ORIGINAL ARTICLE Endocrine Research The Effect of Gonadotropin Withdrawal and Stimulation with Human Chorionic Gonadotropin on Intratesticular Androstenedione 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 Reproduction 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) testosterone (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 dehydroepiandrosterone (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 gonadotropins in 23 normal men with the GnRH antagonist acyline 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 hormones 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 spermatogenesis, IT In the context of low IT T, such as with experimental male T concentrations are known to be 100–200 times greater hormonal contraception regimens, it is possible that other than those in the serum (1–6). However, these high con- IT androgens, 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 man 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 hormone 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 dihydrotestosterone (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 infertility 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 androgen-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.