0021-972X/79/4902-0278$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1979 by The Endocrine Society
Vol. 49, No. 2 Printed in U.S.A.
Kinetics of Human Chorionic Gonadotropin-Induced Steroidogenic Response of the Human Testis. I. Plasma Testosterone: Implications for Human Chorionic Gonadotropin Stimulation Test* JOSfi M. SAEZ AND MAGUELONE G. FOREST Unite de Recherches sur le Controle Hormonal des Activites Cellulaires, INSERM U. 162, and Unite de Recherches Endocriniennes et Metaboliques chez I'Enfant, INSERM U. 34, Hopital Debrousse, 69322 Lyon Cedex 1, France
h and a second iv injection of hCG did not induce a significant increase. The delayed peak of plasma testosterone (2.2 ± 0.2-fold of control) was seen about 24 h later than that in the first protocol. Four subjects were studied in the third protocol, which consisted of four iv injections of hCG on days 0, 5, 10, and 15. Basal testosterone levels on days 5, 10, and 15 were significantly higher (P < 0.001) than those of control. Moreover, the hCG injection on days 5 and 10 produced, within 2-4 h, a significant increment of plasma testosterone (P < 0.01). These results suggest that in man, as in other species, hCG might induce a testicular steroidogenic desensitization. Moreover, our data raise question of the rational of daily hCG injection as used in the current protocols investigating human testicular function. (J Clin Endocrinol Metab 49: 278, 1979)
ABSTRACT. The profiles of plasma testosterone and hCG in normal adult men were studied after the administration of 6000 IU hCG under three different protocols. In the first protocol, seven subjects received a single im injection. Plasma testosterone increased sharply (1.6 ± 0.1-fold) within 4 h. Then testosterone decreased slightly and remained at a plateau level for at least 24 h. A delayed peak of testosterone (2.4 ± 0.3-fold) was seen between 72-96 h. Thereafter, testosterone declined and reached the initial levels at 144 h. In the second protocol, six subjects received two iv injections of hCG at 24-h intervals. The initial increment of plasma testosterone after the first injection was similar to that seen in the first protocol despite the fact that plasma hCG levels were 5-8 times higher in this case. At 24 h, testosterone levels were again lower than those observed at 2-4
T
HE STIMULATORY effect of hCG on human testicular endocrine function has been known for many years (1). Administration of HCG is followed by an increase in plasma levels of both androgens and estrogens (2-14). However, the magnitude of the plasma testosterone peak and the time at which the peak appears have not been well established. The differences among the results reported by several groups could be related to variations in the doses of hCG, the number of injections, and the time at which the blood was sampled for testosterone determination. Recently, it has been shown that in rats a single injection of a high dose of hCG, in addition to the acute stimulation of testicular steroidogenesis, induces a temporary state of steroidogenic refractoriness to gonadotropin stimulation (15). Since this hCG-induced rat Leydig
cell steroidogenic refractoriness is dose and time dependent (16, 17), we hypothesize that some of the conflicting results concerning the response of human testis to hCG might be related to such a desensitization phenomenon. The aim of this work was, first, to determine the kinetics of human testicular responsiveness to hCG stimulation and, second, to determine whether hCG induces testicular desensitization in humans. Materials and Methods Subjects and protocols
Received January 29, 1979. Address requests for reprints to: Dr. Jose M. Saez, Unite de Recherches "controle hormonal des activites cellulaires," Hopital Debrousse, 29 rue Soeur Bouvier, 69322 Lyon Cedex 1, France. * This work was supported by DGRST Grant 78-7-0361, INSERM Grant ATP-33-76-35, the Fondation pour la Recherche Medicale, and Universite Claude-Bernard.
Thirteen normal adult male volunteers were studied under ambulatory conditions. Their ages and body surface areas as well as the protocol used for each subject are indicated in Table 1. In protocol I, subjects received a single im injection of 6000 IU hCG (Pregnyl, Endo Laboratories, Montreal, Quebec, Canada) at about 0900 h. Samples of heparinized blood were drawn before and 1, 2, 4, 6, 8, and 12 h after hCG administration and then every day for 6 days. In protocol II, subjects received two iv injections of 6000 IU hCG at 24-h intervals. Blood was drawn at 0,1, 2, 4, 6, 8,12, and 24 h after each injection and then every day for 7 days. Subjects studied with protocol III received 6000 IU hCG iv on days 0, 5, 10, and 15. Blood was sampled before
278
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HUMAN TESTICULAR RESPONSIVENESS TO hCG TABLE 1. Subjects and protocols Subject no.
