Luteal estradiol pre-treatment coordinates follicular growth during controlled ovarian hyperstimulation with GnRH antagonists

Renato Fanchin1,3, Laurent Salomon1, Altina Castelo-Branco1, François Olivennes1, Nelly Frydman2 and René Frydman1

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The purpose of this study was to investigate whether luteal estradiol (E2) administration reduces follicular size discrepancies and enhances ovarian response in recombinant FSH (r-FSH)/GnRH antagonist protocols. METHODS: We studied prospectively 90 IVF-embryo transfer (ET) candidates who were randomly pre-treated with 17{beta}-E2 (4 mg/day) from day 20 until next cycle day 2 (E2 group, n = 47) or served as controls (control group, n = 43). On day 3, all women started r-FSH treatment. A single 3 mg dose of GnRH antagonist was administered eventually according to follicular maturation. Outcome measures were magnitude of size discrepancy of growing follicles on day 8 of r-FSH treatment and number of follicles >=16 mm in diameter on the day of HCG. RESULTS: On day 8, follicles were smaller (9.9 ± 2.5 versus 10.9 ± 3.4 mm, P < 0.001) and their size discrepancies attenuated (P < 0.001) in the E2 group compared with the control group. This was associated with more >=16 mm follicles, mature oocytes and embryos in the E2 group. CONCLUSIONS: 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. Coordination of follicular development optimizes ovarian response to r-FSH/GnRH antagonist protocols and may constitute an attractive approach to improving their outcome.

Key words: controlled ovarian stimulation/estradiol/follicular synchronization/ovarian response


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During controlled ovarian hyperstimulation (COH), most of the early antral follicles are required to grow coordinately in response to exogenous gonadotrophins to accomplish simultaneous functional and morphological maturation. Marked follicular size discrepancies at the end of COH may be counterproductive since they imply that a substantial fraction of FSH-sensitive follicles fail to undergo satisfactory maturation. This phenomenon potentially reduces the number of viable oocytes and embryos and the probability of conception (Devreker et al., 1999Go; Opsahl et al., 2001Go). In line with this, defective coordination of antral follicle growth during COH may constitute a plausible explanation for the putative poorer IVF-embryo transfer (ET) outcome with GnRH antagonist (Al-Inany and Aboulghar, 2001Go) or short GnRH agonist (Tan et al., 1992Go; Cramer et al., 1999Go) protocols compared with long GnRH agonist protocols.

Although complex mechanisms inherent to each follicle act together to determine its sensitivity to FSH and developmental competence (McNatty et al., 1983Go; Gougeon, 1996Go; Fauser and Van Heusden, 1997Go), 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, 1983Go; Fanchin et al., 2003aGo). 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., 1987Go; Roseff et al., 1989Go). During the luteal–follicular transition, FSH preserves early antral follicles from atresia and ensures their subsequent growth (Chun et al., 1996Go). 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., 1996Go). Since larger follicles are more FSH responsive than are smaller follicles (Hillier et al., 1980Go; McNatty et al., 1983Go), 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 luteal–follicular 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., 2003aGo).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
We prospectively studied 100 female volunteers, 25–38 years of age. All participants met the following inclusion criteria: (i) age <=38 years; (ii) regular, ovulatory menstrual cycles every 25–35 days; (iii) both ovaries present; (iv) no current or past diseases affecting ovaries or gonadotrophin or sex steroid secretion, clearance or excretion; (v) body mass indexes ranging from 18 to 27 kg/m2; (vi) no hormone therapy during the past 6 weeks; and (vii) adequate visualization of both ovaries in transvaginal ultrasound scans. Indications for IVF-ET were male factor (58%), tubal factor (27%), endometriosis (2%) or unexplained infertility (13%). ICSI was programmed in 46% of cases. An informed consent was obtained from all women, and this investigation received the approval of our internal Institutional Review Board. Due to personal reasons (n = 4) or major protocol violation (n = 6), 10 women were excluded from the analysis. The population analysed was therefore limited to 90 participants.

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{beta}-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., 1998Go; Fanchin et al., 2003aGo). Patients included in the control group (n = 43) remained untreated during the luteal phase.



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Figure 1. Study protocol. Horizontal black bars represent menstrual bleeding. Note that in the luteal E2 group, r-FSH treatment was started on the day after 17{beta}-E2 discontinuation.

