Use of cetrorelix in combination with clomiphene citrate and gonadotrophins: a suitable approach to friendly IVF?

Human Reproduction Vol.17, No.8 pp. 2022 2026, 2002 Use of cetrorelix in combination with clomiphene citrate and gonadotrophins: a suitable approach to friendly IVF? J.B.Engel, M.Ludwig 1, R.Felberbaum, C.Albano, P.Devroey and K.Diedrich Division of Reproductive Medicine and Gynecologic Endocrinology, Department of Gynecology and Obstetrics, University Clinic, Ratzeburger Allee 160, 23538 Lübeck, Germany 1 To whom correspondence should be addressed. E-mail: ludwig_m@t-online.de BACKGROUND: With the recently introduced GnRH antagonists, soft stimulation protocols on the basis of clomiphene pretreatment should be possible as the pituitary remains fully sensitive at the beginning of the cycle. METHODS: A prospective trial was carried out on 107 patients undergoing IVF treatment using the multiple dose GnRH antagonist protocol (cetrorelix), clomiphene citrate, and either (n 54) or recombinant FSH (rfsh) (n 53). Different stimulation protocols were used to find the most appropriate one for clinical application. RESULTS: Both treatment groups, and rfsh, yielded comparable results concerning gonadotrophin dose, stimulation days and pregnancy rate. A mean number of 6.34 4.4 metaphase II oocytes was retrieved and a mean number of 2.45 0.65 embryos was transferred. However, the overall rate of premature LH surges was 21.5% (defined as measurement of LH >10 IU/l and progesterone >1 ng/ml) which is unacceptable for clinical practice. CONCLUSIONS: Increasing the daily cetrorelix dose from 0.25 to 0.5 mg might decrease the number of premature LH surges. Soft stimulation protocols with clomiphene should be used cautiously. Key words: cetrorelix/clomiphene/cos/friendly IVF/GnRH antagonist Introduction The recently introduced GnRH antagonists offer possibilities to create innovative ovarian stimulation regimens with lower chances of side effects (Ludwig et al., 2000). The occurence of a premature LH surge is 1% without compromizing fertilization, cleavage and pregnancy rates (Albano et al., 1997, 2000; Felberbaum et al., 2000; Olivennes et al., 2000). The GnRH antagonists are administered midcycle, either according to the single (Olivennes et al., 1994) or the multipledose protocol (Diedrich et al., 1994). Thus at the beginning of the controlled ovarian stimulation (COS) cycle the pituitary is fully susceptible to GnRH pulses. This should permit the revival of stimulation protocols of the pre-agonist era using e.g. clomiphene citrate (CC) (Quigley, 1984). CC is cheap and simple to administer. It has been in use for four decades and is still the first-line therapy in normogonadotrophic anovulatory women. The combination of CC treatment in the early follicular phase and subsequent, overlapping, gonadotrophin stimulation has been a standard therapy in the past (Quigley, 1984; Lehmann et al., 1988). Due to the synergistic effect of these compounds the amount of gonadotrophin required is lower and so are the costs (Ronen et al., 1988; Tummon et al., 1992). In addition the gonadotrophins counteract the detrimental effects of the CC to the endometrium (Ronen et al., 1988). Due to the high rate of premature LH surges, and therefore the high cancellation rate, this stimulation regimen was abandoned when GnRH agonists were introduced in IVF. This study was designed to investigate the possibility of using CC in combination with gonadotrophins in a prospective GnRH antagonist protocol. More specifically, it was carried out to evaluate the equivalence of rfsh and with regard to the rate of HCG administration. Materials and methods Study design This prospective, open, bicentric, phase II trial was carried out on a total of 107 patients ( group: n 54; rfsh group: n 53) between June 1997 and October 1999. It was originally done in a randomized fashion, to compare the use of recombinant FSH and. However, since the study protocol was changed several times during the study course, the randomization procedure can no longer be considered appropriate. We therefore analysed the study as a prospective non-randomized trial. Approval was given by the ethical committees of the Medical University of Lübeck and the Brussels Dutch speaking Free University. Inclusion and exclusion criteria Healthy female partners of infertile couples were included after having given written informed consent. They were between 18 39 years of age at the time of screening with normal menstrual cycles with a range of 24 35 days and an intra-individual variation of 3 days. All had a normal uterus, confirmed by ultrasound. Exclusion 2022 European Society of Human Reproduction and Embryology

