FERTILITY AND STERILITY
Vol. 52, No.4, Octoher 1989
Printed on acid-free paper in US.A.
Copyright ('J 1989 The American Fertility Society
Effects of gonadotropin-releasing hormone (GnRH) agonist on pituitary and ovarian responses to pulsatile GnRH therapy in polycystic ovarian disease*t Eric S. Surrey, M.D.:j:II§ Dominique de Ziegler, M.D.lI John K. H. Lu, Ph.D. II
R. Jeffrey Chang, M.D.# Howard L. Judd, M.D. II
Cedars-Sinai Medical Center and University of California, Los Angeles, California
Nine clomiphene citrate-resistant polycystic ovarian disease (PCOD) patients received intravenous gonadotropin-releasing hormone (GnRH) pulses before and immediately after 1 month of GnRH agonist (GnRH-a) therapy. Circulating gonadotropin and ovarian steroid levels, as well as follicular development, were measured throughout therapy.ResuIts were compared with those obtained from five hypogonadotropic patients treated with GnRH pulses only who ovulated during six of seven treatment cycles. Only two PCOD patients ovulated normally with GnRH pulses before GnRH-a therapy. Aberrant gonadotropin and ovarian steroid secretory patterns were noted in the others. After GnRH -a, gonadotropin and ovarian steroid hormone levels were similar to those of the hypogonadotropic patients. Subsequent secretory responses to GnRH pulses were partially normalized. However, only two additional PCOD patients ovulated. Fertil Steril 52: 547,1989
The pulsatile, intravenous administration of gonadotropin-releasing hormone (GnRH) is a highly successful method of inducing ovulation in patients suffering from amenorrhea secondary to hypothalamic dysfunction. 1 ,2 Use of pulsatile GnRH in patients with polycystic ovarian disease (peOD) Received November 28, 1988; revised and accepted June 1, 1989. * Presented in part at the 34th Annual Meeting of the Society for Gynecologic Investigation, Atlanta, Georgia, March 18 to 21,1987. t Supported in part by a grant from the Ortho Pharmaceutical Company, Raritan, New Jersey, and by grant RR00865 from the United States Public Health Service, Bethesda, Maryland. Cedars-Sinai Medical Center. § Reprint requests: Eric S. Surrey, M.D., Cedars-Sinai Medical Center, 444 South San Vicente Boulevard, Suite 1102, Los Angeles, California 90048. II Department of Obstetrics and Gynecology, University of California, Los Angeles, California. 11 Present address: Department of Obstetrics and Gynecology, Maternite Hospital A. Beciere, Clamart, France. # Present address: Department of Obstetrics and Gynecology, School of Medicine, University of California, Davis, Davis, California.
has resulted in less encouraging results, characterized by inconsistent ovulatory patterns and low pregnancy rates. 3 ,4 In an attempt to identify the mechanisms underlying this discrepancy, we have examined the pituitary and subsequent ovarian responses to chronic pulsatile GnRH both in patients with peOD and hypothalamic amenorrhea. We have attempted to improve responses by reproducing the hypoestrogenic and hypogonadotropic state of hypothalamic amenorrhea with the administration of a highly potent agonist of GnRH (GnRH-a) to peOD patients for 1 month before reinstitution of pulsatile GnRH therapy.
*
Vol. 52, No.4, October 1989
MATERIALS AND METHODS Subjects
Two groups of subjects were studied. The fi-rBt group consisted of nine patients with peOD, all of whom were attempting pregnancy and were selected based on classical signs of hirsutism and chronic anovulation. All had failed clomiphene ciSurrey et al.
Agonist effect on GnRH pulse therapy
547
trate therapy. The second group consisted of five anovulatory patients with hypothalamic amenorrhea based on long-standing amenorrhea, low levels of gonadotropins, absence of radiological evidence of a pituitary lesion, and failure to respond with withdrawal bleeding to a progesterone (P) challenge test. Three were attempting pregnancy. Serum prolactin levels were normal in all patients. The mean ± standard error (SE) measurements of body mass index were similar in the groups of patients with PCOD (22.8 ± 7.4 [SE] kg/m 2) and those with hypothalamic amenorrhea (21.0 ± 1.5 kg/m 2). Each subject desiring pregnancy had undergone a thorough infertility evaluation that was normal with the exception of the ovulatory defects. None of the patients had received exogenous hormone for 30 days before entry into the study.
