1 Infertility Clinic, The Family Federation of Finland, Kalevankatu 16, FIN-00100 Helsinki and 2 Departments I and II of Obstetrics and Gynaecology, University Central Hospital of Helsinki, Helsinki, Finland
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Abstract |
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Key words: embryo transfer/follicle-stimulating hormone/in-vitro fertilization/intracytoplasmic sperm injection/pregnancy rate
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Introduction |
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A number of studies comparing recombinant (rec) FSH and urinary-derived gonadotrophins administered for ovulation induction (Coelingh Bennink et al., 1998; Messinis et al., 1998
), or in women undergoing assisted reproduction techniques (Hedon et al., 1995
; Out et al., 1995
; Recombinant Human FSH Study Group, 1995
; Aboulghar et al., 1996
; Ubaldi et al., 1996
; Bergh et al., 1997
; Jansen et al., 1998
), have been performed. In most (Hedon et al., 1995
; Out et al., 1995
; Aboulghar et al., 1996
; Ubaldi et al., 1996
; Bergh et al., 1997
; Jansen et al., 1998
) but not in all studies (Recombinant Human FSH Study Group 1995
), recFSH appeared to be more effective than urinary FSH. Significantly higher numbers of follicles were recruited, oocytes collected and embryos obtained after a shorter duration of treatment with a lower total dose of recFSH. Clinical pregnancy rates in recFSH cycles have been comparable (Hedon et al., 1995
; Out et al., 1995
; Aboulghar et al., 1996
; Ubaldi et al., 1996
; Bergh et al., 1997
), or even higher (Out et al., 1997
; Jansen et al., 1998
) than in cycles with urinary FSH when frozenthawed embryo replacement cycles were included.
Two recFSH preparations with slightly different carbohydrate compositions are currently on the market, follitropin (Gonal-F®; Ares Serono, Geneva, Switzerland) and follitropin ß (Puregon®; NV Organon, Oss, the Netherlands). It has been proposed that differences in the isoform profiles of the two recFSH preparations could influence their clinical effectiveness (de Leeuw et al., 1996
; Olijve et al., 1996
; Shoham and Insler, 1996
). However, information about the possible superiority of one or the other is still under debate.
Controversy exists as regards the appropriate dose of gonadotrophin to be used to induce multiple follicular development but at the same time to avoid ovarian hyperstimulation syndrome (OHSS). Currently, starting doses of 150 IU/day or 225 IU/day are widely used. However, in the light of the possibly higher bioactivity of recFSH over urinary FSH (Out et al., 1995; Bergh et al., 1997
; Coelingh Bennink et al., 1998
; Devroey et al., 1998
; Jansen et al., 1998
), a lower dose may be appropriate, at least in women at a high risk of OHSS.
We designed an assessor-blind, randomized study to compare the efficacy of the two recFSH preparations for stimulating multiple follicular development in women undergoing conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Furthermore, we evaluated the total ongoing pregnancy rates, including frozenthawed embryo replacement cycles.
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Materials and methods |
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Pituitary down-regulation with buserelin (Suprecur®, Hoechst Ab, Frankfurt, Germany, 900 µg/day intranasally) was started 1 week before an expected menstrual period. After 2 weeks, down-regulation was confirmed by the absence of ovarian follicles >12 mm in diameter, endometrial thickness <5 mm (both layers) in vaginal ultrasonographic examination and/or serum oestradiol concentration <0.2 nmol/l. If pituitary down-regulation was not achieved within 2 weeks, the dose of buserelin was increased to 1200 µg/day intranasally.
Patients were randomly allocated to ovarian stimulation with either Gonal-F® or Puregon® according to a computer-generated randomization list. The medication was given in a blind fashion for the administering physician. Both drugs were administered by daily subcutaneous injections at a starting dose of 150 IU. In women with a poor response (fewer than five follicles) in the previous treatment cycle, the starting dose was 300 IU/day. Simultaneously with the start of recFSH treatment, the dose of buserelin was reduced (450 µg/day) and gonadotrophin-releasing hormone agonist (GnRHa) was administered until the day of human chorionic gonadotrophin (HCG) injection. After 5 days, the dose of recFSH was adjusted according to serum oestradiol measurements and vaginal ultrasound examinations. HCG (5000 IU; Profasi®, Ares Serono in the Gonal-F® group and Pregnyl® in the Puregon® group) was given when the three largest follicles exceeded 18 mm in diameter.
