Effects of gonadotrophin-releasing hormone agonists on human ovarian steroid secretion in vivo and in vitro—results of a prospective, randomized in-vitro fertilization study

J. Dor1, D. Bider, A. Shulman, J. Levron, S. Shine, S. Mashiach and J. Rabinovici

IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel Hashomer, Israel


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this prospective randomized study was to compare the effects of two gonadotrophin-releasing hormone (GnRH) agonists, buserelin and triptorelin, on human ovarian follicular steroidogenesis, oocyte fertilization and IVF treatment outcome. Ovulatory, healthy women undergoing IVF were treated either with human menopausal gonadotrophin (HMG) alone or with HMG and one of the two GnRH agonists. Serum and follicular fluid hormonal concentrations and cultures of luteinizing granulosa cells obtained during follicular aspiration were analysed. GnRH agonist treatment significantly affected steroidogenesis both in serum and follicular fluid. In follicular fluid, progesterone and oestradiol concentrations were significantly elevated while testosterone concentrations were significantly lower in the triptorelin group. The ratios of testosterone/progesterone, oestradiol/progesterone but not oestradiol/testosterone concentrations were significantly affected by GnRH agonist administration. Similarly, the steroidogenic activity of luteinizing granulosa cells in vitro was significantly decreased in women treated with GnRH agonists. Women treated with GnRH agonists had significantly more fertilized oocytes and cleaving embryos. The results indicate a marked effect of GnRH agonists on the pattern of ovarian follicular steroidogenesis that cannot be explained solely by changes in gonadotrophin concentrations.

Key words: GnRH agonists/granulosa cells/IVF/ovary/steroidogenesis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gonadotrophin-releasing hormone (GnRH) agonists are widely used in reproductive medicine to implement down-regulation of pituitary gonadotrophin receptors. The ensuing endogenous hypogonadotrophic state results in a marked decrease in cancellations of ovulation induction cycles for IVF and facilitates patient scheduling (Dor et al., 1992aGo). However, administration of a GnRH agonist resulting in pituitary desensitization also leads to an increase in the number of ampoules of gonadotrophins and in the duration of treatment needed to evoke an adequate ovarian response (Lipitz et al., 1989Go; van de-Helder et al., 1990Go). This altered response has been generally attributed either to the suppression of endogenous pituitary gonadotrophin concentrations that accompany GnRH analogue administration or to a putative direct action of GnRH analogues on the ovary.

Numerous studies have demonstrated a possible direct modulatory role of GnRH and its analogues on ovarian function in non-human species. GnRH and its analogues affect in-vitro steroidogenesis, atresia and apoptotic cell death of granulosa cells of several animal species (Hsueh and Erickson, 1979Go; Ledwitz-Rigby, 1990Go; Wickings et al., 1990Go; Liu et al., 1991Go; Billig et al., 1994Go; Erickson et al., 1994Go; Mori et al., 1994Go; Sirotkin et al., 1994Go). Further, Olofsson et al. reported the presence and regulation of GnRH receptor mRNA on murine granulosa cells (Olofsson et al., 1995Go).

Recent studies indicate that GnRH and its analogues also directly affect differentiation of human granulosa cells (Guerrero et al., 1993Go; Gaetje, 1994Go; Uemura et al., 1994Go) and that GnRH receptor mRNA can be detected in these cells (Peng et al., 1994Go; Minaretzis et al., 1995Go). Although most authors reported an inhibitory effect of GnRH analogues on ovarian steroidogenesis in vitro (Guerrero et al., 1993Go; Gaetje, 1994Go; Uemura et al., 1994Go), one study described a stimulatory effect on aromatase activity (Bussenot et al., 1993Go) and another study failed to show any effect (Fabbri et al., 1996Go). Further, despite similar pituitary effects different GnRH analogues elicited variable ovarian steroidogenic responses (Bussenot et al., 1993Go; Lockwood et al., 1995Go), suggesting different effects of the analogues on the pituitary and ovarian GnRH receptors (Bussenot et al., 1993Go).

