Therapeutic use of gonadotropin-releasing hormone (GnRH) was first reported in anovulatory women 1 and later in men with hypogonadotropic

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1 FERTILITY AND STERILITY Copyright 1978 The American Fertility Society Vol. 3, No. 6, December 1978 Printed in U.SA. GONADOTROPIN AND TESTOSTERONE SECRETION IN NORMAL HUMAN MALES AFI'ER STIMULATION WITH GONADOTROPIN-RELEASING HORMONE (GnRH) OR POTENT GnRH ANALOGS USING DIFFERENT MODES OF APPLICATION* JOACHIM HAPP, M.D.t UDO HARTMANN* THOMAS WEBER UWE CORDES, M.D. JtlRGEN BEYER, M.D. Department of Endocrinology, Second Medical Clinic, University of Mainz, Mainz, Federal Republic of Germany Gonadotropin-releasing hormone (GnRH) and some potent long-acting GnRH analogs, applied by different routes of administration, were tested in six healthy human males. The effects on gonadotropin secretion were compared with the one after intravenous (i.v.) bolus injection of 25 pg of GnRH. The net increase ofluteinizing hormone (a LH) in serum produced by 25 pg of GnRH i.v. was matched by subcutaneous (s.c.) injection of 1 pg ofgnrh, dissolved in 2% gelatin or without gelatin; 5 pg of D-Ser (TBU)6-des-Gly 1 -GnRH-ethylamide i.v.; 5 pg of D-Leu 6-des Gly1- GnRH-ethylamide i.v.; and 5 pg of n-trp 6 -des Gly 1 -GnRH-ethylamide given pernasally (p.n.). D-Leu 6-des-Gly 1-GnRH-ethylamide, 5 pg p.n., produced one-half such increase as did also multiple p.n. administrations of 2 pg of GnRH every 2 hours. With 1 pg of GnRH and all analogs, elevation of serum LH lasted for about 7 to 9 hours. The longest elevation was observed with GnRH dissolved in gelatin and with n-ser (TBU) 6-des-Gly1-GnRH-ethylamide, as reflected by the greatest areas under the curves of net increase. The longer the duration of the action on LH secretion, the higher was the observed increase in follicle-stimulating hormone (FSH). Correlation between effect on LH secretion and testosterone secretion was not found. By infusion of 1 pglkglhour of D-Leu 6 -des-gly 1 -GnRH-ethylamide in two men over a period of 15 hours, a plateau of gonadotropin levels was reached within 7 to 9 hours. These plateau levels, especially of FSH, were higher than after bolus injection or p.n. application. Fertil Steril3:666, 1978 Therapeutic use of gonadotropin-releasing hormone (GnRH) was first reported in anovulatory women 1 and later in men with hypogonadotropic Received April 3, 1978; revised July 31, 1978; accepted August 7' *Supported in part by Grant BE 61/3 from the Deutsche Forschungsgemeinschaft. treprint requests: Dr. Joachim Happ, II. Med. Klinik und Poliklinik, Johannes Gutenberg-Universitat, Langenbeckstrasse 1, D-65 Mainz, Federal Republic of Germany. *The results of this study were the subject of the thesis of U.H. 666 hypogonadism.2 Another very important application was introduced with the successful GnRH therapy in cryptorchidism. 3 In view of simplification of the GnRH therapy, several modes of application 4 7 and long-acting analogs8-12 have already been tested. Comprehensive studies of this kind with intraindividual comparison have not, to our knowledge, been conducted. In the present study, GnRH and some of its potent long-acting analogs were compared intraindividually and modes of application which are practical in long-term treatment were investi-

2 Vol. 3, No.6 GONADOTROPIN SECRETION AFTER GnRH STIMULATION. I 667 gated. The doses used in this study are based on the results of previous investigations of the acute effects or on the results of therapeutic experiences METHODS Gonadotropin secretion after stimulation with GnRH or some of its long-acting analogs as well as different routes of application of the substances were investigated in six healthy human males between 2 and 3 years of age who had given their informed consent. The synthetic decapeptide GnRH and the nonapeptides, D-Ser (TBU)6-des-Gly 1 -GnRHethylamide (D-Ser(TBU)6-des-Gly1-GnRH-EA), D-Leu6-des-Gly1-GnRH-EA, and D-Trp6-des Gly'-GnRH-EA were given. The