Age (yr)
Body surface area (m2)
Protocol
1 2 3 4 5 6 7 8 9 10 11 12 13
30 25 33 37 31 31 43 32 33 25 25 23 23
1.88 1.64 1.71 1.68 1.96 1.81 1.62 1.77 1.79 1.73 1.77 1.78 1.86
I I I I I and II I and II I and II (twice) II II III III III III
Mean ± SD
30 ± 2
1.77 ± 0.02
and 2 and 4 h after each hCG injection. When the same subjects were studied with protocols I and II, the interval between the two studies was more than 1 month. Subject 7 was studied three times, once with protocol I and twice with protocol II. Each study in this subject was separated by more than 6 months. Results of the two trials in subject 7 with protocol II were combined before calculation of the mean ± SD of the group.
279
levels declined and only reached control values 6 days after the hCG injection. The mean second peak of testosterone appeared slightly wider due to the fact that this peak did not occur at the same time in all subjects (one at 48 h, four at 72 h, and two at 96 h). The mean apparent half-life of hCG in three subjects was 32 h (Fig. 1), which is longer than that reported by Rizkallat et al. (21) after iv injection of hCG. This difference most likely results from a slow release of the im injected hCG into the circulation. Protocol II The modifications of the levels of plasma testosterone after hCG administration were also expressed as the percent variations of the control levels since those varied between subjects (range, 4.5-7.4 ng/ml). After the first iv injection of hCG, a sharp increase in plasma testosterone was observed within the first 4 hours (Fig. 2, bottom panel). Testosterone levels at 24 h were still higher than
Methods Plasma testosterone was measured in triplicate by a specific RIA after purification by celite column chromatography (18, 19). To avoid interassay variations, all plasma samples from a single subject were measured in the same series. Sensitivity of the method was 7 pg/sample and the intraassay coefficient of variation was 5.6%. Plasma hCG was measured by RIA (20). The reagents were provided by CEA (Gif-sur-Yvette, France). The sensitivity of the assay varied from 0.75-1 mlU/ml. The statistical analysis of the results was performed using Student's t unpaired and paired tests.
Results Protocol I Basal plasma testosterone levels in the seven subjects studied was 6.0 ± 0.6 (mean ± SD), ranging between 3.29.6 ng/ml. Therefore, to give more homogeneity to the results, the variations of plasma testosterone after hCG injection have been calculated for each subject as the percentage of his own basal levels. After hCG administration, a peak of plasma testosterone was observed within the first 2 h (Fig. 1, bottom panel). Thereafter, testosterone levels declined slightly and remained at a plateau for at least 24 h, in spite of high plasma hCG levels during this period (Fig. 1, top panel). A second peak of plasma testosterone was observed between 72-96 h at the time when plasma hCG levels were less than one fourth those observed at 4 h. Thereafter, testosterone
25 50'" 70 100 150 HOURS after hCG INJECTION 6000 IU im. FIG. 1. Effects of a single injection of 6000 IU hCG im (protocol I) on plasma levels of hCG {upper panel) and testosterone {lower panel). The values are the mean ± SE of three subjects (hCG) or seven subjects (testosterone).
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280
JCE&M • 1979 Vol 49 • No 2
SAEZ AND FOREST
The disappearance of hCG from plasma had at least two slopes, the first with a ti/2 of about 5 h, the second with a ti/2 of about 22 h (Fig. 2). These results are similar to those reported previously (21). Protocol HI
25 50 70 100 150 H O U R S a f t e r the first h C G i n j e c t i o n 6 0 0 0 IU i.v.
FIG. 2. Effects of two iv injections of 6000 IU hCG at 24-h intervals (protocol II) on plasma levels of hCG (upper panel) and testosterone (lower panel). The values are the mean ± SE of three subjects (hCG) or five subjects (testosterone).
the controls but were lower than those observed 2-4 h after hCG. This decline in plasma testosterone could result from the dramatic decrease of plasma hCG during this period (Fig. 2, top panel). However, this explanation cannot completely account for this phenomenon, since a second administration of hCG did not significantly increase plasma testosterone (P > 0.05; Student's unpaired and paired t tests) despite the fact that plasma hCG reached levels higher than those observed after the first injection. Plasma testosterone began to increase progressively at 48 h to reach a second peak at 96 h. This peak was significantly higher than that observed 2-4 h after hCG (P < 0.05). As in protocol I, not all subjects presented the secondary peak at the same time (two at 72 h, two at 96 h, and two at 120 h). The time of occurrence of this secondary peak of plasma testosterone seems to be reproducible for each subject and related to the time of the last hCG injection [i.e. in subject 7 testosterone peaked 96 h after either the single injection of hCG (protocol I) or the second hCG administration (protocol II)]. Likewise, in subject 6, testosterone peaked in both protocols 72 h after the last hCG injection (data not shown).
