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ORIGINAL ARTICLE

Endocrine Research

The Effect of Withdrawal and Stimulation with Human Chorionic Gonadotropin on Intratesticular and DHEA in Normal Men

M. Y. Roth, S. T. Page, K. Lin, B. D. Anawalt, A. M. Matsumoto, B. Marck,

W. J. Bremner, and J. K. Amory Downloaded from https://academic.oup.com/jcem/article/96/4/1175/2720870 by guest on 02 October 2021

Departments of Internal Medicine (M.Y.R., S.T.P., B.D.A., A.M.M., W.J.B., J.K.A.) and Obstetrics and Gynecology (K.L.) and Center for Research in and Contraception (M.Y.R., S.T.P., B.D.A., A.M.M., W.J.B., J.K.A.), University of Washington, Seattle, Washington 91895; and Geriatric Research (B.M.), Education and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98105

Introduction: Concentrations of intratesticular (IT) (T) are known to be 100–200 times those of serum T; however, the IT concentrations of T’s precursors, their testicular to serum gra- dients, gonadotropin dependence, and response to stimulation with human chorionic gonado- tropin (hCG) have not been studied in detail. We hypothesized that serum and IT androstenedione (ADD) and IT (DHEA) would be significantly suppressed by the adminis- tration of a GnRH antagonist and increased when stimulated by hCG, without a similar suppression of serum DHEA.

Methods: We suppressed in 23 normal men with the GnRH antagonist and randomly assigned them to one of four doses of hCG, 0, 15, 60, or 125 IU sc every other day for 10 d. Blood and IT fluid for the measurement of serum and IT were obtained at baseline and after 10 d of treatment.

Results: Baseline IT ADD [median (25th, 75th percentile)] was 629 (308, 860) nmol/liter, and IT DHEA was 564 (411, 879) nmol/liter, which were 175 and 27 times higher than their respective serum concentrations. IT ADD and IT DHEA were suppressed by 98 and 82%, respectively, by acyline and significantly increased with hCG administration. Likewise, serum ADD was suppressed by 50%, but serum DHEA was unchanged.

Discussion: ADD and DHEA are highly concentrated within the human testes compared with serum. Serum and IT ADD and IT DHEA are markedly suppressed with GnRH administration and stimulated by hCG, but serum DHEA is not, suggesting that most circulating DHEA is not of testicular origin. (J Clin Endocrinol Metab 96: 1175–1181, 2011)

ntratesticular (IT) testosterone (T) is required for sper- lower concentrations of IT T in both rats (7) and men (8). I matogenesis. In men with normal , IT In the context of low IT T, such as with experimental male T concentrations are known to be 100–200 times greater regimens, it is possible that other than those in the serum (1–6). However, these high con- IT , such as androstenedione (ADD) and dehy- centrations of IT T are not essential for spermatogenesis droepiandrosterone (DHEA), may play a role in support- because spermatogenesis has been observed with much ing spermatogenesis. Previous work has suggested that

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: ADD, Androstenedione; DHEA, dehydroepiandrosterone; DHT, dihydrotes- Printed in U.S.A. tosterone; hCG, human chorionic gonadotropin; IT, intratesticular; T, testosterone. Copyright © 2011 by The Endocrine Society doi: 10.1210/jc.2010-2518 Received October 25, 2010. Accepted January 10, 2011. First Published Online February 2, 2011

J Clin Endocrinol Metab, April 2011, 96(4):1175–1181 jcem.endojournals.org 1175 1176 Roth et al. hCG and Intratesticular Steroid Biosynthesis J Clin Endocrinol Metab, April 2011, 96(4):1175–1181