first two substances (Hoe-471 and Hoe-766) were kindly provided by Hoechst AG, Frankfurt, Germany; the third substance (L-665-2) and the last substance were provided in bulk by Serono GmbH, Freiburg, Germany. According to IUPAC,16 the nomenclature of the three analogs is as follows: [D-Ser (Bu 1 ) 6] GnRH( 1-9)nonapeptide-EA, [o-leu 6] GnRH(1-9)nonapeptide-EA,Iand [o-trp6] GnRH (1-9)nonapeptide-EA. The bulk ware of the last analog was diluted in saline immediately before use so that 1 -tl contained 25 1-Lg of the analog. The following doses and routes of administration were used (tests 1 to 8; see Fig. 6): 25 1-Lg of GnRH were given by intravenous (i.v.) bolus injection, 1 1-Lg ofgnrh or the same dose dissolved in 1 -tl of 2% Haemaccel (Depot luteinizing hormone-releasing hormone [Depot LH-RH 2] were administered subcutaneously (s.c.). Haemaccel consists of cross-linked polymers of enzymatically digested gelatin. The Depot preparation was provided by Hoechst AG. GnRH (2 1-Lg) was applied pernasally (p.n.) every 2 hours for a total of six times. For administration, a nasal spray was used as reported elsewhere.14 D-Ser(TBU)6-des Gly1-GnRH-EA or D-Leu6-des-Gly1-GnRH-EA (5-tg) was given by i.v. bolus injection. The latter substance or D-Trp6-des-Gly1-GnRH-EA (5 1-Lg) was administered intranasally; 25 1-Lg of D-Leu6-des-Gly1-GnRH-EA dissolved in 1 -tl of aqueous solution were contained in so-called "nasioles," and the dose of 5 1-tg was administered as previously described. 15 D-Trp6-des Gly1- GnRH-EA (5 1-Lg), 25 1-Lg dissolved in 1 -tl of saline, was administered by using a micropipette.12 Finally, o-leu6-des-g:ly 1 -GnRH-EA dissolved in saline was infused in two men at a rate of 1 -tglhour over a period of 15 hours (test 9). Serum luteinizing hormone (LH) and folliclestimulating hormone (FSH) were measured 1 hour before stimulation, every 3 minutes for 2 hours after stimulation, and thereafter at hourly intervals, except in tests 2 and 3 (which were terminated 1 hour earlier) and in tests 1, 4, and 9. In test 1, blood samples were obtained once before and 3 and 6 minutes after stimulation. In test 4, the sampling times were 6, 3, and minutes before and 2, 3, 45, 6, 9, and 12 minutes after the first and the sixth doses; in test 9, from 15 minutes before (at at least hourly intervals) until the end of the infusion time. Mean basal levels (MBL) were calculated from the single basal values. The gonadotropin measurements were done by radioimmunoassay (RIA) and the values expressed in milliunits per milliliter of the Second International Reference Preparation human menopausal gonadotropin (2nd IRP-HMG) as previously described. 1 Serum testosterone (T) levels were measured by the use of the Testosteron RIA-Kit Serono following a slightly modified method. ForT measurements in tests 2 and 3, blood samples were obtained 1 hour and immediately before stimulation and pooled, and 1, 2, 4, and 5 hours after stimulation. In tests 5 to 8, samples were obtained before and 1, 2, 4, and 6 hours after stimulation; in test 4, before and 1 hour after the first and sixth stimulation; and in test 9, before and 1, 2, 4, 6, 8, 1, and 15 hours from the beginning of the infusion. Maximal net increases (a) of the obtained values, and areas under the curves of the net increases were calculated as previously described. 1 LH, FSH and T increases were integrated over 6 hours after stimulation and, since the maximal effect of LH and T secretion was reported to occur 9 minutes after LH peak levels in serum,4 only areas of LH increase up to 4lh hours after stimulation were used for correlation with T secretion. The i.v. bolus injection of25 1-Lg ofgnrh, as it is used for routine diagnosis, served in these studies as a reference. Since i.v. injections are not suited for therapy, s.c. administration of GnRH and, in particular, of the long-acting galenic preparation of GnRH was tested. Pernasal application was studied in view of simplification ofgnrh therapy. Repetitive p.n. administration of the short-acting synthetic GnRH and single administration of potent long-acting analogs were compared. For comparison of potency of the two analogs, D-Ser (TBU)6-des-Gly1-GnRH-EA and D-Leu6-des-Gly1-

