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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 10 5544-5550
Copyright © 2005 by The Endocrine Society

Aging in Healthy Men Impairs Recombinant Human Luteinizing Hormone (LH)-Stimulated Testosterone Secretion Monitored under a Two-Day Intravenous Pulsatile LH Clamp

Peter Y. Liu, Paul Y. Takahashi, Pamela D. Roebuck, Ali Iranmanesh and Johannes D. Veldhuis

Endocrine Research Unit (P.Y.L., P.D.R., J.D.V.), Department of Internal Medicine, Mayo School of Graduate Medical Education, General Clinical Research Center, and Department of Internal Medicine (P.Y.T.), Division of Primary Care Internal Medicine, Mayo Clinic, Rochester, Minnesota 55905; and Salem Veterans Affairs Hospital (A.I.), Salem, Virginia 24153

Address all correspondence and requests for reprints to: Johannes D. Veldhuis, Endocrine Research Unit, Department of Internal Medicine, Mayo School of Graduate Medical Education, General Clinical Research Center, Mayo Clinic, Rochester, Minnesota 55905. E-mail: veldhuis.johannes{at}mayo.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Testosterone (Te) depletion in aging men in principle could reflect deficits in the hypothalamus, pituitary gland, or testis. Available pharmacological studies of possible failure of Leydig cell steroidogenesis remain inconclusive.

Objective: The objective of the study was to assess Te secretion in older and young men in response to near physiological LH stimulation.

Intervention: Pulsatile iv infusion of recombinant human LH was administered for 2 d to stimulate Te secretion during suppression of endogenous LH concentrations with a potent selective GnRH receptor antagonist (ganirelix).

Subjects/Context: Healthy older (aged 60–73 yr, n = 8) and young (19–30 yr, n = 13) men were studied in an academic setting.

Measures: Pulsatile LH and Te concentrations on the second day of exogenous LH stimulation were measured.

Results: Serum ganirelix concentrations and infused LH pulse increments were similar by age. In contrast, older subjects manifested: 1) reduced mean Te concentrations (P = 0.016), Te peak heights (P = 0.014), increments (P = 0.010), summed areas (P < 0.013), and interpeak Te concentrations (P = 0.023); 2) decreased Te to LH concentration ratios (P = 0.002); 3) diminished LH-Te feed-forward synchrony (P = 0.020); and 4) a blunted amplitude (P = 0.036) and advanced phase (P = 0.013) of diurnal Te rhythms.

Conclusion: A novel regimen of pulsatile LH stimulation for 48 h during GnRH receptor blockade unmasks deficits in pulsatile, basal, synchronous, and nyctohemeral Te secretion in healthy older men. These findings do not exclude concomitant defects in GnRH outflow and/or Te-negative feedback in the aging male.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
AGING MEN EXHIBIT a 35–50% decline in the incremental amplitude and area of LH pulses and a commensurate fall in free and bioavailable testosterone (Te) concentrations (1, 2, 3, 4, 5, 6, 7). Combined decreases in the mass of LH and Te secretory bursts resemble the clinical syndrome of hypogonadotropic hypogonadism (8, 9, 10, 11, 12, 13, 14, 15, 16). Available studies, however, do not exclude additional deficits in the older male. For example, gonadotrope responsiveness appears to be preserved in the older male because exogenous pulses of GnRH evoke normal or accentuated LH secretion (17, 18, 19, 20). On the other hand, GnRH-induced amplification of pulsatile LH secretion over an interval of 14 d fails to normalize total, free, or bioavailable Te concentrations (19). This outcome is difficult to interpret, inasmuch as aging may reduce the bioactivity of secreted LH as estimated in vitro via Te production by Leydig cells (9, 16, 21). Injection of human chorionic gonadotropin (hCG) does not elicit maximal Te secretion in elderly individuals (8, 22, 23, 24, 25). However, the pharmacological effects of hCG differ from the physiological actions of LH pulses because hCG rapidly down-regulates Leydig-cell Te biosynthesis in the human and experimental animal (26, 27, 28, 29, 30, 31, 32). Such limitations preclude definitive conclusions regarding putative primary testicular failure in older men.

