Hormonal and biochemical changes during treatment of endometriosis with the luteinizing hormone-releasing

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1 FERTILITY AND STERILITY Copyright" 199 The American Fertility Society Vol. 54, No.2, August 199 Printed on acid-free paper in U.S.A. Hormonal and biochemical changes during treatment of endometriosis with the luteinizing hormone-releasing hormone (LH-RH) agonist [D-Trp6,des-Gly-NH21 ]LH-RH ethylamide Andre Dupont, M.D., Ph.D.*t:!: Pierre Dupont, M.D. Alain Belanger, Ph.D.:!: Jacques Mailoux, M.D. Leonello Cusan, M.D., Ph.D.*:!: Fernand Labrie, M.D., Ph.D.*:!: Centre Hospitalier de L'Universite Laval (CHUL) Research Center, Quebec, Quebec, Canada The effect of medical oophorectomy induced by treatment with the luteinizing hormone-releasing hormone (LH-RH) agonist [D-Trp6,des-Gly-NHlO]LH-RH ethylamide was studied in 34 patients with laparoscopically proven endometriosis. Tamoxifen was administered during the 1st month of therapy to prevent flare-up of the disease during the estrogen surge. Fifteen women had a decrease of their laparoscopy scores translated into an improvement in the stage of disease, whereas in 12 others, the decrease in their scores was not enough to allow a change of disease stage. The 2nd laparoscopy was not performed in 7 women. Medical oophorectomy, after daily injection of the LH -RH agonist (LH -RH -a), was accompanied by low levels of circulating estradiol. The serum concentration of all.:1 4-3-ketosteroids was significantly decreased during medical oophorectomy, whereas the level of circulating.:1 6-3fJ-hydroxysteroids was not altered except for pregnen- lone. The present data indicate that medical oophorectomy induced by an LH-RH-a in association with tamoxifen is a very efficient and well tolerated therapy in endometriosis. Fertil Steril54:227, 199 Endometriosis is the 2nd most frequent gynecological cal menopause in women as well as in experimental disorder after leiomyoma. In 198, 97, animals. 3 Similar observations were reported hospital discharges for females of 15 to 44 years of age indicate endometriosis as first diagnosis. 1 The prevalence of endometriosis has been estimated to be 3% to 5% in infertile couples.2 Since endometrial tissue requires estrogens for its growth and proliferation, a state of hypoestrogenism results in atrophy and regression of endometriosis as observed after either natural or surgiwith androgens: methyltestosterone was partially effective in relieving dysmenorrhea and abdominal pain, but at high dosages, important masculinizing signs appeared. In addition, ovulation was not consistently inhibited, and treatment with androgens raised the possibility of virilization of the female fetus or genitourinary teratogenicity.2 A widely used drug in the treatment of endometriosis, namely danazol, possesses androgenic, anabolic, Received December 4, 1989; revised and accepted April 3, 199. progestational, and antiestrogenic properties. 4 Recently, it has been demonstrated that the administration of danazol increases serum-free and total * Clinical Endocrine Research Unit. t Reprint requests: Andre Dupont, M.D., Ph.D., Laboratory androgen levels, and it has been suggested that its of Molecular Endocrinology, CHUL Research Center, 275, Laurier Boulevard, Quebec G 1 V 4G2, Quebec, Canada. clinical efficacy might be explained by increased :j: Laboratory of Molecular Endocrinology. levels offree testosterone (T).5 Department of Gynecology. After our observations that chronic administra- Vol. 54, No.2, August 199 Dupont et al. LH-RH-a in endometriosis 227

2 tion of luteinizing hormone-releasing hormone agonists (LH-RH-a) led to an inhibition oftesticular and ovarian functions characterized by a loss of LH receptors6,7 and blockage of steroidogenesiss,9 in male and female rats, a similar loss of ovarian gonadotropin functions in women was also observed.1 The effect of LH -RH -a as medical oophorectomy in the treatment of endometriosis has been the subject of several studies. However, most of the time, measurement of steroids has been limited to estradiol (E 2 ) and progesterone (p).n-13 The present study was designed to assess in detail the changes of ovarian and adrenal steroids during the 9 months of LH -RH -a therapy and the 6 months of recovery after cessation of therapy. MATERIALS AND METHODS Beginning in June 1986, 34 women with laparoscopically proven endometriosis were entered into the study after written informed consent. All patients had endometriosis staged according to the revised American Fertility Society criteria14 within 3 months before enrollment in the study. Average age at entry in the