3 668 HAPPETAL. December LH- FSH (mu/ml) T (ng /1 ml) n=6 MBL h FIG. 1. Serum concentrations of gonadotropins and testosterone after subcutaneous administration of 1 JJ.g of GnRH (e) and of the same dose dissolved in Haemaccel () in healthy human males. GnRH-EA, on which interest has lately focused, i.v. administration was used to exclude as much as possible the influences of absorption, diffusion, and distribution. Finally, the dynamics of gonadotropin secretion under constant stimulation with a GnRH analog were studied by the infusion ofd-leu 6 -des-gly 1 -GnRH-EA. For statistical analysis Wilcoxon's matched-pairs signed-rank test 17 was used. A probability level of 5% or less was considered significant. RESULTS The characteristics of a routine GnRH test are well known. The maximal increase of serum LH was observed 3 minutes after the i.v. bolus injection and that of serum FSH after 6 minutes. A rapid decrease in the LH serum concentration was recorded as early as 1 hour after the injection. Administration of 1 J.tg of GnRH s.c. (Fig. 1), compared with the i.v. injection of25 J.tg, produced a slower increase of the serum gonadotropins. The maximal levels after 1 J.tg given s.c. were about the same as with 25 J.tg given i.v., but with 1 J.tg s.c. the duration of stimulation was much longer. Extrapolation of the LH secretion curve suggests an increased gonadotropin secretion over 7 hours. After the injection of GnRH, a small initial de- crease of the mean T serum level was observed which was followed by a continuous increase of the serum concentration until a plateau was reached after 4 hours. An important prolongation of the effect was obviously achieved by Haemaccel. A gonadotropin increase after Depot LH-RH 2 was markedly retarded and secretion prolonged. Extrapolation of the LH secretion curve suggests that the basal levels were presumably reached after about 9 hours. Starting from a basal level higher than in the previous test, the mean T level initially decreased and showed only a small transitory increase 2 hours after stimulation. When six doses of 2 J.tg were given pernasally at intervals of2 hours (Fig. 2), the first single dose showed no significant effect; after repeated administrations elevated serum levels of the gonadotropins, especially of LH, were seen before the sixth dose and a significant stimulating effect of the ON $ e FSH i rf-t-1 +I +I _j1 T (ng /1 ml) 4 sootr---.j--1 &th dose ft n:6 ln=5 rl n:6 n=4 2 MBL h FIG. 2. Serum concentrations of gonadotropins and testosterone after intranasal administration of2 JJ.g ofgnrh every 2 hours in healthy human males.

4 Vol. 3, No.6 GONADOTROPIN SECRETION AFI'ER GnRH STIMULATION. I LH- FSH (mu/ ml) n=6 2 OL--+-_ T (ng/1 ml) MBL h FIG. 3. Serum concentrations of gonadotropins and testosterone after intravenous administration of 5 #Lg of D Ser (TBU)8-des-Gly1-GnRH-EA (e) and of 5,.g of o-leu8-des Gly1-GnRH-EA () in healthy human males. single dose of GnRH on LH and FSH secretion could be demonstrated. At the time of the sixth dose, serum T levels were still about as high as in the morning. At the dose level of5 /Lg given i.v., D-Ser(TBU)6- des-gly1-gnrh-ea showed itself in regard to the LH increase in serum to be significantly more effective than D-Leu 6 -des-gly1-gnrh-ea (Fig. 3). With both substances, two peaks of gonadotropin secretion were observed: the first maximum of LH secretion after 1 hour, the second one with D-Ser(TBU)6-des-Gly1-GnRH-EA after 3 hours, and with D-Leu 6 -des-gly1-gnrh-ea after 4 hours. The second peak was more pronounced with D-Ser(TBU) 6 -des-gly1-gnrh-ea. Extrapolation of the secretion curves suggests that the gonadotropin stimulation lasts for about 9 to 1 hours with D-Ser(TBU)6 -des-gly 1-GnRH-EA and for 7 or 8 hours with D-Leu6-des-Gly1- GnRH-EA. Serum T, starting