1 Department of Obstetrics and Gynaecology, University of Thessalia, Larissa, 2 Clinical Chemistry Laboratory, General Hospital G.Gennimatas, Athens and 3 Department of Obstetrics and Gynaecology, University of Athens, Athens, Greece
4 To whom correspondence should be addressed. E-mail: messinis{at}med.uth.gr
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Abstract |
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Key words: FSH/GnRH antagonist/LH/LH surge/luteinization
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Introduction |
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Therefore, any protocols that use the least amount of GnRH antagonists would be less costly and, if live birth rates were not affected, could be to patients advantage. So far, no study has investigated the effect of less frequent than the daily administration of small doses of a GnRH antagonist on the occurrence of the endogenous LH surge and premature luteinization during ovulation induction with FSH. The present study was undertaken as a preliminary investigation to determine, in women during treatment with FSH in combination with the GnRH antagonist ganirelix, whether LH values were similar with injection daily and every other day.
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Materials and methods |
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Ovarian stimulation protocol included the s.c. administration of recombinant FSH (Puregon 150 IU/0.5 ml; Organon, Athens, Greece) at the dose of 150 IU per day from cycle days 2 to 5. The dose of FSH was then individually adjusted according to ovarian response. On cycle day 7, i.e. the 6th day of FSH treatment, the GnRH antagonist ganirelix (Orgalutran 0.25 mg/0.5 ml; Organon) was added s.c. at multiple doses of 0.25 mg each (09:00). In group 1, the antagonist was given to the women on a daily basis, one dose every day, and in group 2 on alternate days, one dose every other day, i.e on days 7, 9, 11 etc.
HCG was injected i.m. at a single dose of 10000 IU (Pregnyl ampoules, 5000 IU per ampoule; Organon) when the leading follicle was 1820 mm with at least three follicles >16 mm in diameter and serum estradiol (E2) concentrations not >3000 pg/ml. Oocyte recovery was performed 3536 h after the administration of HCG. Embryo transfer was performed 2 days after the oocyte recovery. The luteal phase was supported in all women with micronized progesterone given intravaginally at the dose of 600 mg/day (Utrogestan capsules, 100 mg per capsule; Faran, Athens, Greece).
In both groups, blood samples were obtained from all women (09:00) on cycle day 2 before the onset of FSH treatment and then on days 5 and 7 and every day thereafter until the administration of HCG. Each time the blood samples were obtained before the administration of the antagonist and/or FSH. All blood samples were centrifuged and the serum was stored at 20°C until FSH, LH, E2 and progesterone were assayed.
Hormone assays
Measurement of FSH, LH and E2 was done using a Chemiluminescent Microparticle Immunoassay (Architect FSH, Architect LH and Architect Estradiol respectively; Abbott Laboratories). The results are expressed as IU/l for FSH and LH and as pg/ml for E2. Progesterone was measured in serum using a Microparticle Enzyme Immunoassay (AxSYM Progesterone; Abbott Laboratories, USA). The results are expressed as ng/ml. The lower limits of detection for FSH, LH, E2 and progesterone were 0.05 IU/l, 0.07 IU/l, 17.9 pg/ml and 0.2 ng/ml respectively. Inter- and intra-assay coefficients of variation were 3.1 and 3.4%, 2.0 and 3.4%, 4.5 and 6.0% and 6.0 and 6.7% respectively. The laboratory analysts were blinded to study allocation.
Data analysis
Sample size calculation
The sample size was based on the primary outcome measure, i.e. the frequency of LH values 10 IU/l during the whole stimulation period. We hypothesized that with alternate day treatment the frequency of LH values
10 IU/l would be
50% of that seen with daily treatment. In previous studies LH rises were found in 2.728.5% of stimulation cycles with gonadotrophins alone or combined with clomiphene citrate (Albano et al., 2000
; Borm and Mannaerts, 2000
; Felberbaum et al., 2000
; Tavaniotou et al., 2003
). With power of 80%, to detect reductions of
50% in frequencies of 6, 14, 30 and 60% respectively would require 603, 244, 98 and 35 patients in each group, assuming a significance level of 0.05 based on the difference in two proportions. An interim analysis was planned (with
= 0.001) after 35 patients were recruited in each study arm to evaluate final sample size.
