A prospective, randomized comparison of two starting doses of recombinant FSH in combination with cetrorelix in women undergoing ovarian stimulation for IVF/ICSI

M. Wikland1,4, C. Bergh2, K. Borg1, T. Hillensjö1, C.M. Howles3, A. Knutsson2, L. Nilsson2 and M. Wood1

1 Fertilitetscentrum, Carlander's Hospital, Göteborg, Sweden, 2 The IVF Unit, Department of Obstetrics/Gynecology, Sahlgrenska University Hospital, Göteborg, Sweden and 3 Serono International, 15bis Chemin des Mines, 1202 Geneva, Switzerland


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: A prospective randomized study was carried out in two centres to compare the number of oocytes retrieved after two different starting doses of recombinant human FSH (rhFSH) (Gonal-F®) in women undergoing ovarian stimulation for IVF/intracytoplasmic sperm injection (ICSI) cycles using the multiple dose regimen of the gonadotrophin-releasing hormone (GnRH) antagonist cetrorelix (Cetrotide®) to prevent induction of the premature LH surge. METHODS: Sixty women were randomized to receive rhFSH 150 IU (`low'), and 60 women to receive rhFSH 225 IU (`high') as the starting dose for the first 5 days of stimulation. From stimulation day 6 and onwards, including the day of human chorionic gonadotrophin (HCG) administration, the women received 0.25 mg of cetrorelix as a daily dose. The primary endpoint was the number of oocytes retrieved. RESULTS: The mean number (± SD) of oocytes was 9.1 ± 4.4 and 11.0 ± 4.6 in the `low' and `high' groups respectively (P = 0.024). The mean number of 75 IU ampoules of rhFSH was significantly lower in the `low' group (23.0 ± 6.3 versus 30.5 ± 5.6, P < 0.0001). The ongoing pregnancy rate per started cycle and per embryo transfer were 25.9 and 28.8% versus 25.4 and 26.8% respectively in the `low' and `high' rhFSH groups (P = NS). CONCLUSIONS: When using a starting dose of 225 IU rhFSH combined with the multiple dose of 0.25 mg cetrorelix from stimulation day 6, significantly more oocytes were obtained than with a starting dose of 150 IU rhFSH.

Key words: assisted reproduction/GnRH antagonist/ICSI/IVF/ovarian stimulation/recombinant FSH


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
It is well established that elevated concentrations of LH are associated with decreased fertility rates in women undergoing ovarian stimulation before IVF procedures (Stanger and Yovich, 1985Go; Howles et al.1986Go, 1987Go). Consequently, the use of long protocol gonadotrophin-releasing hormone (GnRH) agonists to down-regulate the pituitary has become routine practice in most centres (Felberbaum and Diedrich, 1999Go; Huirne et al.1999Go).

Although GnRH antagonists were developed at the same time as the GnRH agonists, the early compounds induced histamine release and their clinical use was limited, therefore, by allergic side-effects ( Hahn et al.1985Go; Reissmann et al.1995Go). However, the development of `third-generation' GnRH antagonists appears to have overcome this problem (Felberbaum and Diedrich, 1999Go). These antagonists have potential advantages over the GnRH agonists for the suppression of LH secretion during ovarian stimulation protocols. In particular, they can suppress the secretion of gonadotrophins within a few hours of administration. Since the effect is due to a dose-dependent competitive inhibition of the GnRH receptors, it can be reversed by agonistic GnRH analogues (Felberbaum and Diedrich, 1999Go). The relatively rapid onset and offset of effect allows the use of GnRH antagonists to be limited to that part of the ovarian stimulation cycle in which the suppression of a premature LH surge is necessary. It therefore offers the potential for more convenient treatment of women undergoing ovarian hyperstimulation protocols.