DHEA may support spermatogenesis in rats (9, 10), either similar suppression of serum DHEA. In addition, we hy- from an adrenal or testicular source, in which case it may pothesized that IT ADD and IT DHEA would be much function as a paracrine stimulatory signal. Nonetheless, lower than IT T at baseline, reflecting their rapid conver- the IT concentrations of these androgenic precursors of T sion to T in the testes. biosynthesis in have not been studied in detail. Previous studies examining IT ADD and IT DHEA re- lied on testicular tissue obtained either at the time of or- Subjects and Methods chidectomy from prostate cancer patients or from testic- ular biopsies of infertile patients (11–15). Only two Subjects studies enrolled normal controls to further characterize The study design has been reported previously (19). Briefly, healthy men, 18–50 yr old, with normal serum gonadotro-

the IT hormonal milieu (16, 17), but the testicular biopsies Downloaded from https://academic.oup.com/jcem/article/96/4/1175/2720870 by guest on 02 October 2021 pins, serum T concentrations, and normal seminal fluid anal- in these studies involved the use of general anesthesia, yses were enrolled. After enrollment, subjects were assigned to which can affect steroidogenesis by suppressing LH secre- one of the treatment groups by a random number sequence tion from the pituitary (18). Additionally, because of the and also randomized to the order of the unilateral testicular rarity of testicular biopsy in normal men, these studies fine-needle aspirations (right vs. left testis on d 1 vs. d 10). All involved small numbers of subjects, which may have ad- subjects had a baseline testicular fine-needle aspiration on d 1, which was performed using a scrotal block with 1% lidocaine versely affected the precision of their estimates for IT ADD buffered 1:10 with sodium bicarbonate injected into the sper- and IT DHEA. matic cord. Next, a blood sample was obtained for assessment To overcome this limitation, Jarow and colleagues (2) of serum hormones, and a unilateral testicular aspiration was developed a minimally invasive fine-needle aspiration performed as previously described (2, 6, 19, 20). After the technique that allows for sampling of IT fluid in normal testicular aspiration on d 1, all subjects received a sc injection of the GnRH antagonist acyline (NeoMPS, San Diego, CA) at men without the requirement for general anesthesia. This a dose of 300 ␮g/kg into the abdominal skin. Subjects then technique makes it possible to assess the IT con- received the first dose of hCG (Pregnyl; Organon, Roseland, centrations in normal, healthy fertile men without under- NJ) based on treatment group randomization: group 1 re- lying medical problems such as prostate cancer or infer- ceived placebo hCG (normal saline) sc every other day for five tility. Several studies have since used fine-needle aspiration doses, group 2 received 15 IU hCG sc every other day for five doses, group 3 received 60 IU hCG sc every other day for to study IT hormones, but these studies have focused on IT five doses, and group 4 received 125 IU hCG sc every other day T and IT (DHT) (2, 4, 6), and the for five doses. On d 10, subjects underwent a testicular fine- IT concentration of T precursors such as IT ADD and IT needle aspiration of the other testis, following the same pro- DHEA has not been studied using this technique. tocol outlined above for d 1. On d 40, subjects had a follow-up Knowledge regarding the concentrations of IT ADD visit to ensure their testicular examination, serum and semen parameters had all returned to normal. The University of and IT DHEA and their regulation by LH may provide Washington Institutional Review Board approved the study, insights into the role of these hormones in spermatogen- and all subjects provided written, informed consent before esis, particularly in the setting of low IT T, as may be study procedures. The study was registered in advance on observed in some men with and/or during treat- www.clinicaltrials.gov as NCT 00839319. ment with experimental forms of male hormonal contra- ception. In addition, knowledge of IT ADD and IT DHEA Measurements may enhance our understanding of T biosynthesis in vivo Testicular fluid samples were immediately placed on ice ϫ and could aid in the development of novel inhibitors of T and centrifuged at 300 g to remove any aspirated cells; the supernatant fluid was decanted and stored at Ϫ70 C. Serum biosynthesis. Such inhibitors could be useful in the treat- was stored at Ϫ20 C. Testicular fluid and serum samples were ment of -sensitive disease or improve the efficacy assayed simultaneously for T, ADD, and DHEA by liquid of male hormonal contraceptives. chromatography-tandem mass spectrometry on a Waters Therefore, to improve our understanding of testicular Aquity UPLC coupled with a Micromass Premiere-XE tandem steroidogenesis in normal men, we measured IT T, IT quadrupole mass spectrometer (Waters Corp., Milford, MA) using a modification of our previously described method (6, ADD, and IT DHEA by fine-needle aspiration in a large 19, 21). The midrange pooled intra- and interassay coeffi- group of healthy, fertile men. Measurements were per- cients of variation were 4.9 and 7.4% for T, 3.5 and 20.6% formed before and after suppression of gonadotropins for ADD, and 7.6 and 15.4% for DHEA. The assay sensitivity with the GnRH antagonist acyline and restimulation with was less than 0.1 pmol/liter for T, less than 0.03 nmol/liter for low doses of human chorionic gonadotropin (hCG). We ADD, and less than 0.07 nmol/liter for DHEA. Serum LH and FSH concentrations were quantified by im- hypothesized that serum and IT ADD and DHEA would munofluorometric assay (8). The sensitivity of the LH assay suppress with administration of a GnRH antagonist and was 0.019 IU/liter, and the intra- and interassay coefficients of increase when stimulated by low doses of hCG, without a variation for a midrange pooled value of 1.2 IU/liter was 3.2 J Clin Endocrinol Metab, April 2011, 96(4):1175–1181 jcem.endojournals.org 1177