Therefore, the current study was aimed to clarify and define further the effects of two different GnRH agonists in use for induction of ovulation for IVF on: (i) human ovarian follicular steroidogenesis both in vivo and in vitro; and (ii) on oocyte fertilization and IVF treatment outcome. To this end, a prospective randomized study of three different ovulation induction regimens in infertile women with normal ovarian function undergoing IVF was performed. Serum and follicular fluid hormonal concentrations and cultures of luteinizing granulosa cells obtained during follicular aspiration and oocyte recovery served to compare the in-vivo to the in-vitro pattern of steroidogenesis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
Seventy-four infertile patients in good general health, aged 20–36 years (mean ± SEM: 29.5 ± 0.4) with regular ovulatory cycles (25–35 days duration) and with a diagnosis of either tubal or unexplained infertility were recruited prospectively for this study. Informed consent was obtained from all patients prior to inclusion and the study protocol had been approved by the local ethical committee on human research.

Protocol
Patients were randomly allocated according to a prospective randomization list maintained by a person not involved in the study to one of three different study protocols. Data reported in this study were obtained only from women who underwent oocyte retrieval. The 26 women allocated to the human menopausal gonadotrophin (HMG) treatment protocol, received daily 225 IU FSH and 225 IU LH (Pergonal; Teva, Petah Tikva, Israel) from day 3 of the cycle. Twenty-four women were allocated to the second treatment protocol: D-Ser (TBU)6-ethylamide-luteinizing hormone-releasing hormone (LHRH) (Buserelin; Aventis Pharma, Frankfurt, Germany)/HMG. Buserelin was administered intranasally at a daily dosage of 600 µg, starting on day 2 of the cycle, followed by HMG from day 15 of the same cycle after hormonal and ultrasonic demonstration of ovarian down-regulation. Twenty-four women were allocated to the third treatment protocol D-TRP-GnRH [3.2 mg CR triptorelin (Ferring, Kiel, Germany)/HMG]. Triptorelin was administered i.m. on day 2 of the cycle followed by HMG from day 15 of the same cycle, after hormonal and ultrasonic demonstration of ovarian quiescence. After 5 days of HMG administration ovarian response was evaluated repeatedly in all patients by serum oestradiol concentrations and vaginal ultrasonographic scans to define ovarian follicular development. The dosage of HMG was adjusted on an individual basis according to clinical judgement as published previously (Dor et al., 1990Go, 1992cGo). When follicles reached a diameter >18 mm and serum oestradiol concentrations were >400 pg/ml, human chorionic gonadotrophin (HCG, 10 000 IU; Chorigon, Teva, Petah Tikva, Israel) was administered. Ultrasonographic-guided follicular aspiration for oocyte retrieval was performed 34–35 h after HCG administration.

Sample collection
Blood samples were drawn during the ovarian stimulation phase of the study and on the day of oocyte retrieval for determination of serum oestradiol, progesterone, testosterone, LH and FSH concentrations. Before oocyte retrieval, the size and number of follicles for each ovary was recorded. Clear follicular fluids (uncontaminated with blood) from at least three leading follicles containing oocytes were obtained from each woman and after centrifugation for 10 min at 190 g were frozen at –70°C for determination of oestradiol, progesterone, testosterone, LH and FSH concentrations.

Cell cultures
Luteinizing granulosa cells were obtained from each patient during oocyte retrieval and cultured as previously described with and without gonadotrophins (Pariente et al., 1990Go; Dor et al., 1992bGo).

Laboratory methods
The stored daily serum samples were used for the determination of oestradiol, progesterone and testosterone. All follicular fluid was assayed after dilution with low steroid serum, when appropriate. Oestradiol, progesterone and testosterone were measured by use of solid phase radioimmunoassay kits provided by Diagnostic Products Corporation (Santa Monica, CA, USA). The intra-assay and inter-assay coefficients of variation (CV) respectively varied between 7 and 10% for oestradiol, 3.6 and 11% for progesterone, and 6 and 9.5% for testosterone. LH and FSH in follicular fluid were measured by Microparticle Enzyme Immunoassay (MEIA; Diagnostic Products Corp.). Sensitivity to LH and FSH was 0.5 and 0.2 mIU/ml respectively.

Statistical analysis
Results are reported as means ± SEM. Differences between the treatment groups were assessed by Student's t-test or by Kruskal–Wallis one-way analysis of variance (ANOVA) on ranks and pairwise multiple comparison procedures (Dunn's method). Differences assigned a P value of < 0.05 were regarded as statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background data
The clinical data for the women allocated to the three treatment protocols were similar (Table IGo).