Statistical analysis
Hormone values were logarithmically transformed to attain a normal distribution (one sample KolmogorovSmirnov test). However, the arithmetic means with SEM of values are presented. Statistical analysis was performed by c2-test with continuity correction, Students t-test and one-way analysis of variance for repeated measurements. The statistical software package used was NCSS 2001 (Number Cruncher Statistical Systems, Kaysville, UT, USA).
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Results |
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Serum FSH and E2 values increased significantly in both groups from cycle day 2 to day 12 (P < 0.05) with no significant difference between the two groups at any point (Figure 1a). LH values (mean ± SEM) increased significantly on day 7 in both groups (P < 0.05) and further on day 9 in group 2 (6.00 ± 1.04 IU/l) (P < 0.01) and were then significantly higher than in group 1 (P < 0.01, Figure 1a). For the rest of the time, LH concentrations showed fluctuations. Serum progesterone values (mean ± SEM) were low up to cycle day 10 with no difference between group 1 and group 2 (Figure 1a). On cycle day 10 (1.91 ± 0.32 and 1.55 ± 0.20 ng/ml respectively), progesterone values were significantly higher than on days 2 (0.64 ± 0.08 and 0.56 ± 0.05 ng/ml respectively) and 5 (0.80 ± 0.25 and 0.75 ± 0.19 ng/ml respectively). A significant increase in serum progesterone concentrations was seen on days 11 and 12 as compared to day 10 in both groups (P < 0.01). When hormone values were analysed in relation to the day of HCG administration (day 0), the patterns of changes were similar to those described above (Figure 1b). E2 concentrations were significantly higher in group 2 than in group 1 on days 2, 1 and +1 (P < 0.05, Figure 1b).
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Analysis of LH and progesterone values in individual patients showed a similar trend in the two groups (Figure 2). In the interim analysis of the primary outcome, LH values 10 IU/l were seen in 13 patients of group 1 (35.1%) and in 14 patients of group 2 (38.8%) (P = 0.929). To evaluate the difference between 35.1 and 38.8% would require 2148 patients in each group, and the study was terminated at this point. In eight women of group 1 (21.6%) and 10 women of group 2 (27.7%) the LH rises were seen during the treatment with the antagonist. Luteinization, i.e. serum progesterone values >2 ng/ml, occurred before the onset of the antagonist administration in three of the five women of group 1 and in two of the four women of group 2, while during the antagonist treatment it occurred only in one patient of each group (12.5 versus 10.0%) (Table II). Overall, only 2.7% of the women of each group showed luteinization during the administration of ganirelix. When the two groups were combined, the percentage of women with high LH (
10 IU/l) who showed luteinization during the GnRH antagonist administration (two out of 18 women, 11.1%) was significantly lower than that before the onset of the administration (five out of nine women, 55.5%) (P = 0.044). Peak LH values in patients with luteinization were 13.0, 53.7, 11.8 and 11.9 IU/l in group 1 and 27.7, 21.3 and 14.0 IU/l in group 2. The increase in serum progesterone levels in these patients was maintained at >4 ng/ml until the HCG administration (maximum values 10 ng/ml). None of the seven patients with increased progesterone values became pregnant. However, two women of group 1 and three women of group 2 with LH rises
10 IU/l and no progesterone increase became pregnant after embryo transfer.
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Table III shows the results of IVF/ICSI treatment. There were no significant differences in the total dose of FSH used, the duration of treatment, the number of follicles, the number of oocytes recovered, fertilization, implantation and clinical pregnancy rates between the two groups. However, the total dose of the GnRH antagonist was significantly smaller in group 2 than in group 1 (P < 0.001). In total, there were six pregnancies in group 1 and seven in group 2 with only two multiples (twins) in group 1. So far, there are three term deliveries, one of them twins, in group 1 and four term deliveries in group 2 and the neonates are normal. All rest pregnancies are ongoing.
