hcg PREPARATIONS have been used extensively

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1 001-97X/8/ $0.00/0 Journal of Clinial Endorinology and Metabolism Copyright 198 by The Endorine Soiety Vol. 55, No. 1 Printed in U.S.A. Testiular Responsiveness to Chroni Human Chorioni Gonadotropin Administration in Hypogonadotropi Hypogonadism ROSARIO D'AGATA, ENZO VICARI, ANTONIA ALIFFI, GRAZIA MAUGERI, ALESSANDRO MONGIOl, AND SALVATORE GULIZIA Endorinology Unit, Andrology Center, Department of Internal Mediine, Catania Medial Shool, University of Catania, Catania, Italy ABSTRACT. Steroidogeni responsiveness to long term hcg administration (1500 U three times a week for months) was haraterized in 8 males with hypogonadotropi hypogonadism (HH). During hcg treatment, testosterone (T), whih was in the prepuberal range under basal onditions, rose onsiderably to the upper end of the normal range and remained at that level during the months of observation. A.5-fold inrease was observed in serum levels of 17/?-estradiol (E) an inrement less than seen with T. The inrement in 17a-hydroxyprogesterone was also lower than that in T throughout the study; thus, the 17a-hydroxyprogesterone to T ratio, despite ontinuous hcg administration, remained low. Serum androstenedione was slightly inreased during hcg therapy. No signifiant hanges were observed in serum levels of dehydroepiandrosterone. These data indiate that ontinuous long term hcg administration stimulated T levels in HH, with a relatively small hange in E. The kinetis of the T and E responses to 000 U hcg, evaluated after months of therapy, indiated that the testiular response was markedly redued. No inrement in T levels was observed at 4 h; the maximal response ourred at 48 h. This pattern of T response supports the idea that partial testiular desensitization ours in HH patients reeiving hroni treatment with hcg. (J Clin Endorinol Metab 55: 7,198) hcg PREPARATIONS have been used extensively in patients with hypogonadotropi hypogonadism (HH) as exogenous replaement for gonadotropin defiieny. The linial improvement whih aompanies suh treatment indiates that testiular steroidogenesis in HH is well preserved and responds normally to hcg stimulation. However, steroidogeni responsiveness to exogenous hcg administration in HH has not been extensively evaluated. Two reent reports postulate a qualitative and quantitative differene in the aute steroidogeni response to hcg ompared to normals (1, ). Besides inreasing androgens, the administration of pharmaologial doses of hcg resulted in paradoxially large inreases in estrogens and C-1 androgen preursors in men [progesterone and 17a-hydroxyprogesterone (17OHP)] (,4). In animals, hcg administration redued the speifi reeptors in the membranes of Leydig ells (5,). Estrogen inreases and dereases in speifi reeptors in Leydig ells are well established as side effets of hcg administration. The pratial impliation of suh reent findings during this therapy in humans is still Reeived Otober,1981. Address requests for reprints to: Dr. R. D'Agata, Unita Endorina, Instituto di Patologia Media I, Ospedale Garibaldi, 9514 Catania, Italy. unknown, sine few studies have been made on the testiular effets of prolonged use. We undertook our study with the aim of defining the effets of hroni hcg treatment on C-1, C-19, and C- steroids and on the dynamis of the testosterone (T) and 17/?-estradiol (E) responses to hcg hallenge in HH. Materials and Methods Eight patients with seletive HH, aged -1 yr, were studied. All subjets had eunuhoid habitus, prepubertal testiular volume (Table 1), and T blood onentrations within the prepuberal range (Fig. 1). None had hyposmia or ryptorhidism. Family history revealed that their diseases represented isolated sporadi ourrenes. The patients were given hcg (Profasi, Serono, Rome, Italy) for orretion of androgen defiieny symptoms (1500 U three times a week) for almost yr. Hormonal levels were measured before (basal) and at 4, 8, 1, and months during hcg administration. During therapy, blood was sampled 48 h after drug injetion. Previous mediation, as reported in Table 1, had been omitted for at least months before this study began (Table 1). To evaluate the dynamis of T and E responses during hroni hcg administration, 000 U hcg were given to five patients 48 h after their last injetion of 1500 U hcg at the end of replaement therapy. Blood samples were olleted daily 7

2 CHRONIC hcg AND STEROIDOGENIC RESPONSIVENESS 77 TABLE 1. Clinial details and basal LH and FSH onentrations (mean ± SE) in our patients Case No Age (yr) Duration of previous therapy (months) (hcg) 7 (hcg + hmg) 4 (hcg) 19 (hcg + hmg) 4 (hcg + hmg) hmg, Human menopausal gonadotropin. Normal value in healthy adults,.8 ± 0.. Normal value in healthy adults,. ± 0.. e No therapy. Duration of hormone therapy withdrawal before study (months) 9 1 Testis size (ml) Right 4 5- Left LH (mui/ml) a 1. ± ± ± ± 0.1 l±0.4 l±0.5.5 ± ± 0. FSH (mui/ml) 1. ± ± ± ± ± ± ± ± 0.1 during the next days. In these subjets, due to the limited serum available, other hormones were not measured. Plasma T, 17OHP, and androstenedione (A) were measured by RIA using paper hromatography (7, 8) (intraassay oeffiients of variations were 11% or less for eah of these assays, and