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Table I. Clinical data of the patients
 
Follicular stimulation phase
Women in the HMG/buserelin and HMG/triptorelin groups were treated longer and needed significantly more ampoules of HMG than women in the HMG alone group (Table IGo). During the follicular stimulation phase, mean serum oestradiol and progesterone concentrations followed a similar pattern and they were not different between the treatment groups.

Day of oocyte retrieval
Serum
On the day of oocyte retrieval, mean serum oestradiol, testosterone and progesterone concentrations did not differ between the three groups (Table IIGo). However, when analysing the pattern of steroidogenesis by calculating the ratio of testosterone versus progesterone (testosterone/progesterone) and oestradiol versus progesterone (oestradiol/progesterone) significant differences were observed: the mean testosterone/progesterone and oestradiol/progesterone ratios of the triptorelin group were significantly lower than that of the HMG alone group. The mean ratios of the buserelin group were between the two groups but did not reach statistical significance. In contrast, the oestradiol/testosterone ratio, a measure of the aromatase activity, was not significantly different between the three groups.


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Table II. Serum concentrations of ovarian steroids on the day of oocyte retrieval
 
Follicular fluid
Mean follicular fluid steroid concentrations as well as the pattern of steroidogenesis was markedly affected by GnRH agonist treatment (Table IIIGo). Mean follicular fluid progesterone concentrations were significantly higher in the HMG/triptorelin group than in the other two groups. In contrast, mean follicular fluid testosterone and oestradiol concentrations were significantly higher in the HMG alone group and in the buserelin group than in the triptorelin group. These differences were accentuated when the ratios of the steroids were calculated. The mean follicular fluid testosterone/progesterone, oestradiol/progesterone ratios of the HMG/triptorelin group were significantly lower than those of the other two groups. In contrast, the oestradiol/testosterone ratios of the two agonist groups were similar in all groups. Follicular fluid gonadotrophin concentrations were significantly different between the three groups (Table IIIGo). While mean FSH concentrations were similar, mean LH concentrations were significantly higher in the HMG alone group than in the two GnRH agonist groups. However, no differences were found between the two GnRH agonist groups.


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Table III. Follicular fluid concentrations of ovarian steroids and gonadotrophins on the day of oocyte retrieval
 
Luteinized granulosa cell culture
Oestradiol secretion from luteinized granulosa cells was time- and group-dependent. Unstimulated as well as FSH-stimulated oestradiol secretion decreased gradually from day 2 to 4 after oocyte retrieval (Figure 1Go). In addition, FSH-stimulated oestradiol secretion was significantly decreased in granulosa cells from women who received GnRH agonist treatment prior to oocyte retrieval (Figure 1Go). These differences were not observed in cultures that were not stimulated by FSH administrations.



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Figure 1. Granulosa cells were obtained from three groups of patients during oocyte retreival after patients were treated with HMG alone, buserelin and HMG or triptorelin and HMG. Oestradiol secretion by granulosa cells in vitro on day 2 and day 4 with or without FSH. Cells (30 000 viable cells) were cultured in serum-free medium containing 10–6 mol/l androstenedione and FSH (100 IU/l) when indicated. The concentration of oestradiol in the medium was measured by radioimmunoassay. Data are the mean ± SEM. The media were changed every 48 h, at which time fresh hormones and substrate were added to the appropriate culture. *P = NS for cells without FSH. **P = 0.003 for cells with FSH on day 2. ***P = 0.005 for cells with FSH on day 4. Analysis of variance (for repeated measures). Conversion factor of 3.671 for ng/ml to nmol/l. HMG = human gonadotrophin.

 
Results of IVF treatment
Despite differences of clinical significance in the mean number of oocytes retrieved for each group, these differences did not reach statistical significance: 9.2 ± 0.9, 13.6 ± 2.9 and 14.1 ± 1.7 oocytes for the HMG alone, buserelin/HMG and triptorelin/HMG groups respectively. The fertilization rate, somewhat lower in the HMG group, did not differ between the three groups. However, women in the two GnRH agonist groups had significantly more fertilized oocytes as well as three pronucleate oocytes than women in the HMG group (P = 0.04, one-way ANOVA) (Table IVGo).


