1 Academic Hospital `Vrije Universiteit' Amsterdam, Department of Obstetrics and Gynaecology, Subdivision of Reproductive Endocrinology and Fertility/IVF Centre, Amsterdam, The Netherlands, 2 Centre for Infertility, University Hospital, De Pintelaan 185, B-9000 Gent, Belgium, 3 Catholic University of Louvain, Faculty of Medicine, Department of Gynaecology, St Luc's University Hospital, Avenue Hippocrate 10, 1200 Brussels, Belgium, 4 Academic Hospital St Radboud, Department of Obstetrics and Gynaecology, G. Grooteplein Zuid 16, 6525 GA Nijmegen, The Netherlands, and 5 Centre for Reproductive Medicine, University Hospital of the Dutch speaking Brussels Free University, Laarbeeklaan 101, B-1090 Brussels, Belgium
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Abstract |
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Key words: highly purified uhFSH/ICSI/IVF/multiple follicular development/rhFSH
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Introduction |
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There remains, nevertheless, some degree of controversy over the most appropriate method of ovarian stimulation. One commonly used protocolselected as the basis for the present studyinvolves long desensitization with a gonadotrophin-releasing hormone (GnRH) agonist and the daily use of FSH to bring about multiple follicular development. Desensitization suppresses production of endogenous gonadotrophins and the spontaneous, mid-cycle LH surge. As a result, there are fewer cancelled cycles, higher numbers of oocytes and more pregnancies (Smitz et al., 1987; Loumaye, 1990
; Hughes et al., 1992
). Until recently, FSH could only be obtained from human menopausal gonadotrophin (HMG) or other urine-derived preparations. These are impure and require the collection of vast quantities of urine, leading to an unreliable supply and, perhaps most importantly, batch-to-batch inconsistency. Gonal-F, the first recombinant human FSH (rhFSH) preparation avoids these problems due to advances in recombinant biotechnology. Gonal-F, with a specific activity of more than 10 000 IU/mg protein (Loumaye et al., 1995
) is devoid of any LH activity, and it is this high purity which is thought to confer safety and tolerability advantages over urine-derived human FSH (uhFSH) (Redfearn et al., 1995
; Albano et al., 1996
).
In clinical terms rhFSH, in comparison with highly purified uhFSH, is associated with improved stimulation of multifollicular development, without any increase in the risk of the ovarian hyperstimulation syndrome (OHSS) (Out et al., 1995; Bergh et al., 1997
; Frydman et al., 1998
; Khalaf et al., 1998
). It is not yet fully understood why rhFSH should be more effective than urinary preparations of FSH, though differences in pharmaceutical formulation and the presence of slightly more basic isoforms of FSH in rhFSH (de Leeuw et al., 1996
; D'Antonio et al., 1999
) have been proposed. The batch-to batch consistency, however, as mentioned previously, is probably also important; rhFSH contains far fewer degraded forms of FSH than high-purity uhFSH (about 4% versus 40%) (Gonal F monograph; Serono data on file; personal communication).
There remains a question mark over the optimal starting dose of rhFSH, and another over the possibility of reducing overall rhFSH consumption. These issues are important not only from a clinical perspective, but also from an economic one. An earlier study (Camier et al., 1998) suggested that a starting dose of 150 IU/day can be as effective as one of 225 IU/day, in terms of oocyte retrieval, fertilization and pregnancy. By contrast, another study (Abu-Heija et al., 1995
) has shown the 150 IU/day dose to be inferior to one of 225 IU/day. In terms of FSH consumption, previous studies (Bergh et al., 1997
; Frydman et al., 1998
), which used a starting dose of 150 IU/day Gonal-F®, found an overall reduction in the number of FSH treatment days and numbers of ampoules used, compared with 150 IU/day highly purified uhFSH (Metrodin HP®). Thus, overall, the data suggest that rhFSH is likely to be advantageous from a clinical and costbenefit perspective. The latter is currently being investigated using a sophisticated pharmacoeconomic model.