interassay variation was 1% or less). Plasma E was evaluated by RIA using speifi antiserum after purifiation on a Sephadex LH-0 olumn. Coeffiients for intra- and interassay variations were 8% and 1%, respetively. Dehydroepiandrosterone (DHEA) was measured in ether-extrated samples using a speifi antiserum. Intra- and interassay oeffiients were 11.1% and 1.5%, respetively. In both the hroni and aute studies, all samples from eah subjet were measured in the same assay. Differenes were statistially evaluated by analysis of variane. Results in the text and figures are expressed as the mean ±SE. Results The mean (±SE) basal T onentration in HH subjets was in the prepuberal range (45 ± 8 ng/dl); E levels were also below normal (4.4 ± 0.4 vs. 1.8 ± 1 pg/ml in ontrols; P < 0.01; Fig. 1). The mean basal 17OHP value was 5. ±.4 (SE) ng/dl, not statistially different from that of eugonadal men (107 ±11 ng/dl). In four of eight patients, basal 170HP values were, as expeted, very low (8.4 ±.7 ng/dl). Basal A was 8.9 ± 1 (SE) VS. 11 ± 95 ng/dl in normals, and DHEA levels were within the normal range (Fig. 1). Effet of hroni hcg administration on plasma T and E levels After 4 months of hcg treatment, blood onentrations of T had inreased onsiderably (11-fold; P < 0.01) and were already within the normal range (Fig. 1). A further onsistent inrease was noted after 8 months of therapy (1-fold above the basal value). Subsequent blood onentrations remained similar to those observed after 8 months of treatment. After an initial.5-fold rise in E levels (P < 0.01) during the first 4 months, no further marked hanges were noted (Fig. 1) in plasma E. The magnitudine of the rise was thus muh greater for T than for E. Effet on DHEA, A, and 170HP levels No signifiant variations above basal values were observed in DHEA levels (Fig. 1). The levels of A were slightly inreased, ahieving mean values 1.-, 1.-, and 1.-fold above basal levels at 8, 1, and months, respetively. The mean 170HP levels after hcg administration were higher than the basal value (Fig. 1), but the differenes beame signifiant from 8 months on (P < 0.0). Sine the 170HP inrease was muh lower than the T rise, the 170HP to T ratios 4, 8, 1, and months after hcg treatment were very low (mean ± SE, 0.1 ± 0.0, 0.14 ± 0.0, 0.1 ± 0.01, and 0.1 ± 0.01, respetively), whih suggests that no aumulation of C-1 preursors ourred. Interestingly, the relative steroid ratios did not hange during the long period of study, hene demonstrating that no steroidogeni variations took plae with inreased time of treatment Aute responses of T and E% to hcg during hroni hcg therapy The dynamis of T and E responsiveness were best seen during the period of frequent sampling after an aute administration of 000 U hcg, 48 h after the last hroni dose of hcg. Figure shows learly that T levels remained virtually unhanged after 4 h and rose at 48 h, when they were slightly higher than those observed during hroni therapy (1.1 times the basal value). In two subjets, no hanges were observed in serum T after the

3 78 D'AGATA ET AL. JCE & M 198 Vol 55 No *~ ! *" 1 0 J hcg i I" THERAPY -r o o oa IU hcg ^0144 ^ 00-1 O r» < I I L 9 CM LU < >^BHB< < H UJ I 400- Q 00 0 J Ht- BASAL MONTHS 1 FIG. 1. Serum blood onentrations of T (nanograms per dl), E (piograms per ml), 170HP (nanograms per dl), A (nanograms per dl), and DHEA (nanograms per dl) before (basal) and during long term hcg therapy in our patients. All values are the mean ± SEM. The dashed lines enompass the normal range. 000-U dose of hcg. After 48 h, T levels gradually dereased to a nadir of 00 ± 04 (SE) ng/100 ml at 144 h, the lower limit of our normal values. Hene, the pattern of testiular responsiveness to 000 U hcg indiates a redued response of the steroid to the trophi stimulus, with a delayed T rise. The response peak for E was observed within 4 h, and the E levels tended to plateau for the next days, followed by a fall in blood levels, with a nadir at 144 h (mean ± SE, 5.4 ± 1.9 pg/ml) Hours After hcg FIG.. Kinetis of T and E responses to 000 U hcg in five hypogonadotropi patients. All values are the mean ± SE. Disussion This report shows that hcg hronially administered to HH patients brought about a predominant inrease in T and minor inreases in E levels. It is noteworthy that after the initial rises observed at 4 and 8 months of therapy with hcg, serum T remained at a plateau despite ontinuous hcg administration. The hcg ourse also augmented the serum 170HP and A onentrations. Compared to the T inrement, the rises of A 4 preursors (170HP and A) were less signifiant, resulting in low preursor to T ratios. This steroidogeni responsiveness pattern is quite different from that found in normal males. In normal subjets, an aute load with hcg/human LH resulted in an exaggerated inrease in E, with a

