A prospective, randomized clinical trial comparing 150 IU recombinant follicle stimulating hormone (Puregon®) and 225 IU highly purified urinary follicle stimulating hormone (Metrodin-HP®) in a fixed-dose regimen in women undergoing ovarian stimulation

Eric H.M. Hoomans1,5, Anders Nyboe Andersen2, Anne Loft2, Robert A. Leerentveld3, Anton A. van Kamp3 and Herbert Zech4

1 N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands, 2 University Hospital of Copenhagen (Rigshospitalet), The Fertility Clinic 5021, DK-2100 Copenhagen, Denmark, 3 Isala Clinics – location Sophia, Department of Obstetrics and Gynaecology, Dr C.A.Heesweg 2, 8025 AB Zwolle, The Netherlands and 4 Institut für in Vitro Fertilisierung und Embryo Transfer, Römerstrasse 2, A-6900 Bregenz, Austria


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A prospective, randomized, open, multicentre (n = 3) study was conducted to compare the efficacy and efficiency of a fixed daily dose of 150 IU (3x50 IU) recombinant follicle stimulating hormone (recFSH, Puregon®) and 225 IU (3x75 IU) highly purified urinary FSH (uFSH-HP, Metrodin-HP®) in women undergoing ovarian stimulation prior to in-vitro fertilization treatment. A total of 165 women were treated with FSH, 83 subjects with recFSH and 82 subjects with uFSH-HP. In the recFSH group a mean number of 8.8 oocytes were retrieved, compared with 9.8 in the uFSH-HP group (not statistically significant). In the recFSH group, a significantly lower total dose was required compared to the uFSH-HP group, 1479 versus 2139 IU, respectively (P < 0.0001; 95% confidence interval –747 to –572). Treatment with recFSH resulted in a significantly higher embryo development rate (69.6 versus 56.2%; P = 0.003) and more embryos accessible for the embryo freezing programme (3.3 versus 2.0; P = 0.02) compared to uFSH-HP. The vital pregnancy rate per cycle started was 30.2 versus 28.3% in the recombinant and urinary FSH group, respectively. It is concluded that treatment outcome of a fixed daily dose of 150 IU recFSH is comparable to a fixed daily dose of 225 IU uFSH-HP. However, a significantly lower total dose was needed in the recFSH group (nearly 700 IU less).

Key words: IVF/Metrodin-HP®/Puregon®/recombinant FSH/urinary FSH


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
At present, assisted reproduction technologies such as in-vitro fertilization (IVF) require ovarian stimulation to increase the number of oocytes, and hence the chances for a successful treatment outcome. Stimulation regimens generally include pituitary down-regulation followed by treatment with gonadotrophins (Porter et al., 1984Go).

Previously, gonadotrophin preparations derived from the urine of postmenopausal women were used. This source implies a number of disadvantages, such as batch-to-batch inconsistency, no absolute source control, dependence on large amounts of urine, low specific activity, low purity [<5% follicle stimulating hormone (FSH)], and the additional presence of luteinizing hormone (LH) (Vandervorst and Devroey, 1997Go). Purity has been improved by the introduction of highly purified urinary FSH preparations (uFSH-HP), of which 95% of the protein content consists of FSH and the residual LH content is <0.1 IU per 1000 IU FSH (Flamigni et al., 1994Go). A further major advance was achieved by the development of recombinant FSH (recFSH). Using a Chinese hamster ovary (CHO) cell line transfected with the genes encoding human FSH, a nearly 100% pure FSH preparation without any LH contamination was obtained (Puregon®, follitropin beta) (Olijve et al., 1996Go).

A large-scale clinical trial programme, comparing recombinant and urinary FSH, demonstrated an increased potency of recombinant FSH in terms of the number of oocytes retrieved, embryos obtained and the total gonadotrophin dose needed (Out et al., 1995Go). Due to its higher in-vivo bioactivity, it could be speculated that with lower dosages of recFSH at least similar results could be obtained compared with uFSH.

