, Carmona F, Vanrell JA, Balasch J 2003

LH serum levels during ovarian stimulation as predictors of ovarian response and assisted reproduction

outcome in down-regulated women stimulated with recombinant FSH. Human Reproduction 18 2689-2697.

Rombauts L, Healy D, Norman RJ 2006 A comparative randomized trial to assess the impact of oral contraceptive

pretreatment on follicular growth and hormone profi les in GnRH antagonist-treated patients. Human

Reproduction 21 95-103.

Sauer MV, Thornton MH, Schoolcraft W, and Frishman GN 2004 Comparative effi cacy and safety of cetrorelix

with or without mid-cycle recombinant LH and leuprolide acetate for inhibition of premature LH surges in

assisted reproduction. Reproductive BioMedicine Online 9 487-493.

Shapiro DB, Mitchell-Leef D, Carter M, Nagy ZP 2005 Ganirelix acetate use in normal- and poor-prognosis patients

and the impact of estradiol patterns. Fertility and Sterility 83 666-670.

Shoham Z 2002 The clinical therapeutic window for luteinizing hormone in controlled ovarian stimulation.

Fertility and Sterility 771170-1177.

Sullivan MW, Stewart-Akers A, Krasnow JS, Berga SL, Zeleznik AJ 1999 Ovarian responses in women to recombinant

follicle-stimulating hormone and luteinizing hormone (LH): a role for LH in the fi nal stages of follicular

maturation. Journal of Clinical Endocrinology and Metabolism 84 228-232.

Vlaisavljevic V, Reljic M, Lovrec VG, Kovacic B 2003 Comparable effectiveness using fl exible single-dose GnRH

antagonist (cetrorelix) and single-dose long GnRH agonist (goserelin) protocol for IVF cycles--a prospective,

randomized study. Reproductive BioMedicine Online 7 301-308.

Westergaard LG, Laursen SB, and Andersen CY 2000 Increased risk of early pregnancy loss by profound suppression

of luteinizing hormone during ovarian stimulation in normogonadotrophic women undergoing assisted

reproduction. Human Reproduction 15 1003-1008.

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Searching for the optimal comparison of GnRH antagonists and GnRH agonists

Part 2

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Cetrorelix in an OC-pretreated stimulation cycle comparing with buserelin in IVF/ICSI patients treated with rhFSH: a randomized, multicentre, phase IIIb study

JAF Huirne, JN Hugues, C Pirard, F Fischl, JC Sage,

JL Pouly, A Obruca, D Braat, AC van Loenen, CB Lambalk

Human Reproduction 2006: 21; 1408-1415

8

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ABSTRACT

Background The aim of this study was to assess the non-inferiority of an oral contraceptive (OC)-pretreated cetrorelix regimen and a buserelin regimen in IVF/ICSI patients treated with rhFSH in terms of total number of oocytes retrieved.Materials and methodsIn a multicentre, randomized study, 182 patients were randomized to receive cetrorelix with OC pretreatment (n = 91) or to receive buserelin (n = 91). The cetrorelix group started with daily OCs on cycle day 5 and continued for 21–28 days. Cetrorelix (0.25 mg) was given daily from stimulation day 6 up to and including the day of rhCG administration. The buserelin group started with buserelin (500 μg/day) for at least 10 days until down-regulation was achieved, after which the dose was reduced to daily 200 μg up to and including the day of rhCG administration. rhFSH was started in both groups on a Friday, in the cetrorelix group 5 days after the last OC pill intake. Both regimens were followed by a standard IVF or ICSI procedure. The primary effi cacy endpoint was the number of oocytes retrieved per patient. ResultsThe number of oocytes, cancellation rates, rhFSH requirements, number of oocyte retrievals during the weekend or public holiday and number of pregnancies were similar in both groups. Both treatment regimens were well tolerated. ConclusionsCetrorelix pretreated with OCs resulted in similar number of oocytes retrieved compared with a long buserelin protocol. Both regimens were well tolerated and allowed scheduling of the oocyte retrieval, with only small number of retrievals falling on a weekend or public holiday.

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INTRODUCTION

GnRH antagonists have recently been introduced in assisted reproduction cycles to prevent premature luteinization. The third generation antagonists are safe with low histamine releasing properties and a lower number of side effects in comparison with the long

agonist protocols (European and Middle East Orgalutran Study Group, 2001; Fluker et al., 2001). Owing to the ability of GnRH antagonists to inhibit gonadotrophin secretion rapidly without exerting any stimulation, less injections are required in comparison with the long agonist protocol which improves patient convenience (Huirne and Lambalk, 2001). Although this antagonist regimen seems to be an attractive alternative to the long agonist protocol,

several drawbacks became clear in the fi rst comparative phase III studies. The pregnancy rates were not different in the various individual studies. But a meta-analysis, including the fi rst fi ve comparative studies indicated that the number of clinical pregnancies might possibly be lower in the antagonist-only protocols (Al-Inany and Aboulghar, 2002). In the individual studies, the number of oocytes was lower with antagonist treatment (Albano et al., 2000 Borm and Mannaerts, 2000; European and Middle East Orgalutran Study Group, 2001; Fluker et al., 2001; Al-Inany and Aboulghar, 2002). Therefore, we hypothesized that the higher number of oocyts retrieved in the agonist groups may play a role in the possibly higher pregnancy rates. Number of oocytes retrieved may be related to improved pregnancy rates, possibly via better embryo selection (Templeton and Morris, 1998). Furthermore, the initiation of FSH administration in a GnRH antagonist regimen is cycle dependent. It is mostly started on day 2 or 3 of the natural cycle, which makes the treatment planning and scheduling diffi cult (Huirne and Lambalk, 2001) and increases the clinical site workload. Therefore, we postulated that oral contraceptive (OC) pills could be used to improve the results of the GnRH antagonist protocols in terms of number of oocytes and treatment

scheduling. A few publications have addressed outcomes of GnRH antagonist cycles in combination with OCs. All confi rmed the feasibility and the convenience of this regimen (Obruca et al., 2000; Huirne et al., 2006). Using oestradiol (E2) or OCs, the endogenous FSH rise which normally occurs during the luteo-follicular transition in a natural cycle can be attenuated (de Ziegler et al., 1998; van Heusden and Fauser, 1999; Fanchin et al., 2003). In a previous study, we demonstrated that OC pretreatment induces a more homogeneous follicular cohort resulting in delayed hCG administration, thus an extended FSH window leading to more oocytes retrieved in comparison with a GnRH antagonist-only regimen (Huirne et al., 2006). We hypothesized that the OC pretreatment in GnRH antagonist cycles will improve follicular homogeneity and number of oocytes rendering these regimen equal to the long agonist protocol in terms of IVF results. The additional advantage of OC pretreatment is that a withdrawal bleeding can be induced to adjust the treatment cycle to the patient’s and centre’s time plan. In a previous study, we demonstrated that the IVF results were independent of the duration of OCs pretreatment in GnRH antagonist cycles (Huirne et al., 2006). We further hypothesized that OC pretreatment in GnRH antagonist

cycles allows oocyte retrieval scheduling with only minor numbers of oocyte retrievals during the weekend or public holiday comparable with the long agonist protocol.

The aim of this study was to assess the non-inferiority of cetrorelix in patients pretreated with OCs with a long buserelin protocol in terms of number of oocytes retrieved in IVF/ICSI patients treated with rhFSH. In addition, we assessed the number of oocyte retrievals occurring on weekends/holidays as well as safety aspects.

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MATERIALS AND METHODS

Study design and patient’s characteristicsThis randomized controlled phase IIIb study was conducted in eight European IVF centres. The protocol was approved in all centres by the respective Ethics Committees. One hundred and eighty two patients were randomly allocated to undergo an OC treatment followed by a GnRH antagonist regimen (cetrorelix group) or a GnRH agonist long protocol without OC (buserelin group). The treatment assigned to each patient was determined according to a computer-generated concealed randomization list. Randomization was performed by centre. Patients needed to have a regular IVF/ICSI indication, a male partner with viable sperm in the ejaculate (testicular biopsy or epididymal sperm was not allowed), aged between 18 and 39 and given their informed consent. Patients with any previous assisted reproduction treatment (ART) cycles with less than three oocytes or three or more consecutive ART cycles without a clinical pregnancy or patients with any contraindication to ART, gonadotrophins or OC pills were excluded from the study. Patients with a signifi cant systemic disease were also excluded.

Treatment protocolOn cycle days 1–5 of menses, patients were randomized to receive either OC pretreatment (cetrorelix group) or not (buserelin group) (Figure 1). In the cetrorelix group, patients started with daily OC pills (Microgynon 30®, Schering, Berlin, Germany, containing 30μg ethinyl E2 and 150 μg levenorgestrel), within 5 days of menses onset and was continued for 21–28 days, stopping on a Sunday. In this group, daily cetrorelix, Serono International, Geneva, Switzerland (0.25 mg, Serono International, Geneva, Switzerland subcutaneously) was started on stimulation day 6 and continued up to and including the day of rhCG administration. The buserelin group started with daily buserelin, Advantis Pharma, Frankfurt, Germany (500 μg, subcutaneously) at the mid-luteal phase of a natural menstrual cycle (on days 18–22 of the pre-ART cycle) for at least 10 days until down-regulation was achieved, after which the dose was reduced to 200 μg/day. Daily administration was continued up to and including the day of rhCG administration. rhFSH (Gonal-F®, Serono, Inetrnational, Geneva, Switzerland) was started on the fi fth day (Friday) after the last OC pill in the cetrorelix group. In the buserelin group, rhFSH was started on a Friday as soon as down-regulation was achieved. In both groups, rhFSH was administered daily, subcutaneously, up to the day of hCG administration. The starting dose of rhFSH 150–225 IU, according to the study centre’s standard practice, was maintained for 5 days, after which it could be adjusted by steps of 75 IU, according to the ovarian response up to a maximal dose of 450 IU/day. rhCG 250 μg (=6500 IU) (Ovitrelle®, Serono, International, Geneva, Switzerland) was injected as soon as the largest follicle reached a mean diameter ≥18 mm and at least two other follicles of a mean diameter ≥16 mm. A transvaginal oocyte retrieval was performed 34–38 h after rhCG administration under ultrasound guidance, followed by a standard IVF or ICSI procedure. No more than 2–3 embryos were replaced either 2–3 days or 5–6 (blastocyst transfer) days after oocyte retrieval, according to centre’s standard procedure and country regulations. Remaining embryos were cryopreserved if the quality was suffi cient enough. After oocyte retrieval, intravaginal natural progesterone (three times daily 200 mg Progestan®, Organon, Oss, The Netherlands) was started as luteal support. This was continued up to a negative

pregnancy test or during the fi rst 3 weeks of pregnancy.

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Figure 1. Treatment schedule. Schematic overview of the treatment regimens. In the cetrorelix group, daily 0.25 mg cetrorelix was pretreated with daily oral contraceptive (OC) pills starting within 5 days of menses onset and was to be continued for 21–28 days, stopping on a Sunday; the Buserelin group started with daily Buserelin (500 μg) at the mid-luteal phase of a spontaneous menstrual cycle [on days 18–22 of the pre-assisted reproduction treatment (ART) cycle] for at least 10 days until down-regulation was achieved, after which the dose was reduced to 200 μg/day. All patients were undergoing ovarian stimulation with rhFSH. S1, stimulation day 1; S6, stimulation day 6.

AssessmentsNot more than 3 months before randomization, gynaecological, physical and ultrasound examination was performed to test eligibility. After giving written consent, a transvaginal ultrasound was performed on cycle day 2 or 3 to measure the antral follicular count and to exclude the presence of ovarian cysts or other abnormalities. A blood sample was taken to perform a pregnancy test. On stimulation day 1, 6 and on each day thereafter that the patient was seen in the clinic while undergoing ovarian stimulation and on the day of rhCG administration, blood samples were taken in the morning before any drugs were given, to assess E2 levels. On the same day, transvaginal ultrasounds was performed to monitor the number and size of the follicles and the endometrial thickness. The oocyte and embryo quality assessment was based on morphological criteria. In case of ICSI, the cumulus cells

were removed to assess nuclear maturity. Date, time, dose of rhFSH injections were recorded daily on personal diary cards as were the side effects or local skin reactions. On days 15–20 after rhCG administration, a blood sample was taken to assess hCG value: if >10 IU/l, the test was repeated 2–4 days later to confi rm pregnancy. Pregnancy was defi ned as continuing increase in serum hCG. In that case, 4 and 10 weeks after embryo

transfer, ultrasound was performed to assess the number of fetal sacs and heart activity. Clinical pregnancy was defi ned as the presence of a fetal sac, with or without heart activity. Ongoing pregnancy as a positive heart activity at a gestational age of 12 weeks. The results from cryopreserved embryos were not included in this study.

StatisticsThe primary effi cacy endpoint was the number of oocytes retrieved per patient. Secondary endpoints were total number of oocyte retrievals performed on weekend or public holiday, cancellation rate, drug requirements, total number of (good quality) embryos, implantation and pregnancy rates.

OC rhFSHCetrorelix

rhCG

5 day pill-free interval

Ovumpick-up

Embryo transfer

Buserelin 0.5mg 0.2mg

rhFSH

Friday S1

S6

CetrorelixGroup(n=91)

BuserelinGroup(n=91)

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As this was a non-inferiority study, the patient population used for the analyses of the primary effi cacy endpoint was defi ned as all randomized patients who received at least one injection of buserelin or took OCs, without major deviation to the protocol (per protocol). For the analysis of the secondary endpoints, all patients allocated were included in the intention to treat (ITT) population.

As the randomization was stratifi ed by centre, it was taken into account in the analyses of the non-inferiority testing. If no convergence of the statistical model could be achieved because of sparse data, pooling of centres was performed. The centre factor was used either as a covariate for parametric models or as a strata variable for non-parametric methods. The treatment by covariate interactions were assessed, and if one of these terms was signifi cant, then the overall comparison of the treatments was interpreted with caution and separate analyses for each strata of the covariate was done. Binary endpoints were compared using Cochran–Mantel–Haenszel (CMH) test of general association or a two-sided Fisher’s exact test, depending on the distribution of the variable. Continuous and discrete endpoints were analysed using analysis of variance (ANOVA). If deemed appropriate to include a covariate, analysis of covariance (ANCOVA) was used. Categorical parameters may have been investigated by using CMH testing or Fisher’s exact tests, whichever was appropriate. All statistical tests were performed two sided at the signifi cance level of 5%, with the exception of the primary endpoint (the number of ooyctes retrieved) which was performed using a one-sided 97.5% confi dence interval.

Sample size calculationThe number of oocytes retrieved was the primary effi cacy endpoint of the study. Sample size calculation was performed, under the assumptions that the non-inferiority margin (also called clinically irrelevant difference) is equal to three oocytes and the estimated standard deviation is equal to 5.9 (obtained from previous studies in which the number of oocytes retrieved was calculated and a one-sided alpha level of 0.025 is used (which was used to

compute the overall 97.5% confi dence interval of the difference between the two treatment groups). With the objective to demonstrate that the difference in average number of oocytes retrieved between the cetrorelix and the buserelin groups would not exceed three, the power for a comparison between the two groups would be equal to 87% for 75 evaluable patients in each treatment groups (for an allocation of 1:1 and a total sample size of 150). To allocate at least 150 patients per protocol, an additional 10% to cover possible dropping out were planned to allocate. However, recruitment was concurrently carried out in eight centres, with each having complete blocks, making it impossible to stop the recruitment exactly after the allocation of 165 patients.

