Post-Prandial Liver Glucose Metabolism in Polycystic Ovarian Syndrome (PCOS) and Understanding Standard of Care Medications
Conditions: Polycystic Ovarian Syndrome, Obesity, Hepatic Steatosis
Sponsor: University of Colorado, Denver

 
Title: Post-Prandial Liver Glucose Metabolism in Polycystic 
Ovarian Syndrome (PCOS) and understanding Standard of 
Care Medications 
 
Version Date: 8/17/2017 
NCT03041129 
 

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w 
COMIRB Protocol 
 
PROTOCOL #: 
16-2399  
 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
Project Title: 
Post-Prandial Liver Glucose Metabolism in Polycystic Ovarian Syndrome 
(PCOS) and understanding Standard of Care Medications 
 
 
Version Date: 8/17/2017 
 
Principal Investigator:  Melanie Cree Green, MD, PhD  
 
 
 
 
  
 
I. Hypotheses and Specific Aims:   
 
HYPOTHESIS:  
Obese girls with hepatic steatosis (HS) will have hepatic glucose metabolism that is upregulated 
towards de novo lipogenesis (DNL) via multiple intrahepatic pathways, compared to PCOS without 
HS and obese controls without HS. HS will be related to relative post-prandial hyperglycemia. Post-
prandial hyperglycemia will be made worse or remain unchanged with oral contraceptives and 
better with metformin.  
 
Specific Aims: 
Aim 1: In obese girls with PCOS, post-prandial hepatic substrate metabolism will differ by HS 
status and treatment regimen and can be assessed with just an oral tracer protocol combined with 
NMR isotopmer analysis. 
Hypothesis: Girls with HS will have upregulated post-prandial hepatic non-glycogenic metabolism 
compared to those without HS. This will be unchanged by oral contraceptives, and decreased with 
metformin. 
Rationale: A marker of whole body DNL was increased in girls with HS, and preliminary work from 
my K23 project indicates that direct measures of DNL are elevated, but upstream alterations in 
intrahepatic metabolism are unclear. Oral contraceptives do not mitigate metabolic risk, whereas 
metformin decreases HS. 
Methods: A 6.5 hour OGTT with an oral U-C13glycerol tracer will be paired with NMR isotopomer 
analysis of serum samples to describe flux through the hepatic pentose phosphate pathway, TCA 
cycle and DNL pathways in girls with PCOS receiving lifestyle only, metformin or oral contraceptive 
treatment, and obese girls with regular menses receiving lifestyle therapy. 
 
Aim 2: Describe the optimal timing for identifying metabolomics profiles associated with risk for T2D 
and HS. 
Hypothesis: Metabolomics profiles between girls with HS and worse IR will differ the most between 
3-6 hours.  
Rationale: Altered glucose and fat metabolism is most apparent in the post-prandial state, in terms 
of insulin resistance, β-cell failure, hyperglycemia and DNL. Our preliminary data indicates that 
glucose and insulin concentrations are most different between 3-4 hours following a glucose load. 
The optimal timing post-drink to describe metabolomics differences is not known.  
Methods: Targeted metabolomics profiles will be performed at intervals during the OGTT to 
determine time points that provide the biggest difference in girls with HS, hyperglycemia, 
inadequate β-cell response and IR. Specific targets will likely be free fatty acid and DNL markers, 
and will be determined from on ongoing pilot study. 
 
II. Background and Significance:  
a) Overall background: 

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Polycystic Ovarian Syndrome (PCOS) affects 6-10% of U.S. women, with an estimated 
economic burden of $4 billion, and is increasing in prevalence in parallel with the obesity 
epidemic1,2. PCOS includes elevated androgens, and higher rates of insulin resistance (IR), type 2 
diabetes (T2D), nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD) 3-6. The 
increasing rates of obesity-related PCOS are a major contributor to the earlier onset and rising 
incidence of T2D, NAFLD and CVD3-7. Current PCOS therapies are marginally efficacious, and one 
of them, oral contraceptives, may adversely affect CVD risk 8-14. Despite the high prevalence and 
serious morbidity associated with PCOS, a gap exists in the current therapeutic options.  
b) Mechanisms of insulin resistance in PCOS 
Alterations in hepatic metabolism may be central to IR and cardiometabolic disease in PCOS. 
An estimated 50-70% of obese women 
with PCOS have NAFLD, compared to 
20-30% of obese women without PCOS 
7,15. Furthermore, obese women with 
PCOS and NAFLD are more IR than 
those without NAFLD, indicating a link 
between NAFLD and worsening IR in 
PCOS 4. Liver enzyme concentrations, as 
a marker of NAFLD, independently predict 
dysglycemia and T2D onset16, and a new 
NAFLD medication decreased 
dysglycemia17, arguing for a tight 
connection between glycemia and liver 
health. In obese non-PCOS youth, 
NAFLD correlates with adipose, hepatic 
and muscle IR, is worsened by fructose 
consumption, and reversed by weight 
loss18-21. Animal models of primary 
hepatic IR demonstrate the causal role of hepatic dysfunction in the development of NAFLD, T2D 
and CVD 22,23. IR in muscle, liver and adipose tissue are reported in PCOS adults24-27, and we 
found the same in girls with PCOS, despite their young age. The synergy between obesity and 
hyperandrogenism relate to alterations in fat metabolism, inflammation, NAFLD and IR in PCOS 
adults28-30. Our proposed mechanism for development of excess liver fat from adipose and hepatic 
IR is shown in Figure 1. It is possible that in the girls with PCOS and HS, the energy production 
from the TCA and pentose phosphate pathways are shifter towards DNL, whereas in girls without 
PCOS they may be shifted towards glycogenolysis. An understanding of the complex physiology 
between tissue-specific IR, hepatic fat and androgens in PCOS is lacking. Addressing this gap is 
my intermediate goal and is an aim this protocol. 
 
c) Current research methods to assess IR      
Research methods to assess early tissue-specific IR require complex protocols and few 
models exist which incorporate oral feedings31-33. Current data for evaluating both hepatic and 
adipose IR have employed intravenous (IV) approaches, which fail to account for contributions of 
hormones such as glucagon, glucagon like peptide (GLP-1) or leptin, which may play a crucial role 
in hepatic and adipose signaling20. Further, the maximum exogenous insulin doses that can be 
administered safely IV often produce lower serum insulin concentrations than generated 
endogenously following an oral glucose load, and the prolonged fasting required for IV studies is at 
the limit of tolerability in youth. In our current study, we are addressing this by developing an OGTT 
model of hepatic glucose suppression, lipolysis suppression and peripheral IR, in conjunction with 
directly measuring DNL. However, this study does not allow for measuring intrahepatic processes 
and detailed metabolomic analysis. This type of analyses is needed as there are several new 
medication classes that may be useful in PCOS. Thus, a gap exists in the methodology to assess 
hepatic glucose metabolism and adipose IR in a comprehensive, minimally-invasive, yet 
physiologic setting. Addressing this gap is the first step towards addressing my long terms goals 
and is a focus of this protocol. 
Figure 1: Proposed Mechanism of NAFLD in 
PCOS 
Mechanisms influenced by hyperandrogenism and unique in 
PCOS vs. T2D are in grey. Items with an * will be studied in this 
grant. HDNL = hepatic de novo lipogenesis, VAT=visceral adipose 
tissue, FFA=free fatty acids, GLP-1=Glucagon Like Peptide 

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A. Preliminary Studies/Progress Report:   
SA1: Optimize novel minimally-invasive physiologic methods to study liver metabolism  
     SA 1 Pilot Data on Liver fat and Insulin Resistance in youth with T2D: In our recent adolescent 
studies,       
we examined the differential tissue-specific 
expression of IR in youth with T2D and PCOS 
utilizing a sophisticated 3 stage hyperinsulinemic 
euglycemic clamp with multiple stable isotope 
tracers. By contrasting patterns of pathology 
between PCOS, obese controls and T2D we are 
uncovering subtle differences suggesting unique 
mechanisms of hepatic IR in PCOS. T2D youth 
appear similar to T2D adults in having adipose, 
hepatic and muscle IR. However, the IR is more 
severe than in adults, with unexpectedly progressed 
markers of CVD and NAFLD at diagnosis despite 
their young age 34. Adipose IR in our T2D youth is 
exhibited by a persistent glycerol rate of 
appearance (Ra) despite very high induced insulin 
concentrations (200 mU/L) and hepatic IR is 
demonstrated by a doubling in the insulin 
concentration required for 50% suppression of 
glucose Ra (IC50 Ra)(Figure 2A). This IR translates 
to persistent elevations in serum free fatty acids 
(FFA) and glucose in these individuals. Elevated 
hepatic fat content (Figure 2B) correlates with both 
elevated serum FFA concentrations 
and glycerol Ra in T2D, indicating 
that hepatic steatosis is related to 
adipocyte IR. Muscle IR, assessed 
by glucose clearance rate, is very 
tightly correlated with both glycerol 
Ra and serum FFA concentrations 
during hyperinsulinemia, indicating 
that adipocyte IR may impact muscle 
IR as well. Finally, our T2D youth 
have muscle mitochondrial 
dysfunction as assessed with 31P 
magnetic resonance spectroscopy 
(MRS), which relates to muscle IR. 
Pilot Data on Liver fat and Insulin 
Resistance in youth with PCOS:       
Obese girls with PCOS have moderate adipose, hepatic (Figure 2A) and muscle IR relative to 
obese controls, but are not as IR as T2D girls. Adipokines such as adiponectin are similarly low in 
PCOS and T2D girls, whereas leptin is higher in PCOS than even T2D (Table 1). Unlike T2D, 
muscle mitochondrial function is not impaired in PCOS compared to obese controls. PCOS girls 
have increased hepatically-derived serum triglycerides, liver enzymes as elevated as in T2D, and 
MRI-assessed rates of hepatic steatosis even higher than in T2D girls (Figure 2B). Hepatic fat in 
PCOS girls is weakly associated to markers of overall lipolysis, but is more closely related to 
visceral adipose tissue (VAT) and plasma markers of hepatic de novo lipogenesis (FFA n7, Table 
1). Further, during IV-induced hyperinsulinemia during the clamp, girls with PCOS have lower rates 
of carbohydrate oxidation than obese controls (0.013±0.0001 vs. 0.017±0.002 mmol/min/kg), 
indicating preferential storing of glucose as evidence of metabolic inflexibility. Finally, markers of 
Figure 2: Hepatic Measures in Girls 
A   Hepatic Insulin Resistance 
B    Hepatic Fat Percentage 
Table 1: Serum Markers in Obese Girls 
 
Control 
PCOS 
T2D 
Number 
19 
38 
40 
AST (IU/L) 
19±2 
36±2 
32±2 
ALT (IU/L) 
29±3 
38±3 
40±2 
Triglycerides (mg/dl) 
78±8 
129±7 
207±7 
Adiponectin (ng/dL) 
8.4±0.9 
5.8±0.4 
5.7±0.1 
Leptin (ng/dL) 
37±6 
43±3 
36±1 
CRP (ng/dL) 
1.7±1.4 
4.3±0.6 
4.1±0.1 
HbA1C (%) 
5.1±0.2 
5.4±0.3 
7.2±0.4 
Oral Disposition Index (oDI) 
5.2±2.2 
3.2±0.5 
N/A 
Fasting FFA (mmol/l) 
523±37 
629±22 
N/A 
FFA n7 (nmol/g) 
29±13  
45±15 
N/A 

