Conditions: Polycystic Ovary Syndrome
Keywords: Inflammation Hyperandrogenism Anovulation Insulin Resistance
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 60
Healthy volunteers: yes

Brief summary:
Polycystic Ovary Syndrome (PCOS) is characterized by hyperandrogenism, ovulatory dysfunction and polycystic ovaries. Insulin resistance (IR) is a common feature of PCOS, and the resultant hyperinsulinemia is theorized to promote hyperandrogenism in the disorder. However, 30-50% of women with PCOS who are lean do not have insulin resistance. Women with PCOS also exhibit chronic low-grade inflammation. In PCOS, glucose ingestion activates nuclear factor ĸB (NFĸB), the cardinal signal of inflammation culminating in upregulation of the inflammation pathway within mononuclear cells (MNC). This phenomenon is independent of excess adiposity and is highly correlated with circulating androgens. In addition, in vitro exposure to proinflammatory stimuli is capable of directly stimulating ovarian theca cell androgen production. Nonacetylated salicylates suppress NFĸB activation and are well tolerated in humans.

The proposed research is a randomized double-blind placebo-controlled study of 90 women with PCOS. Forty-five subjects with PCOS (15 lean without IR), 15 lean with IR and 15 obese) receiving salsalate, a nonacetylated salicylate, at an oral dose of 3-4 gm daily for 12 weeks will be compared with 45 age- and body-composition-matched control women with PCOS receiving placebo. The overarching hypothesis is that inflammation contributes to ovarian dysfunction, independent of excess adiposity or IR.

The specific aims are, I: To examine the effect of salsalate administration on the ovarian capacity to secrete androgen and on insulin sensitivity in PCOS. II: To examine the effect of salsalate administration on the inflammatory response of mononuclear cells induced by lipid ingestion and glucose infusion in PCOS. The approach involves evaluation of ovarian androgen secretion in response to human chorionic gonadotropin (HCG) administration and insulin sensitivity during the euglycemic phase of a two-step pancreatic clamp along with ovulation monitoring before and after salsalate administration. The inflammatory response of MNC to lipid ingestion and the hyperglycemic phase of the two-step clamp will also be evaluated during treatment by measuring reactive oxygen species, the mRNA and protein content of inflammation markers, NFĸB activation and cytokine release in culture.

The investigators expect that women with PCOS receiving salsalate will exhibit decreased ovarian androgen secretion and reduced inflammation regardless of adiposity or IR status. These results will be significant if they show a causal contribution of inflammation to ovarian dysfunction in PCOS, thus improving our understanding of the pathogenesis of PCOS, opening previously unexplored therapeutic avenues that are not necessarily dependent on improving IR, and guiding the design of future studies aimed at determining what interventions will optimally attenuate inflammation in PCOS to reduce medical disease and enhance fertility.

Detailed description:
PCOS is characterized by hyperandrogenism, ovarian dysfunction and polycystic ovarian morphology. Obesity and IR are common features of PCOS. Under the current model of pathophysiology of PCOS, the compensatory hyperinsulinemia of IR is the primary driver of hyperandrogenism. This concept was born from the cross-sectional observation that insulin is positively correlated with androgens in obese women with PCOS, and is supported by reports of increased androgen production from theca cells obtained from obese women with PCOS following insulin exposure in vitro and increases in circulating androgens in women with PCOS following insulin infusion in vivo. However, these in vitro - in vivo responses were elicited with supraphysiological insulin concentrations. Physiological insulin infusion on the other hand does not augment androgen levels in PCOS. The current model also does not explain the cause of hyperandrogenism and ovarian dysfunction in the 30-50% of women with PCOS who are lean and lack IR. Thus, some other factor contributes to these abnormalities in PCOS.

The investigators have shown that ingestion of glucose and saturated fat elicits an inflammatory response from circulating MNC in lean women with PCOS who lack excess abdominal adiposity. The hallmark of this response is increased activation of NFĸB, the cardinal signal of inflammation. These findings illustrate the separate and discrete role of MNC in manifesting inflammation in PCOS and that MNC are an excellent model to assess systemic inflammation in PCOS.