Therefore, a fixed dose of 150 IU recFSH (3x50 IU) was compared with 225 IU (3x75 IU) of uFSH-HP, in women undergoing ovarian stimulation, to test the hypothesis that a 50 IU ampoule of recFSH has a similar bioactivity as a 75 IU ampoule of uFSH-HP. A fixed-dose regimen was chosen to prevent any bias between the two treatment groups due to dose adaptations during the treatment cycle.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Patient selection
Infertile women were recruited according to the following inclusion/exclusion criteria. Inclusion criteria: patients had to be in good physical and mental health, at least 18 and at most 39 years of age at the time of screening, and have a body mass index between 18 and 29 kg/m2. All females needed to have normal ovulatory cycles with a mean length of 24–35 days, and their cause of infertility had to be potentially treatable by IVF. Exclusion criteria: women with infertility caused by endocrine abnormalities such as hyperprolactinaemia, polycystic ovarian syndrome and absence of ovarian function, and women with more than two previous IVF, gamete intra-Fallopian transfer (GIFT) or zygote intra-Fallopian transfer (ZIFT) attempts or previous IVF, GIFT or ZIFT attempts in which less than five oocytes were retrieved. Women with a history of (within 12 months) or current abuse of alcohol or drugs or women to whom investigational drugs were administered within 3 months prior to screening were not allowed to enter the study. Sperm characteristics defined as less than 10x106 sperm cells/ml, or <40% of sperm cells of normal morphology or motility were not allowed (World Health Organization, 1992Go).

Study design
This was a prospective, randomized, open, multicentre (n = 3) study comparing recombinant human FSH (Puregon®; N.V. Organon, Oss, The Netherlands, batch no. CP 096011) and Metrodin-HP® (urofollitropin; Ares-Serono, Geneva, Switzerland, batch no. 96C29). The objective of this study was to assess the efficacy and efficiency of a fixed daily dose of 150 IU recFSH (3x50 IU) with a fixed daily dose of 225 IU uFSH-HP (3x75 IU) in pituitary-suppressed women undergoing ovarian stimulation and IVF.

The study was performed between October 1996 and April 1998, in three specialized infertility clinics in Austria, Denmark and The Netherlands. The study was approved by the Ethics Committees of the study centres and each subject had given informed consent before participating in the study. The study was conducted in full compliance with the Declaration of Helsinki (and revisions), ICH Harmonized Tripartite Guidelines, Guideline for Good Clinical Practice, and local rules.

Pre-treatment with buserelin for pituitary down-regulation started on the first day of the menstruation or in the mid-luteal phase. When serum oestradiol concentrations were <200 pmol/l, and no ovarian cyst larger than 10 mm had developed, daily treatment was started with either three ampoules of 50 IU recFSH or three ampoules of 75 IU uFSH-HP, both given by s.c. injection. Treatment was continued until at least three follicles >=17 mm had developed, with a maximum of 3 weeks. Two 5000 IU ampoules of human chorionic gonadotrophin (HCG) (Pregnyl®, N.V. Organon, Oss, The Netherlands, batch nos. CP 096090 and 096032) were given i.m. to trigger ovulation. After oocyte retrieval and IVF, a maximum of three embryos was allowed to be replaced. Luteal phase support was given according to the preference of the treatment centre.

Assessments
At screening, demographic and other subject characteristics were obtained. The medical history was obtained and a general medical and gynaecological examination (including an ultrasound scan) was performed. In addition, routine blood biochemistry and haematology, sperm analysis, and serum prolactin and FSH measurements were carried out according to routine procedures of the individual study centres. At the start of FSH treatment and on the day of HCG injection, serum concentrations of oestradiol, progesterone, FSH, and LH were measured using locally applied assays, since none of these assessments were on primary endpoints and it was assumed that randomization of the study groups would result in an equal distribution of between-centre assay variations. Monitoring included frequent vaginal ultrasound investigations and oestradiol measurements.

