Geneva, Switzerland
Correspondence: E.Loumaye, Ares-Serono Scientific and Medical Communication Department, 15 bis Chemin des Mines, CH-1202 Geneva, Switzerland
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Abstract |
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Key words: follicular maturation/IVF/ovulation induction/rHCG/uHCG
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Introduction |
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Due to its similarity to LH, urine-derived HCG (uHCG) has been used clinically in both men and women for around 40 years. In hypogonadotrophic men, in combination with other gonadotrophins, it is used to stimulate testicular endocrine function. In women, it was used initially to trigger ovulation and luteinization in anovulatory or oligo-anovulatory patients. For the last 20 years it has also been used in women undergoing ovulation induction for assisted reproduction treatment, to bring about final ovarian maturation and to support the corpus luteum. Modern assisted reproduction treatment protocols commonly use long-acting gonadotrophin-releasing hormone (GnRH) agonists for pituitary down-regulation, which suppress endogenous gonadotrophin production, improving folliculogenesis and inhibiting the endogenous mid-cycle LH surge. This has resulted in fewer cancelled cycles, higher numbers of oocytes and more pregnancies (Smitz et al., 1987; Loumaye, 1990
; Hughes et al., 1992
). In these women, therefore, uHCG is used as a surrogate LH surge. In some countries, uHCG is also used in women who are at risk of recurrent miscarriage or threatened abortion.
Despite the widespread use of uHCG, commercial preparations suffer from the same disadvantages as other urine-derived gonadotrophins. Namely, they require the collection of vast quantities of urinea poor quality starting materialleading to unreliable supply and, perhaps most importantly, batch-to-batch inconsistency. Studies with other urinary-derived gonadotrophin preparations (e.g. FSH) have shown that this inconsistency can lead to undesirable variations in response, not just between patients, but also within the same patient from cycle to cycle (Polson et al., 1987). Additionally, contamination of preparations by non-HCG proteins may lead to unpredictable, adverse immunological reactions (Albano, 1996
; Whitmann-Elia, 1998).
The advent of recombinant DNA technology has now permitted the development of a recombinant form of HCG (rHCG), which is very pure, and whose production is independent of urine collection. It is produced in genetically engineered Chinese hamster ovary (CHO) cells, into which the genes encoding for the and ß subunits of HCG have been introduced (Recombinant human FSH product development group, 1998
). Pre-clinical studies in monkeys and rats have confirmed the safety of rHCG (Ares-Serono International, Geneva, Switzerland, data on file). Preliminary clinical studies in volunteers have also shown the product to be well tolerated and have demonstrated that the pharmacokinetic profile of rHCG is similar to that of uHCG. Both show linearity over a 50020 000 IU dose and a terminal elimination half-life of ~30 h (Ares-Serono International, data on file). Traditionally, urine-derived HCG is calibrated in terms of biological activity with an in-vivo bioassay (rat seminal vesicle weight gain). The biological activity of rHCG has been assessed and the consistency of the production process has allowed the definition of a constant relationship between the protein mass of rHCG (expressed in µg) and its biological activity. The conclusion was that 250 µg rHCG correspond to ~5000 IU uHCG.
The use of other recombinant gonadotrophins suggests that these products may have a better bioefficiency than urinary-derived products, in women undergoing assisted reproduction treatment (Out et al., 1996; Daya and Gunby, 1999
). This may also be the case with rHCG (Penarrubia et al., 1999
).
The aims of this study were to evaluate the efficacy and safety of rHCG and uHCG, both administered s.c. for inducing final oocyte maturation and initiation of follicular luteinization. The study was carried out in women who had undergone pituitary desensitization in preparation for IVF and embryo transfer.
