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The antiprogestin RU486 delays the midcycle gonadotropin surge and ovulation in gonadotropin-releasing hormone-induced cycles†
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~~~19§W:f_":~":~rinOlo9Y FERTILITY AND STERILITY Copyright
©
Vol. 62, No.1, July 1994
Printed on acid-free paper in U. S. A.
1994 The American Fertility Society
The antiprogestin RU486 delays the midcycle gonadotropin surge and ovulation in gonadotropin-releasing hormone-induced cyclest
Marcelo C. Batista, M.D.*:j:§ Tannia P. Cartledge, R.N. II Ann W. Zellmer, R.N. II
Lynnette K. Nieman, M.D.:j:~ D. Lynn Loriaux, M.D., Ph.D.:j:** George R. Merriam, M.D.:j:tt
National Institute of Child Health and Human Development, and Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland
Objective: To investigate whether the antiprogestin RU486 acts primarily on the hypothalamus to delay the midcycle gonadotropin surge and thus gain insight into the site(s) of action of P in the control of ovulation. Design: Prospective, crossover, single-blinded clinical study. Setting: Outpatient clinic in an academic research environment. Patients: Women with hypothalamic amenorrhea. Interventions: RU486 or a placebo was given orally at a low dose of 1 mg/d for 5 days, starting when the dominant follicle reached 14 to 16 mm, to women with hypothalamic amenorrhea undergoing ovulation induction with GnRH pulses of unvarying frequency and dose. Blood samples and ovarian ultrasounds were obtained daily in the late follicular phase and every 3 to 4 days in the remainder of the cycle. Main Outcome Measures: Follicular diameter and plasma levels of LH, FSH, E 2 , and P. Results: RU486 consistently delayed the timing of the midcycle gonadotropin surge and ovulation. Gonadotropin and steroid levels were suppressed during RU486 treatment, but follicular growth progressed normally in most patients. Conclusions: RU486 does not act primarily on the hypothalamus to delay ovulation. Rather, this compound appears to antagonize P at the pituitary level to suppress gonadotropin and steroid hormone secretion. P may thus act on the pituitary, independent of any hypothalamic effects, to regulate the timing of the midcycle gonadotropin surge and ovulation. Fertil Steril 1994;62:28-34 Key Words: RU486, progesterone, ovulation, gonadotropin, gonadotropin-releasing hormone
The antiprogestin RU486 provides a powerful tool to study the physiology of P action throughout Received September 24, 1993; revised and accepted March 7, 1994. * Supported in part by grant 86/2520-2, Funda<;ao de Amparo 'it Pesquisa do Estado de Sao Paulo, Sao Paulo, Brazil. t Presented in part at the 71st Annual Meeting of the Endocrine Society, Seattle, Washington, June 21 to 24, 1989. :j: Developmental Endocrinology Branch, National Institute of Child Health and Human Development. § Present address: Department of Endocrinology, Hospital das Clinicas, University of Sao Paulo, Sao Paulo, Brazil. II Nursing Department, Warren Grant Magnuson Clinical Center.
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Effects of RU486 on GnRH-induced cycles
the menstrual cycle (1). In a previous study, we gave RU486 orally at the relatively low dose of 1 mgjd for 5 days during the late follicular phase of normally cycling women to investigate the role of P in
1'[ Reprint requests: Lynnette K. Nieman, M.D., National Institutes of Health, Building 10, Room 10N262, Bethesda, Maryland 20892 (FAX: 301-402-0574). ** Present address: Division of Endocrinology and Metabolism, Oregon Health Sciences University, Portland, Oregon. tt Present address: Research Service, American Lake Veterans Affairs Medical Center, and Division of Endocrinology, University of Washington School of Medicine, Seattle, Washington.
Fertility and Sterility
the regulation of the midcycle gonadotropin surge (2). This compound delayed the timing of the gonadotropin surge and ovulation without suppressing folliculogenesis or E2 secretion (2). Progesterone concentrations, however, remained low during RU486 treatment and started to rise only after the compound was discontinued (2). These effects could be reversed by adding small doses of P to RU486 therapy after the emergence of a mature follicle, suggesting that P plays an important, if not critical, role in the initiation ofthe midcycle gonadotropin surge (2). RU486 may thus delay ovulation by acting directly on the ovary to suppress an ovarian progestational signal or by antagonizing P action at the level of the hypothalamus or pituitary (2). In the present study, we investigated whether RU486 acts primarily on the hypothalamus to delay the midcycle gonadotropin surge and thus gain insight into the site(s) of action of P in the control of ovulation. We thus evaluated the effects of RU486 on the timing of the gonadotropin surge and ovulation in women with hypothalamic amenorrhea undergoing ovulation induction with GnRH pulses of unvarying frequency and dose. This human model is analogous to the "hypophysiotropic clamp" system used by Nakai et al. (3) to study the control of gonadotropin secretion in monkeys. It allows the administration of fixed amounts of pulsatile GnRH throughout the menstrual cycle and is sufficient to support an ovulatory gonadotropin surge in women with hypothalamic amenorrhea, whether endogenous GnRH is present partially or absent totally (4). It thus provides a constant GnRH rhythm that could bridge over and counteract any suppressive effects of RU486 on endogenous GnRH secretion that might be responsible for the delay in ovulation previously seen in normally cycling women (2). Under these conditions, any effects of RU486 on the timing of ovulation should reflect primarily its action on the pituitary-ovarian axis.
normal physical and pelvic examination, with welldeveloped secondary sexual characteristics and absence of hirsutism or ovarian enlargement; no evidence of a pituitary or hypothalamic lesion, as evidenced by a normal computerized tomography or magnetic resonance imaging of the hypothalamicpituitary area; normal serum levels of triiodothyronine (88 to 124 ng/dL [1.4 to 1.9 nmoljL]), T4 (6.2 to 8.8 JLg/dL [80 to 113 nmoljL]), free T4 (1.0 to 1.4 ng/dL [13 to 18 pmoljL]), and thyroid-stimulating hormone (0.7 to 3.3 JLU/mL or mUlL), except in patient 2, who had borderline or slightly reduced triiodothyronine (80 ng/dL [1.2 nmoljL]), T4 (3.8 JLg/dL [49 nmoljL]), and free T4 (0.8 ng/dL [10 pmol/L]) concentrations; normal or low basal plasma levels ofLH (5 to 6 mlU/mL or IU/L) , FSH (5 to 12 mlU/mL or IU/L), PRL (2 to 12 ng/mL or JLg/L), T (0.3 to 0.4 ng/mL [1.0 to 1.4 nmoljL]), and E2 (12 to 38 pg/mL [44 to 139 pmol/L]). Five women had normal gonadotropin responses to an 100 JLg IV bolus of GnRH; patient 4 did not undergo this test, because she had responded previously with ovulation to pulsatile GnRH therapy. The diagnosis of hypothalamic amenorrhea was attributed to postviral meningitis (patient 1), low body weight and psychological stress (patients 2 and 3), or psychological stress alone (patient 4). No clear cause was found in the other two women. The patients had not taken sex steroid hormones in the past 6 months. All used barrier methods of contraception or abstained from sexual intercourse during the study. Pregnancy was excluded by a plasma ,B-hCG concentration ~ 2 ng/mL (the detection limit for pregnancy in this assay) at the beginning of each study cycle. The study was approved by the Food and Drug Administration and by the Clinical Research Subpanel of the National Institute of Child Health and Human Development. Informed consent was obtained from each subject. Study Design
MATERIALS AND METHODS Patient Population
Six patients with hypothalamic amenorrhea, aged 26 to 34 years (mean ± SD, 30.7 ± 3.0 years), volunteered for the study. They had the following clinical and laboratory findings: secondary amenorrhea of 1 to 14 years duration; body weight ranging from 73% to 114% of ideal value (57.8 ± 11.0 kg; range, 45 to 74 kg); no history of intensive exercise; Vol. 62, No.1, July 1994
All women were induced to ovulate with pulsatile GnRH therapy administered SC via portable infusion pumps (Zyklomat or Pulsamat; Ferring Laboratories, Suffern, NY). Patients 1, 2, 3, 4, and 6 received pulses of 200 ng/kg every 90 minutes throughout the entire protocol, including the priming and study cycles. Patient 5 did not respond well to this dose and subsequently was treated with pulses of 400 ng/kg every 120 minutes throughout the priming and study cycles. Thus, in each subject, Batista et al.
