Age diminishes the testicular steroidogenic response to repeated intravenous pulses of recombinant human LH during acute GnRH-receptor blockade in healthy men
Johannes D. Veldhuis,1
Nathan J. D. Veldhuis,2
Daniel M. Keenan,2 and
Ali Iranmanesh3
1Endocrine Research Unit, Department of Internal Medicine, Mayo School of Graduate Medical Education, General Clinical Research Center, Mayo Clinic, Rochester, Minnesota; 2Departments of Bioethics and Statistics, University of Virginia, Charlottesville; and 3Endocrine Service, Research and Development, Salem Veterans Affairs Medical Center, Salem, Virginia
Submitted 31 August 2004
; accepted in final form 17 November 2004
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ABSTRACT
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Testosterone (Te) concentrations fall gradually in healthy aging men. Postulated mechanisms include relative failure of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and/or gonadal Te secretion. Available methods to test Leydig cell Te production include pharmacological stimulation with human chorionic gonadotropin (hCG). We reasoned that physiological lutropic signaling could be mimicked by pulsatile infusion of recombinant human (rh) LH during acute suppression of LH secretion. To this end, we studied eight young (ages 1930 yr) and seven older (ages 6173 yr) men in an experimental paradigm comprising 1) inhibition of overnight LH secretion with a potent selective GnRH-receptor antagonist (ganirelix, 2 mg sc), 2) intravenous infusion of consecutive pulses of rh LH (50 IU every 2 h), and 3) chemiluminometric assay of LH and Te concentrations sampled every 10 min for 26 h. Statistical analyses revealed that 1) ganirelix suppressed LH and Te equally (> 75% median inhibition) in young and older men, 2) infused LH pulse profiles did not differ by age, and 3) successive intravenous pulses of rh LH increased concentrations of free Te (ng/dl) to 4.6 ± 0.38 (young) and 2.1 ± 0.14 (older; P < 0.001) and bioavailable Te (ng/dl) to 337 ± 20 (young) and 209 ± 16 (older; P = 0.002). Thus controlled pulsatile rh LH drive that emulates physiological LH pulses unmasks significant impairment of short-term Leydig cell steroidogenesis in aging men. Whether more prolonged pulsatile LH stimulation would normalize this inferred defect is unknown.
luteinizing hormone; Leydig cell; aging; male; human
HEALTHY MEN EXHIBIT a 3050% decline in free and bioavailable testosterone (Te) concentrations in the sixth and later decades of life (10, 22, 24, 29, 38). Waning Te production in the aging male has been verified by direct sampling of the human spermatic vein, cross-sectional meta-analysis, and longitudinal studies (68, 18). The primary cause is not known. Experimental data in the brown Norway rat indicate that aging reduces testis expression of specific genes encoding cholesterol transport and steroidogenic proteins (17). In this species, in vitro or in vivo exposure of Leydig cells from older animals to luteinizing hormone (LH) does not restore diminished Te secretion over the short term (40). No comparable insights are available at present in the human.
Clinical investigations have disclosed several attributes of the aging hypothalamo-pituitary-gonadal axis: 1) minimal or no elevation in LH concentrations concomitantly with low Te availability (2, 32, 37), 2) variable accentuation of LH release in response to a single intravenous pulse of gonadotropin-releasing hormone (GnRH) (36), and 3) lower total Te concentrations after intramuscular injection of human chorionic gonadotropin (hCG) (2, 9, 15, 23, 28). In the last regard, hCG is a pharmacological lutropic stimulus that assesses maximal Te secretion but downregulates Leydig cell responsiveness (5, 25).
As an alternative strategy to assess the role of gonadal mechanism(s) in mediating reduced Te production in older men, the present study implements a two-step protocol of overnight suppression of LH secretion followed by pulsatile intravenous infusion of recombinant human (rh) LH. According to the null hypothesis, young and older men should achieve comparable rh LH-stimulated Te concentrations if Leydig cell steroidogenesis does not diminish with age.
