1 Farah Hospital, Amman, Jordan and 2 Reproductive Endocrinology Center, University of Bologna, Via Massarenti 13 40138, Bologna, Italy
3 To whom correspondence should be addressed. e-mail: marco.filicori{at}unibo.it
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Abstract |
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Key words: highly purified hMG/ICSI/LH/ovulation induction/recombinant FSH
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Introduction |
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The purification process of HP hMG allows its administration through the subcutaneous route with an incidence of local cutaneous reactions comparable with recombinant products. A recent large multicentre trial comparing HP hMG with rFSH in 727 treatment cycles has shown that these two drugs appear to be equally effective when employed in assisted reproductive technology programmes (European and Israeli Study Group, 2002). However, that study did not provide information as to the exact hormone profiles resulting from the administration of HP hMG and rFSH. Furthermore, the multicentre trial format employed in this study, the use of both IVF and ICSI as assisted reproductive technology procedures, the heterogeneous pituitary suppression regimens, and the flexible gonadotrophin dosages employed limited the potential for discriminating the features of each hormone preparation examined.
Thus, we elected to plan a study with more uniform procedures in a narrowly defined patient population aiming to characterize more precisely the response to a fixed regimen of either HP hMG or rFSH in closely monitored infertile patients undergoing assisted reproduction treatment. One critical feature of our protocol was the choice to administer a constant FSH dose (150 IU/day) for 14 consecutive days (or less if full response was achieved sooner) to all patients, without response-driven adjustments; this experimental approach was previously applied in several studies (Filicori et al., 1999b
; 2001; European Recombinant Human LH Study Group, 1998
). In this study we chose ICSI as the sole assisted reproductive technique as we wanted unequivocally to document oocyte maturity on the day of oocyte retrieval and to eliminate the occurrence of fertilization failures dependent on factors unrelated to the quality of the ovarian stimulation.
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Materials and methods |
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Study design
The primary outcome measures chosen for this study were (i) the duration of gonadotrophin treatment (days) and (ii) the amount of gonadotrophin needed to achieve comparable levels of folliculogenesis. Secondary outcome measures included term pregnancy rates. The sample size (Pocock, 1983) was based on a previous similar trial that compared HP FSH to hMG (Filicori et al., 2001
); according to this estimate the trial was considered large enough to provide reliable answers to the questions addressed, with 22 patients in each treatment group (t-test, based on difference between treatments of 3.5 and SD of 4). With 50 patients in each treatment group and using a significance level of 5%, the power of the study to detect a similar difference between treatments as seen in the previous trial (using a comparable assessment of variability) was estimated to be >99%. Following the CONSORT guidelines (Moher et al., 2001
), the design and the execution of the patient randomization procedure were kept separate; the randomization sequence and the sealed envelopes containing the treatment group assignments were prepared at the University of Bologna and then sent to Farah Hospital where the patient selection and randomization process was conducted by separate personnel. A total of 150 women were screened for eligibility for the trial and 105 were found to meet the defined entry criteria and were able to receive the GnRH agonist (see below for details), but five patients failed to menstruate after GnRH agonist administration and thus could not be randomized to receive the trial medications. Therefore, 100 patients were randomized to enter the study between August 2000 and May 2001. The randomization process was conducted with sealed envelopes, containing assignment of 50 subjects to each treatment group.