Neither basal testosterone levels nor the levels found during the first hours of hCG stimulation was different from the values observed at the same times with protocols I and II (Table 2). Likewise, testosterone levels 5 days after hCG were not significantly different from those found in the delayed peak of protocols I and II and were very similar to those observed 5 days after the second injection of hCG in protocol II (11.9 ± 1.4 vs. 12.4 ± 2.2; Table 2). A second injection of hCG on day 5 induced a significant increase of plasma testosterone within 2 h (Fig. 3). On day 10, basal testosterone levels as well as the acute response to a third injection of hCG were similar to those observed on day 5. On the other hand, on day 15, basal and post-hCG testosterone levels were somewhat lower than those on days 5 and 10. In addition, on day 15, the post-hCG levels were not significantly different from the basal values of the same day. Table 2 summarizes the main data obtained with the three protocols. Acute testicular steroidogenic responsiveness to hCG was similar with the three protocols despite the fact that plasma hCG levels after iv administration (protocols II and III) were 5- to 8-fold higher than those after im injection. For each protocol, the delayed testosterone peaks were significantly higher than those observed at 2-4 h.
Discussion The stimulatory effect of hCG on testicular testosterone production has been well documented, and this property has been used to evaluate the testicular reserve in TABLE 2. Mean (±SD) plasma testosterone concentrations (nanograms per ml) under basal conditions and after hCG injection No. Proof tocol subjects I II III Total
7 5 4 17
h After hCG administration Basal 2-4
6.0 ± 6.1 ± 6.7 ± 6.2 ±
1.6 1.1 0.8 1.2
24
26-28
72-120
9.4 ± 1.6' 8.2 ± 1.0" 13.5 ± 1.3*'c 10.0 ± 2.8' 8.3 ± 0.8° 9.0 ± 1.5" 12.4 ± 2.26'c 9.8 ± 1.2' 11.9 ± 1.46c 9.9 ± 2.0' 13.1 ±2.0 ft
Vol. 49, No. 2 Printed in U.S.A.
Kinetics of Human Chorionic Gonadotropin-Induced Steroidogenic Response of the Human Testis. I. Plasma Testosterone: Implications for Human Chorionic Gonadotropin Stimulation Test* JOSfi M. SAEZ AND MAGUELONE G. FOREST Unite de Recherches sur le Controle Hormonal des Activites Cellulaires, INSERM U. 162, and Unite de Recherches Endocriniennes et Metaboliques chez I'Enfant, INSERM U. 34, Hopital Debrousse, 69322 Lyon Cedex 1, France
h and a second iv injection of hCG did not induce a significant increase. The delayed peak of plasma testosterone (2.2 ± 0.2-fold of control) was seen about 24 h later than that in the first protocol. Four subjects were studied in the third protocol, which consisted of four iv injections of hCG on days 0, 5, 10, and 15. Basal testosterone levels on days 5, 10, and 15 were significantly higher (P < 0.001) than those of control. Moreover, the hCG injection on days 5 and 10 produced, within 2-4 h, a significant increment of plasma testosterone (P < 0.01). These results suggest that in man, as in other species, hCG might induce a testicular steroidogenic desensitization. Moreover, our data raise question of the rational of daily hCG injection as used in the current protocols investigating human testicular function. (J Clin Endocrinol Metab 49: 278, 1979)
ABSTRACT. The profiles of plasma testosterone and hCG in normal adult men were studied after the administration of 6000 IU hCG under three different protocols. In the first protocol, seven subjects received a single im injection. Plasma testosterone increased sharply (1.6 ± 0.1-fold) within 4 h. Then testosterone decreased slightly and remained at a plateau level for at least 24 h. A delayed peak of testosterone (2.4 ± 0.3-fold) was seen between 72-96 h. Thereafter, testosterone declined and reached the initial levels at 144 h. In the second protocol, six subjects received two iv injections of hCG at 24-h intervals. The initial increment of plasma testosterone after the first injection was similar to that seen in the first protocol despite the fact that plasma hCG levels were 5-8 times higher in this case. At 24 h, testosterone levels were again lower than those observed at 2-4
T