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METHODS
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Clinical protocol.
The study cohorts comprised eight young men (ages 1930 yr, with body mass indexes of 2126 kg/m2) and seven older men (ages 6173 yr, with body mass indexes of 2229 kg/m2). Each subject provided voluntary written informed consent approved by the Institutional Review Board. The study protocol was reviewed by the General Clinical Research Center (GCRC), the National Institutes of Health, and the United States Food and Drug Administration. Entry criteria included an unremarkable medical history, physical examination, and biochemical measures of renal, hepatic, hematologic, and metabolic function and a normal prostate-specific antigen. All subjects had normal fasting serum concentrations of LH, follicle-stimulating hormone (FSH), prolactin, Te, estradiol, insulin-like growth factor (IGF-I), thyroxine, and thyroid-stimulating hormone (TSH) (19, 20, 37). Exclusion criteria included acute or chronic organic disease, alcohol or drug abuse, psychiatric illness, use of any systemic prescription medications, and failure to provide consent. Volunteers were reimbursed for the time spent in participation.
Subjects were admitted to the GCRC in the late afternoon, provided supper at 1800, and given a single subcutaneous injection of the GnRH-receptor antagonist ganirelix (2.0 mg) at 2200. This dose suppresses LH concentrations maximally in young women (1, 27). On the basis of earlier time-course analyses, >75% inhibition of LH and Te concentrations emerges after 68 h and persists for 2428 h from the time of ganirelix injection (4). To stimulate Te secretion, the first of seven consecutive intravenous injections of rh LH was given at 0800 (10 h after ganirelix administration). The infusion paradigm comprised rh LH (50 IU Serono Laboratory Standard, equivalent to 20 IU First and 15.3 IU Second International Reference Preparation) diluted in sterile water and injected intravenously via commercial pump in 6-min square-wave pulses every 2 h. The last dose was infused at 2000.
Blood was withdrawn in 2 ml samples from a contralateral forearm vein every 10 min for a total of 26 h, beginning at 2000 (2 h before ganirelix administration), until 2200 the next day (2 h after the last rh LH pulse; Fig. 1A).

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Fig. 1. A: schema of study protocol. The selective gonadotropin-releasing hormone (GnRH)-receptor antagonist ganirelix (2 mg sc) was administered at 2200 to suppress luteinizing hormone (LH) and testosterone (Te) secretion. Beginning 10 h later at 0800, recombinant human (rh) LH was infused iv every 2 h for 14 h as 7 consecutive 6-min square-wave pulses (50 IU Serono standard = 15.3 IU Second International Reference Preparation). Blood was sampled every 10 min for 2 h before and 24 h after ganirelix administration for later assay of LH and total Te concentrations. B: illustrative LH (left) and total Te (right) concentrations monitored every 10 min for 26 h, beginning 2 h before and continuing 24 h after sc ganirelix injection (solid arrow at 130 min) in one young (top) and one older (bottom) man. Discrete pulses of rh LH were infused iv 2 h apart (beginning at 730 min, open arrow).
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Hormone assays.
Serum LH and Te concentrations were quantitated in duplicate in each 10-min sample in each subject as a batch using an automated random-access chemiluminescence-based assay (ACS:180; Chiron Diagnostic, Walpole, MA) (34). The LH reference standard is the Second World Health Organization International Standard 80/552. Median within and between-assay coefficients of variation were 5.1 and 6.8%, respectively. Assay sensitivity is 0.05 IU/l at 2.5 SD above hypopituitary serum. Sensitivity and intra- and interassay precision of the chemiluminescent Te assay were 18 ng/dl and 5.2 and 6.5%, respectively.
Free and bioavailable Te assays.
