Androgen Synthesis in the Gonadotropin

ORIGINAL
E n d o c r i n e
ARTICLE
R e s e a r c h
Androgen Synthesis in the Gonadotropin-Suppressed
Human Testes Can Be Markedly Suppressed by
Ketoconazole
M. Y. Roth, J. J. S. Nya-Ngatchou, K. Lin, S. T. Page, B. D. Anawalt,
A. M. Matsumoto, B. T. Marck, W. J. Bremner, and J. K. Amory
Departments of Internal Medicine (M.Y.R., J.J.S.N.-N., 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.,
J.J.S.N.-N., S.T.P., B.D.A., A.M.M., W.J.B., J.K.A.), University of Washington, Seattle, Washington 98195;
and Geriatric Research, Education, and Clinical Center (A.M.M., B.T.M.), Veterans Affairs Puget Sound
Health Care System, Seattle, Washington 98108
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Context: The concentration of intratesticular testosterone (IT-T) required for human spermatogenesis is unknown because spermatogenesis can persist despite the markedly reduced IT-T concentrations observed with LH suppression. Methods to lower IT-T further are needed to determine
the relationship between IT-T and spermatogenesis.
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Objective: The objective of the study was to determine the effect of inhibiting the synthesis and
metabolism of testosterone (T) on IT-T in gonadotropin-suppressed human testes.
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Design/Setting/Patients: Forty normal men participated in a blinded, placebo-controlled, randomized trial at an academic center.
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Intervention/Outcome Measures: All men were first administered the GnRH antagonist acyline to
suppress LH. Forty-eight hours after acyline administration, subjects were randomly assigned to
placebo, ketoconazole (to inhibit T synthesis) at 400 or 800 mg, dutasteride (to inhibit T metabolism) 2.5 mg, or anastrazole (to inhibit T metabolism) 1 mg, daily for 7 days (n ⫽ 8/group).
Intratesticular steroid concentrations were measured 48 hours after acyline administration alone
and again after 7 days of combination treatment.
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Results: After 7 days of combination treatment, the median IT-T (25th, 75th percentile) in the
placebo group was 14 (8.0, 21.2) ng/mL. IT-T was reduced to 3.7 (2.5, 7.1) ng/mL in the ketoconazole
400 mg group and 1.7 (0.8, 4.0) ng/mL in the ketoconazole 800 mg group (P ⬍ .001 vs placebo for
both comparisons). IT-T concentrations in the dutasteride and anastrazole groups were similar to
placebo.
Conclusion: Combining inhibition of steroidogenesis with gonadotropin suppression lowers IT-T
more than gonadotropin suppression alone. This combination might be useful to determine the
minimum IT-T concentration necessary for human spermatogenesis, information essential for developing male hormonal contraceptives. (J Clin Endocrinol Metab 98: 1198 –1206, 2013)
ale hormonal contraceptive strategies, which rely
on the administration of exogenous testosterone
(T) to suppress the secretion of pituitary gonadotropins,
achieve azoospermia in 60%–70% of men (1, 2). Com-
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binations of progestins and T further suppress gonadotropins and achieve azoospermia in up to 90% of men.
Nevertheless, some men fail to completely suppress spermatogenesis on these regimens (3–5). Understanding why
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
Received October 4, 2012. Accepted December 21, 2012.
First Published Online January 24, 2013
Abbreviations: ADD, androstenedione; BMI, body mass index; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; E2, estradiol; IT, intratesticular; 17-OHP, 17-hydroxyprogesterone; T, testosterone.
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J Clin Endocrinol Metab, March 2013, 98(3):1198 –1206
doi: 10.1210/jc.2012-3527
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tosterone) to 22 ng/mL, a 95%–97% reduction from baseline concentrations (19). In this study, we hypothesized
that in the presence of profound gonadotropin suppression (induced by acyline), inhibition of testosterone synthesis with ketoconazole, an inhibitor of the 17,20 lyase
enzyme, (20, 21) would suppress, and inhibition of the
metabolism of testosterone, with the 5␣-reductase inhibitor dutasteride or the aromatase inhibitor anastrozole,
would increase IT-T, relative to gonadotropin suppression
alone. Therefore, we conducted a prospective, randomized, blinded, placebo-controlled, 5-arm interventional
study in normal men of the GnRH antagonist acyline
alone or in combination with ketoconazole (at 2 doses),
dutasteride, or anastrazole to determine the effect of these
medications on the concentration of IT-T and other intratesticular steroids.
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Subjects and Methods
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Subjects
Healthy men, aged 18 –50 years were recruited for this study
using newspaper and online advertisements. All subjects provided written informed consent prior to the screening evaluation.