Endpoints
The primary efficacy variable was the number of oocytes retrieved, and the primary efficiency outcome variable was the total dose of gonadotropin administered. Secondary variables included the number of follicles >=11, >=13, >=15 and >=17 mm at the day of HCG administration, levels of FSH, LH, progesterone and oestradiol at the day of HCG administration, treatment length, number of ampoules used, number of mature oocytes, the number and quality of transferable embryos, embryo development rate, clinical pregnancy rate, implantation rate, miscarriage rate, vital pregnancy rate, and the incidence of ovarian hyperstimulation syndrome (OHSS).

Classification of oocytes as mature or immature and embryos as type 1, 2, 3 or 4 was done according to previously published criteria (Staessen et al., 1989Go). Type 1, 2 and 3 embryos were considered transferable embryos. The embryo development rate was defined as the number of transferable embryos divided by the total number of oocytes incubated with semen. The implantation rate was defined as the number of gestational sacs seen on ultrasound examination divided by the total number of embryos replaced. All pregnancies assessed by a positive biochemical pregnancy test and/or by ultrasound (with or without proof of heart activity) were considered as clinical pregnancies. A pregnancy was considered vital when heart activity was present as assessed by ovarian ultrasound at 6 weeks post embryo transfer.

Sample size
With 80 subjects included in each treatment group and assuming a SD of 450 IU for the total dose of FSH administered and a SD of 6.4 oocytes for the total number of oocytes retrieved, a difference of 200 IU gonadotrophin dose and 2.8 oocytes could be detected between the two treatment groups with a power of 80% using a two-sided t-test with a significance threshold of 5%.

Statistical methods
For non-dichotomous variables, analysis of variance (ANOVA) was performed with centre and treatment group as fixed factors. If ANOVA was not applicable, Wilcoxon's rank sum test was performed. To communicate the Wilcoxon results in clinically meaningful entities, Cochran's method adjusted for centre was used. Dichotomous variables were analysed by Mantel–Haenszel statistics, adjusted for centre. A treatment difference was considered statistically significant if the calculated two-sided P value was <=0.05. An interaction between treatment and centre was considered statistically significant if the corresponding P value was <=0.10.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
A total of 169 women (recFSH: n = 84, uFSH-HP: n = 85) were randomized in three centres and 165 women started FSH treatment (recFSH: n = 83, uFSH-HP: n = 82). The number of subjects treated with FSH per centre was 18, 99 and 48 respectively.

Both treatment groups were comparable in demographic and infertility characteristics (Table IGo). The mean duration of infertility was 5.2 years for the recFSH group and 5.7 years for the uFSH-HP group, respectively. Tubal disease was the main cause of infertility in both treatment groups. All women were pituitary-suppressed (oestradiol <200 pmol/l) at the start of FSH treatment. Mean oestradiol concentrations prior to FSH treatment were 108 and 105 pmol/l in the recFSH group and uFSH-HP group, respectively. The corresponding FSH and LH concentrations were 4.2 versus 4.1 IU/l and 2.1 versus 2.3 IU/l respectively. Sperm analysis of the male partners revealed a mean sperm concentration of 138.8x106 spermatozoa/ml, with 56.8% normal morphology and 62.2% progressive motility (Table IIGo).


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Table I. Demographic and infertility characteristics
 

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Table II. Male infertility characteristics
 
Primary endpoints
The results of the main efficacy parameters are given in Tables III and IV.GoGo In the Puregon® 150 IU group, a mean number (adjusted for centre) of 8.8 oocytes were retrieved, compared to 9.8 in the Metrodin-HP® 225 IU group. The treatment difference of 1.0 was not statistically significantly different.