TABLE 1. Median baseline characteristics and serum and IT hormones of 23 participants by treatment group (25th, 75th interquartile range)

Group 1, Group 2, Group 3, Group 4, 0IUhCG 15 IU hCG 60 IU hCG 125 IU hCG All subjects (23 ؍ n) (5 ؍ n) (5 ؍ n) (7 ؍ n) (6 ؍ n) Age (yr) 21 (20, 26) 25 (20,29) 22 (20, 24) 22 (21, 26) 22 (20,26) Body mass index 24.8 (23.6, 26.3) 24.1 (23.2, 26.7) 24.9 (21.2, 26.3) 25.8 (22.9, 26) 24.9 (23.2, 26.3) (kg/m2) Serum hormones LH (IU/liter) 3.5 (3.1, 4.8) 3 (2.6, 4.9) 3.4 (3.4, 4.9) 2.9 (2.3, 3.7) 3.4 (2.6, 4.9) FSH (IU/liter) 2.7 (1.2, 3.4) 2.4 (2, 2.8) 2.6 (2.2, 3.2) 2.2 (1.9, 2.5) 2.4 (1.9, 3.1) T (nmol/liter) 13 (11.3, 16.9) 15 (11.4, 20.9) 14.2 (12.7, 14.9) 16.8 (14.4, 18.6) 14.5 (11.4, 17.3) Downloaded from https://academic.oup.com/jcem/article/96/4/1175/2720870 by guest on 02 October 2021 ADD (nmol/liter) 4.2 (3.6, 5) 3.8 (3.3, 6.8) 3.5 (3.3, 3.7) 2.6 (2.5, 3.4) 3.6 (3.3, 4.5) DHEA (nmol/liter) 31.9 (16.1, 43.2) 25.5 (11.8, 33.1) 20.9 (19, 24.2) 16.1 (13.9, 18.2) 20.8 (16, 32.8) SHBG (nmol/liter) 24 (16, 30) 35 (21, 43) 28 (26, 39) 30 (21, 37) 21 (15, 33) IT hormones T (nmol/liter) 3467 (2508, 3839) 2425 (1700, 3380) 1821 (1753, 2412) 3502 (2305, 3959) 2449 (1820, 3669) ADD (nmol/liter) 530 (265, 645) 811 (246, 956) 629 (336, 766) 395 (352, 808) 629 (308, 860) DHEA (nmol/liter) 650 (454, 879) 526 (411, 911) 458 (393, 565) 676 (518, 693) 564 (411, 879)

