0021-972X/91/7303-0644$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 73, No. 3 Printed in U.S.A.
Follicular Arrest during the Midfollicular Phase of the Menstrual Cycle: A Gonadotropin-Releasing Hormone Antagonist Imposed Follicular-Follicular Transition* L. M. KETTELt, S. J. ROSEFF, T. C. CHIU, M. L. BANGAH, W. VALE, J. RIVIER, H. G. BURGER, AND S. S. C. YEN* Department of Reproductive Medicine, School of Medicine, and the General Clinical Research Center, University of California-San Diego, and the Peptide Biology Laboratory, the Salk Institute (W. V., J.R.), La Jolla, California 92093; and Prince Henry's Institute of Medical Research, South Melbourne, 3205 Victoria, Australia
5.4 ± 0.5; P < 0.01) and LH pulse amplitude (5.5 ± 0.7 to 2.4 ± 0.3 IU/L; P < 0.01) in response to Nal-Glu antagonist. The number of LH pulses was reduced (36%), but pulses remained discernible. Concentrations of FSH (10.8 ± 1.4 to 5.9 ± 0.4 IU/ L; P < 0.05), E2 (322.7 ± 71.9 to 84.8 ± 7.7 pmol/L; P < 0.01) and i-INH (284.0 ± 25.9 to 164.4 ± 7.5 U/L; P < 0.01) decreased concomitantly. Within 24-48 h of the last injection of Nal-Glu, all hormones had returned to pretreatment levels. This was followed by normal functional expression of follicular growth and maturation, as reflected by an increase in E2 and i-INH levels, timely ovulation, and normal luteal function. These findings indicate that an approximately 50% decline in gonadotropin support to the dominant follicle leads to functional arrest, but not demise, of the developing follicle(s) without triggering new folliculogenesis. The follicular apparatus retained its ability to reinitiate its original functionality once appropriate gondotropin inputs were reinstated. (J Clin Endocrinol Metab 73: 644-649, 1991)
ABSTRACT. The functional dependency of the dominant follicle on pulsatile gonadotropin inputs was evaluated by using a GnRH antagonist as a probe. Hormonal dynamics, particularly the relationship of FSH, estradiol, and inhibin, during and after the withdrawal of GnRH receptor blockade achieved by treatment with Nal-Glu GnRH antagonist (50 Mg/kg, im) for 3 days in the midfollicular phase of the cycle (days 7-9) were ascertained. Daily blood samples were obtained for LH, FSH, estradiol (E2), progesterone, and immunoreactive inhibin (i-INH) measurements by RIA during 2 consecutive (control and treatment) cycles in 12 women. In 5 women, LH pulsatility was assessed by 10-min blood sampling for 12 h before, during, and after Nal-Glu treatment. The administration of Nal-Glu prolonged both follicular phase (14.0 ± 0.5 vs. 19.7 ± 0.8 days; P < 0.0001) and total cycle length (28.1 ± 0.5 vs. 34.1 ± 1.2 days; P < 0.0001). Gonadotropin suppression (50-60%) was achieved, as reflected by a marked decrease in mean LH levels (14.3 ± 1.9 to
I
T IS NOW established that hypothalamic GnRHmediated pulsatile gonadotropin release is essential for orderly folliculogenesis and maturation (1). Physiological changes in the pulsatile pattern of gonadotropin secretion are essential for the maintenance of ovulatory menstrual cycles (2-4). Aberrations of pulsatile gonadotropin secretion, in both amplitude and frequency, may result in follicular growth that is disordered, subnormal, or arrested (5-8). While the time course for follicular
recruitment and the selection of the dominant follicle (days 5-7 of the follicular phase) is well characterized (9), the functional relationship between pulsatile gonadotropin inputs and the intraovarian processes of ongoing follicular maturation remain unclear (10). Until recently, in vivo assessment of the dependency on gonadotropins in the maintenance of folliculogenesis in the human was not possible. The availability of rapidacting GnRH antagonists has provided an important probe for such studies. Several years ago, we demonstrated that the administration of the 4F-antagonist for 3 days during the midfollicular phase of the cycle resulted in follicular collapse (11). Because of the relatively low potency and short duration of action (6-8 h) of the 4Fantagonist, it was difficult to determine the precise degree of gonadotropin suppression throughout the 3-day course of treatment. The new generation of GnRH antagonists, particularly the Nal-Glu antagonist, possess greater biological effect
Received December 3,1990. Address all correspondence and requests for reprints to: Dr. S. S. C. Yen, Department of Reproductive Medicine (0802), University of California-San Diego, La Jolla, California 92093-0802. * This work was supported by NIH NICHHD Center Grant HD12303-12, (S. Y.), Grant HD-13527, the Hearst Foundation (W. V., J. R.), the Andrew W. Mellon Foundation, the National Health and Medical Research Council of Australia, (H. G. B.), and in part by Grant MO1-00827 from the General Clinical Research Branch, NIH. The research was conducted by the Clayton Foundation for Research, California Division. t Andrew W. Mellon Foundation Faculty Scholar. X Clayton Foundation Investigator.
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FOLLICULAR-FOLLICULAR TRANSITION and a longer duration of action. A single injection (50 Atg/kg, im) results in gonadotropin suppression for more than 24 h in postmenopausal women (12). The degree of gonadotropin suppression by Nal-Glu antagonist remains incomplete (50-60% reduction for LH and 3040% reduction for FSH) independent of the dose. These observations made in the hypergonadotropic state were identical to those found in early follicular phase women (13), midluteal phase women (14), and men (15). We took advantage of the relatively incomplete, but predictable, gonadotropin suppression by Nal-Glu antagonist to define the effect of gonadotropin deprivation on the newly selected dominant follicle in midfollicular phase women. The specific aim of our study was to ascertain the hormonal dynamics, particularly the relationship of FSH, estradiol, and inhibin, during and after withdrawal of GnRH receptor blockade by Nal-Glu antagonist. The hormonal events of this follicular-follicular transition may provide insight into the functionality of the dominant follicle and clues into the aberrations that take place in conditions of chronic anovulation.