Several studies with the GnRH antagonist cetrorelix have now demonstrated its efficacy in assisted reproductive treatment (ART) cycles. A dose-finding study found that a single daily dose of 0.25 mg cetrorelix from day 6 of ovarian stimulation with gonadotrophins to the day of human chorionic gonadotrophin (HCG) administration, safely and effectively prevented premature LH surges. The pregnancy rate in women treated with this protocol was 30% ( Albano et al.1996Go, 1997Go). In addition, a randomized, comparative study found that a greater proportion of women who started treatment in cycles in which 0.25 mg cetrorelix daily was used to suppress LH secretion, reached the day of HCG injection compared with those who used buserelin, 600 µg daily, according to the long protocol. In addition, significantly fewer small follicles were seen in the cetrorelix group, and the incidence of severe ovarian hyperstimulation stimulation syndrome (OHSS) was also significantly lower in this group compared with those who received buserelin (Felberbaum and Diedrich, 1999Go).

Most studies of GnRH antagonists in ART cycles have been performed using FSH at a starting dose of 150 IU daily, and a somewhat lower number of oocytes have been retrieved than in agonist cycles (Borm and Mannaerts, 2000Go). There are no randomized controlled studies which have compared the effect of different starting doses of FSH when a GnRH antagonist is used to prevent premature LH surges.

The objective of the present study was to investigate whether 150 IU or 225 IU of recombinant human FSH (rhFSH) resulted in a similar number of oocytes in IVF/intracytoplasmic sperm injection (ICSI) cycles using a multiple dose regimen of cetrorelix to avoid premature LH surges.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
A total of 120 women was recruited between September 15 and December 20, 1999. Women were eligible for inclusion if they were between 20 and 39 years of age, had regular menstrual cycles of 25–32 days, two normal ovaries and a normal uterine cavity as judged by ultrasonography, and were being treated for infertility due to tubal, male or idiopathic factors or mild endometriosis. In addition, the women should not have undergone more than three previous ART attempts, or had any ovarian stimulation in the 3 months prior to study entry. All patients were required to give written informed consent prior to study entry. Exclusion criteria included body mass index (BMI) >30 kg/m2, previous history of severe OHSS, previous failure of IVF or ICSI treatment due to poor response to gonadotrophin therapy (fewer than three mature follicles) or to ICSI failure. Further exclusion criteria were a history of abnormal gynaecological bleeding of undetermined origin, any contraindication to pregnancy, or the presence of a clinically significant systemic disease.

Study design
This was a prospective, randomized, parallel group, multicentre study (The IVF Unit, Sahlgrenska University Hospital, Göteborg, Sweden and Fertilitetscentrum, Carlander's Hospital, Göteborg, Sweden) to assess the efficacy and safety of two different starting doses of rhFSH (follitropin {alpha}, Gonal-F®; Serono Laboratories, Aubonne, Switzerland) in combination with a fixed daily dose of the GnRH antagonist cetrorelix (Cetrotide®; Asta Medica, Frankfurt, Germany) from day 6 of stimulation in a natural cycle in women undergoing ovulation induction for ART procedures.

The treatment protocol is summarized in Figure 1Go. Ovarian stimulation was by rhFSH (supplied in 75 IU ampoules), and patients were randomized to receive a starting dose of either 150 IU (`low') or 225 IU (`high') rhFSH beginning on day 2 or 3 of the menstrual cycle. The dose was fixed for the first 5 days of stimulation. Suppression of LH was provided by daily injection between 08:00 and 12:00 of 0.25 mg cetrorelix s.c. from day 6 of the stimulation cycle until the day of HCG administration. Ovarian response was monitored by ultrasound on day 6 and on days 9 and 10, with additional ultrasound scans when needed. From day 6 of stimulation, the dose of rhFSH could be altered by increasing or decreasing the dose by one or two ampoules of 75 IU, as judged by the clinician and based on the number and size of developing follicles. When the three largest follicles measured >=18 mm, final ovarian maturation was triggered with a single s.c. injection of HCG 10 000 IU (Profasi®; Serono Laboratories). Oocytes were retrieved 34–38 h after injection of HCG and were fertilized in vitro according to standard procedures.