RESULTS

Baseline characteristics and cancellationsA total of 216 patients were screened for eligibility, and 182 patients were enrolled in the study (Figure 2) and received at least one dose of either OC or buserelin’s; 91 received OC pills and 91 received buserelin. In the cetrorelix group, two patients did not start with rhFSH after OC intake; one patient turned out to be hepatitis B positive, and another patient was excluded because of non-compliance before fi rst cetrorelix dose. In the buserelin group, six patients did not start with rhFSH after buserelin administration; two patients became pregnant before rhFSH administration, in three patients desensitization failed and one

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patient decided to withdraw because of failed confi dence in buserelin. One hundred and seventy patients received rhCG. In the cetrorelix group, one patient did not receive rhCG for personal reasons, and two others had insuffi cient follicular response and did not reach the rhCG criteria of which one patient received rhCG followed by IUI. In the buserelin group, one patient stopped rhFSH because of stimulation failure. All together, 169 patients had an oocyte retrieval, 85 in the cetrorelix and 84 in the buserelin group. In the cetrorelix group, 81 patients had embryos transferred, one patient had no mature oocytes and three had no viable embryos. In the buserelin group, 79 patients had an embryo transfer, three patients had no (mature) oocytes and two patients had no fertilization (Figure 2). Overall, the number of patients receiving hCG, oocyte retrieval and an embryo transfer were comparable in both arms.

Figure 2. The fl ow of the participants through each stage of the trial

The mean age of the study population was 32.5 (±4.0) years, with a range of 20–39 years. The two treatment groups were similar with regard to demographics and baseline characteristics except for body mass index which was slightly higher in the cetrorelix group (Table I). All P values are adjusted for the centre effect.

Eligible patients who give informed consent

OCs and Cetrorelix group (n=91)

had oocyte retrieval (n=85) had oocyte retrieval (n=84)

analysed per ITT (n=91)

Patients assessed for eligibility (n=216)

Buserelin group (n=91)

received rhFSH (n=89)

received OCs (n=91) received Buserelin (n=91)

received rhFSH (n=85)

received cetrorelix (n=89)

received rhCG (n=86) received rhCG (n=84)

analysed per ITT (n=91)

had embryo transfer (n=81) had embryo transfer (n=79)

were randomized (n=182)

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Randomization group

Cetrorelix group(with OC)

Buserelin group(no OC)

P value

Number of patients (ITT) 91 91

Age (years) 32.8 (3.8) 32.2 (4.2) 0.30

Body mass index (kg/m2) 23.7 (4.0) 22.6 (3.5) 0.04

Race 1.00

White 87 (95.6%) 86 (94.5%)

Black 3 ( 3.3%) 3 ( 3.3%)

Other 1 ( 1.1%) 2 ( 2.2%)

Primary infertility 51 (56.0%) 59 (64.8%) 0.22

Smoking habits (% smokers) 17 (18.7%) 15 (16.5%) 0.85

Duration of infertility (years) 3.9 (2.8) 3.6 (2.4) 0.48

Type of infertilitya: 0.76

Male factor 53 (58.2%) 50 (54.9%)

Tubal factor 24 (26.4%) 26 (28.6%)

Endometriosis 9 ( 9.9%) 6 ( 6.6%)

Unexplaned 8 ( 8.8%) 17 (18.7%)

Other 4 ( 4.4%) 5 ( 5.5%)

Number of previous ART attempts 0.48

0 49 (53.8%) 58 (63.7%)

1 24 (26.4%) 15 (16.5%)

2 14 (15.4%) 15 (16.5%)

3 4 ( 4.4%) 3 (3.3%)

Number of follicles ≤ 11 mm on CD2 or 3 10.1 (7.7) 9.7 (7.1) 0.61

Endometrial thickness (mm) on CD2 or 3 4.9 (2.2) 4.7 (2.1) 0.38

FSH (IU/L) on CD2 or 3 7.2 (2.2) 7.4 (3.3) 0.61

Oestradiol (pmol/L) on CD2 or 3 138 (55) 148 (103) 0.45

Table I. Baseline characteristics. Values are mean (± standard deviation). Analyses were performed per intention to treat (ITT). CD=cycle day. aMore than one options is possible in one patient.

Effi cacy resultsThe mean duration of OC intake in the cetrorelix group was 24 (±2.7) days, with a range of 15–31 days. The mean duration of cetrorelix administration was 5.3 (±1.8) days, with a range of 2–10 days. Duration of GnRH agonist administration was 25.4 ± 4.4 days (range 14–38 days). The stimulation period and FSH requirement were similar in both groups (Table II). Also no differences were observed between the two groups in number of follicles ≥11 mm, ≥16 mm or the endometrial thickness on S1, S6 and day of hCG. All P values were >0.58 (Figures 3 and 4). The number of patients having oocyte retrievals at the weekend or on public holidays was low and similar in both groups, 5 (5.9%) in the cetrorelix and 6 (7.1%) in the buserelin group (p=0.74).

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Randomization group

Cetrorelix group(with OC)

Buserelin group(no OC)

P value

Number of patients (PP) 90 85

Duration of FSH treatment (days) 10.1 (1.8) 10.3 (1.6) 0.49

Total dose of Gonal-F (IU) 1919 (638) 1949 (773) 0.81Table II. Drug requirements mean (± standard deviation). Analyses were performed per protocol (PP), using analysis of variance (ANOVA) with treatment and centre as a factor. S6=stimulation day 6. aANOVA with treatment and center as a factor.

Figure 3. Endometrial thickness. Mean endometrial thickness, with standard deviations (error bars) on stimulation day 1 (S1), 6 (S6) and day of hCG administration (rhCGd). Analyses were performed on an intention to treat (ITT) basis, and P values were all >0.05.

Figure 4. Follicular growth pattern. Number of follicles ≥11 mm and ≥16 mm on stimulation day 1 (S1), stimulation day 6 (S6) and on the day of rhCG administration (rhCGd). Values are mean with standard deviations (error bars). Analyses were performed on an intention to treat basis, and P values were all >0.05.

0

2

4

6

8

10

12

S1 S6 r-hCGd

Endo

met

rial t

hick

ness

(mm

) (m

m)

Cetrorelix groupBuserelin group

02468

101214161820

S1 S6 hCGd

Num

ber o

f fol

licle

s >

10

Cetrorelix

Buserelin

Num

ber o

f fol

licle

s ≥

11 m

m

0123456789

10

S1 S6 hCGd

Num

ber o

f fol

licle

s >

15 m

m

CetrorelixBuserelin

Num

ber o

f fol

licle

s ≥

16 m

m

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In the per protocol data set, there was no signifi cant difference between the two treatment groups in the number of oocytes retrieved. The mean number of oocytes retrieved was 11.2 (±7.1) for the cetrorelix and 11.3 (±6.8) for the buserelin group. As the lower boundary of the one-sided 97.5% confi dence interval for the difference between the two treatment groups was –2.19, the two treatment groups were declared as non-inferior. Results for the ITT data set were also similar: the two treatment groups were not statistically different for the number of oocytes retrieved [11.4 (±7.3)] in the cetrorelix group and [10.9 (±10.9)] in the buserelin group. The estimate was 0.47, with the lower boundary of the one-sided 97.5% confi dence interval for the difference between the two groups is of –1.51.

Number of intact oocytes, number of metaphase II oocytes, the number of embryos obtained, the mean number of embryos transferred and the number of patients in which blastocysts were transferred were not different between the two groups (Table III). We could

not observe differences between the two groups with respect to implantation, clinical and ongoing pregnancy rates although this study was not powered to assess these points in detail (Table III). Overall, multiple clinical pregnancies (all twin) occurred in 11 of 52 pregnant patients (21.2%) and were similar in both groups, six in the cetrorelix group and fi ve in the buserelin group. There were three clinical miscarriages reported (one in the cetrorelix group and two in the buserelin group); one of these was a partial miscarriage of a twin pregnancy. Of the 37 ongoing pregnancies, there were three patients who had adverse pregnancy outcomes, all in the buserelin group. One patient suffered a fetal death in utero; in one patient, trisomy 21 was diagnosed, and the pregnancy was aborted. One patient with a twin pregnancy had a fetus selectively removed by embryo reduction because of trisomy 21 but also lost the second fetus because of an immature delivery. Finally, there were 34 births, 17 in each treatment group, resulting in 43 live babies, 23 from the cetrorelix-treated women and 20 from buserelin-treated women.

The E2 level on hCG day were signifi cantly lower in the cetrorelix group compared with the buserelin group, 5439 (±2743) versus 9443 (±2789) pmol/l, respectively (p<0.001). The E2 levels on S1 and S6 were not different (Figure 5).

Figure 5. Mean oestradiol (E2) levels (pmol/l). Oestradiol levels were measured on stimulation day 1 (S1), 6 (S6) and the day of rhCG administration (rhCGd). The error bars represent the standard deviation. Analyses were performed on an intention to treat (ITT) basis. *P < 0.05.

-2000

0

2000

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Randomization group

Cetrorelix group(with OC)

Buserelin group(no OC)

P value

Number of patients (ITT) 91 91

Oocyte retrieval on a weekend or public holidaya

5 (5.9 %) 6 (7.1%) 0.74

Number of oocytes retrieveda 11.4 (7.3) 10.9 (10.9) 0.64

Number of intact oocytesa 10.8 (7.1) 10.8 (6.5) 0.91

% of oocytes inseminateda 90.3 89.5 0.67

Number of metaphase-II oocytesb 8.9 (5.8) 8.8 (4.4) 0.95

Number of embryos (2pn) 6.2 (4.3) 6.5 (4.0) 0.73

Fertilization rate (%)c 56.7% 57.9% 0.59

Number of good quality embryos (grI,II)a 3.2 (2.5) 3.1 (2.9) 0.73

Number of embryos replaced/ETd 2.1 (0.6) 2.1 (0.4) 0.52

Number of patients with blastocyst transferd 12 (13.2%) 13 (14.3%) 0.78

Number of embryos cryopreservedd 1.3 (2.8) 1.2 (4.2) 0.85

Positive pregnancy test (hCG >5 IU/l) 25 (27.5%) 27 (29.7%) 0.72

Clinical pregnancies 19 (20.9%) 22 (24.2%) 0.72

Singleton 12 (63.2%) 16 (72.7%)

Multiple 6 (31.6%) 5 (22.7%)

Ectopic 1 (5.3%) 1 (4.5%)

Ongoing pregnancies 17 (18.7%) 20 (22%) 0.71

Mean Implantation rate (%)e 14.3% 17.3% 0.55

Live birth rate f 17 (18.7%) 17 (18.7%) 1.00

Live baby rate 23 (25.3%) 20 (22%) 0.73

Table III. IVF outcome. Values shown are mean (± standard deviation). Analyses were performed per intention to treat (ITT) unless others stated. Analysis of Variance with treatment and center as factors or Fisher’s Exact tests were used. S1 = stimulation day 1; ET = embryo transfer. a Assessed if oocyte retrieval was performed. b Assessed if ICSI was performed. c FR is fertilization rate =number of embryos/number of oocytes inseminated per patient. d Analyses were performed in case at least one embryo was obtained. e Implantation rate = number of fetal sacs/number of embryos replaced; mean number of implantation rate is the mean of the individual implantation rates. f Live birth rate is number of deliveries with one or more live babies.

Safety evaluationOver the course of the study, 88 of the 182 patients (48.4%) reported at least one adverse event (AE). About 41.8% in the cetrorelix group and 54.9% in the buserelin group (P = 0.10). Before the stimulation with rhFSH, signifi cantly less AEs were reported in the cetrorelix group versus the buserelin group (Table IV). The number of patients with at least one reported AE during stimulation, and up to 30 days after rhCG injection were similar in the cetrorelix and buserelin group. The majority of the AEs were mild in severity (237 of 259 events) and possibly

related to study drug (146 of 259 events). The reported possibly related AEs were signifi cantly less in the cetrorelix group (15) compared with the buserelin group (37) (P < 0.001).

The probably-related AEs were similar in both groups (11 in cetrorelix group and 10 in buserelin

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162

group). When the AEs were examined by both severity and drug relationship, signifi cantly

fewer patients in the cetrorelix group (14.3%) had mild, possibly related AEs compared with the buserelin group (39.6%) (P < 0.001). Three serious AEs were reported in both groups: in each treatment group, there was one patient with moderate ovarian hyperstimulation syndrome (OHSS), one patient with an ectopic pregnancy and one patient with abdominal pain. Additionally, in three patients, mild OHSS was reported, one in the cetrorelix and two in the buserelin group. There were no cancellations because of risk of OHSS recorded during the study. The most affected system organ classes with 10 or more reported AEs in the total study are summarized in Table IV. The main reported AEs were gastrointestinal disorders (21.4%), like nausea, abdominal discomfort, distension, pain, constipation and nausea, followed by nervous system disorders (20.9%) which was mainly headache. Some patients reported dizziness. Reported general disorders and administration site condition (18.7%) were fatigue, injection site bruising and pruritus. Reported reproductive system and breast disorders (13.7%) were breast tenderness and enlargement, ovarian cyst formation and enlargement. Reported musculoskeletal disorders were arthralgia and back pain. Reported psychiatric disorders mainly occurred in the buserelin group and varied widely, including abnormal dreams, aggression, crying, mood alterations, sleep disorders, decreased libido and depression. In the cetrorelix group, there were signifi cantly fewer AEs from the nervous system class and the reproductive system and breast disorders class compared with the buserelin group (Table IV). The other organ class systems with less then 10 reported AEs were ear, eye, immune system, infections, musculoskeletal, respiratory and vascular organ class.

Randomization group

Cetrorelix group(with OC)

Buserelin group(no OC)

P value

Number of patients (ITT) 91 91

Any AE (number of patients; % of patients)

83 (38; 41.8%) 176 (50; 54.9%) 0.10

General and administration site conditions 20 (12 ;13.2%) 32 (22 ;24.2%) 0.09

Gastrointestinal disorders 26 (18;19.8%) 37 (21;23.1%) 1.00

Musculoskeletal disorders 3 (3;3.3%) 8 (8;8.8%) 0.21

Nervous system disorders 16 (13;14.3%) 37 (25;27.5%) 0.04∗Psychiatric disorders 3 (3;3.3%) 15 (9;9.9%) 0.13

Reproductive system and breast disorders 7 (7;7.7%) 26 (18;19.8%) 0.03∗Table IV. Adverse events occurring during the study by organ system affected. Values are total number of reported adverse events (AEs), (total number of patients in which these events were reported; percentage of patients in which these events were reported). Analyses were performed in all patients receiving at least one medication (i.e.ITT). Fisher’s Exact tests were used. Organ system is only reported if the total number of reported AEs in both arms is >10. * p<0.05.

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DISCUSSION

The aim of this study was to assess the non-inferiority of an OC-pretreated cetrorelix regimen compared with a long agonist buserelin regimen in terms of effi cacy (total number of oocytes), number of oocyte retrievals on weekend or public holidays and safety in IVF/ICSI patients treated with rhFSH.