Effects of RU486 on GnRH-induced cycles
29
a
the dose of GnRH was the same in the placebo and RU486 cycles. The study consisted of one or two priming cycles followed by two study cycles. Women were allowed to proceed into the study cycles only after exhibiting an ovulatory priming cycle, as documented by luteal P levels> 5 ng/mL (16 nmoljL). Patients 2, 3 5 and 6 fulfilled this requirement in the first p~i~ing cycle (length of 21 to 29 days, peak luteal P levels of9 to 22 ng/mL [29 to 70 nmoljL]), whereas patient 1 ovulated only in the second priming cycle (length of 27 days, peak luteal P level of 18 ng/mL [57 nmoljL]). Patient 4 was treated with GnRH pulses of 200 ng/kg given SC every 90 minutes throughout two cycles before entering our study as part of another protocol conducted at the National Institutes of Health (Bethesda, Maryland). Because ovulation was documented in her second GnRH-induced cycle (length of 27 days, peak luteal P level of22 ng/mL [70 nmoljL]), this served as the priming cycle for our study. All women received a placebo capsule orally during the first study cycle, whereas RU486 (Roussel UCLAF, Romainville, France) was prescribed orally at a dose of 1 mg/d during the second study cycle. Thus, placebo always preceded RU486 treatment so as to avoid any long-standing effects of RU486 on the placebo cycle. Both compounds were administered for 5 days, starting when the dominant follicle reached 14 to 16 mm. In each subject, every attempt was made to begin placebo and RU486 administration at the same follicular diameter. The patients but not the investigators were blinded to the treatment given in each study cycle. Plasma levels of LH, FSH, E 2 , and/or P were monitored weekly in the priming cycles. In the study cycles, hormone measurements and ovarian ultrasound examinations were obtained daily after the dominant follicle exceeded 12 mm in diameter and continued until 1 day after follicular collapse. Blood samples were drawn every 3 to 4 days during the remainder of the study cycles. Plasma concentrations of LH (5), FSH (5), E2 (6), and P (7) were measured by RIA. The intra-assay and interassay coefficients of variation were <12% and 17%, respectively, for all hormones. Ovarian ultrasound examinations were performed with a 5.0 MHz vaginal transducer, except in patient 2, who was scanned with a 3.5 MHz abdominal probe. Follicular development was evaluated by measuring the anteroposterior, longitudinal, and/ or transverse diameter of the dominant follicle and taking the mean of at least two measurements. Col30
Batista et al.
Effects of RU486 on GnRH·induced cycles
lapse of the dominant follicle, suggesting ovulation, was defined as the complete disappearance of the follicle, significant reduction of volume associated with thickening and irregularity of the wall, or replacement of the follicle by a multiechoic mass (8,9). Data Analysis
The follicular phase was defined as the days elapsed between the onset of previous menses and the day of the LH peak. The luteal phase was defined as the days after the LH peak until the day before next menses. Because both RU486 and placebo were started at similar stages of development of the dominant follicle, the midcycle gonadotropin surge and ovulation were timed according to the onset of either compound rather than to the first day of menses. This helped eliminate intraindividual and interindividual variation in the timing of follicular recruitment that could otherwise blur the interpretation of results. Data are presented as means ± SE. Paired two-tailed t-tests were used to compare results between placebo and RU486 cycles. Differences were considered significant at P < 0.05. RESULTS Effects of RU486 on the Timing of Ovulation
The effects of RU486 on the timing of ovulation were evaluated by grouping data from all six patients. RU486 consistently delayed the timing of the midcycle gonadotropin surge by a mean of 3.3 ± 0.7 days (range, 2 to 6 days; P = 0.005 compared with placebo; Fig. 1). The LH and FSH peaks
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Day of LH Surge Relative to First Day of Placebo or RU 486 Administration
Figure 1 Day of the midcycle LH peak relative to the first day (day 1) of placebo or RU486 administration in six women with hypothalamic amenorrhea treated with pulsatile GnRH. PIa· cebo or RU486 (1 mg/d) were given orally for 5 days (arrows), starting when the dominant follicle reached 14 to 16 mm. Sym· boIs connected with a line represent the same subject.
Fertility and Sterility
Table 1 Effects of RU486 or Placebo Administration on Peak Preovulatory Levels of LH, FSH, E 2, and Follicular Diameter in Six Women With Hypothalamic Amenorrhea Treated with Pulsatile GnRH* Parameter LH (mIU/mL):j: FSH (mIU/mL):j: E2 (pg/mL) II Follicular diameter (mm)
Peak levels placebo
Peak levels RU486
Expected peakt RU486
55.9 ± 6.8 17.8 ± 1.2 423.0 ± 68.1
117.0 ± 35.2 20.1 ± 3.3 334.8 ± 52.3
20.9 ± 4.3§ 6.3 ± 1.5§ 153.4 ± 31.9§
23.9 ± 1.4
24.9 ± 0.9
on the last 2 days of placebo administration and on the first 2 days after discontinuing RU486 (Fig. 1). Because of their uniform response, their data are presented as means ± SE (Fig. 2). As opposed to the
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20.3 ± 1.6
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* Values are means ± SE; RU486 (1 mg/d) or placebo were given for 5 days starting when the dominant follicle reached 14 to 16 mm. t Measurements obtained on the day of RU486 treatment corresponding to the day of placebo administration when maximum values occurred (e.g., if peak LH levels occurred on day 4 of placebo administration, "expected" peak values would correspond to LH concentrations on day 4 of RU486 treatment). :j: Conversion factor to SI units, 1.0. § Significantly different compared with peak levels in the placebo cycles, P < 0.01. II Conversion factor to SI units, 3.671.
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occurred simultaneously and were followed by the collapse of the dominant follicle within 3 days_ The duration of the follicular phase (15.3 ± 1.3 versus 17.7 ± 1.2 days), luteal phase (14.0 ± 0.4 versus 13.8 ± 0.9 days), and entire menstrual cycle (29.3 ± 1.3 versus 31.5 ± 1.5 days) was not statistically different in the placebo and RU486 groups.