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Figure 1. Summary of the treatment protocol for women undergoing ovarian stimulation with recombinant human FSH (rhFSH; Gonal-F®) in cycles using multiple dose cetrorelix (Cetrotide®) to suppress LH secretion. OPU = oocyte pick-up (retrieval).

 
A maximum of two embryos were replaced 48–72 h after oocyte retrieval. Three patients requested single embryo transfer in order to avoid the risk of multiple pregnancy. Luteal support was provided according to the clinic's usual practice, and consisted of either micronized progesterone 1200 mg intravaginally from the day of oocyte retrieval, or progesterone in oil, 50 mg daily, given i.m. When there were fewer than 15 follicles >12 mm diameter on the day of HCG administration, patients in one centre received HCG (Profasi®) 1225 IU every 3–4 days, starting on the day of embryo transfer.

Treatment was discontinued in any patient who had an excessive ovarian response which was considered to indicate a risk of OHSS or if there was any serious adverse reaction to treatment.

Parameters evaluated
The primary endpoint was the number of oocytes retrieved from women who received HCG. A number of secondary endpoints were also evaluated. Endpoints related to stimulation parameters were: the total number of ampoules of rhFSH (75 IU) required for each cycle in which HCG was administered; the number of patients who required an increase in the dose of rhFSH; the number of days of rhFSH treatment; and the duration of cetrorelix treatment and the number of follicles >10 mm on the day of HCG administration. Endpoints relating to treatment outcome included: the number of two-pronuclear (2PN) oocytes; the number of cleaved embryos; the number of embryos which were replaced; the number of cryopreserved embryos; the total number of good quality embryos as judged at day 2 or 3 after insemination (i.e. the number of transferred embryos plus the number of frozen embryos); and the number of pregnancies.

Assessments
Follicular size and number were measured by vaginal ultrasonography. The follicular size was given as the mean of the two largest diameters in the same plane and perpendicular to each other ( Wikland et al.1994Go).

Blood samples for retrospective analysis of oestradiol and LH were collected on day 6 of FSH stimulation prior to the injection of cetrorelix. Additional measurements of oestradiol were performed when it was judged to be important for clinical evaluation of the ovarian response.

The hormone assays were performed by similar (but not identical) methods at the two centres. At the Fertilitetscentrum, oestradiol and LH were determined by Immulite Automated Analyser (DPC, Los Angeles, California, USA) using a solid-phase chemoilluminescent enzyme immunometric assay. At the IVF unit of the Sahlgrenska Hospital, oestradiol and LH concentrations were measured using the AxSystem (Abbott Laboratories, Diagnostics Division, Abbott Park, Illinois, USA), which uses microparticle enzyme immunoassay (MEIA) technology. Oestradiol concentrations were expressed in pmol/l, and LH in IU/l.

Oocyte maturity was assessed according to established criteria, as germinal vesicle (GV) stage, metaphase I, metaphase II, atretic or unknown. Classification of embryos was performed according to published criteria (Steer et al., 1992Go).

Sample size
The clinical equivalence of starting doses of rhFSH 150 and 225 IU was to be declared if the limits of the 90% confidence interval (CI) for the difference in the mean number of oocytes retrieved in the two groups included ±3 oocytes. To demonstrate this equivalence with a probability of 95%, using a two-sided 90% CI interval for difference in the mean number of oocytes retrieved, 54 evaluable patients per group were required (at least 120 patients in total to allow for any drop-outs).

Statistical analyses
The analysis was performed on an intention to treat (ITT) basis, taking into account all randomized patients who followed the protocol. All computations were performed using version 6.10 of SAS for Windows (SAS Institute, Cary, NC, USA). Descriptive statistics (mean ± SD) have been reported for efficacy criteria. Analysis of variance (ANOVA) was performed for all comparisons on the raw data. Models with terms for centre and treatment were fitted; no interactions were included.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patient demographics
Each study centre recruited a total of 60 patients, who were randomized equally between the two treatment groups (i.e. 30 patients per group). In the `low' FSH group, one patient became pregnant without treatment between randomization and start of FSH. One patient in each group was excluded because of a protocol violation related to the randomized dose of rhFSH. After exclusion of these patients, there were 117 evaluable patients.