Subjects were included if they had a normal history and physical
examination [body mass index (BMI) 19 –32 kg/m2], a normal
andrological history, a normal testicular volume measured by
Prader orchidometer, normal random cortisol, normal prostatespecific antigen, normal serum gonadotropins, and serum testosterone concentrations. Exclusion criteria included poor general health, history of liver disease or adrenal insufficiency, BMI
greater than 32 kg/m2, abnormal blood test results including
prostate-specific antigen greater than 4.0 ng/mL, active skin conditions that would prevent the use of testosterone gel, active
alcohol or drug abuse, a history of testicular or scrotal surgery,
chronic pain syndrome, use of glucocorticoids or medications
that are contraindicated with ketoconazole use (including terfenadine, astemizole, cisapride, budesonide, felodipine, fluticasone, lovastatin, midazolam, sildenafil, or vardenafil), a known
bleeding disorder, or the use of medications that affect bleeding
time (such as aspirin or warfarin). All subjects agreed to use a
reliable form of contraception during the study.
The study design is illustrated in Figure 1. Briefly, after enrollment, a baseline blood sample was obtained from all subjects
for serum hormones and safety laboratory tests prior to administration of acyline (NeoMPS, San Diego, California) 300 ␮g/kg
by sc injection. All subjects were administered T gel daily to
maintain normal serum T concentrations throughout the treatment period. Subjects were instructed in the proper use and application of 1% testosterone gel, Testim (Auxilium Pharmaceuticals Inc, Malvern, Pennsylvania) 5 g daily for 10 days. In
addition, the assessment of vital signs, adverse events, and concomitant medications occurred at every study visit.
Subjects returned on day 3 and were randomized to the side
of unilateral testicular fine-needle aspirations (right vs left on day
3 vs day 10) by random number sequence. Local anesthesia was
provided using 1% buffered lidocaine injected into the spermatic
cord. We obtained a peripheral blood sample for quantification
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some men fail to completely suppress spermatogenesis
despite profound inhibition of gonadotropin secretion
is a significant barrier to male hormonal contraceptive
development.
One possible explanation for this failure is that some
men on male hormonal contraceptives continue to have
low concentrations of intratesticular (IT) testosterone that
are permissive for spermatogenesis (6). The LH receptor
knockout mouse may be a useful model for understanding
this phenomenon because these animals produce functional sperm despite an absence of LH signaling (7). Interestingly, spermatogenesis in these animals can be completely abrogated by the addition of the androgen receptor
antagonist flutamide, suggesting that the process is androgen dependent. Similarly, in the rat quantitatively and
qualitatively normal spermatogenesis can occur despite
dramatic reductions in IT-T (8). In male hormonal contraceptive studies, some men continue to maintain spermatogenesis despite profound gonadotropin suppression,
which decreases IT-T by 95% (6). In these men, IT-T still
exceeds serum T concentrations. As a result, the quantitative relationship between low concentrations of IT-T
and spermatogenesis in humans has not been defined.
From the standpoint of the development of male hormonal
contraceptives, it is possible that greater reductions in
IT-T may result in more consistent suppression of spermatogenesis and higher rates of azoospermia.
In addition to IT-T, intratesticular dihydrotestosterone
(DHT) and estradiol (E2) may play a role in the maintenance of spermatogenesis in the low IT-T environment
created by male hormonal contraceptive regimens (6, 9).
For example, some men have a dramatic drop in sperm
concentrations associated with the chronic administration
of 5␣-reductase inhibitors such as finasteride and dutasteride (10), suggesting that spermatogenesis in some men
might depend on intratesticular DHT. Conversely, aromatase inhibitors that block the metabolism of T to E2 have
been used for the treatment of male infertility (11–13), and
high serum E2 concentrations have been associated with suppression of gonadotropins and spermatogenesis (14). Establishing a method for suppressing DHT and E2 concentrations within the testis could lead to a greater understanding
of the differential roles of these hormones on spermatogenesis in men and might help in the development of more effective male hormonal contraceptives.
In our previous work, we have examined the effect of
gonadotropin suppression on IT-T using the administration of acyline, a GnRH antagonist (15), coupled with
testicular fine-needle aspiration to obtain intratesticular
fluid for the measurement of IT-T (16 –19). Acyline administration alone decreases IT-T from a median of 715
ng/mL (more than 100 times serum concentrations of tes-
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Roth et al
Human Intratesticular Steroidogenesis
J Clin Endocrinol Metab, March 2013, 98(3):1198 –1206
consin). DHT 16,16,17-d3 and estradiol
16,16,17-d3 were purchased from
CDN Isotopes (Que´bec, Canada). DHEA
2,2,3,4,4,6-d6 was purchased from
Unilateral testicular fine-needle
Cambridge Isotope Laboratory (Anaspiration
dover, Massachusetts). Androstenedione 19,19,19-d3 was purchased from
Daily oral medication
BDG Synthesis (Wellington, New
Group 1: placebo pill
Zealand).
