Conditions: Polycystic Ovary Syndrome Obstructive Sleep Apnea
Keywords: PCOS OSA Metabolism Diabetes
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 18
Healthy volunteers: no

Brief summary:
The purpose of this study is to look at the metabolic (use of energy) and hormonal features of sleep problems in women with polycystic ovary syndrome (PCOS).

Detailed description:
The prevalence of obesity and chronic sleep loss are at record levels among Americans and evidence continues to emerge to support a causal link between the two conditions. Metabolic abnormalities related to sleep disruption are particularly evident in individuals with obstructive sleep apnea (OSA), a disorder traditionally associated with male gender. While more prevalent in men, OSA is underrecognized in women in part because its clinical and polysomnographic features differ from those of men. Women with polycystic ovary syndrome (PCOS) are particularly susceptible to OSA with at least a 5-fold higher risk for its development compared to obese women without PCOS. This study will enroll obese women with PCOS, with and without OSA. Those with OSA will be randomized to receive CPAP or to receive depot leuprolide to suppress ovarian steroid output over 12 weeks, reassessed at 6 weeks, and then randomized (double-blind, placebo controlled) to 6 weeks of either micronized estrogen + placebo or micronized progestin + placebo. The independent effects of androgen, estrogen, and progesterone on OSA and metabolic function will be assessed. In addition, primary human adipocytes will be prepared from fat biopsies obtained from subjects. Insulin sensitivity will be determined by phospho-specific immunoblotting in conjunction with glucose uptake and anti-lipolysis assays. In parallel, adipocytes from these subjects will be cultured for 1-5 days prior to metabolic assays to ascertain if removal of from circulating factors will improve insulin signaling, or if insulin resistance persists in vitro. Finally, there will be an interface with the Metabolomics Laboratory at Duke University (C. Newgard, Lab Director), and metabolomics assessment will be done on blood and urine samples.

Arms:
arm 1: Randomized to receive depot Lupron for 6 weeks. Then randomized again to receive estrogen plus placebo for another 6 weeks.

Interventions: intervention 1: Depot Lupron followed by estrogen plus placebo
Intervention descriptions: intervention 1: A single intramuscular dose of depot lupron (11.25 mg). Six weeks after injection, subjects will receive daily oral doses of estrogen (2mg) plus placebo for six weeks.

Protocol document:
Prot_SAP_000.pdf: 
 
 
 
 
Cover page 
 
 
 
 
 
 
 
Official Title on ClinicalTrials.gov:   PCOS, Sleep Apnea and Metabolic Risk in Women 
  
ClinicalTrials.gov ID NCT number: 
  NCT00696111 
 
Document Date:    
 