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CVD, including carotid plaque development and exercise tolerance are similar between PCOS and 
T2D, indicating that PCOS status may be a significant risk factor for CVD even in youth. In 
summary, in youth with PCOS, excess liver fat may relate to adipose IR, hepatic IR and metabolic 
inflexibility with increased de novo lipogenesis from carbohydrates. Targeting improved 
understanding of these pathologic processes in a physiologic model is a logical step for reducing 
progression of T2D, CVD and NAFLD in PCOS youth. 
      Preliminary data from oral glucose tolerance 
tests in girls with PCOS: 
Obese girls with PCOS have high 2 hour 
oral glucose tolerance test (OGTT) glucose and 
insulin concentrations (Figure 3 A and B). Of note, 
the PCOS OGTT insulin concentrations are higher 
than those achieved during the clamp 245±40IU/ml). 
Despite the relative hyperinsulinemia, they have 
poor insulin secretion relative to IR, i.e. a lower 
OGTT-derived oral disposition index (oDI) vs. obese 
controls (Table 1) indicating the presence of both β-
cell dysfunction and IR. Girls with PCOS and an oDI 
of <1, an established risk for T2D35, have half the 
insulin sensitivity vs. those with an oDI of >1 
(5.3±2.1 vs. 10.8±0.8 mg/kg lean/min). Thus it may 
be that inadequate insulin secretion during an OGTT 
allows for persistent lipolysis and gluconeogenesis, 
which increases hepatic fat content and worsens 
muscle IR. GLP-1 and leptin, both hormones 
affected by oral ingestion, may be involved in this 
physiologic interplay, which is completely ignored 
with hyperinsulinemic clamp IV glucose delivery. 
The combination of isotope tracers and oral nutrient 
delivery is required to answer this critical question. 
This data is one of the primary reasons that we need 
additional oral models.  
 
 
 
Prolonged OGTT in obese girls: 
In our current protocol, we initially started with an OGTT with 4 hours of post-prandial monitoring. 
However, we found that at 4 hours, the insulin and glucose concentrations were still not returned to 
fasting levels. We thus amended our protocol and are now conducting monitoring out to 6.5 hours. 
By this time, the concentrations have returned to fasting levels.  
 
 
Figure 3: OGTT Plasma Results 
A Serum glucose concentrations 
B Serum Insulin Concentrations 
 
 

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Additionally, in the initial work describing this method in healthy lean men, serum metabolite 
profiles at 4 hours have not yet returned to baseline, and thus especially in our patients we will 
need to follow their metabolites out to 6.5 hours.  
 
Preliminary data from polysomnograms in girls with PCOS:                   
We had a pilot study to perform a limited number of inpatient polysomnograms in another of our 
current protocols, 14-0542, which has identical inclusion criteria. Just analyzed results from 14-
0542 indicate the there is a strong relationship between obstructive sleep apnea (OSA) defined as 
an apnea hypopnea index (AHI) of 5 events per hour or greater and fatty liver disease. Girls with 
OSA also had higher serum triglycerides and glucoses 2 hours after the glucola drink, despite 
similar BMI’s (Table 1).  
 
Mean AHI 
(apnea/hour) 
BMI 
%ile 
Hepatic fat 
fraction 
(>5.5%= fatty 
liver) 
Serum TG, 
mg/dL (>150 
abnormal) 
2 hour glucose, 
mg/dL (>140 pre-
diabetes) 
AHI 
>5, 
N=13 
14.6±3.5 
98±1 
10.5±2.6 
162±19 
153±6 
AHI< 
5, 
N=13 
1.3±0.4 
97±1 
5.3±1 
110±12 
131±5 
 
Further, at least 50% of the girls studied had mild to moderate obstructive sleep apnea, and a few 
had severe sleep apnea, although there was a bias to performing sleep studies in girls with 
symptoms suggestive of OSA, so this is not a true prevalence estimate from this obese female 
population. Clinically, from the PI’s PCOS clinic, approximate 1/3 of obese girls with PCOS are 
requiring continuous positive airway pressure therapy for OSA. Thus, it may be that the presence 
and severity of sleep apnea is a confounder for our primary outcome of altered hepatic metabolism. 
Accordingly, we need to quantify the presence and severity of OSA in all of our subjects to be 
enrolled in 16-2399. 
Traditionally, inpatient overnight polysomnograms are performed in youth, to determine 
OSA. However, through our experience in performing these with protocol 14-0542, these can only 
be performed at Children’s Hospital Colorado on Thursday nights, which greatly limits scheduling 
and enrollment. Additionally, whereas the price for these was $500 a piece, this has now been 

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changed, and is currently nearly $2,000 per patient, as a research price. Due to these limitations, 
we have sought alternative means of quantifying the degree and severity of OSA.  
A wearable sleep device, WatchPAT, designed for home use has been utilized for the last 
5 years clinically in adults. In July of 2016, the FDA approved the use of this device down to the 
age of 12 specifically for the oxygen saturation and apnea hypopnea index, but not the respiratory 
disturbance index (>17 years only). We have included the device company provided general 
description of the device, as well as the descriptions of limitation and use with individuals 12-17 
years. As many of the limitations of pediatric use pertain to placement and utilization of the device, 
we will perform the sleep study while the patients are having their already planned and approved 
overnight stay in the hospital, and will have the device placed by our study personnel, who will be 
trained in correct placement and interpretation. Our study team also has extensive experience with 
similar devices from this company, and they are a supportive and easy to work with company. 
Known obstructive sleep apnea or treatment with CPAP will not be an exclusion criteria, 
however, those individuals already utilizing CPAP at home will be requested to bring it for 
admission and use it during the WatchPAT study, so that we capture their typical home sleep 
patterns. 
 
III. Research Methods 
 
A.  Outcome Measure(s):   
Primary Outcome Measure(s): Activity ratios of the hepatic pentose phosphate pathway, TCA cycle 
and TG synthesis. 
Secondary Outcome Measure(s):  Peripheral IR as assessed with a 6.5 hour OGTT, measures of 
SNS activity, Hepatic fat content, hepatic phosphate profile. Targeted glucose and fat metabolism 
metabolomics, and untargeted exploratory analysis, gut microbiota profile. 
Likely contributors to above measures: Lipid/glucose markers:(fasting C-peptide and lipid panel, 
HbA1c); hepatic markers:(c-reactive protein, glucagon, GLP-1 and leptin at baseline, 5 min and 30 
min post glucose load to asses change with OG, adiponectin, AST, ALT, GGT), sex-
steroids:(DHEAS, free and total testosterone, sex hormone binding globulin, progesterone, 
estradiol); Body size and composition: (BMI, waist/hip ratio, DEXA, hepatic visceral fat via MRI36-
39), Whole body fat oxidation at rest and following glucose ingestion as measured with a metabolic 
cart; Physical activity/ diet:(accelerometer, activity survey (3DPAR); Food frequency 
survey).Questionnaires for presence of obstructive sleep apnea. Questionnaires for perceived 
mental strengths and difficulty (note there is no assessment of suicidality on this tool). Obstructive 
Sleep Apnea, to be assessed with an overnight sleep study via WatchPAT. 
 
B. Description of Population to be Enrolled:   
Study staff aims to enroll 20 obese girls with regular menstrual cycles, 20 girls with un-treated 
PCOS, 20 girls with PCOS treated with at least 6 months of Metformin and 20 girls with PCOS 
treated with at least 6 months of oral contraceptives. The PCOS groups will be compared to 20 
obese non-PCOS controls.  This is the number of participants needed to be completed statistically, 
thus more subjects may be enrolled, to allow for screen failures and dropouts. Total enrollment will 
be up to 100 participants. 
 
Ethnic Categories 
Gender 
Females 
Males 
Total 
Hispanic or Latino 
40 
0 
40 
Not Hispanic or Latino 
60 
0 
60 
Ethnic Categories: Total of All Subjects 
100 
0 
100 
  
Racial Categories 
 
 
 
American Indian/Alaska Native 
5 
0 
5 
Asian 
10 
0 
10 

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Native Hawaiian or Other Pacific Islander 
10 
0 
10 
Black or African American 
32 
0 
32 
White 
43 
0 
43 
Racial Categories: Total of All Subjects* 
100 
0 
100 
 
Inclusion Criteria: 
1) Female 
2) Ages 12-21 
3) Sedentary- less than 2.5 hours of moderate (jogging, swimming etc) exercise a 
week. 
4) BMI equal or greater than the 90th percentile for age and gender 
 
5) For PCOS groups: (NIH definition) irregular menstrual cycles at least 1.5 years 
after menarche and either clinical evidence of hyperandogenism or elevated 
Testosterone (above the norms for age/tanner stage) at time of screening or 
documented prior to initiation of therapy for OCP and metformin groups. 
 
6) For PCOS groups: patients un-treated or currently treated with either Metformin 
1500-2000 mg a day or oral contraception (30-35 mcg ethynyl estradiol a day) for at 
least 6 months, with > 80% adherence confirmed via refill frequency from pharmacy. 
 
7) For non-PCOS groups: regular menstrual cycles at least 1.5 years after menarche and no 
clinical evidence of hyperandrogenism 
 
Exclusion Criteria: 
1. Use of medications known to affect insulin sensitivity: oral glucocorticoids 
within 10 days, atypical antipsychotics, immunosuppressant agents, HIV 
medications. Nexplanon, Depo-Provera or Mirena progesterone only 
contraceptives. Dermal patch or vaginal ring contraception methods. For 
controls only: metformin or oral contraception. 
2. Currently pregnant or breastfeeding women. Development of pregnancy 
during the study period will necessitate withdrawal from the study. 
3. Severe illness requiring hospitalization within 60 days 
4. Diabetes, defined as Hemoglobin A1C > 6.4% 
5. BMI percentile less than the 90th percentile for age and sex. Weight >325 lbs 
or <84 lbs. 
6. Anemia, defined as Hemoglobin < 10 mg/dL 
7. Diagnosed major psychiatric or developmental disorder limiting informed 
consent 
8. Implanted metal devices that are not compatible with MRI 
9. Use of blood pressure medications 
10. Known liver disease other than NAFLD or AST or ALT >150 mg/mL 
 
Rationale for Inclusion of Non-PCOS Subjects  
Obese adolescents are at risk of T2D and its complications, reduced exercise capacity, increased 
liver and visceral fat, muscle dysfunction and cardiovascular dysfunction therefore the tests 

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performed screening for each of these problems provide useful information to these subjects. 
Likewise, sedentary subjects of any weight are at increased risk of reduced bone mineral density 
(BMD), T2D, reduced exercise capacity, increased liver and visceral fat, muscle dysfunction and 
cardiovascular dysfunction. Therefore, all of the subjects may benefit from the results of DEXA, 
glucose testing and exercise prescription and dietary counseling.  
 
C. Study Design and Research Methods   
 
 
 
 
The study consists of 3 visits - 2 outpatient visits and one overnight inpatient visit. Inpatient visit will 
last approximately 24 hours, outpatient screening visit approximately 2 hours, outpatient MRI visit 
approximately 2 hours. The 3 visits would be completed within 4 months’ time. 
 
