1 Centre for Reproductive Medicine, Jean Verdier Hospital, A.P.-H.P., 93143 Bondy University Paris XIII, 2 ART Centre, Clinique du Blanc Mesnil, 93150 Le Blanc Mesnil and 3 IVF Centre, Aquitaine Santé Médecine de la Reproduction, 33 523 Bruges Cedex, France
4 To whom correspondence should be addressed. Email: isabelle.cedrin-durnerin{at}jvr.ap-hop-paris.fr
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Abstract |
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Key words: assisted reproductive technology/controlled ovarian stimulation/GnRH antagonist/rLH
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Introduction |
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Both multiple dose and single dose protocols proved to be effective in IVF/ICSI cycles (Olivennes et al., 2002). However, the optimal timing for GnRH antagonist administration remains to be determined as well as the consequences of this administration on the hormonal ovarian status. Indeed, a minimum threshold of LH secretion has to be maintained to ensure adequate steroidogenesis and folliculogenesis (European Recombinant Human LH Study Group, 1998
). Furthermore, antagonists are usually administered when follicles become increasingly responsive to LH due to the acquisition of LH receptors on granulosa cells and when LH may operate with FSH to achieve full follicular maturity and oocyte competence (Hillier, 2001
). As a consequence, it may be assumed that the dramatic decrease in LH secretion induced by GnRH administration is likely to be detrimental for the follicleoocyte complex. As the inhibitory effect of GnRH antagonists upon LH secretion has been shown to be dose dependent (Ganirelix Dose-finding Study Group, 1998
), the decrease in serum LH values is sharper and more consistent using a single high dose protocol than a multiple low dose one (Reissman et al., 2000
). Besides, it has been shown in the literature that measurement of the serum LH value is not valid for assessing residual LH bioactivity (Jaakkola et al., 1990
) or for defining a sub-group of patients that might benefit from exogenous LH administration (Peñarrubia et al., 2003
). For that reason, supplementation with exogenous recombinant human (r)LH has been suggested as an alternative way to evaluate indirectly the consequences of LH depletion.
Therefore, this study was designed to assess prospectively the effects of recombinant rLH supplementation on the main ovarian parameters and on the outcome of assisted reproduction cycles in patients undergoing IVF/ICSI cycles and using a single dose GnRH antagonist protocol.
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Materials and methods |
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Protocol
Pre-treatment with an oral contraceptive pill (levonorgestrel 0.15 mg + ethynylestradiol 30 µg) was given during the cycle prior to the IVF/ICSI procedure. Three days after pill discontinuation (S1), stimulation was started by daily injection of 150300 IU of rFSH (Gonal F; Serono SA, France). The starting dose was chosen according to patients' age (150 IU/day, age <35 years; 225 IU/day, age >35 years), BMI (starting dose increased by 75 IU/day if BMI was 2730 kg/m2) and ovarian responsiveness in previous cycles. This dose was maintained constant for 5 days. From day 6 of the stimulation (S6), rFSH doses were individually adjusted according to hormonal determinations and ultrasound data.
When the leading follicle reached 1416 mm diameter, patients received a single injection of cetrorelix 3 mg (Cetrotide; Serono SA) and received or not, according to randomization, a daily injection of 75 IU rLH (Luveris; Serono SA) from the time of cetrorelix injection up to hCG administration. In both groups, rFSH dose adjustment was not allowed at the time of cetrorelix administration. If criteria for triggering ovulation were not met within 4 days after cetrorelix administration, additional injections of cetrorelix 0.25 mg per day were performed until hCG administration.
An injection of 10 000 IU hCG (Gonadotrophines Chorioniques Endo; Organon, France) was given when at least three mature (>17 mm) follicles were obtained and oocyte retrieval was performed 36 h later.
Luteal phase was supported by vaginal administration of progesterone 400 mg/day (Utrogestan; Besins International, France) from the day of ovarian puncture to the day of pregnancy test. If a pregnancy occurred, progesterone administration was extended up to the evidence of fetal heart activity at ultrasound.
Objectives and outcomes
The possible deleterious effect of LH depletion induced by antagonist administration on the follicleoocyte complex was considered to be clinically relevant if a difference in the number of embryos available for transfer could be observed between patients supplemented or not with rLH. Therefore, the primary outcome measure was the number of total embryos per patient. Secondary outcomes were serum estradiol, LH and progesterone values on the day of hCG administration, numbers of total and mature oocytes, pregnancy rate per treated cycle (positive hCG test), implantation rate per embryo (number of gestational sacs on ultrasound scan/number of transferred embryos) and ongoing pregnancy rate (pregnancy >12 weeks of gestational age).
Sample size
The estimated sample size to detect a difference of 1.5 embryos between groups was 200 patients (one-sided, P<0.05 considered significant). As randomization was not performed on the day of cetrorelix administration, a larger number of patients was included (114 in the group supplemented with rLH and 104 in the group without rLH) to take into account cancellations before cetrorelix administration (Figure 1). Indeed, eight patients were cancelled for a poor ovarian response before cetrorelix administration (six in rLH allocated group and two in the other group) and one for omitting FSH injections for several days (rFSH allocated patient). Six other patients could not be included in the analysis due to the lack of cetrorelix administration (n=4 with two pregnancies in rFSH allocated patients) or to a late administration of cetrorelix on the day of hCG (n=2, one in each group). Therefore, the number of analysed patients was 107 in the group supplemented with rLH and 96 in the group without rLH.