Ultrasound-guided transvaginal follicle aspiration was performed 3638 h after HCG injection. The oocytes were cultured in Medi-Cult Universal IVF Medium (Medi-Cult A/S, Copenhagen, Denmark) and fertilized in vitro (Moilanen et al., 1999). The meiotic stage of all oocytes was assessed 1720 h after insemination.
For ICSI, the cumulus cells were removed and assessment of the meiotic maturation stage was made. Only MII-stage oocytes were injected. The presence of two pronuclei (PN) and polar bodies was confirmed 1720 h after insemination or after microinjection. The cleavage state of the normally fertilized oocytes was assessed 48 h after retrieval. Embryo morphology was assessed as previously described by Veeck (Veeck, 1991): grade 1: no anucleate fragments, symmetrical blastomeres; grade 2: fragmentation <10% of volume; grade 3: 1020% fragmentation; grade 4: 2060% fragmentation (Veeck, 1991
). One or two embryos were replaced 2 or 3 days after oocyte retrieval.
Supernumerary embryos were frozen at either the PN or cleavage stage using a slow freezingthawing protocol with 1.5 mol/l 1,2-propanediol and 0.1 mol/l sucrose as cryoprotectants in a programmable cryomachine (Kryo 10 Series II, Planer Products Ltd, Sunbury-on-Thames, UK) with manual seeding at 8°C.
Luteal support was provided for 3 weeks by giving 600 mg micronized vaginal progesterone daily (Lugesteron®, Leiras, Turku, Finland). Serum HCG was assessed 14 days after embryo transfer. If the pregnancy test result was positive, the viability of the pregnancy was assessed by transvaginal ultrasonographic (TVS) examination 45 weeks after embryo transfer. Clinical pregnancies were defined gestations with observable embryonic sacs on TVS.
For frozen embryo transfer, the embryos were thawed either on the previous day of replacement or on the day of replacement by warming and gradual removal of the propanediol and sucrose. The embryos were then replaced either during a natural cycle or during hormone replacement treatment using buserelin combined with exogenous oestradiol and progesterone [Suprecur® 450 (Hoechst, Frankfurt, Germany) intranasally, Progynova®, 46 mg/day (Schering, Berlin, Germany), Lugesteron®, 600 mg/day)].
According to power analysis, to confirm better efficacy of either recFSH preparation a difference of more than three in the mean number of oocytes collected per subject had to be demonstrated. Hence, a study population of 282 women was calculated to be necessary to reveal the better efficacy of either recFSH preparation as regards the number of oocytes retrieved, with 95% probability.
Hormone assays
Serum oestradiol concentrations were measured using solid phase fluoroimmunoassay (Delfia, Wallac, Turku, Finland). The intra- and inter-assay coefficients of variation were 3.810.0% and 3.69.7% respectively.
The results are expressed as mean ± SEM. Data comparisons between the study groups were made by 2 test and by analysis of variance after logarithmic transformation when appropriate.
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Results |
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No significant differences emerged between the Gonal-F® and Puregon® groups according to the starting dose with respect to serum oestradiol concentrations on days 6 and 9, in endometrial thickness or in the number of recruited follicles of 12 mm in diameter on day 9 of the recFSH treatment (Table II
). The mean duration of stimulation and the total amount of recFSH medication used were comparable. No significant differences were observed in the mean number of oocytes collected in the Gonal-F® or Puregon® groups (starting dose 150 IU/day: 13.0 ± 0.7 versus 12.4 ± 0.7, not significant; starting dose 300 IU/day: 6.1 ± 1.6 versus 7.1 ± 1.1, not significant, Table II
).
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The proportion of mature oocytes was similar in the Gonal-F® and Puregon® groups, as were the rates of normal fertilization and embryo cleavage (Table IV). No overall differences were observed between IVF and ICSI cycles in the rates of 2PN formation or the number of cleaved embryos (data not shown). The medication used did not affect the grades of embryos or the proportion of fast dividing embryos in IVF or ICSI (data not shown).