 
On the first day of E2 discontinuation (cycle day 3) in the E2-treated group or on cycle day 3 in controls, r-FSH treatment (Gonal-F, Serono Laboratories, Saint Cloud, France) was started at a fixed dose set at 225 IU/day, s.c., for 5 days. Further r-FSH administration was adjusted according to usual parameters of follicle growth determined by serum E2 levels and ultrasound monitoring. When at least one follicle exceeded 13 mm in diameter (de Jong et al., 2000Go), a potent, third-generation GnRH antagonist, cetrorelix acetate (Cetrotide, 3 mg, Serono Laboratories), was administered s.c. in a single dose at 8 p.m. An i.m. injection of 10 000 IU of HCG (Gonadotrophine Chorionique ‘Endo’, Organon Laboratories, Saint-Denis, France) was performed when at least five follicles >=16 mm in diameter were obtained. Follicular sizes (mean of two orthogonal diameters) >20 mm in diameter were avoided as far as possible. Cycle cancellation for inadequate ovarian response to COH was decided when fewer than five follicles >=12 mm in diameter were observed after 10 days of r-FSH treatment. Oocytes were retrieved 36 h after HCG administration by transvaginal ultrasound-guided aspiration. Adequate embryo quality was defined as embryos having uniform sized and shaped blastomeres, ooplasm having no granularity and a maximum fragmentation of 10%. All ETs were performed 2 days after oocyte retrieval using a Frydman catheter (CCD Laboratories, Paris, France). The luteal phase was supported with 400 mg of micronized progesterone (Estima Gé, Effik Laboratories, Bièvres, France) administered daily (200 mg in the morning, 200 mg in the evening) by the vaginal route starting on the day of ET.

Ultrasound scans were performed using a 4.5–7.2 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 Student’s t-test when data distribution was normal, or the Mann–Whitney 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, 1960Go). 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Population characteristics
The population included in the E2-treated and in control groups was similar with regard to women’s ages (median, 33 years, range 26–38 versus 33 years, range 25–38, respectively), indications for IVF-ET (male factor, 62% versus 54%; tubal factor, 21% versus 32%; endometriosis, 2% versus 2%; unexplained infertility, 15% versus 12%, respectively), duration of infertility (4.3 ± 0.2 versus 4.1 ± 0.2 years, respectively), rank of the current IVF-ET attempt (2.6 ± 0.3 versus 2.1 ± 0.2, respectively), average menstrual cycle length (29.5 ± 0.4 versus 29.4 ± 0.4 days, respectively) and ovarian status assessment by day 3 serum FSH (6.1 ± 0.2 versus 6.2 ± 0.2 mIU/ml, respectively) and E2 (31.6 ± 2.4 versus 29.1 ± 2.6 pg/ml, respectively) measurements performed within 3 months before inclusion in the protocol. Luteal E2 treatment lasted 11.3 ± 0.6 days. This treatment was well tolerated by patients, who did not report any unwanted side effects. Luteal E2 administration did not alter the expected onset of menstrual bleeding.

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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Table I. Follicular development and embryological results in women pre-treated or not with E2 during the luteal phase
 
Hormonal dynamics
Hormonal dynamics during COH are depicted in Figure 2. As an expected result of luteal E2 administration, serum E2 levels remained higher in the E2-treated than in the control group (139 ± 54 versus 34 ± 8 pg/ml) at baseline. Conversely, on days 6 and 8, serum E2 reached slightly, but significantly, lower levels in patients pre-treated with luteal E2, probably reflecting a slower pace of follicle development. On the day of HCG, however, serum E2 levels in the luteal E2 group tended to exceed control values, which is in keeping with the larger number of mature follicles obtained in that group. Serum progesterone and LH levels remained similar in both groups throughout r-FSH treatment but, as for E2, progesterone showed a trend to higher levels in the luteal E2 group compared with the control group on the day of HCG administration.