One recent approach to investigating Leydig cell Te secretion in healthy young adults entailed overnight administration of a selective and potent GnRH receptor antagonist to suppress pituitary LH secretion followed the next day by iv infusion of pulses of recombinant human (rh) LH to reinstate Te secretion (33). In contradistinction, administration of a GnRH agonist to down-regulate the gonadal axis for 4–6 wk markedly impaired testicular steroidogenic responses to the same schedule of rh LH pulses (34). Based on this background, the present study adopts the strategy of acute administration of a GnRH receptor antagonist and pulsatile iv infusion of rhLH over 48 h to test the null hypothesis that age does not impair Leydig cell Te secretion driven by near-physiological LH stimuli.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Clinical protocol

The study cohorts comprised 13 young men (aged 19–30 yr with body mass indices of 23–32 kg/m2) and eight older men (aged 60–73 yr with body mass indices of 24–32 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 U.S. 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. Subjects had normal fasting serum concentrations of LH, FSH, prolactin, testosterone, estradiol, IGF-I, and TSH (11, 12, 19). Exclusion criteria included acute or chronic organic disease, alcohol or drug abuse, psychiatric illness, use of any systemic prescription medications, and failure to provide written informed consent. Volunteers were reimbursed for the time spent in participation.

Subjects were given a single sc injection of the GnRH receptor antagonist, ganirelix (2.0 mg), at 0800 h followed by 0.75 mg sc twice daily for three additional injections. The loading dose of ganirelix suppresses LH concentrations maximally in young women and men (35). Prior time-course analyses disclosed more than 75% inhibition of LH and Te concentrations within 6–8 h of the loading dose and continued inhibition for an additional 18–24 h (33). Pilot studies in 13 young men were used to establish the foregoing twice-daily ganirelix dose (see Results). To stimulate Te secretion, a total of 24 consecutive iv injections of rhLH (50 IU Serono Laboratory Standard, equivalent to 20 IU First and 15.3 IU Second International Reference Preparation diluted in sterile water) were given via commercial pump each as a 6-min square-wave pulse every 2 h. The dose of rhLH was optimized earlier to yield LH pulses in the upper quartile of the normal young-adult range (33, 34). The LH preparation has been described and used in reproductive therapy in men and women (36, 37, 38).

Blood sampling was conducted in the GCRC over the second 24 h of the infusion protocol. Volunteers slept in the GCRC to allow overnight adaptation before repetitive blood sampling on the second day and night. Blood was withdrawn in 2-ml samples from a contralateral forearm vein every 10 min for a total of 24 h beginning at 0800 h (24 h after the loading dose of ganirelix).

Hormone assays

Serum LH and Te concentrations were quantitated in duplicate in each 10-min sample as a batch for any given subject using an automated random-access chemiluminescence-based assay (ACS:180, Chiron Corp. Diagnostic, Walpole, MA) (39). 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, in the LH concentration range analyzed here. Assay sensitivity is 0.05 IU/liter at 2.5 SD above hypopituitary serum. Sensitivity and intra- and interassay precision of the chemiluminescent Te assay were 25 ng/dl and 5.2 and 6.5%, respectively. Te measurements correlated strongly (r2 = 0.985) with values quantitated by tandem gas chromatography-mass spectrometry. Ganirelix concentrations were measured by RIA as recently reported (40).

Assay of free and bioavailable Te concentrations

Free Te was measured by equilibrium dialysis and (non-SHBG bound) Te after 50% ammonium-sulfate precipitation, as reported (19). To verify free Te and bioavailable Te measurements, calculations employed individual total Te, albumin and SHBG concentrations, assuming association constants of 3.6 x 104 M–1 (albumin) and 1 x 109 M–1 (SHBG) as described elsewhere (41). Regression of calculated values on measured data yielded correlation coefficient and slope values of r = 0.937 and slope = 1.03 ± 0.04 (SD) (free Te) and r = 0.941 and slope = 0.96 ± 0.05 (bioavailable Te).