 
  June 15, 2023 
 


PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
A. SPECIFIC AIMS
Polycystic ovary syndrome (PCOS) affects between 5 and 8% of women making it one of the most
common endocrinopathies in women (3, 4). The disorder typically has its onset at puberty with evidence of
excessive androgen production, obesity, and insulin resistance. Women with PCOS are more insulin
resistant than weight-matched controls (5) and have an exceptionally high prevalence (6) of early-onset
impaired glucose tolerance (30 - 40 percent), and type 2 diabetes (up to 10 percent) (7, 8). In addition, the
rate of deterioration in glucose tolerance is accelerated in PCOS (7, 9). Vascular disorders are also
common in this population due, at least in part, to disordered lipid metabolism. Hypertriglyceridemia,
increased levels of very-low-density lipoprotein (VLDL) cholesterol, low-density lipoprotein (LDL)
cholesterol, and decreased levels of high-derisity lipoprotein (HDL) cholesterol are typical (10) (11). While
insulin resistance contributes to glucose intolerance and dyslipidemia, it has become increasingly clear that
other factors must be involved in the pathogenesis of the metabolic derangements so commonly seen in
PCOS.
Over the past several years, chronic decreases in sleep duration and/or quality have been identified as
a risk for the development of a number of metabolic derangements (12-14) that are strikingly similar to
those seen in PCOS (3, 10, 15). Specifically, decreased sleep quality due to obstructive sleep apnea
(OSA) has been causally linked to insulin resistance, glucose intolerance, dyslipidemia and hypertension
independent of body mass index (BMI) (12-14). Until recently however, it had not been recognized that
OSA is present in a disproportionate number of women with PCOS: the risk for OSA is at least 5-
fold higher, and perhaps as much as thirty-fold higher (16), compared to the risk in similarly obese
women without PCOS.
It was initially thought that hyperandrogenemia or obesity are the key determinants of the high
prevalence of OSA in PCOS. However, both published data (16) as well as results of our Preliminary
Studies are not consistent with this hypothesis. We have found that plasma concentrations of free
testosterone are virtually identical in PCOS women with and without OSA. In addition, measures of OSA
severity, such as the apnea-hypopnea index (AHI), do not correlate with either the degree of androgen
elevation or with body mass index (BMI) among PCOS women with OSA.
Both progesterone and estrogen reportedly "protect" against the development of OSA (17, 18) (19-24)
(25), and levels of these sex steroids are typically low in PCOS relative to those in normal cycling women.
Thus, we will test the hypothesis that circulating progesterone and estrogen levels are significant
determinants in the development of OSA among hyperandrogenic women with PCOS. Using a
novel paradigm that permits us to independently modulate sex steroid concentrations, we will be
able to address the specific and independent effects of progesterone and estrogen in the
pathogenesis of OSA among women with PCOS.
OSA appears to be an underrecognized, yet significant factor in the pathogenesis of metabolic
derangements in PCOS. In a recent study, we showed that measures of insulin resistance and glucose
tolerance in PCOS women are strongly predicted by the severity of OSA (15). These intriguing findings
suggest that there may be two "subtypes" of women with PCOS, i.e. those with and those without OSA,
and that these two subtypes may be associated with distinct metabolic and endocrine alterations. PCOS
women with OSA may be at much higher risk for diabetes and cardiovascular disease than PCOS women
without OSA and may benefit from therapeutic interventions targeted to decrease the severity of OSA.
Thus, a major goal of the present project is to contrast the metabolic and hormonal features of
these two subtypes of PCOS, explore causative mechanisms for the high prevalence of OSA in
PCOS, and test the hypothesis that continuous positive airway pressure (CPAP) treatment may
decrease the risk of diabetes and other cardiovascular and metabolic abnormalities in PCOS
women with OSA.
Specific Aim 1. To determine if women with PCOS who have OSA differ from those without OSA as
a consequence of differences in circulating concentrations of the sex-steroids estrogen and
progesterone. We hypothesize that in comparison to PCOS women without OSA, those who have OSA
will have lower circulating levels of estradiol, progesterone, or both estradiol and progesterone at baseline.
We will further test our hypothesis by comparing stimulated steroid levels in these two groups. Specifically,
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
we will compare the ovarian steroidogenic response to a single dose of the GnRH agonist leuprolide in
PCOS women with and those without OSA. Developed by our group, the GnRH agonist test has become a
novel method to assess ovarian steroidogenic function.
Specific Aim 2. To determine if OSA improves in women with PCOS treated with estrogen or
progesterone. For these studies, we will enroll women with PCOS who have OSA. Subjects will have a
detailed baseline metabolic and metabolomic profile together with a baseline polysomnogram. Subjects will
then receive a single dose of depot-leuprolide in order to suppress ovarian production of estrogen,
progestin, and androgen over a period of 3 months. Six weeks after administration of depot-leuprolide,
subjects will have repeated assessment of metabolic, metabolomic, and sleep measures to determine if
these outcomes are altered by the combined suppression of ovarian sex steroids. Subsequently, subjects
will be randomized in a double-blind placebo controlled fashion to one of two treatment arms for a period of
six weeks. Treatment arms are: estrogen plus placebo or progesterone plus placebo. At the end of this
second six weeks, subjects will have metabolic, metabolomic, and sleep measures repeated. Primary
outcome measures will be compared both within and between treatment arms.
Specific Aim 3. To determine if metabolic disturbances present in PCOS women with OSA are
ameliorated by the treatment of OSA with continuous positive airway pressure (CPAP). The
presence of OSA in women with PCOS has a significant negative effect upon metabolic measures. In
particular, the presence of OSA in PCOS is associated with hyperinsulinemia, elevations in plasma
glucose, and lipid abnormalities that include higher levels of triglycerides and LDL-cholesterol. OSA in
PCOS is also associated with hypercortisolemia that persists throughout both day and night. Results of our
preliminary studies indicate that CPAP treatment of OSA results in attenuation of cortisol levels not only
during sleep, but throughout waking hours as well. This aim will serve to test the hypothesis that correction
of OSA in PCOS will lead to improved metabolic function which can be attributed, at least in part, to a
reduction in circulating levels of cortisol.
Relationship of the present Project to Projects 1. 3. 4. and the Metabolomics Laboratory in this
SCOR Proposal
The studies planned in this Project will integrate with and complement those of Projects 1, 3, and 4 as
outlined below. Metabolomics profiling is integrated into all SCOR Projects; specific interactions with the
current Project are detailed below.
1. Insights into the basis for gender differences in normal sleep and pathologic sleep (OSA) will be
facilitated by interactions between the present Project and Project 1 (Van Cauter. PI). In Project 1.
metabolic measures, slow wave sleep (SWS), rapid-eye movement (REM) sleep, and non-REM sleep
will be characterized in obese men and obese women, with and without OSA. Those with OSA will be
treated with CPAP and reevaluated for sleep and metabolic outcomes. In the present Project, similarly
obese women with PCOS, with or without OSA, will be characterized as above. Those with OSA will
then be randomized to CPAP treatment or to receive medication designed to modify the sex steroid
milieu. This integrated study design will permit us to compare normal sleep to sleep disrupted by OSA
in obese men and women and obese women with PCOS. The present project will benefit from the
ability to distinguish the independent effects of gender (men vs women) from circulating sex steroid
concentrations (PCOS vs non-PCOS). The effects of CPAP treatment of OSA on sleep and metabolic
outcomes can also be compared between groups studied in Projects 1 and 2.
2. Elevation in triglyceride (TG) concentration is a common feature of the metabolic abnormalities
associated with obesity and the increase in cardiovascular risk resulting from a given rise in plasma TG
concentration is approximately 2-fold greater in women than in men. Project 3 (Mittendorfer. PI) and the
present Project are linked by a shared hypothesis, i.e., that the hyperandrogenemia and relative
progesterone deficiency are important factors in the pathogenesis of TG elevation (Project 3) as well as
OSA (present Project 2) in women with PCOS. Detailed studies of TG kinetics will be undertaken
before and after modulation of plasma progesterone or testosterone concentrations in women.
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Ehrmann. David A.
PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle):.
3. In Project 4 (Brady. PI) primary human adipocytes will be prepared from fat biopsies obtained from
PCOS women with and without OSA as well as from PCOS women with OSA before and after
treatment with either CPAP or hormonal modulation. In vitro assessment of adipocyte insulin sensitivity
will be determined by phospho-specific immunoblotting in conjunction with glucose uptake and anti-
lipolysis assays. In parallel, adipocytes from these subjects will be cultured for 1-5 days prior to
metabolic assays, to ascertain if removal of adipocytes from circulating factors will improve insulin
signaling.
4. This Project, along will all others in this SCOR proposal, will provide samples of blood and urine to the
Metabolomics Laboratory at Duke University (C. Newqard. Director) for highly sensitive and specific
assays, as described in the overall Introduction to this proposal. This collaboration presents a unique
opportunity for this Project to determine if metabolomic measures in women with PCOS and OSA differ
from those of PCOS women without OSA. In addition, it will be possible to determine if interventions
with CPAP or hormonal modulation are associated with changes in these same measures. This
methodology provides a powerful tool to examine traditional hormones (e.g., insulin, adiponectin,
ghrelin, resistin) and inflammatory markers (e.g., CRP, IL-6, TNFalpha), as well as less traditional, but
potentially significant, measures such as free fatty acids and acylcarnitines (a complete list of analytes
is provided in the overall introduction to this application). The sophisticated methodologies used by the
Metabolomics Laboratory have already elucidated a previously unsuspected metabolic pathway in the
regulation of glucose stimulated insulin secretion in the pancreas, a lipid-derived metabolite involved in
the mediation of insulin resistance in muscle, and products of branch-chained amino acid metabolism
that appear to provide a "signature" of human obesity. Finally, the Laboratory has found that there is
heritability in the metabolic profiles in families with a high incidence of early-onset cardiovascular
disease. Thus, the collaborative interaction between this Project and the Metabolomics Laboratory
offers both real and potential opportunities to define metabolic pathways involved in the pathogenesis
of PCOS and OSA.
A summary of abbreviations used throughout this application is provided below.
A1C 
Glycohemoglobin
ACTH 
Adrenocorticotropin
AMI 
Apnea-Hypopnea index
BMI 
Body mass index
Cl 
Confidence interval
CPAP 
Continuous positive airway pressure
CRP 
C-reactive protein
DMAS 
Dehydroepiandrosterone sulfate
fslVGTT Frequently-sampled intravenous glucose
tolerance test
FSH 
Follicle stimulating hormone
FTI 
Free thyroxine index
GnRH 
Gonadotropin releasing hormone
HDL 
High density lipoprotein
HOMA 
Homeostasis model assessment
HPA 
Hypothalamic-pituitary-adrenal
HSD 
Hydroxysteroid dehydrogenase
IL 
Interleukin
LDL 
Low density lipoprotein
LH 
Luteinizing hormone
OSA 
Obstructive sleep apnea
PCOS Polycystic ovary syndrome
REM 
Rapid eye movement
SHBG Sex hormone binding globulin
SWA 
Slow wave activity
SWS 
Slow wave sleep
T 
Testosterone
TG 
Triglyceride
TNF 
Tumor necrosis factor
TT4 
Total thyroxine
VLDL Very low density lipoprotein
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
B. BACKGROUND AND SIGNIFICANCE
PCOS affects approximately 5-8% of women in the United States and typically manifests at the time of
puberty with menstrual irregularity, hirsutism, and obesity (4). The ability to diagnose PCOS at an early age
has important implications, since those affected have a substantial risk for subsequent development of a
number of metabolic (5, 26) and cardiovascular (11, 27, 28) disorders. Specifically, women with PCOS
have among the highest reported rates of early-onset impaired glucose tolerance and type 2 diabetes (7, 8)
as well as a substantial increase in risk for hypertension (29), dyslipidemia (30, 31), coronary (30) and
other vascular disorders (32-34). An important addition to this list of health risks is obstructive sleep apnea
(OSA) which is present in a disproportionate number of women with PCOS. The risk for OSA is at least 5-
fold higher (see Preliminary Studies), and perhaps as much as thirty-fold higher in PCOS (16), than in
similarly obese women. Results of our Preliminary Studies suggest that there may in fact be two "subtypes"
of women with PCOS - those with OSA and those without OSA - and that these two subtypes may be
associated with distinct metabolic and endocrine alterations. PCOS women with OSA may be at much
higher risk for diabetes and cardiovascular disease than PCOS women without OSA and may benefit from
therapeutic interventions targeted to decrease the severity of OSA. Most importantly, nearly all published
studies that have characterized metabolic and cardiovascular abnormalities in PCOS have not controlled
for the potential impact of OSA and chronic sleep loss.
The Background that follows is intended to provide an overview of the prevalence of chronic sleep loss and