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Table IV. Clinical IVF outcome in the three study groups
 
The increase in the number of fertilized oocytes resulted in significantly more embryos (Table IVGo). The slightly higher pregnancy rate as well as the delivery rate in this study in the two GnRH agonist groups did not reach statistical significance (Table IVGo).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of this study indicate that administration of GnRH agonists to infertile women with normal ovarian function undergoing IVF treatment affects ovarian follicular steroidogenesis both in vivo and in vitro. The relative abundance of steroidogenic ovarian hormones in serum as well as in follicular fluid was significantly altered by the administration of the GnRH analogues (Tables II and IIIGoGo). In serum, mean concentrations of progesterone, testosterone and oestradiol on the day of oocyte retrieval were not statistically different. However, the mean ratios of serum concentrations of testosterone versus progesterone and of oestradiol versus progesterone indicate that during administration of a GnRH agonist, the transfer of ovarian C21 progestagens to C19 androgens was decreased (Table IIGo). The ratios between the concentrations of progesterone and testosterone and between progesterone and oestradiol, the main C21, C19 and C18 ovarian steroid hormones were used to examine indirectly the steroidogenic activity of the follicular apparatus. Thus, the progesterone/testosterone ratio is a rough measure of 17-hydroxylase/desmolase activity and the oestradiol/testosterone ratio serves as a rough measure of aromatase activity. The effect of GnRH agonists on aromatase activity expressed as the ratio of oestradiol versus testosterone was less pronounced. In a previous study on women undergoing ovulation induction for intrauterine insemination similar changes in mean serum testosterone and oestradiol concentrations were observed after treatment with leuprorelin, a GnRH agonist (Filicori et al., 1996Go). However, although in contrast to the present study the authors did not calculate the effects of the GnRH agonist on the different steps of steroidogenesis, their results seem also to indicate an inhibitory effect on metabolization of C21 (progestagens) to C19 (androgens) steroids (Filicori et al., 1996Go).

Serum oestradiol concentrations serve as a major endpoint of treatment and therefore might depend on treatment decisions. In this study, the mean oestradiol concentrations of all three study groups were similar. However, to achieve these similar concentrations significantly more HMG was administered to women in the GnRH analogue groups (Table IGo). In addition, the number of aspirated follicles was slightly higher in the two GnRH agonist groups. This indicates that the oestradiol secretion per follicle was lower in the agonist groups. This finding serves as additional evidence for decreased transfer of C21 to C19 steroids in the GnRH agonist treated women. The findings in follicular fluid were even more pronounced than those found in serum. Women treated with the GnRH agonist triptorelin had significantly higher mean concentrations of progesterone and lower mean concentrations of testosterone and oestradiol in follicular fluid. Follicular conversion of progesterone to testosterone, of progesterone to oestradiol but not of testosterone to oestradiol was decreased in both GnRH agonist groups (Table IIIGo). These findings supported and explained the parallel findings in serum and suggest that during GnRH agonist administration follicular progesterone production is increased and conversion of C21 progesterone into C19 androgens and C18 oestrogens is inhibited.

The alterations in follicular steroidogenesis seen in this study could be attributed either to changes in circulating gonadotrophin concentrations or to a possible direct ovarian action of GnRH analogues. Long-term administration of GnRH analogues decreases circulating concentrations of pituitary gonadotrophins in women undergoing ovulation induction. Exogenous administration of gonadotrophins is necessary to achieve adequate ovarian stimulation. In this study, mean ovarian follicular fluid concentrations of FSH were similar in all three groups while mean follicular fluid concentrations of LH were significantly higher in the HMG group (Table IIIGo). Progesterone production, affected mainly by LH and HCG (Rabinovici, 1993Go), was highest in the triptorelin group which had the lowest mean LH concentrations, while aromatase activity, affected mainly by FSH (Rabinovici, 1993Go) was lowest in the HMG group despite similar FSH concentrations. Thus, the differential effects on the steroidogenic steps seen in this study are difficult to explain by the known actions of LH and FSH. These findings suggest therefore, some additional, direct or indirect effects not mediated by gonadotrophins, possibly by the GnRH agonists, on follicular steroidogenesis.