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Materials and methods |
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Patients were excluded from the study if they had (i) had an extra-uterine pregnancy in the previous 3 months; (ii) a previous IVF or gamete intra-Fallopian transfer (GIFT) failure due to poor response to gonadotrophins or a problem of sperm fertilization; (iii) polycystic ovarian syndrome (PCOS); (iv) a previous history of severe ovarian hyperstimulation syndrome (OHSS); (v) abnormal gynaecological bleeding of unknown origin; (vi) a male partner with leukospermia (>2x106/ml) or clinical signs of infection detected in a semen analysis within the last 2 months; (vii) a previous history of intolerance to any of the agents used in the study; or (viii) a clinically significant condition/disease or active substance abuse.
Concomitant medication for an existing condition or for an inter-current illness was allowed at the discretion of the investigator, though other hormonal treatments, anti-inflammatory medication and psychotropic agents with a known effect on ovulation (e.g. neuroleptics) were to be avoided. Patients requiring these medications were only permitted to continue in the trial at the investigator's discretion.
Study design
This was a multicentre, randomized, double-blind, double-dummy, parallel-group study carried out at nine centres throughout Europe and in Israel. The objectives of the study were to compare the efficacy and safety of Ovidrel® (rHCG, Laboratoires Serono SA, Geneva, Switzerland) with Profasi® (uHCG, Laboratoires Serono), both administered s.c., during one cycle of ovulation induction for IVF/ICSI. The study was conducted in accordance with the requirements of the Declaration of Helsinki and approved by the Independent Ethics Committees of the participating centres.
Drug administration
Patients underwent pituitary desensitization with 400 µg intranasal nafarelin (Monsanto Searle, Bucks, UK), twice daily, for 1025 days, starting in accordance with the centre's own practice. Pituitary desensitization was confirmed by an ultrasound scan (no evidence of ovarian activity, endometrial thickness <10 mm) and oestradiol concentrations (50 pg/ml). Recombinant FSH (Gonal-F®; Laboratoires Serono, Geneva, Switzerland) treatment was initiated after down-regulation was confirmed, and was administered once daily as a subcutaneous injection. The starting dose was in accordance with each centre's normal practice and the history of the patient (26x75 IU ampoules, 150450 IU/day). The dose could be adjusted according to ovarian response as judged by ultrasound and by plasma oestradiol concentrations, but could not exceed a dose of 450 IU/day up to a maximum cumulative dose of 7500 IU.
HCG (either rHCG or uHCG) was administered to the patient within 24 h after the last dose of rFHS and nafarelin, when the following criteria were met: at least one follicle with a mean diameter of 18 mm plus at least two others with mean diameters of 16 mm, and oestradiol concentrations within the acceptable range for the number of follicles present (~150 pg/ml per follicle, i.e. 540 pmol/ml per follicle). Provided the ovarian response was not excessive, each patient received two s.c. injections: either 250 µg rHCG (vial) plus uHCG placebo (ampoule) or 5000 IU uHCG (ampoule) plus rHCG placebo (vial). Micronized natural progesterone (600 mg/day) was administered by the vaginal route as luteal phase support, starting after oocyte retrieval. Progesterone treatment was continued until menstruation or for at least the first 3 weeks after diagnosis of pregnancy.
Oocyte retrieval and fertilization
Oocytes were retrieved and counted 3438 h after HCG administration. The cumulus oophorus maturity (very immature, immature, mature, very mature, post-mature or luteinized) was assessed and at selected centres, where ICSI was to be performed, the cumulus cells were removed and an assessment of oocyte nuclear maturity (germinal vesicle, metaphase I, metaphase II, atretic or degenerative) was made. Oocytes were fertilized in vitro, and the numbers of one pronuclear (1PN), 2PN and multipronucleate eggs on day 1 after oocyte retrieval were recorded. On days 2 or 3, the number of blastomeres, embryo grading and the outcome of each embryo (transferred, frozen or discarded) were recorded. Up to three embryos were replaced, 23 days after oocyte retrieval.