This large, multicentre, study compared Gonal-F® with Metrodin HP® for ovarian stimulation in women undergoing IVF or intracytoplasmic sperm injection (ICSI). The main objectives were to compare ovarian response between groups and to evaluate this response in relation to the consumption of FSH. The data have already been published in summary form as part of a meta-analysis in which data from 12 different trials were pooled (Daya and Gunby, 1999).
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Materials and methods |
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Patients were excluded from the study if they had: (i) abnormal gynaecological bleeding of undetermined origin; (ii) a previous IVF or ICSI failure due to a poor response to gonadotrophins or a previous ICSI failure due to a problem of sperm fertilization (<20%); (iii) a previous history of severe OHSS; (iv) a male partner with azoospermia and clinical signs of infection detected in a semen analysis within the past 12 months; (v) a clinically significant condition/disease; or (vi) microsurgical epididymal sperm aspiration, testicular sperm extraction or percutaneous epididymal sperm aspiration procedures. The most common causes of infertility were male factor (~50%) and tubal pathology (23%).
Concomitant medication for an existing condition or for an inter-current illness was allowed at the investigator's discretion, though other hormonal treatments, anti-inflammatory medication and psychotropic agents with a known effect on ovulation (e.g. neuroleptics) were to be avoided. Patients requiring these agents were only permitted to continue in the trial at the investigator's discretion.
Study design
This was an assessor-blind, randomized, parallel-group study carried out at 30 centres throughout Belgium and The Netherlands. The study was designed to compare the efficacy and safety of Gonal-F® (Serono Benelux b.v., Koninginnegracht 28, 2514 AB, The Netherlands) with Metrodin HP® (Serono Benelux b.v.) during one cycle of ovarian stimulation for IVF/ICSI. To achieve assessor blinding, all medication used was dispensed from a central pharmacy in The Netherlands. Preprinted prescriptions were faxed to the pharmacy from participating clinics. Randomization lists were held at the pharmacy and patients were randomized after receipt of a prescription from a participating centre. Allocation to one of the two groups was equal at each centre. The study was conducted in accordance with the requirements of the Declaration of Helsinki and Good Clinical Practice (GCP) guidelines.
Patients underwent pituitary desensitization with either 1.0 mg s.c. leuprolide (Lucrin®, Abbott b.v., Siriusdreef 51, 2132 WT Hoofddorp, The Netherlands), once daily, or 900 µg intranasal buserelin (Suprefact®, Aventis Pharma b.v., Brussels, Belgium), divided in three applications over the day, starting in the midluteal phase of the cycle preceding the stimulation cycle. GnRH treatment was continued until the day of HCG administration, but could not exceed 5 weeks. Patients were considered to be desensitized if an ultrasound scan showed no evidence of ovarian activity together with an endometrial thickness of <7 mm, and if serum oestradiol concentrations were 50 pg/ml (185 pmol/l).
After desensitization, patients received either Gonal-F® or Metrodin HP® administered once daily, s.c., starting at a fixed dose of 150 IU/day for the first 6 days. Ovarian response was assessed on day 7, and the results were used to evaluate whether the dose needed to be reduced. Ovarian response was regarded as high if in total 20 follicles and/or oestradiol concentrations already >5000 pmol/l were detected. If the response was satisfactory, patients continued on a fixed dose of 150 IU FSH/day until HCG was administered. Patients with a poor response were withdrawn and, in cases of an excessive response, patients judged to be at risk of OHSS had oocyte retrieval and any resultant embryos frozen for replacement in a subsequent cycle.
GnRH agonist (GnRHa) and FSH were administered until the largest follicle had a mean diameter of 18 mm, there were at least two other follicles with a diameter of
16 mm and if the patient's oestradiol concentration was within an acceptable range (500 pmol oestradiol/follicle). HCG (Profasi®, Serono Benelux b.v.) was administered s.c. at a dose of 10 000 IU, within 24 h of the last dose of GnRHa and FSH.