Analytical methods

To limit assumptions about the injected LH waveform and the shape of induced Te pulses, model-free Cluster analysis was used (42). The relevant end points were mean, absolute peak (maximum), incremental amplitude (peak minus nadir), peak area (integral of peak above mean of preceding and subsequent nadir values), and interpulse nadir concentrations of LH and Te. Conservative pulse-detection parameters (<5% false-positive errors) for LH and Te peaks included two-by-two test cluster sizes and a threshold of t = 2.0 to identify consecutive upstrokes and downstrokes in the time series (19, 43).

Cross-approximate entropy (cross-ApEn)

Cross-ApEn is a scale- and model-independent two-variable regularity statistic used to quantitate the relative pattern synchrony of coupled time series (44, 45). Clinical experiments establish that changes in two-hormone synchrony monitor feedback and/or feed-forward adaptations within an interlinked axis with high sensitivity and specificity (46, 47, 48). To normalize comparisons among subjects, cross-ApEn is computed on the paired original time series (observed cross-ApEn) and then recalculated after each series in a pair is shuffled randomly [rearranged in order without replacement or loss (random cross-ApEn)]. Repetition of the permutation procedure 1000 times allows calculation of the mean and SD of random cross-ApEn for a given series length and assay pair. A normalized distributional measure is then the number of SD (z scores) separating observed from mean random cross-ApEn. Higher absolute z scores denote more synchronous patterns.

Cosine regression

Cosine regression analysis was applied to quantitate the 24-h rhythmic amplitude (50% of the zenith-to-nadir difference), mesor (mean value about which the rhythm varies), and acrophase (clock time of daily maximum) of LH and Te concentration profiles (49).

Statistical methods

Data are presented as the mean ± SEM. Age-related contrasts were examined via the rank-sum (Wilcoxon two-sample) test (50). Statistical significance was construed for P < 0.05. Bonferroni correction was used when making two or more comparisons that were not independent by a priori hypothesis (51).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
In pilot dose-finding experiments, a loading dose of 2.0 mg ganirelix sc followed by ganirelix 0.5 mg either once or twice-daily for 5 d failed to maintain 24-h mean LH and Te concentrations consistently below a priori thresholds of 2 IU/liter and 385 ng/dl, respectively, on the fifth day (n = 8 subjects). In contrast, ganirelix 2 mg sc followed by 0.75 mg twice-daily suppressed concentrations of LH and total Te into the foregoing predetermined range at 0800 h on the second of 5 d (n = 4 men; Fig. 1Go). The latter ganirelix schedule was applied in one additional volunteer, who underwent blood sampling every 10 min during the time interval 24–48 h. Mean LH and Te concentrations in this subject were 0.61 IU/liter (normal range 2–12) and 52 ng/dl (normal range 385–950), respectively. In the total group of 34 men studied (13 in the dose-finding phase), adverse events comprised occasional local skin erythema, mild tenderness, and less than 1 cm edema at the site of ganirelix injection. There were no untoward systemic signs or symptoms.



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  		FIG. 1. Serum hormone concentrations measured at 0800 h at baseline (d 0) and after 24 and 48 h of administering ganirelix (a GnRH receptor antagonist) twice daily (0.75 mg) after a loading dose (2 mg sc). Horizontal interrupted lines denote lower 2.5% limits in normal young men. Data are the mean ± SEM (n = 4 young men). To convert Te (nanograms per deciliter) and estradiol (picograms per milliliter) to nanomoles per liter and picomoles per liter, multiply by 0.0347 and 3.67, respectively.

 
Baseline total Te concentrations in the young and older cohorts were 570 ± 74 and 450 ± 56 ng/dl (P = NS) and LH concentrations 5.3 ± 0.40 and 5.0 ± 0.57 IU/liter (P = NS), respectively. Corresponding free Te concentrations were 12.5 ± 1.6 (young) and 8.7 ± 1.5 ng/dl (older) (P < 0.02).

Figure 2Go illustrates 24-h profiles of serum LH and total Te concentrations in a young (aged 29 yr) and older (aged 73 yr) volunteer sampled every 10 min on the second day of concurrent ganirelix injections and pulsatile rh LH infusion. For comparison, d 2 LH and Te data are shown in the subject given ganirelix 2 mg sc followed by 0.75 mg twice-daily for 48 h. Visual inspection revealed distinct LH pulses every 2 h in the two individuals receiving the rhLH infusion; normal and reduced 24-h Te concentrations in the young and older subjects, respectively; and significant (> 80%) but not total suppression of LH and Te secretion by ganirelix alone.