trial was 27 (19 to 38 years), whereas the median was 26 years of age. Nineteen subjects were previously treated with danazol, 4 others had received oral contraceptive pills, and 11 were previously untreated. None of the women had received danazol or sex steroids within 3 months before starting LH -RH -a treatment. Complete clinical, gynecological, biochemical, radiological, and endocrinological evaluations (including laparoscopy for diagnosis and staging of endometriosis) according to the revised American Fertility Society criteria14 were performed before therapy and after 9 months in a week of arrest of therapy. Among the 37 women, 5 decided on their own to stop therapy, and 2 refused the second laparoscopy after completion of treatment-in spite of clinical improvement. Pretreatment evaluation as well as clinical followup included gynecological history, recording of pelvic and low back pain, dysmenorrhea, dyspareunia, and secondary effects. Blood withdrawal was performed in the morning of midluteal or late luteal phases before treatment and thereafter at.5, 1, 2, 3, 4, 6, 9, 12, and 15 months of therapy. Ovarian and adrenal steroids were determined by a radioimmunoassay (RIA) after separation on LH-2 columns as previously described.15,16 Pituitary hormones were measured by RIA as described. 1 A negative Ii-subunit human chorionic gonado- Table 1 Staging of Endometriosis in Patients Before and After 9 Months of LH-RH-a Therapy No. of patients Stage 1st Laparoscopy 2nd Laparoscopy 4 (14.8) I 3 (11.1)" 12 (44.4) II 17 (62.9) 6 (22.2) III 2 (7.4) () IV 5 (18.5) 5 (18.5) Excluded b 7 " Values in parentheses are percents. b Two women declined 2nd laparoscopy, and five opted to stop therapy. tropin was required at study entry. Clinicallaboratory tests before, during, and after treatment included complete blood count, biochemistry analysis, and urinalysis. For preventing disease flare-up, patients received tamoxifen 1 mg orally, every 12 hours, during the 1st month of therapy with the LH-RH-a [D-Trp6, des-gly-nh2 1 ]LH-RH ethylamide. The LH -RH -a was injected subcutaneously at a daily dose of 5 f.lg in the morning for 1 month followed by 25 f.lg/d for 8 months. Statistical Analysis Radioimmunoassay data were analyzed using a program based on model II of Rodbard and Lewald.17 Univariate analysis of repeated-measured was performed for comparing hormonal data before, during, and after LH-RH-a therapy.1s Results are presented as means ± standard error of the mean (SEM). RESULTS To evaluate the success of therapy, only data from subjects having had the two laparoscopies before and after treatment were used (Table 1). Regression of endometriotic implants is quantitated by the laparoscopic score determined at the time of the second laparoscopy. From a total of 27 subjects, 15 women had a decrease of laparoscopic score translated into a change in stage of the disease. Among the 4 women in whom endometriotic lesions completely disappeared after LH -RH-a treatment, 3 were previously classified as stage II and 1 stage I. On the other hand, 9 patients determined as having stage II before therapy became stage I. Among the 2 women having stage III disease, 1 became stage I, whereas the other was classified as stage II at 9 months of therapy. In the re- 228 Dupont et ai. LH-RH-a in endometriosis Fertility and Sterility

3 Table 2 Effects of a 9-Month Therapy With an LH -RH -a on Serum Concentration of Gonadotropins and Steroids Before, During, Months LH FSH P 17-HP 17.6 ± ± ± ± ± ± ± ± ± ±.4.6± ± ±.8 7. ±.6.67 ± ± ±.7 8.7±.5.75 ± ± ±.7 8.8± ± ± ± ±.5.8± ± ± ± ± ± ± ± ± ± ± ± ± ±.27 a 4 -dione T E2 El Pregnenolone 3.29±.5.9 ±.1 483± ± ± ± ±.17 1,718 ± ± ± ±.4.68 ±.Q7 94± ± ± ± ±.9 61± 8 16± ± ±.37.55±.7 75± 1 158± ± ± ±.8 75± 9 137± ± ± ±.6 74± 8 127± ± ± ±.11 75± 1 197± ± ± ±.8 163± 66 26± ± ±.54.68± ± ± ±.34 maining 12 women, 1 at stage I, 5 at stage II, and 5 at stage IV, the laparoscopic score decrease was not sufficient to permit a change of disease stage. With regard to baseline clinical symptoms, in respectively 8%,73%,5%, and 75% of the patients, low back pain, pelvic pain, dyspareunia, and dysmenorrhea were present. The 4 symptoms of endometriosis were rapidly and markedly improved during the course of treatment. Low back pain, pelvic pain, and dyspareunia were respectively at 8%, 23%, and 18% after only 2 months of treatment. The number of patients with severe and very severe symptoms fell quickly during the first 15 days of treatment in spite of the increase in serum estrogen levels. However, mild pelvic pain remained in about 3% of the women until the 6th month of treatment. As adverse effects, hot flushes