 
 
 
 
 
 
 
 
 
 
 
Study Calendar 
Visit 1 – 
Screen 
Visit 2 –MRI 
Visit 3 – 
Overnight 
Consenting and Eligibility Assessment 
X 
 
 
History & Physical 
X 
 
 
Intravenous Blood Draw 
X 
 
X 
Finger Stick Blood Draw 
 
X 
 
Urine Pregnancy Test 
 
 
X 
Accelerometer Teaching  
X 
 
 
Gut Bacteria Collection 
 
 
X 
Questionnaires- SEARCH Food frequency, Activity 
3DPAR, Strengths and Difficulties and Sleep 
assessments 
 
 
X 
DEXA Scan (located in Leprino building) 
 
 
X 
Oral Glycerol Tracer 
 
 
X 
WatchPAT sleep study 
 
 
X 
EndoPat and Dynapulse 
 
X 
 
MRI of abdomen and liver,  
 
X 
 
P MRS of Liver (pending scheduling) 
 
X 
 
Metabolic Cart 
 
 
X 
Total Time of visit (approximately) 
2 Hours 
2 Hours 
24 Hours 
Location of Visit 
CHCO 
CTRC 
Outpatient 
UCD Outpatient 
Brain Imaging 
Center 
CHCO Inpatient 
Hospital 

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Overall enrollment procedure plan per group 
 
Test 
Group 
Screening 
MRI/MRS 
Overnight 
stay 
Oral Glycerol 
tracer  
DEXA 
Untreated 
PCOS 
25 
20 
20 
20 
20 
PCOS w/ 
Metformin 
25 
20 
20 
20 
20 
PCOS w/  
OCP’s 
25 
20 
20 
20 
20 
Obese 
Control 
25 
20 
20 
20 
20 
Total 
100 
80 
80 
80 
80 
 
VISIT 1 (SCREEN VISIT) 
Pediatric CTRC Outpatient unit:  Participants will begin with a medical screening and physical 
exam for inclusion/exclusion criteria evaluation.  During this visit, patients will review and complete 
consent documents, have demographics and medical history confirmed, assess allergies and 
further evaluation of inclusion/exclusion criteria, have blood samples drawn, and have 
anthropometrics completed.  HbA1c, ALT, AST, CBC panel and testosterone panel will be drawn at 
the beginning of the visit after consent in all subjects. PCOS subjects will have more labs 
performed for confirmation of PCOS status, if not performed previously.  
 
Screening lab test 
Purpose for test 
HbA1C 
Rule out type 2 diabetes, if > 6.4% subject to be 
excluded 
ALT, AST 
Ensure no severe liver disease, if >150 IU 
subject to be excluded normal subject is 
excluded 
Hemoglobin & Hematocrit (part of CBC) 
If subject is Anemic, they will be excluded 
Testosterone  
Test for hyperandrogenism – required to meet 
NIH criteria for PCOS 
Optional PCOS labs 
PCOS status must be confirmed prior to enrollment in PCOS group. Referring physicians often do 
not perform the entire recommend work-up for oligomenorrhea (per 2013 Endocrine Society 
Clinical Guidelines for PCOS). The values are typically expected in PCOS publications 
TSH, total T4 
Ensure no hypo or hyperthyroidism causing 
amenorrhea 
LH, FSH 
Rule out primary ovarian failure 
17-hydroxyprogesterone 
Rule out late onset congenital adrenal 
hyperplasia 
DHEAS 
Rule out adrenal tumor 
Prolactin 
Rule out prolactin secreting brain tumor 
 
 
 

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VISIT 2 (MRI/CV VISIT) 
Overall Plan: UCD Research MRI (Brain Imaging Center): Subjects will be asked to fast for 4-6 
hours prior to this visit. The imaging will be of the liver. Two different studies may be conducted. 
One will be standard MRI of the mid abdomen to assess the amount of subcutaneous fat, visceral 
fat and percent liver fat. The second type of scan is optional pending scheduling. It will be 31P 
spectroscopy to measure the concentrations of phosphate molecules including PDE, PME and 
PCr. This will be done twice, before and 30-45 min following a standard 75 gram glucose load. The 
MRI time is approximately an hour in a 2 hour visit. Vascular Endothelial function and Heart Rate 
Variability will be assessed with Endopat and Dynapulse, which take approximately 30 minutes. 
 
31P MRS of the Liver and Abdominal Imaging (pending scheduling) 
       MRS Data acquisition: Imaging and MRS will be performed on a Seimens 2.9 Tesla MRI 
magnet. A custom 1H/31P abdominal coil will be used for imaging and MRS (Clinical MR Solutions, 
Brookfield, WI) as in our previous 31P work40. The coil will be a concentric probe with an inner coil 
16 cm in diameter (for 31P) and a 20 cm outer coil (for 1H scout imaging and shimming).  A 2 cm x 2 
cm x 2 cm area of focus is found in the liver in homogenous tissue for MRS, similar to our previous 
studies38. A 31P MRS scan will then be performed for baseline measurements. We will continue to 
work with our current collaborators Mark Brown, PhD, Assistant Professor, Department of 
Radiology, UC Denver Anschutz and Bradley Newcomer, PhD, Professor, Department of 
Radiography, University of Alabama at Birmingham to optimize the MR signal collection. Subjects 
will then consume 75 grams of glucose and 25 grams of fructose and the scan will be repeated 
every 10 min for an hour. Visceral adiposity will be measured using the gold standard of an MRI 
slice at L4-L5. Hepatic fat fraction will be performed using modification of the Dixon method as in 
our previous studies34. Hepatic fibrosis will be measured with a fibroscan sequence. 
 
MRS Data Analysis: For the 31P data, peak positions and areas of interest [phosphocreatine (PCr), 
inorganic free phosphate (Pi), β-ATP(3 peaks), α-ATP(2 peaks), γ-ATP(2 peaks), and PME] will be 
determined by time domain fitting with jMRUi 41,42, utilizing AMARES (A Method of Accurate, 
Robust and Efficient Spectral fitting), a nonlinear least-square-fitting algorithm using our previously 
built prior knowledge files 43. We have utilized this method for muscle 31P analysis for the previous 
6 years, and have extensive experience with this analysis.  Percent PME relative to all other 
phosphate peaks will be calculated before and every 10 min following a glucose load, and percent 
suppression calculated.  The adipose data will be analyzed by Collaborate Ann Scherzinger, PhD, 
as in previous protocols (COMIRB #’s 10-1288 and 14-0542). 
 
Gut Bacteria Collection: The gut microbiome consists of the microorganisms, predominantly 
bacteria, that inhabit the gastrointestinal tract and are estimated to outnumber mammalian cells by 
up to a factor of 10, and their genes outnumber human genes by a factor of over 100 [69]. It is 
possible that gut microbiota contribute to the development of NAFLD, and this has not been 
explored in PCOS. We will collect a sample to define the microbiota in girls who are already 
undergoing extensive metabolic profiling.  The gut microbiota will be collected with BBL culture 
swabs (Becton, Dickinson and Company, Sparks, Maryland) one week prior to visit 3. Fecal 
samples will be collected from the first bowel movement of any day the week before visit 3 and 
stored in the freezer. Fecal samples are being collected for present and future research markers in 
the mircrobiome. Fecal samples are routinely collected in research and pose little risk to subjects. 
For all samples, bacterial DNA will be extracted from the swab using established methods and the 
V4 region of 16S bacterial rRNA will be amplified using previously published primers and PCR 
conditions. [66, 67, 68] To provide a full picture of microbial diversity in the gut, we have combined 
phylogenetic and Operation Taxonomic Units (OTC)-based methods for comparing communities. 
Grouping bacterial rRNA sequences by similarity is important for asking questions about which 
particular species, genera, phyla, etc, contribute to differences between samples. To choose 
OTUs, groups of similar 16S bacterial rRNA sequences are identified, and candidate OTUs are 
identified as sets of sequences connected to each other. Candidate OTUs are considered valid if 
the average density of connection is above 70% (i.e., if 70% of the possible pairwise connections 
between sequences in the set exist. 

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Accelerometer: One week prior to visit 3, the subject will be provided two accelerometers (GT3X 
BT by Actigraph and ActiWatch by Philips Respironics) to be worn for seven days to measure level 
of habitual physical activity, which affects insulin sensitivity, and sleep patterns. Accelerometers are 
effective tools for the objective measurement of physical activity 44 because they have the ability to 
continuously record physical activity data and such data can be used to estimate METs of activity. 
They provide more detailed information than pedometers, which only measure walking steps, and 
help get around the recall bias of questionnaires. We are currently using the GT3X BT Actigraph in 
adolescents in our other diabetes studies; therefore, we are familiar with their use in this population 
and have the necessary computer software and interpretation skills. The Actiwatch is being used 
as a tool for objective measurement of sleep patterns. The Actiwatch is fitted with a LED monitor 
that detects multiple spectrums of light to better assess sleep patterns in this population. 
 
*One week prior to visit 3, participant will receive via mail the accelerometer, sleep diary and 
stool sample collection kit (Gut bacteria collection)* 
 
 
 
 
 
 
 
 
 
 
Figure 4: Proposed Oral Glucose Challenge 
 
 
The study will have 2 physiologic states. 1. A basal fasted state, and 2. A post-prandial state following a 
glucose load. The labelled glycerol is given anywhere from 3 to 3.5 hours after the glucose load, as hepatic 
glucose output needs to be high enough to detect the incorporated carbon from the glycerol. 

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VISIT 3 (ORAL GLUCOSE TOLERANCE TEST WITH ORAL GLYCEROL TRACER)  
 
Pediatric CTRC Inpatient unit:  Subjects will be asked not to have caffeine or exercise for 3 days 
prior to this visit. During admission, subjects will be provided with a study diet dinner and snack. 
Patients will be admitted to the Pediatric CTRC for a monitored overnight fast. Subjects will be 
questioned regarding changes in concomitant medications and medical history, height, weight, BP.  
A DEXA scan will be performed to assess body composition. A urine pregnancy test will be 
done on all female subjects prior to the DEXA scan.  If a female subject is confirmed to be 
pregnant, she will be withdrawn from the study and referred to her primary care physician for 
follow-up. 
The following morning, a blood sample for baseline metabolic labs will be drawn the 
modified OGTT will be completed. DNA extract-and-hold samples for future genetic analysis 
associated with hepatic steatosis will also be drawn and stored at this time. Additional blood will be 
drawn and stored for future inflammatory markers as well as for targeted and untargeted 
metabolomics analysis.  
WatchPAT: During the hospital overnight stay, trained study staff will place the WatchPAT 
sleep monitor. The primary measures will be for oxygen saturation and apnea hypopnea index 
(AHI). Each participant will wear the watch with a one-time use finger cuff as recommended by the 
FDA..The watch will be placed by 8 PM, and will be removed the following morning. 
 
