Testicular histology following chronic gonadotropin-releasing hormone agonist treatment

Transcription

1 FERTILITY AND STERILITY Copyright 1984 The American Fertility Society Printed in U.8A. Testicular histology following chronic gonadotropin-releasing hormone agonist treatment Jacob Rajfer, M.D.*t Ronald S. Swerdloff, M.D.:j: David M. Heber, M.D., Ph.D.:j: University of California, Los Angeles (UCLA) School of Medicine, and Harbor/UCLA Medical Center, Torrance, California The histologic appearance of the testes of men exposed to chronic gonadotropinreleasing hormone agonist (GnRH-A) therapy has not been previously documented. Herein, we report on the histologic features of the testes of four patients with disseminated prostatic carcinoma who received at least 1 year of daily treatment with (D-Leu 6, des-gly-nh/o, proethylamide 9 )-GnRH (leuprolide, Abbott Laboratories, North Chicago, IL) for their disease, and subsequently underwent bilateral orchiectomy. In marked contrast to the testes from five control patients, the testes of these agonist-treated patients demonstrated absence of spermatogenesis, Leydig cell hypoplasia, and Leydig cell inactivity. These data provide direct histologic evidence that the chronic administration of GnRH agonists may be suitable as a potential male contraceptive. Fertil Steril 42:765, 1984 Superactive analogs of gonadotropin-releasing hormone (GnRH) are presently being utilized in the treatment of a variety of endocrine disorders because of their ability to paradoxically suppress testosterone (T) production to castrate levels. I - 3 In men, it is believed that these GnRH agonists (GnRH-A) have their effect directly on the pituitary gland, which results in the suppression of luteinizing hormone (LH) and follicle-stimulating hormone (FSH); and this, in turn, results in the suppression of T production by the testes. 4 Whereas testis histology after GnRH analog treatment of subhuman species has been published, the morphologic changes in the human Received February 27, 1984; revised and accepted July 11, *Division of Urology, Department of Surgery. treprint requests: Jacob Rajfer, M.D., Box 5, Harbor/UCLA Medical Center, 1000 W. Carson Street, Torrance, California :j:division of Endocrinology, Department of Medicine. testis as a result of chronic exposure to these agents have not been previously documented. This report examines the testicular histologic features of four patients subjected to at least 1 year of daily treatment with 1 mg of (D-Leu 6, des-gly NH 2 1O, proethylamide 9 )-GnRH for disseminated prostatic cancer. For comparison, the testicular histologic features were evaluated in five patients who were orchiectomized during the same time period as their GnRH-A-treated cohorts for the same disease but had not had any prior hormonal therapy. MATERIALS AND METHODS Five previously untreated patients and four patients who received subcutaneously 1 mg of (D Leu 6, des-gly-nh/o, proethylamide 9 )-GnRH (leuprolide, Abbott Laboratories, North Chicago, IL) daily for at least 1 year for metastatic prostatic carcinoma underwent bilateral orchiectomy for progression of their metastatic disease. All the Rajfer et ai. GnRH agonists and testis histology 765

2 Table 1. Plasma LH, FSH, and T in GnRH-A Patients Patient LH Day 0 Day 180 Day 0 FSH Day 180 Day 0 Day 180 T mlulml < mlulml nglml < agonist-treated patients had initially achieved a clinical response to therapy associated with decreased serum T concentrations. Despite continued treatment and persistence of suppressed T concentrations at castrate levels, the tumors of these individuals underwent secondary growth and escape from therapy. The average age of the five control patients was 79 years (range, 67 to 87), whereas for the four GnRH-A-treated patients it was 71 years (range, 64 to 83). Plasma FSH, LH, and T (Table 1) were measured at day 0 and day 180 of therapy by radioimmunoassay as previously described. 5, 6 All four GnRH-A-treated patients were known to have castrate levels of plasma T at day 180 of therapy (Table 1),4 and all four achieved a clinical remission. Portions of the testicular tissue were placed in either 10% formalin or Bouin's solution. In addition, one testis of one control subject and the testis of one GnRH-A-treated patient was placed in 2.5% glutaraldehyde for electron microscopy. For light microscopy, the tissues were stained with hematoxylin and eosin and/or toluidine blue. Electron microscopy was performed in the laboratory of Dr. Luciano Zamboni by embedding the tissues in Epon and staining them with uranyl acetate. Representative sections were taken from each testis. RESULTS The testes of four of the five control patients with advanced prostatic carcinoma whose ages averaged 79 years demonstrated an adequate number of germinal cells in all stages of maturation from spermatogonia to mature spermatozoa (Figs. 1 to 3). When examined, spermatozoa were also seen in the epididymides of these four control patients (Fig. 4). In the fifth control patient (82 years of age), spermatogenesis was present up to the spermatocyte stage, but no sperm were seen within the lumina of the seminiferous tubules (Fig. 5). The Sertoli cells appeared normal in all five control patients. In the interstitium, there appeared to be an adequate number of Leydig cells in all five control patients. By electron mi- Figure 1 Histologic appearance of a testis from a control patient (76 years of age) demonstrating all stages of spermatogenesis, including spermatozoa (x 250). Figure 2 Histologic appearance of a testis from a control patient (67 years of age) demonstrating spermatozoa in the lumen of the seminiferous tubules. Leydig cells (arrows) are present in adequate numbers (x 250). 766 Rajfer et ai. GnRH agonists and testis histology Fertility and Sterility