and 12.5%, respectively. The sensitivity of the FSH assay was technique. No corrections were made for multiple compari- 0.016 IU/liter, and the intra- and interassay coefficients of sons. All statistical analyses were performed using STATA variation were 2.9 and 6.1% for a midrange pooled value of version 10.0 (StataCorp, College Station, TX). For all com- 0.96 IU/liter. Serum hCG was measured by immunofluoro- parisons, an ␣ of Ͻ0.05 was considered significant. metric assay (Delfia, Wallac, Inc., Turku, Finland). The hCG assay used in this study is specific for the intact heterodimer and was calibrated against the against the 4th International Standard for Chorionic Gonadotropin (75/589) (22). The in- Results tra- and interassay coefficients of variation for hCG were 3.4 and 3.7%, respectively, and the lower limit of detection was Subjects less than 1 IU/liter. All samples for all subjects were batched A description of the number of subjects screened and and measured in a single assay. enrolled for this study, along with data for IT T and IT DHT in a larger group, has been previously published Statistical analysis (19). There were no serious adverse events during the Due to nonnormality, the data are expressed as medians ␮ and 25th and 75th percentiles. Six subjects (one each in groups study. Median testicular aspirate volume was 10 l both 1 and 2 and two each in groups 3 and 4) had serum LH values at baseline and after 10 d of treatment. above 1.2 IU/liter on d 7. These subjects were excluded from further analysis because their IT hormones were affected by normal serum concentrations of LH. Therefore, analysis of Baseline hormone concentrations baseline and end-of-treatment hormone concentrations was Baseline serum and IT hormone concentrations and performed on the 23 subjects who suppressed serum LH below demographics are reported in Table 1. IT ADD was the lower limit of the normal range. Due to nonnormality, roughly 25% of IT T, and IT DHEA was 23% of IT T. comparisons of hormone concentrations between groups were At baseline, median IT T was 169 times higher than performed in a nonparametric fashion using Kruskal-Wallis ANOVA with a Wilcoxon rank-sum post hoc test. Correla- serum T, IT ADD was 175 times higher than serum tions between serum hormone levels and IT hormones, and ADD, and median IT DHEA was 27 times higher than between IT hormones, were performed using the Spearman serum DHEA. There were no statistically significant

TABLE 2. Posttreatment median serum and IT hormones of 23 participants by treatment group (25th, 75th interquartile range)

Group 1, Group 2, Group 3, Group 4, (5 ؍ IU hCG (n 125 (5 ؍ IU hCG (n 60 (7 ؍ IU hCG (n 15 (6 ؍ IU hCG (n 0 LH (IU/liter) 0.13 (0.07, 0.17) 0.26 (0.14, 0.65) 0.16 (0.16, 0.49) 0.33 (0.28, 0.39) FSH (IU/liter) 0.37 (0.17, 0.44) 0.27 (0.26, 0.6) 0.27 (0.24, 0.29) 0.28 (0.25, 0.31) T (nmol/liter) 0.46 (0.39, 0.67) 1.1 (0.39, 1.2) 3.1 (1.1, 3.8) 8.2 (6.3, 12) ADD (nmol/liter) 2.3 (1.6, 2.9) 1.6 (1.5, 4.7) 2.1 (2.0, 2.5) 2.6 (2.5, 3.4) DHEA (nmol/liter) 20.8 (15.2, 27.8) 13.0 (10.5, 23.4) 13.8 (13.6, 23.3) 11.1 (9.9, 13.9) IT T (nmol/liter) 77 (40, 122) 104 (66, 244) 174 (139, 319) 923 (895, 1017) 1178 Roth et al. hCG and Intratesticular Steroid Biosynthesis J Clin Endocrinol Metab, April 2011, 96(4):1175–1181 differences between any of the treatment groups for any of the measurements at baseline. IT hormone concentrations were strongly correlated with one another at baseline. In particular, IT T correlated strongly with both IT ADD (r ϭ 0.48; P ϭ 0.02) and IT DHEA (r ϭ 0.76; P Ͻ 0.01), and IT ADD correlated with IT DHEA (r ϭ 0.79; P Ͻ 0.001). Neither IT ADD nor IT DHEA correlated significantly with serum T at baseline. In addition, neither IT ADD nor IT DHEA correlated with serum ADD or serum DHEA at baseline. Lastly, none of the IT hormones correlated with serum LH, FSH, DHT, Downloaded from https://academic.oup.com/jcem/article/96/4/1175/2720870 by guest on 02 October 2021 , 17-hydroxyprogesterone, SHBG, age, or body mass index at baseline.

