Division of Reproductive Medicine, Department of Obstetrics and Gynaecology, Vrije Universiteit medical centre (VUmc), Amsterdam, The Netherlands
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, VUmc, PO Box 7075, 1007 MB Amsterdam, The Netherlands. Email: cb.lambalk{at}vumc.nl
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Abstract |
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Key words: antide/dose-finding/GnRH antagonist/IVF/LH
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Introduction |
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Materials and methods |
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Study design
A phase II, single center study, conducted in two phases: a double-blind phase with two parallel treatment groups was followed by an open phase (see Figure 1). In the double-blind phase 60 patients were randomized to two different treatment groups (A: 2 mg/2 ml, B: 1 mg/ml). To improve patients' convenience, the 2 mg in group A was given as two injections of 1 mg/ml antide, one injection in the morning and one in the evening, since we expected that one injection with a volume of 2 ml would be too painful. Patients in group B received placebo in the morning and 1 mg/ml antide in the evening. Since none of the two groups turned out to be a failure group (i.e.with 2 LH surges) we decided to add an open phase in which three additional treatment groups with lower antide dosages were studied (0.5 mg/0.5 ml, 0.5 mg/ml, 0.25 mg/ml), administered once daily. The additional arms were added in a consecutive order and patients were enrolled in a chronological way. New evidence which became available after the start of this study suggested that the bio-availability of antide increases after dilution in larger volumes of glucose 5% (Data on file, Serono, Geneva). Therefore in two groups (0.5 mg/ml and 0.25 mg/ml) antide was diluted in larger volumes of glucose 5% solution; 0.5 mg and 0.25 mg in 1 ml, respectively. This means that in two arms 0.5 mg antide was administered but in group C it was diluted in 1.0 ml of glucose 5% solution and in group D it was diluted in 0.5 ml of glucose 5% solution.
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Masking
Treatment packs for the double-blind phase of the study were prepared according to the randomization list by Serono International (Geneva, Switzerland). Patient packs, containing antide/placebo or antide/antide vials, were labeled with unique study identification numbers, provided by Serono International (Geneva, Switzerland); placebo vials contained a sterile isotonic aqueous solution. When eligible, patients were enrolled to the study by one of the two responsible trained researchers and received a unique study number in a chronological order at the start of the first stimulation day. The code was not known to the executors of the study. Assignment to group A or B was therefore double blind, assignment to group C, D or E was dependent on the chronological entry of the study.
Treatment protocol
The treatment protocol is illustrated in Figure 2. On day 2 or 3 of a spontaneous menstruation, r-hFSH (Gonal-F®, Serono, Aubonne, Switzerland) was given as a single daily s.c. injection. The starting dose varied between 150300 IU, depending on previous ovarian response, but was fixed for the first 5 days. After this period, depending on ovarian response as assessed by daily ultrasound, the r-hFSH dose could be adjusted. All antide, placebo and r-hFSH injections from stimulation day 6 (S6) onwards, were given subcutaneously, by a trained research professional. From stimulation day 6 onward, up to and including the day of recombinant human chronic gonadotropin (r-hCG; Ovitrelle®, Serono) administration, daily antide was given. Recombinant hCG was administered as soon as one follicle 18 and two follicles were
16 mm. Thirty-six hours after r-hCG administration, ovum pick-up (OPU) was performed transvaginally and ultrasound guided. The OPU was followed by IVF with or without ICSI, a maximum of three embryos were replaced 23 days thereafter. Luteal support (200 mg progesterone vaginally, three times daily) was started 1 day after OPU until the third week of pregnancy or a negative pregnancy test.
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Local tolerance after the different injections was assessed 60 min and 12 h after each antide injection. Pain, itching, tenderness, redness, swelling and bruising were recorded on a 4 point scale (none, mild, moderate and severe) of a diary card. All other side effects were reported daily on this diary card as well.
Serum assessment
Blood samples were processed to serum immediately after collection and stored at 20°C. Routine haematology, biochemistry and urine assessment were performed by the local laboratory (The Central Laboratory of the VUmc) using commercially available immunometric assays. To detect LH surges, all morning samples were assessed daily by this local laboratory to measure the LH levels, using immunometric assay kits from Amerlite (Amerlite, Amersham, Bucks, UK). An LH surge was defined as LH >10 IU/l and P4 >2 ng/ml using this assay. Halfway through the study we were forced to change the assay, since Amerlite assays were not available anymore. We decided to use Delphia (Dephia, Wallac, Finland) assays. During the transition period of the assays, we assessed LH levels using both assays in 89 patients. Excellent correlation was observed between the two assays (R=0.98). A regression analyses revealed that the coefficient of LH was 1.24 using Delphia assay in comparison to Amerlite assay, thus the LH cut-off level 10 IU/l assessed by Amerlite was equivalent to 12.4 IU/l if assessed by Delphia assay.