We have demonstrated that in terms of length of stimulation, follicle and oocyte, yield the combination of OC with a GnRH antagonist was comparable with a long GnRH agonist treatment. Several phase III studies consequently report signifi cantly less oocytes retrieved in the GnRH antagonist groups compared with the long agonist protocols (Albano et al., 2000; Borm and Mannaerts, 2000; European and Middle East Orgalutran Study Group,

2001; Fluker et al., 2001; Huirne and Lambalk, 2001; Al-Inany and Aboulghar, 2002). The timing of gonadotrophin suppression was totally different in these regimens, and this has probably played a role in follicular development and number of oocytes retrieved. GnRH analogue administration is usually started in the luteal phase of the preceding cycle in the long agonist protocol. Desensitization resulting in profound suppression of the endogenous gonadotrophins allows early antral follicles to grow more synchronized, when exogenous gonadotrophins are being given with more simultaneous maturation as result. In GnRH antagonist-only protocols, endogenous FSH during luteo-follicular

transition is not suppressed because the antagonist administration is mostly started from stimulation day 6 onwards. If the endogenous FSH rise is not suppressed, marked follicular size discrepancies will lead to the administration of hCG dependent on the size of the leading follicle(s) while a number of follicles is still immature. Thus consequently, a shorter stimulation period and lower number of oocytes at time of retrieval is a feature of GnRH antagonist IVF/ICSI cycles as shown in the various phase III studies (Albano et al., 2000; Borm and Mannaerts, 2000; European and Middle East Orgalutran Study Group, 2001; Fluker et al., 2001; Al-Inany and Aboulghar, 2002). Not only GnRH agonists but also estrogens alone and estrogens in combination with progestagens in the form of OCs are able to suppress and attenuate the endogenous FSH rise (de Ziegler et al., 1998; van Heusden and Fauser, 1999). E2 or OC pretreatment are therefore possible tools in the prevention of untimely and uncoordinated development of the follicular cohort in GnRH antagonist regimens (Fanchin et al., 2003; Huirne et al., 2006). This study confi rms that OC-pretreated regimens

are feasible with similar effi cacy in terms of number oocytes compared with a long GnRH agonist protocol. The three-oocyte margin at the time selected for this study was based on discussion between the investigators and European and US regulatory authorities. The results of this study do not indicate any difference in pregnancy rates. With the number of oocytes selected as primary endpoint, this study has only limited value with respect to

pregnancy and implantation rates, but it can be of use in future meta-analyses. Our results are in agreement with the fi ndings of a study comparing the same strategies in polycystic ovary syndrome (PCOS) patients (Hwang et al., 2004) and another study comparing an OC-pretreated fl exible GnRH antagonist protocol to a long GnRH agonist protocol, showing similar number of oocytes, embryos and pregnancy rates (Barmat et al., 2005). A very recent

study comparing a fi xed GnRH antagonist regimen with or without OC pretreatment reported a signifi cantly higher early pregnancy loss after OC pretreatment (Kolibianakis et al., 2006). So far, only a few studies assessed the effect of GnRH antagonists versus GnRH agonists on endometrial development and reported confl icting results (Saadat et al., 2004; Simon et al., 2005). Further studies are needed to study the effect of OC pretreatment on endometrial

development, and proper designed meta-analyses and future adequately powered studies

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are needed to proof the idea that the combined OC-antagonist strategy overcomes the possible disadvantage of antagonist use over agonist use in terms of pregnancy outcome

and its effect on implantation.

Another relative disadvantage of GnRH antagonist-only protocols in IVF is the limited ability to plan the start of the treatment which is cycle dependent. When stimulation is started on cycle day 2 or 3, it turns out that both initiation of treatment and oocyte retrieval occurred equally distributed on all week days (Huirne et al., 2005). The current study clearly indicates that OC pretreatment allows accurate planning of the initiation of FSH treatment, such that weekend retrievals occurred only in a very limited number of patients, comparable with the long GnRH agonist schedule. Another way of preventing oocyte retrieval from occurring at the weekend in GnRH antagonist cycles by delaying the hCG injection does not seem an attractive option since it was demonstrated that one day delay was associated with lower pregnancy rates (Kolibianakis et al., 2004). Therefore, planning by scheduling a withdrawal bleeding with OC pretreatment in combination with timely injected hCG could be advantageous.

Both treatment regimens in our study were well tolerated, with a similar duration of the stimulation period, but with signifi cantly lower number of GnRH analogue injections with signifi cant less reported side effects in the cetrorelix group before rFSH is started.

In conclusion, it seems that the combination of OC with antagonist largely overcomes several disadvantages of GnRH antagonist application in IVF that were claimed in the past compared with long agonist regimens. The mean number of oocytes retrieved were similar in OC-pretreated cetrorelix group and the long buserelin group with only a small number of oocyte retrievals occurring at the weekend or on public holidays. In our opinion, this combined strategy, in particular given the signifi cant reduction of required

injections and in part as a result of the signifi cant reduction in reported side effects before stimulation, provides a good alternative for the long GnRH agonist protocol in the prevention of premature luteinization in ART.

Acknowledgements The authors thank Serono for their support of the study, in particular R. Percival, C. Howles and L. O’Dea for the clinical aspects of the study design, D.W. Warne and S. Robson-Lasserre

for the statistical design and analysis, J. Griffi ths and R. Duran for data management, V. Hild and G. Decosterd for the study management and M. Leynaert and A. Zrener for the study

monitoring.

REFERENCES

Albano C, Felberbaum RE, Smitz J, Riethmuller-Winzen H, Engel J, Diedrich K, Devroey P and The European

Cetrorelix Study Group (2000) Ovarian stimulation with HMG: results of a prospective randomized phase III

European study comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and

the LHRH-agonist buserelin. Hum Reprod 15,526–531.

Al-Inany H and Aboulghar M (2002) GnRH antagonist in assisted reproduction: a Cochrane review. Hum Reprod

17,874–885.

Barmat LI, Chantilis SJ, Hurst BS and Dickey RP (2005) A randomised prospective trial comparing gonadotropin-

releasing hormone (GnRH) antagonist/recombinant follicle-stimulating hormone (rFSH) versus GnRH-agonist/

rFSH in women pre-treated with oral contraceptives before in vitro fertilization. Fertil Steril 83,321–330.

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Borm G and Mannaerts B (2000) Treatment with the gonadotrophin-releasing hormone antagonist ganirelix

in women undergoing ovarian stimulation with recombinant follicle stimulating hormone is effective,

safe and convenient: results of a controlled, randomized, multicentre trial. The European Orgalutran Study

Group. Hum Reprod 15,1490–1498.

European and Middle East Orgalutran Study Group (2001) Comparable clinical outcome using the GnRH

antagonist ganirelix or a long protocol of the GnRH agonist triptorelin for the prevention of premature LH

surges in women undergoing ovarian stimulation. Hum Reprod 16,644–651.

Fanchin R, Salomon L, Caselo-Branco A, Olivennes F, Frydman N and Frydman R (2003) Luteal estradiol pre-

treatment coordinates follicular growth during controlled ovarian hyperstimulation with GnRH antagonists.

Hum Reprod 18,2698–2703.

Fluker M, Grifo J, Leader A, Levy M, Meldrum D, Muasher SJ, Rinehart J, Rosenwaks Z, Scott RT Jr, Schoolcraft W

et al. (2001) Effi cacy and safety of ganirelix acetate versus leuprolide acetate in women undergoing controlled

ovarian hyperstimulation. Fertil Steril 75,38–45.

van Heusden AM and Fauser BCJM (1999) Activity of the pituitary-ovarian axis in the pill-free interval during use

of low-dose combined oral contraceptives. Contraception 59,237–243.

Huirne JA and Lambalk CB (2001) Gonadotropin-releasing-hormone-receptor antagonists. Lancet 358,1793–

1803.

Huirne JA, Van Loenen ACD, Schats R, Mcdonnell J, Hompes PGA, Schoemaker J, Homburg R and Lambalk CB

(2004) Dose-fi nding study of daily gonadotropin-releasing hormone (GnRH) antagonist for the prevention

of premature luteinizing hormone surges in IVF/ICSI patients: antide and hormone levels. Hum Reprod

19,2206–2215.

Huirne JA, Van Loenen ACD, Schats R, Mcdonnell J, Hompes PGA, Schoemaker J, Homburg R and Lambalk CB

(2005) Dose-fi nding study of daily gonadotropin-releasing hormone (GnRH) antagonist for the prevention

of premature luteinizing hormone surges in IVF/ICSI patients: optimal changes in LH and progesterone for

clinical pregnancy. Hum Reprod 20,359–367.

Huirne JA, Van Loenen ACD, Donnez J, Pirand C, Homburg R, Schats R, McDonnell J and Lambalk CB (2006)

Follicular development in IVF/ICSI patients treated with a GnRH antagonist and rhFSH with or without

pretreatment with an oral contraceptive pill; a randomized study. RBM Online 13, 235-245.

Hwang JL, Seow KM, Lin YH, Huang LW, Hsieh BC, Tsai YL, Wu GJ, Huang SC, Chen CY, Chen PH et al. (2004) Ovarian

stimulation by concomitant administration of cetrorelix acetate and HMG following Diane-35 pre-treatment

for patients with polycystic ovary syndrome: a prospective randomised study. Hum Reprod 19,1993–2004.

Kolibianakis EM, Albano C, Camus M, Tournaye H, Van Steirteghem AC and Devroey P (2004) Prolongation of the

follicular phase 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

82,102–107.

Kolibianakis EM, Papanikolaou EG, Camus M, Tournaye H, Van Steirteghem AC and Devroey P (2006) Effect of oral

contraceptive pill pretreatment on ongoing pregnancy rates in patients stimulated with GnRH antagonists

and recombinant FSH for IVF. A randomized controlled trial. Hum Reprod 21,352–357.

Obruca A, Fischl F and Huber JC (2000) Scheduling OPU in GnRH antagonist cycles. J Reprod Fertil 4,37.

Saadat P, Boostanfar R, Slater CC, Tourgeman DE, Stanczyk FZ and Paulson RJ (2004) Accelerated endometrial

maturation in the luteal phase of cycles utilizing controlled ovarian hyperstimulation: impact of

gonadotropin-releasing hormone agonists versus antagonists. Fertil Steril 82,167–171.

SimonC, Oberye J, Bellver J, Vidal C, Bosch E, Horcajadas JA, Murphy C, Adams S, Riesewijk A, Mannaerts B et al.

(2005) Similar endometrial development in oocyte donors treated with either high- or standard-dose GnRH

antagonist compared to treatment with a GnRH agonist or in natural cycles. Hum Reprod 20,3318–3327.

Templeton A and Morris JK (1998) Reducing the risk of multiple births by transfer of two embryos after in vitro

fertilization. N Engl J Med 339,573–577.

de Ziegler D, Jaaskelainen AS, Brioschi PA, Fanchin R and Bulletti C (1998) Synchronization of endogenous and

exogenous FSH stimuli in controlled ovarian hyperstimulation (COH). Hum Reprod 13,561–564.

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General discussionAre GnRH antagonists compareble to agonists in IVF?: a debate

JAF Huirne, R Homburg, CB Lambalk

Submitted

9

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ABSTRACT

We believe that appropriate comparison of optimal GnRH agonist and antagonist regimens has not been performed yet. Currently available meta-analysis included all comparative studies between GnRH agonists and antagonists performed so far, including less than optimal GnRH antagonist regimens. After critical appraisal of the various studied GnRH antagonist regimens in terms of follicular development and IVF outcome, we postulate that early suppression of endogenous FSH results in optimal follicular development. Additionally, stable and early suppression of LH and progestrone levels during the entire period of stimulation may be an advantage for implantation and pregnancy outcome. In this respect, single dose and particularly fl exible protocols seem to be less advantageous. Early FSH and LH suppression can be achieved by early GnRH antagonist administration (stimulation day 1) or by OC pretreatment. A meta-analysis including four studies comparing the long GnRH agonist protocol with OC-pretreated fi xed GnRH antagonist regimen; could not identify differences in number of oocytes retrieved and clinical pregnancies. More studies comparing long GnRH agonist protocols with “long” GnRH antagonist protocols, with enough power to identify differences in pregnancy rates, are required before appropriate comparison can be made.

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The fi rst in-vitro fertilization (IVF) therapies were performed in natural unstimulated IVF cycles. Nowadays gonadotrophins are given to induce multiple follicular development and GnRH analogues for the prevention of premature LH surges in IVF. Without the use of GnRH analogues LH surges occur in about 20% of hyperstimulated IVF patients (Edwards et al., 1996; Janssens et al., 2000). Preventing LH surges using GnRH analogues improves oocyte yield with more embryos, allowing better selection, leading to an increase in pregnancy rates (Templeton et al., 1998). It took 15 years of experience with the GnRH agonists in IVF to identify the optimal protocol (the long protocol starting in the midluteal phase of the preceeding cycle) with regard to the best IVF results in a general population (Daya, 2000; Huirne et al., 2004a). GnRH agonist administration causes gonadotrophin suppression via pituitary desensitization, after an initial short period of gonadotrophin hypersecretion. In contrast, GnRH antagonists cause immediate and rapid gonadotrophin suppression, by competitive occupancy of the GnRH receptor and therefore intuitively a more logical choice to use in IVF for the prevention premature LH surges. Theoretically, GnRH antagonists could be administered at any time during the early or mid-follicular phase of a treatment cycle to prevent a premature LH surge. With this in mind, around a decade ago, the fi rst dose-fi nding studies in IVF started GnRH antagonist medication on a fi xed day late in the follicular phase (Albano et al., 1997; Ganirelix dose-fi nding study group, 1998; Olivennes et al., 1998; Huirne et al., 2004b). Initially two general approaches emerged; 1) the single dose protocol, in which one injection is administered late in the follicular phase around stimulation day 7 or 8, and 2) the multiple dose regimen in which the antagonist is administered daily from stimulation day 6 onwards. Soon, comparative studies with long GnRH agonist protocols were initiated, without certainty about the possible optimal GnRH antagonist administration strategy (Albano et al., 2000; Borm and Mannaerts, 2000; Olivennes et al., 2000; European Middle East Orgalutran study group, 2001; Fluker et al., 2001). Several drawbacks of the GnRH antagonist regimens emerged with these fi rst phase III studies. 1) The numbers of oocytes retrieved were in favour of the long GnRH agonist arms compared to the fi xed multiple dose day 6 and fi xed single dose GnRH antagonist regimen (Albano et al., 2000; Borm and Mannaerts, 2000; Olivennes et al., 2000; European Middle East Orgalutran study group, 2001; Fluker et al., 2001; Roulier et al., 2003) 2) The initiation of FSH administration in a GnRH antagonist regimen is cycle dependent. It is mostly started on day 2 or 3 of the natural cycle which made treatment planning and scheduling more diffi cult (Huirne and Lambalk, 2001; Huirne et al., 2005). 3) Although the pregnancy rates were not different in various individual studies, a meta-analysis including the fi rst 5 comparative studies of fi xed GnRH antagonist protocols compared with long agonist protocols, indicated 5% less clinical pregnancies in the antagonist groups (Al-Inany and Aboulghar, 2002). The initial reported results of these comparative studies, together with the results of national large database evaluations (Devaux et al., 2004; Griesinger et al., 2005a), which were not in favour of the GnRH antagonist, made the GnRH antagonist for many clinicians a second choice. Is this justifi ed? Analysis of the national IVF registry in Germany from 2000 to 2003 demonstrated that GnRH antagonists were often utilized in cycles with an unfavourable a priori prognosis, in patients with advanced age and with a higher number of previous unfavourable cycles (Griesinger et al., 2005a). Subanalysis of patients with equal demographic and clinical features resulted in similar pregnancy rates independent of whether GnRH agonists or antagonists were used (Engel et al., 2006). Furthermore, several comments can be made with respect to the design of the fi rst published meta-analyses. It included one study, with different starting doses of FSH in the comparative arms (see table 1), which possibly confounded the effects (Olivennes et al., 2000). Differences were not statistically signifi cant any longer in an analysis that left out this study.

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ner

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lon

g M

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in IV

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D=

sin

gle

do

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=fl

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=o

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ho

rio

n g

on

ado

tro

ph

in.

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Since then over 200 clinical trials involving GnRH antagonists in IVF have been published, so it is a good time to compare results to those achieved with the long GnRH agonist protocol, the most commonly adopted protocol for assisted reproductive treatement cycles worldwide. Table 1 and 2 provide an overview of all currently available comparative RCT’s (published as full papers), including their design and regimen used, with or without oral contraceptive (OC) pretreatment. Very recently two meta-analyses have been published with confl icting results (Al-Inany et al., 2006; Kolibianakis et al., 2006a). Both studies reported highly signifi cantly shorter duration of stimulation (-1.5 and -1.1 days) and less oocytes (-1.6 and -1.1) retrieved using GnRH antagonists compared to agonists. The fi rst meta-analysis including 27 relevant published papers, abstracts and proceedings, showed signifi cant differences with respect to clinical- (OR=0.84, 95% CI 0.72-0.97) and ongoing pregnancy/live birth rate (OR=0.82, 95% CI 0.69-0.98) in favour of the agonist regimen (Al-Inany et al., 2006). However the quality, method of design and analysis could not be assessed for all used individual papers. The other meta-analysis, with 22 RCT’s published as full papers in peer reviewed journals, could not identify signifi cant differences with respect to the probability of live birth independent of population studied, gonadotrophin type used for stimulation, type of agonist protocol or whether a fi xed or fl exible GnRH antagonist regimen was used (Kolibianakis et al., 2006a). Thus the question is still unanswered; what is the current place of the GnRH antagonist in IVF/ICSI?

