1 Departments of Obstetrics and Gynaecology and 2 Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, Ontario, Canada
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Abstract |
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Key words: assisted reproductive technology/IVF/metaanalysis/recombinant FSH
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Introduction |
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Human FSH is a glycoprotein hormone consisting of two non-co-valently linked, non-identical protein chains ( and ß subunits) to each of which are attached two complex carbohydrate structures. Endogenously secreted FSH consists of a family of isoforms with identical primary structures, but with variable composition of the carbohydrate side-chains and sialic acid residues (Chappel, 1995
). This heterogeneity is responsible for differences in plasma half-life and consequent biological activity of the isoforms (Chappel, 1995
).
Until recently, the main source of exogenous FSH was urine of postmenopausal women. Menotrophin [human menopausal gonadotrophin (HMG)], which consists of a mixture of FSH, LH and urinary proteins, was the first such preparation. Two decades later, a further refinement in the extraction process permitted the removal of LH by absorbing it onto an antibody column. The resulting urofollitrophin, although essentially devoid of LH activity, still contained urinary protein contaminants. Over the last decade, further advances in purification techniques, using monoclonal antibodies, enabled the extraction of FSH from urine to produce highly purified (HP) urofollitrophin from which all of the contaminant proteins and LH had been removed leaving only FSH activity. Recently, biotechnology has made available a recombinant preparation of FSH (rFSH), produced by inserting the genes encoding for and ß subunits of FSH into expression vectors that are transfected into a Chinese hamster ovary cell line (Howles, 1996
). The use of mammalian cells for this purpose is necessary because glycosylation is required to ensure full biological activity of the protein (Howles, 1996
). There are two rFSH preparations currently available for clinical use: follitrophin alpha, marketed as Gonal-F® by Ares-Serono, Geneva, Switzerland, and follitrophin beta, marketed as Puregon® or Follistim® by NV Organon, Oss, The Netherlands. Although both preparations have been developed using the same technique, the post-translation glycosylation process and purification procedures are not identical (Olijve et al., 1996
). The purification procedure used for follitrophin alpha includes the use of immunochromatographic methods, whereas purification of follitrophin beta does not involve immunological methods (Olijve et al., 1996
).
The purity and batch-to-batch consistency of rFSH makes it an attractive alternative to urinary FSH (uFSH). In a combined analysis of three randomized trials, rFSH was observed to be associated with a significantly higher pregnancy rate in IVF treatment cycles compared to urinary gonadotrophins (Out et al., 1997). However, in this study, the trials selected were limited to those in which follitrophin beta was used. Furthermore, in one of the trials, rFSH use was compared with HMG (Jansen and Van Os, 1996
; Out et al., 1996
). The observation that HMG is not as efficacious as FSH (Daya et al., 1995a
,b
; Daya, 1998
) suggests that the inclusion of this trial is likely to have biased the overall conclusion of the combined analysis. Therefore, the purpose of this study was to review the evidence from all randomized trials comparing rFSH (both follitrophin alpha and follitrophin beta) with uFSH to evaluate the relative efficacy with respect to clinical pregnancy rates in treatment cycles with IVF or ICSI.
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Materials and methods |
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The Excerpta Medica CD: Fertility data base, which contains publications in human reproduction and all publications in the Excerpta Medica Data base (Embase) related to obstetrics and gynaecology, was searched, covering the period 1985 to October 1998. The search was performed on titles and abstracts using `recombinant FSH', `IVF' and `randomized' as keywords.
The bibliographies of relevant publications and review articles were scanned, and abstracts of major scientific meetings from 1992 to 1999 were hand-searched. The Cochrane Menstrual Disorders and Subfertility Specialized Register was also searched. When necessary, authors of relevant abstracts were contacted for detailed data on their studies. Peer consultation was sought for any remaining articles. Finally, the pharmaceutical companies that manufacture the gonadotropin preparations were consulted for additional information.
Reports of clinical trials were selected only if they met the inclusion criteria and if the outcome information was provided in sufficient detail to enable the data to be pooled.
Study inclusion
This systematic review was limited to trials reporting random allocation to rFSH (either follitrophin alpha or follitrophin beta) or uFSH (either urofollitrophin or urofollitrophin HP) for ovarian stimulation in infertile women undergoing treatment with IVF or ICSI. Trials were included whether or not the stimulation protocol included pituitary down-regulation with gonadotrophin releasing hormone agonists (GnRHa). The primary outcome of interest was the clinical pregnancy rate (usually defined as a gestational sac seen by ultrasonography), which was calculated per treatment cycle commenced.