Details of Stable Glycerol Isotope Tracer studies with an OGTT: After the overnight fast at 
approximately 730AM, subjects will consume a 75 grams glucose load with an additional 25 grams 
of fructose to stimulate hepatic de novo lipogenesis. An 3 hours later, following consumption of the 
glucola/fructose mixture, 50 mg/kg of an oral UC13-glycerol tracer will be consumed mixed with 
water. Analysis of serum glucose isotopomers will be performed by our collaborator Craig Malloy at 
UT Southwestern, using NMR isopomomer analysis and pathway modeling software. Lipolysis will 
be modeled using FFA concentrations and whole body IR by a time modified version of the 
Matsuda model. 
Using a metabolic cart and hood, resting VO2 (ml/kg/min) and VCO2 (ml/kg/min) 
measurements (REE) will be collected the morning of the OGTT prior to the start of the OGTT, as 
well a, 30 min after the start of the OGTT, 3 hours and 30 min after the start of the OGTT and 5 
hours and 30 min after the start of the OGTT. This is required to determine what portion of ingested 
carbohydrates are subject to oxidative and non-oxidative glucose disposal45. 
 
Study diet: Variations in diet, activity and circadian rhythms affect metabolism 34. Therefore, OGTT 
studies will be performed in the AM fasting, in the follicular phase where possible, preceded by 3  
days of no strenuous physical activity and 2 meals of a fixed macronutrient, high carbohydrate 
(65% carbohydrate, 20% fat, 15% protein), fixed grams of fructose, calculated as Females: ([8.365 
 (weight in kg) + 465 (height in m) + 200] X Activity Factor x 1.25), dinner and snack provided by 
the Colorado CCTSI metabolic kitchen (similar to our previous studies34). 1.25 x weight 
maintenance was chosen as this as most similar to our subjects food consumption based on pilot 
subject’s food frequency questionnaires and optimal for detection of hepatic glucose Ra 46. 
 
Purpose for lab test to be drawn: 
OGTT Labs 
Purpose 
Lipids and glycerol samples for tracer analysis 
Determination of hepatic metabolism flux 
Glucose  
Determination of IR 
Insulin 
Determination IR 
FFA 
Measure of lipolysis 

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Glycerol 
Measure of lipolysis 
Glucagon 
Gut hormone known to influence hepatic IR 
GLP-1 
Gut hormone known to influence hepatic IR 
CRP 
Marker of inflammation, know to effect IR 
Leptin 
Gut hormone known to influence hepatic IR 
Adiponectin 
Adipokine thought to influence adipose IR 
Estradiol 
Known to effect IR and HDNL 
Progesterone 
Demonstrate the subject is in the follicular 
phase of cycle, required for publication 
Metabolomics 
Correlation with hepatic metabolism flux 
1 sample for genetic analysis 
Measure for common polymorphisms 
associated with hepatic steatosis47,48 
Stored blood 
Future markers of glucose and fat metabolism, 
CVD or hormones related to PCOS 
 
Purpose for questionnaires being done: 
Questionnaire  
Description 
Study Validity 
Adolescent Sleep Hygiene 
Scale 
Measurement of 
sleep 
patterns/habits 
Cronbach’s alpha ranges from .46-.74; total 
scale alpha = .80 
LeBourgeois et al. 2005. The Relationship 
Between Reported Sleep Quality and Sleep 
Hygiene in Italian and American 
Adolescents 
Center for Epidemiological 
Studies Depression (CES-D) 
Measuring for 
depression 
See Table 3 below, adapted from: 
Stockings et al. 2015. Symptom screening 
scales for detecting major depressive 
disorder in children and adolescents: A 
systematic review and meta-analysis of 
reliability validity and diagnostic utility 
Cleveland Adolescent 
Sleepiness Questionnaire 
Measurement of 
sleepiness during 
a typical week 
alpha = .89 
Spilsbury et al. 2007. The Cleveland 
Adolescent Sleepiness Questionnaire: A 
New Measure to Assess Excessive 
Daytime Sleepiness in Adolescents 
SDQ: Strengths and Difficulties 
Questionnaire 
Behavioral 
screening 
questionnaire 
The internal reliability of the various self 
report scales was assessed using 
Cronbach’s alpha coefficient. This was 0.82 
for the total difficulties, 0.75 for emotional 
symptoms, 0.72 for conduct problems, 0.69 
for hyperactivity, 0.65 for prosocial 
behaviour, and 0.61 for peer problems. 
Goodman et al. 2003. The Strengths and 
Difficulties Questionnaire: a pilot study on 

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the validity of the self-report version 
Sleep Disturbances Scale for 
Children 
Gain 
understanding of 
sleep-wake 
rhythm and any 
problems in sleep 
behavior 
Internal consistency ranged from .71-.79l 
test-retest reliability r = .71 
Bruni et al. 1996. The Sleep Disturbance 
Scale for Children (SDSC)Construct ion 
and validation of an instrument to evaluate 
sleep disturbances in childhood and 
adolescence 
3DPAR: Activities Scale 
Measuring activity 
in the 3 days 
previous as a 
typical activity 
score 
Interrater and test-retest reliability was 0.99 
and 0.98, respectively (P < 0.01). The 
correlation between relative energy 
expenditure from the PDPAR (kcal.kg-1.l.d-
1) and pedometer and Caltrac counts was 
0.88 (P < 0.01) and 0.77 (P < 0.01), 
respectively. The correlation between 
percentage heart rate range (HRmax-HR-
rest) and mean energy expenditure from 
the PDPAR was 0.53 (P < 0.01). The 
correlation between 1-min heart rates > 
50% HRR sustained for 20 min and the 
number of 30-min blocks with a relative 
energy expenditure of at least four 
metabolic equivalent tasks (MET) was 0.63 
(P < 0.01). The PDPAR provides valid and 
reliable estimates of physical activity and 
also accurately identifies bouts of moderate 
to vigorous activity. 
Weston et al. 1997 Validation of an 
instrument for measurement of physical 
activity in youth 
 
Food Frequency Questionnaire 
Measuring typical 
food intake over 
previous seven 
days. 
The mean correlations, adjusted for 
measurement error, of food groups and 
nutrients between the FFQ and true usual 
intake were 0·41 and 0·38, respectively, 
with 57 % of food groups and 70 % of 
nutrients exhibiting correlations >0·35. 
Correlations were high for low-fat dairy 
(0·80), sugar-sweetened beverages (0·54), 
cholesterol (0·59) and saturated fat (0·51), 
while correlations were poor for high-fibre 
bread and cereal (0·16) and folate (0·11). 
Reliability of FFQ intake based on two FFQ 
administrations was also reasonable, with 
54 % of Pearson correlation coefficients 
≥0·5. Reliability was high for low-fat dairy 
(0·7), vegetables (0·6), carbohydrates, 
fibre, folate and vitamin C (all 0·5), but less 
than desirable for low-fat poultry and high-
fibre bread, cereal, rice and pasta (0·2-0·3).  
Liese et al. 2015 Relative validity and 

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reliability of an FFQ in youth with type 1 
diabetes 
First described in 2006:  
Mayer-Davis et al. 2006. Search FFQ 
Dietary Intake among Youth with Diabetes: 
The SEARCH for Diabetes in Youth Study 
 
Optional 7 Day Food Log 
Optional record of 
7-day diet to aid in 
food recall. Added 
per participant 
request to help fill 
out food 
frequency 
questionnaire. 
N/A 
Actigraphy Daily Sleep Diary  
 
Recording 
bedtime/wake 
time during 
actigraphy. 
Needed to 
corroborate watch 
collected data 
N/A 
 

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LIFESTYLE PRESCRIPTION 
The final visit will conclude with education regarding the importance of physical activity, diet and 
lifestyle modification to mediate the risks associated with sedentary lifestyle and an exercise 
prescription designed to increase physical activity. The exercise information and prescription are 
the standard of care used in our Children’s Hospital Colorado Pediatric Metabolic Syndrome Clinic, 
designed by the Children’s Hospital Colorado Pediatric Exercise Physiologist. Families will be 
provided with standard information about follow up care with their primary care provider and 
contact information for Children’s Hospital Colorado Diabetes and Child Health Clinics if needed 
regarding any abnormal study findings. In addition, the subject/family will be provided with copies of 
their study lab results, DEXA scan and physical activity monitoring. Study staff will also call the 
family within 6 months of after completion of the study to check-in on the recommended follow up 
care and answer any questions about test results. A results letter will be provided to the family 
about clinically relevant results obtained during the study. Participants will be encouraged to 
contact study staff with questions regarding the results letter. 
 
 
 

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Follow-up from Sleep study results: 
As discussed above, we anticipate that approximately 30-40% of our participants will have 
an abnormal apnea hyponea index (AHI), requiring some type of follow-up. We have worked with 
Drs. Ann Halbower, Stephen Hawkins and Ben Hughes, our primary pediatric sleep pulmonologists 
to develop a post-study follow-up algorithm. Of note, Dr. Hallbower is currently working with Kaiser 
to verify the accuracy of the results from the WatchPAT device as compared to inpatient 
polysomnograms in children younger than 12, and is very familiar with this device, it’s output and 
limitations. Our youth fall into a grey zone in terms of what is an abnormal sleep study, as pediatric 
criteria are defined for less than 12, and adult for 18 or older. The international accepted clinical 
criteria are listed below, as well as the American Academy of Sleep Medicine’s recommendations 
of how to handle age 12-17: 
 
Pediatrics: 
 
Mild OSA 
Moderate OSA 
Severe OSA 
1 to 4.9 
5 to 9.9 
>10 
Adults 
Mild OSA 
Moderate OSA 
Severe OSA 
5 to 14.9 + symptoms 
15-30 
>30 
 
 
Since the recommendations are not concrete for the 12-17 year old range, we sought to follow 
what is being done in clinical practice at Children’s Hospital Colorado sleep clinics, were a patient 
to have an inpatient polysomnogram. Sleep studies are read within 5 business days, and we will 
adhere to this same turnaround timeline. In terms of interpretation, currently, the pediatric 
guidelines are being applied for the 12-17 year old age group but 2 is considered normal in this 
age, and thus our post-study for the 12-17 year olds will follow this, and are shown below. 
Approximately half of the participants have a patient relationship with the PI Dr. Green, and thus 
she can order F/U evaluation if needed, and if not, Dr. Green will request the follow-up be arranged 
by the primary care provider. We have 2 algorithms by age. 

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For participants 12-17 years of age: 
 
 
 
 
For participants ≥18 years of age: 
 
 
 
 
       
 
SUBJECT RECRUITMENT/CONSENT/PAYMENT 
1. Subject Recruitment Plan 
Subjects will be recruited from pediatric endocrine, PCOS, Lifestyle, adolescent and gynecology 
clinics, and from the community. We receive 4-8 new PCOS referrals a month, showing the 
feasibility of recruiting the required subjects. Further, we enrolled >100 obese girls in studies in the 
last 60 months. The PI and Co-I’s have a treatment relationship with girls from clinic or subjects 

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can call from study advertisements. Protected health information will only be accessible by study 
investigators. The initial patient contact will be made by personnel who have a treatment 
relationship with the subject. Our past studies have consented participants and asked if they’d like 
to be re-contacted for future research studies.  Furthermore, after the conclusion of our previous 
studies in PCOS (this protocol, 14-0542 and 10-1288), participants may have been prescribed 
treatment with OCP or Metformin, therefore study staff may aim to re-contact them had those 
participants agreed to be re-contacted and still meet inclusion criteria of the current study. 
 