Measures of efficacy
The primary efficacy endpoint was the number of oocytes retrieved per patient following HCG administration. Additionally, the following secondary endpoints were recorded: number of patients who received HCG with at least one oocyte retrieved, number of oocytes retrieved per number of follicles >10 mm diameter on the day of HCG, number of mature oocytes, number of 2PN fertilized oocytes, number of 2PN cleaved embryos, serum progesterone and HCG concentrations on day 1 post-HCG, on the day of oocyte retrieval, on the day of embryo transfer and on day 67 post-HCG, implantation rate per embryo transferred, luteal phase endometrial thickness, number of biochemical and clinical pregnancies, number of multiple pregnancies, abortion rate and number of live births.
Safety evaluations
Physical examination and clinical laboratory measurements, adverse events (including incidence and severity of injection-site reactions, OHSS and development of antibodies to HCG), premature discontinuations and concomitant medications were summarized for all patients who took medication in this study. Adverse events were recorded on the case report form (CRF) on the basis of the patient's or physician's observations and coded using the World Health Organization (WHO) assisted reproduction technology system (WHO Adverse Reaction Terminology, 1996). An adverse event was defined as an undesirable medical experience, whether considered as related or not to the investigational product, which represented an adverse change from the patient's pretreatment condition. An adverse event was classified as serious if it was fatal or life-threatening, was permanently disabling, required inpatient or prolonged hospitalization, or was a congenital anomaly, cancer or overdose. A treatment-related adverse event was one that was judged by the investigator to be probably or possibly related to the study drug.
Patient withdrawals
Patients could be withdrawn from the study at any time for the following reasons: poor response to the stimulation protocol, not meeting the criteria for pituitary suppression after 25 days of nafarelin administration, at risk of OHSS, a serious adverse event, a protocol violation, non-compliance, an administrative reason, discovery of ineligibility, pregnancy, or at the patient's or physician's own request.
Sample size and statistical methods
A 90% confidence interval (CI) was used to display the difference in the mean numbers of oocytes retrieved between the treatment groups. The clinical equivalence of rHCG and uHCG was declared if the limits of the 90% CI fell within the prespecified range of ±3 oocytes. A total of 172 patients was required to demonstrate this equivalence with a probability of 97.5%.
Statistical analyses were performed on the following defined populations: `all patients' population (those who received HCG, regardless of adherence to protocol), and the `evaluable patients' population (those in the all patients population who were not considered to be major protocol violators). All analyses were performed using the SAS version 6.12 statistical package.
The comparability of the two treatment groups at baseline was checked for all demographic, medical history and clinical examination data, and both this and the efficacy data were evaluated using summary statistics (n, mean, SD, median and range for continuous variables and frequencies and percentages for categorical data). Binary outcomes were analysed by logistic regression or Fisher's exact test; continuous variables were analysed by t-tests, analysis of co-variance (ANCOVA) or analysis of variance (ANOVA) on raw or ranked data.
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Results |
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The two treatment groups were generally comparable for demographic and baseline characteristics. Of the 190 patients treated with HCG, 118 had primary infertility (62%). The mean infertility duration was 4 years and ranged from 1 to 15 years. There were no significant differences in the type or duration of infertility between the two treatment groups.
Tubal factors (79 patients, 42%), male factors (62 patients, 33%) and `unexplained' (54 patients, 28%) were the main causes of infertility and endometriosis (15 patients, 8%) was the other cause noted. In total, 171 patients noted only one factor (67 tubal, 49 unknown, 46 male, nine endometriosis) and 19 had two or more causes (seven tubal + male, six unknown + male, one endometriosis + male, four tubal + endometriosis, one tubal + endometriosis + male).
Menstrual cycles were regular for all patients except for eight (five rHCG, three uHCG). Cycle length varied between 24 and 46 days.
There was no difference between the two treatment groups in the number of patients who had had a previous non-assisted reproduction treatment pregnancy or a previous miscarriage. A total of 66 patients had had at least one pregnancy.