Demographic characteristics are shown in Table IGo. There were no significant differences between the two treatment groups with respect to their demographic characteristics. The most common cause of infertility in both groups was male factor, accounting for 28 (48.3%) of patients in the `low' rhFSH group and 30 (50.8%) of those in the `high' rhFSH group.


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Table I. Demographic characteristics of patients
 
Stimulation characteristics
In the `low' FSH group, 57 patients received HCG and had oocyte retrieval performed, but one patient did not receive HCG due to poor follicular development. In the `high' FSH group, all 59 patients received HCG and had oocyte retrieval. In the `low' FSH group, four patients did not reach embryo transfer due to absence of fertilization or poor embryo quality; the corresponding number in the `high' FSH group was three.

The results of stimulation are presented in Table IIGo. The average duration of treatment with rhFSH and cetrorelix did not differ between the groups, nor did the average LH concentration and number of follicles on day 6. The number of follicles falling into different size categories were compared in the two groups on day 6 and 9 of FSH. On day 6 there were more follicles between 10–14 mm in the `high' FSH group. By stimulation day 9, this trend now achieved statistical significance, with there also being one more follicle in the 15–17 mm category in the high-dose group. Overall, the total number of follicles >=10 mm on day 6 was 5.3 ± 3.1 versus 6.3 ± 3.3 (P = NS) in the two groups, and for follicles >=15 mm the corresponding values were 0.3 ± 0.7 versus 0.4 ± 0.8 (P = NS). On day 9, the number of follicles >=10 mm was 8.8 ± 4.3 versus 11.6 ± 4.6 (P = 0.012) and >=15 mm, 3.3 ± 2.5 versus 4.7 ± 2.7 (P = 0.024) in the two groups respectively. Also, on the day of HCG administration the number of large follicles (>10 mm) and the concentration of oestradiol were higher in the `high' rhFSH group. As might be expected, the total amount of rhFSH was higher in the `high' FSH group than in the `low' FSH group.


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Table II. Stimulation characteristics
 
Most importantly, the mean number of oocytes retrieved (primary endpoint), was significantly higher (P = 0.0235) for the `high' rhFSH group (11.0 ± 4.6) than for the `low' rhFSH group (9.1 ± 4.4); when considering the 90% CI for the treatment difference [–3.3 to –0.5], the predefined limits of ±3 do not fall entirely within the interval, and so equivalence has not been shown.

In addition, an analysis was made on the number of oocytes retrieved in relation to the day of starting stimulation with rhFSH (day 2 or 3 of the menstrual cycle). For the `low' rhFSH group, the mean numbers of oocytes were for day 2 and for day 3 were 9.1 ± 3.9 and 9.1 ± 4.7 respectively. For the `high' rhFSH group, the mean number of oocytes were 11.2 ± 4.7 and 10.8 ± 4.7 respectively. Overall, the effect of changing from day 2 to day 3 was not significant.

In the `low' rhFSH group, 28 patients completed the study without any change in dose, while three patients had their dose decreased and 26 had an increase (Table IIIGo). The corresponding numbers for the `high' rhFSH group were 35 unchanged, seven decreased and 17 increased.


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Table III. Cycles in which an adjustment was made in the dose of rhFSH
 
Fertilization and treatment outcomes
The most common method of fertilization was by ICSI, which was used in 30 (52.6%) cycles in the `low' rhFSH group and in 32 (54.2%) cycles in the `high' rhFSH group, accounting for 53% of all cycles in which HCG was administered. Conventional IVF, or a combination of IVF and ICSI (50% of oocytes fertilized by each procedure), was used in 20 (35.1%) and seven (12.3%) cycles respectively in the `low' rhFSH group, and in 19 (32.2%) and eight (13.6%) cycles respectively in the `high' rhFSH group.

