Hyperandrogenemia and Altered Day-night LH Pulse Patterns

This is a randomized, placebo-controlled, double-blinded crossover study to test the
following hypothesis: In mid- to late pubertal girls with hyperandrogenism (HA), acute
progesterone suppression of waking LH pulse frequency is greater after 2 weeks of
spironolactone pretreatment compared to after 2 weeks of placebo pretreatment. We will only
study mid- to late pubertal girls with HA (i.e., girls who would be candidates for
therapeutic spironolactone use). Subjects will complete two 18-hour Clinical Research Unit
(CRU) admissions in separate menstrual cycles. Subjects will be randomized to be pretreated
for 2 weeks with either oral spironolactone (50 mg twice daily) or placebo prior to the first
CRU admission. Immediately before and during each CRU admission, oral micronized progesterone
(0.8 mg/kg/dose) will be given at 0700, 1500, 2300, and 0700 h. During each CRU admission,
blood will be obtained every 10 minutes through an indwelling iv catheter from 1800 to 1200
h. This will allow full characterization of pulsatile LH secretion in addition to other
hormone measurements. Formal polysomnography will be performed during CRU admissions. A
second CRU admission (performed at least 2 months later given blood withdrawal limits) will
be identical to the first except that placebo pretreatment will exchanged for spironolactone
pretreatment or vice versa (treatment crossover). The primary endpoint is LH pulse frequency
while awake. (LH pulse frequency while asleep is an important secondary endpoint.) The wake
LH pulse frequency data from the spironolactone and placebo admissions will be analyzed via a
hierarchical linear mixed model (HLMM). The admission (spironolactone vs. placebo) will
represent the fixed effect factor of the HLMM. Random effects will be utilized to account for
the hierarchical variance-covariance structure of the two-period cross-over design. Wake LH
pulse frequency in response to exogenous progesterone will be compared between the
spironolactone admission and the placebo admission via a linear contrast of the HLMM least
squares LH pulse frequency means. A similar analysis will be performed for sleep-related LH
pulse frequency. Using published and preliminary data, we determined that, if 16 mid- to late
pubertal girls with HA complete both admissions, we should have at least an 80% chance of
detecting a 0.35 pulse/hour mean within-subject difference in wake LH pulse frequency between
the spironolactone and placebo admissions with a two-sided false positive rejection rate of
no more than 0.05.

Inclusion Criteria:

- Mid- to late pubertal adolescent girl (at least Tanner breast stage 3, but no more
than 2 years postmenarcheal)

- Hyperandrogenism, defined as a serum (calculated) free testosterone concentration
greater than the Tanner stage-specific reference range and/or unequivocal evidence for
hirsutism

- General good health (excepting overweight, obesity, hyperandrogenism, and
adequately-treated hypothyroidism)

- Capable of and willing to provide informed assent (adolescents under age 16 years)
and/or consent (adolescents over age 16 years; custodial parents or guardians of all
adolescent volunteers)

- Willing to strictly avoid pregnancy with use of reliable non-hormonal methods during
the study period

Exclusion Criteria:

- Inability/incapacity to provide informed consent

- Males will be excluded (hyperandrogenism is unique to females)

- Obesity resulting from a well-defined endocrinopathy or genetic syndrome

- Positive pregnancy test or current lactation

- Evidence for non-physiologic or non-PCOS causes of hyperandrogenism and/or anovulation

- Evidence of virilization (e.g., rapidly progressive hirsutism, deepening of the voice,
clitoromegaly)

- Total testosterone > 150 ng/dl, which suggests the possibility of virilizing ovarian
or adrenal tumor

- DHEA-S elevation > 1.5 times the upper reference range limit. Mild elevations may be
seen in adolescent HA and in PCOS, and will be accepted in these groups.

- Early morning 17-hydroxyprogesterone > 200 ng/dl measured in the follicular phase,
which suggests the possibility of congenital adrenal hyperplasia (if elevated during
the luteal phase, the 17-hydroxyprogesterone will be repeated during the follicular
phase). NOTE: If a 17-hydroxyprogesterone > 200 ng/dl is confirmed on repeat testing,
an ACTH stimulated 17-hydroxyprogesterone < 1000 ng/dl will be required for study
participation.

- Abnormal thyroid stimulating hormone (TSH): Note that subjects with stable and
adequately treated primary hypothyroidism, reflected by normal TSH values, will not be
excluded.

- Hyperprolactinemia > 20% higher than the upper limit of normal. Mild prolactin
elevations may be seen in adolescents and women with HA/PCOS, and elevations within
20% higher than the upper limit of normal will be accepted in this group.

- History and/or physical exam findings suggestive of Cushing's syndrome, adrenal
insufficiency, or acromegaly

- History and/or physical exam findings suggestive of hypogonadotropic hypogonadism
(e.g., symptoms of estrogen deficiency) including functional hypothalamic amenorrhea
(which may be suggested by a constellation of symptoms including restrictive eating
patterns, excessive exercise, psychological stress, etc.)

- Persistent hematocrit < 36% and hemoglobin < 12 g/dl.

- Severe thrombocytopenia (platelets < 50,000 cells/microliter) or leukopenia (total
white blood count < 4,000 cells/microliter)

- Previous diagnosis of diabetes, fasting glucose > or = 126 mg/dl, or a hemoglobin A1c
> or = 6.5%

- Persistent liver panel abnormalities, with two exceptions. Mild bilirubin elevations
will be accepted in the setting of known Gilbert's syndrome. Also, mild transaminase
elevations may be seen in obesity/HA/PCOS; therefore, elevations < 1.5 times the upper
limit of normal will be accepted in this group.

- Significant history of cardiac or pulmonary dysfunction (e.g., known or suspected
congestive heart failure, asthma requiring intermittent systemic corticosteroids,
etc.)

- Decreased renal function evidenced by GFR < 60 ml/min/1.73m2

- A personal history of breast, ovarian, or endometrial cancer

- History of any other cancer diagnosis and/or treatment (with the exception of basal
cell or squamous cell skin carcinoma) unless they have remained clinically disease
free (based on appropriate surveillance) for five years

- History of allergy to micronized progesterone or spironolactone

- Body mass index (BMI)-for-age percentile < 5% (underweight)

- Due to the amount of blood being drawn, adolescent volunteers with body weight < 25 kg
will be excluded.

- Restrictions on use of other drugs or treatments: No medications known to affect the
reproductive system, glucose metabolism, lipid metabolism, or blood pressure can be
taken in the 2 months prior to the screening visit and in the 3 months prior to the
start of the study medications. Such medications include oral contraceptive pills,
progestins, metformin, systemic glucocorticoids, some antipsychotic medications, and
sympathomimetics/stimulants (e.g., methylphenidate).