Materials and Methods Twelve normal cycling women (aged 28-37; mean, 33 yr) participated in 16 studies. Four women repeated the study after a 3-month recovery interval, and data are analyzed as the composite of these 16 data sets. All women were of normal weight for height and had documented cycle lengths of 26-32 days. None of the women had taken any hormonal medications for 3 months before enrollment and used barrier contraceptives throughout the study period. The project was approved by the Committee on Investigations Involving Human Subjects of the University of California-San Diego, and informed written consent was obtained from each subject before enrollment. Daily blood samples were obtained during two consecutive cycles; the first cycle served as the control cycle, and the second as the treatment cycle. Beginning in the midfollicular phase of the second cycle (day 7, 8, or 9), each woman was given daily injections of [Ac-D 2 Nal 1 ,D 4 ClPhe 2 ,D 3 Pal 3 ,Arg 5 , DG1U6(AA),DAla10]GnRH antagonist (Nal-Glu; 50 Mg/kg, im) for 3 consecutive days. To ensure that none of the subjects was in the immediate preovulatory stage before receiving Nal-Glu, a pelvic ultrasound (ADR 4000S sector scanner, 3.5-mHz transducer, ADR, Tempe, AZ) was performed to assure the absence of an ovarian follicle(s) greater than 11 mm at the greatest diameter. The dose chosen for Nal-Glu antagonist was based on our previous experience, in which we observed maximal suppression of LH (>50%) lasting more than 24 h (12, 14). In five women, LH pulsatility was assessed by 10-min blood sampling for 12 h (0800-2000 h) 1 day before, during the first day, and 1 day after Nal-Glu treatment. For these studies, the women were admitted to the Clinical Research Center of the University of California-San Diego Medical Center after an overnight fast. Blood was drawn through an indwelling venous catheter placed 1 h before the initiation of the study. Subjects were not allowed to sleep during the day, and tobacco and
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caffeine were prohibited. For the study performed during treatment, blood was sampled for 1 h before receiving Nal-Glu antagonist and continued for 12 h afterward. For the purpose of data analysis, the follicular phase was defined as those days from the first day of menstrual bleeding to the day before the LH peak. The luteal phase was defined as the day after the LH peak to the day before uterine bleeding. Serum LH, FSH, estradiol (E2), and progesterone (P4) determinations were measured in duplicate by previously characterized RIA (16-18). The intra- and interassay coefficients of variation (CVs) for the RIAs were, respectively: LH, 2.8% and 8.7%; FSH, 1.4% and 6.0%; E2, 2.7% and 9.3%; and P4) 2.7% and 9.1%. Serum immunoreactive inhibin (i-INH) levels were determined in duplicate by a heterologous RIA using antiserum raised against highly purified bovine 3IK inhibin (no. 1989), [125I]31K bovine inhibin as a tracer, and a human serum pool (MR1) obtained from women undergoing ovarian hyperstimulation as standard. This standard was calibrated against a human follicular fluid inhibin preparation (no. 382) of known bioassay potency (19). There is no cross-reactivity with a variety of inhibin-related peptides, including bovine activin-A (20). Recent data have demonstrated a significant cross-reactivity between the antibody used in this RIA and both recombinant free a-subunit (21) and the subunit precursor protein, pro-ac, from bovine follicular fluid (22). The implications of this cross-reactivity in the RIA system for measurements of inhibin concentrations are presently unknown. However, when serum concentrations of inhibin, as detected by this RIA, were compared to levels measured in a sensitive sheep pituitary cell bioassay the results were similar (23). A serum volume of 100 /JL was used for the assay, and the lower limit of sensitivity for the assay was 143 U/L. The average minimum intraassay CV was 4.0%, while the interassay CVs were 14.4%, 6.3%, and 7.5% in the upper, mid-, and lower regions of the standard curve, respectively. Significant LH pulses were identified using the Cluster computer pulse detection algorithm (24, 25). A cluster configuration of 2 X 1 (two values for a nadir and one for a peak) with a t statistic of 2.1 for both up-stroke and down-stroke was chosen to limit the detection of false positive and false negative pulses to less than 5%. Since the variance of a hormone determination is a curvilinear function of the hormone level, a second degree polynomial regression analysis was performed relating the SD to the hormone concentration. This estimate of measurement error was employed within the Cluster program to assess the significance of hormone concentration changes. The resulting second degree coefficient, first degree coefficient, and intercept for LH values between 0-70 IU/L were 0.0011, 0.0095, and 0.5352, respectively. The LH pulse frequency was defined as the number of pulses per U observation time (12 h). The pulse amplitude was defined as the difference in hormone level between the cluster peak and the preceding nadir. For each individual subject, the pulse amplitude used in analysis represents the mean of pulse amplitudes for the 12-h data series. Data were analyzed by two-factor analysis of variance with repeated measures, followed by Dunnett's test. Correlation coefficients were generated as Pearson product-moment correlations. Paired t tests were used to analyze attributes within
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KETTEL ET AL.
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groups. Results are presented as the mean ± SE, and P < 0.05 was considered significant.
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Results Mean (±SE) daily serum concentrations of LH, FSH, i-INH, E2, and P 4 during two consecutive cycles are displayed in Fig. 1. Compared with the control cycle (first cycle), Nal-Glu antagonist given for 3 days in the midfollicular phase of the second cycle prolonged both follicular phase (14.0 ± 0.5 to 19.7 ± 0.8 days; P < 0.0001) and total cycle (28.1 ± 0.5 vs. 34.1 ± 1.2 days; P < 0.0001) length. Luteal phase length was unchanged (13.1 ± 0.4 vs. 13.4 ± 0.6 days). After the first injection of Nal-Glu antagonist, there was a 62.2% suppression of LH levels and a 45.3% suppression of FSH levels from baseline values. A concomitant decrease in E2 (73.7%) and i-INH (42.1%) levels occurred (Fig. 2). This degree of suppression was maintained throughout the 3-day treatment period. Within 24-48 h after Nal-Glu was discontinued, the serum levels of each of these hormones had returned to baseline, with a substantial rebound (P< 0.01) in FSH secretion lasting 48 h (Fig. 3). The effect of Nal-Glu antagonist on LH pulsatile release was determined in five women who had frequent sampling studies (Fig. 4). Compared to baseline values on day 6 of the menstrual cycle, there was a significant decrease in LH pulse amplitude (5.5 ± 0.7 to 2.4 ± 0.3 IU/L; P < 0.01) after Nal-Glu treatment. LH pulse NAL-GLU
FIG. 1. Mean (±SE) daily serum LH, FSH, E2, P4) and i-INH concentrations during control and treatment cycles in 12 women. Data are centered around the LH peak. Nal-Glu GnRH antagonist (50 fig/kg, im) was administered for 3 days in the midfollicular phase of the second cycle. • * , P < 0.0001.