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Table III. Number of oocytes retrieved
 

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Table IV. Total follicle stimulating hormone dose administered (IU)
 
The total gonadotrophin dose needed to reach the criteria for HCG administration was significantly lower in the recFSH group compared to the uFSH-HP group [mean adjusted for centre: 1479 versus 2139 IU, P < 0.0001; 95% confidence interval –747 to –572].

Secondary parameters
Results of the secondary parameters are given in Table V. On the day of HCG administration, the mean number of follicles >=15 mm and >=17 mm were 8.2 versus 8.6 and 4.4 versus 4.6 in the recFSH group versus uFSH-HP group, respectively. Endocrinological parameters measured on the day of HCG administration showed significantly higher values for serum FSH (17.2 versus 8.8 IU/l, P < 0.001) in the uFSH-HP 225 IU group compared to the recFSH 150 IU group; serum oestradiol concentrations (6214 versus 6356 pmol/l) were comparable for both treatment groups.

On average, treatment length was similar in the two groups, 9.9 days in the recFSH group as compared to 9.6 days in the uFSH-HP group. As ampoules of 50 IU were used for recFSH and ampoules of 75 IU for uFSH-HP in a fixed daily dose regimen of 3 ampoules, the mean number of ampoules utilized was comparable for recFSH and uFSH-HP, being 29.6 and 28.6 respectively.

After oocyte retrieval, the number of mature oocytes recovered was 5.1 versus 4.8 in the recombinant and urinary FSH group respectively.

In total, 70 women treated with recFSH and 71 women treated with uFSH-HP received conventional IVF and had an embryo transfer. The number of embryos obtained (6.5 versus 5.9, P = 0.33) as well as the number of transferable embryos (6.4 versus 5.4, P = 0.09) appeared higher in the Puregon® group, although no statistical significance was reached. A significantly higher embryo development rate was found in the recFSH group compared to uFSH-HP (69.6 versus 56.2%, P = 0.003). The number of embryos replaced (2.0 in both groups) were the same for recFSH and uFSH-HP. One embryo more (3.3 versus 2.0, P = 0.02) was frozen in the recFSH group as compared to the uFSH-HP group.

Following embryo transfer, the clinical pregnancy rate was comparable between the recombinant and urinary FSH group, both per started cycle (38.6 versus 33.2%) and per embryo transfer (45.6 versus 37.9%). In total, seven women treated with the recFSH and four women treated with uFSH-HP had a miscarriage (9.9 and 5.7% per embryo transfer, respectively).

The vital pregnancy rates per cycle started and per embryo transfer were 30.2 versus 28.3% and 35.6 versus 32.3% in the recFSH and uFSH-HP groups, respectively (not statistically significant). The implantation rate was 23% in both treatment groups.

Cycle cancellations
Of the 83 women that started recFSH treatment, 81 had an oocyte retrieval (98%) and 70 an embryo transfer (84%). Eighty (98%) women out of 82 that started uFSH-HP treatment had an oocyte retrieval and 71 (87%) had an embryo transfer.

Overall, the cancellation rate was comparable between both treatment groups, being 15.7% and 13.4% for recombinant and urinary FSH, respectively. In the recFSH group, insufficient ovarian response (n = 3), no fertilization (n = 6), risk of hyperstimulation (n = 1) and other (n = 3) were reported. In the uFSH-HP group, the reasons for cancellation were insufficient ovarian response (n = 2), no fertilization (n = 4), risk of hyperstimulation (n = 1) and other (n = 4).