HE STIMULATORY effect of hCG on human testicular endocrine function has been known for many years (1). Administration of HCG is followed by an increase in plasma levels of both androgens and estrogens (2-14). However, the magnitude of the plasma testosterone peak and the time at which the peak appears have not been well established. The differences among the results reported by several groups could be related to variations in the doses of hCG, the number of injections, and the time at which the blood was sampled for testosterone determination. Recently, it has been shown that in rats a single injection of a high dose of hCG, in addition to the acute stimulation of testicular steroidogenesis, induces a temporary state of steroidogenic refractoriness to gonadotropin stimulation (15). Since this hCG-induced rat Leydig
cell steroidogenic refractoriness is dose and time dependent (16, 17), we hypothesize that some of the conflicting results concerning the response of human testis to hCG might be related to such a desensitization phenomenon. The aim of this work was, first, to determine the kinetics of human testicular responsiveness to hCG stimulation and, second, to determine whether hCG induces testicular desensitization in humans. Materials and Methods Subjects and protocols
Received January 29, 1979. Address requests for reprints to: Dr. Jose M. Saez, Unite de Recherches "controle hormonal des activites cellulaires," Hopital Debrousse, 29 rue Soeur Bouvier, 69322 Lyon Cedex 1, France. * This work was supported by DGRST Grant 78-7-0361, INSERM Grant ATP-33-76-35, the Fondation pour la Recherche Medicale, and Universite Claude-Bernard.
Thirteen normal adult male volunteers were studied under ambulatory conditions. Their ages and body surface areas as well as the protocol used for each subject are indicated in Table 1. In protocol I, subjects received a single im injection of 6000 IU hCG (Pregnyl, Endo Laboratories, Montreal, Quebec, Canada) at about 0900 h. Samples of heparinized blood were drawn before and 1, 2, 4, 6, 8, and 12 h after hCG administration and then every day for 6 days. In protocol II, subjects received two iv injections of 6000 IU hCG at 24-h intervals. Blood was drawn at 0,1, 2, 4, 6, 8,12, and 24 h after each injection and then every day for 7 days. Subjects studied with protocol III received 6000 IU hCG iv on days 0, 5, 10, and 15. Blood was sampled before
278
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HUMAN TESTICULAR RESPONSIVENESS TO hCG TABLE 1. Subjects and protocols Subject no.
Age (yr)
Body surface area (m2)
Protocol
1 2 3 4 5 6 7 8 9 10 11 12 13
30 25 33 37 31 31 43 32 33 25 25 23 23
1.88 1.64 1.71 1.68 1.96 1.81 1.62 1.77 1.79 1.73 1.77 1.78 1.86
I I I I I and II I and II I and II (twice) II II III III III III
Mean ± SD
30 ± 2
1.77 ± 0.02
and 2 and 4 h after each hCG injection. When the same subjects were studied with protocols I and II, the interval between the two studies was more than 1 month. Subject 7 was studied three times, once with protocol I and twice with protocol II. Each study in this subject was separated by more than 6 months. Results of the two trials in subject 7 with protocol II were combined before calculation of the mean ± SD of the group.
279
levels declined and only reached control values 6 days after the hCG injection. The mean second peak of testosterone appeared slightly wider due to the fact that this peak did not occur at the same time in all subjects (one at 48 h, four at 72 h, and two at 96 h). The mean apparent half-life of hCG in three subjects was 32 h (Fig. 1), which is longer than that reported by Rizkallat et al. (21) after iv injection of hCG. This difference most likely results from a slow release of the im injected hCG into the circulation. Protocol II The modifications of the levels of plasma testosterone after hCG administration were also expressed as the percent variations of the control levels since those varied between subjects (range, 4.5-7.4 ng/ml). After the first iv injection of hCG, a sharp increase in plasma testosterone was observed within the first 4 hours (Fig. 2, bottom panel). Testosterone levels at 24 h were still higher than
Methods Plasma testosterone was measured in triplicate by a specific RIA after purification by celite column chromatography (18, 19). To avoid interassay variations, all plasma samples from a single subject were measured in the same series. Sensitivity of the method was 7 pg/sample and the intraassay coefficient of variation was 5.6%. Plasma hCG was measured by RIA (20). The reagents were provided by CEA (Gif-sur-Yvette, France). The sensitivity of the assay varied from 0.75-1 mlU/ml. The statistical analysis of the results was performed using Student's t unpaired and paired tests.