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m
£
200
OL Baseline Max A
FIG. 2. Mean (±SE) serum LH, FSH, E2, and i-INH levels at baseline and maximal change (displayed as concentrations and percent change) after receiving Nal-Glu GnRH antagonist (50 ^g/kg, im). *, P < 0.05; **, P < 0.01.
frequency did not change significantly, but there were fewer pulses detected during Nal-Glu treatment (10.8 ± 1.6 to 7.0 ± 2.8 pulses/12 h). LH pulse frequency (9.6 ± 1.5 pulses/12 h) and pulse amplitude (5.2 ± 0.7 IU/L) were restored within 24 h of the last injection (Fig. 5). Although FSH is also released in a pulsatile fashion, the long serum half-life of this glycoprotein makes detection and meaningful interpretation of FSH pulses difficult. The fluctuations of FSH levels were attenuated by NalGlu antagonist in parallel to that of LH, resulting in a significant decrease in mean FSH values (10.8 ± 1.4 to 5.9 ± 0.4 IU/L; P < 0.05), with restoration 24 h after completion of Nal-Glu antagonist treatment (Fig. 4). After recovery from Nal-Glu suppression, a prompt reinitiation of follicular growth and development occurred, as reflected by a rapid increase in E2 and i-INH, timely ovulation, and normal luteal function (Fig. 6). Because the drug was initiated in the midfollicular phase of the cycle (day 7, 8, or 9), the baseline concentrations of E2 varied from woman to woman (194-756 pmol/L; mean, 368.9 ± 52.9 pmol/L). The time course for recovery from Nal-Glu-induced gonadotropin suppression to the subsequent LH surge was inversely correlated with pretreatment E2 levels (r = -0.60; P < 0.02), but not with age, weight, or baseline or recovery gonadotropin levels. No subjects experienced significant adverse effects
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FOLLICULAR-FOLLICULAR TRANSITION NAL-GLU V
V
V
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£2 • — • CONTROL O—O TREATMENT
§ 500 a OL
i-INH 300
100
-
2
0
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4
DAYS
FIG. 3. Mean (±SE) changes in daily serum FSH, E2, and i-INH in 12 women before, during, and after the administration of Nal-Glu GnRH antagonist (50 /xg/kg, im) for 3 days in the midfollicular phase. Data are centered around the first day of Nal-Glu administration (day 0) and the corresponding values in the control cycle.
from the Nal-Glu antagonist. A small area of erythema and induration accompanied by some mild discomfort around the injection site occurred in eight of the subjects. This usually resolved within 2 h. The other four women had no discernible reaction. In no subject was the pain or discomfort considerable enough to discontinue the investigation, and most women experienced a decrease in discomfort with each succeeding injection. No urticarial reactions were noted.
Discussion Nal-Glu GnRH antagonist (50 Mg/kg, im) administered to midfollicular phase women maximally reduced mean
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serum levels of immunoreactive LH and FSH by 62.2% and 45.3%, respectively. The rapid attenuation of LH release after administration of Nal-Glu antagonist was accompanied by a 35% decline in LH pulse frequency and a 56% fall in LH pulse amplitude. These data corroborate similar studies performed in normal cycling women given Nal-Glu antagonist in the early follicular phase (13) and normal men in whom the Nal-Glu antagonist (5 mg, sc) induced a 25% and 56% decline in LH pulse frequency and amplitude, respectively (15). The Nal-Glu antagonist-induced gonadotropin suppression during the midfollicular phase was maintained throughout the 3 treatment days, with a corresponding decrease in serum E2 and i-INH levels. Previous studies using the same GnRH antagonist in postmenopausal women demonstrated 53% and 21% decreases in serum immunoreactive LH and FSH, respectively (12). The partial decrease in serum gonadotropin levels would suggest either an incomplete blockade of pituitary GnRH receptors by Nal-Glu antagonist or non-GnRHdependent gonadotropin secretion. The percent decline in FSH secretion was less than that in LH secretion in all subjects. This is consistent with all previous studies using GnRH antagonists and implies a significant nonGnRH-dependent control mechanism for FSH secretion. Activin, a /3-/3 dimer of inhibin subunits, has been shown to stimulate FSH secretion from pituitary cells in culture, even after GnRH desensitization (26, 27). Since information on circulating activin is not presently available, the possibility that this represents a significant regulator for FSH secretion remains speculative. After Nal-Glu antagonist, serum concentrations of FSH rebounded to levels significantly higher than either pretreatment or control cycle values. This FSH increment was associated with low serum levels of E2 and i-INH, which may be instrumental in the disinhibition of FSH release. When pretreatment levels of E2 and iINH were reached 2 days after discontinuation of GnRH antagonist treatment, levels of FSH declined to corresponding control cycle concentrations. Levels of E2 and i-INH fell very low after Nal-Glu antagonist administration and remained so throughout treatment. Recovery from Nal-Glu antagonist-imposed TREATMENT
MAL-OLU
FIG. 4. Pulsatile activity of LH and FSH in a subject 1 day before (BASELINE), during (TREATMENT) and 1 day after (RECOVERY) receiving Nal-Glu GnRH antagonist (50 /ig/kg, im) for 3 days in the midfollicular phase of the menstrual cycle. Asterisks indicate LH pulses. 0800
1200
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2100 0800
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2000
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KETTEL ET AL. 15
• ••
2 10 w
UJ CO
3
UJ
Q
4
I 2 Baseline
Treatment
Recovery
FIG. 5. Mean (±SE) LH pulse frequency and amplitude in five women before (Baseline), during (Treatment), and after (Recovery) receiving Nal-Glu GnRH antagonist (50 Mg/kg, im). **, P < 0.01.
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GnRH receptor blockade was rapid, with gonadotropin, E2, and i-INH levels returning to pretreatment midfollicular concentrations within 48 h after the last injection. Thereafter, progressive increases in E2 and i-INH concentrations occurred, with midcycle surge and luteal phase hormonal profiles identical to those of control cycle studies and previous reports (28). The time required to achieve the subsequent LH surge was inversely correlated with pretreatment levels of E2, a finding consistent with the heterogeneous functional status of the follicles before Nal-Glu administration. It is apparent from the present study that there was functional arrest of the dominant follicle, in that the endocrine expression of the follicle was restored in a timely fashion after GnRH antagonist was discontinued. Ovarian hormones, E2 and i-INH, were suppressed to a much greater degree than gonadotropins, with levels reaching those in postmenopausal women. One explanation for this phenomenon is a greater decline in levels of bioactive gonadotropin after administration of the Nal-Glu GnRH antagonist. In studies using a shorter acting compound, the 4F-antagonist, in both men (29) and women (30), the bioactive to immunoreactive ratios of LH and FSH declined. Nal-Glu antagonist administration in postmenopausal women also induces a greater decrease in bioactive LH and FSH and reduces the bioactive/ratio ratio by 50% and 27%, respectively (12). In these studies both the degree and the duration of suppression were greater for bioactive gonadotropin. It is possible that the discrepancy between bio- and immunoactive LH and FSH is due to the inability of antibodies to recognize the biologically active portion of the glycoprotein molecules in the RIA system. Alternatively, a preferential decline in the secretion of biologically active glycoprotein isoform occurred after blockade of GnRH receptors (31). In summary, after an approximately 50% decrease in gonadotropin support to the dominant follicle for 3 days in the midfollicular phase of the menstrual cycle, arrest of ongoing follicular maturation occurred. However, the follicular apparatus retained its ability to reinitiate its original functionality once appropriate gonadotropin inputs were reinstated with subsequent cycle dynamics appropriate for the original follicular status. Our findings provide in vivo information on the robust nature of the developing follicle and its ability to retain its cellular endowments during gonadotropin deprivation.
