Effects of Dehydroepiandrosterone Therapy on Pubic Hair Growth ...

1184 Binder et al. DHEA Replacement in Young Females J Clin Endocrinol Metab, April 2009, 94(4):1182–1190
99.5%/aerosol 0.5%. For the assessment <strong>of</strong> compliance, unused capsules
were counted at each visit.
Efficacy assessment
In the current trial, we tested the hypothesis that oral administration
<strong>of</strong> 25 mg DHEA is able to promote impaired pubic hair growth (primary
endpoint) and to decrease psychological distress (secondary endpoint 1),
both caused by central adrenal insufficiency. In addition, normalization
<strong>of</strong> adrenal androgen levels in serum 2 h after drug intake and in a 24-h
urine collection were tested (secondary endpoint 2). Efficacy measurements
were performed at baseline, and at 6 and 12 months during
therapy.
The primary outcome measure with respect to efficacy was the proportion
<strong>of</strong> patients with a change to a more mature pubic hair stage from
baseline to month 12, as measured by Tanner stages. For clinical purposes,
Tanner has separated the pubic hair development into five stages
from stage PH1 (infantile) to stage PH5 (adult) (17) (Fig. 1). This staging
is routinely used by pediatricians. All five clinical investigators involved
are experienced pediatric endocrinologists who are very familiar with the
staging <strong>of</strong> pubic hair according to Tanner.
Secondary outcome measures were positive changes (calculated as
the difference <strong>of</strong> each score) in well-being and mood from baseline to
month 12 as measured by the German versions <strong>of</strong> the Symptom Check-
List-90-R (SCL-90-R) and <strong>of</strong> the Centre for Epidemiological Studies-
Depression Scale (CES-D). The SCL-90-R is a widely used, well-validated
psychological status symptom inventory that measures the subjectively
experienced distress by 90 given somatic and mental symptoms. The
individual tested reports the symptoms experienced during the last 7 d.
This inventory allows a multidimensional analysis and contains repeated
measurements. The German version <strong>of</strong> SCL-90-R is well established, and
reference data for schoolchildren and adolescents are available (18, 19).
The other test used, the CES-D, is one <strong>of</strong> the most frequently used and
well-validated self-report screening tool for depressive symptoms.
CES-D consists <strong>of</strong> 20 items assessing the frequency <strong>of</strong> depressive symp-
FIG. 1. Participant flow throughout the study.
toms during the preceding week. The German version <strong>of</strong> the CES-D is
well established, and reference data for schoolchildren and adolescents
are available (20, 21).
Additional secondary outcome measures were the change to normalization
<strong>of</strong> serum androgen levels (DHEAS, androstenedione, testosterone)
2 h after drug intake and <strong>of</strong> the amount <strong>of</strong> urinary excreted androgens
[DHEA, DHEA and M (16-hydroxylated downstream metabolites),
5-androstene-3,17-diol (ADIOL), C19 (total androgen metabolites)].
Serum samples were collected from the nonfasted patient in the morning
before 1000 h during each visit (0, 6, and 12 months), around 2 h after
oral administration <strong>of</strong> DHEA at visits 6 and 12 months. The samples
were collectively assayed at the end <strong>of</strong> the trial to exclude changes <strong>of</strong>
measurements due to interassay variability. Serum DHEAS levels were
measured using an automated chemiluminescence assay system (Immulite;
DPC Biermann GmbH, Bad Nauheim, Germany). Intraassay coefficients
ranged from 4.8–8.8%. Total serum testosterone was measured
by solid-phase 125J RIA in unextracted serum (Coat-A-Count total testosterone;
Diagnostic Products Corp., Los Angeles, CA). Cross-reactivity
to natural steroids was by far less than 1%, to 5-dihydrotestosterone
3%. The intraassay coefficient was 6.0% according to the manufacturer.
Androstenedione serum levels were determined by Active Androstenedione
RIA (Diagnostics Systems Laboratories, Sinsheim, Germany). Cross-reactivity
to natural androgens is less than 0.33%. The intraassay coefficient
was 5.6% according to the manufacturer. The measurements <strong>of</strong> androstanediol
glucuronide levels in serum were performed using androstanediol
glucuronide RIA DSL-6000 (Diagnostics Systems Laboratories).
Intraassay coefficients range from 4.2–8.2%. Reference values for the
hormone assays used were taken from three references (22–24). Serum
SHBG levels were determined using the Immulite 1000 SHBG assay
(Immulite). Intraassay coefficients ranged from 4.1–7.7% according to
the manufacturer.
Urinary pr<strong>of</strong>iling was performed at 0 and 12 months. Patients and
parents received instruction and written guidance to ensure compliance
in the 24 h-urine collection that was performed at home by each participant
before the visits. The urinary steroid pr<strong>of</strong>iles were determined using
quantitative data produced by gas chromatography (GC)-mass spectrometry
analysis according to the method described previously (25). In
brief, free and conjugated urinary steroids were extracted by solid-phase
extraction, and the conjugates were enzymatically hydrolyzed. Thereafter,
steroids were again recovered by solid-phase extraction and derivatized
to form methyloxime-trimethylsilyl ethers. GC was performed
using an Optima-1 fused silica column (Machery-Nagel, Dueren, Germany)
and helium as carrier gas. The GC (Agilent 6890 Series GC; Agilent
7683 Series Injector; Agilent Technologies, Inc., Palo Alto, CA) was
directly interfaced to a mass selective detector (Agilent 5973N MSD)
operated in selected ion monitoring mode.
Daily urinary excretion rates were determined for DHEA, ADIOL,
and the sum <strong>of</strong> DHEA and its 16-hydroxylated downstream metabolites
16-hydroxy-DHEA and 3,16,17-androstenetriol (DHEA
and M), reflecting major adrenarchal secretion products. Overall androgen
metabolite excretion (C19) was determined as the sum <strong>of</strong>
androsterone, etiocholanolone, ADIOL, DHEA, 16-hydroxy-
DHEA, and 3,16,17-androstenetriol.
Safety assessments
The clinical assessment performed at every visit included physical
examination with measurement <strong>of</strong> height, weight, blood pressure, serum
chemistry, and complete blood count. We especially looked for the occurrence
<strong>of</strong> acne and <strong>of</strong> hirsutism, which was quantified by the score
proposed by Ferriman and Gallwey (26). Adverse events were monitored
and recorded throughout the study according to Good Clinical Practice
guidelines.
Sample size
We estimated that the probability for a change <strong>of</strong> the pubic stage
would be 5% in the placebo group and 50% in the DHEA group, according
to the data from two small uncontrolled case studies on DHEAS