For definitive analyses of all hormone and antide levels, as presented in this report, all serum samples (taken three times daily) were assessed retrospectively by LCG Bioscience Services LTD. E2 was measured using Sorin Radioimmunoassay, P4 using DPC Coat-a-count, RIA solid phase coated tube separation, FSH and LH using Serono MAIAclone IRMA. The lower limit of quantification for LH was 1 IU/l. For the retrospective analyses we defined an LH surge as LH >12.4 IU/l and P4 >2 ng/ml in one or more samples, taking all samples (three times daily) into account from S6 until hCG administration day, equivalent to the cut-off levels using the Delphia assays. The retrospective centralized analysis of serum antide levels were performed by Woods Assay (RIA), all samples were analyzed in triplicate, 1 µg/l is the Limit of Quantification.
Outcome measures
The primary endpoint of this study was to determine the minimal effective dose, defined as the lowest dose group in which fewer than two LH surges occurred. Secondary endpoints were drug requirements, serum hormone and antide levels and safety aspects.
Statistical analysis
The treatment groups were compared depending on the nature of the variables, [i.e the analysis of variance (ANOVA) or the analysis of covariance (ANCOVA), Chi-square tests, Fisher's exact test or non-parametric ranking methods like KruskallWallis and MannWhitney U tests]. Results are reported as mean±SD. Correlations were calculated using Pearson's correlation coefficient. P<0.05 was considered to be statistically significant. Analyses were conducted on an intention to treat base. An overall dose-response test for trend with the treatment groups were performed on all efficacy data. The statistical hypothesis of no treatment effect was tested against the ordered alternative hypothesis that at least one antide dose is superior to the lowest dose group and that the response is decreased, or at least equal to those of the previous group when decreasing the dose. The statistical methods used to test this hypothesis were directly related to the nature of the variable, i.e. ANOVA with linear contrast, JonckheereTerpstra test or a CochranArmitage test. To identify different covariates on pharmacokinetic parameters, univariate analysis within NONMEM was used.
Total exposure to antide and hormone levels was expressed as area under the curves (AUC) during antagonist administration (i.e. S6-hCG day). For this calculation, the sum of the mean daily levels of all days during antide treatment were taken.
To calculate the induced change in serum levels in comparison to the basal level on stimulation day 6 (the moment on which the antagonist was started), AUC was calculated after subtraction of the basal level on S6 of all samples, defined as AUCS6.
The sample size was not calculated; we based our groups size on clinically relevant arguments. More than one LH surge per 30 patients was considered to be unacceptable for clinical use in IVF patients. Therefore the number of patients was intended to be 30 per group, unless more than one LH surge occurred.
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Results |
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Stimulation phase
The mean duration of antide and r-hFSH administration was 5 (range 29) and 9 days (range 613), respectively. This was comparable between the groups (P=0.89) (see Table II). The mean Gonal-F® starting dose was similar in the various dose groups and varied between 223 and 238 IU (P=0.78). Additionally, the mean dose Gonal-F® per day and the mean total dose of Gonal-F® were also similar in the various dose groups (P=0.98 and 0.91, respectively). Because of insufficient response, the Gonal-F® dose was more often increased in the higher antide dose groups (P=0.04).
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FSH, oestradiol and progesterone levels during stimulation
On the day of hCG, the FSH levels were higher in group A (2.0 mg/ml) and group B (1.0 mg/ml) in comparison to the lower dose groups (P=0.05). The total AUC of the FSH levels were not significantly different between the individual treatment groups (P=0.25) (see Table IV).
The steroid hormone levels are presented in Table V. E2 levels increased in all groups during antide administration (see Figure 4). The mean E2 levels/day and the induced E2 AUC, adjusted for baseline levels on S6 (E2-AUCS6) were proportionally dose-related to the treatment group (P=0.009 and <0.001, respectively), with the highest E2 levels in the lowest antide dose groups. The same was so for the calculated E2 levels per follicles >10 mm on the day of hCG administration, with the highest E2 levels in the lowest dose group (0.25 mg) and the lowest E2 levels in the highest antide dose group (2.0 mg), 501 (228) and 388 (168) pmol/l/follicle, respectively (P=0.05).
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Safety and tolerance
Overall, antide was well tolerated, with none of the patients experiencing a serious adverse event related to medication. The most frequently recorded side effects were general disorders (especially fatigue and headache) and gastro-intestinal disorders (abdominal pain and nausea). 83.1% of all patients recorded at least one local skin reaction (redness, tenderness, itching, bruising or swelling) with transient redness (25%) and tenderness at the injection site (22.2%) being most frequent. Most patients considered the local reactions to be mild. The local reactions mostly resolved spontaneously within 1 h.8.8% of patients had at least one moderate local reaction occurring 1 h after antide injection and were mostly found in treatment groups A and B. No severe local reactions were observed 1 h after antide injection. In the minimal effective dosegroup (D: 0.5 mg/ml), a moderate local reaction observed 1 h after the injections was observed in only one patient. The biochemical and hematological parameters measured in all patients were similar before and after the treatment cycle (data not shown).
