Delivery rates following IVF treatment, using two recombinant FSH preparations for ovarian stimulation

Jonas Harlin,1, A. Aanesen, G. Csemiczky, H. Wramsby and G. Fried

Reproductive Medical Centre, Department of Women and Child Health, Division of Obstetrics and Gynaecology, Karolinska Hospital, Stockholm, Sweden


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
BACKGROUND: A prospective, semi-randomized, open, clinical study was conducted to compare ovarian response, pregnancy outcome and delivery rates using two recombinant FSH preparations (Gonal-F® and Puregon®) for IVF. METHODS: We analysed stimulation parameters and outcome data in 812 initiated treatment cycles where 292 women used Gonal-F and 200 used Puregon. Embryo transfer was carried out in 676 cycles. In the two preparation groups we also compared 170 women previously treated with FSH for IVF with 266 previously untreated. RESULTS: The pregnancy rate with Gonal-F was 26% and with Puregon 28%. Delivery rates were identical, 22%. Clinical pregnancy and delivery rates per cycle with embryo transfer in earlier untreated women were 29.0 and 24.0%, whereas in previously treated women they were 23.5 and 18.8% respectively. After repeated cycles, delivery frequencies consecutively decreased, independent of the FSH preparation used. CONCLUSION: Gonal-F and Puregon seem to be equally potent in achieving follicular development and equally effective, in terms of delivery rates, for use in an IVF programme. Repeated cycles resulted in equally large consecutive decreases in delivery rates, regardless of preparation choice, but were considered worthwhile for up to three stimulation cycles in selected patients.

Key words: delivery rates/ovarian stimulation/recombinant FSH


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
FSH preparations, manufactured by means of recombinant DNA technology, presently replace previously used drugs extracted from urine in assisted reproduction techniques, including controlled ovarian stimulation for IVF. The new preparations possess a high level of purity, a high batch to batch consistency, absence of contaminating proteins found in urinary extracts and power to stimulate ovarian follicle development as effectively as their urinary predecessors (Anonymous, 1995Go).

Two further advantages of recombinant human follicle stimulating hormone (rhFSH) include limitless availability and suitability for s.c. self-administration.

Before introduction to the market, the available preparations, Gonal-F® (Serono) and Puregon® (Organon) had gone through extensive clinical trials (Loumaye et al., 1993Go; Devroey et al., 1994Go), which were followed by a considerable number of studies, both comparing highly purified urinary preparations with rhFSH (Out et al., 1995Go, 1997Go; Bergh et al., 1997Go) and comparing the two manufacturers' products (Tulppala et al., 1999Go; Harlin et al., 2000Go; Brinsden et al., 2000Go). In some studies rhFSH was shown to be more potent than urinary preparations and Puregon was presumed to be somewhat more potent than Gonal-F. Therefore Puregon was marketed at different dosages (i.e. 50, 100 and 150 IU) compared with the `standard' size of 75 and 150 IU per ampoule. Recommended doses of Puregon were also slightly lower than for Gonal-F. In most studies on rhFSH, high pregnancy rates are reported. From the treated couple's point of view, only the ultimate result of the treatment, i.e. delivery of a healthy child, is the most important factor. It has been suggested that there might be a difference between the two preparations in terms of stimulation outcome. Since 1997 only recombinant gonadotrophins have been readily available in Sweden, giving us the opportunity to perform an extensive study of possible clinical differences of the two products and their use in IVF, including final stimulation outcomes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Study population
The study consisted of 676 completed cycles in 436 individuals consecutively treated at our clinic from February 1997 until September 1999. The population was unselected, and all patients signed an informed consent form. Of these patients, 170 had been previously stimulated, both at our clinic and elsewhere, with urinary derived FSH for IVF. During the same period of time another 136 started cycles in 122 women were cancelled. After one or more unsuccessful treatment cycles, 56 of these couples were not treated further, due to insufficient ovarian response and/or failed fertilization, and in ten cases due to infectious disease of female or male partner. Only four patients treated during the period were excluded from the study because of concomitant growth hormone therapy. The age of the women included ranged from 22 to 38 years at their first stimulation cycle. No couples were accepted for treatment if they had a common biological child, but children from previous relationships did not disqualify them. Characteristics of the patients in the study are given in Table IGo. The allocation to Gonal-F or Puregon was based on the odd or even days in the date of birth (year, month, day). Forty of the individuals agreed to change preparation after two treatment cycles, for the purpose of a cross-over study. The intervals between repeated treatment cycles were never shorter than 3 months and no longer than 11 months. The male partners included in the study had either normal or subnormal sperm counts.