Figure 1. Variations in duration and initiation time of the GnRH antagonist in different GnRH antagonist protocols. MD=multiple dose, SD=single dose, CDI=cycle day 1, S1=stimulation day 1, S6=stimulation day 6, S7=stimulationday 7 and hCG=day of human chorion gonadotrophin administration.

fixed SD day 7- 8

flexible � 14 mm

flexible � 15 mm

fixed day 1

preceeding luteal phase

fixed MD day 6

CD1

delayed hCG

hCGS1 S6 S7

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172

After critical appraisal of currently available studies using GnRH antagonists, we think that the differences in reported outcome measurements could be the consequence of the large variation of employed GnRH antagonist regimens (see fi gure 1). In this respect it is likely that two phenomena play an important role to facilitate optimal IVF results when GnRH analogues are used:1) Stable and low LH and progesterone levels throughout the stimulation phase to achieve optimal conditions for implantation2) Sustained low levels of endogenous FSH before stimulation is started to allow optimal synchronization of the follicular cohort.

Figure 2a synchronized follicular development after FSH administration in a long GnRH antagonist regimen. The long GnRH agonist protocol suppresses endogeneous FSH levels, leading to a follicular cohort of all small follicles at the initiation of FSH administration without leading larger follicles. After exogeneous FSH administration, FSH levels remain above the threshold, resulting in a synchronized follicular development. As soon as one or two follicles meet the hCG administration criteria, most follicles will be of more or less similar size and sensitive for hCG.

if 2 or 3 follicles �17mm FSH

window

CD21 CD1 hCGd S1 S3 S5 S7 S9 S11 S13

FSH levels

FSH threshold

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THE OPTIMAL GNRH ANALOGUE REGIMEN

Long versus short or ultra-short GnRH agonist regimenMany treatment schedules with the use of GnRH agonists in IVF therapy have been designed and studied (for review see Huirne et al., 2004). Several investigators tried to shorten the duration of GnRH agonist administration by later administration or early cessation. However, the long protocol (starting in the midluteal phase of the preceeding cycle) gave the best IVF results with regard to oocyte yield and pregnancy rates (Daya, 2000). This protocol induces profound suppression of endogenous release of gonadotrophins during the early follicular phase, allowing the early antral follicles to grow co-ordinately in response to exogenous gonadotrophins to accomplish simultaneous maturation (see fi gure 2a). This leads to an extended widening of the FSH window, increased FSH requirement and in the end more mature follicles and retrieved oocytes (Daya, 2000).

Fixed versus fl exible and short versus long GnRH antagonist regimensThe initially developed GnRH antagonist regimens started relatively late in the follicular phase on a fi xed day, mostly stimulation day 6. Under these circumstances, the luteo-follicular transitory rise of endogenous FSH starts the stimulation of a cohort of follicles that vary in stage of development. Subsequently exogenous FSH allows further development of a few leading large follicles and several smaller follicles (Albano et al., 2000; Borm and Mannaerts, 2000; European Middle East Orgalutran study group, 2001; Fluker et al., 2001; Huirne et al., 2004a; Huirne et al., 2005). As the criteria to administer hCG are based on the size of the leading largest follicles, consequently a number of follicles will still be immature at that time (see fi gure 2b). Logically, the stimulation period will be shorter with less FSH required but also the number of oocytes will be reduced compared to the long GnRH agonist protocol (Albano et al., 2000; Borm and Mannaerts, 2000; Olivennes et., 2000; European Middle East Orgalutran study group, 2001; Fluker et al., 2001). We think that the relatively higher levels of FSH during the early follicular phase in various initially developed GnRH antagonist regimens, results in less synchronization of the follicular cohort with less oocytes retrieved (see fi gure 2b). One could argue that the slightly smaller number of oocytes retrieved cannot be held responsible for possible differences in pregnancies since the number of good quality embryos was not different. Yet another feature of the originally employed GnRH antagonist protocols may have contributed negatively. It is likely that the higher LH, oestradiol and progesterone levels during the early follicular phase in most of these GnRH antagonist regimens compared to the long agonist regimen (see fi gure 3a and b) may play a role. Signifi cantly lower ongoing pregnancy rates are seen in patients with elevated progesterone at initiation of stimulation of fi xed day 6 GnRH antagonist cycles (Kolibianakis et al., 2004b). The level of LH suppression 2 days after commencement of GnRH antagonist therapy in a fi xed day 6 protocol is possibly associated with ongoing pregnancy, the higher the LH levels the lower the probability of achieving an ongoing pregnancy (Kolibianakis et al., 2004a). Possibly early closure of the implantation window occurs (Develioglu et al., 1999) through earlier expression of progesterone receptors in the follicular phase and down regulation of estrogen receptors by the exposure to supraphysiological steroid hormone levels (Kolibianakis et al., 2002; Papanikolaou et al., 2005). These fi ndings support the proposed facilitating/activating mode of hormonal control of endometrial receptivity (de Ziegler, 1995). According to this theory, once endometrium is primed by oestradiol the duration of progesterone exposure is the crucial point leading to a receptive endometrium.

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174

Figure 2b follicular development in a fi xed day 6 GnRH antagonist regimen without OC-pretreatment. Endogeneous FSH levels are not suppressed during the early follicular phase. The luteofollicular transition induces FSH levels above the threshold for a short period until hormonal feedback occurs, leading to the initiation of follicular growth of a few leading follicles. After exogeneous FSH administration, FSH levels arise above threshold again and will initiate several additional follicles to grow. As soon as the leading follicles meet the hCG criteria, several other follicles will be of smaller sizes and may not be sensitive for hCG yet. Such an asynchronized cohort may therefore result in less oocytes retrieved, compared to the long agonist protocol.

Other studies could not fi nd an effect of the absolute LH concentrations on stimulation day 8 or the day of hCG adminstration during a fi xed GnRH antagonist regimen on ovarian response and IVF outcome (Bosch et al., 2003; Penarrubia et al., 2003; Merviel et al., 2004). Differences between the various studies with regard to the level of LH suppression, study populations and type of GnRH antagonist regimen used, may play a role in the confl icting

hCGd S1 CD1 S3 S5 S7 S9

FSH window

hCG if 2 or 3 follicles �17mm

FSH window

CD21

FSH threshold

FSH levels

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results. Furthermore one study indicates that the stability of LH levels rather than absolute LH values are associated with clinical pregnancy as no pregnancies occurred if the LH and progesterone levels changed too markedly (either increase or decrease) during GnRH antagonist administration (Huirne et al., 2005). In order to reduce the number of antagonist injections and the duration of stimulation, fl exible protocols were developed. Instead of starting with the GnRH antagonist on a fi xed day, administration was made dependent on the follicular size. GnRH antagonist injections were started as soon as the follicles reach a size of ≥ 14, 15 or 16mm after 5 days of stimulation (Ludwig et al., 2002; Hohmann et al., 2003; Kolibianakis et al., 2003b; Escudero et al., 2004; Klipstein et al., 2004; Mochtar et al., 2004). Overall this implies that almost 50 percent of the patients will start with a GnRH antagonist beyond day 6 of FSH stimulation (Kolibianakis et al., 2003b; Escudero et al., 2004; Mochtar et al., 2004;). Such protocols in particular allow higher LH, oestradiol and progesterone levels, especially when antagonist treatment is started beyond day 6 and are associated with lower pregnancy rates (Kolibianakis et al., 2003b) whereas an earlier start (CD4 or 5) of GnRH antagonists in these protocols is associated with improved pregnancy rates (Lainas et al., 2005). In this respect, starting with a GnRH antagonist on day 1 compared to day 6 will even further decrease the exposure to LH and oestradiol during the early follicular phase (Kolibianakis et al., 2003a). The long GnRH agonist protocol is favourable to fl exible start antagonist protocols with respect to the number of follicles on the day of hCG and number of oocytes retrieved ( Hohmann et al., 2003; Weghofer et al., 2004; Ragni et al., 2005). Again asynchronous follicle development through absent suppression of early endogenous FSH secretion, could explain this (see fi gure 2 and 3a and 3b). Overall it seems that the low gonadotrophin levels prior to stimulation created by the long agonist protocol are of particular favourable value with regard to IVF/ICSI yield and outcome.

Oral contraceptive (OCs) pretreatment versus GnRH antagonist only protocolsIn order to make the starting time of hormonal stimulation in antagonist cycles for IVF predictable, oral contraceptive pre-treatment has been suggested and evaluated over the past several years. In some studies gonadotrophin administration was started 2 or 3 days (Cedrin-Durnerin et al., 2004; Cheung et al., 2005; Bahceci et al., 2005; Huirne et al., 2006b; Huirne et al., 2006c; Rombauts et al., 2006) and others, 4 or 5 days after OC withdrawal (Obruca A et al., 2000; Vlaisavljevic et al., 2003; Hwang et al., 2004; Sauer et al., 2004; Barmat et al., 2005; Huirne et al., 2006a; Koichi et al., 2006; Kolibianakis et al., 2006b) in either fl exible or fi xed GnRH antagonist protocols. OC pretreatment using GnRH antagonists with subsequent starting of FSH 2 or 3 days after the last OC intake is associated with deep suppression of LH and FSH levels and improved synchronization of the follicular cohort development compared to GnRH antagonist only protocols (Huirne et al., 2006b; Rombauts et al., 2006). Whereas this effect is not seen when FSH stimulation was started on day 5 after the last OCP (Obruca A et al., 2000; Kolibianakis et al., 2006b). Appearently, timing the start of exogenous gonadotrophin administration after OCP-pretreatment affects follicular development (Cedrin-Durnerin et al., 2006). Figures 3c and 3d show a schematic presentation of the expected patterns of LH and FSH levels during the follicular phase if stimulation is started on day 2 or day 5 after the last OC intake. Straight forward comparison of starting with gonadotrophin administration on day 2 versus day 5 after the last OC intake indeed showed stronger gonadotrophin suppression and less large follicles in the early stimulation period if stimulation was started earlier after OC withdrawal (Huirne et al., 2006c). The drawbacks of OC pretreatment are that the stimulation period is increased and more gonadotrophins are needed. Several RCT’s comparing OC pretreated GnRH antagonist with long agonist protocols could not observe

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General discussion

176

signifi cant differences with respect to number of oocytes retrieved and pregnancy rates (Hwang et al., 2004; Sauer et al., 2004; Barmat et al., 2005; Cheung et al., 2005; Bahceci et al., 2005; Huirne et al., 2006a; Rombauts et al., 2006). Although some studies indicate lower implantation rates after OC pretreatment (Huirne et al., 2006b; Rombauts et al., 2006) or increased pregnancy loss compared to GnRH antagonist only regimens (Kolibianakis et al., 2006b). Similar luteal endometrial development was found in OCs pretreated fl exible GnRH antagonist protocol in comparison to a long GnRH agonist protocol (Saadat et al., 2004) or a short GnRH agonist protocol (Schmidt et al., 2005) and in a fi xed day 6 antagonist compared to a long agonist protocol (Simon et al., 2005).

Figure 3. Schematic overview of expected FSH and LH concentrations in various GnRH analogue regimens. 3a and b are regimens without oral contraceptive pill (OCP) pretreatment: 3a long GnRH agonist protocol and 3b Fixed day 6 GnRH antagonist protocol. 3c FSH is started 2 days after the last OCP and 3d FSH is started 5 days after the last OCP in a fi xed day 6 GnRH antagonist regimen.

GNRH AGONISTS AND ANTAGONISTS: have we compared them in the optimal way?

Taken all together, the optimal GnRH analogue regimens seem to be regimens ensuring stable FSH and LH suppression during the entire stimulation period. In this respect, the long agonist or long fi xed antagonist regimen seemed to be preferred (i.e. long OC-pretreated fi xed

hCG

FSH

FSH window

FSH window

CD21 CD1 CD2 CD3

S1 S3 S5 S7 S9 S11

GnRH agonist

FSH threshold

antagonist

hCG FSH window

FSH window

CD21 CD1 CD2 CD3

S1 S3 S5 S7 S9

FSH

FSH threshold

antagonist

FSH leve

hCG

FSH window

OCP +2

S1 S3 S5 S7 S9 S11

FSH OCP’s

OCP+1

FSH threshold

antagonist

hCG FSH

window

FSH window

OCP +3

OCP’s

OCP+1

OCP+2

FSH

S1 S3 S5 S7 S9

OCP+4

OCP+5

FSH threshold

3a 3c

3b 3d

FSH levels LH levels

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GnRH antagonist protocol or a long GnRH antagonist protocol from S1 onwards). We stress that an optimal comparison of GnRH agonists versus antagonist requires the comparison of the optimal regimens of both compounds. So far, only a few individual studies compared the long GnRH agonist protocol with OC-pretreated fi xed GnRH antagonist protocols (see table 2). Most individual RCT’s comparing OC-pretreated GnRH antagonist (fi xed or fl exible) with a long GnRH agonist protocol, could not identify signifi cant differences in number of oocytes retrieved and pregnancy rates in a general IVF population (Vlaisavljevic et al., 2003; Hwang et al., 2004; Sauer et al., 2004; Barmat et al., 2005; Huirne et al., 2006a; Rombauts et al., 2006), PCO patients ( Bahceci et al., 2005) or poor responders (Cheung et al., 2005) (see table 4a and b).

To further explore our idea that the OC pretreated GnRH antagonist regimen is comparable to the long GnRH agonist protocol, we performed a systematic review including only studies comparing OC pretreated GnRH antagonist with long GnRH agonist regimens. The purpose of this systematic review was to assess, among patients undergoing ovarian stimulation in GnRH analogue IVF or ICSI cycles, whether GnRH long agonist- or OC-pretreated fi xed GnRH antagonist- or OC-pretreated fl exible antagonist regimens resulted in signifi cantly different number of oocytes and clinical pregnancies. In August 2006, the bibliographic database MEDLINE, EMBASE and Cochrane were searched using the following keywords: GnRH antagonist; GnRH agonist, cetrorelix, ganirelix, antide, and limited to “human”, “female” and “clinical trial”. All 180 studies comparing GnRH agonists with GnRH antagonists were studied, and were considered for inclusion where OC pretreatment in the antagonist arms was used. Despite the risk of publication bias, meeting proceedings were not considered because unpublished studies cannot be adequately evaluated for their design and quality which is essential to address our research question. Most abstracts do not report their GnRH antagonist regimen in detail and whether OC pretreatment is given. All published randomized controlled trials addressing the research question were included regardless of the sample size, language of publication or day of FSH commencement. Primary outcome parameter was the clinical pregnancy rate (defi ned as intra-uterine pregnancy confi rmed by ultrasound at a gestational age of 6 weeks) per randomized patient. Secondary outcome parameter was the number of oocytes retrieved per ovum-pick up. If the defi nition of the pregnancy rates used or any other information was not clear, we contacted the authors for additional information. Information was also requested from the corresponding authors regarding pregnancies in patients who were randomized but did not start any treatment. If we did not obtain the additional information these patients were considered as not pregnant. One study reported the number of oocytes retrieved as median and range. We were not able to obtain the mean value and standard deviations. Therefore this study was not included for calculation of the weighted mean differences of number of oocytes retrieved (Barmat et al., 2005). The clinical characteristics of included studies are presented in table II. Patient populations were divided into PCOS, poor responders or unspecifi ed/general population of infertile couples undergoing IVF or ICSI. The clinical pregnancy rates for each study were expressed as an odds ratio with 95% confi dence intervals (CI). These results were combined for meta-analysis with Comprehensive Meta-analysis software (STATA 9) using the Mantel Haenzel method. The results with respect to the number of oocytes were presented as the mean weighted differences with 95% CI. Results: 8 studies, incorporating 932 patients, that met the requirements could be indentifi ed, with a mean number of 108 patients per study (see table 2).