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Effects of RU486 on Follicular and Hormonal Dynamics
There were no differences in peak preovulatory levels of LH, FSH, E z, and follicular diameter between placebo and RU486 cycles when all six women were analyzed as a group (Table 1). However, comparison of results on the day of maximum values during placebo administration with results on the corresponding day of RU486 treatment (the "expected" day of peak values) showed that RU486 suppressed all parameters except for follicular diameter (Table 1). The rate of follicular maturation varied substantially among patients, even in the placebo (i.e., control) cycle. This was attributed in part to the heterogeneous response of the reproductive axis to GnRH stimulation. To minimize blurring of the data due to the variable rates of follicular development, the time course of follicular growth and hormonal changes was analyzed after dividing patients into two groups, i.e., those with normal or prolonged folliculogenesis. Patients with Normal Folliculogenesis
Patients 1 to 4 had similar, normal rates offollicular maturation, with gonadotropin surges occurring Vol. 62, No. I, July 1994
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Figure 2 Effects of placebo or RU486 administration in four women with hypothalamic amenorrhea treated with pulsatile GnRH. Placebo or RU486 (1 mg/d) were given orally for 5 days (arrows), starting when the dominant follicle reached 14 to 16 mm. Data represent means ± SE levels of LH (A), FSH (B), follicular diameter (C), E2 (D), and P (E) plotted relative to the first day (day 1) of placebo or RU486 administration. *Significant difference (P < 0.05) between placebo and RU486 cycles. Conversion factor to SI units for LH and FSH levels, 1; for E2 levels, 3.671; and P levels, 3.180. Batista et al.
Effects of RU486 on GnRH-induced cycles
31
ascending curves seen in the placebo group, mean LH and FSH levels remained stable or declined during RU486 treatment, peaking only after the drug was stopped (Fig. 2A and B). Follicular growth progressed at a similar rate in both study cycles up to the day of follicular collapse in the placebo group (Fig. 2C). In contrast, E2 concentrations rose steadily during placebo administration but failed to do so during RU486 treatment (Fig. 2D). In both study cycles, follicular diameter and E2 levels reached maximum values within 2 days of the LH peak. As opposed to rising concentrations seen during placebo administration, P levels remained low during RU486 treatment (Fig. 2E). They started to increase within 1 day ofthe LH surge and peaked at similar luteal levels in placebo (25.6 ± 3.3 ngjmL [81.4 ± 10.5 nmol/L]) and RU486 cycles (19.7 ± 5.1 ngjmL [62.6 ± 16.2 nmol/L]). Patients with Prolonged Folliculogenesis
Patients 5 and 6 had more prolonged folliculogenesis and ovulated only after placebo and RU486 were discontinued (Fig. 1). Because of their heterogeneous response, their data were analyzed individually (graphs not shown). The time course of hormonal changes progressed at a slower rate in these two women but was otherwise similar to the one seen in patients 1 to 4. Thus, LH and FSH levels showed minor fluctuations but did not change significantly during RU486 treatment. A gonadotropin surge of reduced or normal magnitude (LH of 27 and 79 mlUjmL or IUjL; and FSH of 16 and 25 mlU jmL or IU jL) occurred 6 and 12 days, respectively, after the compound was stopped. Estradiol levels failed to rise during RU486 administration but increased rapidly after the antiprogestin was discontinued, peaking at 154 and 403 pgjmL (565 and 1479 pmol/L) within 2 days of the LH surge. Progesterone concentrations remained low or declined during RU486 treatment. They rose within 1 day ofthe LH surge (i.e., after stopping RU486) and peaked at luteal levels of 7 and 17 ngjmL (22 and 54 nmol/L). In contrast to the other four women who exhibited normal folliculogenesis in both study cycles, follicular diameter increased slightly or remained unchanged during RU486 treatment in patients 5 and 6, progressing at a much slower rate than during placebo administration. After the antiprogestin was discontinued, follicular diameter paralleled the rise in E2 concentrations and peaked at 24 and 28 mm just before ovulation. 32
Batista et al.
Effects of RU486 on GnRH-induced cycles
DISCUSSION
The primary site(s) at which P acts to regulate the midcycle gonadotropin surge has not been defined fully. In rhesus monkeys with hypothalamic lesions receiving unvarying pulses of GnRH, P advances the initiation of E 2-induced gonadotropin surges, suggesting that a major part of this effect is exerted at the level of the pituitary gland (10). Several lines of evidence, however, indicate that the hypothalamus also may be a site of P action. In ovariectomized, estrogen-primed monkeys, P appears to activate neural substrates in the ventral hypothalamus to increase the frequency and amplitude of LH pulses and thus induce a gonadotropin peak (11, 12). This process may be suppressed with anesthetics such as pentobarbitone, presumably by blocking the effects of P on the brain (13). Additional evidence of the role of the hypothalamus has been provided by studies showing that GnRH concentrations increase at the time ofthe preovulatory LH surge in peripheral and pituitary stalk plasma of human and nonhuman primates (14, 15). In the present study, we investigated whether the antiprogestin RU486 can delay the midcycle gonadotropin surge independent of hypothalamic inhibition' in an attempt to assess the site(s) of action of P in the control of ovulation. We thus treated women with hypothalamic amenorrhea, who have abnormal or absent GnRH secretion, with exogenous GnRH pulses of unvarying frequency and dose to provide a constant GnRH rhythm sufficient to induce ovulation and counteract any hypothalamic suppressive effects of RU486 (4). The latter compound was given at low doses in the late follicular phase of these GnRH -controlled ovulatory cycles to evaluate primarily its effects on the pituitary-ovarian axis. We chose to recruit women with functional hypothalamic amenorrhea and not those with Kallmann's syndrome (i.e., with congenital absence of GnRH secretion and thus ideal for this protocol) because ofthe rarity of the latter group. Although it is possible that patients who participated in this study had some endogenous hypothalamic activity that could influence our results (4), several steps were taken to minimize this problem. Thus, we only selected women with long-standing amenorrhea to limit the chance of spontaneous return of hypothalamic function during the protocol. In addition, the priming cycles before entering the study cycles helped reduce the possibility of exaggerated pituitary responses to GnRH therapy due to prolonged deprivation of this hormone. Fertility and Sterility
When given at the same dose and regimen as previously reported in normally cycling women (2), RU486 consistently delayed the timing of the midcycle gonadotropin surge and ovulation despite the administration of constant pulsatile GnRH signals. The magnitude of the delay was similar in GnRHstimulated and spontaneous cycles (3.3 ± 0.7 versus 2.8 ± 0.5 days, P not significant by unpaired t-test), demonstrating that this effect of RU486 was not altered by replacing or overriding endogenous hypothalamic signals with pulsatile GnRH therapy. Although these results do not exclude a concurrent action of RU486 or its agonist P on the hypothalamus, they indicate that effects on the pituitaryovarian axis are sufficient to explain the delay in ovulation induced by RU486. Thus, the hypothalamus does not seem to play a critical role in this process. In GnRH -stimulated cycles, LH and FSH levels increased during placebo administration but remained unchanged or decreased during RU486 treatment, suggesting that this compound acts on the pituitary to inhibit gonadotropin secretion. In normally cycling women, RU486 suppressed LH and FSH levels when administered at a single dose of 10 mg during the midfollicular or late follicular phase (16). This appears to represent