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Table I. Characteristics of patients in the study. Values are given as means ± SEM or as percentages
 
Study protocol and method
All patients followed our standard long GnRH agonist–FSH protocol for stimulation as follows. GnRH agonist (Suprefact®; Svenska Hoechst AB, Stockholm, Sweden) was administered as a nasal spray 6x200 µg/day, starting on day 21 of a menstrual cycle, or after a gestagen induced bleeding in cases with oligomenorrhoea, and was reduced to half the dose when rhFSH injections were commenced. After down-regulation was verified by measurement of an endometrium <=3 mm, using vaginal ultrasound scanning (Siemens Sonoline S1/200), 75–300 IU/day of rhFSH (Gonal-F; Serono Nordic AB, Sollentuna, Sweden or Puregon (Follistim® in the USA); Organon Sweden AB, Göteborg, Sweden) were administered s.c.

Individual adjustment of the starting dose was performed according to the same rules in both groups. The initial dose of rhFSH was generally 150 IU/day up to age 35 for both preparations and 225 IU/day of Gonal-F or 200 IU of Puregon above this age. Known poor responders, with insufficient follicle development in a previous cycle using 225–300 IU daily as shown by 0–3 oocytes at previous oocyte retrieval, started 63 cycles with 300–450 IU rhFSH and 13 cycles of 11 patients with polycystic ovary syndrome were started with <=75 IU. The dose was raised by 37.5–150 IU in 49 Gonal-F cycles due to slow ovarian response, and lowered by 37.5–150 IU in 52 cycles at risk of ovarian hyperstimulation syndrome (OHSS). Corresponding numbers in Puregon® cycles were a raised dose by 50–150 IU in 24 cases and a lowered dose by 50–150 IU in 27 cases. Most adjustments were made 7–9 days after the start of injections.

Follicular development and endometrial growth were monitored by vaginal ultrasonography in combination with blood samples for 17ß-estradiol (E2) assays. When an adequate stimulation was achieved, i.e. a controlled increase of serum estradiol and a leading follicle sized at least 17 mm in diameter, 10 000 IU of HCG (Profasi, Serono Nordic AB) was given s.c. Approximately 35 h later oocyte retrieval was performed by transvaginal ultrasound-guided follicle aspiration under local anaesthesia. IVF, embryo transfer and pregnancy follow-up were performed as described elsewhere (Csemiczky et al., 1995Go). Luteal phase support was given, using 400 mg micronized progesterone as vaginal suppositories twice daily until a pregnancy test was performed and, if found positive, was continued 8 weeks after embryo transfer. A serum HCG level above 50 IU/l, assayed at day 17 after embryo transfer, indicated pregnancy, and ultrasonography was used 4 weeks later to verify a clinical pregnancy by the presence of at least one intrauterine fetus with regular heartbeats. Criteria for cycle cancellation were: (i) insufficient increase of E2 levels, even after a raised dose of rhFSH, (ii) fewer than four follicles approaching 17 mm diameter and (iii) imminent risk of developing OHSS, i.e. sharp rise in E2 and >25 follicles.

Hormone assays
The Central Laboratory for Clinical Chemistry at Karolinska Hospital assayed serum 17ß-E2, progesterone and HCG by electrochemiluminescence immunoassay, using reagent kits from Roche Diagnostics GmbH, Mannheim, Germany.

Statistical analysis
Prior to statistical analysis, data were tested for Gaussian distribution using the Kolmogorov–Smirnov test. Since some data were found not to be normally distributed, non-parametric statistics were used for all data. Comparisons between mean values were done using the Mann–Whitney Rank Sum test. Although the standard errors were therefore not used for statistical comparisons, they have been appended to the mean values in the tables. For analysis of nominal data and proportions the {chi}2 test was applied. Tests were performed with the statistical package Stat. View.4.5 (Abacus Concepts Inc., Berkeley, CA, USA). A P value < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Study population
Characteristics of the study population are presented in Table IGo. The age, body mass index (BMI) and causes of infertility of the women in the two preparation groups were not significantly different, however, previously treated subjects were slightly older (mean 34.0) than untreated (33.2) (P < 0.001). The proportions of previously treated and untreated women were not different in the preparation groups. The number of previous treatment cycles before the study of 170 subjects, allotted to Gonal-F and to Puregon, ranges from 1 to 5, mean value 1.9 and 1.8 cycles respectively.