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Discussion |
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These results differ from findings in previous studies in which LH rises of 10 IU/l were seen in a smaller percentage of women, although overall premature luteinization in the present study (2.7%) was not much higher than in previous studies (
1%) (Table IV). In the previous studies, however, a progesterone level >1 ng/ml instead of >2 ng/ml was adopted (Ganirelix Dose-finding Study Group, 1998
; Albano et al., 2000
; Felberbaum et al., 2000
; European and Middle East Orgalutran Study Group, 2001
). This difference in the frequency of LH rises is unlikely to be explained by patients compliance with drug injection or the protocol of GnRH antagonist administration, since in group 1 of our study the protocol was the same as that in the previous studies. The GnRH antagonist was injected s.c. via pre-filled syringes at regular intervals, i.e. in group 1 every morning (09:00). Furthermore, established assays were used for hormone measurements, and to avoid variation all blood samples from each individual patient were assayed in the same batch while the laboratory analysts were blind to study allocation. Regardless of the difference from previous studies, it is interesting that with the minimal effective dose of 0.25 mg ganirelix (Ganirelix Dose-finding Study Group, 1998
), the frequency of endogenous LH surge occurrence and premature luteinization in the present study was similar in the two groups.
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Our data are consistent with the notion that an endogenous LH surge occurs invariably during superovulation induction in women (Messinis and Templeton, 1987; Glasier et al., 1988
) and suggest that the GnRH antagonist at the daily dose of 0.25 mg hardly prevents it. In fact, premature LH rises that could by definition belong to an LH surge (Messinis et al., 1985
) were seen in this study in
50% of the women during the administration of ganirelix. However, these LH rises were followed by luteinization only in two out of 18 cases with LH
10 IU/l (Table II) or in two out of 40 cases with LH
5 IU/l (data not shown). They therefore differ from what has been described previously in superovulated women as attenuated LH surges that always resulted in luteinization (Messinis et al., 1985
; Messinis and Templeton, 1986
). It has been suggested that a gonadotrophin surge-attenuating factor (GnSAF) produced by the overstimulated ovaries is responsible for the attenuation of the LH surge (Messinis and Templeton, 1989
). Since in this study luteinization following a premature LH rise
10 IU/l occurred more frequently before the onset of GnRH antagonist administration than during the administration, it is possible that in FSH-treated cycles, as a result of the synergistic action of the antagonist and GnSAF, the LH surge either is blocked or becomes abortive, i.e. unable to induce luteinization in the vast majority of women. As a matter of fact, pregnancies after embryo transfer occurred in such cases in the present study, although high LH levels in the follicular phase during treatment with a GnRH antagonist are detrimental for clinical outcome (Kolibianakis et al., 2003a
). Increased bioactivity of GnSAF is particularly evident during the first 5 days of FSH-treated cycles but is subsequently counteracted at least in part by E2 (Messinis et al., 1998
). This can explain the first significant rise of LH in the present study at the earliest on cycle day 7. However, due to high rate of LH rise on this day it might be worth starting the administration of the GnRH antagonist at least 1 day earlier as has been already suggested (Kolibianakis et al., 2003a
).
Previous studies have suggested that the GnRH antagonists may affect ovarian steroidogenesis and have an impact on the endometrium and the implantation (Kol et al., 1999; Hernandez, 2000
; Kolibianakis et al., 2003b
). The present study shows a similar pregnancy rate in the two groups, but does not have the power to address this point, since our main purpose was to investigate changes in LH values. Whether the subtle increase in serum progesterone concentrations seen in late follicular phase can affect endometrium maturation needs to be investigated.
In conclusion, in this pragmatic trial, the incidence of premature LH rises during FSH treatment was not significantly different with GnRH antagonist ganirelix 0.25 mg given daily or on alternate days, although the incidence was higher in both groups than in previous efficacy studies. However, premature luteinization was markedly reduced during the period of treatment with the antagonist as compared to the period preceding the onset of its administration. Although underpowered for clinical outcomes, the present study shows that alternate day administration of GnRH antagonist may not increase the risk of premature LH surge, and ethically could be evaluated in larger trials.
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Submitted on December 6, 2005; resubmitted on June 2, 2005; accepted on June 3, 2005.
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