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178

auth

ors

nu

mb

er

of

pat

ien

tsp

op

ula

tio

nty

pe

of

ago

nis

t u

sed

ago

nis

t p

roto

col

typ

e o

f an

tag

on

ist

use

d

anta

go

nis

t p

roto

col

star

t FS

H:

nu

mb

er o

f d

ays

afte

r la

st O

CP

star

tin

gd

ose

FSH

OC

/lo

ng

ag

on

ist

vers

us

OC

/fi x

ed a

nta

go

nis

t p

roto

col

Hw

ang

et a

l. (2

004)

a29

/27

PCO

SB

use

reli

nlo

ng

MD

(±O

C)

Cet

rore

lix

OC

/MD

fi x

ed

(0.1

25m

g/d

O

CP+

4 to

OC

P+9)

(0

.25m

g/d

O

CP+

10 t

o h

CG

d)

415

0 h

MG

Sau

er e

t al

. (20

04)b

25/2

4(2

5/24

/25)

gen

eral

Leu

pro

reli

nO

C/l

on

g SD

Cet

rore

lix

OC

/fi x

ed S

D S

7O

C/fi

xed

SD

S7

+LH

5 22

5 rh

FSH

Ch

eun

g et

al.

(200

5)33

/33

po

or

resp

on

der

sB

use

reli

nO

C/l

on

g M

DC

etro

reli

xO

C/fi

xed

MD

S6

2 o

r 3

300

rhFS

H

Hu

irn

e et

al.

(200

6)91

/91

gen

eral

B

use

reli

nlo

ng

MD

Cet

rore

lix

OC

/fi x

ed M

D S

65

150-

225

rhFS

H

OC

/lo

ng

ag

on

ist

vers

us

OC

/fl e

xib

le a

nta

go

nis

t p

roto

col

Bah

ceci

et

al. (

2005

)75

/73

PCO

SLe

up

roli

nO

C/l

on

g M

DC

etro

reli

xO

C/fl

ex

MD

(≥

14m

m)

315

0-30

0 u

FSH

/hM

GB

arm

at e

t al

. (20

05)

40/4

0ge

ner

alLe

up

roli

nO

C/l

on

g M

DG

anir

elix

OC

/fl e

x M

D

(≥12

mm

)5

300

rhFS

H

Ro

mb

ou

ts e

t al

. (20

06)

111/

111

gen

eral

Naf

arel

inlo

ng

MD

Gan

irel

ixO

C/fl

ex

MD

(≥

14m

m)

220

0 rh

FSH

Ko

ich

i et

al.

(200

6)c

66/6

3/63

ge

ner

alB

use

reli

nO

C/l

on

g M

DC

etro

reli

xO

C/fl

ex

MD

(≥

14m

m)

522

5-30

0uFS

H

Tab

le II

. Ch

arac

teri

stic

s of c

urr

ent p

ub

lish

ed ra

nd

om

ized

co

ntr

olle

d tr

ials

co

mp

arin

g G

nR

H a

go

nis

t an

d a

nta

go

nis

ts in

IVF

incl

ud

ing

OC

pre

trea

tmen

t.

a d

ose

of

anta

go

nis

t w

as i

ncr

ease

d f

rom

0.1

25 t

o 0

.25m

g/d

ay o

n d

ay 1

0 af

ter

last

OC

P. b

3 ar

m s

tud

y; L

euro

lin (

n=

25)

vs C

etro

relix

(n

=24

) v

s C

etro

relix

+LH

(n

=25

). c

3 ar

m s

tud

y; B

use

relin

(n

=66

) vs

Cet

rore

lix,

star

tdo

se 2

25 I

U F

SH,

FSH

in

crea

se t

o 3

00 i

f fo

llicl

e ≥

14m

m (

n=

63)

vers

us

Cet

rore

lix, s

tart

do

se 2

25 IU

FSH

, FSH

dec

reas

ed t

o 7

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Three studies were excluded (see table III); one included oocyte donors (Prapas et al., 2005), one compared an OC pretreated GnRH antagonist with a fl are (short) agonist protocol (Schmidt et al., 2005) and one study did not perform true randomization (Vlaisavljevic et al., 2003).

authors number of patients

population type of agonistused

agonist protocol

type of antagonist used

antagonist protocol

reason exclusion

OC/long agonist versus OC/day fl exible antagonist protocol

Vlaisavljevic et al. (2003)

236/226 general Goserelin OC/long SD

Cetrorelix OC/fl ex MD (≥12-14mm)

inadequate randomization

OC/long agonist versus OC/fi xed antagonist protocol

Prapas et al. (2005)a

75/73 oocyte donors

Triptorelin OC/long MD

Ganirelix OC/fi xed MD S8

oocyte donors

short agonist versus OC/fl exible antagonist

Schmidt et al. (2005)

24/24 poor responders

Leuprolin fl are up MD

Ganirelix OC/ fl ex MD (≥12mm)

short agonist protocol

Table III. excluded studies. aSome women were used twice as donor

All included studies were published within the last 6 years and were adequately randomized, described inclusion criteria and analysed only one cycle per patient. The number of oocytes per cycle are summarized in table IV and the clinical pregnancy rates per started cycle are summarized in table V. Long GnRH agonist protocols were compared with OC-pretreated fi xed GnRH antagonist protocols (table IVa and Va) and with OC-pretreated fl exible GnRH antagonist protocols (table IVb and Vb), seperately.

Table IVa. Number of oocytes in a long agonist and OC pretreated fi xed GnRH antagonist.n/N = outcome/total a mean number of metaphase II oocytes

Study long OC-fixed WMD Weight WMD agonist antag (95%CI ) % (95%CI) n mean(sd) n mean(sd) long agonist flexible antagonist Hwang 2004 27 17.6 (5.9) 25 16.3 (6.4) 17.0 0.21 (-0.33 - 0.76)

Sauer 2004a 23 13.3 (6.5) 21 13.7 (6.8) 14.4 -0.06 (-0.65- 0.53)

Cheung 2005 21 5.6 (4.2) 19 5.9 (3.0) 13.1 -0.82 (-0.70- 0.54)

Huirne 2006 84 10.9 (6.7) 85 11.4 (7.3) 55.5 -0.07 (-0.37- 0.23)

Total 156 149 100 -0.02 (-0.25- 0.20)

Test for heterogeneity chi-square= 0.86, p=0.84 Test for overall effect z=0.20, p=0.84

0 -10 -5 5 10

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General discussion

180

Number of oocytes was similar in the long GnRH agonist protocol compared to the OC- pretreated fi xed GnRH antagonist protocol, with a weighted mean difference (MWD) of -0.02 (95%CI;-0.25 to 0.20). However the number of oocytes was slightly higher in the long agonist protocol compared to the OC-pretreated fl exible GnRH antagonist protocol (MWD: 0.22, 95%CI;0.04 to 0.40). The majority of the included OC-pretreated fl exible GnRH antagonist regimens started with FSH administration 5 days after the last OCP taken, which may play a role in the lower number of oocytes retrieved in the fl exible antagonist regimens despite OC pretreatment (Huirne et al., 2006c). No differences in number of oocytes were found in a sub-analysis including only studies comparing the long agonist protocol with OC-pretreated fi xed or fl exible GnRH antagonist regimen in which stimulation was started 2 or 3 days after the last OCP (MWD:0.11, 95%CI; -0.91 to 0.31).

Table IVb. Number of oocytes in long agonist and OC pretreated fl exible GnRH antagonist. WMD = weighted mean difference.

Although the number of oocytes was lower in the OC-pretreated fl exible protocol in comparison to the long agonist protocol, it could be questioned whether the weighted mean difference of 0.22 oocytes is of any clinical relevance. The calculated weighted mean difference of all OC-pretreated fi xed and fl exible protocols compared to the long agonist protocol was not signifi cantly different when all patients were taken into account in a general IVF population (0.10,95%CI;-0.65 to 0.27).Clinical pregnancy rates were not different between the long GnRH agonist protocol in OC-pretreated fi xed protocols in this (small) meta-analysis (OR 0.99, 95%CI;0.61-1.62) (see table 5a). However signifi cantly more clinical pregnancies were found using long GnRH agonists compared with the OC-pretreated fl exible protocols (OR 1.67, 95%CI 1.18-2.38) (table 5b).

Study long OC-flexible WMD Weight WMD agonist antag (95%CI ) % (95%CI) n mean(sd) n mean(sd) long agonist flexible antagonist

Bahceci 2004 70 21.6 (11.0) 59 19.3 (9.0) 27.7 0.22 (-0.12- 0.57)

Rombauts 2006 101 14.2 (8.1) 107 13.6 (7.5) 45.2 0.08 (-0.20- 0.35)

Koichi 2006 66 14.3 (7.8) 62 11.2 ( 6.2) 27.1 0.44 ( 0.09- 0.79)

Total 237 228 100 0.22 (0.04- 0.40)

Test for heterogeneity chi-square= 2.55, p=0.28 Test for overall effect z=2.32, p=0.02 -

0 -5 -10 5 10

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Table Va Clinical pregnancy rates in long agonist and OC pretreated fi xed GnRH antagonist. n/N = outcome/total

Table Vb Clinical pregnancy rates in long agonist and OC pretreated fl exible GnRH antagonist. n/N = outcome/total. a6.6% in the agonist arm and 19.1% of patients in the antagonist arm never started with “ovulation induction” for personal reasons. bone patient randomized to receive antagonists received agonists and did not became pregnant, she was calculated on ITT base (in the antagonist group).

ConclusionThe results of this meta-analysis indicate that the OC-pretreated fi xed GnRH antagonist regimen is as effective as the long GnRH agonist protocol in IVF with respect to number of oocytes retrieved and pregnancy rates. Thus it seems that indeed the oocyte yield depends on the duration of rhFSH administration and is highest if endogenous gonadotrophins are profoundly suppressed from the early stimulation phase onwards (see fi gure 2).

Study long OC-fixed OR Weight OR agonist antag % (95%CI) Hwang 2004 10/29 10/27 21.2 0.89 (0.30-2.67)

Sauer 2004 11/25 11/24 19.6 0.93 (0.30-2.86)

Cheung 2005 3/33 5/33 14.2 0.56 (0.12-2.56)

Huirne 2006 22/91 19/91 45.0 1.21 (0.60-2.43)

Total 46/178 45/175 100 0.99 (0.61-1.62) Test for heterogeneity chi-square= 0.9, p=0.83 Test for overall effect z=0.02, p=0.98

1 0.1 0.2 5 10

Study long OC-flexible OR Weight OR agonist antag % (95%CI) Bahceci 2004a 41/75 34/73 32.2 1.38 (0.73-2.64)

Barmat 2005b 18/40 16/40 18.2 1.23 (0.51-2.98)

Rombauts 2006 30/111 20/111 30.1 1.69 (0.89-3.20)

Koichi 2006 37/66 21/63 19.5 2.55 (1.25-5.21)

Total 126/292 91/287 100 1.67 (1.18-2.38)

Test for heterogeneity chi-square= 2.14, p=0.54 Test for overall effect z=2.88 p=0.004

1 0.1 0.2 5 10

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182

Furthermore these results underline our idea that stable and early suppression of endogeneous gonadotrophins may be advantageous to achieve pregnancy, which can be achieved by OC pretreatment in a fi xed GnRH antagonist protocol but not in a OC pretreated fl exible protocol. Based on the lower number of side-effects and lower number of required (GnRH analogue) injections with similar ability to schedule ovum pickup, this regimen may be an attractive alternative for the commonly used long GnRH agonist protocol (Huirne et al., 2006a). The reliability of the results of a meta-analysis depends on the quality of the included randomized controlled trials and of its power. The latter was not suffi cient enough in our case. More (larger) randomized controlled trials comparing OC-pretreated fi xed GnRH antagonist regimen or a long (S1) fi xed GnRH antagonist regimen with long GnRH agonist regimens, of suffi cient power to indentify signifi cant differences in pregnancy rates, are required to allow optimal comparison between GnRH agonists and antagonists for its use in IVF or ICSI therapy.

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Epilogue

10

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Epilogue

188

Now that this thesis is complete, it is the time to ask what we have learned and if we reached our goal, were we able to identify the optimal gonadotrophin releasing hormone (GnRH) antagonist regimen to compare it with GnRH agonists for its use in in-vitro fertilization (IVF)?

WHAT HAVE WE LEARNED ABOUT GNRH ANTAGONISTS IN IVF AND WHAT IS

THE OPTIMAL REGIMEN?

First of all, by studying the effect of various dosages of the GnRH antagonist Antide in IVF patients we identifi ed the minimal effective dose to prevent luteinizing hormone (LH) surges in IVF (Chapter 4). However due to marketing reasons, Antide was not further developed and planned phase III studies were cancelled. Our results indicate that from a clinical and scientifi c perspective, there is a place for Antide to broaden the pharmacological armamentarium (Chapter 4 and 5). Besides this, our study was of additional value to study the effect of various induced endogeneous LH levels on endocrinology and implantation; LH levels were dose-dependent (Chapter 4). There is an optimal GnRH antagonist dose for its use in IVF, too high and too low seems to be detrimental for implantation. Favourable clinical pregnancy rates were observed between a particular window of changes in LH levels (Chapter 5). These fi ndings indicate that stable LH levels may be desirable during GnRH antagonist use in IVF, making fl exible or short and late administered GnRH antagonist protocols less optimal. Keeping this in mind several optimal GnRH antagonist strategies were postulated. First of all oral contraceptive (OC) pre-treated fi xed GnRH antagonist protocols to stabilize LH and FSH levels during the early follicular phase or starting with a GnRH antagonist earlier during a stimulation cycle from stimulation day 1 onwards instead of the most used stimulation day 6 onwards. We tested the fi rst strategy. OC pre-treatment, results in lower and stable LH and FSH levels during the early follicular phase and resulted in an optimization of follicular development with in the end more oocytes retrieved (Chapter 6). Unfortunately we could not detect any positive effect on pregnancy rates in a small study comparing GnRH antagonist cycles with or without OC pre-treatment. Gonadotrophin administration was started on day 2 after the last oral contraceptive pill. We postulated that this was caused by too strong suppression of endogenous LH levels or that stimulation was started too soon after the last OC pill which may negatively effect bleeding pattern and implantation (Chapter 6). Therefore we studied the effect of starting with stimulation on day 2 or day 5 after the last OC pill with or without LH addition in a GnRH antagonist regimen in IVF patients (Chapter 7). When starting with gonadotrophin administration on day 2 in comparison to day 5 more steroidal hormonal suppression is present, producing improved synchronization of the follicular cohort without affecting the oocyte yield, although more gonadotrophins are needed. Pregnancy rates in this small study were promising in all tested arms. The addition of LH did not improve IVF outcome and it resulted in less oocytes retrieved (Chapter 7). From an economical point of view OC pre-treatment of a GnRH antagonist cycle, starting with gonadotrophins stimulation on day 5 after the last OC pill and without LH addition seems to be preferred. Due to the insuffi cient power of the last study we need to see this study as an indication that timing of gonadotrophin administration after OC pre-treatment affects follicular development. Additional studies are needed to confi rm our fi ndings and to test the effect on pregnancy outcome.

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IS THERE A PLACE FOR GNRH ANTAGONISTS IN IVF?