Free Te was measured by equilibrium dialysis, precisely as reviewed earlier, and non-sex hormone-binding globulin (SHBG)-bound Te as the 50% ammonium sulfate supernatant, as reported (20). To verify free Te and bioavailable Te measurements, we calculated each value from total Te, albumin, and SHBG concentrations in each subject, assuming association constants of 3.6 x 104 M1 (albumin) and 1 x 109 M1 (SHBG), as described in Ref. 13. The correlation coefficient/slope values were r = +0.937/slope 1.03 ± 0.04 (SD) (free Te) and r = +0.941/slope 0.96 ± 0.05 (bioavailable Te).
Analytic methods.
Mean, absolute peak (maximum), incremental amplitude (peak minus nadir), peak area, and interpulse nadir concentrations of LH and Te were computed by model-free Cluster analysis (35). Conservative pulse detection parameters for infused LH peaks included a two-by-two test cluster size and thresholds of t = 2.0 to identify significant upstrokes and downstrokes in the LH time series (31). The half-lives of distribution and elimination of infused rh LH were quantitated by a new statistically and experimentally validated model of variable-waveform deconvolution analysis (11, 13, 14).
Statistical methods.
Data are presented as means ± SE. Between- and within-subject contrasts were examined via a two-sample and paired Student's t-test, respectively. Derived measurements were subjected to logarithmic transformation first. Statistical significance was construed for P < 0.05.
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RESULTS
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Baseline free Te (ng/dl) was 1.8 ± 0.21 (young) and 0.67 ± 0.10 (older; P < 0.01), and bioavailable Te was 204 ± 47 (young) and 136 ± 22 (older; P = 0.043).
Figure 1B illustrates paired profiles of LH and total Te concentrations monitored every 10 min for 2 h before and 24 h after injection of ganirelix in one young and one older man. Ganirelix lowered LH and Te concentrations in parallel toward nadir values 68 h thereafter. Thus consecutive intravenous pulses of rh LH were given every 2 h, beginning 10 h after ganirelix injection. Nadir LH and Te concentrations were computed as the 2-h mean just before the first rh LH injection (viz., time interval comprising 810 h after the ganirelix dose; see below).
Statistical analyses revealed that the monoexponential rate (half-life) and absolute extent of decline (decrement) of total Te and LH concentrations did not differ by age (Table 1). Neither preganirelix baseline nor postganirelix nadir concentrations of LH or total Te differed by age (Fig. 2). Ganirelix suppressed both hormones by >75% (median values; P < 0.01). Mean (14-h) LH concentrations achieved during pulsatile rh LH infusion were also comparable in young and older men (Table 2). Pulse analysis revealed no effect of age on absolute peak (maxima), incremental peak, integrated peak (area), and interpulse nadir (minima) LH concentrations (Table 2).
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Table 1. Apparent rate of suppression (half-life) and absolute decrement in LH and Te concentrations after Ganirelix injection
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Fig. 2. Comparable 2-h mean baseline (left, before the ganirelix injection) and nadir (right, 810 h post-ganirelix injection) LH and total Te concentrations in 8 young and 7 older men. Data were obtained before starting the rh LH infusion. Data are group means ± SE. *P < 0.01, significant suppression by ganirelix of nadir vs. baseline concentrations in both age cohorts.
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Total Te concentrations rose rapidly in response to successive pulses of rh LH and reached a plateau after three or four (of 7) pulses (Fig. 1B). Therefore, values averaged over the last 4 h (2 pulses) of the 14-h rh LH infusion were used to assess stable Te responses. Figure 3 gives individual plateau concentrations of total and bioavailable Te in each of the 15 subjects studied. Total Te concentrations (ng/dl) increased to 629 ± 52 (young) and 506 ± 29 (older; P = 0.14), and bioavailable Te concentrations (ng/dl) rose to 337 ± 20 and 209 ± 16 in young and older subjects, respectively (P < 0.001). The foregoing differences in total and bioavailable Te measurements were consistent with SHBG concentrations of 41 ± 5.8 (young) and 85 ± 8.2 (older) nmol/l (P < 0.005). Equilibrium dialysis measurements of free Te concentrations (ng/dl) corroborated a significantly smaller plateau (4-h mean) response to rh LH pulses in older than in young men, viz., 4.6 ± 0.38 (young) and 2.1 ± 0.14 (older; P < 0.001).