Oocytes were retrieved 3438 h after HCG administration and their nuclear maturity assessed according to established criteria as germinal vesicle, metaphase I, metaphase II, atretic or unknown. They were then fertilized in vitro, and the resultant embryos graded. Up to three embryos were replaced, 24 days after oocyte retrieval. For ICSI, the cumulus cells were removed and an assessment of oocyte nuclear maturity was made. Any remaining embryos were frozen at the 2- or 4-cell stage. Luteal phase support was initiated after oocyte retrieval using a commercially available progesterone preparation (Progestan®/Utrogestan®; Nourypharma, Oss, The Netherlands) at a dose of 200 mg three times daily, intravaginally. This was continued up to a negative pregnancy test or for at least the first 3 weeks of pregnancy.
Efficacy measures
The primary efficacy endpoints were the total number of 75 IU ampoules of FSH used and the number of follicles 10 mm diameter on the day of HCG administration. Additionally, the following secondary endpoints were recorded: number of patients requiring a decrease in FSH dose; number of days of FSH stimulation; number of oocytes retrieved; number of oocytes retrieved/number of follicles on day of HCG administration; number of two-pronuclear (2PN) fertilized oocytes; number of 2PN cleaved embryos; number of viable embryos (transferred or cryopreserved); implantation rate (both biochemical and clinical pregnancies); number of ongoing pregnancies; number of multiple pregnancies; and number of deliveries.
Safety assessments
Data for the duration of exposure, incidence and severity of adverse events (including injection-site reactions and OHSS), ectopic pregnancies, premature discontinuations and concomitant medications were summarized for all patients who took at least one dose of study medication. Adverse events were recorded on the case report form (CRF) on the basis of the patient's or physician's observations and coded using the COSTART system. An adverse event was defined as an undesirable medical experience, whether considered related or not to the study medication, which represented an adverse change from the patient's pretreatment condition. An adverse event was classified as serious if it was fatal or life-threatening, was permanently disabling, required inpatient or prolonged hospitalization, or was a congenital anomaly, cancer or overdose. A treatment-related adverse event was one which was judged by the investigator to be probably or possibly related to the study drug.
Sample size
This was a combination of two separate studies: the Feronia (Dutch) and Apis (Belgian) multicentre studies. The sample size for each study was that required to demonstrate equivalence of the two study treatments for the two primary efficacy endpoints (mean number of ampoules and mean number of follicles 10 mm). The calculated sample size was 120 patients per treatment group for each study (90% power, one-sided significance level 0.05). Therefore, a minimum of 480 patients had to be recruited for the two studies combined.
Statistics
Statistical analyses (SAS program) were performed on all data available. The set of patients used for the analyses was based on the ITT (intention-to-treat) principle and included all randomized patients who received HCG. The comparability of the two treatment groups was checked for all demographic data, and both this and the efficacy data were evaluated using summary statistics (n, mean, SD, minimum and maximum for continuous variables and frequencies and percentages for categorical data). Differences between groups were analysed using Wilcoxon two-sample or 2 tests. Parametric tests, which are known to have a higher power to detect a statistically significant effect, e.g. analysis of variance on raw data, compared with non-parametric test such as a Wilcoxon two-sample t-test were not used because there was some doubt about ascertaining and confirming the normality and homogeneity of variance, assumptions required to use a parametric test. This was mainly caused by the multicentre design of the study
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Results |
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Embryological characteristics
A total of 442 patients had oocyte retrieval; the embryological characteristics are shown in Table IV. Significantly more oocytes were inseminated in the Gonal-F® group than in the Metrodin HP® group (P < 0.003). The number of 2PN cleaved embryos was similar between groups, as were the numbers of cryopreserved embryos.