were predominantly seen followed by the decrease of libido and vaginal dryness. No patient had to be withdrawn from the study because of liver intolerance or other side effects. After a transient rise in serum E2 and estrone (E 1) during the first 15 days of LH-RH-a therapy, the blood concentration of both steroids decreased to levels similar to those found after oophorectomy and promptly resumed after termination of LH RH -a administration, reaching a normal level 6 months later (Table 2). The serum concentration of all ~4-3-ketosteroids, P, 17a-hydroxyprogesterone (17-HP), androstenedione (~4-dione), and T significatively (P <.5) decreased during treatment (Fig. 1). After cessation oflh-rh-a administration, blood levels of P and ~4-dione returned to the concentrations measured before treatment, whereas T was at 77% of its basal level, and 17- OHP remained low (Fig. 1). No significant changes in serum concentrations of three of the 3/3-hydroxy-5-ene-steroids measured (17 -hydroxypregnenolone, dehydroepiandrosterone [DHEA], and androst-5-ene-3/3,17/3-diol [5-diol]) have been observed during medical oophorectomy (Table 2). However, it is interesting to notice that the blood levels of pregnenolone and a 3/3-hydroxy- 5-ene-steroid remained partially inhibited after termination of therapy. Serum levels of the 5a-reduced androgens dihydrotestosterone, (DHT) (P <.8), androstane- 3a,17/3-diol (P <.7), and androstane-3/3,17/3-diol (P <.1) were slightly diminished throughout the treatment and post-therapy periods. It is interesting to notice that DHT, the main metabolite of T, was still at 65% of its basal level after 6 months of recovery. Moreover, an early decrease in androsterone and 3a-diol levels occurred during the first 2 months of therapy. However, the serum levels of androsterone resumed to pretreatment values at 3 months, whereas androstane-3a,17/3-diol serum 12 1 w 8 ~- w~ 1;;15 6 we 8~ 4 a: Q. 2 o +-,..--,--r---r--r--,---,--, w ~ w I;;::r 8~ a: Z Q.- l: g TIME (months) 4 2 L 1.5 OOO+-"'--'--r-r~~"'--' Figure 1 Serum P, 17-HP, a 4 -dione, and T concentrations before, during, and after a LH-RH-a. Data are means ± SEM. - Vol. 54, No.2, August 199 Dupont et al. LH-RH-a in endometriosis 229

4 and After Treatment (n = 27 patients) 17-H- Androstane- Androstane- Pregnenolone DHEA 5-diol DHT A 3a-17(j-diol 3(j, 17 (j-diol Cortisol Aldosterone 6.52 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.11 levels remained slightly decreased during both treatment and recovery periods (Fig. 2 and Table 2). The serum concentrations of aldosterone and cortisol displayed a similar pattern (Table 2): a rise followed (P <.5) by a return to normal levels, a phenomenon that could be explained by the stimulatory effect of estrogens on transcortin and aldosterone-binding globulin levels. Although not significant, a slight rise in cortisol and aldosterone can be seen after the arrest of therapy. An inverse relationship between LH and folliclestimulating hormone (FSH) secretion is suggested by a 7% (P <.5) inhibition of FSH secretion observed concurrently with a 6% increase in the serum levels of LH after daily injection of an LH RH-a in association with tamoxifen (Table 2). After 2 months of therapy, mean serum FSH levels first increased, then decreased and remained at 4% of the starting blood concentration, whereas 4. w 3. \It z ffi2!iia oe ",;. z «2. 1..! t f t 3. -' g 2.5 ~ 2. wo ~? 1.5 t ze! t ~o;. 1. V'' '".5 z « TIME (months) Figure 2 Serum androsterone and androstane-3a,17(j-diol concentrations before, during, and after an LH-RH-a treatment as described in Figure 1. serum LH levels displayed only a 33% decrease, pointing out the lack of reliability of LH measurement as a parameter of medical ovariectomy. Serum PRL showed a tendency to a slight and transient decrease of serum concentration between months 3 and 9. DISCUSSION The endocrine changes induced by the LH -RHa [D-Trp6,des-Gly-NH 2 1O]LH-RH ethylamide therapy suppressed ovulation and menstrual cycles and improved endometriosis scores at laparoscopy as well as clinical symptoms in most patients. It should be noted that none of the women deteriorated under therapy. Similar results on the efficacy of LH -RH -a on endometriosis have been reported ll - 13 and agree well with the regression of endometriotical lesions observed in our study. The association of tamoxifen to an LH -RH -a during the 1st month of therapy successfully prevents any clinical disease flare, as indicated by the prompt relief of clinical symptoms in spite of the estrogen surge. The concomitant fall of serum FSH and rise in serum LH observed after 15 days of daily administration of the LH-RH-a in association with tamoxifen could be related to changes in the circulating concentration of ovarian steroids. 19 It is of interest to recall that in the treatment of endometriosis, intranasal administration of buserelin acetate started in the early follicular phase of the menstrual cycle has been found to suppress FSH release after 14 days oftherapy, whereas LH secretion was still increased. 2 In fact, Gaspard 21 reported that serum FSH and LH levels, respectively, reached their peaks of maximum secretion during the 1st and the 4th week after the beginning 23 Dupont et al. LH-RH-a in endometriosis Fertility and Sterility

