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The role of GnRH antagonists in assisted reproduction
RBMOnline - Vol 5. Suppl. 1. 14–19 Reproductive BioMedicine Online; www.rbmonline.com/Article/557 on web 27 June 2002
The role of GnRH antagonists in assisted reproduction Pedro N Barri1, Francisca Martinez, Buenaventura Coroleu, Rosa Tur Service of Reproductive Medicine, Department of Obstetrics and Gynecology, Institut Universitari Dexeus, Paseo Bonanova 67, 08017 Barcelona, Spain 1Correspondence: Tel. 34–93 2274700; Fax 34–93 2057966; e-mail: [email protected]
Abstract The different schemes of application of gonadotrophin-releasing hormone (GnRH) antagonists in ovarian stimulation protocols for assisted reproduction are reviewed. Starting from the initial schemes of single and/or multiple doses, the efficacy of GnRH antagonists in ‘soft’ protocols of ovarian stimulation has been evaluated in natural cycles, and the possibility of inducing the final oocyte maturation under antagonists either with native luteinizing hormone-releasing hormone or with GnRH agonists. The largest clinical studies carried out with cetrorelix or with ganirelix and published to date were analysed. Apart from the benefits of reducing the duration of the treatment, gonadotrophin requirements and ovarian hyperstimulation syndrome incidence, there is still a serious debate on the potential deleterious effect of GnRH antagonists on implantation and pregnancy rates. More specific aspects such as cycle programming, application of GnRH antagonists in protocols for poor responders etc., were also reviewed in this survey. Keywords: buserelin, cetrorelix, ganirelix, GnRH antagonist, oocyte maturation, ovarian stimulation
Introduction The replacement or deletion of several amino acids within the native gonadotrophin-releasing hormone (GnRH) molecule has resulted in the development of GnRH agonists and antagonists. The mechanism of action of GnRH agonists consists of the desensitization of the gonadotrophic cells and the downregulation of pituitary receptors, leading to a reversible medical hypophysectomy. However, GnRH agonists produce an initial stimulatory effect on FSH and LH secretion (flare-up). For clinical situations in which an immediate and profound pituitary suppression is needed, new analogues devoid of a stimulatory phase, such as the GnRH antagonists, would be useful.
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Protocols of ovarian stimulation for assisted reproduction techniques with concomitant GnRH antagonist treatment Although the first clinical trials were carried out with the second-generation antagonist Nal-Glu (Ditkoff et al. 1991; Kolb and Paulson, 1996), it could not be used further for clinical purposes because of its histaminergic properties, and the only two preparations currently available are cetrorelix and ganirelix. There are two different schemes of administration for both antagonists.
The synthesis of antagonist analogues began 20 years ago; however, the first preparations were not suitable for clinical use because of their histaminergic side effects. Since then, significant improvements have been achieved by introducing several amino acid substitutions, finally resulting in the synthesis of cetrorelix, the first GnRH antagonist to be marketed worldwide for its efficacy and safety (Reissmann et al., 2000). Later on, new preparations with antagonistic properties became available for clinical use, and ganirelix, a third generation GnRH antagonist, has been applied to treating clinical situations in which a rapid pituitary suppression is desired to prevent premature LH surges in patients undergoing ovarian stimulation for assisted reproduction techniques (The Ganirelix DoseFinding Study Group, 1998; Oberyé et al., 1999).
The multiple dose protocol
New GnRH antagonists are ready for clinical use and, like their precursors, are able to induce a rapid fall in FSH and LH secretion without flare-up and can be administered in mid- or final follicular phase to avoid the premature LH surge or to interrupt an LH surge that has already started.
The single dose protocol
In this protocol, patients are stimulated with different doses of either urinary or recombinant gonadotrophins starting on cycle day 2, and they receive daily doses of 0.25 mg of the antagonist (cetrorelix or ganirelix) starting on stimulation day 6. Based on clinical experience, GnRH antagonist administration starts when there is a risk of an imminent and premature LH surge, according to the follicular response and to the oestradiol concentration. This scheme, the most commonly used, has been applied since the beginning and is still used by the Department of Obstetrics and Gynecology at the Medical University of Lübeck (Diedrich et al., 1994; Felberbaum and Diedrich, 1999).
In order to simplify the stimulation protocol, a French group started to administer a single dose of cetrorelix (5 mg in the first trials and 3 mg in the latest ones) when oestradiol concentrations were between 150 and 200 pg/ml per follicle >14 mm. A second dose was injected 48 h later if ovulation was not triggered. However, this protocol is now defined by a single dose of 3 mg and a protection period of 4 days.
The role of GnRH antagonists in assisted reproduction - PN Barri et al. This protocol has been followed by the French group from Clamart since their initial phase ll studies (Olivennes et al., 1994, 1998) (Figure 1). So far, both protocols have proven comparable in terms of efficacy and safety in IVF patients (Table 1).