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Fig. 3. Exogenously stimulated total (left) and bioavailable (right) Te concentrations in 8 young and 7 older men. Data were obtained after administration of a single dose of ganirelix during the last 2 of 7 consecutive iv pulses of rh LH (Fig. 1). Each value is an individual 4-h mean concentration during the plateau stage of rh LH infusion. P values reflect unpaired parametric comparisons by age.
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To assess intraindividual incremental Te responses to rh LH, we calculated the algebraic difference between the 2-h mean concentration of Te determined after the last pulse of rh LH (interval, 20002200) and that over the same time interval before ganirelix injection (baseline, 20002200). Concentration increments for total Te (ng/dl) were 243 ± 41 (young) and 124 ± 19 (older; P < 0.01); for free Te 3.8 ± 0.31 (young) and 1.2 ± 0.17 (older; P < 0.001); and, as calculated from the raw data shown in Fig. 4, for bioavailable Te 141 ± 33 (young) and 74 ± 18 (older; P < 0.005). Thus older volunteers manifested lower plateau and incremental elevations of bioavailable and free Te concentrations than young subjects during rh LH stimulation. Power estimates are that >80 subjects would need to be studied to detect at >90% power a >30% difference in total Te concentrations at P < 0.05, assuming SHBG concentrations comparable to those reported here (2.1-fold higher in older men).

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Fig. 4. Bioavailable Te concentrations measured in healthy young and older men before ganirelix injection (baseline) and after the last of 7 successive iv pulses of rh LH (post-LH). Each datum is the 2-h mean from 1 subject determined at baseline (20002200) and after the last rh LH stimulus (20002200 the next day). SHBG, sex hormone-binding globulin. P values were estimated by paired parametric contrasts.
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Hormone concentrations are determined jointly by secretion and elimination rates. Therefore, we applied deconvolution analysis to estimate LH kinetics from the pulsatile concentration profiles. Figure 5 shows that age does not affect the calculated rapid- and slow-phase half-lives of elimination of rh LH. The relative partitioning of the two components (ratio of rapid to total amplitude of elimination) was also comparable at 0.43 (young) and 0.32 (older) (median).

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Fig. 5. Rapid (distributional) and slow (elimination) half-lives of rh LH infused iv in 6-min square-wave pulses. Disappearance rates were estimated by deconvolution analysis in 8 young and 7 older men after ganirelix-induced suppression of LH secretion (Fig. 1). Data are means ± SE. P = not significant (NS) denotes P > 0.05 for an age comparison.
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DISCUSSION
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The current investigation examines the impact of age in healthy men on testicular Te secretion by implementing an experimental pulsatile LH clamp. The protocol required overnight suppression of LH (and thereby Te) concentrations using a maximally effective dose of a potent selective GnRH receptor antagonist, followed the next morning by stimulation of Leydig cell Te secretion with consecutive intravenous pulses of rh LH. The strategy of administering a GnRH receptor antagonist acutely (overnight) rather than a GnRH receptor agonist chronically (several weeks) was motivated by prominent loss of Leydig cell steroidogenic responsiveness in the human after more prolonged withdrawal of the tropic effects of LH (4, 21). Under the present stimulation conditions, we demonstrated that healthy older men attain significantly (
50%) lower absolute and incremental free and bioavailable Te concentrations than young men. In contrast, the measured concentrations and calculated kinetics of infused LH did not differ by age. Therefore, we infer that short-term testicular steroidogenic responsiveness to near-physiological LH pulses is significantly attenuated in healthy older men.