A total of 74 patients had had at least one assisted reproduction treatment attempt and there were 123 attempts in all. There was a slight, statistically significant (t-test, P = 0.035) difference between the two treatment groups in the number of patients who had a previous assisted reproduction treatment attempt: 35 patients treated with rHCG had an average of 1.9 assisted reproduction treatment attempts, while 39 patients treated with uHCG had an average of 1.5 attempts. There were no differences between treatment groups in relation to stimulation with rFSH. For the rHCG and uHCG groups respectively, mean rFSH treatment duration was 10.7 versus 10.8 days; total rFSH dose was 2263.1 versus 2285.9 IU and the number of 75 IU rFSH ampoules used was 30.2 versus 30.5.
Oocyte retrieval and fertilization
In total, 186 out of 190 patients had oocytes successfully retrieved. Oocytes were not obtained in four patients (2%) (three rHCG; one uHCG). Comparable numbers of oocytes were retrieved per patient in each treatment group. For the evaluable patients population (n = 172), the mean number of oocytes retrieved per patient (primary efficacy variable) was 11.6 in the rHCG group (n = 88) versus 10.6 in the uHCG group (n = 84) (Table I). The two-sided 90% CI (0.841, +1.515) fell within the limits set for the study (3, +3), thus confirming the equivalence of the two treatments. A similar result was obtained for the all patients population (n = 190). Again, the 90% CI fell within the pre-defined limits of the study (see Table I
).
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In relation to fertilization, the mean numbers of 2PN fertilized oocytes and 2PN cleaved embryos respectively were 6.2 and 5.4 in the rHCG group compared with 5.9 and 4.7 in the uHCG group. The difference between the two treatments was not statistically significant.
Serum progesterone and HCG concentrations
The mean serum progesterone and HCG concentrations, for the evaluable patients population of both treatment groups, on day 1 post-HCG, on the day of oocyte retrieval and on the day of embryo transfer and on day 67 post-HCG are shown in Figure 1. Statistical differences were recorded between treatment groups in favour of rHCG for mean progesterone concentrations at two time points; on day 1 (rHCG, 30.1 nmol/l versus uHCG, 23.3 nmol/l, P = 0.04) and on day 67 (rHCG, 391.9 nmol/l versus uHCG, 315.9 nmol/l, P = 0.03) post-HCG. For HCG, there were statistical differences between treatment groups in favour of rHCG at all time points (see Figure 1
); the greatest statistical difference in mean HCG concentration was recorded on the day of embryo transfer (rHCG, 2.1 µg/l versus 1.6 µg/l, P = 0.0001). It is noteworthy that the number of growing follicles (
11 mm diameter) on the day of HCG administration was comparable in the two treatment groups: 12.0 versus 11.6 for rHCG and uHCG respectively.
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Pregnancies and outcome
As only two of 18 patients (one in each group) who were non-evaluable became pregnant, pregnancies were only analysed for the all patients population. A summary of the pregnancies obtained during the study and their outcome is given in Table II. Overall, there was a good pregnancy rate; a total of 66 pregnancies was recorded among the 190 patients (34.7%), of which 55 (83.3%) were clinical pregnancies and 11 (16.7%) were biochemical. Although the percentage of clinical pregnancies appeared higher in the patients treated with rHCG (33 versus 25%), this difference was not statistically significant.
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There were 47 deliveries, in which 63 babies were born. Of these, 26 deliveries (18 singleton and eight twin) occurred in rHCG patients (34 babies) and 21 (13 singleton and eight twin) in uHCG patients (29 babies). One rHCG patient gave birth to a baby with a congenital abnormality who died, and the outcome of one pregnancy was unknown as the patient was lost to follow-up.