Cancelled cycles
Of all started cycles insufficient follicle development occurred in 30 Gonal-F and in 25 Puregon cycles, and failed fertilization also in 30 and 25 cycles respectively. In each group, seven cycles were cancelled due to threatening ovarian hyperstimulation syndrome and six cycles in each group due to miscellaneous other reasons. Cancellation rates did not differ between the preparation groups. However, a larger proportion of started subjects were dissuaded from further treatment in the Puregon than in the Gonal-F group, due to inadequate follicle development or failed fertilization, although the population characteristics were not different. As a result oocyte retrieval and embryo transfer were possible in 84.8% of Gonal-F and in 81.1% of started Puregon cycles.

Male factor, ICSI and cryopreservation
The sperm counts of the male partners, after swim-up preparation, exceeded 5x106/ml in most cases; however, in 17.5% of the Gonal-F and 16.3% of the Puregon cycles sperm counts of 1x106/ml or less were used (NS). In 85 and 61 cycles respectively, ICSI was performed. In 19 and 13 cycles respectively a concentration procedure of the sperm into a tiny droplet was performed, into which the oocytes were placed. In all other treatment cycles regular IVF was performed. There was no difference in terms of method used between the groups. Cryopreservation and subsequent embryo transfer was possible in nine Gonal-F® cases, resulting in three clinical pregnancies, all spontaneously aborted, and in three Puregon® cycles, all unsuccessful. Eight subjects in the Gonal-F and four in the Puregon group were hospitalized due to moderate ovarian hyperstimulation after embryo transfer. None of them needed intensive care.

Total dose of FSH and duration of stimulation
The overall mean total dose of Gonal-F given was significantly larger than that of Puregon, and the number of days of stimulation were significantly fewer in Gonal-F than in Puregon cycles: 11.8 and 12.5 days respectively (Table IIGo). For previously untreated women the total dose of Gonal-F (mean 2090 IU) was only slightly larger than that of Puregon (2051 IU), but the number of treatment days was significantly different, 11.8 and 12.6 respectively (P < 0.01). For previously treated patients, the total doses of Gonal-F (mean 2456 IU) and Puregon (2159 IU) were different (P < 0.005), but the treatment duration did not differ significantly.


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Table II. Clinical response to ovarian stimulation of 436 women, in 676 cycles, with two preparations of rhFSH. Data given as mean ± SEM
 
Repeated cycles in the same patient using either Gonal-F or Puregon
After the first treatment cycle at our centre, a second treatment cycle was performed in 89 Gonal-F and in 61 Puregon patients. Three consecutive cycles with each preparation were performed in 15 and 10 cases respectively (Table IIIGo). The treatment duration of the first cycle out of two in the Puregon group was significantly longer than in the first Gonal-F cycle, but not in the second. The total doses of rhFSH used did not differ between the cycles or the preparations in these patients.


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Table III. Mean total dose and duration of injections in repeated ovarian stimulation cycles in two or three successive cycles, using each of two rFSH preparations, and in one cycle of each preparation in the same subjects. Values given as means ± SEM
 
Total doses of rhFSH or number of injection days in three repeated cycles did not differ between the two or between the three cycles of each preparation, furthermore no differences were found between the drugs in these respects.

Forty patients consented to a switch from the starting preparation to the alternative after two cycles and the two cycles of each preparation closest to each other were compared. No differences were seen between total doses of rhFSH or of treatment duration.

Outcome in relation to indication for treatment
Analysis of differences between total rhFSH dose in relation to indication for treatment showed that significantly larger total doses were needed in subjects with endometriosis than in those without (P < 0.05). This applied to both preparation groups, in Gonal-F cases a mean of 2757 IU and in Puregon 2858 IU. No differences in total doses were found between other treatment indications.

Subjects who required more than 3000 IU of rhFSH for optimal ovarian stimulation were regarded as poor responders, and their treatments amounted to 15.3% of the Gonal-F cycles and 8.5% of the Puregon cycles (NS). More than 4000 IU were used in 6.2 and 4.1% of all cycles respectively (NS). Previously treated women received larger doses than 3000 IU twice as often as earlier untreated women.