an antagonistic action of RU486, because P given at the same dose and time of the cycle induced opposite changes in gonadotropin concentrations (16). Taken together with these findings, our present data suggest that RU486 antagonizes P at the level of the pituitary gland to suppress LH and FSH secretion and delay the midcycle gonadotropin surge that leads to ovulation. RU486 also inhibited ovarian steroidogenesis and delayed the timing of peak E2 concentrations and preovulatory P rises. The latter events occurred within 2 days of the midcycle gonadotropin surge and had similar magnitudes in placebo and RU486 cycles. Thus, the temporal relationship between steroid and gonadotropin concentrations at midcycle was preserved during RU486 treatment, so that LH and FSH surges began when E2 and P reached normal preovulatory levels. Despite its effects on ovarian steroidogenesis, RU486 did not suppress follicular growth in most patients. However, in two women who could not be distinguished in any other way from the remaining subjects, follicular maturation was prolonged in both RU486 and placebo cycles, suggesting that GnRH replacement therapy was insufficient to induce a completely normal follicular phase. In contrast to the other paVol. 62, No.1, July 1994
tients, the dominant follicle grew at a slower rate when these two women were given RU486 rather than placebo. Thus, it is possible that in the latter subjects the lack of optimal GnRH (hence, gonadotropin) support, associated with a pituitary suppressive effect of RU486, resulted in more severe disruption of follicular maturation than in the other four patients exhibiting normal GnRH-induced folliculogenesis. The effects of RU486 to inhibit ovarian steroidogenesis and, in some instances, also follicular maturation appear to be due to P antagonism at the level of the pituitary gland to suppress gonadotropin secretion. Both the development of the follicle and its hormonal production are dependent on gonadotropin support (17). A less likely explanation is that RU486 acted directly on the ovary to inhibit 3,B-hydroxysteroid dehydrogenase activity and thus impair steroid synthesis (18). This probably would not explain the lower gonadotropin concentrations seen in association with reduced steroid levels during RU486 treatment. Disruption of follicular maturation and suppression of E2 and P secretion were reported previously in normally cycling women treated with substantially higher doses of RU486 in the late follicular phase (19, 20). In contrast, when the compound was given to normal subjects at a lower dose of 1 mg/ din the same regimen used in the present study, E2 secretion appeared unaffected but P concentrations remained low and did not rise during RU486 treatment (2). Differences in RU486 effects in spontaneous and GnRH -controlled cycles may be due to the fact that, in the former cycles, the hypothalamus was intact and thus capable of increasing GnRH secretion to overcome an inhibitory action of RU486 on the pituitary-ovarian axis. In summary, our results show that RU486 delays the midcycle gonadotropin surge and ovulation despite the external administration of constant GnRH signals, arguing against a critical role of the hypothalamus in this process. Rather, this effect appears to be due to P antagonism at the level ofthe pituitary gland to suppress gonadotropin and, subsequently, steroid hormone secretion. These findings support the concept that P can act on the pituitary, independent of effects on the hypothalamus, to regulate the timing of the midcycle gonadotropin surge that leads to ovulation. Acknowledgments. Weare indebted to the 9th Floor Clinic nursing staff of the Clinical Center of the National Institutes of Health (Bethesda, Maryland) for supporting this study and also
Batista et al.
Effects of RU486 on GnRH-induced cycles
33
r to Roussel-UCLAF (Romainville, France) for providing RU486. We are grateful to Helena Fundament, M.D. (University of Sao Paulo, Sao Paulo, Brazil) for assistance with data analysis. We thank Ms. Barbara Filmore (National Institutes of Health, Bethesda, Maryland) for technical assistance.
11.
12. REFERENCES 1. Baulieu EE. RU-486 as an antiprogesterone steroid. From receptor to contragestion and beyond. JAMA 1989; 262:1808-14. 2. Batista MC, Cartledge TP, Zellmer A W, Nieman LK, Merriam GR, Loriaux DL. Evidence for a critical role of progesterone in the regulation of the midcycle gonadotropin surge and ovulation. J Clin Endocrinol Metab 1992;74:56570. 3. Nakai Y, Plant TM, Hess DL, Keogh EJ, Knobil E. On the sites of the negative and positive feedback actions of estradiol in the control of gonadotropin secretion in the rhesus monkey. Endocrinology 1978;102:1008-14. 4. Santoro N, Filicori M, Crowley WF Jr. Hypogonadotropic disorders in men and women: diagnosis and therapy with pulsatile gonadotropin-releasing hormone. Endocr Rev 1986;7:11-23. 5. Odell WD, Rayford PL, Ross GT. Simplified, partially automated method for radioimmunoassay of human thyroid stimulating, growth, luteinizing and follicle stimulating hormones. J Lab Clin Med 1967;70:973-80. 6. Jiang NS, Ryan RJ. Radioimmunoassay for estrogens: a preliminary communication. Mayo Clin Proc 1969;44:461-5. 7. Abraham G E, Swerdloff R, Tulchinsky D, Odell WD. Radioimmunoassay of plasma progesterone. J Clin Endocrinol Metab 1971;32:619-24. 8. Wetzels LCG, Hoogland HJ. Relation between ultrasonographic evidence of ovulation and hormonal parameters: luteinizing hormone surge and initial progesterone rise. Fertil Steril 1982;37:336-41. 9. Queenan JT, O'Brien GD, Bains LM, Simpson J, Collins WP, Campbell S. Ultrasound scanning of ovaries to detect ovulation in women. Fertil Steril 1980;34:99-105. 10. Wildt L, Hutchison JS, Marshall G, Pohl CR, Knobil E. On the site of action of progesterone in the blockade of the es-
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tradiol-induced gonadotropin discharge in the rhesus monkey. Endocrinology 1981;109:1293-4. Terasawa E, Krook C, Eman S, Terasawa E, Krook C, Eman S, et al. Pulsatile luteinizing hormone (LH) release during the progesterone-induced LH surge in the female rhesus monkey. Endocrinology 1987;120:2265-71. Yeoman RR, Terasawa E. An increase in single unit activity of the medial basal hypothalamus occurs during the progesterone-induced luteinizing hormone surge in the female rhesus monkey. Endocrinology 1984;115:2445-52. Terasawa E, Noonan J, Bridson WE. Anaesthesia with pentobarbitone blocks the progesterone-induced luteinizing hormone surge in the ovariectomized rhesus monkey. J EndocrinoI1982;92:327-39. Elkind-Hirsch K, Ravnikar V, Schiff I, Tulchinsky D, Ryan KJ. Determinations of endogenous immunoreactive luteinizing hormone-releasing hormone in human plasma. J Clin Endocrinol Metab 1982;54:602-7. Neill JD, Patton JM, Dailey RA, Tsou RC, Tindall GT. Luteinizing hormone releasing hormone (LHRH) in pituitary stalk blood of rhesus monkeys: relationship to levels of LH release. Endocrinology 1977;101:430-4. Permezel JM, Lenton EA, Roberts I, Cooke ID. Acute effects of progesterone and the antiprogestin RU486 on gonadotropin secretion in the follicular phase of the menstrual cycle. J Clin Endocrinol Metab 1989;68:960-5. Mais V, Kazer RR, Cetel NS, Rivier J, Vale W, Yen SSC. The dependency of folliculogenesis and corpus luteum function on pulsatile gonadotropin secretion in cycling women using a gonadotropin-releasing hormone antagonist as a probe. J Clin Endocrinol Metab 1986;62:1250-5. DiMattina M, Albertson B, Seyler DE, Loriaux DL, Falk RJ. Effect of the antiprogestin RU 486 on progesterone production by cultured granulosa cells: inhibition of the ovarian 31'i-hydroxysteroid dehydrogenase. Contraception 1986; 34:199-206. Liu JH, Garza G, Morris S, Stuenkel C, Ulmann A, Yen SSC. Disruption offollicular maturation and delay of ovulation after administration of the antiprogesterone RU486. J Clin Endocrinol Metab 1987;65:1135-40. Shoupe D, Mishell DR Jr, Page MA, Madkour H, Spitz 1M, Lobo RA. Effects of the antiprogesterone RU486 in normal women. II. Administration in the late follicular phase. Am J Obstet GynecoI1987;157:1421-6.