Why did we put all the effort in searching the optimal GnRH antagonist regimen in IVF if we have a good alternative, the well established long GnRH agonist protocol (Chapter 3)? The most important reason is that GnRH antagonists cause an immediate and direct reversible suppression of LH and FSH levels due to their mechanism of action (Chapter 2). Whereas GnRH agonists needs to be administered for a longer period, pituitary desensitization needs to be induced fi rst, before gonadotrophin suppression occurs. Therefore GnRH antagonist can be administered at a later stage during the treatment cycle, resulting in a shorter treatment period and less injections required in comparison to the long agonist protocol. However the introduction of a more convenient IVF strategy can only be justifi ed if this new strategy is comparable to the best available alternative (i.e. the long GnRH agonist protocol) in terms of IVF outcome. We have the opinion that the current place of GnRH antagonists can only be established in a fair way if the optimal GnRH antagonist regimen is compared with the optimal GnRH agonist (i.e. long agonist) protocol. As discussed earlier (part one of this thesis), an OC pretreated fi xed GnRH antagonist protocol may be such a protocol. This protocol, in which gonadotrophin stimulation was started 5 days after the last OC pill taken, resulted in similar IVF outcome in comparison to the long GnRH agonist protocol (Chapter 8). Thus OC pre-treatment may overcome several drawbacks of the early studied GnRH antagonist only protocols; similar ability to predict oocyte retrieval as using the GnRH agonist in a long protocol with equal number of oocytes retrieved and pregnancy rates, with similar or even lower number of reported side-effects (Chapter 8). After critical appraisal of current literature we observed a negative opinion with respect to the GnRH antagonists in several papers. This is mainly based on current available meta-analyses including inferior GnRH antagonist regimens which were compared to the optimal long GnRH agonist protocol. After critical appraisal of all available literature with respect to the use of GnRH agonists versus GnRH antagonists in IVF we conclude that it is too early to denigrate the GnRH antagonist at such an early stage, even before the optimal GnRH antagonist regimen has been established (chapter 9). A meta-analysis including only studies that compare the optimal long GnRH agonist regimens with OC pretreated fi xed GnRH antagonist regimens confi rmed our positive IVF outcome of this regimen, which in our opinion may be one of the optimal GnRH antagonist strategies. However the power of this meta-analysis is insuffi cient to identify signifi cant differences in clinical pregnancy and life birth rates due to the limited number of studies comparing these regimens so far. This thesis indicates that that optimal GnRH antagonist regimen seems to be comparable to the currently used long agonist protocol, but we are not there yet. This thesis should be seen as a fi rst step for fair comparison of GnRH agonists and antagonist regimens. How to move ahead with GnRH antagonists?

FUTURE PERSPECTIVES

To allow fi nal and optimal comparison between GnRH agonists and antagonists we need an additional effort in further optimization of GnRH antagonist regimens. Larger studies comparing the effect of starting time of stimulation (day 2 versus day 5) should be performed to study its effect on pregnancy rates. Comparative studies between GnRH antagonist regimen starting on stimulation day 1 versus the long GnRH

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agonist regimens should be performed. Although promising results with respect to optimal synchronization of the follicular cohort have been reported for GnRH antagonist administration during the preceding luteal phase, such a regimen does not seem to offer any benefi t from a patients convenience or economical point of view, since this will lead to similar duration of the GnRH analogue administration period. Recently, very promising results have been reported after the administration of a GnRH agonist during the midluteal phase of GnRH agonists but especially during GnRH antagonist regimens (Tesarik et al., 2004 and 2006, Pirard et al., 2005, Hugues et al., 2006). Although we do not understand the working mechanism, the impressive improvement of implantation justifi es exploration of this regimen. However, before this regimen can be introduced on a larger scale several safety aspects for these embyros (including possible direct effects) and for the resulting children should be elucidated (Lambalk and Homburg, 2006). Direct effects or later effects of various assisted reproductive therapies are in general very limited (Janssens et al. 2000). Basic studies addressing this topic using GnRH analogues should gain high priority to allow a bright future for both GnRH agonists and antagonists. Finally, when the above problems have been solved and all topics have been elucidated, optimal comparison between GnRH agonists and antagonists should be performed preferably by a randomized controlled trial of suffi cient power or by a meta-analysis of suffi cient power including only the optimal protocols. Besides its effect on IVF outcome and preferably take home baby rate, cost-effectiveness should also be taken into account.

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11 Summary

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The optimal gonadotrophin releasing hormone (GnRH) antagonist regimen has not been elucidated yet, despite over 200 reported clinical studies using GnRH antagonists. Currently available meta-analysis included all comparative studies between GnRH agonists and antagonists performed so far, including less than optimal GnRH antagonist regimens leading to confl icting results dependent on the selection of studies made. The aim of this thesis was to search for the optimal GnRH antagonist regimen and to compare it with GnRH agonists for its use in in-vitro fertilization (IVF). As outlined in chapter 1, the studies reported in the fi rst part of this thesis (chapter 2 to 8) aim to search for factors which may improve GnRH antagonist regimen in terms of follicular development and oocyte yield. The studies in the second part of this thesis (chapter 8 and 9) aim to search for the optimal comparison of GnRH antagonists with GnRH agonists to evaluate the current place of GnRH antagonists in IVF.

CONSIDERATIONS IN SELECTING THE OPTIMAL GNRH ANTAGONIST REGIMEN

Chapter 2 This chapter addresses the development of GnRH antagonists, their mechanism of action their possible clinical applications in the fi eld of reproduction and beyond.

Pulsatile GnRH stimulates the pituitary secretion of both luteinizing hormone (LH) and follicle stimulating hormone (FSH) and thus controls the hormonal and reproductive function of the gonads. Blockade of GnRH effects may be wanted for a variety of reasons, e.g. to prevent untimely luteinization during assisted reproduction or in the treatment of sex-hormone-dependent disorders. Selective blockade of LH/FSH secretion and subsequent chemical castration have previously been achieved by desensitizing the pituitary to continuously administered GnRH or by giving long-acting GnRH agonists. Only recently have GnRH receptor antagonists, that immediately block GnRH’s effects, been developed for clinical use with acceptable pharmacokinetic, safety, and commercial profi les. We concluded that all current indications for GnRH agonist desensitization may prove to be indications for a GnRH antagonist, including endometriosis, leiomyoma, breast cancer, benign prostatic hypertrophy and prostatic carcinoma and central precocious puberty. However, the best clinical evidence so far has been in assisted reproduction and prostate cancer. Finally, licensed GnRH antagonists are ideal research tools for further exploration of the pathophysiology of the human reproductive system.

Chapter 3This chapter provides an overview of the literature with respect to various drugs and therapeutic regimens (i.e. GnRH agonists and antagonists, urinary and recombinant gonadotrophins) used for ovarian stimulation in IVF.

FSH treatment to induce follicular development in an-ovulating women and multiple folli-cular development for assisted conception has been incorporated in almost all reproductive treatment cycles in the form of either urinary, purifi ed urinary or recombinant preparations. Besides improved tolerance and theoretically lower chances of infection by prions, the latter have proven to be more effective in terms of clinical pregnancy rates, FSH requirement and cost effectiveness. The low-dose step-up protocol to induce monofollicular development, which is applied worldwide, has to compete with the equally effective but

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health economically benefi cial step-down protocol. The long protocol using recombinant FSH 150 IU/day is advocated when using GnRH agonists in IVF or intracytoplasmatic sperm injection (ICSI) treatment. Besides this well adopted strategy, GnRH antagonists with acceptable safety profi le became available which allow alternative treatment strategies with milder approaches with acceptable cancellation rates. Additionally GnRH antagonists allow the use of GnRH agonists to trigger fi nal oocyte maturation and ovulation; the latter require pituitary responsiveness and are therefore excluded in agonist protocols. FSH and LH are both required for appropriate folliculo- and steroidogenesis. In hypogonadotropic women, the addition of LH (human menopausal gonadotrophin, human chorionic gonadotrophin or rLH) is therefore obligatory to achieve appropriate follicular growth and pregnancy. The role of LH in ovulation induction is still a matter of debate, although in GnRH agonistic protocols there seems to be a ‘therapeutic window’; levels that are too high or too low have detrimental effects on IVF outcome. Prospective studies investigating possible direct effects of GnRH analogues, optimal dose-fi nding studies and treatment regimens under different conditions, with or without pharmacological co-administration and for different indications, should be performed to optimise the effi cacy and tailor treatment strategies to individual needs.

Chapter 4The aim of this chapter was to defi ne the minimal effective dose of a new GnRH antagonist antide to suppress LH levels and to prevent premature LH surges in IVF patients.

We performed a prospective parallel, single centre study. The primary endpoint of this study was to determine the minimal effective dose, defi ned as the lowest dose group in which fewer than two LH surges occurred. Secondary endpoints were drug requirements, serum hormone and antide concentrations and safety aspects. This study was conducted in two phases, a double-blind phase (n=60) was followed by an open phase in which 3 additional treatment groups were added with lower antide dosages. In total 144 IVF/ICSI patients were stimulated with rhFSH from cycle day 2 onwards, and co-treated with daily 2 mg/2 ml (n=30), 1 mg/ml (n=30), 0.5 mg/ml (n=31), 0.5 mg/0.5 ml (n=23) and 0.25 mg/ml (n=30) GnRH antagonist (antide) from cycle day 7 onwards. Serum samples were taken three times daily during antide administration to assess antide and hormone levels. Serum antide levels, mean LH and oestradiol levels per day and their area under the curves were dose-related to antide. The bioavailability of antide almost doubled after dilution in larger volumes. LH levels immediately decreased after the fi rst antide injection, pre-injection LH levels gradually increased during GnRH antagonist treatment. LH surges occurred only in the lowest dose groups 0.5 mg/ml (3.2%), 0.5 mg/0.5 ml (6.7%) and 0.25 mg/ml (13.3%). Hence, 0.5 mg/ml is considered to be the minimal effective dose. Antide was overall well tolerated and safe. We concluded that 0.5 mg/ml antide is the minimal effective dose to prevent an untimely LH surge in IVF patients stimulated with rhFSH.

Chapter 5This chapter addresses the relation between various LH and progesterone concentrations, induced by different GnRH antagonist doses, on IVF outcome.

We performed a prospective, single centre study including 144 IVF patients, stimulated with recombinant FSH from cycle day 2, and co-treated with daily GnRH antagonist

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(antide) (2 mg/2 ml, 1 mg/ml, 0.5 mg/ml, 0.5 mg/0.5 ml or 0.25 mg/ml) from cycle day 7 onwards. Serum samples were taken three times daily. Outcome measures were drug requirements, stimulation results, IVF outcome and its relationship to serum and antide concentrations. There were no differences in number of mature follicles (≥11 mm), mean follicular size and endometrial thickness on the day of hCG administration between

the various dose groups. Also no differences were found in number of oocytes retrieved, mean number of metaphase II oocytes per patient, fertilization rate, total number of embryos, good quality embryos (grade I and II) and number of embryos transferred. There was a tendency toward higher pregnancy and implantation rates in the middle dose groups (0.5 and 1.0 mg/ml; not signifi cant). None of the patients in this study

had moderate or severe symptoms associated with OHSS necessitating hospitalization.

Clinical pregnancies were only observed within a particular range of change in LH levels. Based on these results we concluded that excessive or insuffi cient suppression of LH and progesterone levels during GnRH antagonist administration and high progesterone per follicle on the day of hCG administration seems to be associated with impaired clinical pregnancy rates.

Chapter 6The aim of this chapter was to assess the effect of oral contraceptive (OC) pretreatment in a fi xed GnRH antagonist IVF protocol on the coordination of follicular development and the number of oocytes retrieved, in comparison to a control group without OCs.

To address this issue, we randomized 64 IVF patients to start rhFSH on day 2 or 3 after OC withdrawal (OC group) or on day 2 of a natural cycle (control group). From stimulation day 6 onwards, all patients were treated with daily (0.5 mg/ml) GnRH antagonist (antide). The primary effi cacy endpoint was the number of oocytes retrieved. OC pretreatment resulted in signifi cantly lower starting concentrations of FSH, LH and oestradiol and a thinner endometrium. In the early stimulation period, fewer large follicles were found after OC pre-treatment, leading to an extended stimulation period (11.6 versus 8.7 days, p<0.0001) with more follicles on the day of rhCG administration (15.4 versus 12.5, p=0.02) and more oocytes retrieved (13.5 versus 10.2, p< 0.001) as compared with the control group. Based on these results we concluded that OCs pretreatment in a fi xed GnRH antagonist regimen in which rhFSH is started 2 days after the last OCs, improved follicular homogeneity with an extended stimulation period, more rhFSH required and more oocytes retrieved.

Chapter 7Adresses the effect of timing gonadotrophin administration (day 2 versus day 5) after the last oral contraceptives (OCs) taken, with or without rLH addition, in a fi xed GnRH antagonist protocol on hormonal concentrations and follicular development.

We randomized 48 IVF/ICSI patients treated with a fi xed day 6, GnRH antagonist protocol, to start with daily 150 IU rhFSH 2 or 5 days after last OCs taken, with or without addition of 150 IU rhLH/day. The primary effi cacy endpoint was the number of mature follicles (≥11mm) on the day of hCG administration. Starting on day 2 versus day 5 leads to signifi cantly lower starting concentrations of FSH, LH and oestradiol, a thinner endometrium (p<0.001) and less large follicles on S6, an extended stimulation period (8.0 versus 4.8 days, p<0.001) and more rhFSH required (1981 and 1474 IU, respectively) (p<0.001). Hormonal concentrations and number of oocytes inseminated were similar on the day of hCG administration (hCGd).

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Addition of daily rhLH leads to higher LH concentrations (all >1.0 IU/L), less large follicles on hCGd and a lower number of oocytes available for insemination (p<0.001). In conclusion, although the follicular cohort is more uniformly developed if gonadotrophins administration is started 2 versus 5 days after OC withdrawal, this does not lead to an improved oocyte yield despite more administered gonadotrophins. Addition of 150 IU rhLH per day in OC pretreated GnRH antagonist regimen results in higher LH levels but may produce less follicles and oocytes. Based on the results of this small study we postulate that starting with gonadotrophins 5 days after OC withdrawal is to be preferred in view of shorter treatment and less gonadotrophin requirements and preferentially without LH addition.

SEARCHING FOR THE OPTIMAL COMPARISON OF GNRH ANTAGONISTS AND

AGONISTS

Chapter 8The aim of this chapter was to compare the oocyte yield and IVF outcome in patients treated with a long GnRH agonist regimen and an oral contraceptive (OC) pretreated fi xed day 6 GnRH antagonist protocol in which stimulation was started on day 5 after last OCs.

To address this aim, we performed a multicentre, randomized study. 182 patients were randomized to receive cetrorelix with OC pretreatment (n=91) or to receive buserelin (n=91). The cetrorelix group started with daily OCs on cycle day 5, during 21 to 28 days. Cetrorelix (0.25 mg) was given daily from stimulation day 6 up to and including the day of rhCG administration. The buserelin group started with buserelin (500 μg/day) for at least 10 days until down-regulation was achieved, after which the dose was reduced to daily 200 μg up to and including the day of rhCG administration. RhFSH was started in both groups on a Friday, in the cetrorelix group 5 days after last OC intake. Both regimens were followed by a general IVF or ICSI procedure. The primary effi cacy endpoint was the number of oocytes retrieved per patient. Number of oocytes, cancellation rates, rhFSH requirements, number of ovum pick-ups (OPU) in the weekend or public holiday and number of pregnancies were similar in both groups. Both treatment regimens were well tolerated. In conclusion, it seems that the combination of OCs with antagonist largely overcomes several disadvantages of GnRH antagonist application in IVF that were claimed in the past compared with long agonist regimens. The mean number of oocytes retrieved were similar in OC pretreated cetrorelix group and the long buserelin group with only a small number of OPU on weekend or public holidays. In our opinion this combined strategy, in particular given the signifi cant reduction of required injections and in part as result of that signifi cant reduction in reported side effects before stimulation, provides a good alternative for the long GnRH agonist protocol in prevention of premature luteinization in IVF.

Chapter 9The aim of this chapter was to identify the optimal GnRH antagonist regimen in terms of IVF outcome and scheduling ability, to compare with GnRH agonists.

To establish this we reviewed currently available literature with respect to various GnRH antagonist regimens and comparative studies including GnRH agonists and

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antagonists in IVF. We believe that appropriate comparison of optimal GnRH agonist and antagonist regimens has not been performed yet. Currently available meta-analysis included all comparative studies between GnRH agonists and antagonists performed so far, including less than optimal GnRH antagonist regimens. After critical appraisal of the various studied GnRH antagonist regimens in terms of follicular development and IVF outcome, we postulate that early suppression of endogenous FSH results in optimal follicular development. Additionally, stable and early suppression of LH and progestrone levels during the entire period of stimulation may be an advantage for implantation and pregnancy outcome. In this respect, single dose and particularly fl exible protocols seem to be less advantageous. LH addition does not offer benefi ts in terms of pregnancy rates in a general IVF population. Early FSH and LH suppression can be achieved by early GnRH antagonist administration (stimulation day 1) or by OC pretreatment. A meta-analysis including four studies comparing the long GnRH agonist protocol with OC-pretreated fi xed GnRH antagonist regimen; could not identify differences in number of oocytes retrieved and clinical pregnancies. We stress that it is too early to denigrate the GnRH antagonist before the optimal GnRH antagonist protocol has been compared with the optimal GnRH agonist regimen in a prospective randomized fashion. More studies comparing long GnRH agonist protocols with “long” GnRH antagonist protocols, with enough power to identify differences in pregnancy rates, (long or pretreated with oestrogens or oral contraceptives) are required before appropriate comparison can be made.