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Discussion |
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We used the same standard definition for premature luteinization as others in GnRH antagonist dose-finding studies, namely a rise of LH above a predetermined threshold plus a significant increase in serum progesterone values (Albano et al., 1997; Ganirelix dose-finding Study Group, 1998
). According to our definitions, groups D (0.5 mg/0.5 ml) and E (0.25 mg/ml) were considered to be failure dosages, and group C (0.5 mg/ml) appeared to be the minimal effective dose, in which no rises occurred. It should be noted that sampling blood three times per day increases the detection of any significant changes in LH. The design of the study was chosen on ethical grounds, with the aim of minimal exposure to possible premature luteinization. We were aware of the risk of a possible imbalance in baseline characteristics. Retrospectively it appeared this was present as regards type of fertility disorder and pretreatment oestradiol levels between the various groups. Nevertheless, these factors had no effect on pharmacokinetics and hormonal dynamics.
As expected there was a clear dose dependency of LH secretion throughout the stimulation period with lowest levels with the highest antide dosages. Similar dose dependency was seen in the other GnRH antagonist dose-finding studies (Albano et al., 1997; Ganirelix dose-finding Study Group, 1998
). This study clearly demonstrated that the dilution of 0.5 mg antide in a larger volume of glucose 5% increased the bioavailability of antide and resulted in a lower incidence of LH surges. The antide serum levels were almost doubled if the same dose of antide (0.5 mg) was diluted in 1 ml instead of 0.5 ml.
A clear and intriguing observation in our study was the remarkable rise of preinjection LH levels throughout the antide treatment period. It occurred with all dosages, but was particularly clear in the lowest antide dose groups. This gradual increase of basal LH secretion while daily injections of a GnRH antagonist are given seems a common feature with this treatment strategy (Ganirelix dose-finding Study Group, 1998). It can also be seen in healthy unstimulated women (Oberye et al., 1999
). Apparently some escape of pituitary LH secretion takes place, which even occurs while serum concentrations of antide remain constant. One explanation is that the pituitary develops an increased sensitivity to endogenous hypothalamic GnRH under these circumstances. The mechanism by which this takes place remains obscure. Possibly changes in hormonal milieu play a role. Increasing oestradiol levels during stimulation may modify pituitary sensitivity. The gradual escape was indeed most prominent in the lowest antide dose groups, which was associated with the highest oestradiol levels, both in our study and also in the Ganirelix dose-finding study (Ganirelix dose-finding Study Group, 1998
). Alternatively, pituitary GnRH receptors may have been up-regulated by the GnRH antagonist itself as suggested by several authors (Gordon et al., 1994
). A final explanation for the LH increase may be that the GnRH antagonists directly intervene in the ultrashort-loop feedback mechanisms comprising GnRH autoregulation (Krsmanovic et al., 1999
).
The FSH levels on the day of hCG were higher in the highest antide dose groups, while there was a trend of a dose dependency in mean daily FSH levels. The adjustments of daily FSH dose, based on insufficient follicular response, may have been responsible for this. Although similar total FSH quantities were used in all antide dose groups, significantly more patients had r-hFSH dose increases in the highest antide dose groups.
The mean oestradiol levels per day and the induced changes in oestradiol levels during antide administration in comparison to the baseline levels, were inversely related to the antide dose. This is in agreement with an earlier report (Ganirelix dose-finding Study Group, 1998). Similarly, the highest levels of oestradiol production per follicle
11 mm were found in the lowest antide dose group. The dose-related oestradiol response to the GnRH antagonist, independent from the number of follicles, indicates a decrease of its production by granulosa cells, probably as a result of reduced availability of androstendione substrate.
It is often suggested that late follicular progesterone secretion is LH dependent. However, a straightforward LH dependency of progesterone secretion was not obvious in our study. Nevertheless, this relation became clear after correction for numbers of follicle on the day of hCG. The total AUC of progesterone increased by 0.6 ng/ml when LH-AUC increased by 1 IU/L. Apparently, the absolute level of progesterone production is also dependent on the total number of follicles. This means that high progesterone levels, independent of high LH levels, may also be the result of profound follicular growth in the ovary.
Overall, antide was well tolerated and safe in terms of side effects and influence on biochemical and haematological parameters. The reported reactions were all minor and most resolved spontaneously within 1 h. Only 9% of the patients had a moderate or severe local skin reaction 1 h after antide administration during the entire treatment period. This was lower than with other antagonist studies employing the same assessment methods, scales and criteria for these side effects (Ganirelix dose-finding Study Group, 1998; Middle East Orgalutran Study Group, 2001
). This can be explained by the much lower histamine releasing property of antide compared to the clinically available GnRH antagonists (Bajusz et al., 1988
; Rivier et al., 1992
).
In conclusion, 0.5 mg/ml antide is the minimal effective dose to prevent LH surges in hyperstimulated cycles for IVF or ICSI. LH levels increase gradually during GnRH antagonist treatment. The bioavailability increases if antide is diluted in a larger volume. Antide was well tolerated and safe.
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Acknowledgements |
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References |
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Submitted on February 5, 2004; accepted on May 14, 2004.