OSA and the roles played by sex steroids and body fat in the pathogenesis of these conditions. This is
followed by a brief review of the metabolic consequences of OSA, a description of the key metabolic
derangements in PCOS and finally, by a review of the current state of knowledge regarding causes and
consequences of OSA in PCOS.
B1. Chronic sleep loss and obstructive sleep apnea: role of sex steroids and adiposity
Over the past several decades the average duration of sleep 
,_, 
r25
has declined for most Americans. During the 1960's, the mean
sleep duration was between 7 and 8 hours per night; today, the |8_
percentage of both men and women who sleep less than 6 |
hours per night has increased dramatically (1) (Figure B1-1). |7_
Chronic sleep loss imposes a significant negative impact upon
individual health as well as an enormous economic cost to
society (35, 36). A number of studies have reported that
shortened sleep duration is associated with increased mortality
(37, 38). In the Nurses Health Study, it was found that sleeping 
«» "w 
»» ™° '*«> *»o
less than 6 hours per night was associated with an increased
risk of death, even after adjusting for age, smoking, alcohol,
-IS
-10
,
exercise, depression, snoring, obesity, and history of cancer and 
duration in Americans. Drawn from
cardiovascular disease (38). Reduced sleep time has also been 
composite data in (1, 2).
reported as a risk factor for the development of obesity as for
type 2 diabetes (39, 40) (11, 12). Results of the Sleep Heart Health Study showed that subjects sleeping 5
hours or less per night had an adjusted odds ratios for diabetes of 2.51 (95% Cl, 1.57-4.02) when
compared to those who slept 7 to 8 hours per night (40). This trend in shorter sleep duration mirrors the
progressive rise in overweight and obesity in the United States (41) (Figure B1-1) and evidence continues
to emerge to support a causal link between these two conditions. Should either or both trends continue
along their current trajectory, the metabolic and cardiovascular health consequences as well as economic
costs will be staggering.
Obstructive sleep apnea (OSA) is one of the major causes of chronic sleep disruption. It is
characterized by episodic partial or complete upper airway obstruction during sleep leading to intermittent
hypoxia, sleep fragmentation and a reduction in the quantity of deep non-rapid eye movement (NREM)
sleep (stages 3 and 4, commonly referred to as slow wave sleep [SWS]). Sleep disruption resulting in
reduced SWS has been associated with a rise in plasma cortisol levels and interpreted to indicate that
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Ehrmann. David A.
PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle):,
SWS has a "restraining" effect on the hypothalamic-pituitary-adrenal axis (42). Consistent with this is the
finding that pharmacologic augmentation of SWS leads to a significant decline in salivary free cortisol
levels (43).
Current estimates of OSA prevalence in the United States (2, 44) are likely to underestimate the true
prevalence of the disorder since 82% of men, and an even greater (93%) proportion of women with
moderate to severe OSA have not been clinically diagnosed (45). It has been consistently noted that men
have a higher prevalence of OSA compared to women (45). In community based studies, the male:female
ratio is usually between 2:1 and 3:1 (46) in contrast to a ratio of 8:1 in clinic-based studies (47).
OSA has been independently associated with glucose intolerance and insulin resistance even after
adjustments for obesity and age (48-52). Treatment of OSA with CPAP can improve insulin sensitivity (53)
and is associated with a reduction in postprandial glucose and glycohemoglobin levels in individuals with
type 2 diabetes (54).
Differences in concentrations of circulating sex steroids - estrogens, progestins, and androgens -
appear to play an important role in the differences between men and women, both in normal sleep as well
as OSA. However, women tend to be underrepresented in most studies of OSA (Table B1-1).
Table B1-1.
Authors
Punjabi, et. al.
Ip, et. al.
Meslier, et. al.
Punjabi, et. al.
Tassone, et. al.
Harsch, et. al. (CPAP treatment)
Babu, et. al. (CPAP treatment)
Reference #
(49)
(48)
(50)
(51)
(52)
(53)
(54)
Sample Size
150
270
595
2,656
30
40
25
Women in Sample (%)
0
27
0
54
30
15
36
Role of Estrogen and Progesterone. Estrogens and progestins have been generally characterized as
protective against the development of OSA in women. Much of the evidence to support this view is derived
from studies in which sleep was evaluated in relation to pregnancy status (17, 18), age and phase of the
menstrual cycle (19, 20), menopausal status (21), or in response to hormone replacement therapy (21).
Lower estradiol levels have been reported in association with poor sleep quality among women aged 45 -
49 yr (22) and with a higher frequency of apneic events in women across a broader age spectrum of 24 to
72 yr (20). Among post-menopausal women, there was a modest, but statistically significant, decrease in
the occurrence and frequency of sleep apnea in those randomly assigned to receive estrogen replacement
rather than placebo (21).
Progesterone is the key hormone thought to underlie the differences in sleep measures that exist
across the normal menstrual cycle. Progesterone levels are low during the follicular (pre-ovulatory) phase;
post ovulation (luteal phase), when progesterone is synthesized by the corpus luteum, plasma levels rise
by up to two log orders. When sleep measures are obtained and compared between follicular (low
progesterone) and luteal (high progesterone) phases, it is apparent that upper airway resistance is lower
during the luteal phase (19).
The expected rise in progesterone with pregnancy is thought to attenuate the severity of preexisting
OSA as well as to "protect" from its development in women without OSA pre-conception (18). These effects
have been ascribed to levels of progesterone that would normally counterbalance the increase in OSA risk
imparted by pregnancy-associated weight gain. Progesterone is thought to promote its effects through
direct stimulation of respiratory drive via an increased ventilatory response to both hypercapnea and
hypoxia (23, 24). Progesterone may also act to enhance upper airway dilator muscle activity (25) and
reduce airway resistance.
Role of Androgens. Androgens are thought to play a significant role in the sexual dimorphism in sleep
architecture and in the pathogenesis of OSA (55, 56). O'Connor, et al (57) analyzed records of 830 patients
with OSA to determine whether there were differences in polysomnographic features between men and
women, particularly with respect to the distribution of respiratory events during REM and non-REM sleep.
Although the apnea-hypopnea index (AMI) during total sleep time was significantly higher in men compared
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middled: 
Ehrmann. David A.
to women (31.8 ± 1.0 vs 20.2 ± 1.5; P<0.001), the number of respiratory events occurring in REM sleep
was greater in women as reflected by the so-called REM difference (i.e., the difference in the AMI in REM
and AMI in non-REM sleep) in women and men. The REM difference was greater in women than men (28.1
± 1.5 vs. 10.3 ± 1.1; P<0.001) at all levels of severity of sleep apnea. These findings were consistent and
remained significant even after adjustment for the effects of covariates including weight, age, and duration
of apnea. Thus, women with obstructive sleep apnea appear, to have a higher proportion of respiratory
events in REM compared to men, and to have a higher prevalence of apnea occurring mostly during REM.
Several studies have also shown that testosterone influences both neural control of breathing (58) and
upper airway mechanics (59). Zhou et al (60) examined the effect of testosterone on apneic threshold in
women during sleep. Eight normal, healthy, pre-menopausal women were studied before and after
treatment with transdermal testostosterone (5 mg/day) administered in the follicular phase of the menstrual
cycle. The authors concluded that testosterone increases apneic threshold in premenopausal women, thus
leading to breathing instability during sleep.
Role of Body Fat and its Distribution.The risk of OSA is increased as a function of both total body fat mass
as well as body fat distribution. Visceral fat appears to be more metabolically active and the quantity of
visceral fat has been shown to highly correlate with OSA risk (61-63). The relative proportion of visceral fat
to total body fat is higher in obese men compared to obese women. This difference is thought to contribute
to the higher prevalence of OSA in men than women. Factors responsible for gender differences in body fat
distribution include sex steroid concentrations, especially androgens. These factors are particularly relevant
to the pathogenesis of OSA in women with PCOS.
B2. Metabolic Consequences of OSA
As previously noted, OSA is characterized by the combination of episodic sleep disruption and hypoxemia,
each of which can trigger at least three major hormonal responses: activation of the hypothalamic-pituitary-
adrenal (HPA) axis with increased cortisol production/secretion, increased catecholamine output from
sympathetic nervous system stimulation, and increased release of adipokines from adipose tissue. These
responses appear to contribute to the metabolic abnormalities associated with OSA, particularly to the
decline in insulin sensitivity and glucose tolerance.
Hypothalamic-pituitarv-adrenal axis. The onset of sleep is normally characterized by a modest inhibition
of cortisol secretion that is concurrent with slow wave sleep (SWS) and lasts between 60 and 120 min (64).
Nocturnal awakenings are consistently followed by a pulse in cortisol secretion (65) whereas the final
morning awakening (the awakening response) is associated with a rapid rise in cortisol lasting
approximately 60 min (64). Work from our laboratories (66) has shown that partial or total sleep deprivation
results in increases in plasma cortisol levels by 37% and 45%, respectively. Most notably, this elevation is
evidenced on the day following sleep loss and during the time when the HPA axis is usually quiescent.
Profiles of cortisol secretion in patients with OSA have been variably reported as normal in some
studies and abnormal in others (67). In one report, 8 of 28 OSA patients demonstrated a disruption in the
circadian rhythm with cortisol levels that were higher late in the day than in the early morning. This so-
called "inverted" cortisol profile was associated in all cases with abnormal blood pressure regulation. When
compared to obese subjects without OSA, obese subjects with OSA had an exaggerated ACTH response
to the administration of CRH although cortisol responses did not differ between groups (68). Whether
alterations in cortisol metabolism are a cause, consequence, or both in OSA remains unresolved.
B3. Metabolic Abnormalities Associated with PCOS.
Both lipid and non-lipid criteria identify individuals at increased risk for coronary heart disease and type 2
diabetes (69-74). Because women with PCOS have high rates of impaired glucose tolerance and type 2
diabetes (7, 8) as well as a substantial number of risk factors for cardiovascular disease (75), it has been
generally assumed that many are also likely to meet criteria for the "metabolic syndrome". We recently
reported that fully one-third of non-diabetic women with PCOS have developed the metabolic syndrome
well before the end of their fourth decade, and usually prior to the end of their third decade of life. This
prevalence is four times higher than that observed in women between the ages of 20 and 30 years and
twice that of women between ages 30 and 40 years (76). Indeed, the metabolic syndrome prevalence was
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
similar to that in women between the ages of 50 and 60 years (76). We have also found that the
prevalence of the metabolic syndrome is similar across ethnic/racial backgrounds.
Insulin resistance and hyperinsulinemia in PCOS. Even though the molecular basis for insulin resistance in
PCOS remains incompletely understood, it is well documented that the compensatory hyperinsulinemia
contributes both directly and indirectly (77-79) to the increase in plasma androgen concentrations that
characterize PCOS. Insulin acts directly by binding to its cognate receptor on the ovarian thecal cell to
stimulate testosterone synthesis (80). Insulin can also act indirectly to raise the serum concentration of free
testosterone, the level of which does not appear to be tightly regulated in the female, by lowering the serum
concentration of sex hormone binding globulin (SHBG) (79).
Insulin resistance is a central factor in the pathogenesis of the metabolic syndrome in both men and
women and there is ample evidence to support a causal link between hyperinsulinemia and the
characteristic features of PCOS. A reduction of serum insulin levels in women with PCOS results in a
decrease in ovarian androgen biosynthesis, an increased SHBG concentration, and a resultant decrease in
free testosterone concentrations (81, 82). Insulin also plays a key role in the impaired glucose
tolerance/diabetes (81, 82) of PCOS and attenuation of hyperinsulinemia, whether through weight
reduction (83) or administration of either metformin (84, 85) or a thiazolidinedione (86-88) substantially
attenuates the metabolic perturbations of PCOS.
Insulin resistance and impaired glucose tolerance/type 2 diabetes. While obesity is a major factor in the
development of insulin resistance in PCOS, it is now established that a component of insulin resistance in
PCOS is independent of body weight (82, 89). Both lean and obese women with PCOS are more insulin
resistant than their non-PCOS counterparts matched for total and fat-free body mass as documented using
the hyperinsulinemic-euglycemic clamp (82, 89), frequently sampled IVGTT (26, 87, 90-92) and protocols
using a graded glucose infusion (87, 90).
In long-term follow-up studies of women with PCOS there is an increased prevalence of type 2 diabetes
when compared to appropriate controls (29). Two large, prospective studies in PCOS place the prevalence
of IGT between 30-40% and type 2 diabetes between 5-10% (7, 8). These prevalences approach those in