Group 2: ketoconazole 400mg
Group 3: ketoconazole 800mg
Testicular aspirates were immediGroup 4: dutasteride 2.5mg
Follow-up visits
ately placed on ice, centrifuged at 300 ⫻
Group 5: anastrazole 1mg
g, and the supernatant fluid was stored at
⫺70°C. All testicular fluid and serum
samples were assayed for T, DHT, ADD,
Day: 1 2 2 3 3 4 55 6 67 87 9 8 10 9 1017
Day:1
17
40
40
DHEA, and estradiol by liquid chromatography-tandem mass spectrometry on
Testosterone gel 5gm daily
an AB Sciex 5500 QTRAP mass spectrometer (AB Sciex, Framingham, MasFigure 1. Study design. SC, subcutaneous.
sachusetts) using a slight modification of
our previously described method (18,
of serum hormones 2–10 minutes prior to the aspiration. After
22). The intra- and interassay coefficients of variation were 3.5%
the procedure, all subjects were randomized using a random
and 7.7% for testosterone and were 3.5% and 6.3% for DHT.
number sequence by the investigational pharmacists to 1 of 5 oral
The assay sensitivities for serum and IT androgens (T, DHT,
medication groups: group 1 received a placebo pill, group 2 reADD, and DHEA) were less than 10 pg/mL.
ceived ketoconazole 400 mg (Teva Pharmaceuticals, Petah
For the estradiol assay, 25 ␮L of estradiol 16,16,17-d3 (2
Tikva, Israel), group 3 received ketoconazole 800 mg, group 4
ng/mL) diluted in methanol-water (1:1, vol/vol) was added to
received dutasteride 2.5 mg (GlaxoSmithKline, London, United
either 100 ␮L of serum or 100 ␮L of diluted testicular extract in
Kingdom), and group 5 received anastrazole 1 mg (AstraZeneca,
a 13- ⫻ 100-mm screw cap glass tube. Using a screw cap with a
London, United Kingdom), daily for 7 days. Subjects were blinded to
polytetraflouroethylene-faced rubber liner, samples were briefly
their oral treatment group, except those in group 3, who required advortexed and allowed to sit at room temperature for 15 minutes
ditional procedures at the day 10 and day 40 visits.
prior to extraction twice with 2.5 mL of hexane-ethyl acetate
On day 10, we performed a testicular fine-needle aspiration of
(80:20, vol/vol) by slow rotational mixing for 15 minutes. Solthe testis contralateral to the testis aspirated on day 3 and obtained
vent phase was removed after centrifugation for 15 minutes
a peripheral blood sample for quantification of serum hormone
(2800 rpm) and steroid extract evaporated to dryness in a Speedconcentrations 2–10 minutes prior to the aspiration. We have prevac. Steroid extract was vortexed with an additional 0.3 mL of
viously demonstrated a high correlation between intratesticular
hexane-ethyl acetate (80:20, vol/vol) and evaporated to dryness
hormone concentrations in a given man; therefore, we aspirated the
in a Speedvac prior to derivatization. Extracted steroids were
contralateral testis on day 10 to avoid repeat aspiration to the predissolved in 50 ␮L of sodium bicarbonate buffer (100 mmol/L,
viously sampled testis within a short period of time (18, 19).
pH 10.5) and derivatized with 50 ␮L of dansyl chloride (1 g/L in
Subjects in group 3 receiving 800 mg ketoconazole daily comacetone) in a capped 12- ⫻ 75-mm glass tube in a 60oC water
pleted a cosyntropin stimulation test immediately after the tesbath for 10 minutes. After centrifugation for 5 minutes, the derivaticular aspiration on day 10 to assess for the possibility of adrenal
tized sample was transferred to autosampler vial with a clear glass
suppression by high-dose ketoconazole. Subjects received 0.25
conical 300 ␮L insert for liquid chromatography with tandem mass
mg cosyntropin (Amphastar Pharmaceuticals Inc, Rancho Cuspectrometry analysis. For the measurement of serum estradiol, the
camonga, California) injected im into the deltoid muscle, follower limit of detection was 5 pg/mL and the intraassay coefficient
lowed by a repeat blood draw for serum cortisol 30 and 60
of variation was 24%. Unfortunately, we were unable to accurately
minutes after the injection. These subjects had a repeat cosynquantitate the concentration of intratesticular estradiol from our
tropin stimulation test at the day 40 visit.