Posttreatment hormone concentrations Posttreatment serum and IT hormone data are pre- sented in Table 2. Treatment of normal men with acyline alone resulted in a 98% drop in IT ADD from 530 (265, 645) nmol/liter to 8 (2, 9) nmol/liter and an 82% drop in IT DHEA from 650 (454, 879) nmol/liter to 102 (66, 160) nmol/liter. IT T fell by 97% after treatment with acyline from 3467 (2508, 3839) to 77 (40, 122) nmol/liter. After 10 d of treatment with hCG, IT ADD and IT DHEA increased in proportion to hCG dose (Fig. 1, A and B). This effect was significant in the 125-IU hCG group compared with the lower-dose groups, but the 125-IU hCG dose did not fully restore IT ADD and IT DHEA to baseline levels. Treatment of normal men with acyline resulted in a significant reduction in serum ADD of 45% (P ϭ 0.03). Serum ADD remained significantly reduced compared with baseline after 10 d of treatment with the two lower doses of hCG, but the 125-IU dose of hCG returned serum ADD to baseline values. Serum DHEA was reduced in a nonsignificant fashion with both acyline alone and in re- sponse to hCG treatment. IT and serum hormone concentrations after 10 d of treatment with hCG were highly correlated with one an- ϭ Ͻ other. IT T correlated with IT ADD (r 0.95; P 0.01) FIG. 1. Box plot of IT ADD (A) and IT DHEA (B) in gonadotropin- and IT DHEA (r ϭ 0.84; P Ͻ 0.01). In contrast to the suppressed subjects on d 10 by treatment group (n ϭ 6 for the 0-IU situation at baseline, treatment serum T was highly cor- hCG group, n ϭ 7 for the 15-IU hCG group, and n ϭ 5 for the 60-IU hCG group and 125-IU hCG group). related with IT ADD (r ϭ 0.88; P Ͻ 0.01) and IT DHEA ϭ Ͻ (r 0.62; P 0.01), but not with serum ADD or serum ADD and IT DHEA in the human testes of a large group DHEA. Lastly, both IT ADD and IT DHEA correlated of normal healthy men. In addition, we have used - significantly with serum hCG after 10 d of hCG treatment otropin suppression with a GnRH antagonist to examine (Fig. 2, A and B). the concentrations of serum and IT ADD and DHEA in a gonadotropin deprived state and after re-stimulation with various low doses of hCG. This is the first study to measure Discussion IT ADD and IT DHEA in a large number of healthy, fertile men using testicular aspiration. Our results de- In this report, we have used testicular aspiration to deter- scribe the range of IT ADD and IT DHEA concentra- mine the IT concentration of the two main T precursors IT tions in normal men and demonstrate that the IT con- J Clin Endocrinol Metab, April 2011, 96(4):1175–1181 jcem.endojournals.org 1179

IT ADD or IT DHEA play a major role in supporting spermatogenesis in a low IT T setting because these com- pounds are rapidly converted to IT T. In contrast to IT ADD, IT DHEA, although present in a similar concentra- tion to IT ADD at baseline, is less affected by gonadotro- pin suppression, suggesting that conversion of IT ADD to ITTby17␤-hydroxysteroid dehydrogenase is more ro- bust than the conversion of IT DHEA to IT ADD by 3␤- hydroxysteroid dehydrogenase. Although IT DHEA was reduced dramatically with gonadotropin suppression, se-