The investigators have also shown that in PCOS, there is a link between molecular markers of inflammation from MNC and circulating androgens. Chronic suppression of ovarian androgen production does not ameliorate inflammation in lean women with PCOS. However, in vitro exposure of ovarian theca cells to proinflammatory stimuli upregulates CYP17, the androgen producing enzyme and increases testosterone.

Salsalate is an inexpensive, safe, well-tolerated, well-understood anti-inflammatory agent that inhibits NFĸB activation when used at higher doses. The salsalate dose required to achieve a salicylate level in the upper therapeutic range is dependent on body mass. This is achieved in lean individuals using 3.0 gm/day as the maximum dose recommended in the salsalate package insert. Individuals across the obese range (30-40 kg/m2) require \>3.0 gm/day to achieve the same objective. Salsalate and other salicylates have also been shown to decrease IR. However, the ability of salsalate to decrease IR would not be necessary if the beneficial anti-inflammatory effect of salsalate to reduce hyperandrogenism is on the ovaries. In fact, we have shown that in lean insulin-sensitive women with PCOS, salsalate reduces HCG-stimulated ovarian androgen secretion by 44% and normalizes basal testosterone levels. Studies performed by the investigators in MNC also confirm the ability of salsalate to suppress NFĸB activation. Together these observations validate the use of these measurements as endpoints to assess the effects of salsalate to probe the pathophysiology of PCOS.

Salsalate raises circulating insulin due to its ability to decreased insulin clearance from the liver which confounds the assessment of insulin sensitivity from post-treatment hyperinsulinemic-euglycemic clamp studies. Performance of a novel minimal model-based analysis from an insulin-modified frequently-sampled intravenous glucose tolerance test (FS-IVGTT) is able to address this confounding factor. With this approach, hepatic and extrahepatic insulin clearance can be estimated before and after salsalate treatment to obtain measures of insulin sensitivity that take into account the salsalate-induced alteration in insulin clearance.

In this context, the rationale for the proposed study revolves around the concept that in PCOS, inflammation contributes to ovarian dysfunction independent of excess adiposity or IR, and may also improve insulin sensitivity when IR is present. The investigators will undertake a 12-week randomized, double-blind, placebo-controlled trial to test the link between inflammation and ovarian androgen secretion in PCOS unrelated to IR. If this study of pathophysiology demonstrates beneficial effects, this will pave the way for developing novel therapies for ovarian dysfunction in PCOS.

The main objective of this proposal is to evaluate the ability of salsalate to reduce ovarian androgen secretion, induce ovulation and decrease lipid-stimulated inflammation independent of body composition and IR in women with PCOS; and to also improve insulin sensitivity in IR women with PCOS. Effects of salsalate will be assessed based on the following aims:

Specific Aim 1. To examine the effects of salsalate administration on the ovarian capacity to secrete androgens, menstrual function, and insulin sensitivity in PCOS.

The hypothesis for this aim is that salsalate treatment will decrease HCG-stimulated ovarian androgen secretion and induce ovulation in women with PCOS regardless of body composition or IR status; and may also improve insulin sensitivity in IR women with PCOS. The investigators will test this hypothesis in a randomized double-blind placebo-controlled study. The ovarian androgen response to HCG administration will be evaluated in women with PCOS (15 lean with IR, 15 lean without IR and 15 obese) before and after administration of a therapeutic salsalate dose for \~12 weeks compared with women with PCOS receiving placebo for \~12 weeks (15 lean with IR, 15 lean without IR and 15 obese). Ovulation monitoring and assessment of insulin sensitivity during FS-IVGTT will be performed before and after salsalate or placebo administration. It is anticipated that salsalate will reduce HCG-stimulated ovarian androgen secretion, induce ovulation regardless of body composition or IR status when compared with placebo. It is also anticipated that salsalate will increase insulin sensitivity in IR women with PCOS compared with placebo.