limited samples of intratesticular fluid. Therefore, only serum esSubjects returned on day 17 and day 40 for a physical examtradiol concentrations are presented.
ination and blood sampling to confirm that their physical exSerum LH and FSH were quantified by immunofluorometric
aminations, serum hormones, and safety laboratory measureassay (6). Assays were run in duplicate and used 100 ␮L of serum.
ments had returned to normal. The Institutional Review Board
The sensitivity of the LH assay was 0.019 IU/L, and the intra- and
of the University of Washington approved this study protocol
interassay coefficients of variation for a midrange pooled value
prior to study initiation. This trial was registered in advance
of 1.2 IU/L was 3.2% and 12.5%, respectively. The sensitivity of
[www.clinicaltrials.gov (National Clinical Trial number
the FSH assay was 0.016 IU/L, and the intra- and interassay
01215292)].
coefficients of variation were 2.9% and 6.1% for a midrange
pooled value of 0.96 IU/L. Serum 17-hydroxyprogesterone (hyMeasurements
pothesized to be increased in the setting of inhibition of the 17,20
lyase by ketoconazole) was measured in duplicate by RIA (SieTestosterone, DHT, dehydroepiandrosterone (DHEA), andromens Healthcare Diagnostics, Deerfield, Illinois) using 25 ␮L of
stenedione(ADD),andestradiolusedforthestandardswerepurchased
serum with intra- and interassay coefficients of variation of 5.6%
from Cerilliant (Round Rock, Texas). Testosterone 16,16,17-d3
was purchased from Sigma-Aldrich Chemicals (Milwaukee, Wisand 6.4%, respectively, and a lower limit of detection of less than
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Acyline injection 300 mcg/kg SC
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doi: 10.1210/jc.2012-3527
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Results
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Subjects
Fifty-two men were screened for the study and 46 met
all inclusion criteria. Six subjects withdrew from the study
prior to any procedures, and 40 were randomized to the
study (n ⫽ 8/group). All 40 subjects who underwent randomization completed all of the study procedures. Of the
6 men who failed to meet the screening criteria for the
study, 2 subjects had an abnormal genital examination, 1
subject had undergone a prior vasectomy, 1 subject exceeded the BMI criteria, 1 had previously undiagnosed
severe hypertension, and 1 had an unstable psychiatric
disorder. Of the 40 subjects randomized, 31 were Caucasian (27 non-Hispanic and 4 Hispanic), 4 were Asian, 3
were African American, 1 was a Pacific Islander, and 1 was
an American Indian.
One serious adverse event occurred during the study,
involving the development of a testicular hematoma and
scrotal hematocele after a subject’s second testicular aspiration procedure on day 10. This subject underwent operative evacuation of the hematocele and cauterization of
a superficial testicular vessel. Follow-up ultrasounds
showed complete resolution of the testicular hematoma
and hematocele. The subject completed all study procedures and was included in the analysis. In addition, 18
subjects reported 21 nonserious adverse events (Supplemental Table 1, published on The Endocrine Society’s
Journals Online web site at http://jcem.endojournals.org).
All adverse events resolved by study completion. Median
testicular aspirate volume was 17 ␮L at day 3 and 20 ␮L
at day 10 (P ⫽ .7). Testicular aspirate volume did not differ
between treatment groups on day 3 or day 10 (P ⫽ .2 and
P ⫽ .8, respectively) (Supplemental Table 2). Six of the 8
subjects randomized to the ketoconazole 800 mg daily
group had an abnormal cosyntropin stimulation test (defined as peak serum cortisol ⬍ 18 ng/dL) on the day 10
Intratesticular hormones
Forty-eight hours after acyline administration, IT-T in
the group of 40 men was 30 (21, 39) ng/mL. This represents a 96% reduction from the baseline median IT-T concentration for normal men of 715 (486, 1000) ng/mL (15).
There were no significant differences in IT-T between
treatment groups on day 3 (data not shown). On day 10,
after 7 days of medications, IT-T decreased further to a
median of 14 (8.0, 21.2) ng/mL in the placebo group, 3.7
(2.6, 7.2) ng/mL in the 400 mg ketoconazole group, and
1.7 (0.9, 4) ng/mL in the 800 mg ketoconazole group (P ⬍
.001 for both comparisons with placebo) (Figure 2A). The
IT-T concentrations were not significantly different in the
groups receiving dutasteride or anastrazole compared
with placebo and not significantly different from day 3
IT-T concentrations.