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  		FIG. 2. Illustrative profiles of (paired) LH (top) and Te (bottom) concentrations sampled every 10 min for 24 h in a healthy young (A) and older (B) man on the second day of pulsatile iv infusion of rhLH and concomitant ganirelix administration (see Subjects and Methods). For comparison, C gives LH and Te concentrations during ganirelix administration and iv addback of saline in a young subject. Interrupted horizontal lines mark the lower 2.5% normal bounds of LH (2 IU/liter) and Te (385 nanograms per deciliter) concentrations in young adults. Multiply Te values in nanograms per deciliter by 0.0347 to obtain units of nanomoles per liter.

 
Figure 3Go summarizes measures of LH pulsatility in older and young men during the rhLH clamp. Statistical analyses disclosed that the incremental amplitude and area of LH peaks did not differ by age (both P > 0.67). Mean LH concentrations were higher in older than young men due to commensurately higher absolute peak and interpeak nadir LH concentrations (P < 0.01). LH interpeak intervals (minutes) averaged 117 ± 2.2 (young) and 117 ± 3.1 (older) (P = NS), thereby verifying the expected infusion frequency. Serum ganirelix concentrations were similar in young (16 ± 2.6 ng/ml) and older (14 ± 2.3 ng/ml) men.



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  		FIG. 3. Mean (24 h) LH concentrations (top left) determined in 13 young (Y) and eight older (O) men on the second day of pulsatile iv infusion of rhLH and sc injection of ganirelix. LH pulse characteristics include the interpulse nadir concentration (top right), incremental peak amplitude (bottom left), and summed peak area (bottom right). Data are the mean ± SEM. P values reflect unpaired nonparametric contrasts.

 
The effects of rhLH pulses on the mean concentrations and pulsatility of total Te are compared by age in Fig. 4Go. Statistical analysis revealed impaired responses in older men with respect to: 1) mean (24 h) Te concentrations (P = 0.014); 2) interpeak nadir Te concentrations (P = 0.023); 3) Te peak increments (P = 0.010); and 4) total pulsatile Te concentrations (product of Te peak area and peak number) (P = 0.013). Absolute peak Te concentrations were also significantly lower in older (407 ± 68 ng/dl) than young (604 ± 35 ng/dl, P = 0.014) men. In contrast, Te interpulse intervals (minutes) did not differ by age stratum [161 ± 30 (young) and 197 ± 28 (older)].



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  		FIG. 4. Mean (24 h) Te concentrations and Te pulsatility characteristics monitored on the second day of a pulsatile iv rhLH clamp under ganirelix suppression in young (Y) and older (O) men. The data format and statistical presentation are defined in Fig. 3Go. Multiply Te data in nanograms per deciliter by 0.0347 to obtain values in nanomoles per liter.

 
To account for interindividual differences in LH availability, mean 24-h Te concentrations were normalized to concomitant LH concentrations in each subject. The resultant mean Te to LH ratio was 41% lower in older (42 ± 5.9) than young (71 ± 3.7) men (P = 0.002; Fig. 5AGo). Ratios of 24-h free and bioavailable Te to LH concentrations were 46 and 66% lower in older than young men, respectively (both P < 0.002). Moreover, older men exhibited lower free (P < 0.002) and bioavailable (P < 0.001) Te concentrations than young counterparts (Fig. 5BGo).



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  		FIG. 5. Ratios of 24-h mean total, bioavailable, and free Te concentrations to LH concentrations (A) and bioavailable and free Te concentrations (B) on second day of rhLH pulses during ganirelix administration in 13 young and eight older men. See Fig. 3Go for data presentation.