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De zoektocht naar het optimale GnRH-antagonistschema voor de vergelijking met GnRH-agonisten tijdens IVF behandelingen.

Ondanks het gegeven dat in meer dan 200 klinische studies gebruik gemaakt wordt van gonadotrophine releasing hormone (GnRH) antagonisten, is het optimale GnRH- antagonistschema nog niet volledig uitgekristalliseerd. Het doel van dit proefschrift is het optimale GnRH-antagonistschema voor een in-vitro fertilisatie (IVF) behandeling te identifi ceren (deel 1) en deze te vergelijken met GnRH-agonisten (deel 2).

OVERWEGINGEN MET BETREKKING TOT DE SELECTIE VAN HET OPTIMALE GNRH-

ANTAGONIST SCHEMA

Hoofdstuk 2Dit hoofdstuk geeft een overzicht van de ontwikkeling van GnRH-antagonisten, hun werkingsmechanisme en de mogelijke klinische toepassingen.

Pulsatiel afgegeven GnRH is noodzakelijk voor de regulering van hormoon-afgifte en de voortplantingsfuncties van eierstokken en testikels. GnRH stimuleert de hypofysaire afgifte van luteïniserend hormoon (LH) en follikel stimulerend hormoon (FSH). Het blokkeren van de GnRH-werking kan nuttig zijn tijdens een IVF-behandeling om ongewenste LH-pieken en ovulaties te voorkomen. Het kan ook gebruikt worden om geslachtshormoon-afhankelijke aandoeningen te behandelen. GnRH-antagonisten geven een directe onderdrukking van LH- en FSH-afgifte door middel van competatieve blokkade van de GnRH-receptor. GnRH-antagonisten met acceptabele pharmacokinetische- en veiligheidsaspecten zijn nog maar sinds kort beschikbaar. Vrijwel alle mogelijke indicaties voor GnRH-agonisten zijn waarschijnlijk ook van toepassing op GnRH-antagonisten, inclusief endometriose, leiomyomen, borstkanker, benigne en maligne prostaatafwijkingen en centrale pubertas preacox. Succesvolle klinische resultaten zijn tot nu toe vooral onderzocht op het gebied van de geassisteerde voortplanting en prostaatkanker. Tot slot bieden GnRH-antagonisten een ideaal middel om meer inzicht te krijgen in de endocrinologische regulatie-systemen en de pathofysiologie van de voortplanting.

Hoofdstuk 3In het derde hoofdstuk wordt een overzicht gegeven van diverse medicijnen en behandelingsstrategieën die gebruikt kunnen worden in de geassisteerde voortplanting, te weten GnRH-agonisten en -antagonisten, FSH, LH en humaan choriogonadotrophine (hCG).

Bijna altijd worden FSH-preparaten (urinair, urinair gezuiverd of recombinant) gebruikt tijdens geassisteerde voortplanting. Dit kan gebruikt worden ter inductie van folliculaire groei in geval van anovulatie of ter inductie van multi-folliculaire groei bij intra-uteriene inseminatie of IVF-behandelingen. In vergelijking met urinaire preparaten biedt recombinant FSH (rFSH) diverse voordelen: verbeterde tolerantie, theoretisch minder kans op infecties door prionen en betere resultaten met betrekking tot zwangerschapscijfers en kosteneffectiviteit. Het low-dose step-up protocol wordt wereldwijd gebruikt om monofolliculaire groei te induceren. Het step-down protocol is even effectief voor deze indicatie met een kortere

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stimulatieperiode en minder FSH, maar vereist mogelijk wel iets meer expertise. Het lange GnRH-agonistprotocol, in combinatie met dagelijks rFSH, is het meest toegepaste schema tijdens IVF. De beschikbaarheid van veilige GnRH-antagonisten maakt alternatieve behandelingsstrategieën mogelijk, zoals mildere stimulatieschema’s. Zowel FSH als LH zijn nodig voor adequate follikelgroei en hormoonproductie. Behandeling van hypogonadotrope vrouwen vereist de toevoeging van één of andere vorm van LH-activiteit (Human menopausal gonadotrophine, hCG of recombinant LH) voor adequate follikel groei en zwangerschap. Voor normogonadotrope vrouwen is het nog steeds niet geheel duidelijk wat de exacte rol is van LH bij IVF-behandelingen. Er lijkt een optimale range te bestaan, aangezien zowel hoge als lage LH-waarden geassocieerd zijn met slechtere IVF-resultaten. Voor het verder verbeterenvan de geassisteerde voortplanting zijn aanvullende onderzoeken nodig naar optimale behandelingsschema’s en mogelijke directe effecten van GnRH-agonisten en antagonisten.

Hoofdstuk 4Dit hoofdstuk geeft de resultaten weer van een onderzoek naar de minimaal effectieve dosering van de GnRH-antagonist Antide ter preventie van vroegtijdige LH-pieken tijdens een IVF-behandeling.

Het primaire eindpunt van deze prospectieve studie was de minimale effectieve dosering, gedefi nieerd als de laagste dosis met minder dan twee LH-pieken per groep. Deze studie werd in twee fasen uitgevoerd. Een dubbelblinde gerandomiseerde fase met twee groepen (n=60) werd gevolgd door een open fase met drie additionele onderzoeksgroepen, waarin verschillende doseringen werden onderzocht. In totaal werden 144 IVF/ICSI patients gestimuleerd met rFSH vanaf cyclusdag 2. Vanaf cyclusdag 7 werd dagelijks een GnRH antagonist (antide) toegevoegd in verschillende doseringen. Serum-antidespiegels, de gemiddelde LH- en oestradiolspiegels per dag en hun oppervlakte onder de curve waren dosis-afhankelijk van antide. De biologische beschikbaarheid van antide was bijna verdubbeld na oplossing van Antide in grotere volumina. LH-pieken traden alleen op in de laagste doseringsgroepen: 0.5 mg/ml (3.2%), 0.5 mg/0.5 ml (6.7%) and 0.25 mg/ml (13.3%). De minimaal effectieve dosering van antide in IVF is volgens onze defi nitie 0.5 mg/ml. Antide werd goed verdragen en lijkt veilig.

Hoofdstuk 5Hoofdstuk 5 onderzoekt de relatie tussen LH- en progesteronspiegels en IVF-resultaten.

In een studie waarin 144 IVF-patiënten vanaf stimulatiedag 6 behandeld werden met een GnRH-antagonist werd de relatie tussen de verschillende LH- en progesteronspiegels en de IVF-resultaten bestudeerd. Er waren geen verschillen in aantal follikels (≥11 mm), follikelgrootte en endometriumdikte op de dag van hCG toediening tussen de verschillende doseringsgroepen. Eveneens waren er geen verschillen in het aantal (metaphase II-) oocyten, de mate van fertilisatie en het aantal embryo’s van goede kwaliteit. Hogere zwangerschaps- en implantatiecijfers werden gezien in de middelste doserings-groepen (0.5 and 1.0 mg/ml), maar de verschillen waren statistisch gezien niet signifi cant. Klinische zwangerschappen werden alleen waargenomen binnen nauwe grenzen van veranderingen in LH- en progesteronspiegels ten opzichte van de uitgangswaarden. Zwangerschappen werden niet gezien als LH- en progesteron te sterk stegen of te sterk daalden tijdens de behandeling.

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Hoofdstuk 6In hoofdstuk 6 wordt het effect onderzocht van voorbehandeling met een orale anticonceptie (OAC-)pil in een GnRH-antagonistprotocol.

In deze studie werden 64 patiënten gerandomiseerd voor hetzij een GnRH- antagonistbehandeling (controlegroep) hetzij gecombineerd met OAC-voorbehandeling (OAC-groep). Alle patiënten kregen vanaf stimulatiedag 6 dagelijks een GnRH-antagonist (Antide) toegediend. In de controle groep werd rFSH gestart op cyclusdag 2 en in de OAC-groep twee of drie dagen na de laatste OAC-pil. Het aantal oocyten was het primaire eindpunt van deze studie. OAC-voorbehandeling gaf in de vroeg-folliculaire fase signifi cant lagere FSH-, LH- en oestrogeenspiegels en een dunner endometrium. Vroeg in de stimulatiefase werden minder grote follikels gezien na OAC-voorbehandeling, resulterende in een langere stimulatieperiode. Het aantal follikels op de dag van hCG-toediening en het aantal oocyten per punctie waren hoger in de OAC-groep ten opzichte van de controlegroep. Conclusie: OAC-voorbehandeling bevordert homogene follikelgroei, geeft een langere stimulatieduur en meer oocyten.

Hoofdstuk 7Dit hoofdstuk behandelt het effect van het moment van rFSH-toediening (twee dagen versus vijf dagen) na de laatste OAC-inname, met of zonder rLH-toevoeging in een GnRH-antagonistprotocol.

Wij randomiseerden 48 IVF/ICSI patiënten, die behandeld werden met een GnRH- antagonistprotocol, in vier groepen. Alle patiënten kregen dagelijks 150 IU rFSH toegediend vanaf dag 2 of vanaf dag 5 na de laatste OAC-pil met of zonder toevoeging van 150 IU rLH/dag. Het primaire eindpunt was het aantal follikels (≥11mm) op de dag van hCG-toediening. Het starten op dag 2 versus dag 5 met rFSH gaf signifi cant lagere FSH-, LH- en oestradiolspiegels, een dunner endometrium in de vroeg-folliculaire fase en minder grote folllikels op stimulatiedag 6. Ondanks een langere stimulatieperiode en een hogere totale dosis rFSH in de dag 2-groep was er geen toename van het aantal oocyten op de dag van hCG-toediening. Toevoeging van 150 IU rLH per dag in dit schema gaf signifi cant hogere LH-spiegels, maar mogelijk minder follikels en oocyten. Op economische gronden lijkt de voorkeur uit te gaan naar een GnRH-antagonistschema waarbij de stimulatie gestart wordt op de vijfde dag na de laatste OAC-inname.

ZOEKTOCHT NAAR DE OPTIMALE VERGELIJKING VAN GNRH-AGONISTEN EN -

ANTAGONISTEN

Hoofdstuk 8De studie in hoofdstuk 8 vergelijkt de IVF-resultaten van een lang GnRH-agonistprotocol met een GnRH-antagonistschema waarin stimulatie gestart werd op dag 5 na de laatste OAC-inname.

De studie werd uitgevoerd in acht internationale IVF-centra. 182 patiënten werden gerandomiseerd in een OAC-voorbehandelde cetrorelix groep (n=91) en in een busereline groep (n=91). Stimulatie met rFSH werd in beide groepen gestart op een vrijdag. De primaire uitkomst was het aantal oocyten verkregen per patiënt. Deze resultaten waren gelijk in beide onderzoeksgroepen, evenals het aantal puncties op weekend- of feestdagen, aantal

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afgebroken behandelingscycli, hoeveelheid medicatie, aantal embryo’s en de implantatie- en zwangerschapscijfers. Beide behandelingsschema’s werden goed verdragen. Op basis van deze gegevens kwamen we tot de conclusie dat OAC-voorbehandeling mogelijk een oplossing kan bieden voor een deel van de eerder waargenomen nadelen van een antagonist-schema (minder oocyten en cyclusafhankelijkheid) ten opzichte van het lange agonist-schema.

Hoofdstuk 9In hoofdstuk 9 geven we een overzicht van onze kritische beoordeling van de huidige literatuur met betrekking tot diverse GnRH-antagonistschema’s en vergelijkende studies van GnRH-agonisten en -antagonisten tijdens IVF-behandelingen.

We komen tot de conclusie dat de GnRH-antagonist lang niet altijd op de juiste wijze vergeleken is met de GnRH-agonist. Beschikbare meta-analyses omvatten alle vergelijkende studies van GnRH-agonisten met antagonisten, inclusief de minder optimale GnRH-antagonistschema’s. Na kritische evaluatie van de diverse onderzochte GnRH-antagonistschema’s zijn wij van mening dat vroegtijdige en constante FSH- en LH-onderdrukking mogelijk een positief effect hebben op follikelgroei en klinische zwangerschapscijfers. Vanuit dit perspectief lijkt een enkelvoudig doseringsschema van een depotpreparaat of een fl exibel schema op basis van folliculaire ontwikkeling minder aan te bevelen. De toevoeging van LH lijkt geen toegevoegde waarde te hebben. Vroege FSH-en LH-onderdrukking kan worden verkregen middels vroege GnRH-antagonisttoediening (vanaf stimulatiedag 1) of door OAC-voorbehandeling. Een uitgevoerde meta-analyse met vier vergelijkende studies van OAC/GnRH-antagonistschema’s versus lange GnRH-agonistschema’s, liet geen verschillen zien tussen deze twee strategieën met betrekking tot aantal oocyten en klinische zwangerschappen. Wij zijn van mening dat op basis van deze gegevens het te vroeg is om de balans op te maken tussen GnRH-agonisten en -antagonisten in IVF. Hiervoor zijn meer gerandomiseerde studies nodig van voldoende omvang om verschillen in zwangerschapscijfers op te sporen, die de GnRH-antagonist toegepast in het optimale schema vergelijken met het lange GnRH-agonistschema.

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Authors’ affi liations

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D Braat Department of Obstetrics and Gynaecology, University Medical Centre St Radoud, Nijmegen, The NetherlandsJ Donnez Universite Catholique de Louvain, Cliniques Universitaires Saint-Luc, Bruxelles, BelgiumBCJM Fauser Department of Obstetrics and Gynaecology, Erasmus Medical Centre, Rotterdam, The Netherlands. (University Medical Centre, Utrecht*)F Fischl Department of Obstetrics and Gynaecology, Universitätsklinik für Frauenheilkunde, Vienna, AustriaJN Hugues Hôpital Jean Verdier, Reproductive Medicine Centre, University Paris XIII, France

JAF Huirne Department of Obstetrics and Gynaecology, Vrije Universiteit Medical Centre, Amsterdam, The Netherlands. (Kennemer Gasthuis, Haarlem*) R Homburg Department of Obstetrics and Gynaecology, Vrije Universiteit Medical Centre, Amsterdam, The Netherlands PGA Hompes Department of Obstetrics and Gynaecology, Vrije Universiteit Medical Centre, Amsterdam, The NetherlandsCB Lambalk Department of Obstetrics and Gynaecology, Vrije Universiteit Medical Centre, Amsterdam, The Netherlands ACD v Loenen Department of Obstetrics and Gynaecology, Vrije Universiteit Medical Centre, Amsterdam, The NetherlandsNS Macklon Department of Obstetrics and Gynaecology, Erasmus Medical Centre, Rotterdam, The Netherlands. (University Medical Centre, Utrecht*)J McDonnell Department of Obstetrics and Gynaecology, Vrije Universiteit Medical Centre, Amsterdam, The Netherlands A Obruca Kinderwunschzentrum Wien, Vienna, Austria C Pirard Universite Catholique de Louvain, Cliniques Universitaires Saint-Luc, Bruxelles, BelgiumJL Pouly Polyclinique Hotel Dieu, Clermont-Ferrand, FranceJC Sage Tour Belledonne, Grenoble, FranceR Schats Department of Obstetrics and Gynaecology, Vrije Universiteit Medical Centre, Amsterdam, The NetherlandsJ Schoemaker Department of Obstetrics and Gynaecology, Vrije Universiteit Medical Centre, Amsterdam, The Netherlands

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Dankwoord

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Het boek is af, maar pas nadat ik alle mensen bedank die hun bijdrage hebben geleverd. En dat waren er veel! Een aantal mensen wil ik graag nog even persoonlijk noemen.

