Vol. 30. No.4. October 1978 Prinf£d in U.S.A.
FERTILITY AND STERILITY Copyright' 1978 The American Fertility Society
CLINICAL STUDIES WITH D-TRp6-LUTEINIZING HORMONE-RELEASING HORMONE IN MEN WITH HYPOGONADOTROPIC HYPOGONADISM*
CARLOS JARAMILLO JARAMILLO, M.D.t ANICETO L. CHARRO-SALGADO, M.D.t VffiGILIO PEREZ-INFANTE, M.D.t ELENA BORDIU OBANZA, M.D.t FRANCISCO CANO IGLESIAS, M.D.:!: ARTURO FERNANDEZ-CRUZ, M.D.t DAVID H. COY, PH.D.§ ANDREW V. SCHALLY, PH.D.§' Servicio de Metabolismo y Nutrici6n, 1" Cdtedra de Patologia General, and Servicio de Endocrinologia, Cdtedra de Patologia Medica, Hospital Clinico, Facultad de Medicina, Universidad Complutense, Madrid, Spain, and Veterans Administration Hospital and Tulane Uniuersity School of Medicine, New Orleans, Louisiana 70146
Three male patients, diagnosed to have hypogonadotopic hypogonadism, were treated with intramuscular injections of 10 f.Ilf of D-Trp 6-luteinizing hormone-releasing hormone (D-Trp 6-LH-RH) every 8 hours for more than 90 days (13 to 17 weeks). The gonadotropin and testosterone concentrations reached levels twice as great during the treatment as those measured previously and on one occasion increased 5-fold. Before treatment, the patients were clinically in grade 1 on Tanner's scale (Growth at Adolescence, Second Edition. Oxford, Blackwell Scientific Publications, 1962) but after treatment they advanced to grades 2 to 4. Testicular sizes before treatment were between 2 and 2 according to the scale ofWaaler et al. (Acta Paediatr Scand [SupplJ 249:1, 1974) and afterwards between 4 and 10. The penis ofone ofthe patients grew 3 cm. The testicular biopsy after treatment showed significant increases in the number of Leydig and Sertoli cells. After treatment, spermatogonia were present in increased number, together with a much higher population of cells corresponding to additional stages of development (spermatids). We are of the opinion that therapy with D-Trp 6-LH-RH may be beneficial for such patients. Fertil Steril30:430, 1978 Received August 17, 1977; revised April 24, 1978; accepted May 22, 1978. *Supported by a grant from Patronato de Investigaciones Endocrino-Metabolicas, Fundacion General Mediterninea, Madrid, Spain. t Servicio de Metabolismo y Nutricion, 1" Catedra de Patologia General, Hospital Clinico, Madrid, Spain. :j:Servicio de Endocrinologia, Catedra de Patologia Medica, Hospital Clinico, Madrid, Spain. §Veterans Administration Hospital and Tulane University School of Medicine, New Orleans, La. "Reprint requests: Dr. Andrew V. Schally, Endocrine and Polypeptide Laboratory, Veterans Administration Hospital, 1601 Perdido Street, New Orleans, La. 70146.