Hormonal, endometrial and oocyte parameters
Estradiol mean levels were significantly higher during the first part of the stimulation periods in Gonal-F compared with Puregon cycles, but from 3 days preceding oocyte retrieval no differences were seen (Table IIGo). Progesterone mean levels were significantly higher (P < 0.001) in Gonal-F compared with Puregon cycles, measured at oocyte retrieval, but no longer at embryo transfer.

Measurements of endometrial thickness did not differ between the preparation groups.

Development of ovarian follicles, both of smaller and of larger size, was not significantly different between the two groups. The mean number of oocytes retrieved in each preparation group was similar, but highly significant differences were seen in the rate of oocytes fertilized (P < 0.005) and cleaved (P < 0.01), in favour of Gonal-F cycles. An identical number of pre-embryos were transferred in both groups, and never more than two.

Pregnancy and delivery rates in relation to cycle number
The overall pregnancy rate in Gonal-F cycles was 26% and in Puregon cycles 28%, and the delivery rates per cycle were identical for the two preparation groups, 22%. The success rate, in terms of clinical pregnancy and delivery rate per cycle with embryo transfer, was substantially higher in women undergoing their first cycles (Table IVGo). In earlier untreated women a total clinical pregnancy rate of 29.0% and a delivery rate of 24.0% per cycle was observed. Previously treated women attained 23.5 and 18.8%, however, if all previous treatment cycles elsewhere were included, the result was a modest 10.6 and 8.5% respectively. In poor responders the delivery rates of Gonal-F and Puregon cycles were low, i.e. 14.5 and 8.7% respectively. There was no difference in delivery rate per cycle with embryo transfer in relation to method used.


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Table IV. Outcomes after stimulation with two preparations of rFSH, in previously treated and untreated subjects, where oocyte retrieval and embryo transfer were performed. Values in brackets are percentages
 
Delivery followed the first cycle with embryo transfer at a rate of 37.7 and 38.1% per woman stimulated with Gonal-F and Puregon and two consecutive cycles, with either preparation, at a rate of 20.2 and 19.7% respectively (Table VGo). Twenty-five women had three consecutive cycles with either preparation, ending in 12 clinical pregnancies, but only five proceeded to delivery, resulting in a delivery rate per cycle of 6.7%. Twin deliveries occurred in 20.2% after Gonal-F and in 26.7% after Puregon (NS). Two ectopic pregnancies were noted, one in each preparation group.


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Table V. Pregnancy outcome of 436 women after one, two and three stimulation cycles, with each of two rFSH preparations, for IVF
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Study population
The population of the present study was not randomized according to generally applied rules. Allocation by means of date of birth to each of the two preparations was chosen for practical reasons with the assumption that this factor would not be a source of bias in terms of treatment effects. Since there are only 1.9% more odd than even dates per year, it is unclear why such a discrepancy in the size of the groups occurred.

Before the start of the study we had the impression that patients referred from other IVF clinics, after previous unsuccessful treatment, had lower pregnancy rates than earlier untreated patients. Comparing previously treated to untreated women we found the mean age of the former to be significantly higher, however diagnosis and BMI distribution were not different.

Outcome of stimulation in relation to preparation used
Most clinical studies report success in terms of clinical or ongoing pregnancies. The ultimate treatment goal, i.e. percentage of treatment cycles leading to the birth of a child, is not so often reported. In the present study certain differences between the responses of women given two rhFSH preparations were found, but there was no difference in delivery frequency.

The average total doses of Gonal-F and Puregon given to previously untreated women were almost identical, even though the treatment duration was longer in Puregon cycles. In women with earlier treatment, 50.3 and 60.4% were 35 years old or more in the Gonal-F and Puregon group respectively, and one quarter of these women were given more than 3000 IU rhFSH per cycle, and may thus be regarded as relatively `poor responders'. In our standard protocol we routinely start with 225IU Gonal-F or 200 IU Puregon above age 35, in part explaining the larger average total dose of Gonal-F given to these women. Even when cycles with total doses >3000 IU were excluded, previously treated women were found to need larger average doses of rhFSH for optimal stimulation than the earlier untreated group.

