Departments of 1 Obstetrics and Gynecology and Reproductive Medicine and 2 Biology and Genetics of Reproduction, Clamart, France
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology and Reproductive Medicine, Hôpital Antoine Béclère, 157, rue de la Porte de Trivaux, 92141 Clamart, France. e-mail: renato.fanchin{at}abc.ap-hop-paris.fr
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Abstract |
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Key words: controlled ovarian stimulation/estradiol/follicular synchronization/ovarian response
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Introduction |
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Although complex mechanisms inherent to each follicle act together to determine its sensitivity to FSH and developmental competence (McNatty et al., 1983; Gougeon, 1996
; Fauser and Van Heusden, 1997
), asynchronous multi-follicular growth during COH may be a direct consequence of size heterogeneities of early antral follicles during the early follicular phase (Gougeon and Lefevre, 1983
; Fanchin et al., 2003a
). The physiological grounds implicated in this process remain poorly understood. A plausible explanation involves the premature, gradient FSH elevation that occurs during the late luteal phase in the menstrual cycle (Mais et al., 1987
; Roseff et al., 1989
). During the lutealfollicular transition, FSH preserves early antral follicles from atresia and ensures their subsequent growth (Chun et al., 1996
). According to their intrinsic sensitivity to this hormone, some follicles are able to respond to lower FSH levels than others and, therefore, to start their development earlier, during the late luteal phase (Klein et al., 1996
). Since larger follicles are more FSH responsive than are smaller follicles (Hillier et al., 1980
; McNatty et al., 1983
), exogenous gonadotrophin administration is likely to intensify further size discrepancies of growing follicles during COH.
Hence, according to this hypothesis, suppression of luteal FSH secretion could prevent untimely, uncoordinated development of FSH-sensitive follicles during the lutealfollicular transition and foster follicular growth synchronization during COH. In agreement with this, we recently demonstrated that administration of physiological estradiol (E2) doses during the luteal phase effectively controls luteal FSH secretion and synchronizes early antral follicle growth during the ensuing follicular phase, as confirmed by the reduction in their mean diameter and mutual size discrepancies on day 3 (Fanchin et al., 2003a).
Spurred on by these previous results, we decided to verify whether luteal E2 administration could promote the coordination of follicular growth during COH and improve its results. For this, we compared follicular development characteristics during COH with recombinant FSH (r-FSH) and GnRH antagonists in women who were pre-treated or not with E2 during the luteal phase.
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Materials and methods |
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Study protocol
The study protocol is depicted in Figure 1. Women randomly received luteal E2 treatment or served as controls. Those included in the luteal E2 group (n = 47) received micronized 17-E2 oral tablets (4 mg/day; Provamès, Cassenne Laboratories, Puteaux, France), in the evening at 8 p.m., from day 20 of the same cycle until day 2 of their next cycle. We chose a 4 mg/day dose for E2 oral administration because of its reported efficacy in reducing endogenous FSH and in preventing early follicular growth (de Ziegler et al., 1998
; Fanchin et al., 2003a
). Patients included in the control group (n = 43) remained untreated during the luteal phase.
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Ultrasound scans were performed using a 4.57.2 MHz multi-frequency transvaginal probe (Siemens Elegra, Siemens S.A.S., Saint-Denis, France) at 8 a.m. by operators who were not aware of the treatment schedule. In addition to usual ultrasonographic monitoring of COH, by design, all women underwent a detailed ultrasound scan of their ovaries on day 8 of r-FSH treatment. During this examination, the number and sizes (mean of two orthogonal diameters) of antral follicles were evaluated; we considered as growing follicles those
8 mm in diameter. Inter- and intra-analysis coefficients of variation (CVs) for follicular measurements were <5% and their lower limit of detection 0.1 mm, respectively.