3 Figure 3 Histologic appearance of a testis from a control patient (87 years of age) demonstrating sperm in seminiferous tubules. Leydig cells (arrow) are present in adequate numbers (x 250). Figure 4 Epididymis of a control patient demonstrating the presence of spermatozoa (x 250). croscopy, these Leydig cells possess normal subcellular characteristics such as Reinke cystalloids and normal smooth and rough endoplasmic reticulum (Fig. 6). As expected in a testis of this age, there was some interstitial fibrosis. All testes of the four GnRH-A-treated patients demonstrated that the seminiferous tubules were extremely fibrotic and atrophied (Figs. 7 to 10). Occasionally, a germ cell was seen, but most of the cells within the tubules were Sertoli cells. No spermatozoa were ever seen in the testes or epididymides of any of the GnRH-A-treated patients. In these agonist-treated patients, the Leydig cells were decreased in number and frequently showed signs of degeneration. In addition, the mitochondria of these Leydig cells appeared atypical in shape, and there was also a conspicuous absence of Reinke crystalloids and endoplasmic reticulum (Fig. 11). Grossly and microscopically, the testes of the analog-treated patients did not demonstrate any testicular infarcts. croscope. They show a conspicuous absence of both smooth and rough endoplasmic reticulum, which is the subcellular apparatus that is necessary for steroid production. Although morphometric analyses were not conducted in this study, and our population sample was small, the Leydig cells of the agonist-treated patients viewed by electron microscopy nevertheless appeared somewhat smaller in size than those of our control patients, as if understimulated (by LH). In the seminiferous tubules of the analog-treated patients, there is a conspicuous absence of any stage of spermatogenesis, particularly when compared with the control patients. The testes of four of the five control patients contained mature spermatozoa both in the seminiferous tubules and DISCUSSION The histologic appearance of the testes of men subjected to the chronic administration of GnRH A parallel their endocrine profile. For example, the administration of these agents on a chronic basis can induce a hypogonadotropic hypogonadal state,4 and the histologic appearance of the testes certainly is consistent with this hormonal state. First of all, the Leydig cells appear to be inactive, particularly when viewed under the electron mi- Figure 5 Histologic appearance of a testis of the fifth control patient (82 years of age) demonstrating spermatogenesis but no spermatozoa (x 250). Rajfer et ai. GnRH agonists and testis histology 767

4 Figure 6 Electron micrograph of adjoining Leydig cells in the control patient in Figure 1 demonstrating Reinke crystalloid (R) and lipid bodies (L). The smooth and rough endoplasmic reticulum can be seen throughout the cytoplasm. The characteristic nucleolus (No) of each cell can be seen (x 2000). epididymal lumina. In the fifth patient (32 years of age), there was evidence of spermatogenesis, but no sperm were seen. From our data to be published in a complementary manuscript, the intratesticular T levels in these five control patients averaged 95.9 ± 20.1 ng/gm testis, as compared with the four GnRH-A-treated patients (average age, 66 years), in whom the intratesticular T concentration was 6.1 ± 1.1 ng/gm testis (P < 0.001). It is our own assumption that the hypo- gonadal state induced by the GnRH-A is responsible for suppressing spermatogenesis in these agonist-treated patients. These findings in the aged human testis of these GnRH-A-treated patients appear somewhat discordant with those observed clinically in the GnRH-A-treated young, sexually active man and experimentally in the GnRH-A-treated mature male rat. In the latter species, sperm production was not completely suppressed, and Leydig cell size was not altered by GnRH-A treatment. 7 The differences in histologic appearance of the testes of GnRH-A-treated rats and those of man can be attributed to a number of factors, including species differences in the mechanisms by which GnRH-A suppress gonadal function. Nevertheless, the Leydig cells of the analog-treated rodents do demonstrate a number of similar cellular characteristics seen in the testes of our agonisttreated patients, such as an increase in lipid inclusions and atypical mitochondria. 7 In young adult men participating in clinical contraceptive studies using other GnRH-A, sperm production as measured by the sperm count does not appear to be completely suppressed. 8,9 This resultant oligospermia occurs in association with extremely low measurable levels of circulating T and may be explained by the fact that either the GnRH analog was not used for a sufficient period of time or the GnRH analog did not suppress the intratesticular concentration of T sufficiently enough to cause a total cessation of sperm production as found in our analog-treated patients. Alternatively, it is theoretically possible that our GnRH-A- Figure 7 (64 years of age) demonstrating Sertoli cells only (x 250). 768 Rajfer et al. GnRH agonists and testis histology Figure 8 (64 years of age) demonstrating absence of any stage of spermatogenesis (x 250). Fertility and Sterility