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~~~19§W:f_":~":~rinOlo9Y FERTILITY AND STERILITY Copyright
©
Vol. 62, No.1, July 1994
Printed on acid-free paper in U. S. A.
1994 The American Fertility Society
The antiprogestin RU486 delays the midcycle gonadotropin surge and ovulation in gonadotropin-releasing hormone-induced cyclest
Marcelo C. Batista, M.D.*:j:§ Tannia P. Cartledge, R.N. II Ann W. Zellmer, R.N. II
Lynnette K. Nieman, M.D.:j:~ D. Lynn Loriaux, M.D., Ph.D.:j:** George R. Merriam, M.D.:j:tt
National Institute of Child Health and Human Development, and Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland
Objective: To investigate whether the antiprogestin RU486 acts primarily on the hypothalamus to delay the midcycle gonadotropin surge and thus gain insight into the site(s) of action of P in the control of ovulation. Design: Prospective, crossover, single-blinded clinical study. Setting: Outpatient clinic in an academic research environment. Patients: Women with hypothalamic amenorrhea. Interventions: RU486 or a placebo was given orally at a low dose of 1 mg/d for 5 days, starting when the dominant follicle reached 14 to 16 mm, to women with hypothalamic amenorrhea undergoing ovulation induction with GnRH pulses of unvarying frequency and dose. Blood samples and ovarian ultrasounds were obtained daily in the late follicular phase and every 3 to 4 days in the remainder of the cycle. Main Outcome Measures: Follicular diameter and plasma levels of LH, FSH, E 2 , and P. Results: RU486 consistently delayed the timing of the midcycle gonadotropin surge and ovulation. Gonadotropin and steroid levels were suppressed during RU486 treatment, but follicular growth progressed normally in most patients. Conclusions: RU486 does not act primarily on the hypothalamus to delay ovulation. Rather, this compound appears to antagonize P at the pituitary level to suppress gonadotropin and steroid hormone secretion. P may thus act on the pituitary, independent of any hypothalamic effects, to regulate the timing of the midcycle gonadotropin surge and ovulation. Fertil Steril 1994;62:28-34 Key Words: RU486, progesterone, ovulation, gonadotropin, gonadotropin-releasing hormone
The antiprogestin RU486 provides a powerful tool to study the physiology of P action throughout Received September 24, 1993; revised and accepted March 7, 1994. * Supported in part by grant 86/2520-2, Funda<;ao de Amparo 'it Pesquisa do Estado de Sao Paulo, Sao Paulo, Brazil. t Presented in part at the 71st Annual Meeting of the Endocrine Society, Seattle, Washington, June 21 to 24, 1989. :j: Developmental Endocrinology Branch, National Institute of Child Health and Human Development. § Present address: Department of Endocrinology, Hospital das Clinicas, University of Sao Paulo, Sao Paulo, Brazil. II Nursing Department, Warren Grant Magnuson Clinical Center.
28
Batista et al.
Effects of RU486 on GnRH-induced cycles
the menstrual cycle (1). In a previous study, we gave RU486 orally at the relatively low dose of 1 mgjd for 5 days during the late follicular phase of normally cycling women to investigate the role of P in
1'[ Reprint requests: Lynnette K. Nieman, M.D., National Institutes of Health, Building 10, Room 10N262, Bethesda, Maryland 20892 (FAX: 301-402-0574). ** Present address: Division of Endocrinology and Metabolism, Oregon Health Sciences University, Portland, Oregon. tt Present address: Research Service, American Lake Veterans Affairs Medical Center, and Division of Endocrinology, University of Washington School of Medicine, Seattle, Washington.
Fertility and Sterility
the regulation of the midcycle gonadotropin surge (2). This compound delayed the timing of the gonadotropin surge and ovulation without suppressing folliculogenesis or E2 secretion (2). Progesterone concentrations, however, remained low during RU486 treatment and started to rise only after the compound was discontinued (2). These effects could be reversed by adding small doses of P to RU486 therapy after the emergence of a mature follicle, suggesting that P plays an important, if not critical, role in the initiation ofthe midcycle gonadotropin surge (2). RU486 may thus delay ovulation by acting directly on the ovary to suppress an ovarian progestational signal or by antagonizing P action at the level of the hypothalamus or pituitary (2). In the present study, we investigated whether RU486 acts primarily on the hypothalamus to delay the midcycle gonadotropin surge and thus gain insight into the site(s) of action of P in the control of ovulation. We thus evaluated the effects of RU486 on the timing of the gonadotropin surge and ovulation in women with hypothalamic amenorrhea undergoing ovulation induction with GnRH pulses of unvarying frequency and dose. This human model is analogous to the "hypophysiotropic clamp" system used by Nakai et al. (3) to study the control of gonadotropin secretion in monkeys. It allows the administration of fixed amounts of pulsatile GnRH throughout the menstrual cycle and is sufficient to support an ovulatory gonadotropin surge in women with hypothalamic amenorrhea, whether endogenous GnRH is present partially or absent totally (4). It thus provides a constant GnRH rhythm that could bridge over and counteract any suppressive effects of RU486 on endogenous GnRH secretion that might be responsible for the delay in ovulation previously seen in normally cycling women (2). Under these conditions, any effects of RU486 on the timing of ovulation should reflect primarily its action on the pituitary-ovarian axis.