Mortimer et al. l demonstrated that the prolonged administration of LH-RH (500 ILg three times per day) was effective in patients with clinical hypogonadism, resulting in maintenance of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion, elevation of plasma testosterone levels, and restoration oflibido, potency, and induction of spermatogenesis. In a recent study2 we demonstrated that relatively small doses ofD-Trp 6-LH-releasing hormone (D-Trp 6LH-RH) increased gonadotropin release in normal men. Since Heller and Clermont 3 demon430
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D-TRP"-LH-RH IN HYPOGONADAL MEN
strated that the duration of spermatogenesis in humans is at least 74 ± 4 days, we decided to administer the analog over a period of at least 90 days and to study its effects on the germinal and endocrine functions of the testes in three men with hypogonadotropic hypogonadism. MATERIALS AND METHODS
Three subjects with delayed puberty who were genetically XY, with a normal sella turcica by x-ray examination and normal electroencephalograms, were referred for hormonal studies to the metabolic unit by the hospital's clinical endocrinology division. All were duly informed that they were going to be subjected to a period of study while being treated with n-Trp6-LH-RH, and all gave their informed consent. Patient 1. Patient 1 was a 19-year-old male measuring 154 cm and weighing 44 kg. He was eunuchoid in appearance and lacked pubic hair, libido, erections, and pollutions, corresponding to grade Ion Tanner's scale. 4 Testicular sizes on the scale of Waaler et al. 5 were right <2 and left 2 years on the same scale. He had a bone age of 13 years. He had received 5000 IU of human chorionic gonadotropin (HCG) every 3 days for a 6-month period during the previous year. Patient 2. Patient 2 was a 20-year-old male measuring 182 cm and weighing 85 kg. He was anosmic and lacked the following sexual characteristics: beard, moustache, axillary and pubic hair, libido, erections, and pollutions, corresponding to grade 1 on Tanner's scale. 4 Testicular sizes (scale of Waaler et al. 5) were right 2 and left 2 with identical corresponding ages on the same scale. He had a bone age of 13 years. He had received 5000 IU ofHCG every 3 days for a 2-year period 3 years previously. Patient 3. Patient 3 was a 22-year-old male measuring 163 cm and weighing 65 kg. He was eunuchoid in appearance and lacked the following sexual characteristics: beard, moustache, axillary and pubic hair, libido, erections, and pollutions, corresponding to grade 1 on Tanner's scale. 4 Testicular sizes (scale of Waaler et al. 5) were right 2 and left 2, both corresponding to age 2 on the same scale. He had a bone age of 14 years and had been treated with 5000 IU of HCG every 3 days for a period of 6 months 1 year previously. LH and FSH levels were undetectable by radioimmunoassay in all three of the patients, and all three had plasma testosterone levels ranging from 60 to 70 ng/lOO ml. Basal levels of growth hormone, prolactin, thyroid-stimulating
hormone, adrenocorticotropic hormone, T 3 , T 4 , and cortisol were in the normal range. All three patients received 100 mg of clomiphene citrate daily for a 5-day period; in none of the patients was there any detectable change in LH and FSH levels following the administration of clomiphene citrate. One week later, each patient received 10 fJ-g of n-Trp 6LH-RH in a single intravenous bolus injection. The next day, treatment with intramuscular injections of the analog (10 fJ-g every 8 hours) was started. Patient 1 was treated for 15 weeks, patient 2 for 17 weeks, and patient 3 for 13 weeks . Serum samples were obtained before testing (basal values), on the 5th day folloW. ng the administration of clomiphene citrate, on the day of the intravenous n-Trp6-LH-RH injection (0, 30, 60, 120, 180, 240, 360, 480 minutes following injection), and once weekly at 9 A.M. for the length of the treatment cycle, immediately before the next scheduled intramuscular injection. n-Trp6-LH-RH (supplied by Ayerst Laboratories, New York, N. Y.) was dissolved in sterile saline before use. Neither during nor after treatment did any of the patients manifest adverse effects. LH and FSH levels were measured by doubleantibody radioimmunoassay with kits provided by the National Institutes of Health, Bethesda, Md. All samples were measured in duplicate. The results were expressed as mill i-international units per milliliter of serum using as a standard the LER-907 reference preparation. In this assay, the minimal detectable concentrations are 0.8 mIU/ml for LH and 0.6 mIU/ml for FSH. The coefficient of intra-assay variation was less than 5% and that for the interassay variation was less than 15% for both FSH and LH.6, 7 Testosterone levels were measured by a double-antibody radioimmunoassay,S using kits prepared by Serono Laboratories. The minimal detectable dose is 12.5 ng/lOO ml, the intra-assay variation was less than 12%, and that between assays was less than 20%. A testicular biopsy specimen was obtained from each patient before and after treatment. RESULTS
Analytic Data In response to the lO-fJ-g intravenous injection of n-Trp6-LH-RH, gonadotropin levels increased and remained elevated for at least 8 hours (Fig. 1).