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With the starting dose of 150 IU/day, 89.6% of subjects underwent embryo transfer in the Gonal-F® group and 91.8% in the Puregon® group. With the starting dose of 300 IU/day, the respective percentages were 87.5 and 92.3%. One or two embryos were replaced each time. Elective one-embryo transfers accounted for 24.5% of transfers in the Gonal-F® group (starting dose 150 IU/day) and 30.1% of transfers in the Puregon® group (starting dose 150 IU/day). The implantation rates per embryo replaced were 25.6 and 26.3% respectively (not significant). The clinical pregnancy rates per started cycle in the Gonal-F® and Puregon® groups were 33.5 versus 32.9%, and per embryo transfer, 37.4 versus 36.4% (not significant). Of these pregnancies, eight (14.5%) in the Gonal-F® group (starting dose 150 IU/day) and five (9.6%) in the Puregon® group (starting dose 150 IU/day) ended in miscarriage. Two pregnancies in both groups were ectopic. Seven of the 45 (15.6%) ongoing pregnancies in the Gonal-F® group were twin pregnancies, and there were nine (20.0%) among the 45 ongoing pregnancies in the Puregon® group (Figure 1). None of the women receiving Gonal-F® at 300 IU/day became pregnant, whereas in the Puregon® group two singleton ongoing pregnancies were achieved (Figure 1
). Considering the morphology of the best embryo transferred, 90.4 and 91.7% of the pregnancies in the Gonal-F® and Puregon® groups occurred when grade 12 embryos were transferred (not significant). Similar good pregnancy results were shown when on day 2 the fastest dividing embryo was in the 4-cell stage (76.9 versus 79.2%, not significant). Of all 79 single embryo transfers performed, 25 (31.6%) resulted in clinical and 19 (24.1%) in ongoing pregnancies.
Supernumerary embryos were frozen in 63.8% of the treatment cycles with Gonal-F® (starting dose 150 IU/day) and in 69.1% of those with Puregon® (starting dose 150 IU/day) (not significant). In women receiving 300 IU/day, embryo freezing was carried out in 28.6% and 50.0% of the cycles with Gonal-F® and Puregon® respectively. So far, 189 frozen embryos have been thawed in connection with 55 women receiving Gonal-F® and 216 embryos in connection with 62 women receiving Puregon®. The survival of embryos after thawing was comparable in both groups (84.0% in the Gonal-F® group and 79.7% in the Puregon® group, not significant). Implantation rates were 15.9% for frozenthawed embryos after Gonal-F® and 9.1% after Puregon® stimulation (not significant). Of the 19 clinical pregnancies that resulted from frozenthawed embryo transfer cycles in the Gonal-F® group, 13 (68.4%) are ongoing. Similarly, of the 16 pregnancies in the Puregon® group, 15 (93.8%) have progressed beyond 16 weeks of gestation. Ongoing pregnancy rates per started stimulation, when frozen embryo transfers were included were 35.4% in the Gonal-F® 150 IU/day group, and 37.7% in the Puregon® 150 IU/day group (not significant).
Mild abdominal swelling and discomfort was observed in 13 women (7.9%) in the Gonal-F® group and in 12 (7.5%) in the Puregon® group. These women were followed up as outpatients. Of the 13 women in the Gonal-F® group, 12 underwent embryo transfer and nine became pregnant. Similarly, of the 12 women in the Puregon® group, nine underwent embryo transfer and four became pregnant. Four women (2.0%) in the Gonal-F® group and one in the Puregon® group (0.7%) needed hospital treatment for moderate or severe OHSS. Only one of these women underwent embryo transfer and she did not become pregnant. Of the five women with OHSS, three later became pregnant after frozenthawed embryo replacement. All cases of moderate OHSS occurred in women whose recFSH dose was decreased.