IT-DHT decreased with acyline administration to a median concentration of 2.3 (1.5, 3.3) ng/ml, compared with
a normal baseline median IT-DHT of 3.5 (2, 6.1) ng/mL
(19). IT-DHT did not differ between treatment groups at
day 3 (data not shown). After 7 days of medication, ITDHT decreased by 95% in the group receiving dutasteride
to a median of 0.12 (0.09, 0.15) ng/mL, which was significantly lower compared with all other treatment groups
(P ⬍ .05) (Figure 2B).
IT-ADD decreased with acyline administration to a median concentration of 4.9 (2.8, 10.7) ng/mL, compared
with an expected baseline of 179 (88, 246) ng/mL (23).
IT-ADD did not differ between treatment groups after
acyline administration at day 3 (data not shown), but after
10 days, IT-ADD decreased significantly compared with
day 3 in all groups except for those receiving anastrazole.
In addition, IT-ADD in the groups receiving ketoconazole
were significantly suppressed as compared with the other
oral treatment groups with a median of 0.5 (0.3, 0.7)
ng/mL for the ketoconazole 400 mg group and 0.12 (0.1,
0.5) ng/mL for the ketoconazole 800 mg group (Figure
2C). There was no significant difference in the IT-ADD
concentration at day 10 between the ketoconazole groups.
IT-DHEA decreased with acyline administration from
normal median baseline concentration of 162 (118, 253)
ng/mL (23) to 5.3 (3.3, 8.4) ng/mL 48 hours after receiving
acyline. IT-DHEA did not differ between treatment
groups at day 3 (data not shown). After 7 days of oral
ketoconazole (both the 400 and 800 mg groups), IT-DHEA
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Serum and intratesticular hormone concentrations from all
40 subjects were included in the final analysis. Due to nonnormal
distribution, the data are expressed as medians and 25th and
75th percentiles. Comparisons of hormone concentrations between groups were performed using a Kruskal-Wallis ANOVA
with a Wilcoxon rank-sum post hoc test. Comparisons of hormone concentrations within a group were made using a Wilcoxon signed-rank test. No corrections were made for multiple
comparisons. All statistical analyses were performed using
STATA version 10.0 (College Park, Texas). For all comparisons,
an alpha ⬍.05 was considered significant.
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Statistical analysis
visit. On day 10, the median (25th and 75th percentiles)
serum cortisol was 13.5 (10.8, 15.5) ng/dL at 30 minutes
and 14.6 (12.2, 17.8) ng/dL at 60 minutes after cosyntropin stimulation. All 8 subjects in this group had normal
cosyntropin stimulation tests on day 40 (Supplemental
Figure 1).
or
1 ng/mL. All samples for all subjects were batched and measured
in a single assay.
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Human Intratesticular Steroidogenesis
J Clin Endocrinol Metab, March 2013, 98(3):1198 –1206
B
215
22
8
140
7
120
6
IntraTesticular
DHT 5
(ng/ml)
100
Intra- 80
Testicular
Testosterone
(ng/ml) 60
4
3
40
2
*
*
20
1
**
Acyline
Day 3
(n=40)
Acyline + Acyline +
Placebo K-400mg
(n=8)
(n=8)
Acyline
Day 3
(n=40)
Acyline + Acyline + Acyline +
K-800mg Dutasteride Anastrazole
(n=8)
(n=8)
(n=8)
D
65
or
Day 10 Post-Treatment
C
Acyline + Acyline + Acyline + Acyline +
Acyline +
Placebo K-400mg K-800mg Dutasteride Anastrazole
(n=8)
(n=8)
(n=8)
(n=8)
(n=8)
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0
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40
40
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30
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20
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IntraTesticular 15
Androstenedione
(ng/ml)
10
35
30
25
IntraTesticular
DHEA 20
(ng/ml)
15
10
†
*
5
†
*
5
*
*
0
Acyline
Day 3
(n=40)
Acyline + Acyline +
Placebo K-400mg
(n=8)
(n=8)
Acyline + Acyline + Acyline +
K-800mg Dutasteride Anastrazole
(n=8)
(n=8)
(n=8)
Day 10 Post-Treatment
0
Acyline
Day 3
(n=40)
Acyline + Acyline +
Placebo K-400mg
(n=8)
(n=8)
Acyline + Acyline + Acyline +
K-800mg Dutasteride Anastrazole
(n=8)
(n=8)
(n=8)
Day 10 Post-Treatment
Figure 2. A–D, Box plots of IT-T (A), IT-DHT (B), IT-ADD (C), and IT-DHEA (D) in gonadotropin-suppressed subjects on day 3 and day 10 by
treatment group. Baseline median (25th and 75th percentiles) IT-T concentration for normal men is 715 (486, 1000) ng/mL (19). The baseline
median IT-DHT concentration for normal men is shown in gray shaded area (19). The baseline median IT-ADD and IT-DHEA concentrations for
normal men are 179 (88, 246) ng/mL and 162 (118, 253) ng/mL, respectively (23). K400, ketoconazole 400 mg; K800, ketoconazole 800 mg; *
P ⬍ .05 compared with day 3 and all nonketoconazole treatment groups;**P ⬍ .05 compared with day 3 and all other treatment groups; †P ⬍
.05 compared with day 3.