2. Informed Consent Plan 
Appropriately qualified and informed personnel who have completed the COMIRB and HIPPAA 
course requirements will fully explain the study protocol and consent form to the subject and 
guardian verbally in the language they understand.  The explanation will be conducted in a quiet 
environment with adequate time given for the subject and guardian to review the study procedure 
before the commencement of the study.  Asking the subject to explain the study in their own words 
will assess the subject’s understanding.  If non-English speaking subjects are enrolled in the study, 
the investigators will adhere to Section 10C of the COMIRB Instructions for Clinical Investigators 
regarding the consent of these subjects. The consent form will also be translated into Spanish. The 
qualified personnel mentioned above will then obtain written consent from the guardian and assent 
from the subject, co-signed on the consent form, or in subjects who are 18 years or older, direct 
consent. The PI will make a good faith effort to obtain both parent signatures.  The subject and 
guardian will be provided a copy of the consent form for better understanding and record purposes.  
  
3. Special Consent/Assent Plan 
Consent will be obtained from all participants in the study.  Following explanation, all subjects 
below 18 years old will co-sign the consent form in addition to the parents signing the consent form.  
All subjects age 18 or older will sign the standard consent form. 
4. Subject Compensation, Incentives and Rewards  
Subjects will be compensated with Target gift cards during each scheduled visit. The initial visit 
consisting of informed consent and lab draw and the 2nd for the MRI will result in a $50 gift card 
each. The 3rd and final visit consisting of the overnight portion and questionnaires will reward a 
$150 gift card. Compensation for all completed visits will total $250. 
 
D.   Description, Risks and Justification of Procedures and Data Collection 
Tools: 
 
 
1. Blood Sampling 
Description: Blood will be drawn for Complete Blood Count (CBC), HbA1c, total and free 
testosterone, and sex hormone binding globulin. If subjects have not had a full evaluation for 
oligomenorrhea, Prolactin, DHEAS, LH, FSH, TSH, total T4 or 17-OH progesterone may be drawn.  
Risk: Minimal. Risk of pain, bruising at site of blood draw, excessive amount of blood. 
Minimizing Risk: Certain studies at our institution draw over 7ml/kg in 6 weeks, or up to 7 ml/kg in a 
single draw, but include iron supplementation. Otherwise, the routine guidelines in our Pediatric 
CTRC are 2.5ml/kg for a single draw and no more than 5 ml/kg over a 4 week period.  Our baseline 
visit will include 11.5 ml of blood (HbA1c, Hb, Cr, AST, ALT) and 25.5 ml of blood for PCOS 
patients (additional draw for T4, TSH, prolactin, LH, FSH, 17OH progesterone and DHEAS). The 
OGTT visit includes up to 300 ml of blood which will occur within 4 weeks of the initial visit. Thus, 
our OGTT visit is within the NIH Clinical Center guidelines of 9 ml/kg in 6-8 weeks and within 
Children’s Hospital Colorado’s institutional guidelines of 5 ml/kg.  In addition, by study design, 
subjects are screened by our baseline CBC and excluded if anemic, further increasing the safety of 
the study regarding blood draws. We will use a minimum weight cutoff of 38 kg to remain below the 
most conservative pediatric CTRC blood drawing guidelines. This screening also helps to increase 
the safety of the blood draw. In addition, the blood planned to be frozen and held could also be 

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omitted if needed to reduce blood volume for a particular subject. Finally, our CTRC has a system 
to track other studies subjects might enroll in, and we ask during our consent process if the subject 
has been involved in any other studies in the past 6 weeks to avoid excessive blood drawing. 
Justification: Screening laboratory measurements are necessary to assure that patients meet 
inclusion/exclusion criteria before any further study is completed. A CBC is necessary as a 
screening lab, to rule out anemia. A hemoglobin A1c can be used to rule out diabetes. Hormone 
levels of free and total testosterone, and sex hormone binding globulin are needed to categorize 
patients as having PCOS, and prolactin, DHEAS, LH, FSH, TSH, total T4 or 17-OH progesterone 
to rule out other causes of oligomenorrhea, if not done previously.  
 
2. IV Risks 
Description: One peripheral IV will be placed during the OGTT for drawing blood samples. 
Risk: There is temporary discomfort when the needle goes in and 10% of the time there is a small 
amount of bleeding under the skin that may produce a bruise. Rarely, there is a risk of a blood clot 
forming or infection.  We will use a low dose of a medication called heparin to try to prevent blood 
clotting. 
Justification/Minimization: These studies involve sampling blood at multiple time points. Thus, an IV 
is needed, so as to avoid multiple needle sticks. These studies are focused on measured rates of 
change which necessitates the sampling of the same test over time. Proper sterile technique will be 
used with blood draws and IV placement to decrease the infection risk. EMLA cream will be used if 
subject desires to minimize pain of IV. 
 
3. Oral Glucose Tolerance Test (OGTT): 
Description: An OGTT will be performed with multiple blood draws over 6.5 hours. The purpose of 
the OGTT is to provide a controlled oral stimulus to effect changes in lipolysis and hepatic glucose 
release. Subjects will also drink a glucose+fructose drink as part of the liver spectroscopy. 
Risk: The subjects rarely experience nausea within 15 min of consuming the drink, however, the 
amount of carbohydrate is very similar to a large soda, which is regularly consumed by this patient 
population.  
Justification/Minimization: A standard oral challenge is needed to study lipogenesis, lipolysis and 
gluconeognesis in the fed state. We have chosen to start with a standard glucose and fructose 
load, to simplify the mathematical modeling. Dynamic carbohydrate metabolism in youth is made 
more relevant by the recently reported TODAY study, showing a decline in beta cell function in 
youth with newly diagnosed type 2 diabetes that was much more rapid than what has been 
reported in adults, and not prevented by metformin in the majority of the youth49. Our team of 
investigators, CTRC pediatric research nursing staff and physicians are well experienced with the 
OGTT blood draw procedure. A floor nurse located on the 9th floor of CHC will be available during 
our inpatient visits and patients will be distracted by TV or other similar means during the OGTT, to 
minimize queasiness. 
 
4. Stable Isotope Studies:  
Description: Oral stable isotope tracer of glycerol will be utilized to determine rates of intrahepatic 
substrate flux. These are substances normally present or produced in the body, and thus pose no 
more risk than typical glucose infusions. Measurements of these metabolic processes are only able 
to be made with the utilization of stable isotope tracers. 
Risk: We are utilizing an isotope which already exists in all humans, but are simply increasing the 
percentage. We are only giving this medication orally. These are NOT radioactive substances. 
Justification/Minimization: Sterile and pyrogen-free ²C13 glycerol will be obtained form the 
manufacturer and delivered to CHCO IDS. The IDS pharmacist will deliver the tracer to the 9th floor 
inpatient CTRC once ordered by the physician.  
 
5: Finger stick for glucose measurement during MRI: 
Description: A glucometer will be utilized to measure fasting and 1 hour blood sugars following the 
glucose drink in the MRI. (Pending scheduling) 

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Risk: There is a small amount of pain with the finger poke and risk of infection. 
Justification/Minimization: The change in blood sugar needs to be assessed to correlate with changes in 
the glucose-6-phosphate concentrations. The finger will be well cleaned and dried with an alcohol pad. 
One-time use Lancet’s will be utilized to avoid the potential for blood exposure to other patients that has 
occurred with multi-use Lancet devices. 
 
6. Standard Diet  
Description: A dinner and snack will be provided from the CTRC the night prior to the OGTT. The 
diet will be composed of 65% carbohydrates, 25% fat, and 10% protein at 1.25 daily needs and will 
be will be provided by the CTRC.  
Risk: None 
Justification/Minimization: 1.25 x weight maintenance was chosen as this as most similar to our 
subjects food consumption based on pilot subject’s food frequency questionnaires and optimal for 
detection of hepatic Ra 46.  
 
7. Magnetic Resonance Imaging (MRI) 
Description: The MRI will usually be obtained the day of admission to CHC), at the UCD Brain 
Imaging Center on the Fitzsimmons campus. A trained research radiographer who is supervised by 
Dr. Mark Brown, of UCD radiology, will perform an abdominal MRI to obtain hepatic, visceral and 
subcutaneous fat on a 3.0 T whole–body MRI scanner (Siemens MAGNETROM, Malverne, PA). 
Subjects will lie supine while these measurements are obtained, need to hold reasonably still 
during the scan and cannot weigh >325 lbs. A second sequence to measure the amount of fibrosis 
(if any present) in the liver will be performed. In some subjects, a specialized phosphorus coil will 
be utilized to measure the concentration of 31P via MRS to calculate glucose-6-Phosphate 
concentrations before and after the glucose drink. 
Risks: Minimal. Subjects may develop claustrophobia in the magnet. 
Minimizing Risk: The subject is provided with audio protection and optional television to help 
increase comfort. Some subjects might feel claustrophobic while having an MRI and the scan will 
be stopped if it cannot be tolerated. In addition, any subjects with implanted metal that is not 
cleared by the MRI technician may not be able to have the MRI due to the type of magnet involved. 
Justification/Minimization: MRI is a non-invasive and non-radiation method to assess body fat, and 
mitochondrial function. The risks are minimized by assuring patient comfort prior to starting the 
scan, placing eye goggle that plays movies on the subjects. Further, per standard protocol, no 
patient will be placed into the scanner if they do not meet the rigorous safety standards for the MRI, 
including the absence of non-compatible implanted metal. 
 
8. Body Composition 
Description: Body composition will be measured using the DEXA technique and will be used to 
derive fat-free mass and % body fat.  This technique relies on the absorption of dual electron 
wavelengths for the assessment of body fat, lean tissue, and bone mineral density.  During the 
procedure, the subject will be supine on the measurement table, and the arm of the machine will 
slowly pass over their body.  
Risk: Minimal. Radiation exposure 
Justification/Minimization: Body composition is best assed via DEXA, and the amount of muscle mass 
is needed to standardize the OGTT results, since body weight can vary greatly. This procedure will 
deliver the radiation exposure that is 2 times the level of background radiation in Colorado.   
Subjects will be tested for pregnancy immediately prior to DEXA, to ensure that they are not 
pregnant. 
 
9. Endopat and Dynapulse 
Description: The Dynapulse Pathway and the EndoPat system are noninvasive portable systems 
that measure brachial artery distensibility and endothelial function, utilizing a standard 
sphygmomanometer cuff inflated in the same fashion as a sphygmomanometer to obtain blood 
pressure. The instrument derives brachial artery distensibility using the technique of pulse 

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waveform analysis of arterial pressure signals obtained from the sphygmomanometer. 
Measurement of heart rate variability for autonomic tone will be performed using the Endopat 
Risk: This procedure may lead to mild discomfort due to the blood pressure cuff being inflated. 
Justification/Minimization: Endothelial function is a novel measure in PCOS and will aid in the 
determination of cardiovascular dysfunction with this population.  
 
10. Body fat distribution   
Description: Height, weight, waist circumference, and hip circumference will be measured. Body fat 
distribution will be determined using the waist-to-hip ratio where the waist circumference is measured 
1/2 the distance from the xiphoid process to the navel and the hip circumference is measured at the 
level of the greater trochanter. 
Risk: None 
Justification/Minimization: IR has been associated with central obesity, as has hyperandrogenism. 
Whereas we are measuring central obesity with MRI, it is important to see if this simple non-invasive 
measure matches the MRI results, as it is a much simpler measure to follow clinically. 
 