Combination of cetrorelix and clomiphene citrate Figure 1. Soft protocol and modifications. criteria were a previous IVF procedure, presence of polycystic ovarian syndrome, corpus luteum insufficiency, impaired ovarian function, severe endometriosis, submucosal uterine myoma, or a FSH level 10 IU/l at screening. The exclusion criterion of no previous IVF cycles, which was valid for stimulation protocols 1 and 2 (see below), was changed during the course of the study. In stimulation protocols 3 and 4 (see below) patients could be included, if they did not have a history of more than three IVF attempts. Stimulation protocols The stimulation procedure was changed three times. Finally, four different stimulation protocols were analysed. Stimulation protocol 1 (group 1): from cycle day 2 or 3 (treatment day 1) 100 mg CC p.o. was administered once daily for 7 days. On treatment day 6 ovarian stimulation was initiated with three ampoules of (Menogon, Ferring Arzneimittel GmbH, Kiel, Germany) or rfsh (Gonal F, Serono International S.A., Geneva, Switzerland) according to randomization. From treatment day 8 up to and including the day of HCG the gonadotrophins were adjusted to individual patients ovarian response. From treatment day 6 up to and including the day of HCG administration 0.25 mg cetrorelix (Cetrotide, Serono International S.A.) was injected s.c. each day. 10 000 IU HCG was administered when at least one follicle had reached 18 mm diameter. All cycles were carried out using ICSI to allow rating of oocyte maturity. Embryo transfer was performed 48 h after oocyte retrieval. Due to the occurrence of premature LH surges, the stimulation protocol was modified three times. A premature LH surge was defined as LH 10 IU/l and progesterone 1 ng/ml, as was done in all prospective randomized studies with antagonists (Albano et al., 2000; The European Orgalutran Study Group et al., 2000). The procedures were amended as follows (Figure 1). Stimulation protocol 2 (group 2) differed from protocol 1 by the number of CC days, which were reduced from 7 to 5. Stimulation protocol 3 (group 3) was similar to protocol 2, but the initial gonadotrophin dose was reduced from three to two ampoules. In stimulation protocol 4 (group 4) the gonadotrophins were given starting on treatment day 3 up to and including the day of HCG administration. Broca-index The Broca-index was calculated as weight/(height 100) f (f 0.85) (Roche Lexikon Medizin, 1999). Clinical pregnancy A clinical pregnancy was confirmed in case of positive fetal heartbeats in transvaginal sonography 28 35 days after embryo transfer. Statistics Statistical analysis was carried out using the Clopper Pearson-test with double-sided 95% confidence interval. A P-value of 0.05 was considered to be statistically significant. The data are presented as means with SD. Results The and rfsh groups were similar concerning age (30.22 3.67 versus 30.11 3.89 years), Broca-index (1.12 0.16 versus 1.12 0.14) and cause of infertility. Both groups required a similar dose of gonadotrophins (19.94 8.7 versus 19.36 8.9 ampoules) and a similar number of stimulation days (7.09 2.08 versus 6.79 1.95 days). The absolute number of metaphase II oocytes was slightly but not statistically significantly higher in the as compared with the rfsh group (7.2 5.31 versus 5.5 3.75). A total of 77% of the cumulus oocyte complexes in both groups were metaphase II oocytes. The number of embryos obtained was similar (3.64 2.43 versus 3.38 1.87) as was the number of transferred embryos (2.38 0.64 versus 2.40 0.66) in the and rfsh group respectively. Only one case of OHSS grade II occurred in the, and none in the rfsh group. Neither the overall pregnancy rate (25.9 versus 13.2%) nor the pregnancy rate per embryo transfer (29.8 versus 16.7%) was statistically significantly different in both patient collectives treated. Due to the occurrence of premature LH surges the stimulation protocol was modified three times. The results are presented exclusively for the different treatment groups (groups 1 4). Oocytes were obtained from all patients with a premature LH surge. In one of these 2023