from levels around 65 ng/1 ml, showed a constant, but small rise after D-Ser(TBU)6 -des-gly 1-GnRH-EA. Mter n-leu 6 -des-gly1-gnrh-ea, the mean value decreased markedly from an initially higher value than in the previous test; this was followed by a constant rise of serum T. The gonadotropin release after p.n. administration of 5 /Lg ofd-leu6-des-gly1-gnrh-ea (Fig. 4) was comparatively small but lasted for about the same duration as after the i.v. stimulation with 5 /Lg. In three men, LH was barely stimulated and, in two cases, there was no FSH response. Much more pronounced gonadotropin secretion, especially offsh, was observed after p.n. administration of 5 /Lg ofd-trp6-des-gly1-gnrh-ea which, with respect to the results of the extrapolation of the secretion curves, presumably lasted for 7 to 8 hours. The differences in a increase and areas under the secretion curves were significant. Gonadotropin secretion also showed two peaks in these p.n. studies. LH secretion peaks occurred after 1% and 3 hours with n-trp6 -des-gly 1- GnRH-EA and after 1 and 5 hours with D Leu6-des-Gly1-GnRH-EA. The mean serum level oft decreased only after D-Leu 6 -des-gly1- GnRH-EA but, after an initial small decrease, increased slightly with n-trp6-des-gly1- GnRH-EA. Under infusion of 1/Lg of D-Leu 6 -des-gly1- GnRH- EA/hour over 15 hours (Fig. 5), a continuous increase of serum gonadotropins was seen, the LH serum levels reaching a plateau after 7 hours and serum FSH after 9 hours. Starting from levels LH-- FSH----- (mu/ ml) + n=6 o MBL h T ( ng /1 ml) MBL h FIG. 4. Serum concentrations of gonadotropins and testosterone after pernasal administration of 5,.g of D Leu8-des-Gly1-GnRH-EA () and of 5,.g of o-trp8- des-gly'-gnrh-ea (e) in healthy human males.

5 67 HAPPETAL. December LH- FSH (mu/ml) infusion T (ng/1 ml) M.G. M.G. cation of 2 p.,g of GnRH had a remarkably good effect on FSH. The effect of 5 p.,g of o-leu 6 -des Gly1-GnRH-EA p.n. was very small, however. The same relations of effectiveness were observed when comparing the areas under the secretion curves. Exceptions were a not significantly smaller FSH secretion after s.c. injection of 1 p.,g of GnRH with Haemaccel than without it. The short effect of the injection of 25 p.,g of GnRH i. v. was not compared by secretion curve integrals with the other stimulations of much longer duration. No significant differences in T secretion could be observed (Fig. 7), and no significant correlation between the areas under the curves of Ll LH and Ll T could be demonstrated. A relatively good effect on T secretion seemed to be obtained by those stimulations which had a relatively strong initial effect on LH secretion. After looking at the single values oft, one presumes negative correlation between the T increases and the T basal levels. 2 MBL h FIG. 5. Serum concentrations of gonadotropins and testosterone during infusion of 1 ILg of D-Leu 6 -des-gly'"-gnrh-ea per hour in two healthy human males. within the lower part of the normal range in the individual with the higher LH increase, serum T increased during the whole infusion period; the other individual showed a transitory rise of serum T after 2 hours. The results of this study are summarized in Figures 6 and 7, which compare the effectiveness of the different substances and modes of application in terms of maximal net gonadotropin increases and of secretion curve integrals. The same maximal LH serum levels were reached by 25 p.,g of GnRH i.v., 1 p.,g of the same substance s.c., 5p.,g of D-Leu 6 -des-gly1- GnRH-EA, and 5 p.,g of D-Trp 6 -des-gly1-gnrh-ea. The effect of 1 p.,g of GnRH s.c. could be slightly increased by Haemaccel. Most effective were 5 p.,g of o-ser(tb U) 6 -des Gly1-GnRH-EA i.v. Comparatively weak stimulation was caused by 5 p.,g of o-leu 6 -des-gly1- GnRH-EA. The same levels were reached by p.n. application of 2 p.,g of GnRH. As to the FSH increases, principally the same observations were made except for the fact that the stimulations with a relatively short action showed correspondingly smaller increases, and repetitive p.n. appli- DISCUSSION Since GnRH has been used for treatment of various diseases with gonadotropin deficiency I attempts to prolong the rather short action of the decapeptide on gonadotropin secretion were made. A prolongation of action was obtained by the use of modes of application other than the i.v. route. 