Androgen Excess as a Cause for Adipogenic Dysfunction in PCOS Women

Aim 1. Examine mechanisms of subcutaneous abdominal adipose (fat) development in
normal-weight PCOS women vs. BMI- and age-matched normal (control) women.

Subjects and clinical assessment: We will recruit 16 lean (18.5-25 kg/M2) PCOS subjects and
16 age- and BMI-matched controls. Subjects will complete a standardized questionnaire
emphasizing menstrual dating, abnormal hair growth and acne. The questionnaire also will
annotate age, smoking status, medications, surgical history and family histories of excess
hair growth in female relatives and of diabetes in parents or siblings for exclusion criteria
and for inclusion of some basic traits as covariates. Subjects also will undergo a physical
examination; hirsutism will be scored by the modified Ferriman-Gallwey (mFG) method.
Transvaginal sonography (TVUS) will be performed to determine the presence or absence of
polycystic ovaries. A screening blood sample will be obtained for determinations of steroid
hormones, SHBG, TSH, and prolactin.

Women of any ethnicity between the ages of 18 and 35 years will be recruited. PCOS patients
will be diagnosed by 1990 NIH criteria. Controls will have regular menstrual cycles at 21 to
35 day intervals, a luteal phase progesterone (P4) level > 3 ng/mL, and no evidence of
hirsutism, acne, alopecia, polycystic ovaries or endocrine dysfunction. Exclusion criteria
are: present/past history (<1 year) of smoking, cancer, alcohol abuse, drug addiction, severe
depression, or post traumatic stress; diabetes; uncontrolled hypertension (≥ 165/100);
clinically significant hepatic or renal disease, or other major medical illness; signs or
symptoms of infection; recent (within 30 days) use of an experimental device; recent (within
3 months) use of androgens, anabolic steroids or non-steroidal anti-inflammatory drugs;
recent (within 3 months) use of hormonal agents (including birth control pills or insulin
sensitizers); use of the drug warfarin.

Studies will be conducted in the follicular phase in controls and during amenorrhea in PCOS
women. All subjects will undergo a modified frequently-sampled intravenous glucose tolerance
test (FSIGTT). Glucose in the form of a 50% solution (0.3 g/kg) and regular human insulin
(0.03 units/kg) will be injected through an intravenous line at 0 and 20 min, respectively.
Blood will be collected at -20, -15, −5, 0, 2, 4, 8, 19, 22, 30, 40, 50, 70, 90, and 180 min
for glucose and insulin determinations. Mathematical modeling of serial glucose and insulin
determinations will calculate: insulin sensitivity index (SI, i.e. the action of insulin to
accelerate glucose uptake and suppress glucose production), glucose effectiveness index (SG,
i.e. the combined effect of glucose to enhance glucose uptake and suppress endogenous glucose
production at basal insulin levels) and the acute response to glucose (AIRG).

Adipocytes isolation and culture: Approximately 1-2 gms of fat will be obtained from the
lower SC abdomen using standard procedures under local anesthesia. Adipocytes (fat cells) and
surrounding fat tissue will be isolated to measure adipocyte cell number and diameter, lipid
accumulation and function, adiponectin, and stem cell development.

Procedures: All procedures will be performed in normal and PCOS women at the start of study.

i). Venipuncture: Fasting blood will be collected for blood count, chemistry panel, LH, FSH,
total/free T, DHT, A4, DHEAS, E1, E2, anti-mullerian hormone (AMH), SHBG, adiponectin,
leptin, IL-6, lipid profile and free fatty acids (FFAs).

ii ). Body composition: Body composition will be assessed by BMI, waist-to-hip circumference
and DEXA scanning. Girths will be measured at the waist (narrowest section of the torso
between ribs and umbilicus) and hips (largest protrusion of the hip region, above the gluteal
fold). For total body fat and regional fat distribution, whole body scans will be performed,
utilizing DEXA imaging.

iii). Body fat distribution: Total body DEXA will measure abdominal fat (i.e., the area
between the dome of the diaphragm and the top of the hip. Total body DEXA images also will
determine % body fat; fat-free body mass; total body, abdominal, and leg fat; and abdomen/leg
fat mass ratio. The leg region is that area below the top of the hip bone (greater
trochanter).

iv). Ovarian testing: Transvaginal ultrasound will be performed in the follicular phase in
controls and during amenorrhea in PCOS women. Ovarian volume will be calculated. Antral
follicle number, defined as the total follicle number (2-9 mm in diameter) of both ovaries,
will be determined by 1 investigator (D.A.D.). Polycystic ovaries will be identified, with
one such ovary sufficient to define PCO

Aim 2. Determine the role of androgen in SC abdominal ASC dysfunction and its relation to
metabolism in normal-weight PCOS women through androgen. antagonism by clinical use of
flutamide vs. placebo.

Modified FSIGTT and Adipogenic studies: The modified FSIGTT and all adipogenic studies
performed at study initiation will be repeated at the end of the 6-month flutamide vs.
placebo intervention in PCOS women.

Procedures: All procedures performed at study initiation also will be repeated at the end of
the six 28-day cycles of flutamide vs. placebo intervention in PCOS women. In addition,
monthly liver function studies will be performed to detect possible elevations of serum
transaminase levels above the normal range during flutamide vs. placebo therapy.

Menstrual records: Ovulatory frequency will be determined by having subjects keep a daily
menstrual record and collect weekly first morning urine samples for possible progesterone and
creatinine determinations. Urine samples will be frozen for later analysis to determine
evidence of ovulation, as necessary.

Aim 3. Identify epigenetic changes that underlie SC abdominal ASC dysfunction in
normal-weight PCOS women vs. BMI- and age-matched controls.

Subcutaneous abdominal adipose stem cells from PCOS and control women will be grown into
newly-formed fat cells (adipocytes) in the laboratory. Cells will be studied before and after
6-month treatment with placebo vs flutamide for changes in the cell epigenome (methylation,
RNA sequences and histone modification).

Inclusion Criteria:

- Women between the ages of 18 to 35 years.

Groups will be: 16 lean controls and 16 age- and BMI-matched PCOS women randomized to
flutamide vs. placebo for 6 months.

i) Lean patients with PCOS: 16 subjects with PCOS (defined by 1990 NIH criteria [all
Aims]), BMI 18.5-25 kg/m2. This BMI range is defined as normal and has been chosen to
examine underlying mechanisms of PCOS-related androgen excess in the genesis of adipogenic
and ovarian dysfunction, independent of obesity.

ii) Lean control women: 16 healthy subjects, BMI 18.5-25 kg/m2. Controls will have regular
menstrual cycles, and no evidence of hirsutism, acne, alopecia, polycystic ovaries, and/or
endocrine dysfunction. This BMI range has been chosen to match that of the PCOS group.

Exclusion Criteria:

- Exclusion criteria for study participation are: present or past history (<1 years) of
smoking, cancer, alcohol abuse, drug addiction, severe depression, or post traumatic
stress; diabetes; uncontrolled hypertension (≥ 165/100); clinically significant
hepatic or renal disease, or other major medical illness; recent (within 3 months) use
of androgens, anabolic steroids or hormonal agents (including birth control pills or
insulin sensitizers). These exclusion criteria are chosen to avoid effects from
medical conditions, environmental factors or exogenous agents. Women taking the drug
warfarin, CYP active medications, or herbs will be excluded.

The screener will assess the participants response to establish if depression or drug use
exclude participation in this study.

Women taking beta blockers will be excluded.

Women who have taken birth control pills or had a Mirena IUD or used Plan B contraception
during the previous 3 months will be excluded.