rum DHEA was not significantly lower compared with Downloaded from https://academic.oup.com/jcem/article/96/4/1175/2720870 by guest on 02 October 2021 baseline. These data are consistent with the conclusion that a significant fraction of circulating DHEA is adrenal in origin (23). Lastly, in contrast to serum DHEA, serum ADD decreased by more than 50% after treatment with acyline, implying that the testis is the major source of se- rum ADD. The concentrations of IT hormones in our study were higher than those measured by De la Torre (17) and colleagues who analyzed testicular biopsies in men un- der general anesthesia, consistent with the notion that general anesthesia may transiently suppress steroido- genesis (18). Therefore, to avoid this potential con- founder, future studies of IT steroid concentration in normal men should focus on the use of the testicular aspiration under local anesthesia. Our study had several weaknesses. One was that 20% of subjects whose gonadotropins did not suppress com- pletely with acyline were subsequently removed from the analysis. A previous study using acyline (24) showed uni- form suppression of gonadotropins in normal young men. It remains unclear why acyline did not uniformly suppress gonadotropin levels in this study. There were no obvious FIG. 2. Correlations between posttreatment serum hCG and IT ADD differences in subject characteristics between the two stud- (A) and IT DHEA (B) for all subjects receiving hCG (n ϭ 23). The dotted ies that would explain this failure. A second caveat to our line represents the limit of the baseline range. work is the difficulty in controlling for the timing of LH pulses in the baseline measurement of the IT hormones. centrations of these hormones are significantly higher Such pulses have been shown to affect serum concentra- than their serum concentrations, yet are only about tions of sex steroids (25). The ultradian pulsatility of LH 25% the concentration of IT T. is the likely explanation for the absence of a correlation Our findings are in contrast to a previous study that between serum LH and steroid hormones at baseline. In examined IT T and IT ADD in men with varicoceles and contrast, correlations between steroid hormones and hCG found nearly identical concentrations of these hormones are more evident during stimulation likely due to the stable in both testicular biopsy samples and spermatic vein blood serum concentrations of hCG during treatment (26). Due (15). Our results are more similar to those observed pre- to the limited volume of fluid aspirated, we were unable to viously by Rajfer et al. (12) in a smaller sample. In our measure estradiol or other key IT T precursors including study, IT ADD was only 23% of IT T, implying that ADD , pregnenolone, 17-hydroxyprogesterone, is rapidly converted to T in the IT environment. This rel- and 17-hydroxypregnenolone. The measurement of the IT ative preservation of IT T over IT ADD appears to be concentration of these steroids will be the subject of future particularly true in the setting of gonadotropin suppres- research. In addition, although testicular fine-needle as- sion where the IT ADD is reduced to 10% of the concen- piration has been associated with testicular hemorrhage in tration of IT T. These results suggest that it is unlikely that a small number of infertile men (27), testicular fine-needle 1180 Roth et al. hCG and Intratesticular Steroid Biosynthesis J Clin Endocrinol Metab, April 2011, 96(4):1175–1181 aspiration for the measurement of IT steroids appears to man testicular fluid using liquid chromatography tandem mass spec- be safe in normal men, without evidence of testicular hem- trometry. Steroids 69:721–726 5. Matthiesson KL, Stanton PG, O’Donnell L, Meachem SJ, Amory JK, orrhage or any adverse impact on endocrine function as Berger R, Bremner WJ, McLachlan RI 2005 Effects of testosterone assessed by serum hormone measurements. and levonorgestrel combined with a 5␣-reductase inhibitor or go- In conclusion, this study expands the information avail- nadotropin-releasing hormone antagonist on spermatogenesis and able regarding IT androgen concentrations and shows that intratesticular steroid levels in normal men. J Clin Endocrinol Metab 90:5647–5655 IT ADD and IT DHEA are both markedly suppressed with 6. Roth MY, Lin K, Amory JK, Matsumoto AM, Anawalt BD, Snyder gonadotropin suppression and respond similarly to LH- CN, Kalhorn TF, Bremner WJ, Page ST 2010 Serum LH correlates like stimulation with hCG. This information will be useful highly with intratesticular steroid levels in normal men. J Androl in future studies examining the concentration of other T 31:138–145 7. Zirkin BR, Santulli R, Awoniyi CA, Ewing LL 1989 Maintenance of precursors alone and after the administration of enzyme-

