1 Organon Laboratories, Cambridge Science Park, Cambridge, UK, 2 IVF Unit, Carmel Medical Centre, Haifa, 3 IVF Unit, Assaf Harofeh Medical Centre, Zerifin, 4 HaEmek Medical Centre, IVF Unit, Afula, 5 Department of Obstetrics and Gynaecology, Sheba Medical Centre, Tel-Hashomer, and 6 Rabin Medical Centre, Campus Beilinson, Petach Tiqva, Israel
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Abstract |
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Key words: ICSI/IVF/Puregon/randomized clinical trial/recombinant FSH
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Introduction |
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With the arrival of recombinant FSH (rFSH) the issue of milder regimens becomes even more relevant, given the higher potency of rFSH versus urinary FSH (Out et al., 1995; Bergh et al., 1997
) and its perceived high acquisition costs (Meniru, 1999
). The availability of lower presentation units of rFSH (50 IU, 100 IU) also facilitates clinical research as to the feasibility of low-dose protocols. Earlier, it was shown in observational studies that a 100 IU starting dose may be effective, thereby considerably reducing the total dose of administered gonadotrophins (Devroey et al., 1998
; Bergh, 1999
). However, very few double-blind, randomized clinical trials have been performed to show that these milder regimens do not jeopardize the chance for a successful birth and at the same time diminish the chances of side effects occurring. Recently, a double-blind comparison of fixed daily doses of 100 IU and 200 IU of rFSH was performed (Out et al., 1999
). Significantly more oocytes were retrieved in the high-dose group, but the clinical pregnancy rates did not differ. In the current study, this clinical trial was repeated in a different subset of patients, namely those undergoing ICSI and in a different geographical location, Israel.
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Materials and methods |
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Selection of patients
Inclusion criteria were as follows: at least 18 and at most 37 years of age at the time of screening; male infertility (total motile count <5x106 spermatozoa) solvable by ICSI using ejaculatory spermatozoa; normal regular cycles with a mean length of between 24 and 35 days; presence of two ovaries; good physical and mental health; body mass index between 18 and 29 kg/m2; and willing to provide written, informed consent.
Exclusion criteria were: female cause of infertility, except mild endometriosis or a mechanical factor; previous IVF or ICSI cycle(s) after which less than three oocytes were retrieved; previous IVF or ICSI cycle(s) with hospitalization due to ovarian hyperstimulation syndrome (OHSS); more than four previous IVF or ICSI cycles; total fertilization failure in a previous IVF or ICSI cycle; LH/FSH ratio at screening 3; chronic cardiovascular, hepatic, renal or pulmonary disease; history of (within 12 months) or current abuse of alcohol or drugs; and administration of non-registered investigational drugs within 3 months prior to screening.
When all inclusion criteria and none of the exclusion criteria were met, the subject was considered to be eligible.
Study drugs and study procedures
Pre-treatment with intranasal buserelin (3x200 µg, Suprecur®; Hoechst, Frankfurt, Germany) for pituitary down-regulation was started in the midluteal phase. Recombinant FSH (Batch nos. CP 096025 and CP 096149) was supplied as lyophilized spheres in ampoules containing 50 or 100 IU FSH in-vivo bioactivity. For subcutaneous injection, two ampoules were reconstituted with 1 ml solvent. Human chorionic gonadotrophin (HCG) (Pregnyl®; NV Organon, Oss, The Netherlands) in doses of 5000 IU per ampoule was supplied to trigger ovulation. For i.m. injection of HCG, one ampoule was reconstituted with 1 ml solvent.
Demographic and other subject variables were obtained during the admission visit. In addition, the general medical and gynaecological histories were obtained, a general medical and gynaecological examination was performed, and an endocrinological, biochemical and haematological analysis of blood was performed. All general medical, biochemical and haematological measurements were performed according to routine procedures of the individual study centres. Eligible subjects were randomized by receiving a subject number from a randomization list corresponding with patient boxes in which the medication was kept. The 50 and 100 IU ampoules were indistinguishable one from another. The randomization was carried out in blocks of four and was computer-generated by using random numbers.
When down-regulation was achieved (defined as oestradiol serum concentrations <200 pmol/l), treatment with rFSH was started and continued until at least three follicles 17 mm had developed. Dose adaptations were not allowed. HCG (5000 IU) was given to trigger ovulation. After oocyte retrieval and ICSI, a maximum of three embryos were replaced. Progesterone was given as luteal support according to the routine regimens of the centre.
Serum concentrations of oestradiol, progesterone, FSH and LH were measured at baseline (moment of down-regulation) and on the day of HCG injection, using local assays. Cycle monitoring included frequent vaginal ultrasound investigations and oestradiol measurements.
Study end-points
The primary efficacy end-point was the number of oocytes retrieved, and the primary efficiency end-point was the total dose of rFSH used.
Secondary variables included the number of follicles 17,
15,
13 and
11 mm at the day of HCG administration, serum concentrations of FSH, LH, progesterone and oestradiol at the day of HCG administration, treatment duration, embryo development rate, the number of transferable embryos, clinical pregnancy rate, implantation rate, miscarriage rate and vital pregnancy rate.