New protocols of GnRH antagonist treatment The availability of GnRH antagonists in ovarian stimulation protocols for assisted reproduction techniques has led to new strategies of ‘soft’ stimulation in order to obtain a moderate ovarian response and a sufficient number of mature oocytes (Fauser et al., 1999). In this respect it is interesting to note the experience of a Dutch group, which tried a protocol in which ovarian stimulation began on day 5 with 100 or 150 IU/day of recombinant FSH (rFSH) and continued from day 8 combined with a daily dose of 0.25 mg of cetrorelix until optimal follicular maturation was achieved. After treating 15 patients with IVF, the authors obtained normal pregnancy rates and a significant reduction in the total amount of gonadotrophins required compared with a standard long protocol of GnRH agonist (de Jong et al., 2000). Another possibility is the modification of the classic natural cycle with minimal ovarian stimulation. In this way, a French group has reported their experience of 44 IVF ‘natural’ cycles in which a single dose of 0.5 or 1 mg of cetrorelix was administered to normal IVF patients when a leading follicle was >12 mm and the oestradiol concentration was above 150 pg/ml. Simultaneously, a daily dose of 150 IU of human menopausal gonadotrophin (HMG) was given until human chorionic gonadotrophin (HCG) administration. Although there were some cancellations (9%), and no oocytes were obtained in 10 cycles (25%), a promising 32% pregnancy rate per embryo replacement was achieved (Rongières-Bertrand et al., 1999).
antagonist, the final oocyte maturation can be achieved either with native GnRH administration (Felberbaum et al., 1995) or with a single dose of a GnRH agonist (Olivennes et al., 1996) in patients at risk of developing ovarian hyperstimulation syndrome (OHSS). Both strategies have a preventive effect on OHSS, because the endogenous LH secreted has a much shorter half-life than that of HCG (Itzkovitz et al., 2000). The birth of a healthy girl following GnRH agonist treatment for the induction of final oocyte maturation in an IVF/intracytoplasmic sperm injection (ICSI) patient has recently been reported (de Jong et al., 2001).
Clinical experience with GnRH antagonists in ovarian stimulation for assisted reproduction techniques After appropriate dose-finding phase II studies, which confirmed that 0.25 mg was the optimal daily dose for both cetrorelix and ganirelix (Albano et al., 1997; The Ganirelix Dose-Finding Study Group, 1998), several multicentre and multinational phase III trials have been carried out with the two GnRH antagonists commercially available so far.
Clinical experience with cetrorelix Two multicentre studies were conducted to compare the GnRH antagonist cetrorelix with different GnRH agonists. When cetrorelix was compared with buserelin (Albano et al., 2000) and with triptorelin (Olivennes et al., 2000), some differences were observed. When cetrorelix was used, a significant reduction in the duration of the stimulation period and in the gonadotrophin
a Triggering final oocyte maturation with GnRH agonists In standard IVF protocols, HCG is used to induce final oocyte maturation. However, HCG has a longer half-life and a longer biological effect than those of endogenous LH because of its increased carbohydrate content. For this reason, and thanks to the preserved pituitary response that exists under a GnRH Table 1. Efficacy and safety of single and multiple dose Cetrotide® regimens (adapted from Diedrich, 2000). Values are percentages unless otherwise stated. Single dose ITT population (n) 192 HCG administered 93.2 Oocyte retrieval procedure 90.6 ET 80.7 Pregnancy/attempt 23.4 Pregnancies/ET 29.0 Miscarriages 13.3 OHSS grade II–III/cycle 1.0
Multiple dose
b
859 93.7 91.3 82.0 23.1 27.4 15.2 1.2
ITT = intention to treat; ET = embryo transfer; OHSS = ovarian hyperstimulation syndrome.
Figure 1. Schemes showing different protocols of cetrorelix administration: (a) multiple and (b) single dose schedules.
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The role of GnRH antagonists in assisted reproduction - PN Barri et al. Table 2. Cetrorelix multiple dose phase III study: details of ovarian stimulation (adapted from Felberbaum et al., 2000).
Table 3. Cetrorelix multiple dose phase III study: final results (adapted from Felberbaum et al., 2000).
Patients (n) Days of stimulation (mean) Ampoules of HMG (mean) Days of cetrorelix (mean) Premature luteinization (n (%)) Oocyte retrieval procedures (n)
Oocyte retrieval procedures (n) Oocytes (n) Fertilization (IVF or ICSI) (%) (mean ± SD) Embryo quality (excellent + good) (% of fertilized oocytes) Pregnancies (n) Clinical pregnancy/ET (%) Miscarriage (n) Ongoing implantation (%) OHSS (%)
346 10.4 25.2 5.7 3 (0.9) 324
HMG = human menopausal gonadotrophin.
requirements for an adequate ovarian response were observed. As far as fertilization and pregnancy rates are concerned, no significant differences were found between cetrorelix and buserelin or triptorelin. The incidence of OHSS was also significantly reduced when cetrorelix was used (1.1% and 1.7%), compared with the incidence observed either with buserelin (6.5%) or with triptorelin (5.6%). The greatest clinical experience with cetrorelix in assisted reproduction techniques comes from a prospective uncontrolled phase III study in which the present authors were involved (Felberbaum et al., 2000). In this study, 346 IVF patients were enrolled and received HMG for ovarian stimulation; after 6 days of stimulation, a multiple dose protocol of 0.25 mg cetrorelix/day was administered until the induction of ovulation. A mean of 25.2 ampoules of HMG was administered for a mean of 10.4 days, and cetrorelix was administered for a mean time of 5.7 days. Only three cases of raised LH (0.9%) were observed, and 324 patients underwent oocyte retrieval. Mean fertilization rate with either conventional IVF or with ICSI was 59.2%, and 70 pregnancies were obtained (23.6% ongoing pregnancy/embryo transfer, 11% ongoing implantation rate and 17% miscarriage rate). The incidence of severe OHSS was very low (0.6%) (Tables 2 and 3).