normal physical and pelvic examination, with welldeveloped secondary sexual characteristics and absence of hirsutism or ovarian enlargement; no evidence of a pituitary or hypothalamic lesion, as evidenced by a normal computerized tomography or magnetic resonance imaging of the hypothalamicpituitary area; normal serum levels of triiodothyronine (88 to 124 ng/dL [1.4 to 1.9 nmoljL]), T4 (6.2 to 8.8 JLg/dL [80 to 113 nmoljL]), free T4 (1.0 to 1.4 ng/dL [13 to 18 pmoljL]), and thyroid-stimulating hormone (0.7 to 3.3 JLU/mL or mUlL), except in patient 2, who had borderline or slightly reduced triiodothyronine (80 ng/dL [1.2 nmoljL]), T4 (3.8 JLg/dL [49 nmoljL]), and free T4 (0.8 ng/dL [10 pmol/L]) concentrations; normal or low basal plasma levels ofLH (5 to 6 mlU/mL or IU/L) , FSH (5 to 12 mlU/mL or IU/L), PRL (2 to 12 ng/mL or JLg/L), T (0.3 to 0.4 ng/mL [1.0 to 1.4 nmoljL]), and E2 (12 to 38 pg/mL [44 to 139 pmol/L]). Five women had normal gonadotropin responses to an 100 JLg IV bolus of GnRH; patient 4 did not undergo this test, because she had responded previously with ovulation to pulsatile GnRH therapy. The diagnosis of hypothalamic amenorrhea was attributed to postviral meningitis (patient 1), low body weight and psychological stress (patients 2 and 3), or psychological stress alone (patient 4). No clear cause was found in the other two women. The patients had not taken sex steroid hormones in the past 6 months. All used barrier methods of contraception or abstained from sexual intercourse during the study. Pregnancy was excluded by a plasma ,B-hCG concentration ~ 2 ng/mL (the detection limit for pregnancy in this assay) at the beginning of each study cycle. The study was approved by the Food and Drug Administration and by the Clinical Research Subpanel of the National Institute of Child Health and Human Development. Informed consent was obtained from each subject. Study Design
MATERIALS AND METHODS Patient Population
Six patients with hypothalamic amenorrhea, aged 26 to 34 years (mean ± SD, 30.7 ± 3.0 years), volunteered for the study. They had the following clinical and laboratory findings: secondary amenorrhea of 1 to 14 years duration; body weight ranging from 73% to 114% of ideal value (57.8 ± 11.0 kg; range, 45 to 74 kg); no history of intensive exercise; Vol. 62, No.1, July 1994
All women were induced to ovulate with pulsatile GnRH therapy administered SC via portable infusion pumps (Zyklomat or Pulsamat; Ferring Laboratories, Suffern, NY). Patients 1, 2, 3, 4, and 6 received pulses of 200 ng/kg every 90 minutes throughout the entire protocol, including the priming and study cycles. Patient 5 did not respond well to this dose and subsequently was treated with pulses of 400 ng/kg every 120 minutes throughout the priming and study cycles. Thus, in each subject, Batista et al.
Effects of RU486 on GnRH-induced cycles
29
a
the dose of GnRH was the same in the placebo and RU486 cycles. The study consisted of one or two priming cycles followed by two study cycles. Women were allowed to proceed into the study cycles only after exhibiting an ovulatory priming cycle, as documented by luteal P levels> 5 ng/mL (16 nmoljL). Patients 2, 3 5 and 6 fulfilled this requirement in the first p~i~ing cycle (length of 21 to 29 days, peak luteal P levels of9 to 22 ng/mL [29 to 70 nmoljL]), whereas patient 1 ovulated only in the second priming cycle (length of 27 days, peak luteal P level of 18 ng/mL [57 nmoljL]). Patient 4 was treated with GnRH pulses of 200 ng/kg given SC every 90 minutes throughout two cycles before entering our study as part of another protocol conducted at the National Institutes of Health (Bethesda, Maryland). Because ovulation was documented in her second GnRH-induced cycle (length of 27 days, peak luteal P level of22 ng/mL [70 nmoljL]), this served as the priming cycle for our study. All women received a placebo capsule orally during the first study cycle, whereas RU486 (Roussel UCLAF, Romainville, France) was prescribed orally at a dose of 1 mg/d during the second study cycle. Thus, placebo always preceded RU486 treatment so as to avoid any long-standing effects of RU486 on the placebo cycle. Both compounds were administered for 5 days, starting when the dominant follicle reached 14 to 16 mm. In each subject, every attempt was made to begin placebo and RU486 administration at the same follicular diameter. The patients but not the investigators were blinded to the treatment given in each study cycle. Plasma levels of LH, FSH, E 2 , and/or P were monitored weekly in the priming cycles. In the study cycles, hormone measurements and ovarian ultrasound examinations were obtained daily after the dominant follicle exceeded 12 mm in diameter and continued until 1 day after follicular collapse. Blood samples were drawn every 3 to 4 days during the remainder of the study cycles. Plasma concentrations of LH (5), FSH (5), E2 (6), and P (7) were measured by RIA. The intra-assay and interassay coefficients of variation were <12% and 17%, respectively, for all hormones. Ovarian ultrasound examinations were performed with a 5.0 MHz vaginal transducer, except in patient 2, who was scanned with a 3.5 MHz abdominal probe. Follicular development was evaluated by measuring the anteroposterior, longitudinal, and/ or transverse diameter of the dominant follicle and taking the mean of at least two measurements. Col30
Batista et al.
Effects of RU486 on GnRH·induced cycles
lapse of the dominant follicle, suggesting ovulation, was defined as the complete disappearance of the follicle, significant reduction of volume associated with thickening and irregularity of the wall, or replacement of the follicle by a multiechoic mass (8,9). Data Analysis
The follicular phase was defined as the days elapsed between the onset of previous menses and the day of the LH peak. The luteal phase was defined as the days after the LH peak until the day before next menses. Because both RU486 and placebo were started at similar stages of development of the dominant follicle, the midcycle gonadotropin surge and ovulation were timed according to the onset of either compound rather than to the first day of menses. This helped eliminate intraindividual and interindividual variation in the timing of follicular recruitment that could otherwise blur the interpretation of results. Data are presented as means ± SE. Paired two-tailed t-tests were used to compare results between placebo and RU486 cycles. Differences were considered significant at P < 0.05. RESULTS Effects of RU486 on the Timing of Ovulation
The effects of RU486 on the timing of ovulation were evaluated by grouping data from all six patients. RU486 consistently delayed the timing of the midcycle gonadotropin surge by a mean of 3.3 ± 0.7 days (range, 2 to 6 days; P = 0.005 compared with placebo; Fig. 1). The LH and FSH peaks
Placebo 0 RU 486 • 01.01.01.01.01.
o
•
o 2
6
8
10
• 12
14
16
18
Day of LH Surge Relative to First Day of Placebo or RU 486 Administration
Figure 1 Day of the midcycle LH peak relative to the first day (day 1) of placebo or RU486 administration in six women with hypothalamic amenorrhea treated with pulsatile GnRH. PIa· cebo or RU486 (1 mg/d) were given orally for 5 days (arrows), starting when the dominant follicle reached 14 to 16 mm. Sym· boIs connected with a line represent the same subject.