Pima Indians, a population with one of the highest rates of development of type 2 diabetes (93). More
recently, we (7) and others (9) have found that the conversion rates from normal glucose tolerance to IGT
or type 2 diabetes in PCOS are substantially elevated.
R-Cell Dysfunction in PCQS. Because glucose intolerance results only when defects in insulin secretion
and insulin action co-exist (94), we postulated that insulin secretory defects could play an important role in
the propensity to develop diabetes in PCOS. Initial evidence for d-cell dysfunction in PCOS was derived
from analyses of basal and postprandial insulin secretory responses in women with PCOS relative to
weight-matched controls with normal androgen levels (95). The incremental insulin secretory response to
meals was markedly reduced in women with PCOS, resulting from a reduction in the relative amplitude of
meal-related secretory pulses rather than from a reduction in the number of pulses present. This pattern,
which resembled that of type 2 diabetes more than that of simple obesity (96), was striking in that it was
evident in nondiabetic women with PCOS.
Insulin secretion is most appropriately expressed in relation to the magnitude of ambient insulin
resistance. The product of these measures can be quantified (97) (the so-called "disposition index") and
related as a percentile to the hyperbolic relationship for these measures established in normal subjects (97,
98). When first-phase insulin secretion is analyzed in relation to the degree of insulin resistance, women
with PCOS exhibit a significant impairment in li-cell function (86, 91). We have additionally quantified IS-cell
function in PCOS by examining the insulin secretory response to a graded increase in plasma glucose and
by the ability of the li-cell to adjust and respond to induced oscillations in the plasma glucose level (26).
Results from both provocative stimuli were consistent: when expressed in relation to the degree of insulin
resistance, insulin secretion was impaired in PCOS subjects.
Dvslipidemia in PCOS. Women with PCOS are frequently characterized as having elevated triglyceride
(TG) levels, increased levels of VLDL and LDL, and a lower HDL cholesterol (99), a lipid pattern similar to
that seen in patients with type 2 diabetes; The mechanisms responsible for the adverse effects of PCOS on
plasma TG homeostasis are not known. Insulin resistance has been postulated to play a key role in
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
causing hypertriglyceridemia in PCOS. However, we found that treatment with the insulin sensitizing agent
troglitazone markedly improved insulin sensitivity in PCOS women but had little, if any, effect on plasma
TG concentration (100). In addition, lean women with PCOS are found to have normal plasma TG
concentrations despite being hyperinsulinemic (30). We therefore propose that increased plasma TG
concentrations in obese women with PCOS are due, at least in part, to hyperandrogenemia and relative
progesterone deficiency, and further that the presence of OSA has a modulating effect upon triglyceride
metabolism, as will be formally tested in SCOR Project 3 (Mittendorfer, PI).
B4. Obstructive Sleep Apnea in Women with PCOS
Women with PCOS have been documented to develop OSA at rates that equal and may even exceed
those in men. The high prevalence of OSA has been thought to be a function of both elevated levels of
testosterone (a defining feature of PCOS) as well as the obesity that commonly accompanies the disorder.
However, it appears that the high prevalence of OSA in PCOS cannot be fully accounted for on the basis of
these two factors alone. In two studies (16, 101), the severity of sleep apnea did not correlate with BMI and
in a third (102), even after controlling for BMI, PCOS women were 30 times more likely to have sleep
disordered breathing and 9 times more likely than controls to have daytime sleepiness. Insulin resistance
was found to be a stronger predictor of sleep disordered breathing than was age, BMI, or circulating
testosterone concentrations (16). It also appeared that women with PCOS taking oral contraceptives were
less likely to have sleep disordered breathing (16), consistent with recent results from the Sleep Heart
Health Study Research Group in which hormone replacement therapy was associated with a lower
likelihood of sleep disordered breathing among postmenopausal women (103). Finally, women with PCOS
had a significantly higher mean apnea-hypopnea index compared to weight-matched controls (22.5 ± 6.0
vs. 6.7 ± 1.7; PO.01), with the difference being most pronounced in REM sleep (41.3 ± 7.5 vs. 13.5 ± 3.3;
P<0.01) (102). Because the risk imparted by obesity does not appear to be sufficient to fully account for the
high prevalence of sleep disordered breathing in PCOS, additional factors have been invoked including the
hyperandrogenemia (44, 55-57) that is characteristic of PCOS, as discussed below.
Androgen levels in PCOS. In response to stimulation by LH, the ovarian theca cell synthesizes
androstenedione and testosterone. Androstenedione is converted by 17li-hydroxysteroid-dehydrogenase
(176-HSD) to form testosterone or aromatized by the aromatase enzyme (cytochrome P450arom) to form
estrone. Results of studies both in vivo and in vitro (using cultured theca cells) are consistent and suggest
that theca cells from PCOS ovaries are more efficient at converting androgenic precursors to testosterone
than are normal theca cells (30). Clinically, this has been documented using a single, diagnostic dose of a
GnRH agonist such as nafarelin or leuprolide (104). Our studies (104), as well as those of others (105),
have shown that the ovarian steroidogenic response of women with PCOS is more robust than that of
normally cycling women and qualitatively similar to the response seen in normal men. This response has
been used as a diagnostic tool as well as a probe to define the pathogenesis of steroidogenic dysfunction
in PCOS, as is proposed in this Project.
Insulin plays both direct and indirect roles in the pathogenesis of hyperandrogenemia in PCOS. Insulin
acts synergistically with LH to enhance theca cell androgen production. Insulin also inhibits hepatic
synthesis of sex hormone binding globulin (SHBG), the key circulating protein that binds to testosterone,
and thus increases the proportion of testosterone that circulates in the unbound, biologically available or
"free", state.
Results of our Preliminary Studies do not support a major role for hyperandrogenemia in the
pathogenesis of OSA in PCOS. Consequently, it is important to examine alternate hypotheses. We have
proposed that the relative reduction in circulating progesterone concentrations as well as estrogen
concentrations, as discussed below, may contribute to the apparent excess of OSA in PCOS.
Progesterone and estrogen levels in PCOS. In normally cycling women, the luteal phase of the
menstrual cycle is characterized by an increase in progesterone production from the corpus luteum and
consequent slowing of GnRH, and thus LH, pulsatility. In the presence of chronic oligo- or anovulation, as
in PCOS, the normal post-ovulatory rise in progesterone does not occur and the restraint on the GnRH
pulse generator is thus absent (106). Thus, on average, circulating progesterone levels in PCOS women
are lower than those in normally cycling women (107, 108).
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Ehrmann. David A.
PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle):.
Underproduction of ovarian estrogen results from low intraovarian aromatase expression and a
consequent reduction in the production of the estrogens, estrone and estradiol, from their respective
precursor androgens, androstenedione and testosterone. While estrone is also synthesized from peripheral
aromatization (especially in adipose tissue), levels of this steroid are normal or even slightly elevated in
PCOS. However, estrone is a weak estrogen with approximately 1/10th the potency of estradiol (109). In
sum, estrogen levels are subnormal in PCOS (110, 111).
C. PRELIMINARY STUDIES
In this section, data will be presented both from unpublished as well as recently published studies from our
group that support the following:
C1. Overweight and (visceral) obesity are exceptionally common in women with PCOS.
C2. Impaired glucose tolerance and type 2 diabetes are present at an early age and in a disproportionate
number of women with PCOS.
C3. Hypertriglyceridemia is a common lipid abnormality in PCOS and predicts the presence of additional
metabolic defects.
C4. There is a 5 - 6 fold increase in risk for OSA in PCOS.
C5. Neither the degree of androgen elevation nor BMI are significant predictors of OSA severity in PCOS.
C6. Androgen levels are significantly reduced over 3 months in PCOS in response to a single dose of the
GnRH agonist leuprolide.
C7. Insulin sensitivity and glucose tolerance decline in response to suppression of slow wave sleep
C8. Treatment of OSA with CPAP in PCOS results in significant reductions in 24 hr secretory profiles for
both cortisol and norepinephrine.
C1. Overweight and obesity are exceptionally common in women with PCOS.
The University of Chicago Center for PCOS database currently numbers 1062 subjects, 829 of whom are
women with PCOS; the remaining 223 subjects are comprised of their first-degree relatives. Among the
women with PCOS, all have been uniformly evaluated and diagnosed by the P.I. of this Project since the
inception of the database in 1989. These women have all been seen in the Endocrinology clinics at the
University of Chicago and are either physician- or self-referred. The overwhelming majority of these women
(see Figure C1-1) are obese (81%) and a substantial proportion (35.5%) are profoundly obese with a BMI
that exceeds 40.0 kg/m2. These prevalences are consistent with those reported elsewhere in the U.S. (8).
50-
45-
40-
4_»
5 35-
030-
•525-
£20-
u
oi 15-
o.
10-
5-
0-
(IE!
i as -24.9
'-••.'••' ''
25-29.9
\
'"
f.
30-39.9 
>«>
BMI (kg/m2)
Figure C1-1. Distribution of BMI among 829 PCOS
women at the University of Chicago. BMI exceeds
30 kg/m2 in over 80% of PCOS subjects.
Figure C1-2. MRI image at L4-L5 in a representative woman
with PCOS. This technique provides the ability to quantify
the relative proportion of visceral fat.
As reviewed in the Background, the quantity of visceral fat appears to be more highly correlated with
metabolic (insulin resistance) and cardiovascular (dyslipidemia) abnormalities than does either total or
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Ehrmann. David A.
PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle):,
subcutaneous fat mass. We have quantified visceral and subcutaneous adiposity in 12 women with PCOS.
The images from the L4 - L5 level are evaluated with a computer algorithm that employs volume
averaging. A representative example is shown in Figure C1-2. The relative proportion of visceral adiposity
(41.3%) is substantially higher than that reported in normal weight and obese women (112).
C2. Prevalence of impaired glucose tolerance and type 2 diabetes in PCOS
We initially reported (7) that among women with PCOS, glucose tolerance was normal in 55%, while 35%
of women had impaired glucose tolerance, and 10% had type 2 diabetes. Our sample of subjects who has
had an OGTT has more than tripled since that time, but these prevalences remain nearly identical: 55%
have normal glucose tolerance while 39% and 6% have IGT and type 2 diabetes, respectively. Our current
analyses of oral glucose tolerance in 402 women with PCOS are summarized below.
Table C2-1. Oral Glucose Tolerance and Androgen Levels in 402 Women with PCOS
N (%)
Age (yr)
BMI (kg/m2)
Total T (ng/dl)
Free T (pg/ml)
SHBG (nM)
DMAS (mcg/dl)
Normal Glucose Tolerance
222 (55)
28.0 ±0.5
36.2 ± 0.6
72.5 ±2.3
21.1 ±0.6
16.7 ±0.7
160.7 
±7.4
Impaired Glucose Tolerance
156(39)
30.9 ± 0.7 *
38.4 ± 0.6 *
86.4 ± 4.6 *
27.0 ±1.6*
14.1 ±0.9
176.5 ±11.5
Diabetes
24(6)
30.5 ±1.4
39.1 ±1.2
94.3 ±19.2
27.2 ± 4.4
11. 4 ±2.3
166.4 ±31 .7
Data are mean ± SEM. *P<0.05 (ANOVA with Tukey post-hoc correction for multiple comparisons) Normal Glucose
Tolerance vs IGT Groups. T=testosterone; SHBG=sex hormone binding globulin; DHAS=dehydroepiandrosterone sulfate.
These results are consistent with earlier reports substantiating the exceptionally high prevalence of
impaired glucose tolerance ("prediabetes") and frank type 2 diabetes in women with PCOS. The high
prevalence of obesity (see C1, above) in our population is a likely contributor to this finding as well as to
the risk of additional metabolic abnormalities and obstructive sleep apnea (see below).
C3. Hypertriqlyceridemia is common and predictive of additional metabolic defects in PCOS.
Women with PCOS have a substantial number of risk factors for cardiovascular disease in addition to the
high rates of impaired glucose tolerance and type 2 diabetes as noted in Section C2 above. The metabolic
syndrome is defined by both lipid and non-lipid criteria that identify individuals at increased risk for coronary
heart disease and type 2 diabetes. Thus, it has been generally assumed that most women with PCOS are
likely to meet criteria for the metabolic syndrome.
We sought to identify factors that serve as predictors for the metabolic syndrome using data derived
from a cohort of women of diverse geographic and racial/ethnic backgrounds who have been diagnosed
with PCOS by uniform criteria (88). Each individual component of the metabolic syndrome was assessed to
determine its ability to predict the presence (positive predictive value) or absence (negative predictive
value) of the requisite number of remaining components needed to establish the diagnosis of the metabolic
syndrome (Table C3-1). The presence of a fasting plasma glucose of 110 mg/dl or greater had the highest
positive predictive value (84%) for the presence of the metabolic syndrome. However, only 19 subjects met
this criterion. Elevated triglycerides, a much more common finding, also had a high positive predictive
value for the presence of the metabolic syndrome: 98 (83%) of the 118 women with a triglyceride level £
150 mg/dl had at least two additional components of the metabolic syndrome.
Table C3-1. Positive and Negative Predictive Values of Components of the Metabolic Syndrome
Waist Circumference > 88 cm
HDL Cholesterol < 50 mg/dl