Serum hormonal (E2, progesterone and LH) measurements that were performed at baseline (just before the start of r-FSH administration), on days 6 and 8 of r-FSH therapy and on the day of HCG were included in the present analysis. Serum E2 and progesterone levels were determined by an automated multi-analysis system using a chemiluminescence technique (ACS-180, Bayer Diagnostics, Puteaux, France). For E2, functional sensitivity was 15 pg/ml, and intra- and inter-assay CVs were 8 and 9%, respectively. For progesterone, functional sensitivity was 0.1 ng/ml, and intra- and inter-assay CVs were 8 and 9%, respectively. Serum LH was measured by an immunometric technique using an Amerlite kit (Ortho Clinical Diagnostics, Strasbourg, France). Intra- and inter-assay CVs were 5 and 7%, respectively, and the lower limit of detection was 0.1 mIU/ml for LH.
Statistics
The measure of central tendency used was the mean, and the measure of variability was the SEM. Medians and ranges were used when data distribution was non-parametric. Treatment allocation was decided by an independent person according to a computer-generated, blocked randomization list. Comparisons of continuous variables between both groups were performed using the Students t-test when data distribution was normal, or the MannWhitney test when normality could not be confirmed. To evaluate the magnitude of follicular size discrepancies on day 8, we tested the homogeneity of variances by using the Levene test for equal variances (Levene, 1960). This test is less sensitive than F-tests to departures from normality and allows a comparison of dispersion of data around the mean independently of mean values. In addition, SD/mean ratios for follicular sizes on day 8 were calculated. The present study was powered to detect anticipated differences of 0.5 mm for follicular sizes and one mature follicle at >80% power at the 0.05 significance level. A P-value <0.05 was considered statistically significant.
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Results |
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Follicular development and embryological results
Follicular development characteristics and embryological data in both groups are presented in Table I. As expected, the number of growing follicles was similar in the two groups on day 8. In contrast, we observed a significant reduction in the mean follicular sizes on day 8 of r-FSH treatment in the luteal E2 compared with the control group. In addition, calculation of homogeneity of variances indicated a noticeable attenuation of follicular size discrepancies in the luteal E2 group compared with the control group on the same observation day (P < 0.01). In line with this, CVs of follicular sizes on day 8 were slightly, yet significantly, lower in the luteal E2 group than in the controls, which further confirms the attenuation in follicular size disparity after E2 pre-treatment. No woman in either group received GnRH antagonist before day 8. However, both cetrorelix acetate and HCG were administered later in the luteal E2 group compared with the control group. Luteal E2 patients tended to require a higher r-FSH dose than controls, but this difference was not significant (2674 ± 91 versus 2463 ± 100 IU, respectively). Six COH cycles (three in each group) had to be cancelled due to unexpected, inadequate response to COH. On the day of HCG administration, more follicles had exceeded 15 mm in diameter in the E2-treated group compared with controls. Consistently, luteal E2 pre-treatment was associated with more mature oocytes and available embryos than in the control group. ICSI was performed in 43 and 50% of cases included in the luteal E2 and control groups, respectively. Whereas the number of embryos transferred was similar in both groups, a higher prevalence of good quality embryos transferred was observed in women pre-treated with E2 compared with controls (63% versus 44%, respectively, P < 0.03), which probably reflects the improved embryo selection from a larger embryo cohort. Although the present investigation was not powered to detect differences in IVF-ET outcome, we incidentally observed a trend for increased pregnancy rates in the luteal E2 group.
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Discussion |
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Our present results indicated that luteal E2 administration effectively reduces the pace of multi-follicular growth in response to r-FSH. Indeed, we observed a slower increase in serum E2 levels during COH and reduced antral follicle sizes on day 8 in E2-pre-treated COH. Also, follicles took longer to achieve maturation and required later GnRH antagonist and HCG administration in E2-pre-treated patients. These effects may be due to an overall reduction in early antral follicle sizes at the start of r-FSH treatment, as previously demonstrated (Fanchin et al., 2003a).