Retrieved oocytes were classified as oocytes with intact zona pellucida and clear cytoplasm, metaphase II oocytes (extrusion first polar body, no germinal vesicles), metaphase I oocytes (no polar body, no germinal vesicle) and germinal vesicles stage oocytes.
The number and quality of embryos were assessed according to the following criteria: type 1, all blastomeres of equal size without the presence of anucleate fragments; type 2, not all blastomeres of equal size, anucleate fragments present in <20% of volume; type 3, not all blastomeres of equal size, anucleate fragments present in >20% of volume; and type 4, the embryo is totally fragmented. Transferable embryos were defined as embryos of either type 1, 2 or 3.
The embryo development rate was defined as the number of transferable embryos divided by the total number of oocytes injected. The implantation rate was defined as the number of gestational sacs seen on ultrasound examination divided by the total number of embryos replaced. A vital pregnancy was defined as an intrauterine pregnancy with positive heart action. No strict definition of OHSS was given; in the analysis of the occurrence of this syndrome, its incidence was based on the fact that the investigator reported it as such.
Sample size considerations
With 100 subjects included in each treatment group, and assuming a SD of 450 IU of rFSH for the total dose used and a SD of 6.4 oocytes for the number of oocytes retrieved, a difference of 180 IU Puregon and 2.5 oocytes could be detected between the two treatment groups with a power of 80% using a two-sided t-test and a significance threshold of 5% (not adjusted for two primary outcomes).
Statistical analysis
The analysis of variance (ANOVA) was performed for non- dichotomous variables. If the ANOVA was not applicable, the Wilcoxon rank sum test was performed. To communicate the Wilcoxon results in clinically meaningful entities, Cochran's method (adjusted for centre) was used, and a pseudo-interaction test based upon midranks (Koch et al., 1990) was calculated to obtain an impression of the consistency of results across centres. Dichotomous variables were analysed by MantelHaenszel statistics, adjusted for centre.
The statistical analysis included all subjects who received at least one injection of rFSH.
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Results |
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Both groups had comparable demographic characteristics (Table I). The overall mean age was 27.5 years in both groups. The mean age in the low and high-dose groups at the five centres were 27.9 and 27.4, 28.2 and 28.0, 27.0 and 27.1, 25.8 and 27.3, and 26.7 and 26.9 years respectively. The cause of infertility was male in all cases. A tubal factor was also present in five subjects of the 100 IU group and two of the 200 IU group.
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Primary end-points
Results on the primary efficacy and efficiency end-points are listed in Tables II and III. For each started cycle, significantly more oocytes were retrieved in the 200 IU group (12.0 versus 5.7, P < 0.001). However, the total rFSH dose used was considerably lower in the 100 IU group (1121 versus 1875 IU, P < 0.001).
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Secondary end-points
Results on the secondary outcomes are listed in Table IV. The mean number of follicles
11 mm on the day of HCG administration was 13.1 in the high-dose group and 10.6 in the low-dose group (P = 0.01). Correspondingly, significantly more follicles
13 mm and
15 mm had developed in the high-dose group. The numbers of follicles
17 mm were similar in both groups (5.9 and 6.5 in the low-dose and high-dose treated women respectively). On average, the treatment duration was longer in the low-dose group (11.1 days) than in the high-dose group (9.4 days, P < 0.001).
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Significantly more oocytes with intact zona pellucida and clear cytoplasm (11.2 versus 5.2, P < 0.001) and metaphase II oocytes (9.3 versus 4.6) were seen in the high-dose group. The number of metaphase I oocytes was lower in the 100 IU group (0.5 versus 1.1, P < 0.01), as was the number of germinal vesicle stage oocytes (0.3 versus 0.9, P < 0.001).
Spermatozoa used for ICSI had an average (± SD) count of 6.3 ± 13.6 x106/ml in the low-dose group, compared with 6.8 ± 13.8x106/ml (SD 13.8) in the high-dose group (P = NS). Progressive motility was 47.8 and 42.3% respectively (P = NS).
Significantly more transferable embryos were obtained in the 200 IU group (5.6 versus 3.7, P < 0.001). The overall embryo development rate was 64.6 and 60.3% in the low-dose and high-dose groups respectively. The centre-adjusted mean number of embryos transferred was 2.7 in both groups.
Two embryos more (2.9 versus 0.9) were frozen in the 200 IU group than in the 100 IU group.
The clinical pregnancy rates were not statistically significantly different in the two dose groups, both per started cycle (20.3 versus 24.7%) and per embryo transfer (27.6 versus 28.3%). The miscarriage rate in the 100 IU group appeared to be lower than that in the 200 IU group (1.5 versus 9.0% per embryo transfer), but the difference was not significant. Vital pregnancy rates per started cycle were not significantly different (19.2% in the 100 IU group versus 16.9% in the 200 IU group). Per embryo transfer, the vital pregnancy rates were 26.2 and 19.3% in the low- and high-dose groups respectively (P = NS).
Cycle cancellations
Among 91 women who started in the low-dose group, 74 had an oocyte retrieval (81%) and 67 an embryo transfer (74%). In the high-dose group, 86 women out of 88 who started rFSH treatment had an oocyte retrieval (98%), and 78 (89%) had an embryo transfer.