Safety
Overall, the two drugs were well tolerated. After administration of HCG, 22/97 patients in the rHCG group (22.7%) reported 32 adverse events compared with 42/93 patients in the uHCG group (45.1%) reporting 65 events. This difference was statistically significant in favour of rHCG (P = 0.0004). Most of these events (93%) were judged as mild to moderate in severity. Of these 97 events, 80 were judged possibly or probably treatment-related. The most common treatment-related events, in both groups, were injection-site disorders that were reported by the patients, mainly pain, inflammation and bruising. In the rHCG group, only seven patients (7.2%) experienced an injection-site event that was probably treatment-related, compared with 28 patients (30.1%) in the uHCG group. This difference between groups was highly significant (P = 0.0001). Eleven patients, six (6.2%) in the rHCG group and five (5.4%) in the uHCG group, experienced a serious adverse event. Serious treatment-related OHSS was reported for two patients (one in each group); all other serious adverse events were judged as not related to treatment.
The incidence of OHSS was similar in both groups; 13 patients were reported to have suffered from this, seven (7.2%) receiving rHCG and six (6.4%) receiving uHCG. Overall, the injections were well tolerated by patients, though a greater proportion of those who took uHCG compared with those taking rHCG reported local reactions (particularly pain and inflammation). In the uHCG group, 35/93 injections (38%) led to a local reaction compared with 13/97 injections in the rHCG group (13%). This difference was statistically significant in favour of rHCG (P = 0.0001). Moreover, rHCG did not appear to result in more local reactions than placebo, in contrast to uHCG, which did. There were no clinically significant changes in physical examination parameters or in routine clinical laboratory measurements and there were no statistical differences between groups. In total, 152/190 patients who received HCG had samples assayed for anti-HCG antibodies; all were found negative.
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Discussion |
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This study was designed to show equivalence between the two HCG products, and the primary efficacy endpoint (mean number of oocytes/patient) indicated that rHCG was, indeed, as effective as uHCG. However, it is interesting to note that rHCG treatment resulted in statistically higher numbers of mature oocytes, statistically higher day 1 and days 67 progesterone concentrations and statistically higher HCG concentrations at all timepoints, post-HCG. The observation of lower oocyte nuclear maturity in the uHCG group, despite administration of similar doses, is not definitely understood. Urine-derived HCG is known to contain HCG degradation products (Wehmann and Nisula, 1980). This may suggest that HCG degradation products in the urinary preparation have subtle interference with the active HCG molecules and therefore with the HCG-induced cumulusoocyte maturation. The difference observed in progesterone concentrations does not seem to be associated with the number of corpora lutea as indicated by the number of follicles on the day of HCG administration. This may be associated with the slightly higher HCG concentrations reported in the rHCG group, or as suggested above to a difference in the biological activity of rHCG in human compared to that of uHCG (Penarrubia et al., 1999
). Furthermore, clinical pregnancy rates were apparently higher in those patients who had received rHCG (33 versus 25%), but this difference was not statistically significant. The risk of OHSS did not appear to differ between the two products, with a similar, low incidence in both treatment groups.
Most patients reported good tolerance to the two HCG preparations and any side-effects were generally mild to moderate in severity. Nevertheless, the incidence of side-effects was statistically higher in the uHCG group (45 versus 23%, P = 0.0004). Many of these adverse effects were injection-site-related, occurring far more extensively in the uHCG patients; 38% of injections led to a local reaction in uHCG patients, compared with only 13% in the rHCG group (P = 0.0001). Although urinary-derived gonadotrophins are therapeutically effective, the non-hormonal proteins contained in these preparations are known to cause severe local and systemic immunogenic reactions. In women who have suffered such reactions, following injection of urinary-derived gonadotrophinshuman menopausal gonadotrophin, for examplethe administration of rFSH has been associated with good tolerance at the site of injection and reduced immunogenicity (Albano et al., 1996; Whitman-Elia et al., 1998
), although relative costs of rHCG versus uHCG were not specifically addressed in this study. The improved tolerance of recombinant products, including HCG, is likely to lead to higher patient acceptability, which is a clear, clinical advantage.
In conclusion, therefore, this study indicates that rHCG is effective in inducing follicular maturation and early luteinization. Recombinant HCG is associated with more mature oocytes, higher progesterone concentration on days 1 and 67 and improved tolerance and is thus likely to have higher patient acceptability.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 30, 1999; accepted on March 27, 2000.