Clinical experience with ganirelix The efficacy and safety of ganirelix was clinically assessed in three different studies carried out in infertile patients undergoing ovarian stimulation with rFSH (Puregon; Organon Laboratories, Barcelona, Spain). These studies included a total of 1335 patients treated in 43 IVF units according to the following characteristics: European randomized trial comparing ganirelix with buserelin (n = 701) (The European Orgalutran Study Group, 2000); North American randomized trial comparing ganirelix with leuprolide (n = 297) (The North American Ganirelix Study Group, 2001); and European–Middle East
324 297 59.2 ± 25.3 80.5 70 23.6 17 11 0.6
ICSI = intracytoplasmic sperm injection; ET = embryo transfer; OHSS = ovarian hyperstimulation syndrome.
randomized trial comparing ganirelix with triptorelin (n = 337) (The European and Middle East Orgalutran Study Group, 2001). All these studies confirmed that the treatment with ganirelix was significantly shorter than the classic long protocol with GnRH agonists, and the duration of the stimulation phase and the amount of rFSH required were also significantly reduced with the antagonist. The incidence of LH elevation with a dose of 0.25 mg of ganirelix was 1% (Table 4). Compared with the long agonist protocol, the ganirelix protocol resulted in 1–2 fewer follicles and consequently 1–2 fewer oocytes. However, the percentage of metaphase II oocytes was similar in all the groups included in the three studies. The mean number of good quality embryos was almost identical in all the groups, and the ongoing pregnancy and implantation rates were somewhat lower in the first European and in the North American studies but not different in the European–Middle East trial (Mannaerts et al., 2001) (Table 5). The trend towards lower clinical pregnancy rates observed in two studies could be explained by the age of the patients included and by the previous clinical experience with GnRH antagonists that some of the IVF units already had.
Points for debate and discussion It is clear that the application of GnRH antagonists to the standard protocols of ovarian stimulation for assisted reproduction techniques will bring both benefits and drawbacks, and it is time to analyse some of these now.
Table 4. Median (ranges) and treatment difference of mean duration of GnRH analogue and Puregon treatment in the European trial. Restricted to patients with a human chorionic gonadotrophin (HCG) injection (adapted from Mannaerts et al., 2001). European trial
Duration of stimulation (days) Amount of rFSH (IU)
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North American trial
European–Middle East trial
Ganirelix (n = 463)
Buserelin (n = 238)
Ganirelix (n = 198)
Leuprorelin (n = 99)
Ganirelix (n = 226)
Triptorelin (n = 111)
9 (6–18)
10 (6–19)
8 (6–19)
10 (6–16)
9 (6–14)
11 (7–16)
1500 (900–5400)
1800 (900–6450)
1800 (1200–4275)
2025 (1350–5100)
1350 (900–3900)
1800 (1050–4050)
GnRH = gonadotrophin-releasing hormone; rFSH = recombinant FSH.
The role of GnRH antagonists in assisted reproduction - PN Barri et al. Table 5. Clinical outcome of controlled, randomized trials. Oocytes and embryos are expressed as mean values (SD). European trial
No. oocytes per attempt Fertilization rate (%) No. good-quality embryos Implantation rate (%) Ongoing PR per attempt (%)
North American trial
European–Middle East trial Ganirelix (n = 226)
Ganirelix (n = 463)
Buserelin (n = 238)
Ganirelix (n = 198)
Leuprorelin (n = 99)
8.7 (5.6)
9.7 (6.2)
11.7 (6.7)
14.1 (8.2)
62.4
61.9
62.1 3.3 (3.0)
62.1 3.5 (3.2)
4.3 (3.7)
Triptorelin (n = 111)
7.9 (5.1)
9.6 (6.8)
64.0
4.8 (4.0)
64.9
2.7 (2.5)
2.9 (2.4)
15.7
21.8
21.1
26.1
22.9
22.9
20.3
25.7
30.8
36.4
31.0
33.9
PR = pregnancy rate.
Table 6. Ganirelix dose-finding study. Incidence of LH increase, implantation rate and ongoing pregnancy rate per attempt versus dose of gonadotrophin-releasing hormone (GnRH) antagonist. Dose (mg)
0.0625
0.125
0.25
0.50
1.0
2.0
(n = 30)
(n = 65)
(n = 68)
(n = 69)
(n = 64)
(n = 26)
Incidence of LH increase (%) Implantation rate (%) Ongoing pregnancy rate per attempt (%)
16 14.2 23.3
9 16.6 23.1
1 21.9 33.8
0 9.0 10.1
0 8.8 14.1
0 1.5 0
Is it possible to schedule IVF cycles with antagonists?
embryo implantation (Hernandez, 2000; Kol, 2000; Ortmann et al., 2001).
Although programming the schedule of the cycles was easier with GnRH agonists, this can also be achieved with a previous administration of oestrogen pills, which can be useful not only for the programming but also for the homogeneity of the luteal phase prior to the IVF cycle. In this way, the follicular cohort will be more homogeneous, and this could overcome the reduction in the number of follicles and oocytes normally observed when GnRH antagonists are used.
It is clear that from the results of the dose-finding studies there was a dose-related decline in the implantation rates when high doses of ganirelix were used (Table 6). It has been speculated that due to the presence of GnRH receptors in the ovary, the tube and the endometrium, as well as in the oocyte and the embryo, the GnRH antagonists could negatively affect implantation (Raga et al., 1999), although pregnancy rates are not adversely affected in subsequent freeze-thaw cycles (Kol et al., 1999).
Is ovarian function impaired with GnRH antagonists?
However, the differences observed are not significant, and there is a clear need for new prospective and randomized studies in order to clarify this issue (Diedrich et al., 2001b).