Reasons for drop-out before the stage of embryo transfer differed between the two treatment groups. In the low-dose group, insufficient ovarian response (n = 14), premature LH surge and (or) progesterone too high (n = 2), poor quality oocytes at retrieval (n = 1), adverse event (n = 1), no fertilization (n = 2) and other causes (n = 4) were reported. In the high-dose group, the reasons for drop-out were risk for hyperstimulation (n = 1), insufficient ovarian response (n = 1), poor quality oocytes at retrieval (n = 1), adverse events (n = 2), no fertilization (n = 4) and other causes (n = 1).
Safety
Ovarian hyperstimulation syndrome was reported in four cases, all in the 200 IU group, and all patients were hospitalized (4.5%). One other patient in the 200 IU group was hospitalized because of an ectopic pregnancy. No reports of OHSS were made in the 100 IU group.
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Discussion |
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The benefit of using 200 IU to treat these women was that two extra embryos could be frozen. However, four hospitalizations due to OHSS occurred in that group, compared with none in the 100 IU group. Although this study does not prove that OHSS occurs significantly more often in women treated with 200 IU rFSH, caution is nevertheless warranted in this situation. In interpreting these data, it is important to realize that the average age of the women in the current study was relatively low at 27.5 years.
It has been shown in the past, that with a successful cryoprogramme, the augmentation in pregnancy rates achieved by replacing frozenthawed embryos can be clinically relevant (Jones et al., 1997). From that perspective, a high number of embryos available for freezing is attractive. Although in the current study overall fresh pregnancy rates were not different between the groups, it is likely that the pregnancy rate per single ovarian stimulation cycle including frozenthawed embryo replacements will be higher in the high-dose group. However, due to limited drug budgets as imposed by health authorities, insurance companies or hospitals, a low-dose alternative might be more acceptable.
It is difficult to define an `ideal' dose of rFSH based on the findings of the current study. Ovarian sensitivity for gonadotrophins varies among women, and an individualized approach is most likely to result in a sufficient number of oocytes with minimal side effects. In addition, more data are needed on the effects of other daily doses (e.g. 150 IU).
It is important to note that large inter-centre variations were observed in the primary outcome parameters. Even using the same protocol, the number of collected oocytes per started cycle ranged from 2.8 to 7.2 in the 100 IU group and from 9.0 to 19.1 in the high-dose group. This was not due to differences in age of the women treated. However, despite this centretreatment interaction, the difference between the two groups in numbers of oocytes retrieved was highly significant.
Also, there was a discrepancy between the number of large follicles seen by using transvaginal ultrasonography on the day of HCG administration and the number of oocytes actually retrieved. In the 100 IU group, a mean of 9.6 follicles 13 mm were seen on ultrasound, and only 6.9 oocytes were retrieved, whereas in the high-dose group 12.0 oocytes could be collected with 12.2 follicles
13 mm on ultrasound. These data suggest that in women in the low-dose group the oocyte recovery might have been performed more efficiently, assuming that it should be possible to retrieve an oocyte from a follicle of at least 13 mm diameter. It is also possible that, due to difficult counting, the actual number of large follicles was underestimated because many are being seen on ultrasound.
Interestingly, the previously observed increased miscarriage rate in the 100 IU group (13.3 versus 1.2%; Out et al., 1999) was not seen in the current study. In fact, more miscarriages occurred in the 200 IU group (9.0 versus 1.5%), although this difference was not statistically significant. This shows that interpretation of the secondary end-points such as miscarriage rates should be made with caution, as these studies were not set up to address differences in these outcomes. Given the fact that many analyses were carried out, somesuch as the tests on miscarriage ratesmight have been statistically significant purely by chance.
Earlier, a doseresponse relationship between rFSH and number of oocytes recovered was not shown in women between 37 and 39 years of age: increasing the daily dose from 150 IU to 250 IU did not result in more retrievable oocytes (Out et al., 2000). It has been shown that the magnitude of the ovarian response is predictive of IVF outcome. High responders attain greater numbers of recovered oocytes and embryos for transfer, and also experience a higher fresh pregnancy rate than low responders (Hall et al., 1999
). This might be due to better possibilities of selecting the morphologically best embryos for transfer. However, in view of the data from the current study it is also possible that the increased pregnancy rates in high responders are due to a general favourable reproductive performance of these women rather than the distinct result of an ovarian stimulation regimen aimed at maximizing the number of retrievable oocytes.
In conclusion, this study has confirmed earlier observations (Devroey et al., 1998; Bergh, 1999
; Out et al., 1999
) that a substantial proportion of women undergoing ovarian stimulation before ICSI benefit from rFSH doses as low as 100 IU per day. This seems to be associated with a lower incidence of OHSS, as well as lower costs of treatment. The challenge for the future is to investigate patient characteristics that predict ovarian response before treatment, thereby enabling rational decisions to be made on the rFSH starting dose.
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Acknowledgements |
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Notes |
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References |
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Submitted on July 5, 2000; accepted on March 6, 2001.