JARAMILLO JARAMILLO ET AL.
LH 10 JIll O-TRP'-LH-RH mlU/ml.
10 JIll O-TRpl_LH-RH
B 30 60
120 180 240
B 3060 120
FIG. 1. LH and FSH serum levels after the intravenous injection of 10 J.Lg of D_Trp6_ LH-RH .... --e, Patient 1; 0 - 0 , Patient 2; ~ .... ~, patient 3. The shaded area represents the response to the analog in normal men.
The LH response in all three patients was inferior to that observed ina control group of four men ofthe same age as the patients. The smallest response was found in the anosmic patient (patient 2). The FSH response was also found to be reduced in patients 1 and 2, whereas it was increased in patient 3. By the end of the 1st week of the intramuscular treatment cycle, gonadotropin and testosterone levels had risen 2-fold and the following weekly extractions revealed only minor fluctuations from the levels obtained on the 8th day of the treatment (see Table 1).
Clinical Data Treatment with the analog resulted in the following changes in the three patients: Patient 1. Patient 1 grew 3 cm in height and 4 kg in weight, and developed normal appearance and sexual characteristics such as axillary and pubic hair, libido, erections, and nocturnal pollutions. His penis grew 3 cm. He was classified in grade 4 on Tanner's scale. 4 The testicular sizes on
the scale ofWaaler et a1. 5 were 8 right and left 10, with corresponding ages of 13 years right and 15 years left. Patient 2. Patient 2 developed axillary, pubic, leg, and arm hair. He moved to grade 2/3 on Tanner's scale 4 ; the testicular sizes on the scale of Waaler er a1. 5 were 10 right and 6 left with corresponding ages of 14 and 12, respectively. Patient 3. Patient 3 developed axillary and pubic hair and moved up to grade 2 on Tanner's scale. 4 The testicular sizes on the scale of Waaler et al. 5 were 6 right and 4 left, corresponding to ages of 12 and 11, respectively. The clinical and analytic data before and after treatment with D-Trp 6-LH-RH are summarized in Table 1.
Histologic Data The biopsies are summarized in Table 2. According to convention, the symbol + + + + indicates what we consider normal. The basal membrane increased after treatment in all cases, indicating an increase in LH. An increase in the
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D-TRP"-LH-RH IN HYPOGONADAL MEN
TABLE 2. Testicular HistopathologY' Pre- and Post-Treatment with D-TrJII-LH-RH Basal membrane
Patient 1 Pretreatment Post-treatment Patient 2 Pretreatment Post-treatment Patient 3 Pretreatment Post-treatment
Germi- Leydig nal cells cells
+++++ ++ +++++ +++ ++++++ ++ +++++ ++ ++++ ++++/ +++++
Intersti· tial edema
+ ++++ ++ ++++++
++ + +++ ++ +++/ ++++ ++++
a +, Very much decreased; ++, quite decreased; +++, little decreased; + + + +, normal; + + + + +, little increased; + + + + + +, quite increased.