Triglycerides 2 150 mg/dl
Hypertension 2 130 mmHg
systolic or 5 85 mmHg diastolic
Fasting Glucose £110 mg/dl
#with
criterion
287
240
118
78
19
#with
criterion
and a 3
total
criteria
119
115
:•: 
98 ..'
61
16
% with criterion
with MS (Pos.
Predictive
Value)
41%
48%
83%
78%
84%
# without
criterion
74
125
247
290
347
# without
criterion
and S3
total
criteria
71
118
223
228
242
criterion
without MS
(Neg.
Predictive
Value)
96%
94%
90%
79%
70%
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Ehrmann. David A.
PROJECT 2. Ehrmann, P.I.-PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle):_
Our analyses were also notable for the finding that fully one-third of non-diabetic women with PCOS have
developed the metabolic syndrome well before the end of their fourth decade, and usually prior to the end
of their third decade of life. This prevalence is markedly higher than the 6.7% prevalence of metabolic
syndrome reported in women between the ages of 20 and 30 years and the 15% prevalence reported in
women between ages 30 and 40 years from the Third National Health and Nutrition Examination Survey
(NHANES III) (76). Indeed, the prevalence of the metabolic syndrome observed in the study population
approached that seen in women who are usually between the ages of 50 and 60 years (76). Obesity
appeared to have an independent effect on risk for the metabolic syndrome: women in the highest quartile
of BMI had nearly a 14 fold increased chance of having the metabolic syndrome compared to women in the
lowest quartile of BMI and none of the 52 women whose BMI was less than 27.0 kg/m2 met criteria for the
metabolic syndrome.
C4. There is a 5-6 fold increase in risk for OSA in PCOS
We have obtained a polysomnogram and metabolic measures in 49 women with PCOS and 22 women
without PCOS (Controls). Among the 22 Controls, 4 (18%) met criteria for OSA (using the most common
AMI criterion of S 5 events/hr of sleep). In contrast, 27 (55%) of 49 women with PCOS had OSA. The
unadjusted odds ratio of 5.5 for OSA in PCOS is highly significant (p=0.006; 95% Cl 1.6 - 18.7).
Even after adjustment for age and BMI, the odds ratio remains highly significant at 5.5 (p=0.009;
95% Cl 1.5 - 20.6). Within group comparisons are shown in Tables C4-1 and C4-2. The magnitude of
metabolic abnormalities is greater in the presence of OSA as well. Control women with OSA had
significantly higher diastolic blood pressure levels; 2hr glucose levels were also higher, but significance
was lost after correction for multiple comparisons. PCOS women with OSA were significantly higher in age
relative to those without OSA, but it is important to note that these women are only in their second and third
decades of life. In addition, BMI and waist circumference were significantly higher in PCOS women with
compared to those without OSA. As was the case in controls, 2hr glucose levels were also significantly
higher, but significance was lost after correction for multiple comparisons. Finally, those with OSA had
higher fasting insulin concentrations as well as higher levels of fasting triglycerides. Thus, in women with or
without PCOS, the presence of OSA is associated with more profound metabolic abnormalities.
n
Age (yr)
BMI (kg/m")
Waist Circ (cm)
Systolic BP
[mmHg)
Diastolic BP
[mmHg)
Fasting
jlucose (mg/dl)
2hr glucose
(mg/dl)
Fasting Insulin
[pmol/L)
HOMA
A1C (%)
Cholesterol
(mg/dl)
HDL (mg/dl)
Triglycerides
(mg/dl)
LDL (mg/dl)
Total T (ng/dl)
Free T (pg/ml)
.v 
Table C4-1. 
:
Controls
Without
OSA
18(82%)
29.9 ± 1 .2
35.3 ± 1 .5
97.1 ±3.6
114.1 ±3.3
67.6 ± 1 .9
88.3 ±2.2
111.6±6.5
1 1 .6 ± 1 .7
0.4 ±0.1
5.4 ±0.1
163.5 ±6.4
54.8 ± 3.2
76.2 ±13.0
93.7 ±6.5
39.8 ±2.7
9.3 ±0.7
Controls
With
OSA
4(18%)
34.4 ± 1 .9
40.7 ±4.3
11 0.4 ±8.6
134.0 ±6.2
80.3 ±3.8
94.9 ± 5.2
151 .6 ±29.1
11. 8 ±3.2
0.5 ±0.1
5.8 ±0.3
172.0 ±8.6
49.0 ± 5.3
86.5 ±9.2
105.5 ±9.3
28.8 ± 3.4
8.5 ±1.3
P-
value*
0.123
0.160
0.123
0.012
0.007*
0.228
0.046
0.972
0.877
0.067
0.553
0.417
0.712
0.416
0.082
0.614
Table C4-2.
n
Age (yr)
BMI (kg/m')
Waist Circ (cm)
Systolic BP
mmHg)
Diastolic BP
mmHg)
-asting glucose
(mg/dl)
2hr glucose
(mg/dl)
Fasting Insulin
(pmol/L)
HOMA
A1C(%)
Cholesterol
(mg/dl)
HDL (mg/dl)
Triglycerides
(mg/dl)
LDL (mg/dl)
Fotal T (ng/dl)
Free T (pg/ml)
PCOS
Without
OSA
22 (45%)
27.6 ±0.7
35.2 ±1.5
101.9±2.5
121.0 ±2.8
70.6 ± 1 .7
93.1 ±1.6
126.1 ±6.9
16.2 ±2.1
0.6 ±0.1
5.5 ±0.1
184.3 ±5.3
52.5 ±3.0
101.2 ±5.9
11 1.6 ±5.2
72.3 ±6.7
20.6 ±1.5
PCOS
With
OSA
27 (55%)
31 .8 ±1.0
42.2 ±1.1
11 9.4 ±3.0
125.8 ±3.0
75.1 ±1.6
99.0 ±2.4
144.4 ±5.2
23.8 ± 1 .6
0.9 ±0.1
5.6 ±0.1
186.5 ±5.6
46.1 ±1.7
135.2 ±10.3
113.4 ±5.0
70.8 ±4.3
21. 3 ±1.4
P-value*
0.002*
0.001*
0.001*
0.254
0.056
0.046
0.040
0.005*
0.014
0.366
0.774
0.069
0.007*
0.806
0.856
0.736
Data are mean ± SEM. 'Asterisk indicates significance after correction for multiple comparisons (Bonferroni correction).
PHS 398/2590 (Rev. 09/04, Reissued 4/2006)
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle):.
C5. Neither the degree of androqen elevation nor BMI are correlated with OSA severity in PCOS.
Ehrmann. David A.
Strum frm Tntoct*ran» CcnontnOlon «»
Atum» Mypuiwu* 9aOm In PCOS -wannniNlth OSA
Body IU» Indu « Apiwi Hnxvnti bnm
to PCOS women <Mth OSA
V »
• *
Figure C5-1
Figure C5-2
To explore potential relationships between serum free testosterone concentration and BMI with severity of
OSA, we related these measures to the AHI in the 27 PCOS women with OSA discussed under C4, above.
Scatterplots relating AHI to serum free testosterone (Figure C5-1) and AHI to BMI (Figure C5-2) are
notable for the poor correlation between these measures and OSA severity. These results underscore the
need to explore the effects of alternate sex hormones (i.e., progesterone and estrogen) as contributing to
OSA in PCOS.
C6. Androaen levels are significantly reduced without improvement in OSA over after significant
reduction in testosterone concentrations in a subject with PCOS.
Luteinizing Hormone (LH)
Prt-GaXH Agonist
Paa-GnKHtgonin •
urn «oo «ao &00
Clock Time
90-
80-
70
60
e 40-
30-
20-
10
Total Testosterone
fe*4>nftH Agonist
HOD
fcoo KOO ithoo nao 
3300
&oo ex» 
KHOO turn 2,-oc
A « I A I > A H r a i
Clock Time
Figure C6-1 (top panel) depicts serum LH levels
measured every 20 min over 24 hr before (Pre) and
again 3 months after (Post) administration of a single
11.25 mg dose of depot leuprolide in a women with
PCOS and OSA. The LH levels at 3 months are at the
lower limit of detection of the LH assay over the entire
24 hr sampling interval.
Figure C6-1 (lower panel) depicts total testosterone
concentrations measured every 2 hr over 24 hr before
(Pre) and after (Post) administration of a single 11.25
mg dose of depot leuprolide in the same subject.
In light of the complete suppression of LH, circulating
testosterone represents the normal contribution by the
adrenal gland. Although the total testosterone levels
decreased by approximately 50% and were well within
the normal range, the magnitude of obstructive sleep
apnea did not improve in this subject. The AHI was
14.2 events per hour sleep prior to treatment and 13.1
events per hour of sleep at 3 months. These results
represent the response of a single individual. Studies
planned under Specific Aim 2 are therefore designed to
explore these findings in depth.
C7. Insulin sensitivity and glucose tolerance decline in response to suppression of slow wave sleep
We have investigated the effects of all night SWS suppression via induction of microarousals without
decrease in total sleep time on glucose metabolism in healthy young adults. Six young healthy lean adults
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Ehrmann. David A.
PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle):.
(age 20 - 31 yr; BMI 19 - 24 kg/m2) were studied under two conditions (baseline, SWS suppression) in a
randomized order separated at least by 4 weeks. Mean BMI did not change over the study period. Sleep
disorders were ruled out by polysomnography. Undisturbed sleep was recorded on two consecutive
baseline nights (B1, B2). Sleep was continuously monitored and acoustic stimuli (1000-2000Hz, 40-110dB)
were administered during NREM sleep to induce microarousals and suppress SWS for three consecutive
nights (S1, S2, S3). At the end of each condition, subjects had an fslVGTT after an overnight fast. Insulin
sensitivity (SI) was derived from minimal model analysis. Glucose tolerance (Kg) was calculated as the
linear slope of the natural log of plasma glucose between the 5th and 19th minutes after the glucose
injection. Sleep fragmentation decreased the amount of SWS (min; 74.8±7.0 on baseline vs 9.0±2.3 on S1,
13.5±3.5 on S2, 11.8±1.3 on S3; p=0.0001) with maintenance of total sleep time between study conditions
(min; 465.6±8.6 on baseline vs 457.6±6.3 on S1, 467.2±3.0 on S2, 457.0±4.6 on S3; p=0.56). SI was 24%
lower (7.3±0.9 vs 5.5± 0.6, p= 0.046) and Kg was decreased by 25% (2.0±0.3 vs 1.5±0.2, p=0.028) with
SWS suppression compared to baseline. These data suggest that sleep disruption, whether selective for
SWS or the result of OSA, lead to deterioration in insulin sensitivity. Studies proposed in this SCOR
application will follow-up on these findings with a particular focus on the role of counterregulatory hormones
(cortisol, norepinephrine) in mediating the decline in insulin sensitivity related to OSA.
C8. Treatment of OSA in PCOS results in significant reductions in 24 hr secretory profiles of
cortisol and norepinephrine.
OH
14:00 
17:00 20:00 23:00 
2.-00 5:00
Clock Time
Figure C8-1
8:00 
11:00 14
14:00 
17:00 
20:00
23:00 
2:00 
5.OO
Clock Time
Figure C8-2
8*0
11:00 
14:00
We tested the hypothesis that treatment of OSA with CPAP would ameliorate metabolic and hormonal
dysfunction in PCOS. Results obtained in 5 non-diabetic women with PCOS and OSA are shown. All
subjects had a polysomnogram, and 24-hr cortisol profile at baseline and after CPAP treatment for 8
weeks. Compliance was confirmed by built-in CPAP monitors and by subject contact every 2 weeks. For
each individual 24-hr cortisol profile, the nadir was defined as the minimum of a regression curve
calculated using a robust locally weighted procedure with a 2 hr window. For each individual 24 hr
catecholamine (NE) profile, mean levels were calculated using the raw data. Data are mean SEM.
Cortisol=mcg/dL; NE=pg/ml.
Subjects were 33.0 ± 2.5 yr old; mean BMI was 51.0 ± 9.0 kg/m2 and did not change over the study
period. Mean AMI was 17.4 ± 2.2 consistent with moderate OSA. CPAP treatment led to a significant
(p=0.005) reduction in mean cortisol levels over 24-hr (7.7 ± 0.9 vs 10.2 ± 1.0) (Figure C8-1). Significant
reductions were seen in both daytime (7.9 ± 0.9 vs 10.3 ± 0.9, p= 0.003) and nighttime (7.5 ± 0.9 vs 10.1 ±
1.5, p= 0.02) cortisol levels after CPAP treatment. The cortisol nadir decreased by 40% (2.0 ± 0.4 vs 3.3 ±
0.8, p=0.03). The reduction in cortisol nadir was significantly correlated with AHI (r=1.0, p=0.006). Mean 24
hr NE levels decreased by 29% after CPAP (233.7 ± 18.4 vs 330.6 ± 11.6, p= 0.001) (Figure C8-2).
Daytime (260.0 ± 19.2 vs 363.6 ± 16.0, p=0.002) and nighttime (183.7 ± 18.4 vs 267.0 ± 8.0, p= 0.006) NE
levels were decreased after CPAP. OSA is likely to contribute to elevated cortisol and NE levels in women
with PCOS and could play a role in the risk for adverse metabolic alterations in this patient population.
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Ehrmann. David A.
PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle):
D. RESEARCH DESIGN AND METHODS
D1. Study subjects and overview of experimental protocol
PCOS subjects will be recruited from the Endocrinology Clinics of the University of Chicago. All will be at
least 18 but less than 40 yr of age. All recruited subjects will be obese (BMI of at least 30 kg/m2). This BMI
criterion is met in 80.7% of PCOS subjects in our database, as shown in Preliminary Studies. A schematic
of the overall study design for Specific Aims 1 - 3 is presented in Figure D1-1 below. A summary grid of
all testing procedures planned is shown in Table D1. Sample size calculations and statistical
considerations follow and are presented in Section D3, below.
All subjects enrolled in this project will have a confirmed diagnosis of PCOS. A diagnosis of PCOS will
require: 1) the presence of oligo/amenorrhea; 2) hyperandrogenemia, defined by a supranormal plasma
free testosterone level (> 10 pg/ml); 3) hyperandrogenism, as evidenced by infertility, hirsutism, acne, or
androgenetic alopecia; and 4) exclusion of nonclassic 21-hydroxylase deficiency congenital adrenal
Specific Aim 1
Baseline Evaluation and Screen
for OSA in women with PCOS
OSA
Negative
OSA
Positive
Figure D1-1. Schematic of overall
study design for Specific Aims 1-3.
Randomize
I
Sped fie Aim 2
JL
J
Specific Aim 3
# •
\f \l
P I 
+ S § i
•i 
*£ -s '
I 
1 \
I 
i 5
I 
six weeks 
|
Estrogen* Placebo _ J
o
n
£
tu
•s-s
VI
Progesterone + Placebo IB
* ill
si* weeks 
i
CPAP TREATMENT* 6 weeks
1 
1
Snetira 
Era) of Stud,
Evaluation* 
Evaluation*
hyperplasia, Gushing syndrome, hypothyroidism, or significant elevations in serum prolactin. Thus, all
subjects will meet the so-called NIH criteria for PCOS (113) as well as the Rotterdam criteria for PCOS
(114). For at least 2 months before the study, all subjects must not take steroid preparations (including oral
contraceptives), medications known to alter insulin secretion and/or action, or medications known to
influence sleep. All studies will be conducted in the follicular phase of the menstrual cycle (days 1 - 7) or
after 2 or months of amenorrhea. A negative pregnancy test will be required prior to all testing procedures.
Subjects who are known to be diabetic will be excluded; those who prove to be diabetic on screening will
not continue in subsequent protocols. Subjects with hypertension (systolic > 140 mmHg and/or diastolic >
90mmHg) will be included only if well-controlled on stable medication with either ACE inhibitors or diuretics.
Subjects on beta-blockers will be excluded as these drugs affect both insulin sensitivity and sleep. Subjects
must have regular life styles (no shift work, no travel across time zones within 4 weeks of study), and
habitual bedtimes. Other exclusion criteria will be: habitual alcohol use of more than 2 drinks per day and
excessive caffeine intake of more than 300 mg per day.
PHS 398/2590 (Rev. 09/04, Reissued 4/2006)
Page 139
Continuation Format Page


PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle):.
Ehrmann. David A.
Table D1. Summary Grid of Tests
by Specific Aim.
THE PRESENCE OF THE NUMBER "1"
DENOTES PRIMARY OR KEY
OUTCOME FOR SPECIFIC AIM;
NUMBER "2" DENOTES SECONDARY
OUTCOME MEASURE. SAMPLE SIZE
POWERED ON PRIMARY OUTCOME
MEASURE(S).
ALL STATISTICAL ANLYSES WILL BE
CORRECTED FOR MULTIPLE
COMPARISONS.
SAMPLE SIZE
Oral glucose tolerance test (OGTT)
Frequently sampled IVGTT (fslVGTT)
Androgen levels (total, free T, et al.)
Estradiol, Estrone, Progesterone
Gonadotropins (LH, FSH)
Metabolomics profile (blood, urine)
24 hr hormonal profiles (including
Cortisol and Catecholamines)
Plasma catecholamines
Abdominal MRI, Bioimpedance
Adipose tissue biopsy
Serum for adiponectin
Sleep and Activity Assessment
Sleep questionnaires
Wrist activity monitoring
Multiple sleep latency test (MSLT)
Sleep recording/Polysomnography
Wake EEG/Neurobehav. assessments
Daytime blood pressure recording
24 hr heart rate monitoring
GnRH agonist test with steroid
intermediates
SPECIFIC AIM
1
Obese
PCOS
OSA
Negative
23
2
2
1
1
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
Obese
PCOS
OSA
Positive
23
2
2
1
1
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
SPECIFIC AIM
2
Obese PCOS
OSA Positive
Depot
Leuprolide
Estrogen
plus
Placebo
20
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
Depot
Leuprolide
Progesterone
plus
Placebo
20
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
SPECIFIC AIM
3
Obese PCOS
OSA Positive
CPAP
20
2
1
2
2
2
2
1
2
2
2
2
2
2
2
2
1
2
2
2
2
D2. Screening and Specific Aim 1 Procedures
46 subjects will need to be enrolled in Specific Aim 1 in order to ensure a sample size of 20 in the group of
PCOS women without OSA; this figure of 46 subjects is based on an estimated 55% prevalence of OSA
among PCOS women as shown in Preliminary Studies. This calculation accounts for the effects of
expected dropouts from the study. The primary outcome measures for this Aim are the differences in
progesterone and estrogen concentrations between groups.
Following an initial phone screening, eligible subjects will be admitted to the GCRC in the early evening
and will undergo the following screening process as described in Figure D2-1, below.
N.B. 
THE SCREENING PROTOCOL DETAILED BELOW WILL PERTAIN TO ALL SPECIFIC AIMS. BASED UPON THE
RESULTS OF THIS SCREENING, SUBJECTS WILL BE CLASSIFIED AS EITHER OSA NEGATIVE, IN WHICH CASE THE
PHS 398/2590 (Rev. 09/04, Reissued 4/2006)
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
EVALUATION IS COMPLETED, OR WILL BE CLASSIFIED AS OSA POSITIVE. OSA POSITIVE SUBJECTS WILL THEN BE
GIVEN THE OPTION TO ENTER INTO THE PROTOCOLS DETAILED IN SPECIFIC AIM 2 OR SPECIFIC AIM 3. SUBJECTS
WILL BE INFORMED THAT IF THEY CHOOSE TO CONTINUE INTO AIM 2 OR AIM 3, THEY WILL BE RANDOMLY ASSIGNED
USING A RANDOMIZATION SCHEDULE PREPARED GY THE GCRC BIOSTATISTICIAN, 
DR. THEODORE 
KARRISON.
SUBJECTS RANDOMLY ASSIGNED TO SPECIFIC AIM 2 WILL ALSO BE INFORMED THAT FURTHER RANDOMIZATION
WILL OCCUR TO ONE OF TWO TREATMENTS, AS DETAILED BELOW.
Screening and Specific Aim 1 Procedures, cont'd.
Figure D2-1. Schematic of Screening Visit. Clock time is shown along horizontal axis.
I 
I
14 
16
Dinner
1 
1
18 
20
^m 
Full PSG
I
I
I
!
22 
24 
02 
04
OGTT
Hood samples
^^^ 
Hiycirat ovam
Lunch
1 
I 
1 
1 
1
06 
08 
10 
12 
14
Subjects will receive instructions on the logs/scales and practice the psychomotor vigilance test (PVT) to
be used in the study. Subjects will also complete screening questionnaires including the Pittsburgh Sleep
Quality Index, Berlin Questionnaire, Epworth Sleepiness Scale, Center for Epidemiologic Studies-
Depression (CES-D) scale, Functional Outcome of Sleep Questionnaire (FOSQ), and the Beck Scale. An
overnight full polysomnography will be performed to differentiate the subjects with from those without OSA.
For all protocols in this SCOR application the presence of OSA will be defined by an apnea-
hypopnea index (AMI) of greater than 15 events per hour sleep (AHI > 15); the absence of OSA will
require an AHI of less than 5 events per hour sleep (AHI < 5). This criterion is consistent with
current standards and ensures that subjects with periodic limb movement disorder (PLM arousal
index > 1) are excluded from study.
On the day following the full polysomnography, a 2 hr OGTT, a complete history and physical
examination, routine laboratory tests (CBC, electrolytes, renal, liver function tests) and an EKG will be
performed. This screening visit will also serve to familiarize the subject with the study environment and the
experimental procedures.
Once the Screening Visit has been completed if the subject is deemed eligible to continue, they will be
instructed on completing one week of continuous wrist actigraphy at home; subjects will be asked to
maintain a standardized schedule of bedtimes and mealtimes designed in accordance with their usual
habits. They will be asked not to deviate from this schedule by more than 30 minutes. During this pre-study
period, subjects will also be asked to fill out a daily log of sleep and meal times. Subjects who have shown
poor compliance will be given the option to repeat the week of ambulatory recordings. If compliance
remains poor, subjects will be excluded from further study.
After the screening visit and completion of the ambulatory recordings, the subsequent experimental
procedures depicted in Figure D2-2 and described below will occur.
The subjects will have an abdominal MRI exam at 4 pm to quantify visceral and subcutaneous fat. They will
then be admitted to GCRC at 18:00 (day 1) and a standardized dinner will be provided at 19:00. The
subjects will also review the PVT and questionnaires that will be used during the study. After a night of full
polysomnography (day 2), 2 blood samples (12 cc for Metabolomics and 1 cc for detailed lipid profile) and
a urine sample (5 cc for Metabolomics) will be obtained. This will be followed by the subcutaneous fat
biopsy. The subjects will then receive a breakfast. At 9:45, 11:45, 13:45, 15:45 and 17:45, the subjects will
perform the PVT and complete various questionnaires (sleepiness, vigor, mood, hunger and appetite)
followed by a Multiple Sleep Latency Test (MSLT). A lunch will be provided around 14:30, depending on
the MSLT duration. After the 17:45 MSLT, a bioimpedence will be obtained to measure body fat. A dinner
PHS 398/2590 (Rev. 09/04, Reissued 4/2006) 
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Ehrmann. David A.
PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle):,
will be offered around 19:00 after these procedures. After a night of polysomnography (day 3), the subjects
will undergo a frequently sampled intravenous glucose tolerance test (fslVGTT) at approximately 09:00,
depending on their usual bedtimes. Starting at 14:00, 24 hour blood sampling will be initiated, with intervals
of every 15, 30 or 60 minutes for measurements of glucose, C-peptide, insulin, adiponectin, GH, cortisol,
leptin, C-reactive protein (CRP, assayed only every 4 hours), estrogen, testosterone, progesterone, ghrelin
and catecholamines (qSOmin) levels. Free fatty acids (FFA) will be obtained before and, 1 hour and 2 hours
after each meal. FFA will also be obtained every hour during the night. Concomitant with the 24-hour blood
sampling, during the waking period, saliva samples will be obtained every 30 minutes for free cortisol
assay, and daytime systolic and diastolic blood pressures will be measured at 15-minute intervals.
Figure D2-2. Experimental protocol after completion of ambulatory recordings
fcfetabotomcs
Fat biopsy
14
04
06 
08 
1C
14
= dinner, L= lurch. 8 = breakfast M= carbohydrate-rich ireal
IVT. Questionnaires. Muftpte Steep Latency Test OCLT)
Bionrpedence body fat
24-hour blood sarqdng, safcrary cortisol (qSOrrin). bbod pressure during
waking hours, tasks (qZhours): PVT. Questionnaires. Wake BH5 with HR
EOG. EMG and HRV (ERG and chest belt)
Every 2 hours, the subjects will perform the PVT
and 
complete 
the 
visual 
analog 
scales.
Subjects will also have a recording of wake EEC
brain activity and an EKG to assess their heart
rate variability (HRV). Caloric intake will be
controlled with carbohydrate-rich meals served
at 14:00 for lunch, 19:00 for dinner, and 9:00 for
breakfast, after a night of polysomnography. The
subjects will be asked to eat each meal entirely
within 
20 
minutes. 
Accelerometer-based
assessments 
of 
activity 
will 
be 
obtained
continuously during the entire study (Actical). At
the end of the 24-hour blood sampling period
(14:00 on day 4), the subject will receive a
lunch.
At 15:50, a GnRH agonist test will be performed, as depicted in FIGURE D2-3 below.
At 15:50, a 15cc blood sample for LH,
FSH, estradiol, estrone, total and free
testosterone, progesterone, 17-hydroxy-
progesterone, and androstenedione will be
obtained. At 15:59, a 5 cc blood sample
will be taken for LH and FSH. At 16:00,
leuprolide acetate 10 ug/kg will be given
as a single subcutaneous injection. At
16:30, 17:00 and 20:00, 5 cc samples will
be obtained for LH and FSH. A 15 cc
sample will be obtained for estradiol,
estrone, 
total
progesterone,
and free testosterone,
17-hydroxyprogesterone,
Leuprofide
1 0 itgfkg body weight
Bbod |,,
sanpfog
1
16
1
1
' 
1 
1
1 
1 
1 
1 
1 
1 
1 
1 
1 
1
18 
20 
22 
24 
02 
04 
06 
08 
10 
12
LH, FSH, estradiol. estrone. total and free testosterone,
progesterone. 1 7-hydroxyprogesterone and androstenedbne.
LKFSH
L
1
1 
1
14 
16
and androstenedione after 16, 20, 24 hours GnRHa administration, i.e. at 8:00, 12:00 and 16:00.
As depicted in Figure D1-1, it is at this point that subjects will have completed the Screening and Specific
Aim 1 Procedures. Details of procedures for subjects randomized into Specific Aim 2 or Specific Aim 3 are
provided below.
D3. Detail of methods pertaining to Specific Aims 2 and 3
The PCOS women with OSA studied in Aim 1 plus additionally enrolled PCOS women with OSA (for a total
of 60 subjects) will be randomized in a ratio of 2:1 to participate in Aim 2 (n=40) or Aim 3 (n=20). Block
randomization will be performed using computer-generated random numbers. The primary outcomes
measure for Aim 2 is the AMI difference pre- and post-interventions, as detail in Section D3; the primary
outcomes measure for Aim 3 is the change in 24 hr cortisol concentration pre- vs post-CPAP
PHS 398/2590 (Rev. 09/04, Reissued 4/2006)
Page 142
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middled 
Ehrmann. David A.
Subjects randomized to Specific Aim 2 will have suppression of endogenous LH secretion, and thus
ovarian sex-steroid production, by administration of a single intramuscular dose (11.25 mg) of the GnRH
agonist depot leuprolide. Leuprolide is a synthetic nonapeptide analog of naturally occurring gonadotropin-
releasing hormone (GnRH). The analog possesses greater potency than the natural hormone. When the
depot form of leuprolide is administered at a dose of 11.25 mg, there is prolonged (3 month) suppression of
pituitary gonadotropin levels and ovarian steroid levels as documented in the literature (115) and
exemplified in Preliminary Studies. Six-weeks after administration, subjects will return to the GCRC for
repeated evaluation, identical to that conducted at Baseline under Specific Aim 1.
After this midpoint assessment, subjects will again enter into a randomized phase of the protocol. Subjects
will be assigned to receive one of two treatment regimens in a randomized, double-blind, placebo
controlled fashion, for a period of 6 weeks. Subjects will receive daily oral doses of either 2 mg micronized
estradiol (Estrace*) plus placebo -OR- 200 mg micronized progesterone (Prometrium*) plus
placebo.
Rationale for preparation and dose of estrogen and progesterone. Natural estradiol is poorly absorbed
in its native form and undergoes rapid metabolism in a first-pass effect through the liver. Thus,
micronization (micronized estradiol) or acetylation (ethinyl estradiol) is required to achieve adequate blood
hormone levels. Micronized estradiol is extremely well-absorbed and much of the compound is metabolized
to estrone sulfate (65%) and estrone (15%). This "pool" of estrone and estrone sulfate then serves as the
source of regenerated estradiol. At a dose of 2 mg per day, the steady-state levels of estradiol and estrone
are typically 220 pmol/L and 68 pmol/L, respectively (116) and most closely recapitulate those seen in
normal women in the midfollicular phase of the menstrual cycle (110). Because ethinyl estradiol is poorly
converted to estrone, it will not be selected for use in these protocols. Micronized progesterone at a dose of
200 mg per day has been reported to result in attainment of serum progesterone concentrations typically
seen in the late follicular/early luteal phase of the cycle (data provided by manufacturer).
Subjects randomized to Specific Aim 3 will be treated with nasal CPAP for a period of 6 weeks.
Compliance will be documented based upon results provided by a microchip sensor that detects not only if
the unit is powered on, but also if the mask is being worn by the subject. These data cannot be altered by
the individual and there is no access to this microsensor by the user. The subjects who have OSA will
undergo one night of CPAP titration study at the end of the baseline assessment. After 6 wks of home
CPAP, the experimental study outline above will be repeated with CPAP use at night. Subjects whose
average nightly CPAP use is > 5 hr will be considered compliant.
D3. Statistical Considerations
re: Specific Aim 1
With 20 subjects per group and assuming a two-sided alpha level of 0.05, there would be approximately
80% power to detect a 0.89 SD difference in progesterone or estrogen levels between PCOS women
without and PCOS women with OSA. These values are derived from data obtained in a group of 10 PCOS
women without OSA. Using the SD from the preliminary data, this study would be powered to detect a 42%
difference in hormone levels between groups.
46 subjects will need to be enrolled for this Aim in order to ensure a sample size of 20 in the group of
PCOS women without OSA; this figure of 46 subjects is based on an estimated 55% prevalence of OSA
among PCOS women as shown in Preliminary Studies.
Progesterone and estrogen levels will be compared between the two groups using a two sample t test.
A log transformation of the data will be performed if there is evidence of non-normality. Statistical correction
will be made for multiple comparisons between groups.
re: Specific Aims 2 and 3
The PCOS women with OSA studied in Aim 1 plus additionally enrolled PCOS women with OSA (for a total
of 60 subjects) will be randomized in a ratio of 2:1 to participate in Aim 2 (n=40) or Aim 3 (n=20). Block
randomization will be performed using computer-generated random numbers.
•Specific Aim 2
For this Aim, there are three time points of interest: baseline (T1), six weeks after administration of
depot leuprolide (T2), and at the end of study after receiving six weeks of either estrogen or progesterone
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Ehrmann. David A.
PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle):,
(T3). Treatment assignment to either estrogen (n=20) or progesterone (n=20) will be randomized using
computer-generated random numbers.
If the variance in AMI is denoted by a2 and the correlation between pre- and post-treatment AMI by r, then
the variance of the change in AMI is given by 2a2(1-r). With 20 subjects per group and assuming a two-
sided alpha level of 0.05, the Table D3-1 below lists the difference in AMI change detectable between the
two treatment groups with 80% power for varying values of r.
Table D3-1. Difference in AMI change detectable between the two treatment groups with 80% power
for varying values of r.
r
0.25
0.50
0.75
0.90
Effect Size
1.08a
0.89a
0.63a
0.40a
Difference Detectable with a = 8
8.6
7.1
5.0
3.2
Polysomnography results in a group of 17 PCOS women with OSA (defined by a total AHI a 10) indicate
that the mean AHI was 18.4 ± 8 (Mean ± SD). Using the SD from these preliminary data, for example, the
present study would be powered to detect a difference of 0.63 * 8 = 5.04 if the correlation is 0.75 (see
Table D3-1). This would be equivalent to a mean change in AHI of 9.2 (a 50% decrease from baseline
assuming a baseline level of 18.4) in the progesterone treated group versus a mean change of 4.2 (23%
decrease from baseline assuming a baseline level of 18.4) in the estrogen treated group. The change in
AHI (T3 - T1) will be compared between the two groups using a two sample t test. This would amount to
performing repeated measures analysis of variance on AHI and testing for group (progesterone vs.
estrogen), time (pre- vs. post-treatment), and group by time interaction effects. The analysis will be
performed both ways. Additionally, testosterone, estrogen, and progesterone levels at time T2 will be
compared to the levels at T1 using a paired f test.
•Specific Aim 3
For this Aim the primary endpoint is the change in 24 hr cortisol concentration from baseline in response to
CPAP treatment. There are two time points of interest: baseline (T1) and end of study after receiving six
weeks of CPAP (T2). If the variance in cortisol is denoted by o2 and the correlation between pre- and post-
treatment cortisol by r, then the variance of the change in cortisol is given by 2a2(1-r). With 20 subjects and
assuming a two-sided alpha level of 0.05, the Table D3-2 below lists the cortisol change detectable with
80% power for varying values of r.
Table D3-2. Cortisol change detectable with 80% power for varying values of r.
r
0.25
0.50
0.75
0.90
Effect Size
0.77a
0.63d
0.44o
0.28a
Difference Detectable with o = 2.2
1.7
1.4
1.0
0.6
Preliminary data in a small group of OSA+ PCOS subjects indicate that the mean cortisol level was
10.2±2.2 (Mean±SD). Using the SD from the preliminary data, for example, this study would be powered to
detect a difference of 0.63x2.2 = 1.4 if the correlation is 0.50 (see Table 2).
Cortisol levels at time T2 will be compared to the levels at T1 using a paired t test. Additionally, the
association between the changes (T2 - T1) in cortisol and in total AHI with treatment will be assessed
using Pearson correlation coefficients or Spearman rank correlation coefficients, as appropriate.
PHS 398/2590 (Rev. 09/04, Reissued 4/2006)
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea. and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, MiddleV: 
Ehrmann. David A.
D4. Potential pitfalls and plans for their resolution
re: Specific Aim 1. In studies conducted under Specific Aim 1, subjects will be recruited to participate in
a wide variety of procedures over a number of weeks. Recruitment and retention may pose challenges,
however we have longstanding experience with studies of this nature and our recruitment procedures have