4 6 Among parenteral applications, s.c. administration is most practical for self-administration. Further prolongation of the effect by Haemaccel could be demonstrated in all individuals, in contrast to previous reports. 5 From the areas under the secretion curves, stimulation of FSH seemed smaller and, from the net increases, higher with Haemaccel than without it. This is due to the late maximum offsh secretion and the relatively short period of 6 hours over which the serum concentrations were integrated. In order to avoid injections, p.n. administration has been tested. Because of a very poor absorption ofgnrh (between 1% and 4% 7 18), a dose of2 p.,g resulted in only small gonadotropin increases after the first dose but, with repeated administration, an increase of the effect, due to the priming effect of GnRH, 19 2 was reached. After repeated administration, application intervals of 2 hours did not allow the serum gonadotropins to decline to the basal levels. Long-acting, highly potent analogs ofgnrh are of special interest, particularly in view of p.n. therapy. By the use of these substances, reduction

6 Vol. 3, No.6 GONADOTROPIN SECRETION AFTER GnRH STIMULATION. I 671 A LH 1) 25 )-19 I.V. GnRH 2) 1 ).19 S.C. GnRH 3) 1 )-19 S.C. GnRH in Haemaccel@; 4) 2 ).19 p.n. GnRH every 2 hours 5) 5}Jg 1 ll LH 6) 5;.g ov r 6 hours 7) 5 J.l9 8) 5 J.l9 i.v. D- Ser (TBU )6-des-Giy1-G"!'<H <:::. i.v. D-Leu6 -C:es -GtylO_GnRH-EA pn D-Leu6 -des- Gly 1 -GnRH-EA p.n D-Trp6 des- Gty1-GnRH-EA z :z: 4 ' ti.n. 2 e :::> 2 3 E A FSH "' 3 25 z 2 :z: N 15 e 1 ::> E ;; 1ll FSH over 6 hours n= FIG. 6. Gonadotropin net increases (.i) in serum and areas under the curves of net increases (1.1) integrated over 6 hours after stimulation with GnRH and GnRH analogs administered by different routes of the frequency of application and of the waste of substance could be expected. In previous investigations, the characteristics ofd-ser(tbu)6 -des Gly1-GnRH-EA,8 21 o-leu6 -des-gly 1-GnRH EA, and o-trp 6 -des-gly1gnrh-ea 11 have been studied. With all three GnRH analogs, the dynamics of gonadotropin secretion appear surprisingly similar, regardless of the route of administration, except in the case of infusion. Maximal levels generally occurred after more than 1 hour. Slight differences mainly concerned the first peak of the generally double-peaked secretion curve, especially of LH. The first peak seems to be most marked with i.v. bolus administration. As with the decapeptide of GnRH, a small early peak obviously appeared only when high doses of o-leu 6 - des-gly1-gnrh-ea were infused. 9 1 The hypothesis of two pools of LH secretion has been previously descussed. 1 Obviously, long-acting GnRH analogs act less strongly on the quickly releasable pool than does the decapeptide of GnRH. In intraindividual comparison, n-ser(tbu) 6 - des-gly1-gnrh-ea showed itself slightly more potent than D-Leu 6 -des-gly1-gnrh-ea. In previous studies, 9 p.n. administration of 5 f.tg of D-Leu 6 -des-gly1-gnrh-ea was shown to be approximately as effective as 5 f.tg of the substance given s.c., and i.v. administration was shown to be slightly more effective than s.c. administration. In accordance with these findings, T :3 s;. 4 > E c 1 II a a -1 2) 1 }JQ s.c. GnRH 3) 1 }J9 s.c. GnRH in Haemaccel 5) 5 )ojq i.v. D-Ser ( TBU)6-des- Gly1-GnRH-EA 6) 5 }JQ i.v. D- Leu6-des-Giy1-GnRH-EA 7) 5}Jg p.n.d-leu6-des-giy1-gnrh-ea 8) 5)oJg p.n D-Trp6-des- Gly1-GnRH-EA - c: :3 FIG. 7. Areas under the curves of net increases (l.1l of testosterone in serum integrated over 6 hours after stimulation with GnRH and GnRH analogs administered by different routes.