advanced spermatogenic cells in the adult rat testis: quantitative Downloaded from https://academic.oup.com/jcem/article/96/4/1175/2720870 by guest on 02 October 2021 specific inhibitors of steroidogenesis such as relationship to testosterone concentration within the testis. Endo- or . Increased knowledge of in vivo T crinology 124:3043–3049 biosynthesis may have utility in treatment of androgen- 8. Page ST, Kalhorn TF, Bremner WJ, Anawalt BD, Matsumoto AM, Amory JK 2007 Intratesticular androgens and spermatogenesis dur- dependent disease such as prostate cancer and aid in the ing severe gonadotropin suppression induced by male hormonal development of male hormonal contraceptives. contraceptive treatment. J Androl 28:734–741 9. Saxena N, Paul PK 1987 Influence of adrenocortical hormones on the onset of spermatogenesis in rats. Indian J Exp Biol 25:296–301 10. Saxena N, Paul PK 1988 Role of adrenal in maintenance of sper- Acknowledgments matogenesis in rats. Indian J Exp Biol 26:932–936 11. Takahashi J, Higashi Y, LaNasa JA, Yoshida K, Winters SJ, Oshima We thank Ms. Iris Nielsen, Ms. Marilyn Busher, Ms. Dorothy H, Troen P 1983 Studies of the human testis. XVIII. Simultaneous McGuiness, and Ms. Connie Pete for their assistance with this measurement of nine intratesticular steroids: evidence for reduced study as well as our study volunteers without whom this research mitochondrial function in testis of elderly men. J Clin Endocrinol Metab 56:1178–1187 would not be possible. 12. Rajfer J, Sikka SC, Rivera F, Handelsman DJ 1986 Mechanism of inhibition of human testicular steroidogenesis by oral ketoconazole. Address all correspondence and requests for reprints to: J Clin Endocrinol Metab 63:1193–1198 Mara Y. Roth, M.D., University of Washington, 1959 NE 13. Rajfer J, Sikka SC, Swerdloff RS 1987 Lack of a direct effect of Pacific Street, Box 357138, Seattle, Washington, 91895. E- gonadotropin hormone-releasing hormone agonist on human tes- mail: [email protected]. ticular steroidogenesis. J Clin Endocrinol Metab 64:62–67 The National Institute of Child Health and Human Devel- 14. Weusten JJ, Smals AG, Hofman JA, Kloppenborg PW, Benraad TJ opment supported this work through cooperative agreements 1987 Early time sequence in pregnenolone metabolism to testoster- U54 HD-12629 and U54 HD-42454 as part of the specialized one in homogenates of human and rat testis. Endocrinology 120: Cooperative Centers Program in Reproductive Research and the 1909–1913 Cooperative Contraceptive Research Centers Program. M.Y.R. 15. Winters SJ, Takahashi J, Troen P 1999 Secretion of testosterone and is supported, in part, by the Eunice Kennedy Shriver National its delta4 precursor steroids into spermatic vein blood in men with Institute of Child Health and Human Development, Grant K12 varicocele-associated infertility. J Clin Endocrinol Metab 84:997– HD053984. S.T.P. is supported by the National Institute of Ag- 1001 ing, a Division of the National Institutes of Health, Grant K23 16. Marie E, Galeraud-Denis I, Carreau S 2001 Increased testicular ste- AG027238. A.M.M. is supported by the Department of Veterans roid concentrations in patients with idiopathic infertility and normal FSH levels. Arch Androl 47:177–184 Affairs. 17. de la Torre B, Nore´n S, Hedman M, Ritze´n M, Diczfalusy E 1982 Disclosure Summary: The authors have nothing to disclose. Intratesticular and plasma steroid profiles in fertile and infertile men. Int J Androl 5:367–378 18. Glass AR, Smith CE, Kidd GS, Vigersky RA 1978 Response of the References hypothalamic-pituitary-testicular axis to surgery. Fertil Steril 30: 560–564 19. Roth MY, Page ST, Lin K, Anawalt BD, Matsumoto AM, Snyder 1. 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