Validity assessment and data extraction
For many of the trials, additional information concerning the methods of the trial and the outcome data was obtained from the authors. The methodological quality of each trial was assessed using a predetermined scoring system consisting of eight criteria as shown in Table I. Each trial was assessed independently by two reviewers and ranked for its methodological rigor and its potential to introduce bias. The evaluation included how the randomization procedure was undertaken and whether it was concealed, the use of blinding, the presence of cointervention, the completeness of follow-up of trial subjects, whether a sample size calculation had been performed, whether a crossover design was used, in which case only data from the first period (i.e. before crossover) were admissible, and whether the unit of comparison was patient or treatment cycle. Any disagreement between the two reviewers was resolved by consensus whenever possible. In the event of persistent disagreement, a third reviewer was consulted. Data were extracted and checked for accuracy in a second review.
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Effectiveness was evaluated using the Peto modification of the Mantel-Haenszel method (Mantel and Haenszel, 1959; Peto, 1987
), which is a test of overall association across all trials. A test of the homogeneity of treatment effect across all trials was performed (Breslow and Day, 1980
). A nonsignificant result (i.e. lack of heterogeneity) indicates that no trial has an OR that is statistically significantly worse or better than the overall common OR obtained by pooling the data. Only when homogeneity of treatment effect was confirmed were the data pooled using the fixed effects model, otherwise the random effects model was used.
A funnel plot (in which the effect estimate of each trial was plotted against the precision of the effect, calculated as the inverse of its standard error) was used to detect publication bias. The value of the funnel plot is based on the fact that precision in estimating the underlying treatment effect will increase as the sample size of the trial increases (Egger et al., 1997). Thus, results from small trials will have a wide scatter at the bottom of the scatter plot, the spread decreasing as the trials become larger. A symmetrically inverted funnel shape to the scatter plot indicates that publication bias is unlikely.
Subgroup analyses were performed to identify whether the two types of follitrophin (i.e. alpha or beta), and the two types of fertilization procedure (i.e. ICSI or IVF) had any effect on the overall combined result. The data were recoded according to these variables and subjected to logistic regression analysis to identify the model that best predicted clinical pregnancy per started cycle.
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Results |
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The validity scores for methodological rigour of the 12 trials included in the analysis are shown in Table II and indicate that most trials are of moderate to high grading based on the predetermined validity criteria used. The methodological details of these trials are listed in Table III
. Apart from minor differences, the patient profiles were quite similar and representative of the infertile population requesting treatment, and the interventions used conform to the currently accepted standards of care. All trials used GnRHa in a long protocol as part of the ovarian stimulation regimen.
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A funnel plot of the ORs for clinical pregnancy per cycle started, shown in Figure 2, demonstrates that the data are distributed along a symmetrical, inverted-funnel shape, indicating that publication bias was unlikely to be present. Although the median sample size of the trials was 160, the range extended from 55 to 981, with the number in all except two trials (Out et al., 1995
; Schats et al., unpublished results) below 300. Thus, no trial was designed with adequate power to test the null hypothesis of no difference in pregnancy rates between the two gonadotrophin preparations.
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Fertilization procedure |
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Data for ICSI were available from four trials in which the ICSI cycles could be separated from the IVF cycles, and from one trial in which ICSI was the only procedure performed. In the five trials in which ICSI was performed (with an aggregate sample size of 567 cycles), there was no significant heterogeneity of treatment effect (Breslow-Day statistic 1.4, P = 0.84). The common OR was 1.02 (95% CI, 0.721.45, P = 0.92) in favour of rFSH (Figure 3). The risk difference was 0.3% (95% CI, 7.47.9%).
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Follitrophin type and fertilization procedure |
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Logistic regression analysis |
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Discussion |
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The possibility of publication bias influencing the results is a potential concern with any meta-analysis, and every effort has been made to identify all the trials that have been published and as many of the unpublished trials as is humanly possible. The funnel plot is a useful graphical assessment of the likelihood of publication bias being present (Egger et al., 1997). The scatter plot of the odds ratios from the trials in this review demonstrated a symmetrical, inverse-funnel shape (Figure 2
), thereby providing reassurance that selective publication is unlikely to be a source of bias in this meta-analysis.