A large body of evidence supports a possible effect of the GnRH analogues on ovarian steroidogenesis. Several studies demonstrate in-vitro effects of GnRH analogues on granulosa cell differentiation (Guerrero et al., 1993Go; Gaetje, 1994Go; Uemura et al., 1994Go) as well as GnRH receptor mRNA on these cells (Peng et al., 1994Go; Minaretzis et al., 1995Go). It was demonstrated in this study that cells exposed to GnRH analogues respond differently to FSH stimulation as compared to HMG only (Figure 1Go). Triptorelin and buserelin are two GnRH agonists that have been extensively used over the last decade as an adjunct in induction of ovulation for IVF (McLachlan et al., 1986Go; Rolet et al., 1988Go; Brogden et al., 1990Go; Out et al., 1996Go). In this study, both GnRH agonists were used at an adequate dosage and both achieved a similar clinical and laboratory degree of pituitary gonadotrophic down-regulation (Table IIIGo) at a dosage recommended by the manufacturers for adequate pituitary down-regulation. Thus, despite the very similar pituitary effects, these two GnRH agonists did not exhibit identical ovarian effects. The effects of buserelin on ovarian steroidogenesis were generally less pronounced than those of triptorelin, either because of different circulating or ovarian concentrations of the GnRH agonists or because of variable effects of the GnRH agonists on the ovarian GnRH receptor. In a previous study, using cultured human luteinizing granulosa cells some but not all tested GnRH analogues altered oestradiol secretion and cell surface morphology (Bussenot et al., 1993Go). These different effects of different GnRH agonists were attributed to possible differences between pituitary and ovarian GnRH receptors (Bussenot et al., 1993Go). Further, a recent study demonstrated that in-vivo administration of a GnRH agonist or a GnRH antagonist results in differences in in-vitro steroidogenesis of luteinizing granulosa cells (Lin et al., 1999Go).

In this study, the inhibitory effects of GnRH agonists on the production of follicular ovarian steroids were found also in the luteinized granulosa cell cultures (Figure 1Go). Granulosa cells exposed in vivo to GnRH agonists secreted significantly less FSH-stimulated oestradiol than cells that were not exposed to GnRH agonists. Basal steroidogenic activity was not altered by GnRH agonist. Previous studies in cultured murine granulosa cells demonstrated that addition of GnRH decreased FSH-induced steroidogenesis (Hsueh et al., 1984Go). Further, the findings of the current study confirmed a previous study where both GnRH and triptorelin strongly inhibited FSH-mediated function in human granulosa–lutein cells in culture (Furger et al., 1996Go).

Use of GnRH agonists affected also the clinical outcome of IVF: women treated with GnRH agonists had more ovarian follicles, more fertilized oocytes and more embryos for transfer (Table IVGo). Increased follicular testosterone and decreased oestradiol concentrations were found in the past to be correlated with follicular and oocyte state (Dor et al., 1992cGo). Clinical experience has shown that treatment of GnRH analogues in women with decreased ovarian response (low responders) might further impair clinical outcome (Feldberg et al., 1994Go; Faber et al., 1998Go; Surrey et al., 1998Go). It remains to be seen whether the local follicular effects observed in this study are responsible for the impaired clinical outcome in these women.

In conclusion, it was found that GnRH agonist therapy had a significant effect on ovarian follicular steroidogenesis. Ovarian steroids, especially oestradiol, serve as the most significant markers of follicular development during ovulation induction. Thus, the high doses of gonadotrophins necessary to induce ovarian response when GnRH agonists are administered might be at least in part related to this inhibitory effect of GnRH agonists on ovarian steroidogenic activity mediated by ovarian GnRH receptors. Taken together with previous observations, the results presented here seem to support a local action of GnRH agonists on human ovarian steroidogenesis. In addition, the findings indicate that different GnRH agonists differ in their effect on ovarian steroidogenesis. Based on these findings, it is suggested that in the future the choice of the GnRH analogue during IVF could be tailored according to the ovarian pathology (polycystic ovarian disease, low ovarian response).


    Acknowledgments
 
This study was supported in part by grants from the Hoechst Company, Germany.


    Notes
 
1 To whom correspondence should be addressed at: IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel Hashomer, Israel. E-mail: jehoshua{at}netvision.net.il Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Billig, H., Furuta, I. and Hsueh, A.J. (1994) Gonadotropin-releasing hormone directly induces apoptotic cell death in the rat ovary: biochemical and in situ detection of deoxyribonucleic acid fragmentation in granulosa cells. Endocrinology, 134, 245–252.[Abstract]

Brogden, R.N., Buckley, M.M. and Ward, A. (1990) Buserelin. A review of its pharmacodynamic and pharmacokinetic properties, and clinical profile. Drugs, 39, 399–437.[ISI][Medline]

Bussenot, I., Azoulay-Barjonet, C. and Parinaud, J. (1993) Modulation of the steroidogenesis of cultured human granulosa–lutein cells by gonadotropin-releasing hormone analogs. J. Clin. Endocrinol Metab., 76, 1376–1379.[Abstract]