Luteal E2 pre-treatment also fostered follicular growth coordination during COH, as corroborated by the attenuation of follicle size discrepancies on day 8 and the increased number of follicles reaching maturation at once. These follicular effects significantly increased the number of viable oocytes and available embryos. Indeed, the number of embryos produced has been shown to influence IVF-ET outcome positively (Devreker et al., 1999; Opsahl et al., 2001
), possibly through the optimization of embryo selection for ET. Our observation that E2-pre-treated patients had more top quality embryos transferred and showed a trend for improved pregnancy rates is consistent with this hypothesis.
The changes in follicular dynamics and coordination induced by luteal FSH suppression may offer a plausible explanation for the reported improved outcome of long GnRH agonist protocols compared with GnRH antagonist protocols (Borm and Mannaerts, 2000; Al-Inany and Aboulghar, 2001
) or short GnRH agonist protocols (Tan et al., 1992
; Cramer et al., 1999
). Indeed, as in the luteal E2 group, long GnRH agonist protocols usually require longer COH duration and yield more oocytes and embryos than protocols deprived of luteal FSH control (Tan et al., 1992
; Cramer et al., 1999
; Borm and Mannaerts, 2000
; Al-Inany and Aboulghar, 2001
). This suggests that dissimilarities exist in baseline and growing antral follicle characteristics between both COH modalities. Hence, according to this hypothesis, luteal E2 administration could render equivalent the effectiveness of GnRH antagonist compared with long GnRH agonist protocols. Additional studies are, however, required to verify this point. Furthermore, luteal E2 administration presents some advantages over long GnRH agonist protocols, such as simplicity, less side effects and no endogenous gonadotrophin suppression during the early follicular phase of COH. Indeed, immunoreactive LH levels remained at
2 mIU/ml during the first 8 days of r-FSH administration in both groups in the present study. Moreover, the significant FSH rebound reported after E2 discontinuation (de Ziegler et al., 1998
; Fanchin et al., 2003b
) is likely to synergize with exogenous gonadotrophins and further promote multi-follicular development.
The present data also propose alternative insights into the reported improvement of COH outcome with combined oral contraceptive pre-treatment (Gonen et al., 1990; Schoolcraft et al., 1997
). Indeed, due to their potent anti-FSH action, oral contraceptives may exert similar, or even stronger, coordinating effects on early follicular development compared with luteal E2 administration. In line with this, Gonen et al. (1990
) reported an increase in the number of mature follicles and oocytes in clomiphene citrate cycles preceded by oral contraceptives compared with controls. Yet, combined oral contraceptives have putative shortcomings, such as lengthy treatment course, menstrual bleeding postponement and adverse effects, that are not shared by the 10 day administration of physiological 17
-E2 doses performed in the present and in previous studies (Fanchin et al., 2003a
,b). Comparative studies between the effects of oral contraceptive and luteal E2 pre-treatments on follicular cohort characteristics and COH outcome are needed to clarify this issue. Also, the appropriate timing to start exogenous gonadotrophin administration after luteal E2 or oral contraceptive discontinuation remains undefined. Some authors usually start gonadotropin treatment 2 or 3 days after oral contraceptive discontinuation (Gonen et al., 1990
; Schoolcraft et al., 1997
). However, in the present study, r-FSH administration was purposely begun on the day after E2 withdrawal to derive maximum benefit from the follicular size coordination resulting from luteal FSH suppression. The possibility of extrapolating this paradigm to oral contraceptive pre-treatment deserves to be addressed in future studies.
In conclusion, luteal E2 administration reduces the pace of growth, improves size homogeneity of antral follicles on day 8 of r-FSH treatment, and increases the number of follicles reaching maturation at once. This approach represents a potential, more physiological alternative to GnRH agonist or oral contraceptive pre-treatment to synchronize multi-follicular development and improve COH results. However, larger studies are needed to confirm whether follicular growth coordination induced by luteal E2 administration improves IVF-ET pregnancy rates with GnRH antagonist or short GnRH agonist protocols.
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Acknowledgements |
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References |
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Submitted on July 30, 2003; accepted on October 8, 2003.