The intraindividual increment in, but not the absolute plateau of, total Te concentrations after rh LH stimulation was significantly reduced in aging volunteers. On the other hand, both incremental and absolute responses of free and bioavailable Te concentrations were lower in older individuals. The distinction between the plateau response of total and free or bioavailable Te concentrations is consistent with higher SHBG concentrations in older individuals, as verified here (16, 22, 39). An implication of the latter difference is that, because the metabolic clearance rate of total Te is inversely related to the SHBG concentration (39), the actual age-related difference in Te secretion is larger than that inferable from total Te concentrations. In contradistinction, the analytically predicted half-life of free Te does not decrease with increased SHBG availability (13). Thus the observed 54 and 66% reductions in absolute and incremental free Te concentrations, respectively, in older compared with young men should approximately reflect the in vivo age disparity in rh LH-stimulated Te secretion.
Clinical studies have reported normal or minimally elevated LH concentrations and normal or decreased LH bioactivity in healthy, unmedicated, independently living older men (30, 36). More detailed analyses have indicated that, even in the face of comparable mean LH concentrations, elderly volunteers secrete smaller and more frequent pulses of LH in less regular patterns (12, 19, 20, 26, 37). Therefore, a key innovation of the current paradigm is assessment of gonadal steroidogenic responses without the potential confounds of aging-related reduced biopotency, attenuated peak amplitude, more frequent LH pulses, and/or more disorderly release patterns. To this end, we utilized a maximally suppressive amount of the GnRH receptor antagonist, which lowered LH (and Te) concentrations by >75% over 810 h. Earlier dose finding studies allowed validation of a schedule of 6-min bolus intravenous injections of 50 IU rh LH every 2 h to closely approximate the physiological amplitude and frequency of LH pulses in healthy young men (4, 21). The resultant pulsatile LH clamp offered a means not only to assess testis responsiveness but also to estimate the distribution and elimination kinetics of infused LH in young and elderly men. On the basis of a biexponential decay model, we observed that age does not affect the rapid- or slow-phase half-lives of rh LH or their relative contributions to total elimination. The present kinetic estimates agree with earlier independent data: 1) calculated after bolus intravenous injection of pituitary LH extracts in hypopituitary men and 2) reconstructed analytically from spontaneous LH pulses monitored in normal men (13, 33).
By way of validity of the current paradigm, a single dose of the GnRH receptor antagonist lowered overnight LH and total Te concentrations equivalently in the two cohorts. In addition, none of the 15 subjects showed downregulation of testis responsiveness to seven successive pulses of rh LH. This outcome differs from that reported after hCG administration (see INTRODUCTION). Of further relevance to validity, in vitro bioassay of LH has indicated that GnRH receptor antagonists do not directly inhibit Leydig cell Te secretion (3). Our results in the human are congruent with observations in the adult male Norway rat, wherein subcutaneous pulses of ovine LH under systemic Te suppression of LH secretion failed to reinstate in vitro Leydig cell responsiveness in the aged animal (reviewed in Ref. 40).
An unresolved mechanistic question is the precise molecular basis for inferentially reduced LH-stimulated Leydig cell steroidogenesis in the older human. Investigations in the aged male rat have revealed decreased Leydig cell LH receptor number, postreceptor signaling, and expression of specific proteins directing sterol uptake, delivery, and utilization in Te biosynthesis (see introductory section). A recent analytical prediction made noninvasively in humans is that aging diminishes the potency and efficacy of feedforward coupling of endogenous LH pulses to Te secretion by 3550% (13). No detailed analysis of in vivo LH dose responsiveness is available at present in the human or experimental animal to validate this inference directly.
Qualifications in the present study include the relatively short-term (14-h) nature of the stimulation protocol. Whether more prolonged infusion of rh LH pulses would overcome attenuated testicular responsiveness in elderly men remains to be seen. And longitudinal studies will be needed to elucidate the precise age dependency of impaired LH-stimulated Te secretion.
In summary, a paradigm of intermittent intravenous injection of rh LH during GnRH receptor blockade to suppress pituitary LH output unveils attenuation of pulse-stimulated Leydig cell Te secretion in healthy aging men. Reduced testicular responsiveness is evident for both absolute and incremental elevations of free and bioavailable Te concentrations. Further studies will be needed to elucidate the degree of reversibility and the relative age of onset of impaired steroidogenesis in older men.
