FERTILITY AND STERILITY Copyright" 1994 The American Fertility Society Printed on acid-free paper in U. S. A. The hypothalamic-pituitary-ovarian axis in patients with endometriosis is suppressed by leuprolide acetate but not by danazol* Masahiro Sakata, M.D.t Shiro Ohtsuka, M.D., Ph.D. Hirohisa Kurachi, M.D., Ph.D. Akira Miyake, M.D., Ph.D. Naoki Terakawa, M.D., Ph.D.* Osamu Tanizawa, M.D., Ph.D. Department of Obstetrics and Gynecology, Osaka University Medical School, Suita, Osaka, Japan Objective: To investigate the effects ofleuprolide acetate (LA), a GnRH agonist (GnRH-a), and of danazol on the hypothalamic-pituitary-ovarian axis in patients with endometriosis. Design: Ten patients were divided into LA and danazol treatment groups. Measures: Serum levels ofe2, immunoreactive and bioactive LH, pulsatility oflh, and gonadotropins release by GnRH. Changes in serum E2levels by hmg administration during LA treatment. Results: Serum E2 level decreased to near castrated levels during the LA treatment, while it remained unchanged during the danazol treatment. Leuprolide acetate administration resulted in a significant suppression of the serum level of bioactive LH, of the pulsatility of LH release, of the pituitary response to GnRH injection, and of the elevation in the serum E2 level by hmg administration, but danazol treatment did not show these suppressive effects. Conclusion: Our results suggest that the hormonal actions of LA and danazol on endometriosis are different from each other, especially in the suppression of serum E2 level. Fertil Steril 1994;61:432-7 Key Words: Endometriosis, leuprolide acetate, GnRH agonist, danazol, serum E2 level As many as 30% of women, who complain of dysmenorrhea and pelvic pain, have endometriosis, and >50% of women with unexplained infertility are diagnosed with endometriosis by laparoscopy. The incidence of endometriosis tends to be increasing (1). It is well known that ovarian estrogen and progestogen stimulate endometriosis and oophorectomy causes regression of the lesion (2). Therefore, one of the mechanisms of medical treatment Received June 21, 1993; revised and accepted October 29, 1993. * Presented in part at the 41th Japanese Congress of Gynecology and Obstetrics, Okayama, Japan, April 2 to 4,1989. t Reprint requests: Masahiro Sakata M.D., Department of Obstetrics and Gynecology, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka, Japan 565. :j: Present address: Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago, Tottori, Japan. 432 Sakata et al. Hormone action of LA and danazol for endometriosis depends on the suppression of ovarian steroidogenesis. Because it has been shown that GnRH agonist (GnRH-a) results in some physiological changes characteristic of medical castration (3), GnRH-a have been evaluated as a medical regimen for endometriosis. Gonadotropin-releasing hormone agonist induces desensitization of pituitary gland for GnRH and decreases the serum level of bioactive LH (4,5), resulting in the reduction of ovarian estrogen production. However, the detailed mechanism(s) of gonadal steroidogenesis suppression has not been fully elucidated. Danazol, a synthetic derivative of 17 a-ethynyltestosterone, has been widely used in the treatment of endometriosis (2). Although this compound is effective in treating the endometriosis (2), its pharmacological mechanism(s) is not completely elucidated. At first, danazol was introduced as an Fertility and Sterility
antigonadotropic agent (2), and its suppressive effect on gonadotropin secretion was considered to cause the reduction of ovarian steroidogenesis, followed by endometrial atrophy. However, its antigonadotropic effect has been questioned because the basal gonadotropin level was reported not to be altered (5). There are now several studies that suggest that danazol exerts its therapeutic action by inhibiting enzyme activities in the steroidogenesis (2) or by binding to multiple steroid hormone receptors in target tissues (2, 6), especially in the endometrium. In this study, we examined not only basal serum levels of E2, immunoreactive LH, and FSH but also the serum level of bioactive LH, the changes in the pulsatility of gonadotropin secretion, and the response of gonadotropins to GnRH during leuprolide acetate (LA, GnRH -a) and danazol treatments in 10 women with endometriosis. Changes in serum E2 levels after hmg administration was investigated before and during LA in four patients. tion, and the pituitary response to GnRH injection were studied at the early follicular phase of the pretreatment cycle and at the 12th week of both treatments. Pulsatility