Puregon was given in slightly lower doses than Gonal-F, following suggestions from the manufacturer, leading to recruitment and growth of a slightly smaller, although not significant, number of ovarian follicles and lower serum estradiol concentrations during stimulation with Puregon as compared with Gonal-F. Serum progesterone levels at oocyte retrieval, as well as the numbers of oocytes fertilized and cleaved, were significantly lower when Puregon was used. This observation may be related to the smaller average total dose given for Puregon. However, the final fate of all acquired pregnancies unveiled an identical rate of deliveries of 22% per cycle with embryo transfer in both preparation groups. A study comparing the daily dose of 100 and 200 IU Puregon (Out et al., 1999Go) showed positive correlation between the dose of FSH administered and the number of stimulated follicles, with increased estradiol and progesterone levels in high dosage, which is in agreement with our findings.

Although the molecular composition of the two preparations is very similar, Puregon has been shown to contain somewhat less acidic isoforms with shorter elimination time than Gonal-F. This might lead to a slower accumulating FSH-concentration in serum (Balen et al., 1999Go), which possibly could contribute to differences in treatment effects, even if the total doses of the preparations injected were equal.

There was no statistical difference in the numbers of follicles or oocytes retrieved between women treated with the two preparations, but fertilization and cleavage rates of the oocytes were significantly higher in Gonal-F cycles, possibly indicating a higher oocyte quality. An identical number of pre-embryos were used at transfer, leading to overall identical delivery rate. Cryopreservation of surplus pre-embryos, after the evaluation by trained embryologists, was low during the whole time period studied. Notwithstanding access to more cleaved oocytes after Gonal-F stimulation than after Puregon, freezing was not suggested more often after Gonal-F, because the oocyte quality estimated by morphology was not satisfactory.

Outcome in repeated cycles
Treatment success in terms of delivery was lower for the previously treated women, most probably due to a selection with a higher proportion of relatively poor responders. It is well known that fertility is negatively correlated with increasing age, both due to ovarian and to endometrial factors (Meldrum, 1993Go; Navot et al., 1994Go; Hull et al., 1996Go). The implantation rate as a function of age has been reported to be constant up to age 35; thereafter a decrease starts, suggesting lower oocyte quality as a major factor for lower implantation rate, although uterine factors may contribute (Spandorfer et al., 2000Go).

Repeated stimulation cycles with each preparation in the same individuals, as well as both preparations to the same individuals in a cross-over fashion, add to the impression that the two preparations are equipotent. The only difference we detected was a longer stimulation time for Puregon in the first cycle out of two repeated cycles, possibly related to a lower total FSH dose (2092 IU as compared with 2323 IU for Gonal-F). In subjects with three repeated cycles and in cross-over cycles we saw no differences (Table IIIGo).

It has been suggested that the pregnancy rate of consecutive stimulation cycles is unchanged, at least up to three (Meldrum et al., 1998Go); however, a recent report founded on a large body of evidence suggests that IVF success declines in repeated cycles (Crosignani and Rubin, 2000Go). Data in the present study are in agreement with the latter view.


    Conclusion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Recombinant FSH preparations have been compared with urinary derived FSH in a number of studies, with the conclusions drawn that the former are to be preferred, due to a high level of purity, a high batch to batch consistency and to a high clinical efficacy in patients undergoing IVF (Bergh, 1999Go; Daya and Gunby, 1999Go). The three previously published studies comparing in-vitro biopotency of the two available rhFSH preparations have found them to be very similar, and clinically they were equally efficient in terms of clinical pregnancy rates (Tulppala et al., 1999Go; Harlin et al., 2000Go; Brinsden et al., 2000Go).

In the present study, the most extensive to date, we found the two recombinant FSH preparations used for stimulation of an unselected population of infertile women, under 40 years of age, to be equally effective, in terms of delivery rates, for use in an IVF programme. The frequencies of OHSS were low and comparable. The two preparations seem to be equally potent according to ovarian follicular development and hormonal changes in stimulated women. In our study, consecutive repeated cycles resulted in equal delivery rates, generally decreasing with each attempt. We regarded the results rewarding for up to three stimulation cycles in selected cases, regardless of the choice of preparation.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
This study was supported by Swedish Medical Research Council (14X-07614) and Karolinska Institutets Fonder. We thank all staff at Reproductive Medical Center for their contribution to this work.


    Notes
 
1 To whom correspondence should be addressed at: Department of Women and Child Health, Division of Obstetrics and Gynaecology, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: jonas.harlin{at}ks.se Back

Submitted on April 17, 2001; resubmitted on September 10, 2001


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
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accepted on October 26, 2001.