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GRANTS
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This work was supported in part by National Institutes of Health Grants MO1-RR-00847 and RR-00585 to the GCRC of the University of Virginia and Mayo Clinic from the National Center for Research Resources (Rockville, MD), and RO1-AG-023133.
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ACKNOWLEDGMENTS
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We thank Brenda Grisso for performing the immunoassays, the GCRC nursing staff for conducting the research protocols, Dr. Christopher Fox for recruiting several patients, and Dr. Louis St. L. O'Dea and Eduardo Kelly at Serono Laboratories (Norwalk, MA) for donating rh LH under a Food and Drug Administration-approved Investigator New Drug assignment by the authors.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. D. Veldhuis, Endocrine Research Unit, Dept. of Internal Medicine, Mayo School of Graduate Medical Education, General Clinical Research Center, Mayo Clinic, Rochester, MN 55905 (E-mail: veldhuis.johannes{at}mayo.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES
|
---|
- A double-blind, randomized, dose-finding study to assess the efficacy of the gonadotrophin-releasing hormone antagonist ganirelix (Org 37462) to prevent premature luteinizing hormone surges in women undergoing ovarian stimulation with recombinant follicle stimulating hormone (Puregon). The ganirelix dose-finding study group. Hum Reprod 13: 30233031, 1998.[Abstract]
- Baker HWG, Burger HG, de Kretser DM, Hudson B, O'Connor S, Wang C, Mirovics A, Court J, Dunlop M, and Rennie GC. Changes in the pituitary-testicular system with age. Clin Endocrinol (Oxf) 5: 349372, 1976.[ISI][Medline]
- Dufau ML and Veldhuis JD. Pathophysiological relationships between the biological and immunological activities of luteinizing hormone. In: Bailliere's Clinical Endocrinology and Metabolism, edited by Burger HG. Philadelphia, PA: WB Saunders, 1987, p. 153176.
- Fox CR, Veldhuis NJ, Mulligan T, Iranmanesh A, and Veldhuis JD. A novel paradigm of tandem GnRH antagonist administration and pulsatile iv infusion of recombinant human (rh) LH unveils prompt in vivo LH-testosterone secretory coupling in healthy humans. In: 83rd Annual Meeting of the Endocrine Society, Denver, CO, 2001.
- Glass AR and Vigersky RA. Resensitization of testosterone production in men after human chorionic gonadotropin-induced desensitization. J Clin Endocrinol Metab 51: 13951400, 1980.[ISI][Medline]
- Gray A, Berlin JA, McKinlay JB, and Longcope C. An examination of research design effects on the association of testosterone and male aging: results of a meta-analysis. J Clin Epidemiol 44: 671684, 1991.[CrossRef][ISI][Medline]
- Gray A, Feldman HS, McKinlay JB, and Longcope C. Age, disease, and changing sex hormone levels in middle-aged men: results of the Massachusetts Male Aging Study. J Clin Endocrinol Metab 73: 10161025, 1991.[Abstract]
- Harman SM, Metter EJ, Tobin JD, Pearson J, and Blackman MR. Longitudinal effects of aging on serum total and free testosterone levels in healthy men. Baltimore Longitudinal Study of Aging. J Clin Endocrinol Metab 86: 724731, 2001.[Abstract/Free Full Text]
- Harman SM and Tsitouras PD. Reproductive hormones in aging men. I. Measurement of sex steroids, basal luteinizing hormone, and Leydig cell response to human chorionic gonadotropin. J Clin Endocrinol Metab 51: 3540, 1980.[Abstract]
- Hollander N and Hollander VP. The microdetermination of testosterone in human spermatic vein blood. J Clin Endocrinol Metab 18: 966971, 1958.[ISI][Medline]
- Keenan DM, Alexander SL, Irvine CHG, Clarke IJ, Canny BJ, Scott CJ, Tilbrook AJ, Turner AI, and Veldhuis JD. Reconstruction of in vivo time-evolving neuroendocrine dose-response properties unveils admixed deterministic and stochastic elements in interglandular signaling. Proc Natl Acad Sci USA 101: 67406745, 2004.[Abstract/Free Full Text]