12
-4 DAYS FROM LH PEAK
FIG. 6. Mean (±SE) daily serum LH, FSH, E2, P4, and i-INH during control cycle and after recovery from Nal-Glu GnRH antagonist (50 /xg/kg, im), given for 3 days in the midfollicular phase of the treatment cycle. Data are centered around the LH peak.
Acknowledgments The authors are grateful for the excellent technical assistance by the staff of the Clinical Research Center, Pam Malcom, Shannon Petze, and Jeff Wong, and to Dr. A. Lein for advice and assistance.
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FOLLICULAR-FOLLICULAR TRANSITION
References 1. Knobil E. The neuroendocrine control of the menstrual cycle. Recent Prog Horm Res. 1980;36:53-88. 2. Yen SSC, Tsai CC, Naftolin F, Vandenberg G, Ajabor L. Pulsatile patterns of gonadotropin release in subjects with and without ovarian function. J Clin Endocrinol Metab. 1972;34:671-5. 3. Reame N, Sauder SE, Kelch RP, Marshall JC. Pulsatile gonadotropin secretion during the human menstrual cycle: evidence for altered frequency of gonadotropin-releasing hormone secretion. J Clin Endocrinol Metab. 1984;59:328-37. 4. Filicori M, Santoro N, Merriam GR, Crowley WF. Characterization of the physiological pattern of episodic gonadotropin secretion throughout the human menstrual cycle. J Clin Endocrinol Metab. 1986;62:1136-44. 5. Kazer RR, Kessel B, Yen SSC. Circulating luteinizing hormone pulse frequency in women with polycystic ovary syndrome. J Clin Endocrinol Metab. 1987;65:233-6. 6. Berga SL, Mortola JF, Girton L, et al. Neuroendocrine aberrations in women with functional hypothalamic amenorrhea. J Clin Endocrinol Metab. 1989;68:301-7. 7. Loucks AB, Mortola JF, Girton L, Yen SSC. Alterations in hypothalamic-pituitary ovarian (HPO) and hypothalamic-pituitary adrenal (HPA) axes in athletic women. J Clin Endocrinol Metab. 1989;68:402-ll. 8. Soules MR, Clifton DK, Cohen NL, Bremner WJ, Steiner RA. Luteal phase deficiency: abnormal gonadotropin and progesterone secretion patterns. J Clin Endocrinol Metab. 1989;69:813-20. 9. diZerega GS, Hodgen GD. Folliculogenesis in the primate ovarian cycle. Endocr Rev. 1981;2:27-49. 10. Tonetta SA, diZerega GS. Intragonadal regulation of follicular maturation. Endocr Rev. 1989;10:205-29. 11. 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. 12. Mortola JF, Sathanandan M, Pavlou S, et al. Suppression of bioactive and immunoactive follicle stimulating hormone and luteinizing hormone levels by a potent gonadotropin-releasing hormone antagonist: pharmacodynamic studies. Fertil Steril. 1989;51:957-63. 13. Hall JE, Whitcomb RW, Rivier JE, Vale W, Crowley WF. Differential regulation of luteinizing hormone, follicle stimulating hormone, and free a-subunit secretion from the gonadotrope by gonadotropin-releasing hormone (GnRH): evidence from the use of two GnRH antagonists. J Clin Endocrinol Metab. 1990;70:328-35. 14. Roseff SJ, Bangah ML, Kettel LM, et al. Dynamic changes in circulating inhibin levels during the luteal-follicular transition of the human menstrual cycle. J Clin Endocrinol Metab. 1989;69:1033-9. 15. Pavlou SN, Wakefield G, Schlechter NL, et al. Mode of suppression of pituitary and gonadal function after acute or prolonged admin-
16. 17.
18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
28. 29.
30. 31.
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istration of a luteinizing hormone-releasing hormone antagonist in normal men. J Clin Endocrinol Metab. 1989;68:446-54. Yen SSC, Llerena 0, Little B, Pearson OH. Disappearance rates of endogenous luteinizing hormone and chorionic gonadotropin in man. J Clin Endocrinol Metab. 1968;28:1763-7. Yen SSC, Llerena LA, Pearson OH, Littell AS. Disappearance rates of endogenous follicle-stimulating hormone in serum following surgical hypophysectomy in man. J Clin Endocrinol Metab. 1970;30:325-9. Anderson DC, Hopper BR, Lasley BL, Yen SSC. A simple method for the assay of eight steroids in small volumes of plasma. Steroids. 1976;28:179-96. McLachlan RI, Robertson DM, Healy DL, Burger HG, deKretser DM. Circulating immunoreactive inhibin levels during the normal human menstrual cycle. J Clin Endocrinol Metab. 1987;65:954-61. Robertson DM, Tsonis CG, McLachlan RI, et al. Comparison of inhibin immunological and in vitro biological activities in human serum. J Clin Endocrinol Metab. 1988;67:438-43. Schneyer AL, Mason AJ, Burton LE, Ziegner JR, Crowley WF. Immunoreactive inhibin a-subunit in human serum: implications for radioimmunoassay. J Clin Endocrinol Metab. 1990;70:1208-12. Robertson DM, Giacometti M, Foulds LM, et al. Isolation of inhibin a-subunit precursor proteins from bovine follicular fluid. Endocrinology. 1989;125:2141-9. Robertson DM, Tsonis CG, McLachlan RI, et al. Comparison of inhibin immunological and in vitro biological activities in human serum. J Clin Endocrinol Metab. 1988;67:438-43. Veldhuis JD, Johnson ML. Cluster analysis: a simple, versatile, and robust algorithm for endocrine pulse detection. Am J Physiol. 1986;250:E486-93. Veldhuis JD, Johnson ML. A novel general biophysical model for simulating episodic endocrine gland signaling. Am J Physiol. 1988;255:E749-59. Ying S. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev. 1988;9:267-93. Schwall RH, Sxonyi E, Mason AJ, Nikolics K. Activin stimulates secretion of follicle-stimulating hormone from pituitary cells desensitized to gonadotropin-releasing hormone. Biochem Biophys Res Commun. 1988;151:1099-104. McLachlan RI, Robertson DM, Healy DL, Burger HG, deKretser DM. Circulating immunoreactive inhibin levels during the normal human menstrual cycle. J Clin Endocrinol Metab. 1987;65:954-61. Dahl KD, Pavlou SN, Kovacs WH, Hsueh AJW. The changing ratio of serum bioactive to immunoreactive follicle stimulating hormone in normal men following treatment with a potent gonadotropin releasing hormone antagonist. J Clin Endocrinol Metab. 1986;63:792. Kessel B, Dahl KD, Kazer RR, et al. The dependency of bioactive FSH on gonadotropin releasing hormone in hypogonadal and cycling women. J Clin Endocrinol Metab. 1988;66:361-6. Dahl KD, Bicsak TA, Hsueh AJW. Naturally occurring antihormones: secretion of FSH antagonists by women treated with a GnRH analogue. Science. 1988;239:72-4.