J.B.Engel et al. patients, however, no fertilization was achieved. COS had to be cancelled before the HCG injection in four patients. In 12 cases total fertilization failure was found and in one additional case treatment failure was reported without any further comments (Table I). There were no statistically significant differences concerning age, stimulation days, dose of gonadotrophins, oocytes, embryos obtained, embryos transferred and pregnancy rate in the different treatment groups (Tables II and III). However, in group 3 the number of metaphase II oocytes was statistically significantly higher in the patients treated with (P 0.019) than those treated with rfsh. A total of 23 LH surges occurred (21.5%), five of these on stimulation day 6 before Cetrotide was administered. The rates were 27.3, 25.0, 11.0 and 27.5% in the groups 1, 2, 3 and 4 respectively. This rate was always higher in the rfsh as compared with the group, explaining the slightly reduced pregnancy rates in patients treated in the rfsh group (Table IV). Discussion This study was designed to evaluate the clinical reliability of a soft stimulation protocol using CC with subsequent gonadotrophin and GnRH antagonist administration. It was carried out comparing and rfsh in a prospective design. Due to the occurrence of premature LH surges, protocol modifications were performed during the course of the study. The emphasis therefore shifted to the problem of premature LH surges and overall reliability of these protocols instead of Table I. Reasons for premature discontinuation. RFSH Total 7 11 Premature LH surge, HCG injected, no 0 1 (G2) fertilization Cancellation of COS, no HCG-injection 1 3 Insufficient ovarian response 0 2 (G4) Abnormal ovarian response 1 (G3) 0 Monofollicular growth 0 1 (G4) HCG administered, but ART failed 5 7 No or bad fertilization, no transfer 3 (G2, G3, 7 (G1: 2, G2:1, G4) G3: 4) Insemination (no ICSI) 2 (G3, G4) 0 Other 1 (G4) 0 G1, G2, G3, and G4 show from which single group the patients came (Group 1, Group 2, Group 3, Group 4). Groups are defined in Figure 1. the comparison of the two different gonadotrophins. The pregnancy rates in the and the rfsh group were not statistically significantly different. However, the pregnancy rate overall was slightly higher in the as compared with the rfsh group (25.9 versus 13.8%). In parallel, there were more premature LH rises in the rfsh group (26.4 versus 16.7%), which might explain this difference, since it is well known that a premature LH surge will result in a poorer oocyte quality, fertilization and pregnancy rate (Stanger and Yovich, 1985). This is further supported by the fact that in group 4 with a similar rate of premature LH surges (27.2 versus 27.7%), the pregnancy rates were also the same (27.3 versus 27.8%) in the and rfsh groups respectively. With a starting dose of 150 IU FSH the number of ampoules was ~18 per stimulation cycle. Though having a tendency to be lower, the gonadotrophin dose was not substantially different from those reported in the large phase III studies carried out according to the multiple-dose antagonist protocol (Albano et al., 2000). Lowering the daily gonadotrophin dose to 75 IU should be attempted to decrease the absolute gonadotrophin dose. However, the gonadotrophin dose is 50% lower than the amount required in the long protocol (Felberbaum and Diedrich, 1998). The different stimulation protocols (groups 1 4) lead to similar clinical results. So the reduction of CC stimulation days and gonadotrophin dose did not have a negative influence on the treatment outcome. Although there was only one patient with a premature LH surge where the oocytes could not be fertilized, the percentage of premature LH surges was unacceptably high for clinical practice. In group 3 there was Table III. Number of pregnancies in the different study groups. There were no significant differences in pregnancy rates. Groups are defined in Figure 1 RFSH Total number Pregnant (%) Total number Pregnant (%) Group 1 6 2 (33.3) 5 0 Group 2 10 2 (20.0) 10 1 (10.0) Group 3 16 4 (25.0) 20 1 (5.0) Group 4 22 6 (27.2) 18 5 (27.7) Total 54 14 (25.9) 53 7 (13.2) Table II. Demographic data and clinical results. Groups are defined in Figure 1 Group Gonadotrophin Patients (n) Age Stimulation days Gonadotrophin Metaphase II Embryos Embryos (gonadotrophins) (ampoules) oocytes obtained transferred I 6 29.50 3.73 6.7 1.2 25.7 7.8 4.2 2.1 1.8 0.8 1.8 0.8 rfsh 5 29.20 1.30 6.8 0.8 24.0 4.6 5.8 2.4 2.7 0.6 2.7 0.6 II 10 29.20 2.30 6.1 1.6 21.3 6.3 6.2 4.2 2.7 0.7 2.6 0.7 rfsh 10 31.30 3.50 6.0 1.2 22.2 5.6 4.9 2.3 2.8 0.5 2.8 0.5 III 16 30.13 3.24 6.5 2.6 19.9 12.9 7.4 4.0 4.0 2.2 2.5 0.5 rfsh 20 29.30 4.65 5.9 2.3 17.5 12.9 3.9 3.2 3.1 2.4 2.1 0.8 IV 22 30.95 4.45 8.1 1.7 17.8 5.1 8.6 5.8 4.4 3.0 2.4 0.6 rfsh 18 30.61 3.62 8.3 1.2 18.6 4.4 7.9 4.5 4.2 1.7 2.5 0.5 Total 54 30.22 3.67 7.1 2.1 19.9 8.7 7.2 4.8 3.6 2.4 2.4 0.6 rfsh 53 30.11 3.89 6.8 2.0 19.4 8.9 5.5 3.8 3.4 1.9 2.5 0.7 Numbers as mean SD. 2024