11: Accelerometer:  
Description subject will be provided two accelerometers (GT3X BT by Actigraph and ActiWatch by Philips 
Respironics) to be worn for seven days to measure level of habitual physical activity, which affects insulin 
sensitivity, and sleep patterns. The accelerometers will be worn on the participants wrist. The wrist 
position has been validated to hip position actigraphy in this population. 
Risk: There is no risk involved with the accelerometer. 
Justification/Minimization: Accelerometers are effective tools for the objective measurement of 
physical activity 44 because they have the ability to continuously record physical activity data and 
such data can be used to estimate METs of activity. They provide more detailed information than 
pedometers, which only measure walking steps, and help get around the recall bias of 
questionnaires. We are currently using the GT3X BT Actigraph in adolescents in our other diabetes 
studies; therefore, we are familiar with their use in this population and have the necessary 
computer software and interpretation skills.  The Actiwatch is being used as a tool for objective 
measurement of sleep patterns. The Actiwatch is fitted with a LED monitor that records multiple 
spectrums of light to better assess sleep patterns in this population. 
 
12: Metabolic Cart: 
Description: The metabolic cart measures the amount of air that the subject breathes in and out. 
The machine attaches to the subject’s mouth through a tube, or a plastic bubble that is placed over 
the subject’s head. There is the potential for experiencing claustrophobia from having the plastic 
bubble over the subject’s head. A metabolic cart will be utilized multiple times during the OGTT 
study day to measure rates of oxygen consumption and carbon dioxide release. These rates can 
be utilized to calculate rates of carbohydrate and fat oxidation and resting energy expenditure. 
Risk: Minimal risk of claustrophobia. 
Justification/Minimization: These studies are well tolerated by youth, and involve placing a clear plastic 
hood over the subjects head for approximately 20 minutes. The data collected from the baseline study is 
also very useful for assisting obese subjects in determining their true caloric needs, and useful in setting 
dietary goals for weight loss. This piece of information is thus utilized in post-study nutritional counseling. 
 
13. Food Frequency Questionnaire (SEARCH FFQ) 
Description: Customary macronutrient pattern will be ascertained by diet interview at the time of 
admission using a SEARCH FFQ, modified to incorporate common food choices among ethnically 
and regionally diverse youth aged 10-19 participating in another large childhood diabetes study, 
SEARCH (48).  The instrument is self-administered with staff support to provide instructions, 
answer questions, and to review the form after completion, and captures the last week of dietary 
intake.   
Risk: None 
Justification/Minimization: Several of the measurements being assed are affected by prior nutritional 
intake. Furthermore, subjects will receive dietary counseling at the end of the study, and by knowing 

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what their previous dietary pattern is, suggestions for improvement can be tailored to their specific 
dietary habits. 
 
14. 3DPar Questionnaire  
Description: A questionnaire (3DPAR) recalling the physical activity levels of the three previous days 
will be completed at visit 3. screening. 
Risk: None 
Justification/Minimization: Physical activity can directly affect insulin sensitivity, our primary outcome 
measure. The 3DPar is a well validated measure to asses 3 days of physical activity in youth, and 
includes a variety of youth centric activities. 
 
15. Strengths and Difficulties Questionnaire: 
Description: This is a survey which identifies areas in a youth’s life that they believe they are strong or 
weak in dealing with, as a measure of coping skills. Low coping skills have been associated with the 
development of depression. 
Risk: None 
Justification/Minimization: This survey can help identify youth at risk for depression or anxiety, and 
identify poor coping skills. It does not directly assess depression or suicidality. 
 
16. WatchPAT and Questionnaires to assess for Obstructive Sleep Apnea 
Description: The WatchPAT is a noninvasive portable system that measures  the oxygen saturation 
and apnea hypopnea index. Three surveys querying signs and symptoms of obstructive sleep apnea. 
Risk: No risk associated with the questionnaires and the WathchPAT,other than a mild discomfort from 
having to wear the watch and cuff around finger during sleep. It is possible that we will discover that 
the participant has obstructive sleep apnea, and will need to be referred for further clinical care. 
Justification: Obstructive sleep apnea is associated with obesity, and can worsen both fatty liver and 
insulin resistance. Thus the presence of OSA must be accounted for when measuring either of these 
outcomes. These surveys selected are currently being utilized by the NIH multiple center study in 
obese youth at risk for diabetes, and are well validated in youth from multi-ethnic populations.  If OSA 
is suspected during the course of the screen, the subjects will be referred for further evaluation and 
treatment. The WatchPAT is an FDA approved device that can be used specifically for oxygen 
saturation and apnea hypopnea index and is approved in children as young as 12 years of age, within 
the age range of our study population. 
 
17: Gut Bacteria Collection: 
Description: A week prior to visit three, participants will be provided with stool collection swabs to 
collect a small sample of stool from the toilet paper they use after having a bowel movement. 
Risk: Although the risk is minimal, subjects may feel uncomfortable taking a sample of stool from 
the toilet paper following a bowel movement. All participants will be instructed to follow proper 
bathroom etiquette as fecal matter can transmit diseases. 
Justification: Studies in obese individuals with type 2 diabetes have alterations in the gut microbiota 
that may be related to NAFLD. These studies have not been performed in PCOS. 
 
18. Violation of Privacy and Loss of Confidentiality  
Description: These are both risks to which research participants are exposed. The possibility of 
these risks increases when protected health information is collected. Every effort will be made to 
decrease this risk by limiting access to protected health information, storing this information in a 
password protected database, and identifying subjects only by a unique identifier that is kept in a 
separate location in a locked container, traceable only by study personnel. All of the tests involve 
the risk of identifying asymptomatic abnormalities. The study may include risks that are unknown at 
this time. At the screening visit, we will obtain specific consent to contact the subjects pharmacy to 
verify home medication (metformin or OCP only) adherence. This contact increases the risk of loss 
of privacy and confidentiality. 
Justification/Minimization: The best way to externally assure medication adherence for home 
medications is by verifying that the medications have been refilled at an expected interval.  Every 

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effort will be made to decrease the risk of loss of confidentiality by limiting access to protected 
health information, storing this information in a password protected database, and de-identifying 
study specimens.   
 
E. Benefits of the study: 
Benefits to Society: 
PCOS affects 6-15% of the female population in the US, has an estimated $4 billion economic 
burden and the associated irregular periods, obesity, fatty liver disease and excessive facial hair 
are especially socially difficult for teens. Current treatment options for PCOS are limited. Women in 
their 20's and 30's with PCOS already have evidence of cardiovascular disease and diabetes, 
making adolescent studies, when the disease starts, crucial to understanding disease 
development. Current therapies options are limited and minimally efficacious. This understanding 
will lead to the development of more effective early treatments, before diabetes and cardiovascular 
disease develop.  
Knowledge to be gained: 
A better understanding of how NAFLD develops in girls with PCOS could lead to more effective 
treatment strategies. This understanding could ameliorate development of diabetes and heart 
disease for these girls as they become adults, and may also help with many of the health and 
social difficulties teens with PCOS experience. Since PCOS is one of the most common endocrine 
diseases in the US female population, improving PCOS care could have major health implications. 
The data to be generated from this project will be utilized to inform R01 grant applications of 
treatment trials with new medications. We will also begin to learn the effect of the two existing 
medical therapies. 
Individual: Subjects will benefit from in depth testing for pre-diabetes, fatty liver disease and sleep 
disorders that are not clinically offered. They will receive extensive counseling for both dietary and 
exercise lifestyle changes. Similar subjects who completed related protocols (10-1288 and 14-
0542) and received this counseling have higher rates of weight loss upon clinical follow-up than 
those children being seen in obesity clinics alone. 
Benefits to participant: 
1) All subjects will be undergoing measures that can identify insulin resistance, hyperlipidemia, 
NAFLD or early cardiovascular disease. These measures are not typically done within the scope of 
daily pediatric practice, and subjects would likely not otherwise know this information. If they have 
one of these conditions, they will be referred for appropriate follow-up and treatment. If the subjects 
have signs of obstructive sleep apnea, they will be referred to the sleep clinic for appropriate 
evaluation and treatment. 
2) All of the subjects enrolled must be sedentary, with less than 2.5 hours a week of physical 
activity. This is less than the time recommended by the US Preventive Task Force and the 
American College of Sports Medicine for this age group. This lifestyle puts them at risk for several 
diseases including diabetes and cardiac disease later in life, even if they don't have evidence of 
disease at this time. At the end of the study, all subjects will receive counseling on how to increase 
their activity levels by trained study staff. Increased activity has been shown to reverse the risk for 
diseases later in life. 
3) All subjects will complete a 3 day food questionnaire. This will be reviewed with them, and 
healthier food choices and meal planning will be discussed with both the subject and their parent 
by the PI or PRA, all of who are trained in providing diet prescriptions. Additionally, obese subjects 
will be counseled on a weight loss diet. Over 80% of obese adolescents are obese as adults, if they 
do not change their eating habits and lose weight when they are still a teen. 

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4) The participants will benefit from getting a sleep study during their overnight stay at the hospital 
and discovering if they have obstructive sleep apnea. Depending on the results they may be 
referred to sleep clinic for further evaluation of sleep apnea. 
Evidence of Direct Benefit 
We believe that this protocol is in the 405 risk category for pediatric research. Subjects can benefit 
from the above study measurements. Further, at the conclusion of the study, all subjects in the 
protocol will also be given counseling on the benefits of exercise as discussed above, and given an 
exercise prescription. The subjects may also be re-contacted once by phone call to follow-up on 
their study results and exercise recommendations. They may also be followed up in a clinical 
setting if PI notes further benefit of clinical follow up. Sedentary subjects will thus gain direct benefit 
from the study through the benefits of specialized counseling and recommendations for increased 
physical activity that would not otherwise be available to them.  
Sedentary and obese adolescents are at increased risk for diabetes, reduced bone mineral density, 
reduced exercise capacity, cardiovascular dysfunction, and increased visceral and liver fat. 
Therefore, obese non-PCOS subjects may benefit from the results of glucose testing, insulin 
resistance assessment, DEXA, cardiovascular measures, and MRI testing. The DEXA scan can 
detect evidence of osteopenia, which is becoming more prevalent in adolescents, especially those 
who are sedentary females. The MRI of the liver can detect early evidence of fatty liver disease or 
fibrosis. The blood tests done for screening can detect alterations in blood glucose, pubertal sex 
hormones, as well as abnormalities in fasting lipids. The risk of all of these endpoints is increased 
in sedentary subjects, especially those who are also obese, and if left untreated can increase long 
term health risk, thus the benefit of detecting any of these would directly impact both the health and 
the longevity of the individual subject. 
Inclusion criteria is less than 2.5 hours of exercise per week, a level of activity well below that 
recommended by the U.S. Surgeon General for youth (Children and teenagers should exercise for 
1 hour of vigorous physical activity daily and weight-bearing activities that strengthen their bones). 
Numerous studies have linked low activity with development of diabetes, heart disease and insulin 
resistance; all of the end-points which we are studying. Recent studies have shown that sedentary 
lifestyle increases risk of cardiovascular disease and all-cause mortality. Youth in the U.S. now 
suffer from obesity in epidemic proportions, with about 32% of US adolescents currently being 
overweight and 17% being obese.  A sedentary state is an increasingly common problem in the 
U.S., especially for adolescents, as a recent study showed that the most sedentary groups in the 
United States were adolescents and adults over 60 years. Adolescents in this study spent about 
60% of their waking time in sedentary pursuits, making sedentary adolescents a critical group to 
study. The amount of time spent in sedentary behaviors has been independently associated with 
increased risk of weight gain and increased risk of metabolic syndrome, diabetes, and heart 
disease. In light of these links to adverse health outcomes and the continued increase in the 
prevalence of overweight and obesity in the United States, sedentary behaviors have emerged as 
an important target of health promotion and obesity and disease prevention efforts, complementing 
efforts to increase levels physical activity. For this reason, sedentary lifestyle can be considered a 
pre-disease state. 
  Because of their sedentary nature, there are several primary direct benefits to the non-PCOS 
subjects from the OGTT, which justify the risk. The primary benefit from the OGTT would be the 
discovery of pre-diabetes via a non-invasive measurement of glucose tolerance. As mentioned 
before, surrogate measures are unable to detect insulin resistance adequately, and insulin 
resistance is a strong predictor of NAFLD.  Thus, it is very possible that we may find evidence of 
NAFLD in the obese non-PCOS population. Discovery of insulin resistance would enable us to 
recommend education and an exercise and diet plan to treat or prevent further development of 
insulin resistance, diabetes and NAFLD. The direct benefit of the isotopic tracers would be the 
differentiation of whether the liver, and/or the muscle and/or the adipose tissue is involved in the 
insulin resistance. Again, if this were true the individual could benefit from referral to the metabolic 