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Vol. 73, No. 3 Printed in U.S.A.
Follicular Arrest during the Midfollicular Phase of the Menstrual Cycle: A Gonadotropin-Releasing Hormone Antagonist Imposed Follicular-Follicular Transition* L. M. KETTELt, S. J. ROSEFF, T. C. CHIU, M. L. BANGAH, W. VALE, J. RIVIER, H. G. BURGER, AND S. S. C. YEN* Department of Reproductive Medicine, School of Medicine, and the General Clinical Research Center, University of California-San Diego, and the Peptide Biology Laboratory, the Salk Institute (W. V., J.R.), La Jolla, California 92093; and Prince Henry's Institute of Medical Research, South Melbourne, 3205 Victoria, Australia
5.4 ± 0.5; P < 0.01) and LH pulse amplitude (5.5 ± 0.7 to 2.4 ± 0.3 IU/L; P < 0.01) in response to Nal-Glu antagonist. The number of LH pulses was reduced (36%), but pulses remained discernible. Concentrations of FSH (10.8 ± 1.4 to 5.9 ± 0.4 IU/ L; P < 0.05), E2 (322.7 ± 71.9 to 84.8 ± 7.7 pmol/L; P < 0.01) and i-INH (284.0 ± 25.9 to 164.4 ± 7.5 U/L; P < 0.01) decreased concomitantly. Within 24-48 h of the last injection of Nal-Glu, all hormones had returned to pretreatment levels. This was followed by normal functional expression of follicular growth and maturation, as reflected by an increase in E2 and i-INH levels, timely ovulation, and normal luteal function. These findings indicate that an approximately 50% decline in gonadotropin support to the dominant follicle leads to functional arrest, but not demise, of the developing follicle(s) without triggering new folliculogenesis. The follicular apparatus retained its ability to reinitiate its original functionality once appropriate gondotropin inputs were reinstated. (J Clin Endocrinol Metab 73: 644-649, 1991)
ABSTRACT. The functional dependency of the dominant follicle on pulsatile gonadotropin inputs was evaluated by using a GnRH antagonist as a probe. Hormonal dynamics, particularly the relationship of FSH, estradiol, and inhibin, during and after the withdrawal of GnRH receptor blockade achieved by treatment with Nal-Glu GnRH antagonist (50 Mg/kg, im) for 3 days in the midfollicular phase of the cycle (days 7-9) were ascertained. Daily blood samples were obtained for LH, FSH, estradiol (E2), progesterone, and immunoreactive inhibin (i-INH) measurements by RIA during 2 consecutive (control and treatment) cycles in 12 women. In 5 women, LH pulsatility was assessed by 10-min blood sampling for 12 h before, during, and after Nal-Glu treatment. The administration of Nal-Glu prolonged both follicular phase (14.0 ± 0.5 vs. 19.7 ± 0.8 days; P < 0.0001) and total cycle length (28.1 ± 0.5 vs. 34.1 ± 1.2 days; P < 0.0001). Gonadotropin suppression (50-60%) was achieved, as reflected by a marked decrease in mean LH levels (14.3 ± 1.9 to
I
T IS NOW established that hypothalamic GnRHmediated pulsatile gonadotropin release is essential for orderly folliculogenesis and maturation (1). Physiological changes in the pulsatile pattern of gonadotropin secretion are essential for the maintenance of ovulatory menstrual cycles (2-4). Aberrations of pulsatile gonadotropin secretion, in both amplitude and frequency, may result in follicular growth that is disordered, subnormal, or arrested (5-8). While the time course for follicular
recruitment and the selection of the dominant follicle (days 5-7 of the follicular phase) is well characterized (9), the functional relationship between pulsatile gonadotropin inputs and the intraovarian processes of ongoing follicular maturation remain unclear (10). Until recently, in vivo assessment of the dependency on gonadotropins in the maintenance of folliculogenesis in the human was not possible. The availability of rapidacting GnRH antagonists has provided an important probe for such studies. Several years ago, we demonstrated that the administration of the 4F-antagonist for 3 days during the midfollicular phase of the cycle resulted in follicular collapse (11). Because of the relatively low potency and short duration of action (6-8 h) of the 4Fantagonist, it was difficult to determine the precise degree of gonadotropin suppression throughout the 3-day course of treatment. The new generation of GnRH antagonists, particularly the Nal-Glu antagonist, possess greater biological effect
Received December 3,1990. Address all correspondence and requests for reprints to: Dr. S. S. C. Yen, Department of Reproductive Medicine (0802), University of California-San Diego, La Jolla, California 92093-0802. * This work was supported by NIH NICHHD Center Grant HD12303-12, (S. Y.), Grant HD-13527, the Hearst Foundation (W. V., J. R.), the Andrew W. Mellon Foundation, the National Health and Medical Research Council of Australia, (H. G. B.), and in part by Grant MO1-00827 from the General Clinical Research Branch, NIH. The research was conducted by the Clayton Foundation for Research, California Division. t Andrew W. Mellon Foundation Faculty Scholar. X Clayton Foundation Investigator.
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FOLLICULAR-FOLLICULAR TRANSITION and a longer duration of action. A single injection (50 Atg/kg, im) results in gonadotropin suppression for more than 24 h in postmenopausal women (12). The degree of gonadotropin suppression by Nal-Glu antagonist remains incomplete (50-60% reduction for LH and 3040% reduction for FSH) independent of the dose. These observations made in the hypergonadotropic state were identical to those found in early follicular phase women (13), midluteal phase women (14), and men (15). We took advantage of the relatively incomplete, but predictable, gonadotropin suppression by Nal-Glu antagonist to define the effect of gonadotropin deprivation on the newly selected dominant follicle in midfollicular phase women. The specific aim of our study was to ascertain the hormonal dynamics, particularly the relationship of FSH, estradiol, and inhibin, during and after withdrawal of GnRH receptor blockade by Nal-Glu antagonist. The hormonal events of this follicular-follicular transition may provide insight into the functionality of the dominant follicle and clues into the aberrations that take place in conditions of chronic anovulation.