Recent studies have suggested that the use of GnRH antagonists could interfere with normal ovarian function (García Velasco et al., 2001). Follicular growth was unaffected by the dose of the GnRH antagonist employed, but late follicular LH, androstendione and oestradiol showed an antagonist dose-related decrease (de Jong et al., 2001).
Is there a clear reduction in the pregnancy rates obtained with GnRH antagonists?
As far as the luteal phase is concerned, LH concentrations are not reduced differently with antagonists and agonists (Tavaniotou et al., 2001). These data suggest that high doses of GnRH antagonists may interfere with follicular steroidogenesis via some intra-ovarian modifiers, such as vascular endothelial growth factor (VEGF) or insulin-like growth factor (IGF).
Do GnRH antagonists interfere with embryo implantation? Several editorials and review articles have fuelled the controversy of a potential deleterious effect of antagonists on
In most GnRH antagonist studies, there is a trend towards somewhat (although not significantly) reduced pregnancy rates (Balasch, 2001). This has been attributed to the learning curve, although the majority of IVF researchers involved in the different multicentre trials have enough experience. However, one crucial point that may partly explain the reduction observed is the differences in monitoring and laboratory protocols that normally exist when a large number of centres are involved. In order to clarify this question, comparative studies between GnRH agonists and antagonists should be carried out in individual centres with wide IVF experience. In this way the comparison will be valid.
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The role of GnRH antagonists in assisted reproduction - PN Barri et al. Table 7. Gonadotrophin-releasing hormone (GnRH) antagonists in poor responders.
Age (years) FSH (mIU/ml) Oestradiol on day 3 (pg/ml) FSH 1 + 2b (mIU/ml) Ampoules of gonadotrophins Cancellations (n (%)) Oestradiol on day of HCG (mean ± SD) (pg/ml) Metaphase II oocytes (mean ± SD) Fertilization rate (%) Embryos (mean ± SD) Pregnancies (n) Pregnancies/oocyte retrieval (%) Implantation rate (%)
Group A Flare-up: Leuprolide + Gonal-F (n = 15)
Group B Clomiphene + Gonal-F + Cetrotide (n = 15)
39.5 ± 5.9 11.2 ± 8.3 48.3 ± 15
39.3 ± 4.6 10.3 ± 4.7 74.3 ± 26
22.7 ± 10.4 62.7 ± 18.2a
21.2 ± 7.3 30 ± 5.1a
1 (6) 1324 ± 359.8
2 (13) 1441 ± 760
3.5 ± 2.9
5.6 ± 2.6
75 2.1 ± 1.2 2 14.2
73 3.2 ± 0.5 3 15.3
12.5
16.6
a Significantly different (P < 0.05). b Sum of FSH values from day 3 and day 10 (clomiphene test). HCG = human chorionic gonadotrophin.
Can poor responders benefit from protocols with GnRH antagonists? So far the studies that have been conducted in assisted reproduction techniques with GnRH antagonists have mainly included patients who were expected to show a normal ovarian response. It is necessary to find out if patients with previous poor responses could improve their responses under antagonists. A recent study comparing a classic agonistic flareup protocol with a protocol in which a daily dose of 0.25 mg of cetrorelix was administered failed to show any benefit for this group of patients (Akman et al., 2001). The present authors have an ongoing study in which two groups of patients with a previous poor response in at least two different IVF cycles are compared. So far, 15 patients have been randomly allocated to two different ovarian stimulation protocols. Patients in Group A received a classic flare-up protocol combining leuprolide and a daily dose of 225 IU of rFSH (Gonal-F, Serono Laboratories, Madrid, Spain), while patients in Group B were treated with a combination of 100 mg clomiphene citrate/day (Effik Laboratories, Madrid, Spain) from day 5 to day 9 in the cycle, a daily dose of 225 IU of rFSH (Gonal-F) from day 6 onwards and a daily dose of 0.25 mg Cetrotide (Serono Laboratories) when a leading follicle was >14 mm and the oestradiol concentration was above 150 pg/ml.
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The preliminary results of this study show that apart from the expected significant reduction in the gonadotrophin requirements, no other benefits were obtained, and the mean
number of oocytes and embryos, their quality and the pregnancy and implantation rates achieved were similar in both groups (Table 7).
Safety of the protocols with GnRH antagonists The third generation of antagonists is practically free of local reactions and no general side effects have been described. The follow-up of the babies born so far is already significant and the fact that no increase in malformations has been observed is reassuring (Ludwig et al., 2001). Another important clinical finding is the significant reduction in the incidence of OHSS observed with the use of cetrorelix compared with that of buserelin in ovarian stimulation for assisted reproduction techniques (Ludwig et al., 2000).
Conclusion With the current knowledge about the clinical benefits of GnRH antagonists for assisted reproduction techniques, it is time to answer the question ‘Do we have to change our protocols completely and replace the agonists with antagonists?’ The present authors have evaluated most of the controversial data suggesting different benefits and drawbacks of the antagonists and conclude that is too early to propose a radical switch. They agree with the opinion of several authors who have postulated that more randomized controlled studies are needed to validate the potential advantages of antagonists over agonists (Albano et al., 2001). In the near future it is hoped that the pregnancy rates obtained with antagonists may be as good as those achieved with agonists; then, the benefits of reducing gonadotrophin requirements, shortening the stimulation phase and reducing the risk of OHSS will be real advantages (Blumenfeld, 2001). Until this moment, researchers should proceed carefully and not abandon the agonists for the antagonists.
Acknowledgements This work was performed under the auspices of ‘Catedra d’Investigació en Obstetricia i Ginecologia’ of the Department of Obstetrics and Gynaecology, Institut Universitari Dexeus, Universitat Autònoma de Barcelona.