been successful to date; our retention rates in similar studies has been over 90%. Subjects are contacted
on a regular basis by our study coordinator. Subjects also receive monetary compensation for participation
in the protocols. We have successfully recruited subjects in the past through cooperation with other
physicians who care for patients with PCOS and we plan to do so again in the event that recruitment goals
are lagging.
re: Specific Aim 2. Studies in this Aim are anticipated to flow smoothly. Potential problems include drop-
outs related to intolerance/untoward effects of study medication and lack of compliance. It is anticipated
that additional subjects may need to be recruited to replace the drop-outs. Another potential pitfall is that
the degree of testosterone suppression expected after depot-leuprolide administration may be insufficient
(i.e., testosterone levels may remain above the normal female range). If this proves to be the case, we will
use testosterone concentration as a covariate in our analyses related to metabolic changes pre- and post-
leuprolide. Likewise, even though micronized estrogen and progesterone are optimized for oral absorption,
if serum levels of estrogen and progesterone achieved are below the expected range observed in normal
women, we will use these hormone concentrations as covariates in our statistical analyses. Although
transdermal (patch or cream preparations) were an alternative to oral hormone therapy, the assessment of
compliance with their use would have posed challenges. Pill counts, patient contact on a regular basis, and
measurment of hormone levels in blood will be used to ensure and document use of study medication.
re: Specific Aim 3. This protocol requires high levels of commitment from study subjects who must use
CPAP on a regular basis for the duration of the study. Compliance with CPAP use is automatically
recorded and powering on the device can be differentiated from patient use via sensors incorporated into
the CPAP mask. Thus, we will have device-generated compliance records.
D5. Methods of procedure
Wrist activity monitoring
The Actiwatch wrist activity monitor (Mini-Mitter Company, Inc., Sunriver, Oregon) is a miniature device (30
x 30 x 10 mm) that is accurate, sturdy and lightweight (< 68g including batteries). It has an extended
memory capacity. Specially designed hardware and software for data downloading, graphing and analysis
are available for this system. Wrist activity recordings will provide objective estimations of sleep duration for
one week prior to the study.
Polysomnography
Sleep recordings will be performed in the laboratory using a Nihon Kohden (Neurofax EEC- 1100A) digital
EEC acquisition system. Recordings will include electroencephalography (EEC), electrooculography
(EOG), electromyography (EMG), electrocardiography (EKG) signals and a chest belt to evaluate
respiration. Full polysomnography will also include oronasal airflow by thermocouples, respiratory effort
from thoracic and abdominal piezo electric belts, and pulse oximetry. Prior to study sessions, all subjects
will undergo one night of full polysomnography to detect sleep disordered breathing (AMI > 15, no OSA is
defined as AMI < 5) and to exclude periodic limb movement disorder (PLM arousal index > 1). This
screening night will also serve as habituation night. During the experimental protocol, three nights will be
recorded. Each 30-sec epoch of recording will be visually scored as stage Wake, I, II, III, IV or REM
following standard criteria by an experienced rater who will be blinded to the age and gender of the subject
and the study condition. Respiratory events, periodic limb movements and microarousals will be scored
according to established criteria. Spectral analysis of sleep EEG will be performed using specially designed
software (PRANA, PhiTools, Strasbourg, France) in the delta, theta, alpha, sigma, beta and gamma bands.
Sleep questionnaires
The Pittsburgh Sleep Quality Index (PSQI). This index is derived from a self-rated 21 item questionnaire
that assesses sleep quality and disturbances over a 1 month time interval. Seven "component" scores
PHS 398/2590 (Rev. 09/04, Reissued 4/2006) 
Page 145 
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
ranging from 0 to 3 for each component are generated from this survey; one global score (PSQI) is derived
from the sum of these seven scores. A global PSQI score greater than 5 has a diagnostic sensitivity of
89.6% and specificity of 86.5% in distinguishing good and poor sleepers. The seven components include:
subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of
sleeping medication, and daytime dysfunction.
The Berlin Questionnaire. This questionnaire is used to assess risk for sleep apnea through questions
regarding risk factors - snoring behavior, wake time sleepiness or fatigue, and the presence of obesity or
hypertension. This instrument predicts high risk for sleep apnea (an RDI of 5 or greater) with a sensitivity of
0.86, specificity or 0.77, a positive predictive value of 0.89, and a likelihood ratio of 3.2.
The Epworth Sleepiness Scale (ESS). This survey was used to assess the level of chronic daytime
sleepiness. A score of 10 or greater is indicative of excessive daytime sleepiness.
Wake EEG recordings and neurobevioral assessments
The subjects will be instructed to relax without falling asleep and to fixate a point on the wall for 3 min while
breathing at a stable pace and then to close their eyes for 3 min while breathing normally. During the
recording, the subjects will be under constant supervision. Spectral analysis of wake EEG will be
performed using specially designed software (PRANA, PhiTools, Strasbourg, France) in the delta, theta,
alpha, sigma, beta and gamma bands. After artifact removal, a spectral analysis, using a Fast Fourier
Transformation, will be computed for 2-sec epochs for the two EEG derivations with a resolution of 0.5 Hz.
The data will then be averaged for the first and the second 3-min periods in different frequency ranges, e.g.
from low delta (0.5-2 Hz) to gamma (>30 Hz). Measures derived from the wake EEG include the alpha-slow
wave index (ASI) defined as the ratio of EEG power in the alpha band over sum of power in the theta and
delta bands) and the EEG power in the beta range of the wake EEG, a marker of alertness largely distinct
from the ASI. Subjective ratings of sleepiness (Stanford Sleepiness Score), mood and vigor (VAS), Positive
Affect Negative Affect Scale), hunger, appetite for various food categories, will be obtained daily in the mid
to late morning, mid afternoon and evening, together with the wake EEG. Subjective sleep quality and
quantity will be obtained using the Karolinska sleepiness scale every morning upon awakening.
Neurobehavioral function will be measured using 5-min psychomotor vigilance test (PVT).
Multiple sleep latency test
The gold standard to evaluate physiological sleepiness is the multiple sleep latency test (MSLT), a series of
five nap opportunities presented at 2-hour intervals beginning approximately 2 hours after initial (morning)
awakening. Individuals undergoing an MSLT are instructed to allow themselves to fall asleep during the
nap. Subjects are tested under standardized conditions in their street clothes and are not permitted to
remain in bed between nap test sessions. Electrophysiological measures (EEG, EOG, EMG) needed to
detect sleep onset and score sleep stages are recorded during nap opportunities. Unequivocal sleep is an
epoch of stage 2, 3, or 4 or rapid eye movement sleep (REM) sleep. Sleep latency is defined as the
elapsed time from the start of the test to the first 30-sec epoch scored as sleep.
2-hour oral glucose tolerance test (OGTT)
The 2-hour OGTT will be used as a screening test for assessment of glucose tolerance status (normal,
impaired glucose tolerance and diabetes) as well as an outcome measure. After an overnight 12-hour fast,
an intravenous catheter will be placed into an antecubital vein for blood drawing. Baseline samples of
glucose, insulin and C-peptide will be taken at -15 and 0 minutes. At time 0, 75 g of glucose is
administered orally, and blood samples are collected for the measurement of glucose, insulin, and C-
peptide concentrations at 30, 60, 90 and 120 min. Diagnostic measures include baseline and 2-hour
glucose concentrations. Normal glucose tolerance, impaired glucose tolerance and type 2 diabetes will be
defined based upon the plasma glucose concentration at 2 h using the criteria of the Expert Committee on
the Diagnosis and Classification of Diabetes Mellitus of the American Diabetes Association. Specifically, a
diagnosis of normal glucose tolerance, impaired glucose tolerance, or diabetes is assigned if the glucose
level at 2 h is less than 140 mg/dl, between 140 - 200 mg/dl, or 200 mg/dl or more, respectively.
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
Frequently sampled intravenous glucose tolerance test (fslVGTT)
The fslVGTT will be used to estimate insulin sensitivity by the Bergman minimal model as well as the
adequacy of the first phase insulin secretory response to an intravenous glucose bolus. Starting at 09:00
after an overnight fast, blood samples (1 ml) will be drawn every 5 min for 10 min (3 baseline samples), at
which time 0.3 g/kg glucose will be administered as an intravenous bolus. Blood samples will be taken at 2,
3, 4, 5, 6, 8, 10, 12, 15, 19, 21, 22, 24, 26, 28, 30, 40, 50, 60, 70, 90, 100, 120, 140, 180, 210 and 240 min.
At time 20 min, intravenous insulin (0.02 U/kg) will be administered. The following summary measures will
be derived: baseline insulin concentration, first phase area under the insulin curve from 0 min to 19 min;
acute insulin response to glucose (AIRg) from 0 min to 10 min; insulin response expressed as a
percentage of baseline insulin level; glucose tolerance index (Kg), insulin sensitivity index (SI;), and
glucose effectiveness at the basal insulin level (SG). The so-called disposition index (Dl), i.e., AIRg x SI, is
calculated to express beta-cell function in relation to the degree of insulin resistance.
Blood sampling and hormonal assays
24-hour blood sampling will be performed at 15-minute intervals for 2.5 hours after each standardized meal
(served at 9:00,14:00 and 19:00) and for 2 hours after scheduled bedtime, and at 30-minute intervals at all
other times for measurements of glucose, C-peptide, insulin, GH, cortisol, leptin, CRP (assayed every 4
hours), estrogen, testosterone, progesterone, acylated and non-acylated ghrelin Blood samples will be
obtained every hour for catecholamines. FFA will be obtained right before each meal and 1 hour and 2
hours after. FFA will also be obtained every hour during the night. During periods of frequent blood
sampling, a sterile heparin-lock catheter will be inserted in the forearm and the line will be kept patent by a
slow drip (10 cc/hr) of heparinized saline (750 ILJ/dl). All blood samples will be collected through the lateral
arm of a three-way stopcock. During waking hours blood samples will be collected at the bedside. During
sleep hours the intravenous line will be extended and fed through a light-tight port in the wall, thus allowing
disturbance-free blood drawing from a next-door sampling room. The total amount of blood withdrawn
during each of the proposed studies will not exceed 500 ml. Blood samples will be centrifuged immediately
at 4°C and plasma will be frozen and stored at -80°C until assay. For each hormonal variable, all samples
obtained from the same subject will be measured in the same assay. C-peptide, Insulin, GH and cortisol
leptin, CRP (assayed every 4 hours), estrogen, testosterone and progesterone will be measured by
chemiluminescence 
assays using the Immulite Immunochemistry System (Diagnostic 
Products
Corporation). Results with the DPC Immulite, as assessed by our previous experience, or according to
College of American Pathologists surveys reporting use of the instrument in clinical lab settings,
demonstrate sensitivity, accuracy and reproducibility that meet all the requirements of the proposed
research. Ghrelin levels will be measured by RIA (Linco Research, Inc., St. Louis, MO) kit for acylated and
total ghrelin. Sensitivity is 10 pg/mL
Plasma catecholamines (q60min) will be measured using the ESA Coulechem Md 5001 instrument. In
this method, the catechols are first partially purified from plasma by absorption to a silica powder.
Norepinephrine (NE) and epinephrine (E) are separated upon emergence from an HPLC column and
quantified in a partially purified state on the basis of a reversible oxidation reduction as each catecholamine
passes across a set of electrodes. The precision (CVs) of the ESA procedure, at physiologic catechol
levels (NE >100 pg/ml, E 20 - 60 pg/ml) has been found to be, 6.0 -1.8 and 4.8 - 8.0% respectively for NE
and E intra- or inter-assay. Our group has recently published 24-h profiles of catecholamine levels,
showing exquisite delineation of meal responses and age effects.
Triglycerides will be measured using a Serum Triglyceride Determination kit (Sigma-Aldrich, Inc.).
FFA concentrations will be assayed with enzymatic colorimetric kits (Wako Chemicals USA, Richmond,
VA).
Hormonal assays for the GnRHa Test
Estradiol will be assayed by a modification of a Pantex kit method after extraction of plasma with a
sensitivity of 2.8 pg.ml and precision of 11%. Steroid intermediates (17-hydroxyprogesterone and
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle!: 
Ehrmann. David A.
androstenedione) will be measured after extraction, purification by thin-layer chromatography, and RIA.
These methods are sensitive to approximately 25 ng/dl and have a precision averaging 13%. Total
testosterone will be measured by a nonchromatographic method using a Diagnostics Products kit with a
sensitivity of <20 ng/ml and a precision of 11%. Free testosterone will be computed from a competitive
protein binding analysis with a sensitivity of 3 pg/ml, precision of 13%.
Body composition and body fat distribution
Waist circumference and waist/hip ratio will be measured. Bioimpedence will be performed. Body
composition and fat distribution will be assessed by abdominal MRI (112).
Subcutaneous fat biopsy (to be performed by PI in the GCRC)
The abdominal area adjacent to the umbilicus is prepped with betadine solution. An area approximately 12
to 15 cm in diameter is prepped and allowed to air dry. Anesthetic agent is pre-mixed by removing 5cc of
1% plain lidocaine from its bottle and replacing the volume removed with 5cc of 8.4% sodium bicarbonate
solution. Lidocaine field block is performed using 10cc of the premixed lidocaine/bicarbonate mixture. The
area is allowed to become anesthetized for approximately 10 min after the field block, during which time it
is covered with sterile 4x4. An incision approximately 0.5cm long is made parallel to the patients' waistline
in the central portion of the field-blocked area, through the dermis and into the subcutaneous adipose
tissue. Pre-filled 60cc syringes containing 10cc normal saline are used to aspirate the adipose tissue using
a 13 gauge 3-inch hypodermic needle. Gentle aspiration back/forth motion is used to aspirate the adipose
tissue. This is subcutaneous biopsy and the proper angle is approx parallel to the skin and extending up 15
degrees from the plane of the abdomen. Up to 5 syringes are used per abdominal site, depending on the
sample size needed for the study. Steri-strips are used to close the wound site and folded 4x4's are placed
over the strips and secured using Elastoplast. Direct pressure is applied to the wound for 5-10 minutes
immediately afterwards. Ice packs are given to the patient to apply to the area after the procedure.
Assessments in adipocytes (performed by M. Brady, PI - Project 4)
Adipocyte preparation: Adipose sample is weighed and placed in DMEM + 1% BSA (Type V, Sigma
#A7888) + 5 mM glucose + 10 mM Hepes (pH 7.4) (DMEM*). Collagenase stock solution is prepared using
3.5 ml of DMEM*/gram of fat) and 1 mg/ml collagenase (type II, Sigma #C6885). Adipose tissue is minced
with a double pair of scissors, approximately 100-150 strokes. Samples are then incubated for 5 min at
37oC. Cells are transferred to new vials containing collagenase solution and incubated for a further 60-75
min at 37oC, swirling vials every 5 min. Cells are transferred to 15 ml conical tubes containing 10 ml of
DMEM*. Adipocytes are washed 3X using centrifugal centrifugation (120 rpm) using 10 mis of
DMEMVwash. After final wash, excess media is completely removed and packed cells are used for in vitro
assays.
Signaling Assays: 100 ml of packed cells are transferred into 1.5 ml epindorf tubes. 100 ml of DMEM*
containing 2X final concentration of insulin (0-20nM) is added to the tubes. Cells are gently vortexed and
incubated at 37oC for 10 min. Cells are then washed 3X in DMEM (no BSA) and lysed by addition of 200
ml of 2X Laemli buffer. All samples are boiled for 2 min, and then spun at 10,000 rpm for 10 min, RT.
Samples are separated by SDS-PAGE, transferred to nitrocellulose for immunoblotting with appropriate
antibodies.
Metabolic Assays: 200 ml of packed cells are transferred in triplicate into 1.5 ml epindorf tubes. 200 ml of
DMEM* containing 2 mCi 14C-glucose, -/+ 10 nM insulin is added to appropriated tubes. Reactions are
incubated at 37oC for 30 min, and vortexed every 5 min. At the end of the assay, half of the cells are
transferred to glass test tubes and glycogen isolated using ethanol precipitation. Glycogen pellets are dried
overnight, solubilized in 1 ml of water, and 14C-glucose incorporation into glycogen is determined by liquid
scintillation counting. The other half of the reaction is transferred to 7 ml scintillation vials containing 900 ul
of PBS and 4 ml of Betafluor is added to each vial. Vials are capped and shaken vigorously to extract the
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
lipids. The next day, the top 3 ml from each vial are transferred to new vials and radioactivity incorporated
is determined by liquid scintillation counting.
Adipocyte culture: Primary adipocytes are resuspended in DMEM +2% FBS in a 1:1 ratio of cells to media
and allowed to recover at 37oC, 6% CO2 for 30 minutes. After recovery, cells are added to Matrigel
Basement Membrane Matrix in a 2:3 ratio, and 250 ul of cell/Matrigel suspension are plated per well on a
12 well plate. Plates are then placed at 37oC for 30 minutes to allow the matrigel to polymerize, after which
500 ul of DMEM +2% FBS are added per well.
Cardiovascular measures
Systolic and diastolic arterial blood pressure will be measured at 15-min intervals during the daytime from
the non-dominant arm using ambulatory monitoring equipment (Accutracker II, Suntech Medical
Instruments). Resting EKG recordings (Ninon Kohden) using two thoracic electrodes will be used to record