Materials and Methods Twelve normal cycling women (aged 28-37; mean, 33 yr) participated in 16 studies. Four women repeated the study after a 3-month recovery interval, and data are analyzed as the composite of these 16 data sets. All women were of normal weight for height and had documented cycle lengths of 26-32 days. None of the women had taken any hormonal medications for 3 months before enrollment and used barrier contraceptives throughout the study period. The project was approved by the Committee on Investigations Involving Human Subjects of the University of California-San Diego, and informed written consent was obtained from each subject before enrollment. Daily blood samples were obtained during two consecutive cycles; the first cycle served as the control cycle, and the second as the treatment cycle. Beginning in the midfollicular phase of the second cycle (day 7, 8, or 9), each woman was given daily injections of [Ac-D 2 Nal 1 ,D 4 ClPhe 2 ,D 3 Pal 3 ,Arg 5 , DG1U6(AA),DAla10]GnRH antagonist (Nal-Glu; 50 Mg/kg, im) for 3 consecutive days. To ensure that none of the subjects was in the immediate preovulatory stage before receiving Nal-Glu, a pelvic ultrasound (ADR 4000S sector scanner, 3.5-mHz transducer, ADR, Tempe, AZ) was performed to assure the absence of an ovarian follicle(s) greater than 11 mm at the greatest diameter. The dose chosen for Nal-Glu antagonist was based on our previous experience, in which we observed maximal suppression of LH (>50%) lasting more than 24 h (12, 14). In five women, LH pulsatility was assessed by 10-min blood sampling for 12 h (0800-2000 h) 1 day before, during the first day, and 1 day after Nal-Glu treatment. For these studies, the women were admitted to the Clinical Research Center of the University of California-San Diego Medical Center after an overnight fast. Blood was drawn through an indwelling venous catheter placed 1 h before the initiation of the study. Subjects were not allowed to sleep during the day, and tobacco and
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caffeine were prohibited. For the study performed during treatment, blood was sampled for 1 h before receiving Nal-Glu antagonist and continued for 12 h afterward. For the purpose of data analysis, the follicular phase was defined as those days from the first day of menstrual bleeding to the day before the LH peak. The luteal phase was defined as the day after the LH peak to the day before uterine bleeding. Serum LH, FSH, estradiol (E2), and progesterone (P4) determinations were measured in duplicate by previously characterized RIA (16-18). The intra- and interassay coefficients of variation (CVs) for the RIAs were, respectively: LH, 2.8% and 8.7%; FSH, 1.4% and 6.0%; E2, 2.7% and 9.3%; and P4) 2.7% and 9.1%. Serum immunoreactive inhibin (i-INH) levels were determined in duplicate by a heterologous RIA using antiserum raised against highly purified bovine 3IK inhibin (no. 1989), [125I]31K bovine inhibin as a tracer, and a human serum pool (MR1) obtained from women undergoing ovarian hyperstimulation as standard. This standard was calibrated against a human follicular fluid inhibin preparation (no. 382) of known bioassay potency (19). There is no cross-reactivity with a variety of inhibin-related peptides, including bovine activin-A (20). Recent data have demonstrated a significant cross-reactivity between the antibody used in this RIA and both recombinant free a-subunit (21) and the subunit precursor protein, pro-ac, from bovine follicular fluid (22). The implications of this cross-reactivity in the RIA system for measurements of inhibin concentrations are presently unknown. However, when serum concentrations of inhibin, as detected by this RIA, were compared to levels measured in a sensitive sheep pituitary cell bioassay the results were similar (23). A serum volume of 100 /JL was used for the assay, and the lower limit of sensitivity for the assay was 143 U/L. The average minimum intraassay CV was 4.0%, while the interassay CVs were 14.4%, 6.3%, and 7.5% in the upper, mid-, and lower regions of the standard curve, respectively. Significant LH pulses were identified using the Cluster computer pulse detection algorithm (24, 25). A cluster configuration of 2 X 1 (two values for a nadir and one for a peak) with a t statistic of 2.1 for both up-stroke and down-stroke was chosen to limit the detection of false positive and false negative pulses to less than 5%. Since the variance of a hormone determination is a curvilinear function of the hormone level, a second degree polynomial regression analysis was performed relating the SD to the hormone concentration. This estimate of measurement error was employed within the Cluster program to assess the significance of hormone concentration changes. The resulting second degree coefficient, first degree coefficient, and intercept for LH values between 0-70 IU/L were 0.0011, 0.0095, and 0.5352, respectively. The LH pulse frequency was defined as the number of pulses per U observation time (12 h). The pulse amplitude was defined as the difference in hormone level between the cluster peak and the preceding nadir. For each individual subject, the pulse amplitude used in analysis represents the mean of pulse amplitudes for the 12-h data series. Data were analyzed by two-factor analysis of variance with repeated measures, followed by Dunnett's test. Correlation coefficients were generated as Pearson product-moment correlations. Paired t tests were used to analyze attributes within
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KETTEL ET AL.
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groups. Results are presented as the mean ± SE, and P < 0.05 was considered significant.
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Results Mean (±SE) daily serum concentrations of LH, FSH, i-INH, E2, and P 4 during two consecutive cycles are displayed in Fig. 1. Compared with the control cycle (first cycle), Nal-Glu antagonist given for 3 days in the midfollicular phase of the second cycle prolonged both follicular phase (14.0 ± 0.5 to 19.7 ± 0.8 days; P < 0.0001) and total cycle (28.1 ± 0.5 vs. 34.1 ± 1.2 days; P < 0.0001) length. Luteal phase length was unchanged (13.1 ± 0.4 vs. 13.4 ± 0.6 days). After the first injection of Nal-Glu antagonist, there was a 62.2% suppression of LH levels and a 45.3% suppression of FSH levels from baseline values. A concomitant decrease in E2 (73.7%) and i-INH (42.1%) levels occurred (Fig. 2). This degree of suppression was maintained throughout the 3-day treatment period. Within 24-48 h after Nal-Glu was discontinued, the serum levels of each of these hormones had returned to baseline, with a substantial rebound (P< 0.01) in FSH secretion lasting 48 h (Fig. 3). The effect of Nal-Glu antagonist on LH pulsatile release was determined in five women who had frequent sampling studies (Fig. 4). Compared to baseline values on day 6 of the menstrual cycle, there was a significant decrease in LH pulse amplitude (5.5 ± 0.7 to 2.4 ± 0.3 IU/L; P < 0.01) after Nal-Glu treatment. LH pulse NAL-GLU
FIG. 1. Mean (±SE) daily serum LH, FSH, E2, P4) and i-INH concentrations during control and treatment cycles in 12 women. Data are centered around the LH peak. Nal-Glu GnRH antagonist (50 fig/kg, im) was administered for 3 days in the midfollicular phase of the second cycle. • * , P < 0.0001.