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The role of GnRH antagonists in assisted reproduction Pedro N Barri1, Francisca Martinez, Buenaventura Coroleu, Rosa Tur Service of Reproductive Medicine, Department of Obstetrics and Gynecology, Institut Universitari Dexeus, Paseo Bonanova 67, 08017 Barcelona, Spain 1Correspondence: Tel. 34–93 2274700; Fax 34–93 2057966; e-mail: [email protected]
Abstract The different schemes of application of gonadotrophin-releasing hormone (GnRH) antagonists in ovarian stimulation protocols for assisted reproduction are reviewed. Starting from the initial schemes of single and/or multiple doses, the efficacy of GnRH antagonists in ‘soft’ protocols of ovarian stimulation has been evaluated in natural cycles, and the possibility of inducing the final oocyte maturation under antagonists either with native luteinizing hormone-releasing hormone or with GnRH agonists. The largest clinical studies carried out with cetrorelix or with ganirelix and published to date were analysed. Apart from the benefits of reducing the duration of the treatment, gonadotrophin requirements and ovarian hyperstimulation syndrome incidence, there is still a serious debate on the potential deleterious effect of GnRH antagonists on implantation and pregnancy rates. More specific aspects such as cycle programming, application of GnRH antagonists in protocols for poor responders etc., were also reviewed in this survey. Keywords: buserelin, cetrorelix, ganirelix, GnRH antagonist, oocyte maturation, ovarian stimulation
Introduction The replacement or deletion of several amino acids within the native gonadotrophin-releasing hormone (GnRH) molecule has resulted in the development of GnRH agonists and antagonists. The mechanism of action of GnRH agonists consists of the desensitization of the gonadotrophic cells and the downregulation of pituitary receptors, leading to a reversible medical hypophysectomy. However, GnRH agonists produce an initial stimulatory effect on FSH and LH secretion (flare-up). For clinical situations in which an immediate and profound pituitary suppression is needed, new analogues devoid of a stimulatory phase, such as the GnRH antagonists, would be useful.
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Protocols of ovarian stimulation for assisted reproduction techniques with concomitant GnRH antagonist treatment Although the first clinical trials were carried out with the second-generation antagonist Nal-Glu (Ditkoff et al. 1991; Kolb and Paulson, 1996), it could not be used further for clinical purposes because of its histaminergic properties, and the only two preparations currently available are cetrorelix and ganirelix. There are two different schemes of administration for both antagonists.
The synthesis of antagonist analogues began 20 years ago; however, the first preparations were not suitable for clinical use because of their histaminergic side effects. Since then, significant improvements have been achieved by introducing several amino acid substitutions, finally resulting in the synthesis of cetrorelix, the first GnRH antagonist to be marketed worldwide for its efficacy and safety (Reissmann et al., 2000). Later on, new preparations with antagonistic properties became available for clinical use, and ganirelix, a third generation GnRH antagonist, has been applied to treating clinical situations in which a rapid pituitary suppression is desired to prevent premature LH surges in patients undergoing ovarian stimulation for assisted reproduction techniques (The Ganirelix DoseFinding Study Group, 1998; Oberyé et al., 1999).
The multiple dose protocol
New GnRH antagonists are ready for clinical use and, like their precursors, are able to induce a rapid fall in FSH and LH secretion without flare-up and can be administered in mid- or final follicular phase to avoid the premature LH surge or to interrupt an LH surge that has already started.
The single dose protocol
In this protocol, patients are stimulated with different doses of either urinary or recombinant gonadotrophins starting on cycle day 2, and they receive daily doses of 0.25 mg of the antagonist (cetrorelix or ganirelix) starting on stimulation day 6. Based on clinical experience, GnRH antagonist administration starts when there is a risk of an imminent and premature LH surge, according to the follicular response and to the oestradiol concentration. This scheme, the most commonly used, has been applied since the beginning and is still used by the Department of Obstetrics and Gynecology at the Medical University of Lübeck (Diedrich et al., 1994; Felberbaum and Diedrich, 1999).
In order to simplify the stimulation protocol, a French group started to administer a single dose of cetrorelix (5 mg in the first trials and 3 mg in the latest ones) when oestradiol concentrations were between 150 and 200 pg/ml per follicle >14 mm. A second dose was injected 48 h later if ovulation was not triggered. However, this protocol is now defined by a single dose of 3 mg and a protection period of 4 days.
The role of GnRH antagonists in assisted reproduction - PN Barri et al. This protocol has been followed by the French group from Clamart since their initial phase ll studies (Olivennes et al., 1994, 1998) (Figure 1). So far, both protocols have proven comparable in terms of efficacy and safety in IVF patients (Table 1).