of LH secretion was examined in blood samples obtained at 15-minute interval for 4 hours (9:00 A.M. to 1:00 P.M.). A pulse was defined as an increase in LH concentration of :2:2 SD from a nadir (within 30 minutes before the peak) (7). To evaluate the effects of LA and danazol on the pituitary response to exogenous GnRH, 100 J.Lg of GnRH (gonadorelin acetate; Tanabe Seiyaku Company Limited, Tokyo, Japan) was injected IV and blood samples were taken for 2 hours. Human menopausal gonadotropin (150 IV; Organon, Oss, The Netherlands) was administered intramuscularly for 3 consecutive days to estimate the ovarian response before and during LA treatment (hmg test). Blood was obtained on 0,2, and 4 days of the hmg test to determine serum E2 concentrations by RIA. MATERIALS AND METHODS Subjects and Protocol Subjects in this study were 10 regularly menstruating women between 29 and 40 years of age. Endometriosis was diagnosed by laparoscopy in all subjects. Before the study, the guideline for the protocol of this study was approved by Osaka V niversity Medical School, and informed consent was obtained from all subjects. Ten women were divided into two groups. Five women were treated with SC injection of 3.75 mg of LA depot (Takeda Chemical Industry Limited, Osaka, Japan) within 3 days of menstrual period followed by injections once per 4 weeks for 24 weeks. The other five women were treated with danazol (200 mg by mouth, 2 times per day) (Tokyo Tanabe Company Limited, Tokyo, Japan), started on the 1st day of menstrual period and then continuously for :2:16 weeks. None of the patients received other hormonal medications for :2:6 months before and during the period of study. They recorded daily basal body temperature (BBT) during a pretreatment cycle and consecutive treatment period. The blood samples for basal serum levels of E2, LH, and FSH were taken at the early follicular phase of a pretreatment cycle, and every 2 or 4 weeks during the treatment. Blood samples were always taken before LA SC injection. The levels of bioactive LH, the pulsatile patterns of LH secre- Hormone Assays The sera were prepared from blood samples and frozen at -20 C until analyses ofe2, immunoreactive LH, bioactive LH, and FSH. Serum concentrations of E2, immunoreactive LH, and FSH were measured by RIA as described previously (8-10). The sensitivity of RIA of these hormones was as follows: E2, 3 pg per tube; LH, 0.2 miv Second International Reference Preparation of hcg (2nd IRP-hCG) per tube; and FSH, 0.1 miv 2nd IRPhMG per tube. The interassay and intra-assay coefficients of variation (CV) were 9.0% and 8.0% for E2, 10.3% and 8.3% for LH, and 9.6% and 9.0% for FSH, respectively. Serum bioactive LH was determined by measuring the T production in dispersed rat Leydig cells in vitro as described previously (9). Leydig cells were prepared from adult rat testes by collagenase digestion and suspended in Medium 199 containing 0.1 % bovine serum albumin (BSA). Aliquots of Leydig cell suspensions were incubated with various concentrations of standard LH (1st IRP LH code 68/ 40) or with sera at 37 C for 3 hours under 95% air and 5% CO2 with shaking. After the incubation, the mixtures were centrifuged at 1,000 X g for 15 minutes, and aliquots of supernatants were diluted with phosphate buffered saline (PBS) containing 1 % BSA for T RIA. The interassay and intra-assay CV of T assay were 9.4% and 9.0%, respectively. Bioactive LH in sera was assayed by measuring T Sakata et al. Hormone action of LA and danazol 433
production in comparison with that of a standard preparation. The interassay and intra-assay CV of bioactive LH were 14.0% and 8.0%, respectively. Statistical Analysis All values were expressed as means ± SE. Analysis of variance was performed for the statistical analysis if necessary, followed by Student's t-test. RESULTS All 10 patients remained anovulatory during the LA and danazol treatments. Their BBT was biphasic in the pretreatment cycle and monophasic during both treatments. Serum E2 levels markedly increased in three of five patients in the 2nd week of LA treatment. In the 4th week, serum E2 levels decreased to near castrated levels (22.9 ± 4.5 pg/ml) (conversion factor to SI unit, 3.671) in all patients, and remained at this level thereafter (Fig. la). All five women com- plained of hot flushes during the LA treatment. On the contrary, the serum E2levels in the five women remained unchanged during the danazol treatment (Fig. IB). None of them complained of hot flushes. During 24 weeks of the LA treatment, basal levels of serum LH and FSH remained unchanged in all patients (Fig. lc), in contrast to the marked reduction in serum levels of E2. Basal levels of immunoreactive LH and FSH also remained unchanged in all five patients during 20 weeks of danazol treatment (Fig. ID). Immunoreactive LH level at the 12th week of LA treatment (n = 5) was 12.5 ±1.9 miu/ml, which was slightly but not significantly lower than that in the pretreatment cycle (18.5 ± 3.2 miu/ml). However, the level of bioactive LH at the 12th week of LA treatment (7.7 ± 2.0 miu/ml) was significantly (P < 0.05) lower than that in the pretreatment cycle (22.4 ± 5.0 miu/ml). These findings suggested that LA treatment suppressed the level of bioactive LH but not the immunoreactive LH. Levels of A. 250 350 200 024 o 2 4 6 a '0 '2 '4 '6 '8 20 (w) D. 40 ~30..s 220 3 10 O~---------------------- ~ o 2 4 6 8 10 12 14 16 18 20 (w) ~30~ 3 20 ~.--.!10: ~~= J:...--=.~----. ~ OL- -- --.-~-- o 2 4 6 8 10 12 14 16 18 20 (w) -_40...J E 30 c. 1 ~20 -.~ "3,~f?=:.::: =- : ;;;; :;4l0 2 4..,. 20 24 (-J ~30 I~~~ o 2 4 8 12 16 20 24 (w) Figure 1 Serum levels of E2 before and during 24 weeks of LA (A) and 20 weeks of danazol (B) treatments (conversion factor to SI unit, 3.671) and of immunoreactive LH and FSH before and during 24 weeks of LA (C) and 20 weeks of danazol (D) treatments in each patient with endometriosis. Blood samples were obtained in the early follicular phase in a pretreatment cycle (0 week) and every 2 or 4 weeks during the treatments (n = 5 in each group). 434 Sakata et al. Hormone action of LA and danazol Fertility and Sterility
b!~~,~~ ~ r 1;,. ~-; ~:~~!~~ Figure 2 Pulsatile patterns of LH secretion in 4 patients before (left columns) and during (right columns, the 12th week) LA (A) and danazol (B) treatments. Blood samples were obtained at 15-minute intervals from 9:00 A.M. through 1:00 P.M. The pulse was defined as described in Materials and Methods.!3G' ~2 ~ -'\"iv~ '~3 3 1 Case 3... F~~ 1 _ ~ase4 JI:~C=:: o 0 60 120 180240 0 60 120 180 240(.) 13~l~!~!0Jl~ o 60 120 180 240 0 60 120 180 240 <-.) immunoreactive and bioactive LH at the 12th week of danazol treatment (n = 5) were 16.2 ± 2.4 and 32.0 ± 3.3 miu/ml, respectively, both of which were not significantly different from those in the pretreatment cycle (15.3 ± 2.4 and 28.2 ± 1.6 miui ml, respectively). The pulsatile patterns of pituitary LH release before and during LA treatment were studied in blood samples obtained at 15-minute interval for 4 hours. Figure 2A shows individual data obtained in four patients before and during LA treatment. Pulsatile secretion of LH (2.3 ± 0.3 pulses per 4 hours) in the pretreatment cycle disappeared at the 12th week of LA treatment. Contrary to these results in patients treated with LA, pulsatile secretion oflh (2.3 ± 0.3 pulses per 4 hours) observed in the pretreatment cycle were not altered at the 12th week of danazol treatment (Fig. 2B). The pituitary response to 100 Jig of GnRH, administered IV in the pretreatment and during LA treatment, is shown in Figure 3A. Gonadotropin release by 100 Jig of GnRH at the 12th week of LA treatment was markedly suppressed when compared with that in the pretreatment cycle. On the contrary, the gonadotropin release by 100 Jig of GnRH during danazol treatment was not significantly different from that in the pretreatment cycle (Fig.3B). Because LA, but not danazol, suppressed the serum E2 level during treatment, the ability of ovaries to secrete E2 in the patients before and during the LA treatment was evaluated by hmg test. Data from four patients before and at the 12th week of LA treatment showed that the increase in serum E2 levels in response to hmg during LA treatment A. 120 :; E '3 l 90 I 60 'i' en... B.120 :; E '3 l 90 :; 120 ~ ~ :::: 90 160 :t en... 120 :; ~! 90 160 :t en... I 'i'.j o'~------~------c-. 120 (m) o 30 60 30 60 0 30 60 Figure 3 Pituitary response to 100 /lg of GnRH administration in the pretreatment cycle (left), and during (right) LA (A) (n = 5) and danazol (B) (n = 4) treatments. Gonadotropin-releasing hormone (100 /lg) was administered, and blood samples were taken before and 30, 60, and 120 minutes after GnRH administration. Serum levels of LH and FSH were determined by RIA. Sakata et al. Hormone action of LA and danazol 435