Fertility and Sterility
Table 1 Effects of RU486 or Placebo Administration on Peak Preovulatory Levels of LH, FSH, E 2, and Follicular Diameter in Six Women With Hypothalamic Amenorrhea Treated with Pulsatile GnRH* Parameter LH (mIU/mL):j: FSH (mIU/mL):j: E2 (pg/mL) II Follicular diameter (mm)
Peak levels placebo
Peak levels RU486
Expected peakt RU486
55.9 ± 6.8 17.8 ± 1.2 423.0 ± 68.1
117.0 ± 35.2 20.1 ± 3.3 334.8 ± 52.3
20.9 ± 4.3§ 6.3 ± 1.5§ 153.4 ± 31.9§
23.9 ± 1.4
24.9 ± 0.9
on the last 2 days of placebo administration and on the first 2 days after discontinuing RU486 (Fig. 1). Because of their uniform response, their data are presented as means ± SE (Fig. 2). As opposed to the
200
A Placeboc RU 486 •
1&0
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20.3 ± 1.6
~
120
g
80
~
* Values are means ± SE; RU486 (1 mg/d) or placebo were given for 5 days starting when the dominant follicle reached 14 to 16 mm. t Measurements obtained on the day of RU486 treatment corresponding to the day of placebo administration when maximum values occurred (e.g., if peak LH levels occurred on day 4 of placebo administration, "expected" peak values would correspond to LH concentrations on day 4 of RU486 treatment). :j: Conversion factor to SI units, 1.0. § Significantly different compared with peak levels in the placebo cycles, P < 0.01. II Conversion factor to SI units, 3.671.
::I: ...I
40
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15
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10
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30 C
•
25
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occurred simultaneously and were followed by the collapse of the dominant follicle within 3 days_ The duration of the follicular phase (15.3 ± 1.3 versus 17.7 ± 1.2 days), luteal phase (14.0 ± 0.4 versus 13.8 ± 0.9 days), and entire menstrual cycle (29.3 ± 1.3 versus 31.5 ± 1.5 days) was not statistically different in the placebo and RU486 groups.
1
.§.
a; ~
20
.!i::J
10
is
2
3
4
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9 10
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15
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a
Effects of RU486 on Follicular and Hormonal Dynamics
There were no differences in peak preovulatory levels of LH, FSH, E z, and follicular diameter between placebo and RU486 cycles when all six women were analyzed as a group (Table 1). However, comparison of results on the day of maximum values during placebo administration with results on the corresponding day of RU486 treatment (the "expected" day of peak values) showed that RU486 suppressed all parameters except for follicular diameter (Table 1). The rate of follicular maturation varied substantially among patients, even in the placebo (i.e., control) cycle. This was attributed in part to the heterogeneous response of the reproductive axis to GnRH stimulation. To minimize blurring of the data due to the variable rates of follicular development, the time course of follicular growth and hormonal changes was analyzed after dividing patients into two groups, i.e., those with normal or prolonged folliculogenesis. Patients with Normal Folliculogenesis
Patients 1 to 4 had similar, normal rates offollicular maturation, with gonadotropin surges occurring Vol. 62, No. I, July 1994
I&.
1 600
4
5
400
~
300
.;;
200
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Figure 2 Effects of placebo or RU486 administration in four women with hypothalamic amenorrhea treated with pulsatile GnRH. Placebo or RU486 (1 mg/d) were given orally for 5 days (arrows), starting when the dominant follicle reached 14 to 16 mm. Data represent means ± SE levels of LH (A), FSH (B), follicular diameter (C), E2 (D), and P (E) plotted relative to the first day (day 1) of placebo or RU486 administration. *Significant difference (P < 0.05) between placebo and RU486 cycles. Conversion factor to SI units for LH and FSH levels, 1; for E2 levels, 3.671; and P levels, 3.180. Batista et al.
Effects of RU486 on GnRH-induced cycles
31
ascending curves seen in the placebo group, mean LH and FSH levels remained stable or declined during RU486 treatment, peaking only after the drug was stopped (Fig. 2A and B). Follicular growth progressed at a similar rate in both study cycles up to the day of follicular collapse in the placebo group (Fig. 2C). In contrast, E2 concentrations rose steadily during placebo administration but failed to do so during RU486 treatment (Fig. 2D). In both study cycles, follicular diameter and E2 levels reached maximum values within 2 days of the LH peak. As opposed to rising concentrations seen during placebo administration, P levels remained low during RU486 treatment (Fig. 2E). They started to increase within 1 day ofthe LH surge and peaked at similar luteal levels in placebo (25.6 ± 3.3 ngjmL [81.4 ± 10.5 nmol/L]) and RU486 cycles (19.7 ± 5.1 ngjmL [62.6 ± 16.2 nmol/L]). Patients with Prolonged Folliculogenesis
Patients 5 and 6 had more prolonged folliculogenesis and ovulated only after placebo and RU486 were discontinued (Fig. 1). Because of their heterogeneous response, their data were analyzed individually (graphs not shown). The time course of hormonal changes progressed at a slower rate in these two women but was otherwise similar to the one seen in patients 1 to 4. Thus, LH and FSH levels showed minor fluctuations but did not change significantly during RU486 treatment. A gonadotropin surge of reduced or normal magnitude (LH of 27 and 79 mlUjmL or IUjL; and FSH of 16 and 25 mlU jmL or IU jL) occurred 6 and 12 days, respectively, after the compound was stopped. Estradiol levels failed to rise during RU486 administration but increased rapidly after the antiprogestin was discontinued, peaking at 154 and 403 pgjmL (565 and 1479 pmol/L) within 2 days of the LH surge. Progesterone concentrations remained low or declined during RU486 treatment. They rose within 1 day ofthe LH surge (i.e., after stopping RU486) and peaked at luteal levels of 7 and 17 ngjmL (22 and 54 nmol/L). In contrast to the other four women who exhibited normal folliculogenesis in both study cycles, follicular diameter increased slightly or remained unchanged during RU486 treatment in patients 5 and 6, progressing at a much slower rate than during placebo administration. After the antiprogestin was discontinued, follicular diameter paralleled the rise in E2 concentrations and peaked at 24 and 28 mm just before ovulation. 32
Batista et al.
