1 City West IVF, City West House, 12 Caroline Street, Westmead, NSW, Australia 2145 and 2 Fertility Associates, Ascot Integrated Hospital, 90 Greenlane Road East, Remuera, Auckland, New Zealand
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Abstract |
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Key words: assisted reproduction treatment/ovulation induction/recombinant HCG/recombinant FSH/urinary HCG
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Introduction |
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The history of gonadotrophin use when derived from either animal or human tissues has, however, not always been without clinical danger (e.g. antibody formation from pregnant mare serum gonadotrophin; CreutzfeldJacob disease from human pituitary gonadotrophin) so, as recombinant technology evolved, the logic of increasing both a compound's purity and safety could not be ignored. Such a uniform, specific product would mean that drug production would no longer be dependent on the vagaries of urine collection and hormone extraction, allowing commercial production to be adjusted according to market requirements. In addition all urinary contaminants would also be removed. Furthermore this would allow the safe s.c. administration of a compound with less batch-to-batch variation than has been demonstrated for urinary menopausal gonadotrophin preparations (Rodgers et al., 1995) and thus potentially improve biological efficiency. These premises have been extensively reviewed (Fauser, 1998
; Loumaye et al., 1998
; Prevost, 1998
; Daya and Gunby, 1999
).
Recombinant HCG (rHCG) has been manufactured by transfecting non-human cell lines (Chinese hamster ovary cells) with genetic material capable of replicating identical amino acid sequences to the human compound and developed as a pharmaceutical product named Ovidrel®. The clinical uses of HCG are based on its molecular similarity to LH where the first 114 amino acids of each compound share 80% homology. The structural similarity of both compounds is further functionally reflected by the fact that they bind to the same hormone receptor (Pierce and Parson, 1981).
Pre-clinical studies with Ovidrel® in monkeys and rats have demonstrated that it is pharmacologically safe, well tolerated over a large dose range and has no intrinsic toxicity. Similarly, in mice, rHCG triggered germinal vesicle breakdown and extrusion of the first polar body and induced a dramatic increase in progesterone production (Smitz et al., 1998). In limited unpublished clinical studies the linearity of the pharmacokinetics of Ovidrel® after a single administration of increasing doses was comparable to that of uHCG over a 50020 000 IU dose range and the terminal half-life was estimated as ~30 h. A dose of 250 µg Ovidrel® was also established to be equivalent to 5000 IU Profasi® as an ovulatory stimulus in monkeys (information provided by AresSerono International SA).
The object of this study was to assess the safety and efficacy of s.c. rHCG (Ovidrel®) compared with i.m. uHCG (Profasi®) for inducing final oocyte maturation and initiation of follicular luteinization in patients who had undergone pituitary down-regulation and ovulation induction as part of their assisted reproductive treatment.
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Materials and methods |
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Patients were excluded if they had: (i) any clinically significant systemic disease; (ii) a body mass index of >30 kgm2; (iii) polycystic ovarian syndrome (PCOS); (iv) a previous history of severe ovarian hyperstimulation syndrome (OHSS); (v) any medical condition which might interfere with the absorption, distribution, metabolism or excretion of the medications; (vi) a prior or poor response to gonadotrophin therapy or a previous history of intolerance to FSH, gonadotrophin releasing hormone (GnRH) agonists or to HCG; (vii) active substance abuse (including smokers consuming more than 20 cigarettes/day); more than three previous assisted reproductive treatment attempts or any form of infertility treatment in the previous two menstrual cycles or (viii) a male partner with leukospermia or bacterial infection detected in a semen analysis within the last 3 months.
Treatment
Standard assisted reproductive treatment protocols were used for ovarian stimulation. Thus patients received 400 µg of intranasal nafarelin (Synarel; Searle Pharmaceutical Products, Sydney, Australia) twice daily from the mid-luteal phase to achieve down-regulation (oestradiol concentration 180 pmol/l, progesterone <4.0 nmol/l, LH <3.0 IU/l). This was confirmed by measurement of serum concentrations of oestradiol LH and progesterone. If down-regulation could not be achieved after 10 days the patient was withdrawn from the study. On commencing ovarian stimulation with recombinant FSH (rFSH, Gonal-F®) the dose of nafarelin was reduced to 200 µg twice daily.