7 672 HAPPETAL. we here observed a smaller effect with 5 J.Lg given p.n. than with 5 J.Lg given i.v., but in the present study, in contrast to our previous results, some individuals showed especially weak stimulation of the gonadotropins. This might be attributed to individual differences in p.n. absorption. All stimulations with the GnRH analogs,i.v., s.c., or p.n., produced a prolonged increase of the gonadotropins lasting for about 6 to 8 hours or longer, a result which could also be obtained by 1 J.Lg of GnRH dissolved in Haemaccel. Conclusions regarding the frequency of application cannot be simply drawn from the duration of the stimulatory effect. With the decapeptide ofgnrh, the priming effect and refractoriness " 25 have been described as a function of the frequency of stimulation and of the dose used. The same probably holds true for potent GnRH analogs, 26 which phenomenon depends essentially on the sensitiv ity of the hypophysis influenced by the gonadal feedback. 27 Therefore, the optimal frequency of administration has to be tested for every GnRH analog at the dose level used for therapy. Because of the intraindividual variation of the l:!asal T serum levels observed in our experiments, possibly the result of episodic secretion, 28 the subtraction of the area under the basal secretion curve of intraindividual controls with saline from the areas under the curves obtained after the stimulation did not seem advisable. Therefore, the area under the basal value of the same experiment had been subtracted instead. Subtraction of MBL calculated from multiple values obtained by frequent sampling during a control experiment might have supplied more relevant results despite the fact that diurnal variation has not been taken into account. Correlation between LH secretion and T secretion could not be demonstrated by our method of evaluation. This is probably due to the described great variability of the basal T levels, which seemed to determine the magnitude of the increases after stimulation. A negative correlation between basal T levels and T increases is supported by the comparison of reactive T increase in healthy subjects with the one in a patient with idiopathic hypogonadotropic hypogonadism. 13 This patient showed very marked increases in GnRH tests starting from basal T levels at the lower limit of the normal range. Furthermore, in many of the tests during the period of observation, increasing Tlevels formed no definite peak and the maximum may have occurred later; thus more characteristic correlations between LH and T secretion could possibly be demonstrated in some of December 1978 the tests, if the duration of the tests had been extended over a longer period. REFERENCES 1. Kastin AJ, Zarate A, Midgley AR Jr, Canales ES, Schally A V: Ovulation confirmed by pregnancy after infusion of porcine LH-RH. J Clin Endocrinol Metab 33:98, Besser GM, Mortimer CH: Clinical neuroendocrinology. In Frontiers in Neuroendocrinology, Edited by L Martini, W Ganong. New York, Raven Press, 1976, p Bartsch G, Frick J: Therapeutic effects ofluteinizing hormone releasing hormone (LH-RH) in cryptorchidism. Andrologia 6:197, Gonzalez-Barcena D, Kastin AJ, Schalch DS, Bermudez JA, Lee D, Arimura A,. Ruelas J, Zepeda I, Schally AV: Synthetic LH-releasing hormone (LH-RH) administered to normal men by different routes. J Clin Endocrinol Metab 37:481, Kastin AJ, Schally AV, Gonzalez-Barcena D, Coy DH, Miller MC, Porias H, Schalch DS: Clinical comparison of natural LH-RH, synthetic LH-RH, and two analogs oflh-rh. J Clin Endocriul Metab 38:81, Keller PJ, Gerber C: Effect of sublingual, intravenous and intramuscular administration of synthetic LH-releasing hormone in the human. Horm Metab Res 5:64, Dahlen HG, Keller E, Schneider HPG: Linear dose dependent LH release followingintranasally sprayed LRH. Horm Metab Res 6:51, Kuhl H, Kaplan H-G, Taubert H-D: Die Wirkung eines neuen