Specific Aim 2. To examine the effect of salsalate administration on the inflammatory response of mononuclear cells induced by lipid ingestion in PCOS.

The hypothesis for this aim is that salsalate administration will down-regulate inflammatory signal transduction and cytokine production within MNC following lipid ingestion in women with PCOS regardless of body composition or IR status. The investigators will test this hypothesis using the study design described in Aim 1. The inflammatory response of MNC to a cream challenge test will be evaluated in women with PCOS before and after salsalate treatment. It is anticipated that lipid-induced inflammation will decrease with salsalate use regardless of body composition or IR status when compared with placebo.

Arms:
arm 1: n=15 arm 2: n=15 arm 3: n=15 arm 4: n=15 arm 5: n=15 arm 6: n=15

Interventions: intervention 1: Salsalate intervention 2: Placebo
Intervention descriptions: intervention 1: Lean PCOS Arms: Salsalate 1.5 gm PO bid; Obese PCOS Arm: Salsalate 2.0 gm PO bid intervention 2: Appears identical to experimental drug

Protocol document:
Prot_SAP_000.pdf: 
 _____________________________________________________________________________________________  
Treating Inflammation in Polycystic Ovary Syndrome to Ameliorate Ovarian Dysfunction                           Version 17                    
Page 1 of 37                                                                                                                                                  5/9/2022 
Treating Inflammation in Polycystic Ovary Syndrome 
to Ameliorate Ovarian Dysfunction 
 
 
Principal Investigator 
Frank González, M.D. 
Associate Professor of Obstetrics and Gynecology 
Division of Reproductive Endocrinology and Infertility 
Director, Laboratory for Reproductive Endocrine and Inflammation Research 
University of Illinois at Chicago College of Medicine 
820 S. Wood Street, m/c 808 
Chicago, IL 60612 
Telephone:  (312) 413-1984 
Facsimile:  (312) 996-4328 
E-mail:  frgz12@uic.edu 
 
Co-Investigators 
Lauretta Quinn, Ph.D., B.S.N. 
Clinical Professor of Nursing 
University of Illinois at Chicago College of Nursing 
845 S. Damen Avenue, m/c 802, Room 724 
Chicago, IL 60612 
Telephone:  (312) 996-7906 
Facsimile:  (312) 996-4979 
E-mail:  lquinn1@uic.edu 
 
Brian Layden, M.D. 
Associate Professor of Medicine 
University of Illinois at Chicago College of Medicine 
835 Wolcott Street, Suite 625E m/c 640 
Chicago, IL 60612 
Telephone:  (312) 996-6060 
Facsimile:  (312) 413-0437 
E-mail:  blayde1@uic.edu 
 
Study Location 
University of Illinois at Chicago 
Center for Clinical and Translational Science, Clinical Research Center 
912 S. Wood Street, Suite 200-S 
Chicago, IL 60612 
 
Sponsor 
National Institute of Diabetes and Digestive and Kidney Diseases 
31 Center Drive 
Bethesda, MD 20892 
 
IND Number:  113845 
Version:  #17 
Date:  5/9/2022 


 _____________________________________________________________________________________________  
Treating Inflammation in Polycystic Ovary Syndrome to Ameliorate Ovarian Dysfunction                           Version 17                    
Page 2 of 37                                                                                                                                                  5/9/2022 
TABLE OF CONTENTS 
Page 
TABLE OF CONTENTS .................................................................................................. 2 
List of Abbreviations ........................................................................................................ 3 
1.0 Project Summary/Abstract ................................................................................... 4 
5.0 Subject Enrollment................................................................................................ 8 
6.0 Study Design and Procedures ........................................................................... 10 
7.0 Expected Risks/Benefits ..................................................................................... 21 
Risks ............................................................................................................................. 21 
Benefits ........................................................................................................................ 24 
8.0  Data Collection and Management Procedures ................................................ 24 
9.0 Data Analysis ....................................................................................................... 24 
10.0 Quality Control and Quality Assurance ............................................................. 24 
11.0 Data and Safety Monitoring ................................................................................ 24 
12.0 Statistical Considerations .................................................................................. 28 
13.0 Regulatory Requirements ................................................................................... 31 
13.1 
Informed Consent ..................................................................................... 31 
13.2 
Subject Confidentiality ............................................................................. 32 
13.3 
Unanticipated Problems .......................................................................... 32 
14.0 References ........................................................................................................... 33 
Appendices ................................................................................................................... 37 
 