Results Protocol I Basal plasma testosterone levels in the seven subjects studied was 6.0 ± 0.6 (mean ± SD), ranging between 3.29.6 ng/ml. Therefore, to give more homogeneity to the results, the variations of plasma testosterone after hCG injection have been calculated for each subject as the percentage of his own basal levels. After hCG administration, a peak of plasma testosterone was observed within the first 2 h (Fig. 1, bottom panel). Thereafter, testosterone levels declined slightly and remained at a plateau for at least 24 h, in spite of high plasma hCG levels during this period (Fig. 1, top panel). A second peak of plasma testosterone was observed between 72-96 h at the time when plasma hCG levels were less than one fourth those observed at 4 h. Thereafter, testosterone
25 50'" 70 100 150 HOURS after hCG INJECTION 6000 IU im. FIG. 1. Effects of a single injection of 6000 IU hCG im (protocol I) on plasma levels of hCG {upper panel) and testosterone {lower panel). The values are the mean ± SE of three subjects (hCG) or seven subjects (testosterone).
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280
JCE&M • 1979 Vol 49 • No 2
SAEZ AND FOREST
The disappearance of hCG from plasma had at least two slopes, the first with a ti/2 of about 5 h, the second with a ti/2 of about 22 h (Fig. 2). These results are similar to those reported previously (21). Protocol HI
25 50 70 100 150 H O U R S a f t e r the first h C G i n j e c t i o n 6 0 0 0 IU i.v.
FIG. 2. Effects of two iv injections of 6000 IU hCG at 24-h intervals (protocol II) on plasma levels of hCG (upper panel) and testosterone (lower panel). The values are the mean ± SE of three subjects (hCG) or five subjects (testosterone).
the controls but were lower than those observed 2-4 h after hCG. This decline in plasma testosterone could result from the dramatic decrease of plasma hCG during this period (Fig. 2, top panel). However, this explanation cannot completely account for this phenomenon, since a second administration of hCG did not significantly increase plasma testosterone (P > 0.05; Student's unpaired and paired t tests) despite the fact that plasma hCG reached levels higher than those observed after the first injection. Plasma testosterone began to increase progressively at 48 h to reach a second peak at 96 h. This peak was significantly higher than that observed 2-4 h after hCG (P < 0.05). As in protocol I, not all subjects presented the secondary peak at the same time (two at 72 h, two at 96 h, and two at 120 h). The time of occurrence of this secondary peak of plasma testosterone seems to be reproducible for each subject and related to the time of the last hCG injection [i.e. in subject 7 testosterone peaked 96 h after either the single injection of hCG (protocol I) or the second hCG administration (protocol II)]. Likewise, in subject 6, testosterone peaked in both protocols 72 h after the last hCG injection (data not shown).
Neither basal testosterone levels nor the levels found during the first hours of hCG stimulation was different from the values observed at the same times with protocols I and II (Table 2). Likewise, testosterone levels 5 days after hCG were not significantly different from those found in the delayed peak of protocols I and II and were very similar to those observed 5 days after the second injection of hCG in protocol II (11.9 ± 1.4 vs. 12.4 ± 2.2; Table 2). A second injection of hCG on day 5 induced a significant increase of plasma testosterone within 2 h (Fig. 3). On day 10, basal testosterone levels as well as the acute response to a third injection of hCG were similar to those observed on day 5. On the other hand, on day 15, basal and post-hCG testosterone levels were somewhat lower than those on days 5 and 10. In addition, on day 15, the post-hCG levels were not significantly different from the basal values of the same day. Table 2 summarizes the main data obtained with the three protocols. Acute testicular steroidogenic responsiveness to hCG was similar with the three protocols despite the fact that plasma hCG levels after iv administration (protocols II and III) were 5- to 8-fold higher than those after im injection. For each protocol, the delayed testosterone peaks were significantly higher than those observed at 2-4 h.
Discussion The stimulatory effect of hCG on testicular testosterone production has been well documented, and this property has been used to evaluate the testicular reserve in TABLE 2. Mean (±SD) plasma testosterone concentrations (nanograms per ml) under basal conditions and after hCG injection No. Proof tocol subjects I II III Total
7 5 4 17
h After hCG administration Basal 2-4
6.0 ± 6.1 ± 6.7 ± 6.2 ±
1.6 1.1 0.8 1.2
24
26-28
72-120
9.4 ± 1.6' 8.2 ± 1.0" 13.5 ± 1.3*'c 10.0 ± 2.8' 8.3 ± 0.8° 9.0 ± 1.5" 12.4 ± 2.26'c 9.8 ± 1.2' 11.9 ± 1.46c 9.9 ± 2.0' 13.1 ±2.0 ft