Dor, J., Ben-Shlomo, I., Lipitz, S. et al. (1990) Ovarian stimulation with gonadotropin-releasing hormone (GnRH) analogue improves the in vitro fertilization (IVF) pregnancy rate with both transvaginal and laparoscopic oocyte recovery. J. In Vitro Fert. Embryo Transf., 7, 351–354.[ISI][Medline]

Dor, J., Ben-Shlomo, I., Levran, D. et al. (1992a) The relative success of gonadotropin-releasing hormone analogue, clomiphene citrate, and gonadotropin in 1099 cycles of in vitro fertilization. Fertil. Steril., 58, 986–990.[ISI][Medline]

Dor, J., Costritsci, N., Pariente, C. et al. (1992b) Insulin-like growth factor-I and follicle-stimulating hormone suppress insulin-like growth factor binding protein-1 secretion by human granulosa–luteal cells. J. Clin. Endocrinol. Metab., 75, 969–971.[Abstract]

Dor, J., Shulman, A., Pariente, C. et al. (1992c) The effect of gonadotropin-releasing hormone agonist on the ovarian response and in vitro fertilization results in polycystic ovarian syndrome: a prospective study. Fertil. Steril., 57, 366–371.[ISI][Medline]

Erickson, G.F., Li, D., Sadrkhanloo, R. et al. (1994) Extrapituitary actions of gonadotropin-releasing hormone: stimulation of insulin-like growth factor-binding protein-4 and atresia. Endocrinology, 134, 1365–1372.[Abstract]

Fabbri, R., Porcu, E., Pession, A. et al. (1996) The effect of leuprorelin on steroidogenesis of human preovulatory granulosa cells in vitro. J. Assist. Reprod. Genet., 13, 287–292.[ISI][Medline]

Faber, B.M., Mayer, J., Cox, B. et al. (1998) Cessation of gonadotropin-releasing hormone agonist therapy combined with high-dose gonadotropin stimulation yields favorable pregnancy results in low responders. Fertil. Steril., 69, 826–830.[ISI][Medline]

Feldberg, D., Farhi, J., Ashkenazi, J. et al. (1994) Minidose gonadotropin-releasing hormone agonist is the treatment of choice in poor responders with high follicle-stimulating hormone levels. Fertil. Steril., 62, 343–346.[ISI][Medline]

Filicori, M., Flamigni, C., Cognigni, G.E. et al. (1996) Different gonadotropin and leuprorelin ovulation induction regimens markedly affect follicular fluid hormone levels and folliculogenesis. Fertil. Steril., 65, 387–393.[ISI][Medline]

Furger, C., Bourrie, N., Cedard, L. et al. (1996) Gonadotrophin-releasing hormone and triptorelin inhibit the follicle stimulating hormone-induced response in human primary cultured granulosa-lutein cells. Mol. Hum. Reprod., 2, 259–264.[Abstract]

Gaetje, R. (1994) Influence of gonadotrophin releasing hormone (GnRH) and a GnRH-agonist on granulosa cell steroidogenesis. Clin. Exp. Obstet. Gynecol., 21, 164–169.[Medline]

Guerrero, H.E., Stein, P., Asch, R.H. et al. (1993) Effect of a gonadotropin-releasing hormone agonist on luteinizing hormone receptors and steroidogenesis in ovarian cells. Fertil. Steril., 59, 803–808.[ISI][Medline]

Hsueh, A.J. and Erickson, G.F. (1979) Extrapituitary action of gonadotropin-releasing hormone: direct inhibition of ovarian steroidogenesis. Science, 204, 854–855.[ISI][Medline]

Hsueh, A.J., Adashi, E.Y., Jones, P.B. et al. (1984) Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr. Rev., 5, 76–127.[ISI][Medline]

Ledwitz-Rigby, F. (1990) Gonadotropin-releasing hormone inhibition of LH stimulated progesterone secretion by porcine granulosa cells in vitro. Domest. Anim. Endocrinol., 7, 265–272.[ISI][Medline]

Lin, Y., Kahn, J.A. and Hillensjo, T. (1999) Is there a difference in the function of granulosa-luteal cells in patients undergoing in-vitro fertilization either with gonadotrophin-releasing hormone agonist or gonadotrophin-releasing hormone antagonist? Hum. Reprod., 14, 885–888.[Abstract/Free Full Text]