chemical criteria: (1) collapse of a follicle that had demonstrated progressive growth to a minimum mean diameter of 18 mm,5 (2) a preceding rise of serum-luteinizing hormone (LH) levels to >30 mIU/mL, and (3) subsequent sustained rise in serum P levels to a minimum of 3.0 ng/mL. Blood samples were obtained from all patients before the onset of each phase of pulsatile therapy and every 1 to 3 days thereafter. Serum levels of LH, follicle-stimulating hormone (FSH), estrone (E l ), estradiol (E 2), androstenedione (A), testosterone (T), P, and dehydroepiandrosterone (DHEA) were measured by radioimmunoassay (RIA) techniques, which have been previously described. 6,7 Statistical evaluations were performed by Wilcoxon's sign-rank sum test and Student's paired and grouped t-tests where appropriate.
Protocol
RESULTS
Informed consent, as approved by the University Human Subjects Protection Committee, was obtained from each patient before entry into the study. Patients with hypothalamic amenorrhea were administered intravenous (IV) pulses of GnRH (Factrel; Ayerst Laboratories, New York, NY) 5 J-Lg every 90 minutes throughout seven treatment cycles. Gonadotropin-releasing hormone was diluted in normal saline to which Heparin Sodium (Upjohn Company, Kalamazoo, MI), 10 U/mL, was added and then infused through a No. 21-gauge intravenous catheter with a small, portable, programmable pump (Pulsamat; Fering Laboratories, Ridgewood, NJ). The infusion was continued for a minimum of 21 days or for 14 days after ovulation had been documented. The nine subjects with PCOD were treated similarly for one cycle each. Those patients who failed to demonstrate sonographic evidence of normal follicular development and collapse were administered GnRH-a, [D-His6(imBzl), Pro9-NET]GnRH (Histrelin; Ortho Pharmaceuticals, Co., Raritan, NJ), 100 J-Lg self-injected subcutaneously daily at 8:00 A.M. for 30 days. They subsequently underwent a second course of pulsatile intravenous GnRH therapy as described above, commencing the day after the last dosage of agonist. All patients underwent pelvic ultrasonography at the onset of GnRH pulse therapy and at regular intervals thereafter to monitor follicular growth (Model RT 3000, 3.5-MHz vaginal probe; General Electric Company, Milwaukee, WI). Ovulation was defined as follows by both sonographic and bio548
Surrey et al.
Agonist effect on GnRH pulse therapy
Baseline Hormone Concentrations
The mean ± SE levels of gonadotropins and sex steroids measured in the patients with hypothalamic amenorrhea and PCOD (both before and after GnRH-a administration) are demonstrated in Table 1. In the PCOD patients, baseline levels of LH, E l , E 2, and T were significantly higher than those observed in women with hypothalamic amenorrhea. The ratio ofE l to E2 was reversed in PCOD. These endocrine findings were similar to those reported previously and supported the respective clinical diagnoses of PCOD and hypothalamic amenorrhea in our patient groups.l,6,8,9 With the exception of LH, the hormone levels in PCOD patients were similar to baseline values found in patients with hypothalamic amenorrhea after 1 month of GnRH-a (Table 1). There was no significant change in DHEA levels, indicating the absence of an effect of GnRH -a on adrenal androgen secretion. Clinical Responses
Based on biochemical and sonographic criteria, ovulation occurred in six ofthe seven cycles studied in the patients with hypothalamic amenorrhea. Of the three patients attempting to become pregnant, two conceived. The one patient who failed to ovulate responded successfully to pulsatile therapy using a dose of 10 J-Lg in a subsequent cycle, which was beyond the confines ofthis study. Before agonist administration, only two PCOD Fertility and Sterility
Table 1
Mean ± SE Baseline Hormone Concentrations pi
Tk
Patient group
LHc
FSH'
mIU/mL
mIU/ml
pg/mL
pg/mL
ng/mL
pg/mL
pg/mL
ng/mL
Hypothalamic amenorrhea PCODapre-GnRH-a b PCOD post-GnRH-a
7±0.8 43 ± 8.3 d 43 ± 3.9
8± 3.2 15 ± 1.8 7±0.8
16 ± 1.8 71 ± 0.3 d 32 ± 6.7 8
8±2.2 52 ± 6A d 17 ± 5.8 8
0.3± 0.03 0.7±0.2 0.3 ± 0.04 8
729 ± 143 1,548 ± 347 633 ± 121 g
216 ± 39 457 ± 60 d 205 ± 35 8