New protocols of GnRH antagonist treatment The availability of GnRH antagonists in ovarian stimulation protocols for assisted reproduction techniques has led to new strategies of ‘soft’ stimulation in order to obtain a moderate ovarian response and a sufficient number of mature oocytes (Fauser et al., 1999). In this respect it is interesting to note the experience of a Dutch group, which tried a protocol in which ovarian stimulation began on day 5 with 100 or 150 IU/day of recombinant FSH (rFSH) and continued from day 8 combined with a daily dose of 0.25 mg of cetrorelix until optimal follicular maturation was achieved. After treating 15 patients with IVF, the authors obtained normal pregnancy rates and a significant reduction in the total amount of gonadotrophins required compared with a standard long protocol of GnRH agonist (de Jong et al., 2000). Another possibility is the modification of the classic natural cycle with minimal ovarian stimulation. In this way, a French group has reported their experience of 44 IVF ‘natural’ cycles in which a single dose of 0.5 or 1 mg of cetrorelix was administered to normal IVF patients when a leading follicle was >12 mm and the oestradiol concentration was above 150 pg/ml. Simultaneously, a daily dose of 150 IU of human menopausal gonadotrophin (HMG) was given until human chorionic gonadotrophin (HCG) administration. Although there were some cancellations (9%), and no oocytes were obtained in 10 cycles (25%), a promising 32% pregnancy rate per embryo replacement was achieved (Rongières-Bertrand et al., 1999).
antagonist, the final oocyte maturation can be achieved either with native GnRH administration (Felberbaum et al., 1995) or with a single dose of a GnRH agonist (Olivennes et al., 1996) in patients at risk of developing ovarian hyperstimulation syndrome (OHSS). Both strategies have a preventive effect on OHSS, because the endogenous LH secreted has a much shorter half-life than that of HCG (Itzkovitz et al., 2000). The birth of a healthy girl following GnRH agonist treatment for the induction of final oocyte maturation in an IVF/intracytoplasmic sperm injection (ICSI) patient has recently been reported (de Jong et al., 2001).
Clinical experience with GnRH antagonists in ovarian stimulation for assisted reproduction techniques After appropriate dose-finding phase II studies, which confirmed that 0.25 mg was the optimal daily dose for both cetrorelix and ganirelix (Albano et al., 1997; The Ganirelix Dose-Finding Study Group, 1998), several multicentre and multinational phase III trials have been carried out with the two GnRH antagonists commercially available so far.
Clinical experience with cetrorelix Two multicentre studies were conducted to compare the GnRH antagonist cetrorelix with different GnRH agonists. When cetrorelix was compared with buserelin (Albano et al., 2000) and with triptorelin (Olivennes et al., 2000), some differences were observed. When cetrorelix was used, a significant reduction in the duration of the stimulation period and in the gonadotrophin
a Triggering final oocyte maturation with GnRH agonists In standard IVF protocols, HCG is used to induce final oocyte maturation. However, HCG has a longer half-life and a longer biological effect than those of endogenous LH because of its increased carbohydrate content. For this reason, and thanks to the preserved pituitary response that exists under a GnRH Table 1. Efficacy and safety of single and multiple dose Cetrotide® regimens (adapted from Diedrich, 2000). Values are percentages unless otherwise stated. Single dose ITT population (n) 192 HCG administered 93.2 Oocyte retrieval procedure 90.6 ET 80.7 Pregnancy/attempt 23.4 Pregnancies/ET 29.0 Miscarriages 13.3 OHSS grade II–III/cycle 1.0
Multiple dose
b
859 93.7 91.3 82.0 23.1 27.4 15.2 1.2
ITT = intention to treat; ET = embryo transfer; OHSS = ovarian hyperstimulation syndrome.
Figure 1. Schemes showing different protocols of cetrorelix administration: (a) multiple and (b) single dose schedules.
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The role of GnRH antagonists in assisted reproduction - PN Barri et al. Table 2. Cetrorelix multiple dose phase III study: details of ovarian stimulation (adapted from Felberbaum et al., 2000).
Table 3. Cetrorelix multiple dose phase III study: final results (adapted from Felberbaum et al., 2000).
Patients (n) Days of stimulation (mean) Ampoules of HMG (mean) Days of cetrorelix (mean) Premature luteinization (n (%)) Oocyte retrieval procedures (n)
Oocyte retrieval procedures (n) Oocytes (n) Fertilization (IVF or ICSI) (%) (mean ± SD) Embryo quality (excellent + good) (% of fertilized oocytes) Pregnancies (n) Clinical pregnancy/ET (%) Miscarriage (n) Ongoing implantation (%) OHSS (%)
346 10.4 25.2 5.7 3 (0.9) 324
HMG = human menopausal gonadotrophin.
requirements for an adequate ovarian response were observed. As far as fertilization and pregnancy rates are concerned, no significant differences were found between cetrorelix and buserelin or triptorelin. The incidence of OHSS was also significantly reduced when cetrorelix was used (1.1% and 1.7%), compared with the incidence observed either with buserelin (6.5%) or with triptorelin (5.6%). The greatest clinical experience with cetrorelix in assisted reproduction techniques comes from a prospective uncontrolled phase III study in which the present authors were involved (Felberbaum et al., 2000). In this study, 346 IVF patients were enrolled and received HMG for ovarian stimulation; after 6 days of stimulation, a multiple dose protocol of 0.25 mg cetrorelix/day was administered until the induction of ovulation. A mean of 25.2 ampoules of HMG was administered for a mean of 10.4 days, and cetrorelix was administered for a mean time of 5.7 days. Only three cases of raised LH (0.9%) were observed, and 324 patients underwent oocyte retrieval. Mean fertilization rate with either conventional IVF or with ICSI was 59.2%, and 70 pregnancies were obtained (23.6% ongoing pregnancy/embryo transfer, 11% ongoing implantation rate and 17% miscarriage rate). The incidence of severe OHSS was very low (0.6%) (Tables 2 and 3).