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syndrome clinic or the GI clinic to be followed for evidence of hepatic disease that may need to be 
treated if there is any disease progression. 
4) WatchPAT results: The patient population is at high risk for OSA. Untreated OSA is 
thought to contribute to worse fatty liver disease and glucose metabolism. By 
identifying that these youth have a problem, they are then in the position to work with 
their physicians to address this, and potentially improve their metabolic health long-
term. Additionally, treatment of sleep disordered breathing is associated with weight 
loss, and this is a goal for all of these participants.  
 
F. Alternative Treatment 
The alternative is for subjects to not participate in the study 
G. Consideration of Specific Subject Categories 
1. Inclusion of Women 
All subjects will be women, as PCOS only occurs in females. 
2. Inclusion of Minorities 
Every effort will be made to include a diverse subject distribution. PCOS affects Caucasians, 
Hispanics and African Americans equally. 
3. Inclusion of Children 
All subjects will be between ages 12 and 21. Insulin sensitivity needs to be studied in the 
adolescent age group as available is scarce in this age group and it is critical to understand the 
pathophysiology of PCOS in its developing stages.  
IV. Potential Scientific Problems:   
Limitation of Method Development: 
The protocol as described includes the entire subject set, which allow for comparisons between 
obese girls with and without PCOS. OGTT with an oral tracer will be conducted in all subjects. The 
specific points that are to be evaluated are listed below: 
1) OGTT with glycerol tracers 
a. Purpose: Develop an oral stimulus model to assess hepatic substrate flux 
i. Question 1.  Is 6.5 hours post oral load long enough to model hepatic and adipose 
insulin sensitivity?   
1. Rationale: In our current study (14-0542), we have found that 6 hours are required for 
metabolites to return to normal, we do not anticipate this to change greatly with the 
addition of the glycerol tracer.  
2. Measurement. Serum glucose and insulin will be measured post-glucose load for 6.5 
hours in the first 6 subjects. If 6.5 hours post glucose values are within 20 mg/dL for 
glucose and 20 IU/mL for insulin relative to baseline values, 6.5 hours will be 
considered sufficient.  
2) Measurement of Hepatic Substrate flux in youth. 
a. Purpose: Optimize a model to asses hepatic substrate flux in youth looking at 
shifts from pentose phosphate pathway (PEP), TCA cycle and fatty acid synthesis 
i. Rational: utilizing and oral glycerol model with NMR isotopmer analysis is a newer 
technique. It has not been validated in youth. 
ii. Measurement: NMR isotopomer analysis with pathway flux calculations and modeling 
iii. Revision plan if model not correct: 1) increase the amount of tracer given 2) increase 
the frequency of blood draws in the later sampling period. 
 
Overall Project Limitations: 

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1) Subject recruitment is always a potential concern, but is minimized by a streamlined 
recruitment system and experience with this population; the previous, more intensive study 
is well tolerated in youth and enrolled faster than projected. In addition, we had >100 new 
PCOS referrals to our pediatric endocrinology department in 2013 alone, and the PI has a 
clinic specifically for girls with PCOS.  
2) Subject drop-out: We expect reasonably good completion rates due to the non-invasive 
nature of the two study visits and our low dropout rate in our previous studies in control and 
PCOS adolescents using similar procedures. 
 
V. Data Management and Security Plan 
Data Entry   
Data will be entered from paper forms. Once forms are completed, verified and corrected for 
inconsistencies, they will be manually entered at our site using a computerized data management 
system (Redcap).  
Edit Checks    
Computerized data validation routines will be used to enhance data quality and verify the accuracy 
of data within predefined value ranges. These checks include, but are not limited to: (a) initial 
screening of data, using logic and range checks built into data entry screens; (b) cross-form 
functional and consistency checks; and (c) edits assessing the serial integrity of data. 
Disaster Recovery    
Routine data backup will occur on data in conjunction with the Children’s Hospital secure server 
and Redcaps. 
Security and Confidentiality   
All hard copy forms will be de-identified with a study number and filed in a locked cabinet, to which 
only the investigators will have access.  Standard protection against computer hackers is 
implemented. Recovery from natural disasters (water, fire, or electrical) can occur through the 
ability to reconstruct both the database management system and the data from nightly backups.   
VI. 
Data and Safety Monitoring Plan 
The principal investigator and study coordinator will monitor the protocol and the safety of the 
research subjects.  The PI will review all laboratory data and report any abnormal values to the 
patient and guardian and instruct the subject to follow-up with their PCP.  If an abnormal result from 
a research procedure exists, the PI will notify the family and their PCP and refer the participant to 
the appropriate clinic for further evaluation. The PI may also share research results in a reasonable 
and prudent manner with appropriate medical professionals if the participant was seriously injured 
as a result of a procedure or if follow-up of the result of the procedure is in the best interest of the 
participant’s health as determined by a medical professional. If immediate medical follow-up of 
participant required, the PI will share the research results via EPIC when clinically relevant. The PI 
will report adverse events, and any decision to suspend or halt the protocol to CTRC and COMIRB 
immediately.  The PI will also prepare a written report for the yearly continuing review required by 
COMIRB and the CTRC.  There are no other entities that require notification about this protocol.   
 
No protected health information will be collected until the appropriate HIPAA forms are completed. 
The protected health information that will be collected will include: Name and phone number, 
demographic information (DOB, sex, ethnicity, address, etc.), diagnosis (es), history and physical, 
laboratory or tissue studies, radiology studies, procedure results, survey/questionnaire results, 
research visit records, and portions of previous Medical Records that are relevant to this study. 
This information will be accessible only by the study investigators, Federal agencies overseeing 
human subject research, the Colorado Multiple Institutional Review Board, regulatory officials from 
the institution where the research is being conducted to monitor safety and compliance with 
policies. 
 

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A. Adverse Events (AE)  
The OGTT is a standard procedure used in a large number of research studies and settings.  
Adverse events are uncommon when the procedure is done by experienced personnel in an 
appropriate setting.   
 
 
1. Adverse Event Definition 
For the purposes of this study, an Adverse Event (AE) is defined as any significantly abnormal 
physical finding identified on examination and any significantly abnormal laboratory result obtained 
on the patient between visits or at the time of the visit.  Questions answered YES and any new 
abnormal physical findings are pursued by the study staff in order to determine the seriousness of 
the event and the need for further evaluation, follow-up, or referral. If follow-up or referral of 
abnormal research results or procedure is required, the PI will place a referral in EPIC, update the 
PCP on patient condition and reason for referral and contact family to discuss follow-up treatment 
options. 
a) Adverse Event Reporting 
AEs are reported on a standard form that is completed by the study staff at each regular follow-up 
visit and phone interview.  Adverse events reported or ascertained between clinic visits are 
captured and reported at the time of the next scheduled visit. 
 
Pre-existing conditions (that is, conditions present prior to study enrollment) are not considered or 
recorded as AEs or SAEs unless the condition worsens in intensity or frequency after enrollment.  
Likewise, continuing adverse events are not reported as AEs at subsequent visits unless they 
increase in severity or frequency between the visits, they result in criteria for an SAE, and/or they 
resolve between visits. 
B. Serious Adverse Events (SAE) 
1. Serious Adverse Event Definition 
Events are divided into those that are not serious (AE) and those that are serious (SAE).  The 
distinction between an SAE and an AE is a regulatory definition established by the FDA, not a 
clinical definition.  The definition of SAE is not always related to clinical severity of the event.  For 
the purposes of this study an AE is considered a Serious (SAE) when it satisfies any one of the 
following criteria: 
 
• 
The event results in an inpatient hospitalization (any overnight stay associated with an 
admission). 
• 
The event results in the prolongation of a hospital stay. 
• 
The event results in permanent or severe disability. 
• 
The event results in death. 
• 
The event is life-threatening. 
• 
Treatment is required to prevent a serious event. 
 
There have been no SAE’s in the research groups experience in the Pediatric CTRC.  We do not 
anticipate encountering SAE’s; however, we have identified the following as possible SAE’s for the 
purposes of monitoring: 
• 
Infection related to blood draw or IV placement 
 
a) Serious Adverse Event Reporting 
Study patients are instructed to contact the clinic with any serious adverse event meeting the 
above criteria.  Each SAE is recorded on the study form and the PI is informed as soon as possible 
after they occur and preferably within 24 hours of the notification of the clinic staff.  This notification 
should occur even if data are incomplete.  Additional data and follow-up information are 
documented and sent subsequently as an update to the original report.  The PI immediately 
forwards SAE reports to the COMIRB and any other required institutional monitoring committee.   

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C. Subject Discontinuation Criteria 
If a subject experiences any of the following, the subject will be withdrawn from the study. 
1. Inability to complete study procedures 
2. Abnormal screening labs (LFT’s >150 IU, HbA1C>6.4%, Hg <10 mg/dL) 
3. Subject becomes pregnant during study 
 
D. Protocol Stopping Criteria 
If one or more subjects experience any of the SAE’s listed above, the PI will consult with the study 
staff prior to continuing study visits with subjects.  The PI and RSA will consult about the 
significance of the SAE’s and make a recommendation about subject continuation in the study.  
 