5.6 ± 0.7 4.7 ± 0.8 4.2 ±0.8
E/
PCOD, polycystic ovarian disease. GnRH-a, gonadotropin-releasing hormone agonist. C LH, luteinizing hormone. d p < 0.05 versus hypothalamic amenorrhea. e FSH, follicle-stimulating hormone. fE" estrone. a b
patients (peOn 3 and 6) apparently ovulated with pulse therapy, based on both biochemical and sonographic criteria, but without conception. Four others (peOn 4, 5, 7, and 8) had rises of serum P >3 ng/mL, suggestive of luteinization, which occurred in the absence of sonographic evidence of adequate follicular development. Two other patients (peOn 1 and 9) failed to respond adequately by any criterion. The last (peOn 2) developed an ovarian cyst (55 mm) after 8 days of GnRH pulses, and therapy was discontinued. The seven peon patients who failed the initial cycle were treated with a second course of GnRH pulses after 1 month of GnRH -a administration. Only two (peOn 2 and 7) apparently ovulated according to ultrasonic and biochemical criteria. Two additional patients (peOn 4 and 5) manifested evidence of premature luteinization with P rises >3 ng/mL without substantial follicular growth. Two patients failed to respond at all (peOn 1 and 8). The final patient (peOn 9) developed a 2-cm follicular cyst after 20 days of pulse therapy, which then collapsed without subsequent elevation of serum P levels above baseline. Hormonal Response Patterns to Pulsatile GnRH
E2h
8
Vol. 52, No.4, October 1989
DHEAI
P < 0.05 versus PCOD pre-GnRH-a.
E 2 , estradiol. P, progesterone. j A, androstenedione. k T, testosterone. I DHEA, dehydroepiandrosterone. h i
did not respond clinically (peOn 1 and 9) (Fig. 1). Gonadotropin secretion was characterized by a minimal initial elevation of FSH but a progressive and sustained rise of LH for 17 days. The ovarian response to this gonadotropin release was limited to minimal rises of E2 and androgen levels, and no evidence of follicular development, ovulation, or luteinization. A second pattern was seen in the four other patients who also apparently did not ovulate, according to sonographic criteria (peOn 4, 5, 7 and 8) (Fig. 2). This response was characterized by early, short-lived, gonadotropin rises associated with abnormally rapid increases in E2 (from 25 to 460 pg/ mL by the fifth day of therapy). nespite minimal follicular growth as measured by ultrasound, an early rise of P suggestive of luteinization commenced on day 11 of GnRH therapy and was sustained for 12 days thereafter. A third pattern was observed in two patients I GnRH pulses
FSH m I U/ml
The hypothalamic amenorrhea patients who ovulated during therapy displayed the expected pituitary and ovarian responses to GnRH pulses that have been reported by others. 1,2,9 These were characterized by an early rise ofFSH, subsequent elevation of E2 with adequate progressive follicular development, a midcycle LH surge, follicular collapse, and a sustained increase and subsequent decline of P. In contrast, several aberrant patterns of response to pulsatile GnRH were noted in the peon patients before the use of GnRH -a. The first pattern was observed in two of the peon patients who
Ai
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pg Iml
1200
4000
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800
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400
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pg/ml 0--0
1000
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9
13
17
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Figure 1 PCOD 1. Levels of gonadotropins, estrogens, P, and androgens during GnRH treatment cycle before GnRH-a. Note progressive disparity between LH and FSH levels and absence of P rise. No follicular growth was noted by ultrasonography. Surrey et al.
Agonist effect on GnRH pulse therapy
549
p
nO/ml
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~
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12
16
20
24
,
28
DAYS
Figure 2 PCOD 5. Gonadotropin and ovarian sex steroid levels in GnRH cycle before GnRH -a. Note early dramatic rise and subsequent decline of E2 with associated P rise. No follicular development or collapse was noted by ultrasonography.