Clinical experience with ganirelix The efficacy and safety of ganirelix was clinically assessed in three different studies carried out in infertile patients undergoing ovarian stimulation with rFSH (Puregon; Organon Laboratories, Barcelona, Spain). These studies included a total of 1335 patients treated in 43 IVF units according to the following characteristics: European randomized trial comparing ganirelix with buserelin (n = 701) (The European Orgalutran Study Group, 2000); North American randomized trial comparing ganirelix with leuprolide (n = 297) (The North American Ganirelix Study Group, 2001); and European–Middle East
324 297 59.2 ± 25.3 80.5 70 23.6 17 11 0.6
ICSI = intracytoplasmic sperm injection; ET = embryo transfer; OHSS = ovarian hyperstimulation syndrome.
randomized trial comparing ganirelix with triptorelin (n = 337) (The European and Middle East Orgalutran Study Group, 2001). All these studies confirmed that the treatment with ganirelix was significantly shorter than the classic long protocol with GnRH agonists, and the duration of the stimulation phase and the amount of rFSH required were also significantly reduced with the antagonist. The incidence of LH elevation with a dose of 0.25 mg of ganirelix was 1% (Table 4). Compared with the long agonist protocol, the ganirelix protocol resulted in 1–2 fewer follicles and consequently 1–2 fewer oocytes. However, the percentage of metaphase II oocytes was similar in all the groups included in the three studies. The mean number of good quality embryos was almost identical in all the groups, and the ongoing pregnancy and implantation rates were somewhat lower in the first European and in the North American studies but not different in the European–Middle East trial (Mannaerts et al., 2001) (Table 5). The trend towards lower clinical pregnancy rates observed in two studies could be explained by the age of the patients included and by the previous clinical experience with GnRH antagonists that some of the IVF units already had.
Points for debate and discussion It is clear that the application of GnRH antagonists to the standard protocols of ovarian stimulation for assisted reproduction techniques will bring both benefits and drawbacks, and it is time to analyse some of these now.
Table 4. Median (ranges) and treatment difference of mean duration of GnRH analogue and Puregon treatment in the European trial. Restricted to patients with a human chorionic gonadotrophin (HCG) injection (adapted from Mannaerts et al., 2001). European trial
Duration of stimulation (days) Amount of rFSH (IU)
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North American trial
European–Middle East trial
Ganirelix (n = 463)
Buserelin (n = 238)
Ganirelix (n = 198)
Leuprorelin (n = 99)
Ganirelix (n = 226)
Triptorelin (n = 111)
9 (6–18)
10 (6–19)
8 (6–19)
10 (6–16)
9 (6–14)
11 (7–16)
1500 (900–5400)
1800 (900–6450)
1800 (1200–4275)
2025 (1350–5100)
1350 (900–3900)
1800 (1050–4050)
GnRH = gonadotrophin-releasing hormone; rFSH = recombinant FSH.
The role of GnRH antagonists in assisted reproduction - PN Barri et al. Table 5. Clinical outcome of controlled, randomized trials. Oocytes and embryos are expressed as mean values (SD). European trial
No. oocytes per attempt Fertilization rate (%) No. good-quality embryos Implantation rate (%) Ongoing PR per attempt (%)
North American trial
European–Middle East trial Ganirelix (n = 226)
Ganirelix (n = 463)
Buserelin (n = 238)
Ganirelix (n = 198)
Leuprorelin (n = 99)
8.7 (5.6)
9.7 (6.2)
11.7 (6.7)
14.1 (8.2)
62.4
61.9
62.1 3.3 (3.0)
62.1 3.5 (3.2)
4.3 (3.7)
Triptorelin (n = 111)
7.9 (5.1)
9.6 (6.8)
64.0
4.8 (4.0)
64.9
2.7 (2.5)
2.9 (2.4)
15.7
21.8
21.1
26.1
22.9
22.9
20.3
25.7
30.8
36.4
31.0
33.9
PR = pregnancy rate.
Table 6. Ganirelix dose-finding study. Incidence of LH increase, implantation rate and ongoing pregnancy rate per attempt versus dose of gonadotrophin-releasing hormone (GnRH) antagonist. Dose (mg)
0.0625
0.125
0.25
0.50
1.0
2.0
(n = 30)
(n = 65)
(n = 68)
(n = 69)
(n = 64)
(n = 26)
Incidence of LH increase (%) Implantation rate (%) Ongoing pregnancy rate per attempt (%)
16 14.2 23.3
9 16.6 23.1
1 21.9 33.8
0 9.0 10.1
0 8.8 14.1
0 1.5 0
Is it possible to schedule IVF cycles with antagonists?
embryo implantation (Hernandez, 2000; Kol, 2000; Ortmann et al., 2001).
Although programming the schedule of the cycles was easier with GnRH agonists, this can also be achieved with a previous administration of oestrogen pills, which can be useful not only for the programming but also for the homogeneity of the luteal phase prior to the IVF cycle. In this way, the follicular cohort will be more homogeneous, and this could overcome the reduction in the number of follicles and oocytes normally observed when GnRH antagonists are used.
It is clear that from the results of the dose-finding studies there was a dose-related decline in the implantation rates when high doses of ganirelix were used (Table 6). It has been speculated that due to the presence of GnRH receptors in the ovary, the tube and the endometrium, as well as in the oocyte and the embryo, the GnRH antagonists could negatively affect implantation (Raga et al., 1999), although pregnancy rates are not adversely affected in subsequent freeze-thaw cycles (Kol et al., 1999).
Is ovarian function impaired with GnRH antagonists?
However, the differences observed are not significant, and there is a clear need for new prospective and randomized studies in order to clarify this issue (Diedrich et al., 2001b).
Recent studies have suggested that the use of GnRH antagonists could interfere with normal ovarian function (García Velasco et al., 2001). Follicular growth was unaffected by the dose of the GnRH antagonist employed, but late follicular LH, androstendione and oestradiol showed an antagonist dose-related decrease (de Jong et al., 2001).