,...., ,...., C"I ,....,
o ,...., 00
ov o v
number of spermatogonia was seen in the germinal cells of patient 1, and in one of the posttreatment preparations, we noted various spermatids (Figs. 2 and 3). The Leydig cells in patient 3 grew considerably and could. be considered normal in most preparations. In the remaining two patients the increase was more discreet. All patients showed an increase in the number of Sertoli cells, and inpatient 1 this was slightly greater than the normal. Interstitial edema was slight in all subjects. DISCUSSION
The LH and FSH response to LH-RH stimula. tion in hypogonadotropc hypogonadal subjects has been widely studied,9-11 and the therapeutic use of the hypothalamic peptide in the treatment of hypogonadotropic hypogonadism has been established. I In this study, we used D"Trp6-LH-RH analog, which has been proven to have a stronger . LH and FSH liberating capacity than LH-RH in normal males. 2 The gonadotropin response to stimulation v.i th 10 ILg of the analog was lower in the three patients with hypogonadotropic hypogonadism than in the control group. The type of response observed in our patients is indicative of pituitary failure, very possibly a consequency of pituitarycell gonadotropin depletion. Other studies undertaken by Zarate et al.9 and N aftolin et aLIO on LH and FSH response to LH-RH stimulation In hypogonadotropic hypogonadic subjects have also revealed a decreased gonadotropic response. Antaki et aI., 11 however, found normal gonadotropin responses in the patients with hypogonadotropic hypogonadism they studied. These differences are perhaps attributable to individual factors, or are perhaps
JARAMILLO JARAMILLO ET AL.
FIG. 2. Pretreatment biopsy of patient 1 showing a scarcity of Leydig, Sertoli, and germinal cells (spermatogonia) (hematoxylin and eosin, x 25).
a function of the different doses and/or compounds used in the stimulation tests. Zarate et al. 9 also found a persistently low LH and FSH response following treatment with 500 p,g/day ofLH-RH for a 5-day period, causing them to postulate the existence of a pituitary defect in addition to the hypothalamic alteration. Unfortunately, we did not study the gonadotropin response of our subjects to analog stimulation during or following the treatment cycle.
In our treatment cycles using doses of 0 - Trp 6LH-RH 50 times smaller in terms of weight and 4 to 5 times smaller in terms of equivalent LH-RH activity than those used with LH-RH by Mortimer et aI.,l the gonadotropin levels became detectable by radioimmunoassay during 13 to 17 weeks of sustained treatment with no substantial change in pituitary responsiveness during the course of the experiment. This finding also indicates that there was no antibody formation in
Post-treatment biopsy of patient 1 showing increased cellularity of Leydig
(A), Sertoli (B ), and germinal cells (C) clearly corresponding to more advanced stages of
spermatogenesis (spermatids) (toluidine blue, section of a photomicrograph, x400).
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response to the analog, in agreement with the study by Stone et al. 12 With such small doses of D-Trp 6-LH-RH one can also possibly avoid the paradoxical antifertility effects which may manifest themselves in male animals by a decrease in levels of plasma testosterone and a decline in testicular weight and testicular receptors to LH and which are known to occur after prolonged treatment with high doses of some superactive analogs of LH-RH.13, 14 However, the complex problem of dosage regimens with analogs ofLH-RH requires further investigation. On the average, testosterone levels in the three patients doubled and in some cases increased 5fold, there being a clear correlation between the plasma hormone levels and secondary sex changes, such as the growth of pubic and facial hair, testicular growth, and, in one case, penile development. The increase in libido experienced by the patients can be attributed to analoginduced hormonal modifications and/or to direct analog action on the brain 15. 16 or to a placebo effect.17 The fact that one patient who received doses of the analog as small as 10 ILg/8 hours experienced increased libido, erections, and pollutions incicated that these small doses are capable not only of acting on the pituitary and maintaining suitable levels of circulating gonadotropins, but also are capable by endocrine or extraendocrine mechanisms of inducing important psychologic changes in the individual. Biopsies indicated that the analog-induced hormonal changes, maintained during 13 to 17 weeks, are capable of inducing important testicular development from the endocrine and germinal point of view. It cannot be excluded that prolonging the treatment time might make the germinal changes clearer. On the other hand, it is possible that one daily dose of about 25 to 50 ILg administered intramuscularly would be sufficient. It is apparent now, after several clinical studies VIi. th long-acting superactive analogs of LH-RH,18 that for successful therapy the doses and time of administration must be carefully evaluated. In any case, we believe that this new analog of LH-RH could be beneficial in the treatment of individuals with hypogonadotropic hypogonadism of hypothalamic origin.