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Embryo transfer and pregnancy
In the Gonal-F® and Metrodin HP® groups, 202 (82%) and 200 patients (80%) respectively underwent embryo transfer (Table V), with means of 2.1 ± 0.6 and 2.1 ± 0.5 embryos replaced in the two groups respectively. The overall clinical pregnancy rates per oocyte retrieval, per cycle and per embryo transfer were all higher in the patients receiving Gonal-F®, though the differences between groups did not reach statistical significance (Table V
). The overall number of deliveries (live births) was not significantly different when comparing the Gonal-F® group (28% per embryo transferred) with the Metrodin HP® group (21.5% per embryo transferred). However, the difference in clinical pregnancy rate between treatment groups reached statistical significance on
2 analysis for the IVF (40 versus 27.6%, P = 0.037) and IVF + ICSI (80 versus 33.3%, P = 0.040) subgroup patients.
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Discussion |
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The primary endpoints in this study were the total number of 75 IU ampoules of FSH used and the number of follicles 10 mm in diameter on the day of HCG administration. Both treatments induced good follicular recruitment (15.6 ± 8.2 for Gonal-F® versus 13.6 ± 7.1 for Metrodin HP®), confirming the effectiveness of the 150 IU starting dose. The difference between the two groups, however, was statistically significant (P < 0.01) in favour of Gonal-F®. Additionally, the significantly higher level of follicular recruitment with Gonal-F® was produced using statistically fewer 75 IU ampoules of FSH (22.6 ± 5.0 versus 24.3 ± 5.1, P < 0.0002) over a statistically shorter treatment time than with Metrodin HP® (11.6 ± 1.9 days versus 12.4 ± 2.7 days, P < 0.0001). In terms of the numbers of oocytes retrieved, following HCG, Gonal-F® was, again, statistically more effective than Metrodin HP®. A mean of 13.1 ± 7.7 oocytes were retrieved from patients who had received Gonal-F®, compared with only 11.4 ± 7.6 oocytes from patients receiving Metrodin HP® (P < 0.002). Thus, this study clearly demonstrates the higher clinical efficacy of Gonal-F® in inducing multiple follicular development.
Two previous studies (Bergh et al., 1997; Frydman et al., 1998
), which compared Gonal-F® with Metrodin HP®, using an initial starting dose of 150 IU FSH, have demonstrated similar results, providing more support for the higher efficacy of the recombinant product. They (Bergh et al., 1997
; Frydman et al., 1998
) reported statistically lower FSH consumption (P
0.001), combined with statistically higher follicular recruitment (P
0.004) and oocyte retrieval (P
0.001) with Gonal-F®. In a third study (Khalaf et al., 1998
), although a lower FSH consumption was associated with a statistically higher number of aspirated follicles (P < 0.04), the difference in consumption between groups was not statistically different. This was because patients were started on up to 225 IU FSH per day, and not on a fixed dose. Although each centre has its own policy regarding timing and method for changing gonadotrophin dosemeaning that one must be a little cautious in comparing FSH consumption across studiesin the studies by Bergh et al. (1997) and Frydman et al. (1998) respectively, the number of FSH treatment days was 11.0 ± 1.6 and 11.7 ± 1.9 for Gonal-F® compared with 13.5 ± 3.7 and 14.5 ± 3.3 for Metrodin HP® (P
0.001). The numbers of FSH ampoules used in these studies were 21.9 ± 5.1 and 27.6 ± 10.2 for Gonal-F® compared with 31.9 ± 13.4 and 40.7 ± 13.6 for Metrodin HP® (P
0.001). The greater number of ampoules used in one of these studies (Frydman et al., 1998
) compared with the numbers used in the present study and that by Bergh et al. (1997) may be partly explained by Frydman et al.'s use of a depot agonist in about 90% of patients treated, which would have caused a more profound level of desensitization than that following intranasal or s.c. administration of a GnRH agonist.
All of these results are consistent with a previous report (Mannaerts et al., 1996), which described a higher bioactivity for rhFSH compared with urine-derived products, despite similar or lower serum immunoactive FSH concentrations. rhFSH is known to contain slightly more basic isoforms than urine-derived hFSH (de Leeuw et al., 1996
), and these have a higher intrinsic bioactivity and receptor affinity than acidic isoforms in vitro. Whether this is important for in-vivo human bioactivity is not clear (Von Düring et al., 1999
).