 
Cross-ApEn was used as a model- and scale-free measure of the joint synchrony of infused rhLH and secreted Te patterns (45, 47). Higher values denote greater irregularity (more asynchrony) between relevantly paired series. Forward LH-Te cross-ApEn, a measure of stimulus-response coupling, was elevated in elderly men, thus predicting relative disruption of LH-Te feed-forward linkages (P = 0.020). Reverse Te-LH cross-ApEn, a monitor of negative feedback, did not differ by age (P = 0.66). Both inferences were confirmed when cross-ApEn was calculated in distributional terms as SDs (z scores) removed from empirically mean random (n = 1000 shuffled versions of each paired series). In particular, age reduced cross-ApEn of LH-Te feed-forward (P = 0.020) but not Te-LH feedback (Fig. 6Go). Cross-ApEn z scores for Te-LH feedback approached empirically random [median z scores 2.18 (young) and 2.21 (older)], consistent with the experimental paradigm of ganirelix-induced suppression of pituitary LH secretion and replacement with uniform rhLH pulses.



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  		FIG. 6. Cross-ApEn analyses of feed-forward (LH -> Te stimulation) and feedback (Te -> LH inhibition) synchrony on the second day of a pulsatile rh LH and fixed ganirelix clamp. Lower absolute cross-ApEn z scores denote reduced two-hormone joint synchrony in older men. The interrupted line gives the 99% confidence interval for a one-tailed test of the null hypothesis of purely random associations between LH and Te. The z score is the number of SDs by which observed cross-ApEn differs from empirically mean random based on 1000 simulations. See Fig. 3Go for data presentation.

 
Cosine regression analysis of Te concentrations revealed that older compared with young men had a lower mesor (rhythmic mean) (P = 0.009) and amplitude (half the zenith-to-nadir difference) (P = 0.036) and an earlier acrophase (clock time of diurnal maximum) (P = 0.013; Fig. 7Go). In contrast, 24-h LH rhythms did not differ in the two cohorts, except for a higher mesor in older subjects (Table 1Go).



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  		FIG. 7. Twenty-four-hour rhythms of Te concentrations in young (Y) and older (O) men on the second day of a combined rhLH/ganirelix regimen. The mesor is the diurnal rhythmic mean, the amplitude 50% of the daily nadir-to-zenith difference, and the acrophase the clock time of the nyctohemeral maximum. Data presentation is described in the legend of Fig. 3Go.

 

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  		TABLE 1. Nycthemeral rhythmicity of LH concentrations during rhLH infusion

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The present investigation demonstrates that healthy older men fail to achieve Te concentrations equivalent to those in young men in response to 2 d of pulsatile LH stimulation and concomitant suppression of LH secretion with a potent selective GnRH receptor antagonist (ganirelix). In particular, aging men exhibited significantly lower absolute, incremental, and integrated Te peak amplitudes and nadir Te concentrations; decreased total, bioavailable, and free Te/LH ratios; disrupted LH-Te feed-forward synchrony; and attenuated diurnal Te rhythmicity. In contrast, incremental and integrated LH pulse size and mean LH interpulse intervals were comparable by age, consistent with the LH clamp.

The study strategy was based on an earlier acute protocol validated in young men in which one injection of ganirelix was given sc at night to lower LH concentrations and seven consecutive iv pulses of rhLH were infused the next day over a 12-h interval to stimulate Te production (33). In the current paradigm, sustained suppression of LH and Te for 48 h required twice-daily administration of ganirelix at 6-fold the daily dose used in assisted-reproduction programs in women (35, 52). This investigational dosing schedule reduced LH and Te concentrations below the lower 2.5 percentile expected in normal young men. Under LH/Te-depleted conditions, iv pulses of rhLH infused every 2 h normalized mean LH concentrations (7.4 ± 0.26 IU/liter) and Te concentrations (519 ± 25 ng/dl) on the second day of study in young individuals. The regimen also reconstituted physiological measures of absolute, incremental, and integrated peak and nadir concentrations of LH and Te in young volunteers.

Earlier analyses did not reveal any age-related distinctions in LH kinetics (34, 53), although in principle longer-lived isoforms of rh LH might accumulate in older individuals (54). Because some endogenous LH (<1.2 IU/liter) was present as a low baseline under ganirelix blockade, we verified that age significantly reduced mean Te/LH concentration ratios. The latter provide a normalized index of LH drive. Age reduced the Te to LH ratio for all three total, bioavailable, and free Te concentrations. In contrast, age did not determine postinfusion incremental and integrated LH peak amplitudes. The foregoing pulse measures are important because they correlate positively with Te concentrations (11, 33, 34).