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m
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OL Baseline Max A
FIG. 2. Mean (±SE) serum LH, FSH, E2, and i-INH levels at baseline and maximal change (displayed as concentrations and percent change) after receiving Nal-Glu GnRH antagonist (50 ^g/kg, im). *, P < 0.05; **, P < 0.01.
frequency did not change significantly, but there were fewer pulses detected during Nal-Glu treatment (10.8 ± 1.6 to 7.0 ± 2.8 pulses/12 h). LH pulse frequency (9.6 ± 1.5 pulses/12 h) and pulse amplitude (5.2 ± 0.7 IU/L) were restored within 24 h of the last injection (Fig. 5). Although FSH is also released in a pulsatile fashion, the long serum half-life of this glycoprotein makes detection and meaningful interpretation of FSH pulses difficult. The fluctuations of FSH levels were attenuated by NalGlu antagonist in parallel to that of LH, resulting in a significant decrease in mean FSH values (10.8 ± 1.4 to 5.9 ± 0.4 IU/L; P < 0.05), with restoration 24 h after completion of Nal-Glu antagonist treatment (Fig. 4). After recovery from Nal-Glu suppression, a prompt reinitiation of follicular growth and development occurred, as reflected by a rapid increase in E2 and i-INH, timely ovulation, and normal luteal function (Fig. 6). Because the drug was initiated in the midfollicular phase of the cycle (day 7, 8, or 9), the baseline concentrations of E2 varied from woman to woman (194-756 pmol/L; mean, 368.9 ± 52.9 pmol/L). The time course for recovery from Nal-Glu-induced gonadotropin suppression to the subsequent LH surge was inversely correlated with pretreatment E2 levels (r = -0.60; P < 0.02), but not with age, weight, or baseline or recovery gonadotropin levels. No subjects experienced significant adverse effects
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FOLLICULAR-FOLLICULAR TRANSITION NAL-GLU V
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FIG. 3. Mean (±SE) changes in daily serum FSH, E2, and i-INH in 12 women before, during, and after the administration of Nal-Glu GnRH antagonist (50 /xg/kg, im) for 3 days in the midfollicular phase. Data are centered around the first day of Nal-Glu administration (day 0) and the corresponding values in the control cycle.
from the Nal-Glu antagonist. A small area of erythema and induration accompanied by some mild discomfort around the injection site occurred in eight of the subjects. This usually resolved within 2 h. The other four women had no discernible reaction. In no subject was the pain or discomfort considerable enough to discontinue the investigation, and most women experienced a decrease in discomfort with each succeeding injection. No urticarial reactions were noted.
Discussion Nal-Glu GnRH antagonist (50 Mg/kg, im) administered to midfollicular phase women maximally reduced mean
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serum levels of immunoreactive LH and FSH by 62.2% and 45.3%, respectively. The rapid attenuation of LH release after administration of Nal-Glu antagonist was accompanied by a 35% decline in LH pulse frequency and a 56% fall in LH pulse amplitude. These data corroborate similar studies performed in normal cycling women given Nal-Glu antagonist in the early follicular phase (13) and normal men in whom the Nal-Glu antagonist (5 mg, sc) induced a 25% and 56% decline in LH pulse frequency and amplitude, respectively (15). The Nal-Glu antagonist-induced gonadotropin suppression during the midfollicular phase was maintained throughout the 3 treatment days, with a corresponding decrease in serum E2 and i-INH levels. Previous studies using the same GnRH antagonist in postmenopausal women demonstrated 53% and 21% decreases in serum immunoreactive LH and FSH, respectively (12). The partial decrease in serum gonadotropin levels would suggest either an incomplete blockade of pituitary GnRH receptors by Nal-Glu antagonist or non-GnRHdependent gonadotropin secretion. The percent decline in FSH secretion was less than that in LH secretion in all subjects. This is consistent with all previous studies using GnRH antagonists and implies a significant nonGnRH-dependent control mechanism for FSH secretion. Activin, a /3-/3 dimer of inhibin subunits, has been shown to stimulate FSH secretion from pituitary cells in culture, even after GnRH desensitization (26, 27). Since information on circulating activin is not presently available, the possibility that this represents a significant regulator for FSH secretion remains speculative. After Nal-Glu antagonist, serum concentrations of FSH rebounded to levels significantly higher than either pretreatment or control cycle values. This FSH increment was associated with low serum levels of E2 and i-INH, which may be instrumental in the disinhibition of FSH release. When pretreatment levels of E2 and iINH were reached 2 days after discontinuation of GnRH antagonist treatment, levels of FSH declined to corresponding control cycle concentrations. Levels of E2 and i-INH fell very low after Nal-Glu antagonist administration and remained so throughout treatment. Recovery from Nal-Glu antagonist-imposed TREATMENT
MAL-OLU
FIG. 4. Pulsatile activity of LH and FSH in a subject 1 day before (BASELINE), during (TREATMENT) and 1 day after (RECOVERY) receiving Nal-Glu GnRH antagonist (50 /ig/kg, im) for 3 days in the midfollicular phase of the menstrual cycle. Asterisks indicate LH pulses. 0800
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KETTEL ET AL. 15
• ••
2 10 w
UJ CO
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I 2 Baseline
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FIG. 5. Mean (±SE) LH pulse frequency and amplitude in five women before (Baseline), during (Treatment), and after (Recovery) receiving Nal-Glu GnRH antagonist (50 Mg/kg, im). **, P < 0.01.
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GnRH receptor blockade was rapid, with gonadotropin, E2, and i-INH levels returning to pretreatment midfollicular concentrations within 48 h after the last injection. Thereafter, progressive increases in E2 and i-INH concentrations occurred, with midcycle surge and luteal phase hormonal profiles identical to those of control cycle studies and previous reports (28). The time required to achieve the subsequent LH surge was inversely correlated with pretreatment levels of E2, a finding consistent with the heterogeneous functional status of the follicles before Nal-Glu administration. It is apparent from the present study that there was functional arrest of the dominant follicle, in that the endocrine expression of the follicle was restored in a timely fashion after GnRH antagonist was discontinued. Ovarian hormones, E2 and i-INH, were suppressed to a much greater degree than gonadotropins, with levels reaching those in postmenopausal women. One explanation for this phenomenon is a greater decline in levels of bioactive gonadotropin after administration of the Nal-Glu GnRH antagonist. In studies using a shorter acting compound, the 4F-antagonist, in both men (29) and women (30), the bioactive to immunoreactive ratios of LH and FSH declined. Nal-Glu antagonist administration in postmenopausal women also induces a greater decrease in bioactive LH and FSH and reduces the bioactive/ratio ratio by 50% and 27%, respectively (12). In these studies both the degree and the duration of suppression were greater for bioactive gonadotropin. It is possible that the discrepancy between bio- and immunoactive LH and FSH is due to the inability of antibodies to recognize the biologically active portion of the glycoprotein molecules in the RIA system. Alternatively, a preferential decline in the secretion of biologically active glycoprotein isoform occurred after blockade of GnRH receptors (31). In summary, after an approximately 50% decrease in gonadotropin support to the dominant follicle for 3 days in the midfollicular phase of the menstrual cycle, arrest of ongoing follicular maturation occurred. However, the follicular apparatus retained its ability to reinitiate its original functionality once appropriate gonadotropin inputs were reinstated with subsequent cycle dynamics appropriate for the original follicular status. Our findings provide in vivo information on the robust nature of the developing follicle and its ability to retain its cellular endowments during gonadotropin deprivation.