 
 
 
 
 
 
 
 
 


 _____________________________________________________________________________________________  
Treating Inflammation in Polycystic Ovary Syndrome to Ameliorate Ovarian Dysfunction                           Version 17                    
Page 3 of 37                                                                                                                                                  5/9/2022 
LIST OF ABBREVIATIONS  
ACCC                         Academic Computing and Communications Center 
AE                              Adverse Event 
AUC                           Area under the Curve 
BBT                            Basal Body Temperature 
BMI                            Body Mass Index 
BW                             Body Weight 
CCT                            Cream Challenge Test 
CCTS                         Center for Clinical and Translational Science 
CITI                            Collaborative Institutional Training Initiative  
CLIV                            Clearance for an Intravenous Infusion  
CLP                             Clearance of Plasma Insulin 
CLportal                         Clearance for a Portal Infusion 
CRC                            Clinical Research Center 
DEXA                          Dual Energy X-ray Absorptiometry 
DSMB  
 
Data and Safety Monitoring Board 
EMSA                          Electrophoretic Mobility Shift Assay 
FDA 
 
 
Food and Drug Administration 
FEL                              Hepatic Fractional Extraction 
HBSS                          Hank’s Buffered Saline Solution 
HIPAA                        Health Insurance Portability and Accountability Act 
HCG                           Human Chorionic Gonadotropin 
HCG-ST                     Human Chorionic Gonadotropin Stimulation Test 
HPF                           Hepatic Plasma Flow 
ICD                            Informed Consent Document 
IDS 
 
         Investigational Drug Service 
IND 
 
         Investigational New Drug 
ICL                            Integrative Physiology Laboratory 
IR                              Insulin Resistance  
ISR                            Insulin Secretion Rate 
IRB 
 
         Institutional Review Board 
IUD                            Intrauterine Device 
Km                             Hepatic Insulin Delivery Rate  
LH                             Luteinizing Hormone 
MNC                          Mononuclear Cells 
MRI                            Magnetic Resonance Imaging 
NFκB                         Nuclear Factor κB 
OCC                          Outpatient Care Center 
OGTT                        Oral Glucose Tolerance Test 
17OHP                      17-Hydroxyprogesterone 
PHI 
 
          Protected Health Information 
PI 
 
          Principal Investigator 
PCOS                        Polycystic Ovary Syndrome 
ROS                          Reactive Oxygen Species 
RT-PCR                    Real-Time Polymerase Chain Reaction 
SI                               Fractional Glucose Disappearance/Insulin Concentration Unit  
UIC                            University of Illinois at Chicago 
UI Health                   University of Illinois Hospital and Health System 
Vmax                           Maximal Hepatic Degradation Rate  
VP                              Extrahepatic Distribution Volume 


 _____________________________________________________________________________________________  
Treating Inflammation in Polycystic Ovary Syndrome to Ameliorate Ovarian Dysfunction                           Version 17                    
Page 4 of 37                                                                                                                                                  5/9/2022 
1.0 
Project Summary/Abstract 
 
     Polycystic Ovary Syndrome (PCOS) is characterized by hyperandrogenism, ovulatory 
dysfunction and polycystic ovaries.  Insulin resistance (IR) is a common feature of PCOS, and 
the resultant hyperinsulinemia has been theorized to promote hyperandrogenism in the disorder. 
However, 30-50% of women with PCOS who are lean do not have insulin resistance. Women 
with PCOS also exhibit chronic low-grade inflammation.  We have shown that in PCOS, glucose 
ingestion activates nuclear factor κB (NFκB), the cardinal signal of inflammation culminating in 
upregulation of the inflammation pathway within mononuclear cells (MNC).  This phenomenon is 
independent of excess adiposity and is highly correlated with circulating androgens.  In addition, 
in vitro exposure to proinflammatory stimuli is capable of directly stimulating ovarian theca cell 
androgen production.  Salicylate administration has been shown to suppress NFκB activation, 
and the nonacetylated form of salicylate is well tolerated in humans.  
 