Protocol
The Institutional Review Board of Farah Hospital approved the protocol and all patients provided informed consent. Following randomization, patients of groups A and B were assigned to receive rFSH (Gonal-F; Serono Pharmaceuticals Ltd, UK), or HP hMG (Menopur; Ferring Pharmaceuticals A/S, Denmark) respectively. Although the patients and physicians were aware of the treatment allocation, the ultrasound personnel and the laboratory performing hormone assays were blind. Therapy was started in the mid-luteal phase of a spontaneous menstrual cycle with the administration of a single injection of 3.75 mg of depot triptorelin (Decapeptyl LP; IPSEN Biotech, Paris, France). Ovarian stimulation began 14 days thereafter. The gonadotrophin regimens adopted in each group were for group A rFSH
150 IU s.c. daily, and for group B HP hMG 150 IU s.c. daily; the gonadotrophins were administered at 17:0019:00 by a physician or a nurse, once the results of the daily estradiol (E2) assays were known. This gonadotrophin dose was continued until the occurrence of at least three ovarian follicles
18 mm diameter and of serum E2 concentrations >600 pg/ml (final maturation parameters) or for 14 days if the final maturation parameters were not achieved. After the 14th day of treatment, increments of the gonadotrophin dose were allowed (225 IU/day on days 1517 and 300 IU/day on days 1820). When the final maturation parameters were attained, 10 000 IU of hCG (Profasi; Serono Pharmaceuticals Ltd) were administered to trigger final follicular maturation; the oocyte retrieval procedure was performed 35 h later and followed by a standard ICSI technique. No more than two of the embryos obtained were transferred 2 days after ICSI. Spare embryos were frozen and their outcome is not discussed further. The luteal phase was supported with 400 mg twice daily of intravaginal progesterone (Cyclogest; AH Cox & Co. Ltd., Barnstaple, UK) administered on days 314 following hCG.
Monitoring
Treatment monitoring was conducted throughout gonadotrophin administration. Each day, one blood sample was drawn at 08:0009:00 in a standard manner, and two serum aliquots were obtained: E2 was measured daily in one for clinical monitoring while the second was stored at 20°C for later measurements of LH, FSH, hCG, E2, progesterone and testosterone. Transvaginal pelvic ultrasound was performed on gonadotrophin treatment days 0 and 6 and at alternate days thereafter, until pre-ovulatory hCG administration.
Hormone assays
Frozen serum samples were shipped to the University of Bologna where the hormone assays were performed. LH, FSH, E2, progesterone, testosterone, and hCG were measured with chemiluminescence assays (Chiron Diagnostics ACS 180, Italy). The minimal detectable level (MDL) of LH was 0.1 IU/l; the inter-assay coefficients of variation (CV) at low, intermediate, and high levels of the standard curve were 4.9, 6.3 and 5.5% respectively. The in-vitro addition of up to 200 000 IU/l of hCG did not affect LH determinations in this assay, as assessed at multiple levels of the standard curve. The MDL of FSH was 0.3 IU/l; the interassay CV at low, intermediate and high levels of the standard curve were 2.7, 5.0 and 3.2% respectively. The MDL of hCG in this -specific assay was 0.1 IU/l; the inter-assay CV at low, intermediate and high levels of the standard curve were 3.4, 4.5 and 5.1% respectively. The in-vitro addition of up to 200 IU/l of LH did not affect hCG determinations in this assay, as assessed at multiple levels of the standard curve. The MDL of E2 was 10 pg/ml; the interassay CV at low, intermediate and high levels of the standard curve were 5.7, 3.3 and 6.6% respectively. The MDL of progesterone was 0.1 ng/ml; the interassay CV at low, intermediate and high levels of the standard curve were 4.7, 2.3 and 4.1% respectively. The MDL of testosterone was 0.1 ng/ml; the interassay CV at low, intermediate and high levels of the standard curve were 3.5, 2.8 and 3.4% respectively.
Statistical evaluation
Data were expressed as mean ± SEM. Serum hormone levels were calculated in each cycle as area under the curve (AUC). Normality of distribution of continuous variables was assessed with a KolmogorovSmirnov test (with Lilliefors correction). Between-group differences of normally distributed continuous variables were assessed with parametric statistics (Students t-test), while non-parametric statistics (MannWhitney rank sum test) were employed when the normality test was not passed. Between-group differences in non-continuous variables were assessed with the 2 method with Yates correction, if needed.