SHBG concentrations increase and the metabolic clearance of total Te decreases in healthy aging men (2, 55, 55, 56, 57). On the other hand, the calculated half-lives of free and bioavailable Te are independent of age in healthy adults (53). Therefore, demonstrably lower free (by 36%) and bioavailable (by 60%) Te concentrations in older men during pulsatile LH infusion strongly support our inference of diminished testicular steroidogenesis.

The cross-ApEn statistic was used as a sensitive (>90%) and specific (>90%) measure of LH-Te feed-forward (stimulatory) and Te-LH feedback (inhibitory) pattern coordination during the ganirelix/rhLH clamp (44, 45, 47, 48). Cross-ApEn comparisons disclosed less LH-Te feed-forward synchrony in older than young men. This outcome could reflect greater variability in Leydig-cell responses to, or in the systemic delivery of, iv pulses of rhLH (30). In the latter context, univariate ApEn disclosed less orderly LH profiles in the aging cohort, possibly due to less uniform distribution or elimination of infused LH. In contrast, Te-LH feedback cross-ApEn values were similar by age and nearly random as expected.

Cosine regression analysis revealed measurable 24-h rhythms in LH and Te concentrations in young and older men during rh LH infusion. The diurnal variation in LH concentrations in both cohorts was similar and averaged 4.5%, which is somewhat reduced (49). The average nyctohemeral amplitude of Te concentrations was 8.5% in both groups, which is decreased (58). The bases of detectable diurnal LH and Te rhythms in this paradigm are not known. Considerations include residual (ganirelix nonsuppressible) gonadal-axis activity, small daily variations in LH or Te kinetics, and/or day-night differences in testis responsiveness (1).

Several interpretative qualifications are relevant. First, the present design does not exclude the possibility that prolonged pulsatile infusions of rhLH could normalize Te secretion in older men. The paradigm implemented here validates a basis for more extended interventional studies. Second, age-related contrasts were accentuated when free and bioavailable Te concentrations were employed as end points. Third, although incremental and integrated LH pulse size during rhLH infusions did not differ by age, older men had higher mean, nadir, and absolute peak LH concentrations than young subjects. Whether such differences could induce subtle testis down-regulation is not known. Fourth, a theoretical possibility is that age might alter the postinfusion metabolism or biopotency of rhLH (54). However, biosynthetic LH appears to exert consistent stimulatory effects in various clinical applications (36, 37, 38). And, fifth, our demonstration of impaired Leydig cell responsiveness does not exclude other concomitant deficits in GnRH outflow or Te-negative feedback in older men (1).

In summary, healthy older men fail to achieve young adult-like Te concentrations in response to repeated iv pulses of rhLH infused over 2 d during inhibition of underlying LH secretion. The mechanisms include reduced basal, pulsatile, synchronous, and 24-h rhythmic Te secretion and lower total, bioavailable, and free Te concentrations.


   Acknowledgments
 
We thank Pat Roberts for performing the immunoassays, Kris Nunez for supporting manuscript preparation, and the GCRC nursing staff for conducting the research protocols under a Food and Drug Administration-approved investigator new drug assignment by the authors.


   Footnotes
 
This work was supported in part by Grants MO1 RR00585 to the General Clinical Research Center of Mayo Clinic and Foundation from the National Center for Research Resources (Rockville, MD) and RO1 AG023133 from the National Institutes of Health (Bethesda, MD). P.Y.T. was supported by a Mayo Institutional Award and P.Y.L. by a Neil Hamilton Fairley Research Fellowship from the National Health and Medical Research Council of Australia (Grant ID 262025).

Current address for P.Y.L.: Division of Endocrinology, Harbor-UCLA Medical Center, Torrance, California 90509-2910.

First Published Online July 19, 2005

Abbreviations: cross-ApEn, Cross-approximate entropy; hCG, human chorionic gonadotropin; rh, recombinant human; Te, testosterone.

Received April 25, 2005.

Accepted July 12, 2005.


   References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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