Combination of cetrorelix and clomiphene citrate Table IV. Premature LH-surges in the different groups (LH 10/l and progesterone 1ng/ml). Groups are defined in figure 1 rfsh Total number LH surge Total number LH surge Group1 6 1 (16.7) 5 2 (40.0) Group 2 10 1 (10.0) 10 4 (40.0) Group 3 16 1 (6.2) 20 3 (15.0) Group 4 22 6 (27.3) 18 5 (27.8) Total 54 9 (16.7) 53 14 (26.4) a trend towards fewer premature LH surges, although this was not significant. It appears that reducing the duration of CC treatment and the starting dose of gonatrophins was a step in the right direction to reduce the amount of ampoules needed. However, these modifications were not sufficient to prevent a premature LH surge, as clearly demonstrated in group 4. With only one grade II OHSS in the group the side-effects of this regimen are comparable with those in the multiple-dose antagonist protocol (Ludwig et al., 2000). To our knowledge, there is only one other study (Fiedler et al., 2001) dealing with the combination of Cetrotide and clomifene citrate. Fiedler and co-workers used CC with or rfsh in 586 IVF cycles and did not conduct the study in a prospective randomized manner. A total of 295 cycles were performed with and 291 without additional administration of a GnRH antagonist. Pregnancy rates were similar with (23%) and without (21%) an antagonist treatment. LH serum levels were not provided by the authors, therefore the rate of premature LH surges cannot be assessed. Despite the benefits of the presented co-administration of gonadotrophins and CC, the rate of premature LH surges remains a major drawback. This rate might be explained by the physiological mechanism, that the addition of CC with anti-estrogen effects leads, on the hypothalamic level, to the release of more native GnRH by the hypothalamus. An increased sensitivity of the pituitary for GnRH as a direct effect of the CC (Emons et al., 1986) could be another possible explanation for our results. Thus CC pretreated rat pituitary cell cultures required higher GnRH antagonist doses to suppress LH than CC untreated cultures (unpublished data). The elevated rate of premature LH surges can be explained by an incompletely blocked pituitary due to an increased release of GnRH by the hypothalamus and an increased sensitivity of the pituitary for GnRH. Thus, increasing the daily dose of cetrorelix from 0.25 to 0.5 mg could overcome the problem of premature LH surge. On the other hand, a single dose of 3 mg Cetrotide in such a protocol might be an acceptable approach to prevent the premature LH surge. In the study by Zhioua and co-workers, 15 patients were treated using CC/ and a single dose of 3 mg Cetrotide on stimulation day 8 (Zhioua et al., 2000). No premature LH surge occurred. The pregnancy rate was 26.6%. Due to the small patient number this preliminary finding has to be confirmed in a greater number of patients. The presumption of a not fully blocked pituitary, i.e. a partially susceptible pituitary ovary feedback loop, offers an explanation for the slightly elevated number of LH surges in patients treated with rfsh. In this patient group estrogen levels throughout the stimulation tended to be lower than in the group, although this finding was not significant. Thus the negative feedback of the estrogen on the endogenous LH production of the not adequately blocked pituitary was weaker in the rfsh group, which might explain the elevated number of LH surges in this patient group. In conclusion, soft stimulation protocols are a promising approach in assisted reproduction treatments. Nevertheless they have to be applied with caution until the problem of the premature LH surge has been overcome. Until a more reliable protocol can be established, this method of ovarian stimulation should only be done within prospective, clinical studies to avoid disadvantages for the treated patients. So far soft stimulation protocols do not have economic advantages when compared with the single or the multiple dose antagonist protocol. So defining a minimal effective gonadotrophin dose should be a main objective for future studies. References Albano, C., Smitz, J., Camus, M., Riethmüller-Winzen, H., Van Steirteghem, A. and Devroey, P. (1997) Comparison of different doses of gonadotrophinreleasing hormone antagonist Cetrorelix during controlled ovarian hyperstimulation. Fertil. Steril., 67, 917 922. Albano, C., Felberbaum, R.E., Smitz, J, Riethmüller-Winzen, H., Engel, J., Diedrich, K. and Devroey, P. 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