12
-4 DAYS FROM LH PEAK
FIG. 6. Mean (±SE) daily serum LH, FSH, E2, P4, and i-INH during control cycle and after recovery from Nal-Glu GnRH antagonist (50 /xg/kg, im), given for 3 days in the midfollicular phase of the treatment cycle. Data are centered around the LH peak.
Acknowledgments The authors are grateful for the excellent technical assistance by the staff of the Clinical Research Center, Pam Malcom, Shannon Petze, and Jeff Wong, and to Dr. A. Lein for advice and assistance.
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FOLLICULAR-FOLLICULAR TRANSITION
References 1. Knobil E. The neuroendocrine control of the menstrual cycle. Recent Prog Horm Res. 1980;36:53-88. 2. Yen SSC, Tsai CC, Naftolin F, Vandenberg G, Ajabor L. Pulsatile patterns of gonadotropin release in subjects with and without ovarian function. J Clin Endocrinol Metab. 1972;34:671-5. 3. Reame N, Sauder SE, Kelch RP, Marshall JC. Pulsatile gonadotropin secretion during the human menstrual cycle: evidence for altered frequency of gonadotropin-releasing hormone secretion. J Clin Endocrinol Metab. 1984;59:328-37. 4. Filicori M, Santoro N, Merriam GR, Crowley WF. Characterization of the physiological pattern of episodic gonadotropin secretion throughout the human menstrual cycle. J Clin Endocrinol Metab. 1986;62:1136-44. 5. Kazer RR, Kessel B, Yen SSC. Circulating luteinizing hormone pulse frequency in women with polycystic ovary syndrome. J Clin Endocrinol Metab. 1987;65:233-6. 6. Berga SL, Mortola JF, Girton L, et al. Neuroendocrine aberrations in women with functional hypothalamic amenorrhea. J Clin Endocrinol Metab. 1989;68:301-7. 7. Loucks AB, Mortola JF, Girton L, Yen SSC. Alterations in hypothalamic-pituitary ovarian (HPO) and hypothalamic-pituitary adrenal (HPA) axes in athletic women. J Clin Endocrinol Metab. 1989;68:402-ll. 8. Soules MR, Clifton DK, Cohen NL, Bremner WJ, Steiner RA. Luteal phase deficiency: abnormal gonadotropin and progesterone secretion patterns. J Clin Endocrinol Metab. 1989;69:813-20. 9. diZerega GS, Hodgen GD. Folliculogenesis in the primate ovarian cycle. Endocr Rev. 1981;2:27-49. 10. Tonetta SA, diZerega GS. Intragonadal regulation of follicular maturation. Endocr Rev. 1989;10:205-29. 11. 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. 12. Mortola JF, Sathanandan M, Pavlou S, et al. Suppression of bioactive and immunoactive follicle stimulating hormone and luteinizing hormone levels by a potent gonadotropin-releasing hormone antagonist: pharmacodynamic studies. Fertil Steril. 1989;51:957-63. 13. Hall JE, Whitcomb RW, Rivier JE, Vale W, Crowley WF. Differential regulation of luteinizing hormone, follicle stimulating hormone, and free a-subunit secretion from the gonadotrope by gonadotropin-releasing hormone (GnRH): evidence from the use of two GnRH antagonists. J Clin Endocrinol Metab. 1990;70:328-35. 14. Roseff SJ, Bangah ML, Kettel LM, et al. Dynamic changes in circulating inhibin levels during the luteal-follicular transition of the human menstrual cycle. J Clin Endocrinol Metab. 1989;69:1033-9. 15. Pavlou SN, Wakefield G, Schlechter NL, et al. Mode of suppression of pituitary and gonadal function after acute or prolonged admin-
16. 17.
18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
28. 29.
30. 31.
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istration of a luteinizing hormone-releasing hormone antagonist in normal men. J Clin Endocrinol Metab. 1989;68:446-54. Yen SSC, Llerena 0, Little B, Pearson OH. Disappearance rates of endogenous luteinizing hormone and chorionic gonadotropin in man. J Clin Endocrinol Metab. 1968;28:1763-7. Yen SSC, Llerena LA, Pearson OH, Littell AS. Disappearance rates of endogenous follicle-stimulating hormone in serum following surgical hypophysectomy in man. J Clin Endocrinol Metab. 1970;30:325-9. Anderson DC, Hopper BR, Lasley BL, Yen SSC. A simple method for the assay of eight steroids in small volumes of plasma. Steroids. 1976;28:179-96. McLachlan RI, Robertson DM, Healy DL, Burger HG, deKretser DM. Circulating immunoreactive inhibin levels during the normal human menstrual cycle. J Clin Endocrinol Metab. 1987;65:954-61. Robertson DM, Tsonis CG, McLachlan RI, et al. Comparison of inhibin immunological and in vitro biological activities in human serum. J Clin Endocrinol Metab. 1988;67:438-43. Schneyer AL, Mason AJ, Burton LE, Ziegner JR, Crowley WF. Immunoreactive inhibin a-subunit in human serum: implications for radioimmunoassay. J Clin Endocrinol Metab. 1990;70:1208-12. Robertson DM, Giacometti M, Foulds LM, et al. Isolation of inhibin a-subunit precursor proteins from bovine follicular fluid. Endocrinology. 1989;125:2141-9. Robertson DM, Tsonis CG, McLachlan RI, et al. Comparison of inhibin immunological and in vitro biological activities in human serum. J Clin Endocrinol Metab. 1988;67:438-43. Veldhuis JD, Johnson ML. Cluster analysis: a simple, versatile, and robust algorithm for endocrine pulse detection. Am J Physiol. 1986;250:E486-93. Veldhuis JD, Johnson ML. A novel general biophysical model for simulating episodic endocrine gland signaling. Am J Physiol. 1988;255:E749-59. Ying S. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev. 1988;9:267-93. Schwall RH, Sxonyi E, Mason AJ, Nikolics K. Activin stimulates secretion of follicle-stimulating hormone from pituitary cells desensitized to gonadotropin-releasing hormone. Biochem Biophys Res Commun. 1988;151:1099-104. McLachlan RI, Robertson DM, Healy DL, Burger HG, deKretser DM. Circulating immunoreactive inhibin levels during the normal human menstrual cycle. J Clin Endocrinol Metab. 1987;65:954-61. Dahl KD, Pavlou SN, Kovacs WH, Hsueh AJW. The changing ratio of serum bioactive to immunoreactive follicle stimulating hormone in normal men following treatment with a potent gonadotropin releasing hormone antagonist. J Clin Endocrinol Metab. 1986;63:792. Kessel B, Dahl KD, Kazer RR, et al. The dependency of bioactive FSH on gonadotropin releasing hormone in hypogonadal and cycling women. J Clin Endocrinol Metab. 1988;66:361-6. Dahl KD, Bicsak TA, Hsueh AJW. Naturally occurring antihormones: secretion of FSH antagonists by women treated with a GnRH analogue. Science. 1988;239:72-4.
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