Analogs des LH-RH, D-Ser(TBU) 6 -EA 1 -LH-RH, auf die Gonadotropin-Freisetzung bei Mannern. Dtsch Med Wochenschr 11:361, Happ J, Depper M, Weber Th, Kollmann F, Krause U, Beyer J: Gonadotropin secretion after D-Leu 6 - des-gly1-gnrh-ethylamide in normal human males and in prepubertal boys. In Hypothalamic Hormones Chemistry, Physiology and Clinical Applications, Edited by D Gupta, W Voelter. Proceedings of the 2nd European Colloquium on Hypothalamic Hormones Tiibingen, July 26-28, Weinheim, Verlag Chemie, 1978, p HappJ, WeberTh,Krause U,BeyerJ: Gonadotropin secretion after subcutaneous and intraveneous stimulation with D-Leu6 -des-gly 1 -GnRH-ethylamide in healthy human males. Infertility 1:87, Jaramillo C, Charro A, Perez-Infante V, Bordiu E, Coy DH, Schally A V: Physiological and clinical studies with a long acting analog of LH,RH: n-trp"-lh-rh in humans. Presented at the Second International Congress on :Human Reproduction, Tel Aviv, October 23-28, Happ J, Depper M, Weber Th, Hartmann U, Kollmann F, Beyer J: Gonadotropinsekretion nach pemasaler Stimulation mit n-leu 6 -des-gly 1 -GnRH-Athylamid bei gesunden Milnnem und prapubertaren Jungen. Endokrinologie. In press, Happ J, Neubauer M, Egri A, Demisch K, Schoffiing K, Beyer J: GnRH therapy in males with hypogonadotrophic hypogonadism. Horm Metab Res 7:526, Happ J, Kollmann F, Krawehl C, Neubauer M, Krause U, Demisch K, Sandow J, von Rechenberg W, Beyer J: Treatment of cryptorchidism with pernasal gonadotropinreleasing hormone therapy. Fertil Steril 29:546, Happ J, Weber Th, Callensee W, Ermert JA, Eshkol A, Beyer J: Treatment of cryptorchidism with a potent analog

8 Vol. 3, No.6 GONADOTROPIN SECRETION AFTER GnRH STIMULATION. I 673 of gonadotropin-releasing hormone. Fertil Steril 29:552, IUPAC-IUB: The nomenclature of peptide hormones. Commission on Biological Nomenclature, Recommendations J Bioi Chern 25:3215, Wilcoxon F: Individual comparisons by ranking methods. Biometrics 3:119, London DR, Butt WR, Lynch SS, Marshall JC, Owusu S, Robinson WR, Stephenson JM: Hormonal responses to intranasal luteinizing hormone releasing hormone. J Clin Endocrinol Metab 37:829, Riimmler A, Hammerstein J: Time dependent alterations in pituitary responsiveness caused by LH-RH stimulation in man. Acta Endocrinol [Suppl 75] (Kbh) 184:21, Beck LV, Bay M, King D: Evidence from perfusion studies that LRH has priming as well as direct stimulating effects on LH release. Endocrinology [Suppl] 98:A-18, Wiegelmann W, Solbach HG, Kley HK, Nieschlag E, Kriiskemper HL: A new LH-RH analog: D-Ser (TBU) 6 - LH-RH1-9EA 1 effects on gonadotrophin and gonadal steroid secretion in men after intraveneous and intranasal application. Acta Endocrinol [Suppl84] (Kbh) 28:37, Turner D, Turner EA, Aparicio NJ, Schwarzstein L, Coy DH, Schally A V: Response of luteinizing hormone and follicle-stimulating hormone to different doses of D-Leucine-6-LH-RH ethylamide in oligospermic patients. Fertil Steril 27:545, Schneider HPG, Dahlen HG: Evidence for partial refractoriness of the human pituitary. Acta Endocrinol [Suppl] (Kbh) 173:86, Sandow J, Seeger R, Heptner W, Konig W: Pituitary reserve capacity and pituitary responsiveness to LH-RH during the estrus cycle in sheep. Acta Endocrinol (Kbh) [Suppl] 173:84, Garcia MD, Tresguerres JAF, Cortes J, Oriol-Bosch A: Response after repeated gonadotropin-releasing hormone administration in humans. Acta Endocrinol [Suppl] (Kbh) 193:19, Wiegelmann W, Solbach HG, Kley HK, Kriiskemper HL: LH and FSH response to long-term application of an LHRH analog in normal males. Horm Metab Res 9:521, Happ J, Scholz P, Weber Th, 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 gonadotropin-releasing hormone. Fertil Steril 3:674, West CD, Mahajan DK, Chavre VJ, Nabors CJ, Tyler FH: Simultaneous measurement of multiple plasma steroids by radioimmunoassay demonstrating episodic secretion. J Clin Endocrinol Metab 36:123, 1973