Embryological characteristics are shown in Table IVGo. The average number of normally fertilized oocytes on day 1 was 5.7 ± 3.2 in the `low' rhFSH group and 7.1 ± 3.2 in the `high' rhFSH group (P = 0.025). The mean fertilization rates, calculated per patient, were 62.9 ± 24.3% and 67.3 ± 21.3% respectively (P = NS). Within the `low' rhFSH group, there was a mean of 5.2 ± 3.2 cleaved embryos on day 2, and the corresponding number for the `high' rhFSH group was 6.6 ± 3.1 (P = 0.014).


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Table IV. Embryological characteristics
 
A mean of 1.6 ± 2.2 embryos were cryopreserved in the `low' rhFSH group, and 2.1 ± 2.3 in the `high' rhFSH group (P = NS). The total number of good quality embryos (transferred + cryopreserved) per cycle was also comparable between the two groups (3.3 ± 2.5 and 3.9 ± 2.4 for the `low' and `high' rhFSH groups respectively).

Embryo transfer was performed in 53 patients in the `low' rhFSH group, and in 56 patients in the `high' rhFSH group, with averages of 1.9 ± 0.3 and 2.0 ± 0.2 embryos being transferred respectively (P = NS).

The clinical outcome is shown in Table VGo. The ongoing pregnancy rate in the `low' rhFSH group was 25.9% per started cycle, 26.3% per oocyte retrieval, and 28.8% per embryo transfer. In the `high' rhFSH group, the ongoing pregnancy rate was 25.4% per started cycle, 25.4% per oocyte retrieval, and 26.8% per embryo transfer. There were four (19.1%) miscarriages (preclinical and clinical) in the `low' rhFSH group and six (25%) in the `high' rhFSH group. Thus, the ongoing pregnancy rate (>12 weeks gestation) did not differ between groups, nor did the implantation rates (24 and 26% respectively).


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Table V. Outcome of cycles
 
There were two (11.8%) extrauterine pregnancies in the `low' rhFSH group, and three (16.7%) in the `high' rhFSH group. One case of moderate OHSS (in the `high' rhFSH group) requiring hospitalization occurred among a total of 117 cycles (<1%) in which HCG was administered.

Tolerability
The combination of rhFSH and cetrorelix was well tolerated by both groups of patients, and no systemic reactions to cetrorelix were reported. However, minor local reactions at the cetrorelix injection site were reported by 82 of the 117 patients. These reactions included swelling (45%), itching (25%) and redness and pain (both 15%), all of which were transient in nature.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The results of this study have shown that rhFSH at a starting dose of either 150 IU or 225 IU is effective and well tolerated for follicular stimulation before IVF/ICSI in cycles in which a multiple dose regimen of cetrorelix is used to suppress premature LH surges. An average of 10 oocytes were collected, and the quality of oocytes and embryos were excellent. Furthermore, no serious side effects were seen, and there was only one case of moderate OHSS.

The primary endpoint of the study was the number of oocytes retrieved, and a statistical significant difference was found (11.0 versus 9.1; P = 0.024). In a previous multicentre trial using multiple dose cetrorelix regimen combined with HMG 150 IU as a starting dose, an average of 11.6 oocytes was reported ( Albano et al.2000Go). In other multicentre studies using ganirelix, a starting dose of 150 IU rhFSH resulted in an average of 9.1 oocytes (Borm and Mannaerts, 2000Go) and a starting dose of 225 IU rhFSH resulted in 11.6 oocytes (Fulker et al., 2001Go). The number of oocytes retrieved in the current study compares well with the results of these multicentre trials.

Different starting doses of rhFSH have also been studied in cycles with down-regulation using the long agonist protocol. In one recent study (Out et al., 2000Go), no significant difference in the number of oocytes retrieved was found when starting doses of 150 IU or 250 IU rhFSH were compared (9.1 versus 10.6 oocytes), while in another study in which a starting dose of 100 IU was compared with 200 IU of rhFSH, the higher dose resulted in significantly more oocytes (10.6 versus 6.2) (Out et al., 1999Go).