5 of subcutaneous injection of an LH-RH-a. Estradiol rather than circulating gonadotropins should be used as the index of pituitary-ovarian suppression, since immunoreactive LH and FSH concentrations do not accurately reflect the suppression of bioactive gonadotropins during treatment with LH -RH analogs.22 The most interesting changes of ovarian secretion are at day 15 of treatment where both E2 and E2 are reaching a peak, whereas the serum levels of Ll4-3-ketosteroids, P, 17-HP, androstenedione, and T are decreasing. The transient rise in serum estrogens is presumably initiated by an acceleration of 3,8-hydroxysteroid dehydrogenase (3,8- HSD) activity-knowing that this enzymatic activity can be enhanced by LH release23-whereas a fall of Ll4-3-ketosteroids is probably indicative of a deceleration of 3,8-HSD activity, evidence that paracrine/autocrine affects locally produced steroids.24 In fact, 3,8-HSD activity is primarily stimulated as reflected by the rise of serum estrogens, which, in turn, could inhibit 3,8-HSD activity, thus leading to decreased blood levels of the Ll4-3-ketosteroids. With regard to the early decrease of androsterone and androstane-3a,17,8-diol circulating levels and the inhibition of FSH release consecutive to chronic subcutaneous injection of LH -RH -a, it is interesting to recall that serum FSH in rats appears to modulate 5a-reductase activity present in granulosa cells.25 Additional evidence for an inhibitory effect of FSH on this enzyme system in humans is the low rate of conversion of the 5a-reduced androgens into C-19 steroid glucuronides (the usual pathway for their elimination of androgens).26 In summary, E2 more than El appears to be a fairly reliable parameter to evaluate the ovarian function under suppression therapy with an LH-RH-a. Serum concentrations of 17-HP remained unexpected by (low) 6 months after arrest of ovarian suppression therapy, whereas the serum levels of the three other Ll4-3-ketosteroids have or were about to reach their basal levels. REFERENCES 1. 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6 Symposium on Research Methods in Reproductive Endocrinology, Edited by E Diczfalusy. Copenhagen, Bogtrykleriet Forum, 197, p Freund RJ, Littell RC, Spector PC: SAS System for Linear Models, Cary, NC, SAS Institute Inc., 1986, p Labrie F, Drouin J, Ferland L, Lagace L, Beaulieu M, De Lean A, Kelly PA, Caron MG, Raymond V: Mechanism of action of hypothalamic hormones in the anterior pituitary gland and specific modulation of their activity by sex steroids and thyroid hormones. Recent Prog Horm Res 34:25, Franssen AMHW, Kauer FM, Chadha DR, Zijlstra JA, Rolland R: Endometriosis: treatment with gonadotropinreleasing hormone agonist Buserelin. Fertil Steril 51:41, Gaspard J: Traitement de l'endometriose par des analogues agonistes de la gonadoliberine (LH-RH). Rev Med Liege, XLI, 1986, p St-Arnaud R, Lachance R, Dupont A, Labrie F: Serum luteinizing hormone (LH) biological activity in castrated pa- tients with cancer of the prostate receiving a pure antiandrogen and in estrogen-pretreated patients treated with LH-releasing hormone agonist and antiandrogen. J Clin Endocrinol Metab 63:297, Veldhuis JD, Klase PA, Sandow BA, Kolp LA: Progesterone secretion by highly differentiated human granulosa cells isolated from preovulatory graafian follicles induced by exogenous gonadotropins and human chorionic gonadotropin. J Clin Endocrinol Metab 57:87, Tonetta SA, De Vinna RS, dizerega GS: Thecal cell 3-beta hydroxysteroid dehydrogenase activity: modulation by human chorionic gonadotropin, progesterone, estradiol-17ft and dihydrotestosterone. J Steroid Biochem 28:77, Goldring NB, Orly J: Concerted metabolism of steroid hormones produced by cocultured ovarian cell types. J BioI Chem 26:913, Prevost J, Belanger A: Formation of estrogen glucuronides by human granulosa-luteal cells isolated from human menopausal gonadotropin-stimulated cycles for in vitro fertilization. BioI Reprod 38:975, Dupont et al. LH-RH-a in endometriosis Fertility and Sterility