Is there a clear reduction in the pregnancy rates obtained with GnRH antagonists?
As far as the luteal phase is concerned, LH concentrations are not reduced differently with antagonists and agonists (Tavaniotou et al., 2001). These data suggest that high doses of GnRH antagonists may interfere with follicular steroidogenesis via some intra-ovarian modifiers, such as vascular endothelial growth factor (VEGF) or insulin-like growth factor (IGF).
Do GnRH antagonists interfere with embryo implantation? Several editorials and review articles have fuelled the controversy of a potential deleterious effect of antagonists on
In most GnRH antagonist studies, there is a trend towards somewhat (although not significantly) reduced pregnancy rates (Balasch, 2001). This has been attributed to the learning curve, although the majority of IVF researchers involved in the different multicentre trials have enough experience. However, one crucial point that may partly explain the reduction observed is the differences in monitoring and laboratory protocols that normally exist when a large number of centres are involved. In order to clarify this question, comparative studies between GnRH agonists and antagonists should be carried out in individual centres with wide IVF experience. In this way the comparison will be valid.
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The role of GnRH antagonists in assisted reproduction - PN Barri et al. Table 7. Gonadotrophin-releasing hormone (GnRH) antagonists in poor responders.
Age (years) FSH (mIU/ml) Oestradiol on day 3 (pg/ml) FSH 1 + 2b (mIU/ml) Ampoules of gonadotrophins Cancellations (n (%)) Oestradiol on day of HCG (mean ± SD) (pg/ml) Metaphase II oocytes (mean ± SD) Fertilization rate (%) Embryos (mean ± SD) Pregnancies (n) Pregnancies/oocyte retrieval (%) Implantation rate (%)
Group A Flare-up: Leuprolide + Gonal-F (n = 15)
Group B Clomiphene + Gonal-F + Cetrotide (n = 15)
39.5 ± 5.9 11.2 ± 8.3 48.3 ± 15
39.3 ± 4.6 10.3 ± 4.7 74.3 ± 26
22.7 ± 10.4 62.7 ± 18.2a
21.2 ± 7.3 30 ± 5.1a
1 (6) 1324 ± 359.8
2 (13) 1441 ± 760
3.5 ± 2.9
5.6 ± 2.6
75 2.1 ± 1.2 2 14.2
73 3.2 ± 0.5 3 15.3
12.5
16.6
a Significantly different (P < 0.05). b Sum of FSH values from day 3 and day 10 (clomiphene test). HCG = human chorionic gonadotrophin.
Can poor responders benefit from protocols with GnRH antagonists? So far the studies that have been conducted in assisted reproduction techniques with GnRH antagonists have mainly included patients who were expected to show a normal ovarian response. It is necessary to find out if patients with previous poor responses could improve their responses under antagonists. A recent study comparing a classic agonistic flareup protocol with a protocol in which a daily dose of 0.25 mg of cetrorelix was administered failed to show any benefit for this group of patients (Akman et al., 2001). The present authors have an ongoing study in which two groups of patients with a previous poor response in at least two different IVF cycles are compared. So far, 15 patients have been randomly allocated to two different ovarian stimulation protocols. Patients in Group A received a classic flare-up protocol combining leuprolide and a daily dose of 225 IU of rFSH (Gonal-F, Serono Laboratories, Madrid, Spain), while patients in Group B were treated with a combination of 100 mg clomiphene citrate/day (Effik Laboratories, Madrid, Spain) from day 5 to day 9 in the cycle, a daily dose of 225 IU of rFSH (Gonal-F) from day 6 onwards and a daily dose of 0.25 mg Cetrotide (Serono Laboratories) when a leading follicle was >14 mm and the oestradiol concentration was above 150 pg/ml.
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The preliminary results of this study show that apart from the expected significant reduction in the gonadotrophin requirements, no other benefits were obtained, and the mean
number of oocytes and embryos, their quality and the pregnancy and implantation rates achieved were similar in both groups (Table 7).
Safety of the protocols with GnRH antagonists The third generation of antagonists is practically free of local reactions and no general side effects have been described. The follow-up of the babies born so far is already significant and the fact that no increase in malformations has been observed is reassuring (Ludwig et al., 2001). Another important clinical finding is the significant reduction in the incidence of OHSS observed with the use of cetrorelix compared with that of buserelin in ovarian stimulation for assisted reproduction techniques (Ludwig et al., 2000).
Conclusion With the current knowledge about the clinical benefits of GnRH antagonists for assisted reproduction techniques, it is time to answer the question ‘Do we have to change our protocols completely and replace the agonists with antagonists?’ The present authors have evaluated most of the controversial data suggesting different benefits and drawbacks of the antagonists and conclude that is too early to propose a radical switch. They agree with the opinion of several authors who have postulated that more randomized controlled studies are needed to validate the potential advantages of antagonists over agonists (Albano et al., 2001). In the near future it is hoped that the pregnancy rates obtained with antagonists may be as good as those achieved with agonists; then, the benefits of reducing gonadotrophin requirements, shortening the stimulation phase and reducing the risk of OHSS will be real advantages (Blumenfeld, 2001). Until this moment, researchers should proceed carefully and not abandon the agonists for the antagonists.
Acknowledgements This work was performed under the auspices of ‘Catedra d’Investigació en Obstetricia i Ginecologia’ of the Department of Obstetrics and Gynaecology, Institut Universitari Dexeus, Universitat Autònoma de Barcelona.
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