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Hormonal measurements
Hormonal measurements were carried out using commercially available chemiluminescence immunoassays with an automated Elecsys immunoanalyser (ECLIA; Roche Diagnostics, France). The sensitivity of the assay was 0.1 IU/l for FSH and LH. Intra-assay and inter-assay coefficients of variation (CV) were within 3 and 6% and within 3 and 4% respectively for FSH and LH. The sensitivity of the assay was 5 pg/ml and 0.03 ng/ml for E2 and progesterone respectively. Intra-assay and inter-assay CV were 5 and 10% respectively for E2 and 3 and 5% respectively for progesterone.
Statistical analysis
Results are expressed as mean±SD. Statistical analysis was performed using Statview 4.5 (Abacus concept software). Nominal or continuous variables were analysed with 2-test or analysis of variance adjusted for centre as required. P<0.05 was considered as statistically significant.
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Results |
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Discussion |
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Supplementation with exogenous rLH has been chosen as an alternative way to evaluate indirectly the consequences of LH depletion because the interpretation of LH values raises many concerns. First, as it is not usual in most assisted reproduction teams to perform hormonal measurements on a daily basis, and as LH release is pulsatile, the interpretation of a single LH determination is limited. Second, it has been shown that measurement of serum immunoreactive LH value is not valid for assessing residual LH bioactivity (Jaakkola et al., 1990). For that reason the E2:oocyte ratio has been proposed as a biological measurement of LH requirement (Loumaye et al., 1997
). Finally, no clear threshold values of deleterious LH levels could be established during assisted reproduction cycles. In anovulatory patients with hypogonadotrophic hypogonadism treated by rFSH alone, no pregnancy can be obtained without any LH supply while follicular growth and ovulation are preserved (European Recombinant Human LH Study Group, 1998
) except if basal LH values are >1.2 IU/l (O'Dea et al., 2000
). In GnRH agonist down-regulated cycles, the impact of low LH levels on outcome is still a matter of debate despite numerous studies (Filicori et al., 2002
) and no obvious criteria could be established to define whose patients may benefit from adding LH to the stimulation protocol (Shoham, 2002
). In GnRH antagonist-treated cycles where the period of LH decrease is restrained to the end of the follicular phase, the impact of low residual LH levels could be less pronounced. However, low residual LH concentrations and impaired estradiol secretion with increasing doses of antagonist were associated with low implantation rate despite the similar number of oocytes and embryos obtained (Ganirelix Dose-finding Study Group, 1998
). As a direct effect of the antagonist seems uncertain because antagonist concentrations are not detectable at the time of transfer, an endometrial impact of low LH levels has been suggested. The direct action of LH on uterine receptivity has recently been hypothesized in the model of oocyte donation. In spite of adequate estrogen and progesterone preparation of the endometrium, a beneficial effect of hCG administration could be observed in patients with low LH levels (Tesarik et al., 2003
). The trend to lower pregnancy rate we have observed in patients with LH deficiency attested by low E2:oocyte ratio could be explained by this endometrial effect. Therefore, further studies are needed to determine better whether a specific sub-group of patients could benefit from rLH supplementation after GnRH antagonist initiation, although it is not evident to define prospectively the criteria to select these patients.
On the contrary, other authors have suggested that high serum LH levels at the early stage of stimulation might be responsible for advanced endometrial maturation and lower pregnancy rates (Kolibianakis et al., 2002, 2003a
). This observation leads the authors to recommend an early initiation of the antagonist on day 1 of the stimulation (Kolibianakis et al., 2003b
). However, many factors may influence the serum LH values during stimulation protocols. We observed that pre-treatment with oral contraceptive pill decreases basal LH values at the beginning of the stimulation. Therefore, while FSH administration has been demonstrated to lower endogenous LH levels (Hull et al., 1998
), increasing E2 levels after a few days of stimulation may trigger a premature LH surge. At the time of GnRH antagonist administration, we observed lower serum LH levels in our study than those reported in the flexible protocol without pre-treatment. Therefore, oral contraceptive pill pre-treatment could be another way to decrease the area under the curve of LH secretion before GnRH antagonist introduction, but whether it may improve implantation remains to be investigated. Finally, the use of a high dose, long-acting GnRH antagonist could prevent an LH surge more efficiently and help to control further LH secretion. So, both the use of a hormonal pre-treatment and a long-acting antagonist could explain why we did not find any influence of the timing of cetrorelix introduction upon the pregnancy rate.
In conclusion, despite some weaknesses in the study design (two different randomization procedures, no blinding for rLH administration), our findings do not support a systematic supplementation with rLH at the time of GnRH antagonist injection in a flexible protocol. In patients pre-treated with an oral contraceptive pill, the duration of stimulation prior to GnRH antagonist introduction does not seem to be related to the outcome of the cycle.
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Submitted on February 26, 2004; accepted on May 19, 2004.