Effects of RU486 on GnRH-induced cycles
DISCUSSION
The primary site(s) at which P acts to regulate the midcycle gonadotropin surge has not been defined fully. In rhesus monkeys with hypothalamic lesions receiving unvarying pulses of GnRH, P advances the initiation of E 2-induced gonadotropin surges, suggesting that a major part of this effect is exerted at the level of the pituitary gland (10). Several lines of evidence, however, indicate that the hypothalamus also may be a site of P action. In ovariectomized, estrogen-primed monkeys, P appears to activate neural substrates in the ventral hypothalamus to increase the frequency and amplitude of LH pulses and thus induce a gonadotropin peak (11, 12). This process may be suppressed with anesthetics such as pentobarbitone, presumably by blocking the effects of P on the brain (13). Additional evidence of the role of the hypothalamus has been provided by studies showing that GnRH concentrations increase at the time ofthe preovulatory LH surge in peripheral and pituitary stalk plasma of human and nonhuman primates (14, 15). In the present study, we investigated whether the antiprogestin RU486 can delay the midcycle gonadotropin surge independent of hypothalamic inhibition' in an attempt to assess the site(s) of action of P in the control of ovulation. We thus treated women with hypothalamic amenorrhea, who have abnormal or absent GnRH secretion, with exogenous GnRH pulses of unvarying frequency and dose to provide a constant GnRH rhythm sufficient to induce ovulation and counteract any hypothalamic suppressive effects of RU486 (4). The latter compound was given at low doses in the late follicular phase of these GnRH -controlled ovulatory cycles to evaluate primarily its effects on the pituitary-ovarian axis. We chose to recruit women with functional hypothalamic amenorrhea and not those with Kallmann's syndrome (i.e., with congenital absence of GnRH secretion and thus ideal for this protocol) because ofthe rarity of the latter group. Although it is possible that patients who participated in this study had some endogenous hypothalamic activity that could influence our results (4), several steps were taken to minimize this problem. Thus, we only selected women with long-standing amenorrhea to limit the chance of spontaneous return of hypothalamic function during the protocol. In addition, the priming cycles before entering the study cycles helped reduce the possibility of exaggerated pituitary responses to GnRH therapy due to prolonged deprivation of this hormone. Fertility and Sterility
When given at the same dose and regimen as previously reported in normally cycling women (2), RU486 consistently delayed the timing of the midcycle gonadotropin surge and ovulation despite the administration of constant pulsatile GnRH signals. The magnitude of the delay was similar in GnRHstimulated and spontaneous cycles (3.3 ± 0.7 versus 2.8 ± 0.5 days, P not significant by unpaired t-test), demonstrating that this effect of RU486 was not altered by replacing or overriding endogenous hypothalamic signals with pulsatile GnRH therapy. Although these results do not exclude a concurrent action of RU486 or its agonist P on the hypothalamus, they indicate that effects on the pituitaryovarian axis are sufficient to explain the delay in ovulation induced by RU486. Thus, the hypothalamus does not seem to play a critical role in this process. In GnRH -stimulated cycles, LH and FSH levels increased during placebo administration but remained unchanged or decreased during RU486 treatment, suggesting that this compound acts on the pituitary to inhibit gonadotropin secretion. In normally cycling women, RU486 suppressed LH and FSH levels when administered at a single dose of 10 mg during the midfollicular or late follicular phase (16). This appears to represent an antagonistic action of RU486, because P given at the same dose and time of the cycle induced opposite changes in gonadotropin concentrations (16). Taken together with these findings, our present data suggest that RU486 antagonizes P at the level of the pituitary gland to suppress LH and FSH secretion and delay the midcycle gonadotropin surge that leads to ovulation. RU486 also inhibited ovarian steroidogenesis and delayed the timing of peak E2 concentrations and preovulatory P rises. The latter events occurred within 2 days of the midcycle gonadotropin surge and had similar magnitudes in placebo and RU486 cycles. Thus, the temporal relationship between steroid and gonadotropin concentrations at midcycle was preserved during RU486 treatment, so that LH and FSH surges began when E2 and P reached normal preovulatory levels. Despite its effects on ovarian steroidogenesis, RU486 did not suppress follicular growth in most patients. However, in two women who could not be distinguished in any other way from the remaining subjects, follicular maturation was prolonged in both RU486 and placebo cycles, suggesting that GnRH replacement therapy was insufficient to induce a completely normal follicular phase. In contrast to the other paVol. 62, No.1, July 1994
tients, the dominant follicle grew at a slower rate when these two women were given RU486 rather than placebo. Thus, it is possible that in the latter subjects the lack of optimal GnRH (hence, gonadotropin) support, associated with a pituitary suppressive effect of RU486, resulted in more severe disruption of follicular maturation than in the other four patients exhibiting normal GnRH-induced folliculogenesis. The effects of RU486 to inhibit ovarian steroidogenesis and, in some instances, also follicular maturation appear to be due to P antagonism at the level of the pituitary gland to suppress gonadotropin secretion. Both the development of the follicle and its hormonal production are dependent on gonadotropin support (17). A less likely explanation is that RU486 acted directly on the ovary to inhibit 3,B-hydroxysteroid dehydrogenase activity and thus impair steroid synthesis (18). This probably would not explain the lower gonadotropin concentrations seen in association with reduced steroid levels during RU486 treatment. Disruption of follicular maturation and suppression of E2 and P secretion were reported previously in normally cycling women treated with substantially higher doses of RU486 in the late follicular phase (19, 20). In contrast, when the compound was given to normal subjects at a lower dose of 1 mg/ din the same regimen used in the present study, E2 secretion appeared unaffected but P concentrations remained low and did not rise during RU486 treatment (2). Differences in RU486 effects in spontaneous and GnRH -controlled cycles may be due to the fact that, in the former cycles, the hypothalamus was intact and thus capable of increasing GnRH secretion to overcome an inhibitory action of RU486 on the pituitary-ovarian axis. In summary, our results show that RU486 delays the midcycle gonadotropin surge and ovulation despite the external administration of constant GnRH signals, arguing against a critical role of the hypothalamus in this process. Rather, this effect appears to be due to P antagonism at the level ofthe pituitary gland to suppress gonadotropin and, subsequently, steroid hormone secretion. These findings support the concept that P can act on the pituitary, independent of effects on the hypothalamus, to regulate the timing of the midcycle gonadotropin surge that leads to ovulation. Acknowledgments. Weare indebted to the 9th Floor Clinic nursing staff of the Clinical Center of the National Institutes of Health (Bethesda, Maryland) for supporting this study and also
Batista et al.
Effects of RU486 on GnRH-induced cycles
33
r to Roussel-UCLAF (Romainville, France) for providing RU486. We are grateful to Helena Fundament, M.D. (University of Sao Paulo, Sao Paulo, Brazil) for assistance with data analysis. We thank Ms. Barbara Filmore (National Institutes of Health, Bethesda, Maryland) for technical assistance.
11.
12. REFERENCES 1. Baulieu EE. RU-486 as an antiprogesterone steroid. From receptor to contragestion and beyond. JAMA 1989; 262:1808-14. 2. Batista MC, Cartledge TP, Zellmer A W, Nieman LK, Merriam GR, Loriaux DL. Evidence for a critical role of progesterone in the regulation of the midcycle gonadotropin surge and ovulation. J Clin Endocrinol Metab 1992;74:56570. 3. Nakai Y, Plant TM, Hess DL, Keogh EJ, Knobil E. On the sites of the negative and positive feedback actions of estradiol in the control of gonadotropin secretion in the rhesus monkey. Endocrinology 1978;102:1008-14. 4. Santoro N, Filicori M, Crowley WF Jr. Hypogonadotropic disorders in men and women: diagnosis and therapy with pulsatile gonadotropin-releasing hormone. Endocr Rev 1986;7:11-23. 5. Odell WD, Rayford PL, Ross GT. Simplified, partially automated method for radioimmunoassay of human thyroid stimulating, growth, luteinizing and follicle stimulating hormones. J Lab Clin Med 1967;70:973-80. 6. Jiang NS, Ryan RJ. Radioimmunoassay for estrogens: a preliminary communication. Mayo Clin Proc 1969;44:461-5. 7. Abraham G E, Swerdloff R, Tulchinsky D, Odell WD. Radioimmunoassay of plasma progesterone. J Clin Endocrinol Metab 1971;32:619-24. 8. Wetzels LCG, Hoogland HJ. Relation between ultrasonographic evidence of ovulation and hormonal parameters: luteinizing hormone surge and initial progesterone rise. Fertil Steril 1982;37:336-41. 9. Queenan JT, O'Brien GD, Bains LM, Simpson J, Collins WP, Campbell S. Ultrasound scanning of ovaries to detect ovulation in women. Fertil Steril 1980;34:99-105. 10. Wildt L, Hutchison JS, Marshall G, Pohl CR, Knobil E. On the site of action of progesterone in the blockade of the es-
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Fertility and Sterility