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Figure 2. Hormonal dynamics during COH in luteal E2 and control groups. Baseline corresponds to the third day of the cycle (the day after E2 discontinuation in the luteal E2 group), before r-FSH treatment. Days 6 and 8 correspond to the sixth and eighth days of r-FSH treatment, respectively. Differences in serum E2 levels between both groups were statistically significantly at baseline (P < 0.001), on day 6 (P < 0.02) and on day 8 (P < 0.03), but not on the day of HCG. Differences in serum progesterone and LH levels between both groups did not reach statistical signification on any observation day.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study investigated whether E2 pre-treatment during the luteal phase affects developmental characteristics of growing follicles during COH. It aimed at challenging the hypothesis that growth asynchrony of antral follicles is a consequence of the gradual FSH elevation that occurs during the late luteal phase. It was based on a preparatory work of our team (Fanchin et al., 2003aGo) that showed that luteal E2 pre-treatment, presumably through its FSH-suppressive effects, reduces the pace of early follicular growth and attenuates size discrepancies of early antral follicles. In addition, the present study was motivated by our concerns on the poorer COH outcome reported with GnRH antagonist protocols compared with long GnRH agonist protocols (Al-Inany and Aboulghar, 2001Go). This spurred us to look for novel approaches that could optimize the results of GnRH antagonist protocols without compromising their simplicity.

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., 2003aGo).

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., 1999Go; Opsahl et al., 2001Go), 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, 2000Go; Al-Inany and Aboulghar, 2001Go) or short GnRH agonist protocols (Tan et al., 1992Go; Cramer et al., 1999Go). 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., 1992Go; Cramer et al., 1999Go; Borm and Mannaerts, 2000Go; Al-Inany and Aboulghar, 2001Go). 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., 1998Go; Fanchin et al., 2003bGo) 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., 1990Go; Schoolcraft et al., 1997Go). 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. (1990Go) 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{beta}-E2 doses performed in the present and in previous studies (Fanchin et al., 2003aGo,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., 1990Go; Schoolcraft et al., 1997Go). 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.


    Acknowledgements
 
The present paper has been pre-selected for the Established Clinician Award during the 19th ESHRE Annual Meeting that was held in Madrid, Spain, from June 29 to July 2, 2003.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Al-Inany, H. and Aboulghar, M. (2001) Gonadotrophin-releasing hormone antagonists for assisted conception. Cochrane Database Syst. Rev., 4.

Borm, G. and Mannaerts, B. (2000) Treatment with the gonadotrophin-releasing hormone antagonist ganirelix in women undergoing ovarian stimulation with recombinant follicle stimulating hormone is effective, safe and convenient: results of a controlled, randomized, multicentre trial. The European Orgalutran Study Group. Hum. Reprod., 15, 1490–1498.[Abstract/Free Full Text]

Chun, S.Y., Eisenhauer, K.M., Minami, S., Billig, H., Perlas, E. and Hsueh, A.J. (1996) Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor. Endocrinology, 137, 1447–1456.[Abstract]

Cramer, D.W., Powers, D.R., Oskowitz, S.P., Liberman, R.F., Hornstein, M.D., McShane, P.M. and Barbieri, R.L. (1999) Gonadotropin-releasing hormone agonist use in assisted reproduction cycles: the influence of long and short regimens on pregnancy rates. Fertil. Steril., 72, 83–89.[CrossRef][ISI][Medline]

de Jong, D., Macklon, N.S. and Fauser, B.C. (2000) A pilot study involving minimal ovarian stimulation for in vitro fertilization: extending the ‘follicle-stimulating hormone window’ combined with the gonadotropin-releasing hormone antagonist cetrorelix. Fertil. Steril., 73, 1051–1054.[CrossRef][ISI][Medline]

Devreker, F., Pogonici, E., De Maertelaer, V., Revelard, P., Van den Bergh, M. and Englert, Y. (1999) Selection of good embryos for transfer depends on embryo cohort size: implications for the ‘mild ovarian stimulation’ debate. Hum. Reprod., 14, 3002–3008.[Abstract/Free Full Text]

de Ziegler, D., Jaaskelainen, A.S., Brioschi, P.A., Fanchin, R. and Bulletti, C. (1998) Synchronization of endogenous and exogenous FSH stimuli in controlled ovarian hyperstimulation (COH). Hum. Reprod., 13, 561–564.[Abstract]