decreased significantly compared with day 3 and compared
with treatment groups that did not receive ketoconazole to a
median of 1.2 (0.8, 1.4) ng/mL in the 400 mg ketoconazole
group and 0.5 (0.4, 0.7) ng/mL in the 800 mg ketoconazole
group (Figure 2D). There was no significant difference in
IT-DHEA between the ketoconazole groups at day 10.
Serum hormones
Serum gonadotropins decreased significantly with the
administration of acyline. Median serum LH concentrations decreased to the lower limit of detection for all
groups by 48 hours after acyline administration and remained less than 1 IU/L for subjects in all treatment groups
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Table 1. Baseline Characteristics and Serum Hormones of 40 Participants by Treatment Group 关Median (25th, 75th
Interquartile Range)兴
Acyline ⴙ Pla
(n ⴝ 8)
Age, y
BMI, kg/m2
Serum hormones
T, ng/mL
DHT, ng/mL
E2, pg/mL
ADD, ng/mL
DHEA, ng/mL
17-OHP, ng/mL
LH, IU/L
FSH, IU/L
Acyline ⴙ K400
(n ⴝ 8)
Acyline ⴙ K800
(n ⴝ 8)
Acyline ⴙ Dut
(n ⴝ 8)
Acyline ⴙ Ana
(n ⴝ 8)
All Subjects
(n ⴝ 40)
24 (21, 35.5)
24.1 (21.9, 26)
22.5 (20, 26)
22.5 (21.6, 25.6)
22 (20.5, 25.5)
24 (21.5, 25.3)
21.5 (20, 24)
24 (23.6, 26.3)
21.5 (20.5, 24.5)
25 (24, 27)
22 (20, 26)
24.3 (22.3, 25.8)
5.2 (4.6, 6.3)
0.5 (0.37, 0.59)
28.3 (21.3, 39.6)
0.76 (0.52, 1.12)
4.2 (3.3, 6.5)
7.2 (5.9, 9.5)
4.3 (3.3, 5.2)
2.7 (2.3, 2.9)
5.5 (5.2, 6.2)
0.44 (0.35, 0.54)
39 (16.5, 65.8)
0.7 (0.57, 1.1)
3.9 (2.7, 7.1)
6 (4.2, 7.9)
5 (3.1, 6.6)
2.3 (1.5, 3.5)
5.3 (4.7, 6.3)
0.44 (0.35, 0.53)
39 (23.4, 69.4)
0.69 (0.59, 0.76)
4.6 (2.8. 5.9)
4.8 (4.2, 6.7)
5.8 (4.7, 7.4)
2.4 (1.7, 2.8)
5.4 (4.4, 6.4)
0.5 (0.43, 0.89)
24 (20.9, 30)
0.75 (0.5, 1.1)
3.9 (3.2, 6.6)
6.1 (5.1, 9.1)
4.7 (4.2, 5.4)
2.7 (1.8, 3.0)
5.2 (4.2, 5.8)
0.42 (0.3, 0.5)
39.4 (21.1, 51.2)
0.65 (0.6, 0.81)
3.8 (2.8, 4.4)
6 (4.6, 9)
4.9 (4.2, 6.6)
2.3 (1.4, 3)
5.3 (4.8, 6.2)
0.46 (0.36, 0.56)
28.3 (20.9, 53.9)
0.67 (0.57, 0.95)
3.9 (2.9, 6.1)
6.1 (4.7, 8.1)
4.8 (3.9, 6.4)
2.6 (1.7, 3)
Abbreviations: Ana, anastrazole 1 mg; Dut, dutasteride 2.5 mg; K400, ketoconazole 400 mg; K800, ketoconazole 800 mg; Pla, placebo.