     The proposed research is a randomized double-blind placebo-controlled study of 90 women 
with PCOS.  Forty-five subjects with PCOS (15 lean without IR), 15 lean with IR and 15 obese) 
receiving salsalate, a nonacetylated salicylate, at an oral dose of 3-4 gm daily for 12 weeks will 
be compared with 45 age- and body-composition-matched control women with PCOS receiving 
placebo (Fig. 1). The overarching hypothesis is that inflammation contributes to ovarian 
dysfunction, independent of excess adiposity or IR.  
 
     The specific aims are, I: To 
examine the effect of salsalate 
administration on the ovarian 
capacity to secrete androgen on 
and insulin sensitivity in PCOS.  
II: To examine the effect of 
salsalate administration on the 
inflammatory 
response 
of 
mononuclear cells induced by 
lipid 
ingestion 
and 
glucose 
infusion in PCOS. The approach 
involves evaluation of ovarian 
androgen secretion in response 
to human chorionic gonadotropin 
administration 
and 
insulin 
sensitivity 
during 
the 
insulin-
modified frequently sampled intravenous glucose tolerance test (FS-IVGTT) along with ovulation 
monitoring before and after salsalate administration.  The inflammatory response of MNC to lipid 
ingestion will also be evaluated during treatment by measuring reactive oxygen species, the 
mRNA and protein content of inflammation markers, NFκB activation and cytokine release in culture.  
 
     It is our expectation that women with PCOS receiving salsalate will exhibit decreased ovarian 
androgen secretion and reduced inflammation regardless of the degree of adiposity or IR status.  
These results will be significant because they will show a causal contribution of inflammation to 
ovarian dysfunction in PCOS, thereby improving our understanding of the pathogenesis of 
PCOS; open previously unexplored therapeutic avenues that are not necessarily dependent on 
improving IR; and guide the design of future studies aimed at determining what dietary 
modifications, medical regimen or both will optimally attenuate inflammation in PCOS to reduce 
medical disease and enhance fertility.    
  
  Figure 1 
Salsalate
45 Women with PCOS
15 
Obese
15 
Lean
with
Insulin 
Resistance
Placebo
45 Women with PCOS
15 
Obese
15 
Lean 
without
Insulin 
Resistance
15 
Lean
with
Insulin 
Resistance
Oral Lipid Challenge Test         HCG Stimulation Test
Insulin-Modified Intravenous Glucose Tolerance Test
15 
Lean 
without
Insulin 
Resistance
Double-Blind Placebo-Controlled Study 


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 _____________________________________________________________________________________________  
Treating Inflammation in Polycystic Ovary Syndrome to Ameliorate Ovarian Dysfunction                           Version 17                    
Page 6 of 37                                                                                                                                                  5/9/2022 
     Salsalate raises circulating insulin due to its ability to decreased insulin clearance from the 
liver 
which 
confounds 
the 
assessment 
of 
insulin 
sensitivity 
from 
post-treatment 
hyperinsulinemic-euglycemic clamp studies.19,23,26 Performance of a novel minimal model-based 
analysis from an insulin-modified FS-IVGTT is able to address this confounding factor.  With this 
approach, hepatic and extrahepatic insulin clearance can be estimated before and after 
salsalate treatment to obtain measures of insulin sensitivity that take into account the salsalate-
induced alteration in insulin clearance.  The FS-IVGTT using the minimal model approach has 
been used for nearly four decades as a metabolic research tool and has been widely 
published.27-32  A co-investigator (Quinn) is experienced in the performance of the minimal 
model FS-IVGTT.33,34 
 