A limitation of the present study was that data on oestradiol were not available for analysis because all oestradiol determinations were carried out locally in the participating centres, using different assays. Higher oestradiol concentrations on the day of HCG administration have been reported in rFSH-stimulated cycles when compared with those performed with highly purified uFSH in normal (Bergh et al., 1997) and poor (Raga et al., 1999
; De Placido et al., 2000
) responders.
Significantly more oocytes were inseminated in the Gonal-F® group than in the Metrodin HP® group (12.1 ± 6.9 versus 10.1 ± 6.0, P < 0.003). The mean number of 2PN fertilized oocytes was also higher for Gonal-F® (7.2 ± 5.4) than for Metrodin HP® (6.4 ± 4.9), though this difference was not statistically significant. Other variables were similar between groups, including the numbers of 2PN cleaved embryos, fresh embryos transferred and cryopreserved embryos. Possible differences in oocyte maturity between treatment groups were not analysed.
Interestingly, the mean number of 2PN fertilized oocytes was higher for patients undergoing ICSI than for patients undergoing IVF, irrespective of treatment group. This is in contrast to an earlier study (Hamilton et al., 1998) which found a similar degree of fertilization between IVF and ICSI groups, though this study was carried out exclusively in women with non-male factor infertility.
Outcome in terms of pregnancy and live birth is unquestionably the most important criterion for patients and clinicians, and the chances of success are known to be determined by the number of fertilized oocytes, rather than by the numbers of embryos transferred (Templeton and Morris, 1998). In line with the higher number of fertilized oocytes, patients receiving Gonal-F® had a higher clinical pregnancy rate per cycle (25.1 versus 20.1%), per oocyte retrieval (26.3 versus 21.6%) and per embryo transfer (30.2 versus 25.0%), though the differences between groups did not reach statistical significance. These results are in line with previous studies where, in general, pregnancy rates have been found to be higher with Gonal-F® than with Metrodin HP® (Bergh et al., 1997
; Khalaf et al., 1998
), and may also be higher than with Puregon® (Brinsden et al., 1998
, 2000
). It has been suggested that rhFSH may have a directly beneficial effect on oocyte and embryo quality, leading to higher implantation rates (Out et al., 1996
), though this is purely a hypothesis which has not been supported by controlled clinical trial data. The overall number of deliveries was marginally higher in the Gonal-F® group (56) than in the Metrodin HP® group (43).
A recent meta-analysis reviewed the clinical pregnancy rates per cycle started using pooled data from 12 randomized trials comparing rhFSH (either Gonal-F® or Puregon®) with uhFSH in treatment cycles with IVF or ICSI (Daya and Gunby, 1999). In the overall meta-analysis, 1556 cases were allocated to rhFSH and 1319 to uhFSH. Overall, the clinical pregnancy rate per started cycle was found to be higher using rhFSH. The common odds ratio for clinical pregnancy rate per cycle started, obtained by pooling the data using a fixed-effects model, was 1.2 (95% confidence interval, 1.021.42, P = 0.03) in favour of the recombinant product. More specifically, the investigators were able to show that the pregnancy rate with Gonal-F® was statistically significantly higher than with uhFSH in IVF cycles.
In conclusion, Gonal-F® and Metrodin HP®, at a fixed starting dose of 150 IU/day for 6 days, were both highly effective in producing multiple follicular development, and were well tolerated in this group of women undergoing IVF or ICSI. Gonal-F® was shown to be more efficacious, producing a significantly higher level of follicular recruitment and a greater number of oocytes than Metrodin HP®, combined with a statistically lower consumption of FSH.
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Acknowledgments |
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Notes |
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7 To whom correspondence should be addressed at: Academic Hospital `Vrije Universiteit' Amsterdam, Department of Obstetrics and Gynaecology, Subdivision: Reproductive Endocrinology and Fertility/IVF Centre, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. E-mail: r.schats{at}azvu.nl
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References |
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Submitted on January 24, 2000; accepted on May 11, 2000.