Nafarelin and rFSH treatment (not required to remain at a fixed dose but required to be <450 IU/day) were administered until the criteria for induction of ovulation were met (largest follicle 18 mm diameter; presence of at least two other follicles with a mean diameter
16 mm; oestradiol concentration in excess of 550 pmol/follicle; cumulative Gonal-F® dose <7500 IU), the last injections being given within 24 h of rHCG or uHCG. At that time the patient was randomly assigned (according to a computer-generated randomization list) to receive either: (i) a s.c. injection of 250 µg (5000 IU) rHCG (Ovidrel® supplied in vials) and an i.m. injection of placebo (supplied in glass snap-top ampoules) or (ii) an i.m. injection of 5000 IU uHCG (Profasi supplied in ampoules) and a s.c. injection of placebo (supplied in vials). Gonal-F, Ovidrel, Profasi and placebo vials were packaged and supplied by AresSerono International SA, Geneva, Switzerland. Oocytes were retrieved, assessed and inseminated/injected in-vitro and up to three embryos (but usually two) were replaced 23 days later. The luteal phase was supported by progesterone pessaries (200 mg/day; Orion Laboratories, Welshpool, WA, Australia) starting the day of oocyte collection and continuing for 2 weeks or until menstruation. For those women who did not menstruate, blood sampling for a pregnancy test was performed 1821 days after HCG followed by an ultrasound scan around day 42.
Study endpoints
The primary endpoint of the study was comparison of the total number of oocytes retrieved per patient who received either rHCG or uHCG and to show equivalence between the two products. For adherence to the multi-trial protocol, equivalence had been pre-defined as falling within a clinically acceptable range of ± 3 oocytes. This value had been established from the number of oocytes retrieved in a similar patient population to that enrolled in this study and was calculated to give statistical power with the number of patients being studied.
Secondary comparisons included the number of patients with at least one oocyte retrieved; the number of oocytes retrieved per number of follicles aspirated; the number of mature oocytes (i.e. metaphase I plus metaphase II); the number of normally fertilized oocytes; and the number of cleaved embryos (embryo utilization). Similarly endocrine profiles and ultrasonic evaluation of endometrial thickness during treatment was also compared. Total obstetric outcome was also evaluated but could not always be completed because some patients still have embryos cryopreserved. The incidence and severity of adverse events (however minor), local tolerance to rHCG or uHCG administration and the incidence of mild, moderate or severe OHSS in each group was also assessed by clinical, ultrasonographic and biochemical parameters. All data entry, compliance and analysis was performed by contract statisticians (Datapharm Australia Pty Ltd, PO Box 220, Five Dock, NSW 2046, Australia).
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Results |
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Of those patients who met protocol requirements nine did not proceed to embryo transfer, five in the rHCG group and four in the uHCG group. In the rHCG group one was because of failed fertilization, three due to the risk of OHSS and one due to poor quality embryos which were unsuitable for either transfer or cryopreservation. In the uHCG group one woman was discontinued because of failed fertilization and three others due to the risk of OHSS. None of the women at risk or any others in the trial developed OHSS, and the two treatment groups had similar numbers of patients with excess embryos suitable for cryopreservation.
Pre-study comparisons
Demographics
There were no differences between the two HCG treatment groups for any demographic characteristic assessed. The mean (± SD) age of women was 32 ± 4 years, with a range of 2138 years. Most (84.5%) were non-smokers. Their mean (± SD) weight and body mass index (BMI) were 63.2 ± 9.5 kg and 23.3 ± 3.1 kg/m2 respectively.