- Keenan DM and Veldhuis JD. Disruption of the hypothalamic luteinizing hormone-pulsing mechanism in aging men. Am J Physiol Regul Integr Comp Physiol 281: R1917R1924, 2001.[Abstract/Free Full Text]
- Keenan DM and Veldhuis JD. Divergent gonadotropin-gonadal dose-responsive coupling in healthy young and aging men. Am J Physiol Regul Integr Comp Physiol 286: R381R389, 2004.[Abstract/Free Full Text]
- Keenan DM, Veldhuis JD, and Yang R. Joint recovery of pulsatile and basal hormone secretion by stochastic nonlinear random-effects analysis. Am J Physiol Regul Integr Comp Physiol 275: R1939R1949, 1998.[Abstract/Free Full Text]
- Longcope C. The effect of human chorionic gonadotropin on plasma steroid levels in young and old men. Steroids 21: 583592, 1973.[CrossRef][ISI][Medline]
- Longcope C, Goldfield SRW, Brambilla DJ, and McKinlay J. Androgens, estrogens, and sex hormone-binding globulin in middle-aged men. J Clin Endocrinol Metab 71: 14421446, 1990.[Abstract]
- Luo L, Chen H, and Zirkin BR. Are Leydig cell steroidogenic enzymes differentially regulated with aging? J Androl 17: 509515, 1996.[Abstract/Free Full Text]
- Morley JE, Kaiser FE, Perry HM III, Patrick P, Morley PM, Stauber PM, Vellas B, Baumgartner RN, and Garry PJ. Longitudinal changes in testosterone, luteinizing hormone, and follicle-stimulating hormone in healthy older men. Metab Clin Exp 46: 410413, 1997.[ISI][Medline]
- Mulligan T, Iranmanesh A, Gheorghiu S, Godschalk M, and Veldhuis JD. Amplified nocturnal luteinizing hormone (LH) secretory burst frequency with selective attenuation of pulsatile (but not basal) testosterone secretion in healthy aged men: possible Leydig cell desensitization to endogenous LH signalinga clinical research center study. J Clin Endocrinol Metab 80: 30253031, 1995.[Abstract]
- Mulligan T, Iranmanesh A, Kerzner R, Demers LW, and Veldhuis JD. Two-week pulsatile gonadotropin releasing hormone infusion unmasks dual (hypothalamic and Leydig-cell) defects in the healthy aging male gonadotropic axis. Eur J Endocrinol 141: 257266, 1999.[Abstract/Free Full Text]
- Mulligan T, Iranmanesh A, and Veldhuis JD. Pulsatile iv infusion of recombinant human LH in leuprolide-suppressed men unmasks impoverished Leydig-cell secretory responsiveness to midphysiological LH drive in the aging male. J Clin Endocrinol Metab 86: 55475553, 2001.[Abstract/Free Full Text]
- Nankin HR and Calkins JH. Decreased bioavailable testosterone in aging normal and impotent men. J Clin Endocrinol Metab 63: 14181420, 1986.[Abstract]
- Nankin HR, Lin T, and Murono EP. The aging Leydig cell. III. Gonadotropin stimulation in men. J Androl 2: 181186, 1981.[ISI]
- Nieschlag E, Kley HK, Wiegelmann W, Solbach HG, and Kruskemper HL. Age and endocrine function of the testes in adult man. Dtsch Med Wochenschr 98: 12811284, 1973.[ISI][Medline]
- Padron RS, Wischusen J, Hudson B, Burger HG, and de Kretser DM. Prolonged biphasic response of plasma testosterone to single intramuscular injections of human chorionic gonadotropin. J Clin Endocrinol Metab 50: 11001104, 1980.[Abstract]
- Pincus SM, Mulligan T, Iranmanesh A, Gheorghiu S, Godschalk M, and Veldhuis JD. Older males secrete luteinizing hormone and testosterone more irregularly, and jointly more asynchronously, than younger males. Proc Natl Acad Sci USA 93: 1410014105, 1996.[Abstract/Free Full Text]
- Rabinovici J, Rothman P, Monroe SE, Nerenberg C, and Jaffe RB. Endocrine effects and pharmacokinetic characteristics of a potent new gonadotropin-releasing hormone antagonist (Ganirelix) with minimal histamine-releasing properties: studies in postmenopausal women. J Clin Endocrinol Metab 75: 12201225, 1992.[Abstract]
- Reubens R, Dhondt M, and Vermeulen A. Further studies on Leydig cell response to human choriogonadotropin. J Clin Endocrinol Metab 39: 4045, 1976.