were 42 ± 15 and 51 ± 12 pg/ml on days 2 and 4, respectively, which were significantly (P < 0.01) lower than those in a pretreatment cycle (258 ± 70 and 493 ± 129 pg/ml, respectively) (conversion factor to SI unit, 3.671). DISCUSSION Serum E2 levels are markedly reduced to near castrated levels during GnRH -a treatment, and it is suggested that the effects of GnRH -a on endometriosis are mainly through the reduction of E2 levels (8,11). Although suppression ofe2 is reported to be associated with the decrease in serum levels of immunoreactive LH (11), neither the serum levels of immunoreactive LH nor FSH significantly declined below the pretreatment levels in this study and in our previous study (8). These data suggested that suppressed serum E2 levels were not caused by the reduction in serum immunoreactive gonadotropins levels during GnRH -a treatment, and these results were consistent with the previous data in patients with prostate cancer (12). We have shown that the serum levels of bioactive LH at the 12th week of GnRH -a treatment were significantly lower than those in the pretreatment cycle. Similar data have been reported in women with endometriosis (8) and polycystic ovarian disease (PCOD) (4). Ovarian follicular maturation requires pulsatile gonadotropin secretion (8). Our results suggest that the absence of LH pulsatility may be a cause to decrease the serum E2 levels during LA treatment in addition to the reduction in bioactive LH levels. The fact that LH release in response to GnRH was reduced during LA treatment may explain the elimination of the LH pulsatility. Another possible mechanism by which GnRH-a suppresses estrogen production is a direct inhibition of ovarian steroidogenesis; it has been reported that GnRH -a directly decreases ovarian LH receptor level (13) and that GnRH-a impairs steroidogenesis in cultured human granulosa cells (14). In this study, we found that hmg-induced estrogen secretion was reduced during LA treatment. These results were different from those of our previous report (8) that hmg-induced estrogen secretion was not reduced during buserelin treatment. Other reports showed that the ovarian response to exogenous gonadotropins was reduced in patients receiving GnRH-a (15, 16). The discrepancy in the effects of LA and buserelin on the hmg test may result from the fact that LA (SC injection of depot formula) is more potent than buserelin (nasal ad- ministration) to inhibit ovarian steroidogenesis by gonadotropins. All these results suggest that LA has potent inhibitory effects on the hypothalamic-pituitary-ovarian axis. Serum E2 levels during danazol treatment are reported to increase (17), to decrease (18), or not to change (19, 20). In our results, danazol did not change the serum E2 nor serum gonadotropin levels in consistent with the previous reports (5). In addition, we have demonstrated that danazol administration did not change the serum levels ofbioactive LH. The result in this study that pituitary gonadotropin secretion by GnRH administration was not altered during danazol treatment was consistent with the previous report (19). Our result, that the LH pulses did not change before and during danazol administration (400 mg/d), was not consistent with a previous one (21) that the mean frequency of LH pulses decreased. However, they treated the patients with 800 mg/d of danazol, and the different dosage of danazol may explain the inconsistency of their data with ours. All of our data suggest that danazol has very little effect on the hypothalamic-pituitary-ovarian axis. It is also known that danazol administration directly induces endometrial atrophy in 4 weeks without decreasing the serum E2 level (22). Finally, these data suggest that the mechanisms of action of GnRH -a and danazol on endometriosis are different from each other. Moreover, it is reported that combination of LA and danazol has more profound effect on the regression of the experimental endometriosis tissue than LA or danazol alone in the rat (10). These considerations suggest that the combined therapy of GnRH -a and danazol may have more efficient therapeutic potency in patients with endometriosis. REFERENCES 1. Houston DE. Evidence for the risk of pelvic endometriosis by age, race and socioeconomic status. Epidemiol Rev 1984;6:167-91. 2. Barbieri RL, Ryan KJ. Danazol: endocrine pharmacology and therapeutic application. Am J Obstet Gynecol 1981;141:453-65. 3. Lemay A, Quesnel G. Potential new treatment of endometriosis: reversible inhibition of pituitary-ovarian function by chronic intranasal administration of a luteinizing hormone-releasing hormone (LH-RH) agonist. Fertil Steril 1982;38:376-9. 4. Meldrum DR, Tsao Z, Monroe SE, Braunstein GD, Sladek J, Lu JKH, et al. Stimulation of LH fragments with reduced bioactivity following GnRH agonist administration in women. J Clin Endocrinol Metab 1984;58:755-7. 436 Sakata et al. Hormone action of LA and danazol Fertility and Sterility
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