VII. Data Analysis Plan:   
 
Overall Project Statistical Plan: 
 
Power calculation: The primary outcome is hepatic substrate flux pathway percentages measured 
as PEP, TCA and TG synthesis pathways. Power analysis is based on publications utilizing similar 
methods, with the Primary Aim of understanding differences in hepatic metabolism in obese girls 
with PCOS who either have or do not have hepatic steatosis. The reference manuscript included 
13 men without HS, and 6 men with HS50. They found that during hyper-insulinemia, glycerol 
related gluconeogenesis was increased, as was the PEP cycle in individuals with HS compared to 
those without. Utilizing the PEP value, from this paper, the difference between the group means is 
3 µmol.min.kg FFM, and the standard deviation is 2 µmol.min.kg FFM. Differences between the 
two primary groups (PCOS, controls) will be assessed with a t test or for non-normally distributed 
data a non-parametric test such as Mann-Whitney U. With 15 untreated PCOS with HS and 15 
untreated PCOS without HS, we would have a power of 0.97 to detect an alpha of 0.05. Since the 
reference population is men who have more uniform hormone profiles, I am choosing to be 
overpowered should the variability in our population be greater. As the measurement is in kg of fat 
free mass, we will not have to do adjustments with BMI or weight.  However, as African Americans 
are less likely to have HS, and Hispanics more likely to have HS, if ethnicity percentages are 
different between groups, we will need to adjust for ethnicity in our calculations. 
 
Secondary Aims: a) For comparison of the PCOS to control groups, individuals will be matched in 
terms of HS status, so that the comparison is just between the hormonal state.  I am assuming that 
there will be less of a difference between these groups, so with a difference of 2 Umol.min.kg FFM, 
and the standard deviation is 2, with 20 PCOS and 20 controls, we would have a power of 0.87 to 
detect a alpha of 0.05. b) For comparison of treatment with either metformin or OCP’s, we will 
compare the untreated group to the treatments individually. We will not compare the two treatments 
to each other. I am assuming that there will be less of a difference between these groups, so with a 
difference of 2 Umol.min.kg FFM, and the standard deviation is 2, with 30 PCOS and 15 in a 
treatment group, we would have a power of 0.87 to detect a alpha of 0.05. 
 
All measures will be adjusted for OSA status (yes/no) and/or severity of AHI. 
 
Secondary outcomes include a Pearson correlation test of potentially related variables followed by 
targeted multiple regression to assess the relationship between hepatic fat and adipose IR, PEP, 
TCA, TG synthesis pathways.  
 
Non-Isotope OGTT calculations: The Matsuda model of IR will be used but adapted for the 6.5 hour 
OGTT, and B-cell function [insulinogenic index (ΔI30/ΔG30) and (ΔC30/ΔG30) and disposition 
index (1/IFasting × ΔI30/ΔG30) and (1/CFasting ×ΔC30/ΔG30) will be calculated38,49,51,52. 

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Differences between groups will be evaluated with and without controlling for activity and sleep 
status. 
      
Fitting glucose and glycerol concentrations curves:  Using computed data points, glycerol and 
glucose concentrations will be fitted with an appropriate model.  In control subjects, concentration 
typically shows 3 phases in OGTT 53. Cubic polynomial, piecewise-linear, spline, and dynamic 
models have been used for fitting Ra in previous work54,55. We will assume a cubic polynomial 
model; we will validate estimates obtained with the cubic polynomial model by computing 
comparable estimates using at least one other model form. In previous work comparing different 
models (piecewise-linear, cubic polynomial, dynamic), results have been relatively unaffected by 
choice of model.  If we find that this is not the case for our data, then we would certainly consider 
the advantages of adopting a more complex approach, such as the state-space, for describing 
concentration changes. 
 
Metabolomics data: Differences in targeted metabolic concentrations will be compared between 
groups, after appropriate cleaning of the data, as performed by the processing site. Changes in 
metabolites over time will be compared between groups, to determine the times that demonstrate 
the largest differences. Results will also be compared to isotopomer results, to see if a 
metabolomics endpoint can yield similar information to the isotopomer analysis. 
 
Gut Microbiota: The microbiota profile is entirely exploratory, and will be related to status of hepatic 
steatosis and 2 hour glucose status of impaired glucose tolerance or not. 
          
VIII.  Summarize Knowledge to be Gained:  
Overall Expected Results:  We anticipate that we will determine which specific areas of hepatic 
metabolism are upregulated in girls with hepatic steatosis. This data would then provide the 
background evidence for future therapeutic trials. We will also refine this protocol and identify if 
metabolomics can be utilized to measure this type of hepatic energy flux. 
 
Significance: Recent evidence in adults with PCOS indicates that 60-70% have NAFLD7,15. Our 
data shows that > 45% of obese girls with PCOS have NAFLD, likely an underestimate due to 
exclusion criteria of T2D and weight >250 lbs in the previous protocols. The majority of NAFLD 
studies have focused on males, and none have been performed in multi-race cohorts of girls with 
PCOS, despite this high prevalence. NAFLD has been described as the primary driver of 
worsening metabolic syndrome and CVD in obesity across populations, and can progress to 
cirrhosis and liver failure5,6. Thus, understanding the relative contributions of adipose and hepatic 
IR to NAFLD is critical to designing strategies to improve the long term health of these girls and 
reduce their risk of T2D, CVD and liver failure. 
 
IX.  References: 
1. 
Knochenhauer ES, Key TJ, Kahsar-Miller M, Waggoner W, Boots LR, Azziz R. Prevalence 
of the polycystic ovary syndrome in unselected black and white women of the southeastern United 
States: a prospective study. J Clin Endocrinol Metab 1998;83:3078-82. 
2. 
Azziz R, Marin C, Hoq L, Badamgarav E, Song P. Health care-related economic burden of 
the polycystic ovary syndrome during the reproductive life span. J Clin Endocrinol Metab 
2005;90:4650-8. 
3. 
Blank SK, Helm KD, McCartney CR, Marshall JC. Polycystic ovary syndrome in 
adolescence. Ann N Y Acad Sci 2008;1135:76-84. 
4. 
Baranova A, Tran TP, Birerdinc A, Younossi ZM. Systematic review: association of 
polycystic ovary syndrome with metabolic syndrome and non-alcoholic fatty liver disease. 
Alimentary pharmacology & therapeutics 2011;33:801-14. 
5. 
de Groot PC, Dekkers OM, Romijn JA, Dieben SW, Helmerhorst FM. PCOS, coronary 
heart disease, stroke and the influence of obesity: a systematic review and meta-analysis. Human 
reproduction update 2011;17:495-500. 

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6. 
Ehrmann DA, Liljenquist DR, Kasza K, Azziz R, Legro RS, Ghazzi MN. Prevalence and 
predictors of the metabolic syndrome in women with polycystic ovary syndrome. J Clin Endocrinol 
Metab 2006;91:48-53. 
7. 
Gambarin-Gelwan M, Kinkhabwala SV, Schiano TD, Bodian C, Yeh HC, Futterweit W. 
Prevalence of nonalcoholic fatty liver disease in women with polycystic ovary syndrome. Clinical 
gastroenterology and hepatology : the official clinical practice journal of the American 
Gastroenterological Association 2007;5:496-501. 
8. 
De Leo V, Musacchio MC, Palermo V, Di Sabatino A, Morgante G, Petraglia F. Polycystic 
ovary syndrome and metabolic comorbidities: therapeutic options. Drugs Today (Barc) 
2009;45:763-75. 
9. 
Park MH, Kinra S, Ward KJ, White B, Viner RM. Metformin for obesity in children and 
adolescents: a systematic review. Diabetes Care 2009;32:1743-5. 
10. 
Hoeger K, Davidson K, Kochman L, Cherry T, Kopin L, Guzick DS. The impact of 
metformin, oral contraceptives, and lifestyle modification on polycystic ovary syndrome in obese 
adolescent women in two randomized, placebo-controlled clinical trials. J Clin Endocrinol Metab 
2008;93:4299-306. 
11. 
Ibanez L, Diaz M, Sebastiani G, et al. Treatment of androgen excess in adolescent girls: 
ethinylestradiol-cyproteroneacetate versus low-dose pioglitazone-flutamide-metformin. J Clin 
Endocrinol Metab 2011;96:3361-6. 
12. 
Lass N, Kleber M, Winkel K, Wunsch R, Reinehr T. Effect of lifestyle intervention on 
features of polycystic ovarian syndrome, metabolic syndrome, and intima-media thickness in obese 
adolescent girls. J Clin Endocrinol Metab 2011;96:3533-40. 
13. 
Domecq JP, Prutsky G, Mullan RJ, et al. Adverse effects of the common treatments for 
polycystic ovary syndrome: a systematic review and meta-analysis. J Clin Endocrinol Metab 
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14. 
Legro RS, Arslanian SA, Ehrmann DA, et al. Diagnosis and treatment of polycystic ovary 
syndrome: an endocrine society clinical practice guideline. J Clin Endocrinol Metab 2013;98:4565-
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15. 
Cerda C, Perez-Ayuso RM, Riquelme A, et al. Nonalcoholic fatty liver disease in women 
with polycystic ovary syndrome. Journal of hepatology 2007;47:412-7. 
16. 
Vernon G, Baranova A, Younossi ZM. Systematic review: the epidemiology and natural 
history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Alimentary 
pharmacology & therapeutics 2011;34:274-85. 
17. 
Mudaliar S, Henry RR, Sanyal AJ, et al. Efficacy and safety of the farnesoid X receptor 
agonist obeticholic acid in patients with type 2 diabetes and nonalcoholic fatty liver disease. 
Gastroenterology 2013;145:574-82 e1. 
18. 
Fabbrini E, deHaseth D, Deivanayagam S, Mohammed BS, Vitola BE, Klein S. Alterations 
in fatty acid kinetics in obese adolescents with increased intrahepatic triglyceride content. Obesity 
(Silver Spring) 2009;17:25-9. 
19. 
Vitola BE, Deivanayagam S, Stein RI, et al. Weight loss reduces liver fat and improves 
hepatic and skeletal muscle insulin sensitivity in obese adolescents. Obesity (Silver Spring) 
2009;17:1744-8. 
20. 
Kim YO, Schuppan D. When GLP-1 hits the liver: a novel approach for insulin resistance 
and NASH. Am J Physiol Gastrointest Liver Physiol 2012;302:G759-61. 
21. 
N. Santoro EP, B. Pierpont, S. Caprio. Hepatic De Novo Lipognesis in Youth: Role of the 
GCKR rs1260326 Variant. Diabetes 2013;61 Supp. 1:A1287. 
22. 
Biddinger SB, Hernandez-Ono A, Rask-Madsen C, et al. Hepatic insulin resistance is 
sufficient to produce dyslipidemia and susceptibility to atherosclerosis. Cell Metab 2008;7:125-34. 
23. 
Cai D, Yuan M, Frantz DF, et al. Local and systemic insulin resistance resulting from 
hepatic activation of IKK-beta and NF-kappaB. Nat Med 2005;11:183-90. 
24. 
Arslanian SA, Lewy VD, Danadian K. Glucose intolerance in obese adolescents with 
polycystic ovary syndrome: roles of insulin resistance and beta-cell dysfunction and risk of 
cardiovascular disease. J Clin Endocrinol Metab 2001;86:66-71. 

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25. 
Ciaraldi TP, Aroda V, Mudaliar S, Chang RJ, Henry RR. Polycystic ovary syndrome is 
associated with tissue-specific differences in insulin resistance. J Clin Endocrinol Metab 
2009;94:157-63. 
26. 
Dunaif A, Graf M, Mandeli J, Laumas V, Dobrjansky A. Characterization of groups of 
hyperandrogenic women with acanthosis nigricans, impaired glucose tolerance, and/or 
hyperinsulinemia. J Clin Endocrinol Metab 1987;65:499-507. 
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