5 Figure 9 (83 years of age) demonstrating thickening of the tubular lumen and the presence of Sertoli cells only (x 250). Figure 10 (71 years of age) demonstrating thickening of the tubular wall and the presence of Sertoli cells only (x 250). treated patients with prostate cancer were severely oligospermic or azoospermic prior to the initiation of their GnRH treatment (as seen in one of our control patients) because pretreatment sperm counts were not performed in our study. However, three of the four agonist-treated patients had normal pretreatment basal FSH levels (Table 1), and this would suggest, although not unequivocally, that spermatogenesis was present in at least three of these four agonist-treated individuals. The fourth GnRH-A-treated patient had low pretreatment FSH and T levels, suggesting a possible preexisting hypogonadotropic state. In addition, all testes of our control patients, who averaged 79 years of age, demonstrated spermatogenesis; and it would be highly improbable that all four of the GnRH-A-treated patients had preexisting testicular disease sufficient to completely suppress spermatogenesis. Why sperm production in the aged testis and not the young adult testis is.suppressed following the chronic administration of these agents remains to be determined. Data from a number of laboratories, including our own, indicate that the mechanism of action of GnRH-A is more complex than originally believed. Suggested mechanisms of action include suppression of gonadotropin secretion,8-10 altered biologic activity of LH,l1 impaired pulsatile pattern of LH and FSH secretion,12 down-regulatory effects of transiently elevated LH and FSH levels/a and direct inhibitory effects of the GnRH-A at the gonadal level. 14, 15 It seems possible that different mechanisms may apply at different times during the treatment regimen. Despite the complexity of the problem, gonadotropin levels may be suppressed below baseline levels in GnRH-A-treated subjects.4 As a result, we wanted to compare our testicular findings with those from hypophysectomized patients. In surgically Figure 11 Electron micrograph of Leydig cells of a GnRH-A-treated patient demonstrating absence of Reinke crystalloids and endoplasmic reticulum. An increase in interstitial collagen (C) deposition and cytoplasmic lipid inclusions (arrows) can be seen (x 3200). Rajfer et al. GnRH agonists and testis histology 769

