Acute Progesterone Suppression of Wake vs. Sleep Luteinizing Hormone Pulse Frequency in Pubertal Girls With and Without Hyperandrogenism

This is a randomized, placebo-controlled, double-blinded crossover study to test the
following hypotheses: (1) In normal mid- to late pubertal girls without hyperandrogenism
(HA), progesterone acutely suppresses waking LH pulse frequency more than sleep-associated LH
pulse frequency; and (2) compared to normal mid- to late pubertal girls without HA, acute
progesterone suppression of waking LH pulse frequency is impaired in mid- to late pubertal
girls with HA. Studies will be performed in mid- to late pubertal girls (at least Tanner
breast stage 3 but no more than 2 years postmenarcheal). Subjects will complete two 18-hour
Clinical Research Unit (CRU) admissions in separate menstrual cycles. Immediately before and
during the first CRU admission, either oral micronized progesterone (0.8 mg/kg/dose) or
placebo (randomized) will be given at 0700, 1500, 2300, and 0700 h. During the 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. A second CRU admission (performed at least 2 months later given blood
withdrawal limits) will be identical to the first except that placebo will exchanged for
progesterone or vice versa (treatment crossover). The primary endpoint is LH pulse frequency
while awake. (LH pulse frequency while asleep is an important secondary endpoint.) Results in
pubertal girls without HA were recently published (Kim et al, J Clin Endocrinol Metab
2018;103:1112-1121). Data from girls with HA will be compared to recently-published results
in girls without HA. Mean LH pulse frequency while awake will be analyzed via a hierarchical
linear mixed model (HLMM). HA status (HA vs. non-HA), sleep status (wake vs. sleep), and
treatment (progesterone vs. placebo) will represent fixed-effects, along with all associated
interactions. Random effects will be used to account for hierarchical variance-covariance
structure of the crossover study design. With regard to hypothesis testing, the association
between HA status and wake LH pulse frequency will be evaluated via linear contrasts of HLMM
least squares pulse frequency means. The differential impact of exogenous progesterone on
wake LH pulse frequency in pubertal girls with and without HA (primary analysis) will be
evaluated via the same testing method. Using published and preliminary data, we determined
that, if 16 pubertal girls with HA complete both admissions, we should have at least an 80%
chance of detecting a 0.2 pulse/hour differential effect of P4 on wake LH pulse frequency
between the HA and the non-HA groups 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)

- For girls without hyperandrogenism: serum (calculated) free testosterone concentration
within the Tanner stage-specific reference range and the absence of hirsutism

- For girls with hyperandrogenism: 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: Mild prolactin elevations may be seen in 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.)

- 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 such girls.

- 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.

- 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).