CD
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nificant changes in androstenedione or DHEA during
treatment.
or
Discussion
We have demonstrated that the combination of gonadotropin suppression with acyline and inhibition of testosterone biosynthesis with ketoconazole significantly decreases IT-T concentrations more than gonadotropin
suppression alone. This finding suggests that Leydig cells
continue to synthesize testosterone at low concentrations
despite GnRH antagonist treatment and marked suppression of LH. Whether this testosterone synthesis occurs in
response to very low concentrations of LH or is LH independent is unknown. Our previous studies and the work
of others have shown that gonadotropin suppression decreases IT-T concentrations by 90%–95% from baseline
(19, 24). Our current study demonstrates that the combination of acyline and ketoconazole, at a commonly used
treatment dose, can reduce IT-T greater than 99%. Such
concentrations of IT-T are at the low end of the normal
range for serum testosterone and might be associated with
significantly decreased androgen activity in the testes and
greater suppression of spermatogenesis. In the future, to
understand the relationship between intratesticular testosterone and spermatogenesis, we plan to study the combination of acyline and ketoconazole for 4 – 6 months in
normal men. This study will include repeated analyses of
semen and should help determine whether a threshold
concentration necessary for spermatogenesis exists in
man. Such a threshold has been suggested by experiments
in rodents. For example, Zirkin et al (8) studied the relationship between IT-T and spermatogenesis and found
that IT-T concentrations below 5% of baseline were unable to support spermatogenesis. Similar results were obtained by Singh and Handelsman using the gonadotropindeficient hpg mouse model (25).
Co
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at day 10 (Supplemental Figure 2A). Median serum FSH
concentrations decreased to 1 IU/L for all treatment
groups by 48 hours after acyline administration and continued to decrease significantly in all groups at day 10. In
addition, serum FSH decreased significantly in the 2
groups receiving ketoconazole as compared with the other
treatment groups (Supplemental Figure 2B).
Baseline serum hormones and gonadotropins are presented in Table 1. There were no differences between treatment groups at baseline. Serum testosterone decreased
from baseline in all groups after receiving acyline despite
administration of testosterone gel, reaching statistical significance in the placebo group (Figure 3A). However, all
subjects had normal serum testosterone concentrations on
both study day 3 and day 10. In the groups receiving ketoconazole, there were no significant differences between
the serum T and IT-T on day 10. In addition, no statistical
correlation was found between serum T and IT-T at day 10
in any groups.
Serum DHT increased significantly in all subjects after
acyline and testosterone gel administration and remained
elevated compared with baseline in all groups except the
group receiving oral dutasteride, in whom serum DHT
was significantly reduced at day 10 (Figure 3B). Serum
DHT did not differ between day 3 and day 10 for all other
treatment groups. Serum estradiol decreased in all
groups, but the group receiving anastrazole was not
significantly lower compared with the other treatment
groups (Figure 3C).
Serum 17-hydroxyprogesterone (17-OHP) decreased
significantly in all treatment groups at 48 hours and then
rose significantly in the 2 groups receiving ketoconazole as
compared with the other treatment groups (Figure 2D).
Serum androstenedione increased significantly in the dutasteride group, and DHEA was significantly decreased in
the group receiving high-dose ketoconazole (Supplemental Figure 3, A and B), but otherwise there were no sig-
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1204
Roth et al
Human Intratesticular Steroidogenesis
J Clin Endocrinol Metab, March 2013, 98(3):1198 –1206
Acyline + Testosterone Gel daily
B
A
8
Ketoconazole, Dut, Ana or Placebo
Acyline + Testosterone Gel daily
2
*
Ketoconazole, Dut, Ana or Placebo
6
*
1.5
Serum
Testosterone
(ng/ml)
4
Serum
DHT
(ng/ml)
*
*
*
*
*
*
1
*
Acyline + Pla
Acyline + K400
Acyline + K800
Acyline + Dut
Acyline + Ana
2
0
1
3
Acyline + Pla
Acyline + K400
Acyline + K800
Acyline + Dut
Acyline + Ana
0.5
0
10
*
**
1
3
Treatment Day
CD
R
Treatment Day
D
Acyline + Testosterone Gel daily
Ketoconazole, Dut, Ana or Placebo
3.5
da
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50
or
C
3
ut
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iza
40
30
Serum
Estradiol
(pg/ml)
aa
*
20
1
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Acyline + Pla
Acyline + K400
Acyline + K800
Acyline + Dut
Acyline + Ana
10
0
pi
*
3
10
Ketoconazole, Dut, Ana or Placebo
Acyline + Pla
Acyline + K400
Acyline + K800
Acyline + Dut
Acyline + Ana
*
*
*
*
1.5
**
*
*
*
*
*
1
Treatment Day
*
**
2.5
Serum
17-OHP 2
(ng/ml)
10
Acyline + Testosterone Gel daily
0.5
1
**
*
3
10
Treatment Day
Figure 3. A–D, Serum T (A), DHT (B), E2 (C), and 17-OHP (D) at baseline, 48 hours after acyline administration (day 3), and 1 week after oral
medication administration (day 10) by treatment group. Dashed lines represent the normal reference range. Ana, anastrazole 1 mg; Dut,
dutasteride 2.5 mg; K400, ketoconazole 400 mg; K800, ketoconazole 800 mg; Pla, placebo. *P ⬍ .05 compared with baseline; **P ⬍ .05
compared with all other treatment groups at day 10.