(PCOD 3 and 6) (Fig. 3). During the first 5 days of GnRH therapy, dramatic but short-term rises of FSH, LH, E 1 , and E2 were again observed, but without concomitant evidence of either follicular development by ultrasound or premature luteinization as assessed by a rise in serum P levels. Subsequently, gonadotropin levels rose again, followed by a more orderly rise of E 2 • This time, sonographic evidence of adequate follicular development and collapse was observed. The absence of a demonstrable LH surge may have reflected the sampling interval used. A sustained luteal phase elevation of P levels ensued. In the four patients who apparently ovulated after agonist therapy (PCOD 2, 4, 5 and 7), more orderly rises of FSH were observed, followed by slow but progressive increases in estrogen and androgen concentrations, culminating in midcycle LH surges and subsequent rises of P. Of the remaining three patients, two demonstrated endocrine patterns that were essentially unchanged from their initial cycles. The third manifested persistent pituitary and ovarian suppression despite reinstitution of GnRH pulses.
lamic amenorrhea treated with GnRH pulses alone. U sing a standardized protocol for GnRH pulse administration, we were consistently able to achieve orderly follicular development, ovulation, and pregnancy in patients with hypothalamic amenorrhea. In contrast, administration of GnRH pulses to women with PCOD resulted in inconsistent and aberrant responses of the pituitary and ovaries. Although the number of patients studied was relatively small, analysis of each of these patterns may give some insight into the pathophysiology of the disorder. The pattern of gonadotropin secretion elicited by GnRH pulses displayed in Figure 1 was characterized by exaggerated LH and minimal or undetectable FSH release. The dichotomy between baseline LH and FSH levels was exaggerated as therapy progressed, culminating in an absence of folliculogenesis. This phenomenon has been previously described in PCOD patients and attributed to either a primary defect in the hypothalamic-pituitary axis or to disordered feedback effects of abnormal ovarian steroid levels on gonadotropin release.lO The other two patterns of hormone secretion observed in the PCOD patients appeared to reflect primarily abnormal ovarian responses to gonadotropin release induced by GnRH pulses (Figs. 2 and 3). The initial pituitary responses were characterized by only minimal early rises in FSH but draI GnR H pulses
LH mlU/ml
FSH mlU/ml
Ej
_
E2~
pg Iml
pg/ml
DISCUSSION
550
Surrey et al.
Agonist effect on GnRH pulse therapy
200
I
100_
T
In this study, we have attempted to characterize pituitary and subsequent ovarian responses to chronic intravenous pulsatile GnRH therapy in patients with PCOD before and after gonadotropin suppression with GnRH-a and to compare these results with those observed in women with hypotha-
300 400f
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200
OJ
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It o I
2000
'
,
,
9
13
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,
,
17
21.
25
29
33
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O'lulolion DAYS
Figure 3 PCOD 6. Serum gonadotropins and steroid levels in GnRH cycle before GnRH-a. Note early dramatic rise and fall of E2 with subsequent and delayed ovulation on day 22.
Fertility and Sterility
matic and rapid elevations in E2 and E 1. These estrogen rises were not accompanied by corresponding elevations of circulating androgens. Thus it is unlikely that this increase of estrogen could be attributed to peripheral aromatization, but rather, must have represented the direct stimulatory effect of FSH on granulosa cell aromatase. With continued administration of pulsatile GnRH in these PCOD patients, one of two ovarian responses ensued: (1) inadequate folliculogenesis with premature luteinization (Fig. 2) or (2) arrest of folliculogenesis until a second cohort of follicles could be recruited, resulting in a delayed ovulatory event (Fig. 3). These ovarian responses are consistent with the hypothesis that at the onset of GnRH pulse therapy, some follicles within the polycystic ovary were not in a resting state, but rather, were in varying states of maturity as a result of disordered gonadotropin secretion. Thus minimal increments of FSH were able to trigger large premature elevations in E2 with subsequent premature LH release and inhibition of FSH release. Therefore, based on our previously published data, an effort was made to enhance the ovulatory responses of PCOD patients to pulsatile GnRH by attempting to re-create the quiescent hypogonadotropic and hypoestrogenic state of hypothalamic amenorrhea by administration of GnRH-a for 1 month.s,ll Baseline levels ofE1, E 2, A, and T in the PCOD patients were dramatically lowered after GnRH-a to concentrations similar to those obtained in patients with hypothalamic amenorrhea. The persistent elevation of immunoreactive LH likely represented artifact in the form of biologically inactive a-subunits, as has been suggested by Meldrum et a1. 