heart rate and HRV. Time domain measures of HRV including SDNN, NN50, pNN50, r(iNN) will be
calculated using ectopy and artifact free 5-min bins. Spectral power estimated in the high frequency band
(HF component; 0.15 - 0.40 Hz) will be used as a measure of parasympathetic activity. Other measures of
cardiac sympathovagal balance will include spectral power in the low frequency band (LF component; 0.04
- 0.14 Hz) and the LF/HF ratio. The total power in the 0.04 - 0.40 Hz band will be used to normalize
absolute LF and HF values.
Measures of energy expenditure
We will use accelerometer-based assessments (Actical, Mini-Mitter Co. Inc, Bend, OR) to monitor the
stability of activity levels in each study condition during the entire study period. The Actical is a small
lightweight device (17g; 28 x 27 x 10 mm) sensitive to omni-axial accelerations, which are sampled at 32
Hz, converted to activity counts, and integrated over 1-minute epochs. The participants will wear the Actical
around the clock (except when bathing) attached to an elastic waist band above their right iliac crest.
Metabolomics
Blood and urine will be collected, temporarily stored at - 80 C, and sent to the Metabolomics Laboratory at
Duke University. Measurement of analytes in a targeted fashion is achieved by addition of known quantities
of stable isotope-labeled internal standards to an array of sample types, including extracts of cells in
culture, as well as bodily fluids and tissue extracts of model organisms and human subjects. The laboratory
has also recently obtained the new Waters Acquity Ultra Performance LC (UPLC)™ and LCT Premier™
LC/MS systems. The enhanced chromatographic peak capacity and sensitivity of the UPLC, coupled with
the high resolution and mass accuracy of the LCT Premier, allows for "unbiased" analysis of as many as
5000 individual metabolites in a single specimen. Although the identity of most of the analytes is unknown
when using the LC/MS instrument, the Waters MarkerLynx™ statistical analysis software can be used to
define principal components (groups of analytes) that discriminate different samples (e.g., samples taken
before and after a period of drug therapy or before and after an intervention such as continuous positive
airway pressure (CPAP)). Once a set of metabolites has been shown to correlate with functional states, the
identity of the individual analytes can be determined by additional chemical and MS-based analyses.
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PROJECT 2. Ehrmann, P.I.-PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle!: 
Ehrmann. David A.
E. HUMAN SUBJECTS RESEARCH
1. Risks to the Subjects
a. Human Subjects Involvement and Characteristics
• 
Study subjects will include women with PCOS. Subjects will be between 18 and 40
years of age.
• 
Women > 40 years of age will not be recruited to avoid the confounding variable of age
related alterations upon ovarian steroidogenesis. It is anticipated that the majority of
subjects will be aged between 25 and 35 years.
• 
Women with nonclassic 21-hydroxylase deficiency congenital adrenal hyperplasia will
be identified by and AM 17-hydroxyprogesterone blood level. In cases where there is
question, subjects will be advised to undergo an adrenocorticotropin stimulation test.
Women with this disorder will be excluded from further study as will those with
Cushing's syndrome. There will be no exclusion criteria based upon race, but subjects
with systemic illnesses, including heart, renal, liver, or malignant disease will be
excluded. Individuals with known peanut allergy or allergies to medications used in this
study will be excluded.
• 
Pregnancy status will be checked. Pregnant women will be excluded from study.
• 
With the exception of children, the proposed studies will not include special classes of
subjects. Specifically, studies will not include fetuses, neonates, pregnant women,
prisoners, institutionalized individuals.
• 
Individuals known to be diabetic will not be enrolled in studies; those who prove to be
diabetic upon screening will discontinue participation in the protocols.
• 
Subjects with hypertension (systolic > 140 mmHg and/or diastolic > 90mmHg) will be
included only if well-controlled on stable medication with either ACE inhibitors or
diuretics. Subjects on beta-blockers will be excluded as these drugs affect both insulin
sensitivity and sleep.
b. Source of Materials
• 
Research materials to be obtained from study subjects includes blood and urine
samples for hormonal and biochemical measures. Blood samples will be collected for
research purposes.
• 
Fat cells from the subcutaneous tissue will be obtained by biopsy procedure.
• 
Existing records will not be incorporated as part of the study files.
c. Potential Risks
• 
The clinical procedures involve a minimal discomfort associated with venipuncture.
There might be slight bruising at the site that will recover in a short period of time.
There is also slight risk of inflammation of the vein or infection. However, our nurses
who perform these procedures are highly trained and thus these risks are minimal.
The amount of blood withdrawn is minimized to prevent significant blood loss. Baseline
hematocrit will be measured in all subjects to ensure that this risk is not excessive.
• 
There are no known direct or permanent side effects from a single injection of either
diagnostic leuprolide (10mcg/kg body wt) or depot leuprolide (11.25mg). In women the
changes in hormones may change the timing or pattern of the menstrual cycle during
which it is given, or cause transient water retention, breast tenderness, or mood
changes. Occasional patients have reported headache, Gl upset, back pain, mild
fever, or increased libido after a single dose of leuprolide. Swelling and tenderness at
the injection site may develop. The safety of leuprolide during pregnancy and risk of
treatment to an unborn fetus is unknown. Women will be instructed to use an effective
non-hormonal form of contraception, such as condom with spermicidal foam or IUD.
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
• 
Insulin. Insulin administered as part of the fslVTT will raise plasma insulin levels
transiently, but hypoglycemia is rare given that the insulin is administered only after a
glucose bolus has been given as part of the procedure.
• 
Micronized progesterone will not be given to individuals with known peanut allergy
since the capsules are known to contain peanut oil. Likewise, this medication will not
be given to women with undiagnosed vaginal bleeding or a history of deep vein
thrombosis. Patients may experience headache, breast tenderness, bloating, or
depression as a result of use of this medication. Patients will be informed of these risks
and all efforts will be taken to minimize them.
• 
Micronized estrogen will not be given to individuals with known peanut allergy since
the capsules are known to contain peanut oil. Likewise, this medication will not be
given to women with undiagnosed vaginal bleeding or a history of deep vein
thrombosis. Patients may experience nausea, headache, breast tenderness, or
bloating as a result of use of this medication. Patients will be informed of these risks
and all efforts will be taken to minimize them.
• 
The GCRC nurses who perform these procedures are highly trained and thus the risks
directly associated with intravenous puncture and blood withdrawal described above
are minimal. For those subjects randomized to take pioglitazone, monitoring of hepatic
function will be performed as described above. Subjects may choose not to participate
in the study, and may discontinue the study at any time point. Conventional diagnostic
and therapeutic modalities will be made available to all subjects where appropriate. In
the event of physical injury resulting from this research, the University of Chicago
Hospitals will provide emergency care at no cost to the subject, if such care is
necessary. The University of Chicago Hospitals will also provide non-emergency care,
but the Medical Center assumes no responsibility to pay for such care or to provide
financial compensation. Confidentiality is always respected. Specimens are logged by
number according to routine practices of the institution. Data collected are included in
the patients' charts and results of the studies are made available to patients, their legal
guardian, and their referring physicians. Samples are not available to any investigators
other than those listed herein.
2. Adequacy of Protection Against Risks
a. Recruitment and Informed Consent
• 
Efforts will be made to recruit women and minorities to participate in the proposed
studies. Since the disorder being investigated affects women of all ethnicities, attempts
will be made to have subjects represented from all known groups. Community
screenings will be conducted in Chicago neighborhoods known to have high
population densities for individuals of American Indian, Asian, and Hispanic origin. The
University of Chicago has established relationships with Community- and Church-
based organizations throughout the metropolitan region. These organizations will be
targeted for recruitment efforts.
• 
Subjects will be recruited from the Outpatient Clinics at the University of Chicago, and
by advertisement in appropriate newsletters or other media. The nature of the research
project will be described in detail to the subjects and written summaries, in lay terms,
of the research proposed will be provided for review. Written informed consent will be
obtained by the P.I. from the subjects and/or parent/legal guardian where appropriate.
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
The protocols and consent forms have been approved by the Institutional Review
Board at the University of Chicago
• 
Because women are the target population for the study of PCOS, the PI anticipates no
difficulty in recruitment of women to participate in the proposed protocols.
• 
Minority populations have actively participated in the Pi's research program in the past,
with the distribution as noted above. As described above, outreach efforts will be
intensified to ensure enrollment of underrepresented minority populations.
• 
Children are not specifically targeted for the present studies, nor will they be excluded.
It is anticipated that a small number of children may be enrolled who are 2 years post-
menarche. It is further anticipated that fewer than 5% of subjects will be less than 18
years of age.
b. Protection Against Risk
The following will be specifically instituted to protect human subjects from research risks:
• 
subjects with systemic illnesses, including heart, renal, liver, or malignant disease will
be excluded. Pregnant women, women with known peanut allergy or allergy to any
planned study medication will be excluded.
• 
The nature of the research project will be described in detail to the subjects and written
summaries, in lay terms, of the research proposed will be provided for review. Written
informed consent will be obtained by the P.I. from the subjects and/or parent/legal
guardian where appropriate. The protocols and consent forms have been approved by
the Institutional Review Board at the University of Chicago.
• 
The amount of blood withdrawn will be minimized to prevent significant blood loss.
• 
Baseline hematocrit will be measured in all subjects to ensure that this risk is not
excessive. A cooling or heating apparatus will be available in the event that local
venipuncture site swelling develops.
• 
Women will be instructed to use an effective non-hormonal form of contraception, such
as condom with spermicidal foam or IUD for studies that will alter sex-steroid levels.
3. Potential Benefits of the Proposed Research to the Subjects and Others
• 
Subjects will gain knowledge about their ability to secrete insulin and metabolize
glucose. They sill also be informed of their sleep pattern and efficiency and will benefit
from treatment, if needed, for obstructive sleep apnea. Because many of the patients
will be at risk for developing diabetes, they will benefit from frequent clinical
monitoring, should their condition progress during the study period. Patients will benefit
from accurate diagnosis which will guide proper treatment. The information gained
may provide insights into the pathogenesis of type 2 diabetes and may have important
therapeutic implications in the future. The risks involved in ascertaining this information
(see above) are minimal in relation to the potential benefit of the information gained.
4. Importance of the Knowledge to be Gained
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
• 
The proposed studies hold the potential for development of new knowledge regarding
the etiology and pathogenesis of PCOS, obstructive sleep apnea, and the metabolic
derangements associated with these conditions. Because these disorders are
extraordinarily common and because they have significant impact on the health and
well-being of those affected, the proposed studies are of great potential importance.
The insights gained may lead to a better understanding of ways to prevent and/or treat
type 2 diabetes, obstructive sleep apnea, and PCOS.
Inclusion of Women:
Because women are the target population for the study of PCOS, the PI anticipates no difficulty in
recruitment of women to participate in the proposed protocols. The vast majority of the proposed study
population will be comprised of women with PCOS, spanning across a broad spectrum of age, race, and
ethnicity. PCOS typically manifests during the late teen years or early adulthood, but can be evident as
early as age 12 years. The protocols in this proposal will be conducted in women ranging in age from 18 -
40 years. Before age 18 years and after age 40 years, there are significant effects of age upon ovarian
steroidogenesis which can confound analyses in this proposal.
Inclusion of Minorities:
Minority populations have actively participated in the Pi's research program in the past, with the distribution
as noted above. Our studies have included women with PCOS of all racial and ethnic backgrounds. We
have made, and will continue to make, efforts to recruit women from both local and distant sites. We will
advertise in neighborhood publications within the City of Chicago, as well as the surrounding suburbs, and
northwest Indiana. Our recruitment of minorities is facilitated, in part, by our location within the City of
Chicago, our referral base (local, Midwest, and national). Outreach efforts will be intensified to ensure
enrollment of underrepresented minority populations.
Inclusion of Children:
Children will not be included in the proposed studies.
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last, First, Middle): 
Ehrmann. David A.
F. VERTEBRATE ANIMALS
None
G. SELECT AGENT RESEARCH
Not applicable
H. LITERATURE CITED
1. 
Centers for Disease Control and Prevention. 2005 Percentage of adults who reported and average
of s 6 hours of sleep per 24-hour period, by sex and age group - United States, 1985 and 2004.
Morbidity and Mortality Weekly Report 54:933
2. 
Colten HR, Altevogt BM, eds. 2006 Sleep Disorders and Sleep Deprivation: An Unmet Public
Health Problem Washington, D.C.: National Academies Press
3. 
Ehrmann DA 2005 Medical Progress: Polycystic Ovary Syndrome. N Engl J Med 352:1223-1236
4. 
Knochenhauer E, Key T, Kahsar-Miller M, Waggoner W, Boots L, Azziz R 1998 Prevalence of the
polycystic ovary syndrome in unselected black and white women of the southeastern United States:
a prospective study. J Clin Endocrinol Metab 83:3078-3082
5. 
Dunaif A, Segal K, Futterweit W, Dobrjansky A 1989 Profound peripheral insulin resistance,
independent of obesity, in polycystic ovary syndrome. Diabetes 38:1165-1174
6. 
Krosnick A 2000 The diabetes and obesity epidemic among the Pima Indians. N J Med 97:31-37
7. 
Ehrmann D, Barnes R, Rosenfield R, Cavaghan M, Imperial J 1999 Prevalence of impaired glucose
tolerance and diabetes in women with polycystic ovary syndrome. Diabetes Care 22:141-146
8. 
Legro R, Kunselman A, Dodson W, Dunaif A 1999 Prevalence and predictors of risk for type 2
diabetes mellitus and impaired glucose tolerance in polycystic ovary syndrome: a prospective,
controlled study in 254 affected women. J Clin Endocrinol Metab 84:165-169
9. 
Legro RS, Gnatuk CL, Kunselman AR, Dunaif A 2005 Changes in glucose tolerance over time in
women with polycystic ovary syndrome: a controlled study. J Clin Endocrinol Metab 90:3236-3242
10. 
Ehrmann DA, Liljenquist DR, Kasza K, Azziz R, Legro RS, Ghazzi MN 2006 Prevalence and
predictors of the metabolic syndrome in women with polycystic ovary syndrome. J Clin Endocrinol
Metab 91:48-53
11. 
Talbott E, Guzick D, Clerici A, Berga S, Detre K, Weimer K, Kuller L 1995 Coronary heart disease
risk factors in women with polycystic ovary syndrome. Arterioscler Thromb Vase Biol 15:821-826
12. 
Tasali E, Van Cauter E 2002 Sleep-disordered breathing and the current epidemic of obesity:
consequence or contributing factor? Am J Respir Grit Care Med 165:562-563
13. 
Spiegel K, Knutson K, Leproult R, Tasali E, Van Cauter E 2005 Sleep loss: a novel risk factor for
insulin resistance and Type 2 diabetes. J Appl Physiol 99:2008-2019
14. 
Spiegel K, Tasali E, Penev P, Van Cauter E 2004 Brief communication: Sleep curtailment in healthy
young men is associated with decreased leptin levels, elevated ghrelin levels, and increased
hunger and appetite. Ann Intern Med 141:846-850
15. 
Tasali E, Van Cauter E, Ehrmann DA 2006 Relationships between sleep disordered breathing and
glucose metabolism in polycystic ovary syndrome. J Clin Endocrinol Metab 91:36-42
16. 
Vgontzas AN, Legro RS, Bixler EO, Grayev A, Kales A, Chrousos GP 2001 Polycystic ovary
syndrome is associated with obstructive sleep apnea and daytime sleepiness: role of insulin
resistance. J Clin Endocrinol Metab 86:517-520
17. 
Brownell LG, West P, Kryger MH 1986 Breathing during sleep in normal pregnant women. Am Rev
Respir Dis 133:38-41
18. 
Maasilta P, Bachour A, Teramo K, Polo O, Laitinen LA 2001 Sleep-related disordered breathing
during pregnancy in obese women. Chest 120:1448-1454
19. 
Driver HS, McLean H, Kumar DV, Farr N, Day AG, Fitzpatrick MF 2005 The influence of the
menstrual cycle on upper airway resistance and breathing during sleep. Sleep 28:449-456
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PROJECT 2. Ehrmann, P.I. - PCOS, Sleep Apnea, and Metabolic Risk in Women
Principal Investigator/Program Director (Last. First, Middle): 
Ehrmann. David A.
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Ehrmann. David A.
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I. MULTIPLE PI LEADERSHIP PLAN
Not applicable
J. CONSORTIUM/CONTRACTUAL ARRANGEMENTS
Not applicable
K. RESOURCE SHARING
No novel resources will be developed in this application.
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