Lipitz, S., Ben-Rafael, Z., Dor, J. et al. (1989) Suppression with gonadotropin-releasing hormone analogues prior to stimulation with gonadotropins: comparison of three protocols. Gynecol. Obstet. Invest., 28, 31–34.[ISI][Medline]

Liu, Y.X., Hu, Z.Y., Feng, Q. et al. (1991) Paradoxical effect of a GnRH agonist on steroidogenesis in cultured monkey granulosa cells. Sci. China B, 34, 1452–1460.[ISI][Medline]

Lockwood, G.M., Pinkerton, S.M. and Barlow, D.H. (1995) A prospective randomized single-blind comparative trial of nafarelin acetate with buserelin in long-protocol gonadotrophin-releasing hormone analogue controlled in-vitro fertilization cycles [see comments]. Hum. Reprod., 10, 293–298.[Abstract]

McLachlan, R.I., Healy, D.L. and Burger, H.G. (1986) Clinical aspects of LHRH analogues in gynaecology: a review. Br. J. Obstet. Gynaecol., 93, 431–454.[ISI][Medline]

Minaretzis, D., Jakubowski, M., Mortola, J.F. et al. (1995) Gonadotropin-releasing hormone receptor gene expression in human ovary and granulosa–lutein cells. J. Clin. Endocrinol. Metab., 80, 430–434.[Abstract]

Mori, H., Ohkawa, T., Takada, S. et al. (1994) Effects of gonadotropin-releasing hormone agonist on steroidogenesis in the rat ovary. Horm. Res. 41, 14–21.

Olofsson, J.I., Conti, C.C. and Leung, P.C. (1995) Homologous and heterologous regulation of gonadotropin-releasing hormone receptor gene expression in preovulatory rat granulosa cells. Endocrinology, 136, 974–980.[Abstract]

Out, H.J., Mannaerts, B.M., Driessen, S.G. et al. (1996) Recombinant follicle stimulating hormone (rFSH; Puregon) in assisted reproduction: more oocytes, more pregnancies. Results from five comparative studies. Hum. Reprod. Update, 2, 162–171.[Abstract/Free Full Text]

Pariente, C., Rabinovici, J., Lunenfeld, B. et al. (1990) Steroid secretion by granulosa cells isolated from a woman with 17 alpha-hydroxylase deficiency. J. Clin. Endocrinol. Metab., 71, 984–987.[Abstract]

Peng, C., Fan, N.C., Ligier, M. et al. (1994) Expression and regulation of gonadotropin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa–luteal cells. Endocrinology, 135, 1740–1746.[Abstract]

Rabinovici, J. (1993) The differential effects of FSH and LH on the human ovary. Bailliere's Clin. Obstet. Gynaecol., 7, 263–281.[ISI][Medline]

Rolet, F., Gadaud, S., Zorn, J.R. et al. (1988) Ovarian programming and GIFT. Hum. Reprod., 3, 563–565.[Abstract]

Sirotkin, A.V., Tarasenko, L.V., Nitray, J. et al. (1994) Direct action of LHRH and its antagonist on isolated bovine granulosa cells steroidogenesis. J. Endocrinol. Invest., 17, 723–728.[ISI][Medline]

Surrey, E.S., Bower, J., Hill, D.M. et al. (1998) Clinical and endocrine effects of a microdose GnRH agonist flare regimen administered to poor responders who are undergoing in vitro fertilization. Fertil. Steril., 69, 419–424.[ISI][Medline]

Uemura, T., Namiki, T., Kimura, A. et al. (1994) Direct effects of gonadotropin-releasing hormone on the ovary in rats and humans. Horm. Res., 41, 7–13.

van de-Helder, A.B., Helmerhorst, F.M., Blankhart, A. et al. (1990) Comparison of ovarian stimulation regimens for in vitro fertilization (IVE) with and without a gonadotropin-releasing hormone (GnRH) agonist: results of a randomized study. J. In Vitro Fert. Embryo Transf., 7, 358–362; discussion 363–354.[ISI][Medline]

Wickings, E.J., Eidne, K.A., Dixson, A.F. et al. (1990) Gonadotropin-releasing hormone analogs inhibit primate granulosa cell steroidogenesis via a mechanism distinct from that in the rat. Biol. Reprod., 43, 305–311.[Abstract]

Submitted on August 19, 1999; accepted on February 23, 2000.