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Results |
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Discussion |
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Serum levels of hCG across treatment were moderately but significantly elevated among patients treated with HP hMG (Figure 1 and Table II). Conversely, serum LH levels (Figure 1 and Table II) were largely congruent in patients treated with HP hMG or rFSH. These findings partly contrast with the results of other studies where hMG was compared with HP FSH (Filicori et al., 2001
) or rFSH
(Filicori et al., 2003
) following similar drug regimens, as in these studies both daily LH and hCG serum levels were significantly increased in hMG-treated patients. Thus we confirmed that menotropin administration results in measurable hCG amounts in peripheral circulation (Filicori et al., 2001
; 2002a) and that this hormone is a critical contributor to the overall LH activity of these products.
A somewhat unexpected feature of the current investigation was the finding of significantly higher serum levels of immunoreactive FSH in the patients treated with HP hMG than in those receiving rFSH (Figure 1 and Table II), despite the daily administration of nominally equal amounts of FSH (150 IU/day). Baseline (day 0) values of FSH did not differ in the two treatment groups (Figure 1), thus indicating that this difference was not due to an unbalanced contribution of endogenously secreted FSH. This finding also confirmed the results of a different study where rFSH
and hMG were compared (Filicori et al., 2003
); conversely, in a study where the same amounts of human-derived FSH and hMG were administered (150 IU/day) serum FSH concentrations overlapped (Filicori et al., 2001
). Thus, it appears that a difference exists either in the amounts or in the pharmacokinetics of immunoreactive FSH contained in menotropins and rFSH
. Recombinant FSH preparations contain less acidic FSH isoforms than human-derived FSH that are cleared more rapidly from the peripheral circulation, possibly due to variations in sialic acid or sulphate incorporation (Mannaerts et al., 1996
; DAntonio et al., 1999
). Thus, as previously reported (Matikainen et al., 1994
; Mannaerts et al., 1996
), single or repeated administration of rFSH gives significantly lower immunoreactive serum FSH concentrations than the administration of the same human-derived FSH amounts, consistent with the findings of our study.
The finding of significantly higher E2 levels in patients treated with HP hMG (Figure 2 and Table II) seems to confirm indirectly that this preparation contains greater amounts of LH activity. However, the increment in serum E2 could be due to: (i) an increase in the androgen substrate produced by LH activity-stimulated theca cells (which nevertheless could not be detected through peripheral serum measurements of testosterone; Figure 2 and Table II); (ii) to the direct actions of LH activity on the granulosa cell aromatase system of larger follicles that became responsive to LH through the acquisition of specific receptors (Sullivan et al., 1999); (iii) better stimulation of the aromatase system by the increased immunoreactive FSH concentrations found in the HP hMG group; or through a combination of these factors. Our study confirmed a similar increment in E2 levels that was previously detected when hMG and HP FSH were compared (Fried et al., 1996
; Filicori et al., 2001
) and the findings of the recently published multicentre trial comparing HP hMG to rFSH
(European and Israeli Study Group, 2002).
When we analysed the specific features of the ICSI procedure, we could not identify any significant difference in the number and quality of the retrieved oocytes, oocyte maturity, fertilization and pregnancy rates, and pregnancy outcome between patients treated with HP hMG or rFSH. These results again confirm the findings of the only previous investigation that compared HP hMG with rFSH
in assisted reproductive treatment (European and Israeli Study Group, 2002
) and of other studies where the effects of FSH-only preparations were assessed against LH activity-containing menotropins (Edelstein et al., 1990
; Daya et al., 1995
; Teissier et al., 1999
; Ng et al., 2001
; Strehler et al., 2001
).
In conclusion, we compared HP hMG to rFSH for ICSI in a prospective, randomized, controlled trial. We found that COS was shortened and that the drug dose needed to achieve comparable levels of folliculogenesis was reduced in HP hMG-treated patients. Administration of HP hMG also resulted in higher serum levels of LH activity, immunoreactive FSH, and E2 during COS than in rFSH
-treated patients. These features render the use of HP hMG a valuable option for ovulation induction in assisted reproduction programmes.
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Acknowledgements |
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References |
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Submitted on September 5, 2002; resubmitted on January 16, 2003; accepted on February 27, 2003.