4 CHRONIC hcg AND STEROIDOGENIC RESPONSIVENESS 79 relatively smaller rise in T (, 4). The stimulation of aromatase enzyme ativity by hcg (9, 10) may aount for this marked inrease in estrogen. This is ommonly explained as resulting in a relative temporary defet in T biosynthesis, on the one hand, and an aumulation of preursors via suppression of some enzymes involved in its biosynthesis, mainly 17a-hydroxylase, 17,0 desmolase, or 17/?-dehydrogenase ativities, on the other (desensitization) (11-14). Interestingly, a reent report shows the persistene of this blok for 10 days after aute hcg administration (15). Therapy with hcg inreased E levels from below normal to normal levels. hcg stimulates estrogen prodution not only by diretly inreasing the aromatase ativity in the testis (9, 10), but also by raising the level of T, whih is then onverted to E. This latter effet appears more likely to aount for the hcg stimulation of E prodution seen in HH subjets and suggests that long term hcg administration in HH does not inappropriately enhane aromatase ativity. This either implies that hcg does not stimulate aromatase ativity as it does in normal subjets or that muh higher amounts of hormone are neessary in HH. The present data referring to a hroni hcg load are in lose agreement with those reently presented by Wang etal. (1). These investigators reported a prevalent inrease in T levels, with a small inrease in E, in response to human LH infusion, and no aumulation in C-1 and C-19 preursors. Unfortunately, their study was not arried out while patients were on replaement therapy. Sine the testiular response to hcg in HH patients appears to reflet the degree and duration of previous exposure to gonadotropin (1, 17), the testiular steroidogeni responsiveness pattern in HH subjets would be expeted to be similar to that in ontrols after long term gonadotropin replaement. The data presented here show that this was not so, as the pattern remained unhanged even while the patients were reeiving hcg replaement. Hene, our data after hroni stimulation with hcg omplete those of both Wang et al. (1) and Smals et al. (), who, by aute hcg stimulation, demonstrated qualitative deviations in the testiular response in HH subjets. In man, the injetion of a pharmaologial dose of hcg indues an initial aute rise in serum T, followed by a plateau for 4-48 h despite high serum hcg; this is evidene of a temporary and partial inability of Leydig ells to respond to hcg with an inrease in T prodution (desensitization). It is widely aepted that suh desensitization of Leydig ells aounts for their subsequent inability to respond to additional repeated hcg injetions with an inrease in T (15,, 19). Aordingly, the lak of a further inrease in serum T after 8 months of therapy, despite ontinuous hcg administration, might then be explained by the ourrene of partial testiular desensitization to hcg in HH subjets exposed to hroni treatment with hcg. Indiret evidene of self-regulation of testiular responsiveness to gonadotropin has been suggested by the fat that male patients with gonadotropin-produing tumor have normal plasma T levels in spite of the large amounts of hcg produed by these tumours (0, 1). This seems to suggest that there exists a negative regulation of Leydig ell responsiveness by high onentrations of irulating hcg. Another plausible explanation for the lak of further T inrement during hcg therapy might be that the testes of HH subjets have limited steroidogeni apability due to the presene of an inadequate enzyme system. The pattern of T response to the injetion of 000 U hcg is not easy to explain. Hypothetially, it ould be asribed to a temporary mehanism of refratoriness of the testiular response, most likely indued by hroni hcg administration. One might postulate that the T inrease observed 48 h after the administration of 000 U hcg may represent resensitization of the testiular responsiveness to the last injetion of 1500 U hcg, with a peak ouring 7-9 h after injetion. In fat, Glass and Vigersky (15) found that the resensitization phenomenon, with normal rises in 170HP and major T release, takes plae 7 h after the injetion of a desensitizing dose of hcg. Hene, the finding of a T response to 000 U hcg would further favor the idea that desensitization did indeed our in HH subjets reeiving hroni treatment with hcg. Aknowledgments We are grateful to Dr. K. D. Smith for reviewing the manusript. The steroid antisera were generously donated by Mr. G. Bolelli, Bologna, Italy. The skilled tehnial assistane of Mr. D. Reupero is aknowledged. Referenes 1. Wang C, Paulsen CA, Hopper BR, Rebar RW, Yen SSC 1980 Aute steroidogeni responsiveness to human luteinizing hormone in hypogonadotropi hypogonadism. J Clin Endorinol Metab 51:19. Smals AGH, Pieters GFFM, Kloppenborg PWC, Lozekoot DC, Benraad TJ 1980 Lak of a biphasi steroid response to single human horioni gonadotropin administration in patients with isolated gonadotropin defiieny. J Clin Endorinol Metab 50:879. Forest M, Leoq A, Saez JM 1979 Kinetis of human horioni gonadotropin-indued steroidogeni response of the human testis. II. Plasma 17a-hydroxyprogesterone, A 4 -androstenedione, estrone and 17/?-estradiol: evidene for the ation of human horioni gonadotropin on intermediate enzymes impliated in steroid biosynthesis. J. Clin Endorinol Metab 49:84 4. Wang C, Rebar RW, Hopper BR, Yen SSC 1980 Funtional studies of the luteinizing hormone-leydig ell-androgen axis: exaggerated response in C- and C-1 testiular steroids to various modes of luteinizing hormone stimulation. J Clin Endorinol Metab 51:01 5. Hsueh AJW, Dufau ML, Catt KJ 197 Regulation of luteinizing hormone reeptors in testiular interstizial ells by gonadotropin.