Although the patient profiles in the trials were relatively similar, and the IVF and ICSI procedures used were standard, there were differences in the type of gonadotrophin preparations administered. Two different types of follitrophin (i.e. alpha and beta) were compared with two different types of uFSH (i.e. urofollitrophin and urofollitrophin HP), thus producing four pair-wise comparisons. Subgroup analyses of each of these comparisons is not reliable because only one trial compared follitrophin beta with urofollitrophin HP (Andersen et al., 1998). Nevertheless, the common OR and risk differences for clinical pregnancy started per cycle for each of the comparisons are as follows: follitrophin alpha versus urofollitrophin (OR = 1.19, risk difference 2.9%), follitrophin alpha versus urofollitrophin HP, IVF cycles only (OR = 1.42, risk difference 6.1%), follitrophin beta versus urofollitrophin (OR = 1.21, risk difference 3.9%) and follitrophin beta versus urofollitrophin HP (OR = 1.10, risk difference 2.1%). Although some of the differences in the magnitude of these estimates of the treatment effect can be explained by the small number of trials in each comparison, an important factor to be considered is the FSH isoform heterogeneity among the various preparations.
Pituitary gonadotrophins secreted into the circulation consist of a mixture of gonadotrophin molecules with a similar peptide structure, but with wide differences in the carbohydrate moieties (Wide, 1997). When synthesized in the cell, the carbohydrate chains contain sites for the addition of negatively charged groups of either terminal sialic acid residues or sulphate. The number of negatively charged sialic acid and sulphate groups produces variability in the overall charge (as revealed by electrophoresis) of the isoforms. The physiological significance of the charge heterogeneity is not well understood, but is believed to influence the biological properties of the isoform, in terms of its metabolic clearance rate and endocrinological effects at the target organ (Wide, 1997
). The less negatively charged (i.e. more basic) isoforms have a shorter half-life in the circulation, but have increased biological activity (Ulloa-Aguirre et al., 1988
). In contrast, the more negatively charged (i.e. more acidic) forms have a longer half-life but lower bioactivity. These observations suggest that the biological activity of the pharmaceutical preparations of FSH also may be dependent on their charge distribution (Ulloa-Aguirre et al., 1988
).
Based on chromatofocusing studies, the charge distribution patterns for follitrophin beta, urofollitrophin and urofollitrophin HP demonstrated that the amount of material with isoelectric point (pI) <4 was <24%, 40% and 74%, respectively (i.e. progressively more acidic) (Robertson, 1997). The two follitrophins had a similar pattern, except that follitrophin alpha contained glycoforms with a narrower pI band (i.e. 45) than follitrophin beta (3.55.5) (Robertson, 1997
). Thus, if terminal charge pattern dictates in-vivo biological activity, the follitrophins should have greater activity than the urofollitrophins. Unfortunately, information on biological endpoints including oocyte quality, and amount of oestradiol production was not available consistently in all the trials in this meta-analysis, making it difficult to test this hypothesis. Instead, clinical pregnancy was used as a surrogate measure of biological activity. Interestingly, differences in outcome have been reported recently in randomized studies comparing the two follitrophins. The morphological quality of embryos (Phillips et al., 1999
) and the number of `good embryos' obtained (Von During et al., 1999
) were significantly higher with follitrophin alpha compared to follitrophin beta.
The ideal method of evaluating the relative efficacy of the four different gonadotropin preparations is to conduct a four-arm randomized trial with sufficient power and with several biological endpoints, including cycle performance characteristics, oocyte and embryo quality, and incidence of ovarian hyperstimulation syndrome, spontaneous abortion, clinical pregnancy and live birth. Until then, or until more comparative data become available to supplement the data in this meta-analysis, these results indicate that there is a statistically significant difference in clinical pregnancy rates when rFSH is compared with uFSH. This finding, and the knowledge that the recombinant preparations have batch-to-batch consistency, are free from urinary protein contaminants and have the potential of being produced in limitless quantities, indicate that rFSH is more appealing for clinical use than uFSH. A cost-effectiveness analysis is currently being undertaken to determine if there are additional advantages, in terms of cost savings, of using one preparation versus the other.
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Acknowledgments |
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Notes |
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References |
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Submitted on September 21, 1998; accepted on June 24, 1999.