Safety
Five hospitalizations due to serious adverse events were noted in the recFSH group, compared to three in the uFSH-HP group. In the recFSH group, two patients had an ectopic pregnancy, one a torsion of her left ovary which was extirpated, one patient had a miscarriage requiring hospitalization and the remaining one was caused by OHSS. In the uFSH-HP group there was one patient with an ectopic pregnancy and two patients were hospitalized because of OHSS. Drug-related adverse events were reported in 8.4% of women treated with recFSH compared to 12.0% of women treated with uFSH-HP. Local tolerance problems at the injection site occurred in one patient receiving recFSH.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this prospective, randomized study it was shown that a daily dose of 150 IU recFSH (3x50 IU) was as effective as a daily dose of 225 IU uFSH-HP (3x75 IU). The number of oocytes retrieved, treatment duration and the number of ampoules used were similar in both groups. However, a significantly lower total dose was needed in the recFSH group (nearly 700 IU less). These findings support the higher bioactivity of recFSH compared to uFSH-HP as reported before (Out et al., 1995Go)

Treatment with recFSH resulted in a significantly higher embryo development rate (69.6% versus 56.2%; P = 0.003) and more embryos accessible for the embryo freezing programme (3.3 versus 2.0; P = 0.02) compared to uFSH-HP. This can not be attributed to differences in sperm characteristics between couples treated with recFSH or uFSH-HP since these were comparable for both groups. The observed difference in embryo development rate and the number of embryos available for the freezing programme can be explained by the higher proportion of mature to immature oocytes recovered following recFSH treatment compared to uFSH-HP and suggests a favourable impact of recFSH on oocyte and embryo development. This direct beneficial effect was also suggested in a combined analysis of three follitropin beta versus urinary gonadotrophin trials in IVF. In this analysis, a significantly higher pregnancy rate directly following fresh transfer was seen with recFSH with a similar number of embryos replaced in both groups (Out et al., 1997Go). In a study comparing another recFSH and highly purified uFSH-HP using a starting dose of 150 IU for the first 6 days, it was also concluded that recFSH is more effective than uFSH-HP (Bergh et al., 1997Go).

It remains to be answered why recFSH is more effective compared to uFSH-HP. Possible factors which might play a role comprise subtle differences at the carbohydrate level, differences in pharmaceutical formulation, the presence of contaminating proteins in products originating from urine or differences in isohormone composition. With respect to the latter, the isohormone profile of natural FSH is known to vary in relation to sex, stage of the menstrual cycle, stage of life, and origin (pituitary, serum or urinary) (Ulloa-Aguirre et al., 1995Go). Recombinant FSH displays a large proportion of relatively basic isohormones, known to exhibit a higher in-vitro bioactivity compared to acidic isoforms (Matikainen et al., 1994Go). In contrast, urine-derived FSH is known to exhibit a more acidic isohormone profile with a lower in-vitro potency and a longer half-life (Lambert et al., 1995Go; De Leeuw et al., 1996Go; Lambert et al., 1998Go). This seems related to the fact that women in their postmenopausal years predominantly produce acidic isoforms of FSH (Anobile et al., 1998Go). Compared to urinary FSH, highly purified uFSH appears to be even more acidic in nature (Lambert et al., 1995Go). This longer half-life is illustrated by the nearly twice as high immunoreactive serum FSH levels of urinary FSH compared to recFSH on the day of HCG administration, whereas, based on the difference in daily dosage, concentrations only 1.5 times as high could be expected in the uFSH-HP group. This also shows that there is no clear relationship between circulating immunoreactive serum FSH levels and ovarian response.

In this prospective randomized study it should be kept in mind that the population studied in this controlled setting might differ from a general IVF population. However, this certainly does not invalidate the comparison between the drugs and doses tested in this study.

Whether a similar treatment outcome would have been achieved using a lower dose of uFSH-HP cannot be completely ruled out. A recent study with recFSH showed that a similar outcome can be achieved using 150 IU or 225 IU of recFSH in normo-responder women, implying that beyond a certain threshold a higher dose does not result in a better outcome (Camier et al., 1998Go). However, another study comparing 100 IU and 200 IU of recombinant FSH (follitropin beta) clearly demonstrated a dose–response relationship in terms of oocytes and embryos obtained, although the clinical pregnancy rate was similar in the two dose groups (Out et al., 1999Go). In a recently conducted study, comparing 150 IU and 250 IU of recFSH (follitropin beta) in older women (30–39 years) undergoing IVF/intracytoplasmic sperm injection it was shown that the higher dose regimen did not result in a better outcome (manuscript in preparation). These data merely suggest that in normo-responder patients there is a certain threshold window for optimal multiple follicular development. Further research is needed to establish whether certain prognostic markers can enable a rational starting dose decision.