Allereerst wil ik alle patiënten en vrijwilligers danken voor hun enthousiaste deelname aan mijn onderzoek. Ik was onder de indruk. Wekenlang drie keer per dag langskomen om bloed af te staan is een grote opgave. De bereidheid was groter dan ik van tevoren gedacht of gehoopt had!

Prof. dr. J. Schoemaker, beste Joop, u zou mijn promotor worden, maar daarvoor was u te snel en ik te langzaam. In dat ene jaar dat we intensief samenwerkten, heb ik veel van u geleerd. Nogmaals dank, ik wens u en Toke veel geluk.

Dr. R. Schats, beste Roel, dank voor al je steun, in het bijzonder je klinische ondersteuning. Je hebt een gezellige groep om je heen gecreëerd. Ik denk met veel plezier terug aan die tijd. Medewerkers van het IVF centrum, alle verpleegkundigen, secretaresses, labmede-werkers, allen hartelijk dank voor de gezellige tijd en jullie ondersteuning. Jan, ESHRE in Tour gaan we nog eens overdoen. René, Mariet, Julie en Ruth, het was gezellig, enorm bedankt. Lieve Ted en Agnes, zonder jullie hulp was het niet gelukt. Bedankt voor alle klussen die jullie voor me verricht hebben tijdens het onderzoek en zelfs nog daarna. Beste Joseph, dank voor je ondersteuning bij een deel van de statistiek, maar vooral voor je hulp bij het omzetten van diverse databases. Heel veel succes in Australië en natuurlijk bij je eigen promotie.

Medewerkers endolab, dank voor jullie enthousiaste hulp en snelle bepalingen. Die ontplofte koelkast ben ik al vergeten.

Medewerkers afdeling voortplantingsgeneeskunde: alle verpleegkundigen, admini-stratieve medewerkers, Peter, Judith, Ingeborg, Annemiek en Marcel: lieve allemaal, bedankt voor de fi jne samenwerking. Jullie waren altijd vriendelijk en geduldig. Het was heerlijk om bij jullie te werken. Ik hoop de samenwerking in de toekomst voort te kunnen zetten. Lieve Madeleine, dank voor je snelle en accurate ondersteuning bij het afronden van dit proefschrift. Lieve Marleen, we lezen je boekjes nog steeds met veel plezier. Dank voor je altijd oprechte belangstelling.

Mijn onderzoeksmaatjes, lieve allemaal, wat hadden we het gezellig! Samen onderzoek doen, met alle diepte- en hoogtepunten, was vooral erg gezellig en constructief. Lieve Joris, je hebt me behoed voor alle beginnersfouten en alle in’s en out’s van de VEVO en het doen van onderzoek bijgebracht. Echt ontzettend bedankt en we gaan binnenkort zeker wat afspreken! Ben heel benieuwd hoe het met je grote gezin is! Lieve Sheda, het werd vol in kamer 44 toen ik me bij jullie aansloot, maar ik was er erg blij mee. Helaas zie ik je niet meer zo vaak, nu je iets heel anders bent gaan doen. Ik wens je samen met je gezin heel veel geluk en liefde. Beste André, je hebt veel werk verzet met erg veel enthousiasme. Ik wens je heel veel succes, zowel als huisarts als privé. Lieve Hans, je bent echt een maatje, eerst samen tijdens het onderzoek en later gedurende de opleiding. Ik mis je enorm, vooral de dagelijkse update van het menu. Heel veel succes met de ideale maatschap op de ideale plek, of wordt het de colpobus? Lieve Janet, steeds zitten we in het zelfde schuitje, als

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gillende keukenmeiden op de IVF, daarna op VK en de VEVO, samen zwanger en nu samen promoveren. We gaan er een groot feest van maken! Ik hoop dat we nog lang collega’s en vriendinnen mogen blijven. Lieve prinses Sandra, niet voor niets heb ik je gevraagd me bij te staan bij de verdediging, je gevatheid kan nog wel eens van pas komen. We hebben veel gelachen, maar ook samen heel hard gewerkt. Je aanwezigheid werkte zeer stimulerend. Op naar het volgende promotiefeest!

Prof. dr. H.P. van Geijn, beste Herman, u heeft wel geen wezenlijk deel uitgemaakt van dit onderzoek, maar als u me niet had aangenomen voor de opleiding, was ik niet in staat geweest dit onderzoek af te ronden. Ik heb het altijd erg naar mijn zin gehad in de VUmc en hoop dit nog lang voort te kunnen zetten.

Gynaecologen en collega’s arts-assistenten Kennemer Gasthuis, Beste allemaal, het voltooien van “het boekje” is voor jullie niet ongemerkt voorbij gegaan. Dank voor de fl exibiliteit en jullie belangstelling.

Prof. dr. R. Homburg, dear Roy, many thanks for all your humoristic comments, the pleasant discussions and all your corrections of my terrible English. Despite all your efforts it didn’t reach the level where it should be, but I will keep practising. The same can be said about your biking skills, it didn’t reach the level to be expected from a frequent visitor of the Netherlands.

Dr. C.B. Lambalk, lieve Nils, we hadden ideeën voor wel drie proefschriften, maar laten we het eerst toch maar bij een houden. Zonder jou was dit proefschrift er niet gekomen! Al mijn dank gaat naar jou uit voor je meer dan inspirerende begeleiding. Ik kon altijd bij je terecht met grote en kleine problemen. Je enthousiasme voor onderzoek heeft aanstekelijk gewerkt. Maar vooral wil ik je danken voor de persoonlijke begeleiding en je oprechte interesse in de mens achter de onderzoeker. Bij life-events kwam voor jou het onderzoek op de tweede plaats. Je bent en blijft een voorbeeld voor me hoe je in deze hectische wereld altijd eerlijke en plezierige begeleiding kunt geven. Helaas kun je mijn promotor niet zijn. Het wordt tijd dat je benoemd wordt tot hoogleraar, je hebt dit meer dan verdiend!

De leden van de leescommissie, prof. dr. P. Devroey, prof. H.P. van Geijn, dr. A. Hoek, dr. P.G. Hompes, prof. dr. J.N. Hugues, prof. dr. N. Macklon en prof. dr. van der Veen bedank ik

Alle vrienden en vriendinnen, lieve allemaal, echte vriendschap kan gelukkig tegen een stootje, en ook het komende halfjaar zal die nog op de proef worden gesteld. Dank voor al jullie steun en begrip in deze drukke tijden. Lieve Gonneke, ik heb bewondering voor al je energie, enthousiasme, altijd oprechte interesse en steun in moeilijke tijden. Helaas heb je een andere richting gekozen, maar dat heeft ons contact er niet minder op gemaakt. Ik hoop dat we altijd vriendinnen blijven. Lieve Annechien, het begon in Enschede. Wat was dat een geweldige tijd. Altijd kon ik bij je terecht. Dankzij jou en Herbert ben ik nu nog gelukkiger dan ik al was. Ik ben zeer vereerd dat je mijn paranimf wil zijn.

voor het kritisch doornemen van het proefschrift.

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Mijn dierbare familie, lieve pap, de interesse voor de wetenschap begon in de kraamstal. Je kennis van de voortplanting kwam later nog veel van pas, alleen vielen de zwangerschapsresultaten bij de mens wel wat tegen. Daarnaast wil ik jou en mama danken voor alle geboden mogelijkheden en jullie onvoorwaardelijke liefde. Lieve Esther, Jeroen, Job, Diane, dank voor al jullie belangstelling, hulp en geduld als ik er weer eens niet bij kon zijn. Lieve Johan en Olga en andere Haantjes, dank voor jullie warme nest, ik hoop er nog lang in te mogen blijven. Mijn lief klein Woutertje - je wordt nu vast boos als je dit leest want je bent per slot van rekening al vier -. De laptop komt gauw wat meer ter beschikking, maar misschien is het tijd voor een eigen “cuter”. Lieve Floor, mijn kleine mollige vrouwtje, je hebt geen idee waar die moeder van je mee bezig is, maar die stapels papieren vond je geweldig, vooral om te leren scheuren en zelfs soms om op te eten. Maar troost je, het is hierna vast niet voorbij, er zullen altijd stapels blijven, tot groot verdriet van de nette mannen in ons huis. Ik hoop dat we de komende tijd nog meer van elkaar mogen genieten.

Mijn allerliefste Joost, zonder jou was dit niet gelukt. Je onvoorwaardelijke steun en liefde is ongekend en je geduld bij vastgelopen computers onmisbaar. Nooit heb je gemopperd als ik weer eens tussen alle chaos achter mijn laptop zat, al waren daar vaak redenen genoeg voor! Op naar onze volgende uitdaging.

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Curriculum Vitae

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Judith Anna Francisca Huirne werd op 24 november 1968 geboren te Eibergen, als dochter van Willem Huirne en Gerrie Huirne-Veelers. In 1988 behaalde zij haar voorbereidend wetenschappelijk onderwijs diploma aan het Katholiek Drentsch College te Emmen. De studie geneeskunde werd gevolgd aan de Rijks Universiteit te Groningen, alwaar in 1996 het artsexamen werd behaald.

Na haar co-assistentschap was zij werkzaam in het Medisch Spectrum Twente te Enschede, eerst als IVF arts, later als AGNIO Verloskunde en Gynaecologie. Tijdens deze periode werd haar interesse voor wetenschappelijk onderzoek gewekt en verrichtte zij verschillende onderzoeken op het gebied van de foetale magnetocardiografi e.

Vanaf oktober 1998 was zij werkzaam als arts-onderzoeker op de afdeling Voortplantings endocrinologie en vruchtbaarheidsonderzoek van het Vrije Universiteit medisch centrum, waar gestart werd met het wetenschappelijk onderzoek dat aanleiding gaf tot deze dissertatie, onder leiding van Prof.dr. J. Schoemaker.

In 2000 kreeg zij een aanstelling als AGIKO Verloskunde Gynaecologie van het Vrije Universiteit medisch centrum te Amsterdam (april 2000 tot april 2007). De opleiding tot gynaecoloog werd gevolgd in het Kennemer Gasthuis (opleider dr. J. Lips) en in het Vrije Universiteit medisch centrum (opleiders prof.dr. H.P. Van Geijn en prof.dr. H.Brolmann). Het wetenschappelijk onderzoek werd begeleid door Dr. C.B. Lambalk en prof.dr. R.Homburg.

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List of publications

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INTERNATIONAL

Peters MJ, Stinstra JG, Broek SP van den, Huirne JAF, Quartero HWP, Brake HJM ter, Rogalla H. On the fetal magnetocardiogram. Bioelec trochemistry and Bioenergetics 1998: 47; 273-281

Huirne JAF, Seppenwoolde Y, Quartero HWP, Peters MJ. Antenatal magnetocardiographic diagnosis of conduc ting disturbances. In Recent advances in biomagnetism. Editors: T.Yoshimoto et al. Tohoku University press, Sendai, Japan, 1999; 967-970

Stinstra JG, Krooshoop HJG, Muis B, Huirne JAF, Quartero HWP, Peters MJ. The infl uence of the vernix caseosa on fetal cardiograms. In Recent ad vances in biomagnetism. Editors: T. Yoshimoto et al. Tohoku University press, Sendai, Japan, 1999; 1070-1073

Huirne JAF, Lambalk CB, Janssens R, Schoemaker J. GnRH Agonists versus Antagonists, where are we today? In Controverses in Obstetrics and Gynaecology. Editors: Z. Ben-Rafael and Z Shoham. Monduzzi Editore Bologna Italy, 1999; 77-81

Janssens RMJ, Brus L, Cahill DJ, Huirne JA, Schoemaker J, Lambalk CB. Direct ovarian effects and safety aspects of GnRH agonists and antagonists. Human Reprod. Update 2000: 6; 505-518

Huirne JA, Lambalk CB. Gonadotropin-releasing-hormone-receptor antagonists.Lancet 2001: 358; 1793-1804

Loenen ACD van, Huirne JA, Schats R, Hompes P, Lambalk CB - GnRH agonists, antagonits, and assisted conception. Sem Reprod Med. 2002: 20; 349-364

Huirne JA, Lambalk CB, Van Loenen ACD, Schats R, Hompes PGA, Fauser BCJM, Macklon NS. Contemporary pharmacological manipulation in assisted reproduction. Drugs 2004: 63; 1-25

Huirne JA, Schats R. The use of GnRH agonists. In Textbook of assisted reproductive techniques laboratory and clinical perspectives. Editors: DK Gardner, A Weissman, C Howles and Z Shoham. 2004; 599-608

Van Montfrans JM, van Hooff MH, Huirne JA, Tanahatoe SJ, Sadrezadeh S, Martens F, van Vught JM, Lambalk CB. Basal FSH concentrations as a marker of ovarian ageing are not related to pregnancy outcome in a general population of women over 30 years. Hum Reprod 2004: 19; 430-434

Huirne JA, van Loenen AC, Scharts R, McDonnell J, Hompes PG, Schoemaker J, Homburg R, Lambalk CB. Dose-fi nding study of daily gonadotropin-releasing hormone (GnRH) antagonist for the prevention of premature luteinizing hormone surges in IVF/ICSI: antide and hormone levels. Hum Reprod 2004: 19; 2206-2215

Huirne JA, van Loenen AC, Scharts R, McDonnell J, Hompes PG, Schoemaker J, Homburg R, Lambalk CB. Dose-fi nding study of daily gonadotropin-releasing hormone (GnRH) antagonist for the prevention of premature luteinizing hormone surges in IVF/ICSI: Optimal changes in LH and progesterone levels for clinical pregnancy. Hum Reprod 2005: 20; 2359-2367

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Huirne JA, van Loenen AC, Donnez J, Pierer C, Homburg R, Schats R, McDonnell J, Lambalk CB. Effect of an oral contraceptive pill on follicular development in IVF/ICSI patients reciving a GnRH antagonist: a randomized study. RBM-online 2006: 13; 235-245

Huirne JA, Donnez J, Hugues JN, Fischl F, Sage JC, Pouly JL, Braat D, Obruca A, van Loenen AC, Lambalk CB. Cetrorelix in an oral contraceptive-pretreated stimulation cycle compared with buserelin in IVF/ICSI patients treated with rhFSH: a randomized, multicentre, phase IIIb study. Human Reprod. 2006: 21; 1408-1415

Homburg R, Pap H, Brandes M, Huirne JA, Hompes P, Lambalk CB.Endometrial biopsy during induction of ovulation with clomiphene citrate in polycystic ovary syndrome. Gynecol Endocrinol. 2006: 22: 506-10

Huirne JA, van Loenen AC, Donnez J, Pierer C, McDonnell J, Schats R, Homburg R, Lambalk CB. The effect of timing recombinant FSH administration after oral contraceptive pretreatment and the addition of recombinant LH on follicular development and hormonal levels in GnRH antagonist treated IVF/ICSI patients; a pilot study. Reproduction (submitted)

Huirne JA, Homburg R, Lambalk CB. Debate: are GnRH antagonists comparable to agonists in IVF? Human Reproduction (submitted)

DUTCH

Albers JF, Gerstenbluth I, Huirne JAF, Smits IMH. Prevalentie schattingen van chronische aandoeningen op Curaçao; zelfrapportage versus huisartsen registratie. Tijdschrift voor Sociale Gezondheidszorg 1996: 74; 184-190

Quartero HWP, Huirne JAF, Seppenwoolde Y, Peters MJ. Foetale magneti schecardiografi e (fMCG) bij ritmestoornissen van de foetus. Nederlands Tijd schrift voor Obstetrie & Gynaecologie 1998: 111; 37-38

Huirne JAF, Schats R, Hompes PGA, Lambalk CB. Mechanism of action of GnRH analogues. Tijdschrift voor fertiliteitsonderzoek 2002: 2; 26-31

Huirne JAF, Lambalk CB - GnRH-agonisten en antagonisten voor IVF-behandeling en andere indicaties. Freya Magazine 2002: 18; 3-4

Huirne JAF, Lambalk CB. GnRH-antagonist goed alternatief voor lang agonisten-protocol. Fertiliteit in de praktijk 2003;1:19

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