It is generally accepted that, within certain limits, a higher number of oocytes predicts better pregnancy outcome in IVF/ICSI, since it reflects a good ovarian reserve and a more optimal stimulation. Additionally, analysis of the UK Human Fertilisation and Embryology Authority (HFEA) database has clearly determined that the higher the number of oocytes fertilized, the greater the likelihood of a livebirth (Templeton and Morris, 1998Go). Even though many centres currently limit the number of embryos for transfer to one or two to avoid the risks associated with multiple pregnancy (Hazekamp et al., 2000Go), a high yield of oocytes is important since it enables: (i) a better selection of high-quality embryos and blastocysts (Scholtes and Zeilmaker, 1998Go) for transfer; and (ii) more embryos for cryopreservation. Again, it was demonstrated (Templeton and Morris, 1998Go) that from the UK analysis, when more than four oocytes were fertilized, there was no increase in the birth rate for women receiving three transferred embryos as compared with those receiving two, but there was a considerable increase in the rate of multiple births when three embryos were transferred.

The higher starting dose led to a slightly higher number of follicles recruited (although not significant), and follicles began to appear in the >=15 mm category by stimulation day 6. These results indicate that the initiation of 0.25mg cetrorelix daily should be performed by stimulation day 6 so as to avoid an untimely LH surge. Additionally, adopting such a fixed-day regimen will also minimize the need for repeated ultrasound scans.

The small difference in number of oocytes (1.9) obtained in the `low' and `high' FSH groups was not judged to be of clinical significance, and neither was the difference found in the number of usable embryos. However, it must be emphasized that the power to detect difference in clinical outcome was low due to the limited number of patients included.

In conclusion, it appears that cetrorelix represents an improvement for women undergoing ovarian stimulation before IVF/ICSI. Restriction of injections of the antagonist to a few days within the period of rhFSH administration greatly simplifies the treatment protocol, and also reduces the overall exposure to medication. A starting dose of rhFSH of 150 IU or 225 IU may be of minor clinical importance. However, a lower dose of rhFSH should perhaps be considered with regard to the cost for the treatment cycle.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank Dr Wolfgang Hamm at ASTA Medica, Frankfurt, Germany for constructive criticism of the study, and David Warne and Sebastian Gerin at Serono International, Geneva, Switzerland for help with the statistics and constructive discussion and criticism of the manuscript. They also thank ASTA Medica and Serono International for financial support of the study.


    Notes
 
4 To whom correspondence should be addressed at: Fertilitetscentrum, Box 5418, S-402 29, Göteborg, Sweden.E-mail: matts.wikland{at}fcivf.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Albano, C., Smitz, J., Camus, M. et al. (1996) Hormonal profile during the follicular phase of in cycles stimulated with a combination of human menopausal gonadotrophin and gonadotrophin-releasing hormone antagonist (cetrorelix). Hum. Reprod., 11, 2114–2118.[Abstract]

Albano, C., Smitz, J., Camus, M. et al. (1997) Comparison of different doses of gonadotrophin-releasing hormone antagonist cetrorelix during controlled ovarian hyperstimulation. Fertil. Steril., 67, 917–922.[ISI][Medline]

Albano, C., Felberbaum, R.E., Smitz, J. et al. (2000) Ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the luteinizing hormone-releasing hormone (LHRH)-antagonist cetrorelix and the LHRH-agonist buserelin. European Cetrorelix Study Group. Hum. Reprod., 15, 526–531.[Abstract/Free Full Text]

Borm, G. and Mannaerts, B. (2000) Treatment with the gonadotrophin-releasing hormone antagonist ganirelix in women undergoing ovarian stimulation with recombinant follicle stimulating hormone is effective, safe and convenient: results of a controlled, randomized multicentre trial. Hum. Reprod., 15, 1490–1498.[Abstract/Free Full Text]

Felberbaum, R.E. and Diedrich, K. (1999) The use of GnRH antagonists in assisted reproduction technologies. In Lunenfeld, B. (ed.), GnRH Analogues. The State of the Art at the Millennium. The Parthenon Publishing Group, New York, pp. 47–63.