6 hypophysectomized patients, Mancini et al. 16 have categorized the testicular findings into three groups, depending on the severity of the testicular changes. In their patients with the most severe changes (group III), the testes demonstrated the presence of Sertoli cells, an occasional spermatogonium, and an occasional Leydig cell. The authors stated that a surgical hypophysectomy never totally removes the entire pituitary, and some gonadotroph cells may be left behind, a finding confirmed by other investigators. 17, 18 It has also been demonstrated that gonadotroph cells may also be found in the stalk of the pituitary gland.19 This part of the pituitary is not removed during a hypophysectomy. MacLeod et al. 20 reported that in a patient 100 days after hypophysectomy Sertoli cells and an occasional spermatogonial cell could be seen in the testis, but the Leydig cells were absent. In addition, peritubuiar thickening occurred. These findings by Mancini et al.16 and MacLeod et al. 20 are consistent with the assumption that some residual gonadotroph activity persists in the hypophysis of surgically hypophysectomized patients. Lostroh21 has shown in rats that LH antisera given to surgically hypophysectomized animals will produce more atrophy of the germinal epithelium than that seen by hypophysectomy alone. The histologic findings in our four patients are consistent with what one would expect with a hypophysectomy. Like LH antisera, these analogs have the potential for causing a complete chemical hypophysectomy with respect to gonadotropin activity. It has also been recorded that the testes of rodents exposed to chronic GnRH-A treatment may demonstrate testicular infarcts.22 In our four patients on the drug for over 1 year, this experimental finding in the rat was not corroborated in the human. It is known that in addition to its pituitary effects, GnRH-A have a direct effect on the testis of the rats but not on the human testis.23 It is possible that this difference in the mechanism of action of these agents between these two species may be important in the development of testicular infarcts in the rodent. In summary, the testes of men treated chronically with GnRH-A appear to parallel the endocrine profile, in that they appear inactive both in the seminiferous tubules and the interstitial compartment. The fact that spermatogenesis may be completely suppressed by the chronic use of these agents suggests that they deserve further study as a potential male contraceptive. 770 Rajfer et al. GnRH agonists and testis histology REFERENCES 1. Happ J, Scholz P, Weber T, Cordes U, Schramm P, Neubauer M, Beyer J: Gonadotropin secretion in eugonadotropic human males and postmenopausal females under long-term application of a potent analog of gonadotropinreleasing hormone. Fertil Steril 30:674, Smith R, Donald RA, Espiner EA, Stronach SG, Edwards IA: Normal adults and subjects with hypogonadotropic hypogonadism respond differently to D-SER(TBU)B-LH RH-EA'. J Clin Endocrinol Metab 48:167, Bergquist C, Nillius SJ, Bergh T, Skarin G, Wide L: Inhibitory effects on gonadotropin secretion and gonadal function in men during chronic treatment with a potent stimulatory luteinizing hormone-releasing hormone analogue. Acta Endocrinol (Copenh) 91:601, Rajfer J, Swerdloff RS, Heber D: The pituitary gland is the primary site of action of GnRH agonist (D-LeuB, des GlylO-GnRH ethylamide) in the male. Fertil Steril (Abstr) 38:277, Odell WD, Swerdloff RS: Radioimmunoassay of LH and FSH in human serum. In Radioisotopes in Medicine: In Vitro Studies, Edited by RL Hayes, FA Goswitz, DEP Murphy. AEC Symposium, Series 13 (Conference ), Oak Ridge, Tennessee, 1968, p Abraham GE: Handbook of Radioimmunoassay, Vol 5. New York, Marcel Dekker, Inc., 1977, p Irby D, Swerdloff RS, Mikos A, Kerr J, de Kretser D: Ultrastructural changes in Leydig cells after treatment with a gonadotropin-releasing hormone agonist. Submitted for publication 8. Linde R, Doelle GC, Alexander N, Kirchner F, Vale W, Rivier J, Rabin D: Reversible inhibition of testicular steroidogenesis and spermatogenesis by a potent gonadotropin-releasing hormone agonist in normal men. N Engl J Med 305:663, Swerdloff RS, Heber DM, Bhasin S: A new approach to male contraception using combined androgtm and GnRH analog treatment. Ann Intern Med. In press 10. Davies T, Gomez-Pan A, Watson MJ, Mountjoy CQ, Hanker JP, Besser GM, Hall R: Reduced gonadotropin response to releasing hormone after chronic administration to impotent men. Clin Endocrinol (Oxf) 6:213, Kelly S, Labrie F, Dupont A: Loss of LH bioactivity in men treated with an LHRH agonist. Presented at the Sixty-Fifth Annual Meeting of the Endocrine Society, June 8 to 10, 1983, San Antonio, Texas. Abstract McNeil LW, McKenna TJ, Lacroix A, Benveneste R, Rabin D: Seventy-two hour infusion of LHRH in normal men: gonadotropin and testicular steroid responses. J Clin Endocrinol Metab 49:149, Heber DM, Swerdloff RS: Down regulation of pituitary gonadotropin secretion in post menopausal females by continuous gonadotropin-releasing hormone administration. J Clin Endocrinol Metab 52:171, Hsueh AJW, Erickson GF: Extrapituitary inhibition of testicular function by LHRH. Nature 281:66, Evans RM, Doelle GC, Alexander AN, Uderman HD, Rabin D: Gonadotropin and steroid secretory patterns during chronic treatment with a luteinizing hormone-releasing hormone agonist analog in men. J Clin Endocrinol Metab 58:862, Mancini RE, Serguer AC, Lloret AP: The effect of gonadotropins on the testes of hypophysectomized patients. In Fertility and Sterility

7 Gonadotropins 1968, Edited by E Rosenberg. Los Altos, California, Geron-X, 1968, p Sternberger E, Duckett GE: The effect of estrogen or testosterone on initiation and maintenance of spermatogenesis in the rat. Endocrinology 76:1184, Kalra SP, Prasad MRN: Effect of FSH and testosterone propionate on spermatogenesis in immature rats treated with clomiphene. Endocrinology 81:965, Lachelin GC, Yen SSC, Alksne JF: H9rmonal changes following hypophysectomy in humans. Obstet Gynecol 50:333, MacLeod J, Pacianos A, Ray B: The restoration of human spermatogenesis and of the reproductive tract with urinary gonadotropins following hypophysectomy. Fertil Steril 17:7, Lostroh AJ: Regulation of FSH and ICSH (LH) of reproductive function in the immature rat. Endocrinology 85:438, Vickery B: Physiology and antifertility effects of LHRH and agonistic analogs in male animals. In LHRH Peptides as Female and Male Contraceptives, Edited by GI Zatuchni, JD Shelton, JJ Sciarra. Philadelphia, Harper & Row, 1982, p Sharpe RM, Fraser HM: Inhibition of maturational changes in Leydig cell function by treatment of rats with an agonist of LH-RH. J Reproc.l Fertil 60:359, 1980 Rajfer et al. GnRH agonists and testis histology 771