- Stearns EL, MacDonald JA, Kaufman BJ, Padua R, Lucman T, Winter JSD, and Faiman C. Declining testicular function with age, hormonal and clinical correlates. Am J Med 57: 761766, 1974.[CrossRef][ISI][Medline]
- Urban RJ, Evans WS, Rogol AD, Kaiser DL, Johnson ML, and Veldhuis JD. Contemporary aspects of discrete peak detection algorithms. I. The paradigm of the luteinizing hormone pulse signal in men. Endocr Rev 9: 337, 1988.[ISI][Medline]
- Urban RJ, Johnson ML, and Veldhuis JD. In vivo biological validation and biophysical modeling of the sensitivity and positive accuracy of endocrine peak detection: I. The LH pulse signal. Endocrinology 124: 25412547, 1989.[Abstract]
- Urban RJ, Veldhuis JD, Blizzard RM, and Dufau ML. Attenuated release of biologically active luteinizing hormone in healthy aging men. J Clin Invest 81: 10201029, 1988.[ISI][Medline]
- Veldhuis JD, Fraioli F, Rogol AD, and Dufau ML. Metabolic clearance of biologically active luteinizing hormone in man. J Clin Invest 77: 11221128, 1986.[ISI][Medline]
- Veldhuis JD, Iranmanesh A, Mulligan T, and Pincus SM. Disruption of the young-adult synchrony between luteinizing hormone release and oscillations in follicle-stimulating hormone, prolactin, and nocturnal penile tumescence (NPT) in healthy older men. J Clin Endocrinol Metab 84: 34983505, 1999.[Abstract/Free Full Text]
- Veldhuis JD and Johnson ML. Cluster analysis: a simple, versatile and robust algorithm for endocrine pulse detection. Am J Physiol Endocrinol Metab 250: E486E493, 1986.[Abstract/Free Full Text]
- Veldhuis JD, Johnson ML, Keenan D, and Iranmanesh A. The ensemble male hypothalamo-pituitary-gonadal axis. In: Physiological Basis of Aging and Geriatrics (3rd ed.), edited by Timiras PS. Boca Raton, FL: CRC, 2003, p. 213231.
- Veldhuis JD, Urban RJ, Lizarralde G, Johnson ML, and Iranmanesh A. Attenuation of luteinizing hormone secretory burst amplitude is a proximate basis for the hypoandrogenism of healthy aging in men. J Clin Endocrinol Metab 75: 5258, 1992.[ISI]
- Vermeulen A, Rubens R, and Verdonck L. Testosterone secretion and metabolism in male senescence. J Clin Endocrinol Metab 34: 730735, 1972.[ISI][Medline]
- Vermeulen A, Verdonck L, Van der Straeten M, and Orie N. Capacity of the testosterone-binding globulin in human plasma and influence of specific binding of testosterone on its metabolic clearance rate. J Clin Endocrinol Metab 29: 14701480, 1969.[ISI]
- Zirkin BR and Chen H. Regulation of Leydig cell steroidogenic function during aging. Biol Reprod 63: 977981, 2000.[Abstract/Free Full Text]