Both serum and IT-DHT were suppressed significantly
in the group receiving dutasteride, but IT-DHT did not
significantly suppress in the other groups and serum DHT
concentrations actually increased significantly in the other
groups, likely due to the use of testosterone gel, which is
known to increase serum DHT due to 5␣-reduction in the
skin. This study is the first to demonstrate the dramatic
effect of a 5␣-reductase inhibitor, dutasteride, on IT-DHT
concentrations in gonadotropin-suppressed normal men
and contributes to our understanding of the impact of this
drug on intratesticular hormone physiology in men. Unfortunately, we were unable to ascertain a similar effect on
intratesticular estradiol due to technical difficulties performing high-quality measurements of estradiol on the
limited sample volumes obtained by testicular aspiration.
The idea that differences in intratesticular DHT may account for differences in the response to male hormonal
contraceptives was first suggested in 1996 (9). However,
the addition of a 5␣-reductase inhibitor to male hormonal
contraceptive regimens does not significantly improve the
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doi: 10.1210/jc.2012-3527
jcem.endojournals.org
studies designed to determine the minimum concentration
of IT-T necessary for human spermatogenesis, information essential for the further development of male hormonal contraceptives.
Acknowledgments
or
CD
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Address all correspondence and requests for reprints to:
Mara Y. Roth, MD, University of Washington, 1959 NE Pacific Street, Box 357138, Seattle, Washington 98195. E-mail:
[email protected].
This work was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development
through Cooperative Agreement U54 HD-42454 as part of the
Cooperative Contraceptive Research Centers Program. M.Y.R.
is supported, in part, by the Eunice Kennedy Shriver National
Institute of Child Health and Human Development Grant K12
HD053984. J.J.S.N. is supported by the National Institute of
Diabetes and Digestive and Kidney Diseases training grant 5T32
DK007247-35. A.M.M. is supported by the Department of Veterans Affairs.
Disclosure Summary: The authors have nothing to disclose.
da
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rates of suppression of spermatogenesis (26). In contrast,
some men can have dramatic suppression of spermatogenesis with use of a 5␣-reductase inhibitor alone (10).
DHT appears to have a highly variable intraindividual
effect on spermatogenesis, and the potential role for DHT
in supporting spermatogenesis in men with low IT-T remains unknown.
In addition to suppressing IT-T, both doses of ketoconazole significantly suppressed IT-ADD and ITDHEA. This finding is compatible with the known inhibition of the 17,20 lyase by ketoconazole. Similarly,
both groups receiving ketoconazole exhibited significant increases in serum 17-OHP. Interestingly, despite
the marked reduction in IT-ADD, we did not see a significant suppression of serum ADD concentrations with
ketoconazole, possibly due to some conversion of the
exogenously administered T gel back into ADD by 17hydroxysteroid dehydrogenase.
Our study had some limitations. The omission of corrections for multiple comparisons in the statistical analysis
increased the risk of a type I error. In addition, the short
duration of treatment did not allow for determination of
the impact of reducing IT-T on spermatogenesis and limited our ability to determine whether there may have been
adverse effects associated with longer treatment. Lastly,
progestogens are commonly used in male hormonal contraceptive regimens but were not included in our study.
Therefore, we cannot comment on the effect of progestogens on IT-T. This will be the focus of future research.
The study protocol was well tolerated by most men,
except for the subject who experienced bleeding from
the testicular fine-needle aspiration, a rarely reported
complication of this procedure (27). Ketoconazole has
been reported to cause liver inflammation in 0.1%–1%
of patients (28), yet we observed no evidence of liver
inflammation in our subjects. Notably, however, the
800-mg dose mildly suppressed the response to cosyntropin stimulation, indicating a potential long-term
safety concern. For this reason, dexamethasone is administered when ketoconazole is used in treatment of
prostate cancer (29, 30), but this approach would be
unacceptable for healthy men for the purposes of male
contraception. In contrast to ketoconazole, which inhibits the 17,20 lyase, an inhibitor of type III 17␤-hydroxysteroid dehydrogenase may provide more effective suppression of T synthesis without the need for
concomitant glucocorticoid administration (32).
In summary, our results demonstrate that the combination of LH suppression and inhibition of T synthesis or
metabolism can dramatically reduce intratesticular concentrations of sex steroids. In particular, combinations of
acyline and ketoconazole may prove to be useful in future
1205
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