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Discussion |
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The two recFSH preparations currently in use have undergone slightly different purification procedures and are therefore not identical. Although exact molecular differences between the two recFSH hormones have not been determined, both Gonal-F® and Puregon® show slightly more basic FSH isoform profiles than seen with urinary FSH preparations. This, in turn, may bring about differences in their biological activities and clinical effectiveness (Olijve et al., 1996; Shoham and Insler, 1996
). This possibility is supported by the lower oestradiol concentrations and a lower number of follicles >14 mm in diameter on the day of HCG administration in a study comparing Gonal-F® with urinary FSH (Recombinant FSH Study Group, 1995). These findings are clearly in disagreement with those from a larger study comparing Puregon® with urinary FSH (Out et al., 1997
; Jansen et al., 1998
). Recent studies, however, comparing Gonal-F® with highly purified urinary FSH in a large number of subjects confirmed the higher efficacy of Gonal-F® over urinary preparations (Bergh et al., 1997
).
In the present prospective, randomized study we were able to show that Gonal-F® and Puregon® were equally effective in terms of number of oocytes collected, the quality and number of embryos obtained, embryos frozen as well as in ongoing pregnancy rates per cycle (including fresh and frozenthawed cycles). It therefore appears that subtle changes in carbohydrate structure are not reflected in clinical efficacy. Cumulative pregnancy rates are a good indicator of the efficacy of the treatment given, particularly when only a small number of embryos are replaced during one cycle. It is also clear from our study that the availability of a large number of good quality embryos for cryopreservation will ultimately improve the couple's chances for conception.
Multiple pregnancies accompany IVF and ICSI, as approximately every fifth pregnancy is a twin pregnancy (Wennerholm et al., 1991; Rufat et al., 1994
; Söderström-Anttila et al., 1998
). The rates of perinatal mortality and fetal and maternal complications are higher in twin pregnancies than in singleton pregnancies and the incidence of adverse outcome rises with an increasing number of multiples (Wennerholm et al., 1991
; Rufat et al., 1994
; Söderström-Anttila et al., 1998
). The current policy of replacing two instead of three embryos in Nordic countries has resulted in a reduction of high order multiple pregnancies but not in twin pregnancies, without affecting the pregnancy rates (Bergh et al., 1997
; Moilanen et al., 1999
). A radical strategy to reduce the rate of multiple births would be to replace only one embryo. Recent evidence indicates that elective transfer of only one embryo results in acceptable pregnancy rates, particularly in women who have more than one good quality embryo for transfer (Vilska et al., 1999
). This is also evident from the results of the present study as approximately one-third of fresh embryo transfers were elective one-embryo transfers, and a third of these transfers resulted in clinical pregnancy. We therefore recommend one-embryo transfer when good quality embryos are available and the couple is undergoing their first or second IVF or ICSI treatment.
OHSS is a potentially life-threatening complication accompanying up to 7%, usually 0.52.0%, of all IVF or ICSI cycles (Bergh and Lundqvist, 1992; Bergh et al., 1997; Out et al., 1997
; Orvieto and Ben-Rafael, 1998
). High oestradiol concentrations on the day of HCG administration and a high number of follicles aspirated have been associated with an increased risk of developing OHSS (Orvieto and Ben-Rafael, 1998
). Despite the high number of follicles recruited and the high serum oestradiol concentrations seen in this study the incidence of moderate or severe OHSS requiring hospitalization was comparable to that seen in previous studies using either recFSH or urinary FSH preparations (Bergh et al., 1997
; Out et al., 1997
; Orvieto and Ben-Rafael, 1998
). Cancellation of the cycle before HCG administration, or withholding embryo replacement and elective cryopreservation of all embryos, has been recommended in subjects at great risk of OHSS (Rizk and Aboulghar, 1991
). We chose the latter strategy. Our choice did not reduce the incidence of OHSS but it may have lessened its severity, since precipitation of OHSS by HCG produced by the trophoblast was avoided. Furthermore, our results confirmed the experience (Tiitinen et al., 1995
) that pregnancy rates after subsequent replacement of frozenthawed embryos are good. Moderate and severe forms of OHSS in the present study occurred in women who needed a decrease in the dose of recFSH. In this subgroup, the use of a lower dose may have prevented unwanted over-stimulation (Devroey et al., 1998
; Out et al., 1999
). Nevertheless, careful monitoring is essential in preventing OHSS.
In conclusion, the present results clearly demonstrate that Gonal-F® and Puregon® are equally effective and safe in producing multiple follicular growth and provide good chances of pregnancy in a single treatment cycle.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 29, 1999; accepted on August 12, 1999.