5 80 D'AGATA ET AL. JCE & M 198 Vol 55 No 1 Biohem Biophys Res Commun 7:115. Purvis K, Torjesen PA, Hang E, Hansson V 1977 hcg suppression of LH reeptors and responsiveness of testiular tissue to hcg. Mol Cell Endorinol 8:7 7. Vermeulen A 197 Determination of androgens in plasma. In: James VHT, Serio M, Martini L (eds) The Endorine Funtion of the Human Testis. Aademi Press, New York, p Verdonk L, Vermeulen A 197 Radioimmunoassay of 17/?-hydroxy-5a-androstan--one, 4-androstene-,17-dione, dehydroepiandrosterone, 17a-hydroxyprogesterone and progesterone and its appliation to human male plasma. J Steroid Biohem 7:1 9. Canik JA, Makris A, Gunsalus GL, Ryan KJ 1979 Testiular aromatization in immature rats: loalization and stimulation after gonadotropin administration in vivo. Endorinology 104: Valladares LE, Payne AH 1979 Indution of testiular aromatization by luteinizing hormone in mature rats. Endorinology 105: Rodriguez-Rigau LJ, Tholakian RK, Smith KD, Steiberger E1977 Effet of in vivo estrogen administration on in vitro steroid bioonversion in human testis. In: Troen P, Nankin A (eds) The Testis in Normal and Infertile Men. Raven Press, New York, p Cigorraga SB, Sorrel S, Bator J, Catt KJ, Dufau ML 1980 Estrogen dependene of a gonadotropin-indued steroidogeni lesion in rat testiular Leydig ells. J Clin Invest 5:99 1. Kalla NR, Nisula BC, Menard R, Loriaux DL 1980 The effet of estradiol on testiular testosterone biosynthesis. Endorinology 10:5 14. Smals AGH, Pieters GFFM, Drayer JIM, Boers GHJ, Benraad ThJ, Kloppenborg PWC 1980 Tamoxifen suppresses gonadotropinindued 17a-hydroxyprogesterone aumulation in normal men. J Clin Endorinol Metab 51: Glass AR, Vigersky RA 1980 Resensitization of testosterone prodution in men after human horioni gonadotropin-indued desensitization. J Clin Endorinol Metab 51: Santen RJ, Paulsen CA 197 Hypogonadotropi eunuhoidism. II. Gonadal responsiveness to exogenous gonadotropins. J Clin Endorinol Metab : Mahoudeau JA, Valke JC, Briaire H 1975 Dissoiated responses of plasma testosterone and estradiol to human horioni gonadotropin in adult men. J Clin Endorinol Metab 41:1. Saez JM, Forest MG 1979 Kinetis of human horioni gonadotropin-indued steroidogeni response of the human testis. I. Plasma testosterone: impliations for human horioni gonadotropin stimulation test. J Clin Endorinol Metab 49: Smals AGH, Pieters GFFM, Drayer JIM, Benraad TJ, Kloppenborg PWC 1979 Leydig ell responsiveness to single and repeated human horioni gonadotropin administration. J Clin Endorinol Metab 49:1 0. Kirshner MA, Wider JA, Roos GT 1970 Leydig ell funtion in men with gonadotropin produing testiular tumors. J Clin Endorinol Metab 0: Kirshner MA, Cohen FB, Jepersen D 1974 Estrogen prodution and its origin in men with gonadotropin-produing neoplasma. J Clin Endorinol Metab 9:11