12 Some normalization of pituitary and ovarian secretory responses to the reinstitution of GnRH pulse therapy was noted in the PCOD patients after GnRH-a. The enhanced secretion of LH in comparison with FSH (Fig. 1) was not seen. The early dramatic elevations of E2 (Fig. 2) were eliminated in all but one PCOD patient. The incidence of "premature luteinization" was also reduced (40% versus 71%). These data suggested that the aberrant pituitary and ovarian responses demonstrated by these PCOD patients may have been the result of chronically elevated ovarian steroid secretion-a phenomenon rectified by GnRH-a. However, one cannot completely eliminate the possibility that GnRH-a therapy may have corrected abnormal endogenous GnRH pulse patterns as well. Despite the improved endocrine responses Vol. 52, No.4, October 1989
achieved after GnRH -a in the small sample of PCOD patients described in the present study, it is important to note that a normal ovulatory event occurred in only two additional patients who had not previously responded to GnRH pulses. This is not a significant difference. These findings confirm those of Willemsen and co-workers,13 who were only able to achieve ovulation with GnRH stimulation in 20% of patients after GnRH-a in an uncontrolled study. In contrast, Filicori et aI,14 achieved a high ovulation rate with pulsatile GnRH therapy both before and after GnRH-a in a group of six PCOD patients. It is possible that longer suppression of the pituitary-ovarian axis with GnRH-a will result in more typical ovarian responses to GnRH pulse therapy in PCOD patients. However, failure to improve on ovulatory responses in PCOD after suppression of the hypothalamic-pituitary-ovarian axis has been achieved may reflect the presence of an inherent ovarian defect. This issue remains unresolved. Acknowledgments. Histrelin was generously provided by Ortho Pharmaceutical Corporation. Factrel was generously provided by the Ayerst'Laboratories. Reagents for the LH and FSH assays were provided by the National Pituitary Agency, National Institutes of Health. Special thanks to the nurses of the Clinical Research Center for their excellent care, to Ms. Laurie Levine for her typographical skills, and to Ms. Elisa Obnial for her laboratory technical assistance.
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gonadotropins, estrogens, and androgens in polycystic ovarian disease. Am J Obstet GynecolI21:496, 1975 Anderson DC, Hopper BR, Lasley BL, Yen SSC: A simple method for the assay of eight steroids in small volumes of plasma. Steroids 28:179, 1976 Chang RJ, Laufer LR, Meldrum DR, de Fazio J, Lu JKH, Vale WW, Rivier JE, Judd HL: Steroid secretion in polycystic ovarian disease after ovarian suppression by a longacting gonadotropin-releasing hormone agonist. J Clin Endocrinol & Metab 56:897,1983 Valk TW, Corley KP, Kelch RP, Marshall JC: Hypogonadotropic hypogonadism: hormonal responses to low dose pulsatile administration of gonadotropin-releasing hormone. J Clin Endocrinol & Metab 51:730, 1980 RebarR,JuddHL, YenSSC, RakoffJ, VandenburgG,Naftolin F: Characterization of the inappropriate gonadotropin secretion in polycystic ovary syndrome. J Clin Invest 57: 1320,1976 Steingold K, de Ziegler D, Cedars M, Meldrum DR, Lu JKH, Judd HL, Chang RJ: Clinical and hormonal effects
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of chronic gonadotropin-releasing hormone agonist treatment in polycystic ovarian disease. J Clin Endocrinol & Metab 65:773, 1987 12. Meldrum DR, Tsao Z, Monroe SE, Braunstein GD, Sladek J, Lu JKH, Vale W, Rivier J, Judd HL, Chang RJ: Stimulation of LH fragments with reduced bioactivity following GnRH agonist administration in women. J Clin Endocrinol & Metab 58:755, 1984 13. Willemsen WNP, Fremssen AMHLO, Rolland R, Verner HM: The effects of Buserelin on the hormonal states in PCOD. In Gonadotropin Down-Regulation in Gynecologic Practice: Proceedings of an International Symposium held at the University of Nijmegen, The Netherlands, April 25 and 26,1986, Edited by R Rolland, DR Chadha, WNP Willemsen. New York, Alan Liss, 1986, p 377 14. Filicori M, Campaniello E, Michelacci L, Pareschi A, Ferrari P, BoleHi G, Flamigni C: Gonadotropin-releasing hormone (GnRH) analog suppression renders polycystic ovarian disease patients more susceptible to ovulation induction with pulsatile GnRH. J Clin Endocrinol & Metab 66: 327, 1988
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