Infertility history
The two treatment groups were similar for all gynaecological and obstetric parameters with the exception of the type of infertility. Thus there was a higher percentage of patients with primary infertility in the rHCG group (79.5%) than in the uHCG group (57.5%), and a correspondingly higher percentage of patients with secondary fertility in the uHCG group, 42.5% compared with 20.5% in the rHCG group (P = 0.029). The median duration of infertility for all women was 3 years, with male infertility being cited most commonly as the cause of infertility in both treatment groups (55.8% rHCG versus 51.1% uHCG). Male factor infertility was given as the only cause of infertility by 23 (52.3%) couples in the rHCG group and 18 (45.0%) couples in the uHCG group.
Endocrinology and drug use
There were no differences between the treatment groups for any of the initial screening hormone concentrations (mean ± SD, rHCG group versus uHCG group: FSH, 4.6 ± 2.1 versus 4.3 ± 1.9 IU/l; LH, 4.6 ± 2.5 versus 4.9 ± 2.6 IU/l; testosterone, 1.1 ± 0.4 versus 1.2 ± 0.5 nmol/l; oestradiol, 338.7 ± 198.2 versus 371.8 ± 185.8 pmol/l; progesterone, 32.8 ± 33.5 versus 33.8 ± 30.1 nmol/l; prolactin, 16.2 ± 7.7 versus 13.2 ± 6.8 ng/ml). Similarly there were no statistically significant differences between treatment groups in either the duration or total dosages of nafarelin and rFSH used. Thus patients in the rHCG group were given a mean (± SD) total dose of 14 627 ± 2035 µg nafarelin for a mean period of 24 days down-regulation compared to the uHCG group of 15 370 ± 2743 µg for a mean period of 25 days down-regulation, neither dose nor duration being significantly different. Comparing rFSH use, women in the rHCG group received a total dose of 2141 ± 725 IU (i.e. 28.5 ampoules x75 IU) compared to 2067 ± 910 IU (i.e. 27.6 ampoules x75 IU) in the uHCG group during a mean period of 12 days ovarian stimulation in each group.
Study comparisons
Primary efficacy analysis
The number of oocytes retrieved in the two groups was examined using an analysis of covariance model taking into account the number of follicles >10 mm at the baseline ultrasound assessment together with the research unit (Australia or New Zealand) and the couples' infertility type (primary or secondary). All interaction effects (two-, three and four-way) were tested and found to be non-significant and were dropped from the model one by one, using a backwards stepwise method. Infertility and the research unit effect were found to be non-significant and were dropped from the model. Thus, as shown in Table I, in the rHCG group the mean ± SD number of oocytes retrieved was 10.8 ± 4.5 whereas in the uHCG group it was 10.3 ± 5.1, and the least square means ± SEM, after adjusting for the baseline follicle count, were 11.0 ± 0.6 and 10.0 ± 0.6 respectively. The mean difference between the two treatment groups was 0.475, with a 90% confidence interval (CI) of 0.945, 1.895, and the least squares mean difference was 1.046 with a 90% CI of 0.382, 2.474. Since both CI were within the required range of ± 3 the two treatments could be declared equivalent.
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Serum progesterone concentrations were assessed during the luteal phase (Table II) and in both groups concentrations rose consistently. Although there were no significant differences between the peri-ovulatory period, a significant difference became apparent 67 days after HCG with progesterone concentrations being greater (353.2 ± 215.1 versus 234.1 ± 129.4 nmol/l) in the rHCG group. Serum HCG concentrations were also assessed on the same days as progesterone. In both groups the mean HCG concentration fell consistently during the luteal phase (Table II
) but statistically significant differences occurred at all time points (P < 0.05), mean concentrations being greatest in the rHCG group.