Hahn, D.W., McGuire, J.L., Vale, W.W. and Rivier, J. (1985) Reproductive/endocrine and anaphylactoid properties of an LHRH antagonist (ORF-12860). Life Sci., 37, 505–514.[ISI][Medline]

Hazekamp, J., Bergh, C., Wennerholm. U.B. et al. (2000) Avoiding multiple pregnancies in ART: consideration of new strategies. Hum. Reprod.,15, 1217–1219.[Abstract/Free Full Text]

Howles, C.M., Macnamee, M.C., Edwards, R.G. et al. (1986) Effect of high tonic levels of luteinizing hormone on outcome of in vitrofertilisation. Lancet, ii, 521–522.

Howles, C.M., Macnamee, M.C. and Edwards, R.G. (1987) Follicular development and early luteal function of conception and non-conceptional cycles after human in vitro fertilization: endocrine correlates. Hum. Reprod., 2, 17–21.[Abstract]

Huirne, J.A.F., Lambalk, C.B., Janssens, R. and Schoemaker, J. (1999) GnRH agonists versus antagonist, where are we today? In Ben-Rafael, Z. and Shoham, Z. (eds), The First Congress on Controversies in Obstetrics, Gynecology and Infertility. Monduzzi Editore, Bologna, Italy, pp. 77–81.

Out, H.J., Lindenberg, S., Mikkelsen, A.L. et al. ( 1999) A prospective, randomised, double-blind clinical trial to study efficacy of a fixed dose of recombinant follicle stimulating hormone (Puregon) in woman undergoing ovarian stimulation. Hum. Reprod., 14, 622–627.[Abstract/Free Full Text]

Out, H.J., Bratt, D.D., Linsten, B. et al. (2000) Increasing the daily dose of recombinant follicle stimulating hormone (Puregon) does not compensate for the age-related decline in retrievable oocytes after ovarian stimulation. Hum. Reprod., 15, 29–35.[Abstract/Free Full Text]

Reissmann, T., Felberbaum, R., Diedrich, K. et al. (1995) Development and applications of luteinizing hormone-releasing hormone antagonists in the treatment of infertility: an overview. Hum. Reprod., 10, 1974–1981.[Abstract]

Scholtes, M.C. and Zeilmaker, G.H. (1998) Blastocyst transfer in day-5 embryo transfer depends primarily on the number of oocytes retrieved and not on age. Fertil. Steril., 69, 78–83.[ISI][Medline]

Stanger, J.D. and Yovich, J.L. ( 1985) Reduced in-vitro fertilisation of human oocytes from patients with raised basal luteinising hormone levels during the follicular phase. Br. J. Obstet. Gynaecol., 92, 385–393.[ISI][Medline]

Steer, C., Mills, C. and Tan, S.L. (1992) The cumulative embryo score a predictive embryo scoring technique to select the optimal numbers of embryos to transfer in an in vitro fertilization and embryo transfer program. Hum. Reprod., 7, 117–119.[Abstract]

Templeton, A. and Morris, J.K. (1998) Reducing the risk of multiple births by transfer of two embryos after in vitro fertilization. N. Engl. J. Med., 339, 573–577.[Abstract/Free Full Text]

Fulker, M., Grifo, J., Leader, A. et al. (2001) Efficacy and safety of ganirelix acetate (AntagonTN/Orgalutran®) versus leuprolide acetate in women undergoing controlled ovarian hyperstimulation. Fertil. Steril., 75, 38–45.[ISI][Medline]

Wikland, M., Borg, J., Hamberger, L. et al. ( 1994) Simplification of IVF: minimal monitoring and the use of subcutaneous highly purified FSH administration for ovulation induction. Hum. Reprod., 9, 1430–1436.[Abstract]

Submitted on November 17, 2000; accepted on April 19, 2001.