Fanchin, R., Cunha-Filho, J.S., Schonauer, L.M., Kadoch, I.J., Cohen-Bacrie, P. and Frydman, R. (2003a) Coordination of early antral follicles by luteal estradiol administration provides a basis for alternative controlled ovarian hyperstimulation regimens. Fertil. Steril., 79, 316–321.[CrossRef][ISI][Medline]

Fanchin, R., Cunha-Filho, J.S., Schonauer, L.M., Righini, C., de Ziegler, D. and Frydman, R. (2003b) Luteal estradiol administration strengthens the relationship between day 3 follicle-stimulating hormone and inhibin B levels and ovarian follicular status. Fertil. Steril., 79, 585–589.[CrossRef][ISI][Medline]

Fauser, B.C. and Van Heusden, A.M. (1997). Manipulation of human ovarian function: physiological concepts and clinical consequences. Endocr. Rev., 18, 71–106.[Abstract/Free Full Text]

Gonen, Y., Jacobson, W. and Casper, R.F. (1990) Gonadotropin suppression with oral contraceptives before in vitro fertilization. Fertil. Steril., 53, 282–287.[ISI][Medline]

Gougeon, A. (1996) Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr. Rev., 17, 121–155.[ISI][Medline]

Gougeon, A. and Lefevre, B. (1983) Evolution of the diameters of the largest healthy and atretic follicles during the human menstrual cycle. J. Reprod. Fertil., 69, 497–502.[Abstract]

Hillier, S.G., van den Boogaard, A.M., Reichert, L.E., Jr and van Hall, E.V. (1980) Intraovarian sex steroid hormone interactions and the regulation of follicular maturation: aromatization of androgens by human granulose cells in vitro. J. Clin. Endocrinol. Metab., 50, 640–647.[ISI][Medline]

Klein, N.A., Battaglia, D.E., Fujimoto, V.Y., Davis, G.S., Bremner, W.J. and Soules, M.R. (1996) Reproductive aging: accelerated ovarian follicular development associated with a monotropic follicle-stimulating hormone rise in normal older women. J. Clin. Endocrinol. Metab., 81, 1038–1045.[Abstract]

Levene, H. (1960) In Olkin, I. et al. (eds), Contributions to Probability and Statistics: Essays in Honor of Harold Hotelling. Stanford University Press, pp. 278–292.

Mais, V., Cetel, N.S., Muse, K.N., Quigley, M.E., Reid, R.L. and Yen, S.S. (1987) Hormonal dynamics during luteal–follicular transition. J. Clin. Endocrinol. Metab., 64, 1109–1114.[Abstract]

McNatty, K.P., Hillier, S.G., van den Boogaard, A.M., Trimbos-Kemper, T.C., Reichert, L.E., Jr and van Hall, E.V. (1993) Follicular development during the luteal phase of the human menstrual cycle. J. Clin. Endocrinol. Metab., 56, 1022–1031.

Opsahl, M.S., Blauer, K.L., Black, S.H., Lincoln, S.R., Thorsell, L. and Sherins, R.J. (2001) The number of embryos available for transfer predicts successful pregnancy outcome in women over 39 years with normal ovarian hormonal reserve testing. J. Assist. Reprod. Genet., 18, 551–556.[CrossRef][ISI][Medline]

Roseff, S.J., Bangah, M.L., Kettel, L.M., Vale, W., Rivier, J., Burger, H.G. and Yen, S.S. (1989) Dynamic changes in circulating inhibin levels during the luteal–follicular transition of the human menstrual cycle. J. Clin. Endocrinol. Metab., 69, 1033–1039.[Abstract]

Schoolcraft, W., Schlenker, T., Gee, M., Stevens, J. and Wagley, L. (1997) Improved controlled ovarian hyperstimulation in poor responder in vitro fertilization patients with a microdose follicle-stimulating hormone flare, growth hormone protocol. Fertil. Steril., 67, 93–97.[CrossRef][ISI][Medline]

Tan, S.L., Kingsland, C., Campbell, S., Mills, C., Bradfield, J., Alexander, N., Yovich, J. and Jacobs, H.S. (1992) The long protocol of administration of gonadotropin-releasing hormone agonist is superior to the short protocol for ovarian stimulation for in vitro fertilization. Fertil. Steril., 57, 810–814.[ISI][Medline]

Submitted on July 30, 2003; accepted on October 8, 2003.