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Adverse events
There were no clinically significant results for haematology, biochemistry or urine analysis and no clinically significant changes during the study. Adverse events were reviewed overall and were also classified for each patient according to the highest severity of any episode they experienced (Table III). There was only one episode that was severe (i.m. injection site pain; uHCG group) and this resolved on the same day. All other events were considered mild or moderate. Local tolerance to injections was also recorded. As expected from the adverse event analysis, pain was the most reported local injection reaction in the active treatment for both routes, recorded by six patients in each, 13.6% in rHCG and 15% in uHCG. However, in the placebo treatment routes, there appeared to be a higher incidence of pain in the placebo rHCG route (s.c., 12 patients, 30.0%) than in the placebo uHCG route (i.m., two patients, 4.5%), which suggests a possible effect of the injection vehicle or the injection method.
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Discussion |
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These events are also induced by an injection of HCG through exactly the same sequence, the only difference being in the pharmacokinetic profile where the mean duration of the `surge' after an i.m. injection of 5000 IU uHCG is longer than for LH (~96 h) and maximum concentrations may be less (Fischer et al., 1993). Current practice is that
5000 IU uHCG is an acceptable ovulatory dose and that lesser concentrations can lead to reduced oocyte recovery and a lower fertilization rate in assisted reproductive treatments (Abdalla et al., 1987
). Duffy et al. (1996) have also demonstrated in monkeys that rHCG is equally efficacious as uHCG in stimulating the steroidogenic and peptidergic activities of the corpus luteum during simulated early pregnancy. While a bolus of rFSH has also been shown to be equivalent to rHCG for the reinitiation of meiosis in macaque oocytes, it was not capable of sustaining luteal function (Zelinski-Wooten et al., 1998
), a necessity after down-regulation of women undergoing assisted reproductive treatment. Thus the higher mid-luteal concentrations of progesterone after rHCG in this study might offer advantages for sustaining the luteal phase in down-regulated patients, conferring an additional advantage in those at minimal risk of OHSS and confirming the recent suggestion that a re-evaluation of the approach to luteal supplementation might be timely (Tay and Lenton, 1999
).
The primary objective of this phase III clinical trial was to compare the total number of oocytes recovered/retrieved per patient who received either s.c. rHCG or i.m. uHCG. The mean number of oocytes retrieved per patient was 10.8 for the rHCG group and 10.3 for uHCG group and equivalence could therefore be declared, since the two-sided 90% CI for both the raw and adjusted differences between the two treatment groups were within the upper and lower bounds of the pre-defined clinically acceptable range of ± 3 oocytes. In the secondary efficacy analysis there were similarly no significant differences between the two treatment groups in the number of mature oocytes retrieved, the number of normally fertilized oocytes per patient 1 day after collection, the number of cleaved embryos per patient, or in the implantation and pregnancy rates.
Of the only two significant differences between the treatment groups, both were secondary effects and offered potential advantages for rHCG. The progesterone concentrations 67 days after HCG administration were greater in the rHCG group and serum HCG concentrations were higher in the rHCG group than in the uHCG group at all time points during the luteal phase. However, whether this would allow a more favourable luteal environment for implantation will need further study.
With respect to patient use, reported side-effects were minor and generally related to the injection site (pain, inflammation or reaction) but a higher proportion of patients (42.5%) experienced these reactions in the uHCG group (s.c. placebo) compared with the use of rHCG (i.m. placebo; 20.5%). Thus rHCG appears also to show better patient acceptance. Hypersensitivity reactions are a documented complication of urinary-derived gonadotrophin therapy and, in a case report, a patient allergic to uFSH was successfully treated with r-FSH (Whitman-Elia et al., 1998).
To conclude, a s.c. injection of a 250 µg rHCG (Ovidrel®) has been shown to be equivalent to an i.m. injection of 5000 IU uHCG (Profasi®) in inducing final follicular maturation, resumption of meiosis in oocytes and early luteinization in women undergoing ovulation induction with rFSH (Gonal-F®) prior to assisted reproductive treatment. The authors believe that the births resulting from this study are the first reported after IVF and ICSI using this combination of recombinant gonadotrophins, confirming the prediction of Agrawal et al. (1997) in their case report of a single ongoing pregnancy.
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Acknowledgments |
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Notes |
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References |
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Submitted on December 13, 1999; accepted on March 16, 2000.