Acknowledgments. The authors wish to express their appreciation to Drs. O. Vaca and I. Torres for excellent technical assistance, and Dr. Max Gahwyler of Ayerst International for gifts ofD-Trp6-LH-RH.
REFERENCES 1. Mortimer CH, McNeilly AS, Fisher RA, Murray MAF, Besser GM: Gonadotrophin releasing hormone therapy in hypogonadal males with hypothalamic or pituitary dysfunction. Br Med J 4:617,1974 2. Jaramillo Jaramillo C, Perez-Infante V, Lopez-Machi A, Charro Salgado A, Coy DH, Schally AV: Serum LH, FSH and testosterone response to the administration of a new LH-RH analog, D-TRP6_LH-RH, in normal men. Int J Fertil 22:77, 1977 3. Heller CG, Clermont Y: Kinetics of the germinal epithelium in man. Recent Prog Horm Res 20:545, 1964 4. Tanner JM: Growth at Adolescence, Second Edition. Oxford, Blackwell Scientific Publications, 1962 5. Waaler PE, Thorsen T, Stoa KF, Aarskog D: Studies in normal male puberty. Acta Paediatr Scand [Suppl] 249:1, 1974 6. Midgley AR: Radioimmunoassay: a method for human chorionic gonadotropin and human luteinizing hormone. Endocrinology 79:10, 1966 7. Fernandez R: Radioinmunoensayo de gonadotropinas humanas y su aplicacion clinica. Tesis doctoral, Universidad Complutense, Madrid, Septiembre, 1975 8. Ismail AAA, Niswender GD, Midgley AR Jr: Radioimmunoassay of testosterone without chromatography. J Clin Endocrinol Metab 34:177,1972 9. Zarate A, Kastin AJ, Soria J, Canales ES, Schally AV: Effect of synthetic luteinizing hormone-releasing hormone CLH-RH) in two brothers with hypogonadotropic hypogonadism and anosmia. 10. Naftolin F, Harris GW, Borrow M: Effect of purified luteinizing hormone releasing factor on normal and hypogonadotrophic anosmic men. Nature 232:496,1971 11. Antaki A, Somma M, Wynson H, Vson Campenhout, J: Hypothalamic pituitary function in the olfacto-genital syndrome. J Clin Endocrinol Metab 38:1083,1974 12. Stone SC, Jaramillo F, Kastin A, Arimura A, Vargas JR, Schally AV, Dickey RP: Effect of LH-RH on antibody formation, sperm count and plasma levels of gonadotropins and testosterone in normal men. Int J FertilI9:192, 1974 13. Auclair C, Kelly PA, Labrie F, Coy DH, Schally AV: Inhibiton of testicular luteinizing hormone receptor level by treatment with a potent luteinizing hormone-releasing hormone agonist or human chorionic gonadotropin: Biochem Biophys Res Commun 76:855, 1977 14. Sandow J, Rechenberg Wv, Konig W, Hahn M, Jerezabek . G, Fraser H: Physiological studies with highly active analogues of LH-RH. In Second European Colloquium on Hypothalamic Hormones, Tiibingen, July 26-28, 1976, Edited by D Gupta, W Voelter. Hamburg, Verlag Chemie, 1978, p 307 15. Moss RL: Role of hypophysiotropic neurohormones in mediating neural and behavioral events. Fed Proc 36:1978, 1977 16. Kastin AJ, Miller LH, Sandman CA, Schally AV, PlotnikoffNP: CNS and pituitary effects of hypothalamic peptides and MSH. In Essays in Neurochemistry and Neuropharmacology, Vol 1, Edited by MBH Youdim, W Lovenberg, DF Sharman, JR Lagnado. London, John Wiley and Sons, 1977, p 139 . 17. Davies TF, Mountjoy CQ, Gomez-Pan A, Watson MJ, Hanner JP, Besser GM, Hall R: A double-bind cross-over of gonadotrophin releasing hormone in sexually impotent men. J Endocrinol 71:39P, 1976