Recently, there has been much debate about how new developments in IVF treatment can contribute to a better outcome. In line with this, emphasis is frequently placed on optimizing instead of maximizing IVF treatment (`friendly IVF') and on introducing dosing regimens which lower the risk of ovarian stimulation-associated side-effects, especially OHSS and multiple pregnancies (Olivennes and Frydman, 1998Go). Given the increased bioactivity of recFSH, it is highly relevant that certain patients, especially those prone to these risks, might benefit from doses even lower than 150 IU (Devroey et al., 1998Go; Out et al., 1999Go).

From this study, it is apparent that the lower total gonadotrophin exposure with recFSH compared to uFSH-HP also has pharmaco-economical implications in terms of lowering the total treatment costs for treatment with recFSH.

In conclusion, this study has demonstrated that treatment outcome of a fixed daily dose of 150 IU of recFSH is comparable to a fixed daily dose of 225 IU of uFSH-HP. The number of ampoules used was equal for both groups, resulting in a significantly lower dose needed (nearly 700 IU less) with recFSH.


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Table V. Secondary parameters (mean adjusted for centre)
 

    Acknowledgments
 
The authors would like to thank R.Dijkstra, E.Hulskotte, J.van Kuijk, M.van Eeden, P.Knoblauch, M.Kouba and J.Mulders, all from Organon, and K.Rombouts from Quintiles Benelux, for their contribution to this study.


    Notes
 
5 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anobile, C.J., Talbot, J.A., McCann, S.J. et al. (1998) Glycoform composition of serum gonadotrophins through the normal menstrual cycle and in the post-menopausal state. Mol. Hum. Reprod., 4, 631–639.[Abstract]

Bergh, C., Howles, C.M., Borg, K. et al. (1997) Recombinant human follicle stimulating hormone (r-hFSH, Gonal-F) versus highly purified urinary FSH (Metrodin HP) — Results of a randomized comparative study in women undergoing assisted reproductive techniques. Hum. Reprod., 12, 2133–2139.[Abstract]

Camier, B., the French multicentre trialists, Howles, C.M. and Truong, F. (1998) A multicentre, prospective, randomized study to compare a low-dose protocol versus conventional administration of recombinant human follicle stimulating hormone (Gonal-F®) in normo-responder women undergoing IVF/ICSI. Hum Reprod., 13 (Suppl.), Abstract P-058, 159.

De Leeuw, R., Mulders, J., Voortman, G. et al. (1996) Structure–function relationship of recombinant follicle stimulating hormone (Puregon®). Mol. Hum. Reprod., 2, 361–369.[Abstract]

Devroey, P., Tournaye, H., Van Steirteghem, A. et al. (1998) The use of a 100 IU starting dose of recombinant FSH (Puregon®) in in-vitro fertilization. Hum. Reprod., 13, 565–566.[Free Full Text]

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Matikainen, T., Leeuw de, R., Mannaerts, B. and Huthaniemi, I. (1994) Circulating bioactive and immunoreactive recombinant follicle stimulating hormone (Org 32489) after administration to gonadotropin-deficient volunteers. Fertil. Steril., 61, 62–69.[ISI][Medline]

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Out, H.J., Lindenberg, S., Eldar-Geva, T. et al. (1999) A prospective, randomized, double-blind clinical trail to study the efficacy and efficiency of a fixed daily dose of recombinant follicle stimulating hormone (Puregon®) in women undergoing controlled ovarian hyperstimulation. Hum. Reprod., 14, 662–627.

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Submitted on April 23, 1999; accepted on July 1, 1999.