     In this context, the rationale for the proposed study revolves around the concept that in 
PCOS, inflammation contributes to ovarian dysfunction independent of excess adiposity or IR, 
and may also improve insulin sensitivity when IR is present.  We propose to undertake a 12-
week randomized, double-blind, placebo-controlled trial to test the link between inflammation 
and ovarian androgen secretion in PCOS unrelated to IR. If this study of pathophysiology 
demonstrates beneficial effects, this will pave the way for developing novel therapies for ovarian 
dysfunction in PCOS. 
 
3.0 
Objectives / Aims 
 
     The main objective of this proposal is to evaluate the ability of salsalate to reduce ovarian 
androgen secretion, induce ovulation and decrease lipid- and glucose-stimulated inflammation 
independent of body composition and IR in women with PCOS; and to also improve insulin 
sensitivity in IR women with PCOS.  Effects of salsalate will be assessed based on the following 
aims: 
   
Specific Aim 1. To examine the effects of salsalate administration on the ovarian capacity 
to secrete androgens, menstrual function, and insulin sensitivity in PCOS.  
  
     The hypothesis for this aim is that salsalate treatment will decrease HCG-stimulated ovarian 
androgen secretion and induce ovulation in women with PCOS regardless of body composition 
or IR status; and may also improve insulin sensitivity in IR women with PCOS. We will test this 
hypothesis in a randomized double-blind placebo-controlled study. The ovarian androgen 
response to HCG administration will be evaluated in women with PCOS (15 lean with IR, 15 
lean without IR and 15 obese) before and after administration of a therapeutic salsalate dose for 
~12 weeks compared with women with PCOS receiving placebo for ~12 weeks (15 lean with IR, 
15 lean without IR and 15 obese). Ovulation monitoring will occur in all subjects using timed 
progesterone measurements and serial follicular ultrasonography as indicated. Fractional 
glucose disappearance per insulin concentration unit adjusted for insulin clearance will be 
determined as a measure of insulin sensitivity (SI) during a FS-IVGTT before and after salsalate 
or placebo administration. We anticipate that salsalate will reduce the exaggerated ovarian 
androgen response to HCG administration and lead to hormonal and sonographic evidence of 
ovulation regardless of body composition or IR status when compared with placebo. We also 
anticipate that salsalate will increase SI in IR women with PCOS compared with placebo. 
 
Specific Aim 2. To examine the effect of salsalate administration on the inflammatory 
response of mononuclear cells induced by lipid ingestion and glucose infusion in PCOS. 
 
     The hypothesis for this aim is that salsalate administration will down-regulate inflammatory 
signal transduction and cytokine production within MNC following lipid ingestion and glucose 


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infusion in women with PCOS regardless of body composition or IR status. We will test this 
hypothesis using the study design described in Aim 1. The inflammatory response of MNC to a 
cream challenge test will be evaluated in women with PCOS before and after salsalate 
treatment with the following measurements: a) reactive oxygen species (ROS) generation; b) 
NFκB activation; c) mRNA and protein proinflammatory markers; d) cytokine release from 
cultured MNC culture. It is anticipated that lipid- and glucose-induced inflammation will decrease 
with salsalate use regardless of body composition or IR status when compared with placebo.  
 
4.0 
Eligibility 
 
     The protocol will prospectively study healthy reproductive-age women with PCOS recruited 
from clinical practice or various local advertisements (see section 5.0). Volunteers interested in 
study participation will respond by telephone or secure e-mail (see section 5.0).  They will 
subsequently be provided with a detailed study description and undergo an initial pre-screening 
using questions from an eligibility assessment form (attached). During the prescreening, 
volunteers will be asked to provide their name, contact information, age/date of birth, smoking 
status, and information about their current medical conditions, general and gynecological health 
status and medication use. All of the answers obtained will be documented in the eligibility 
assessment form which will be kept in each volunteer’s study booklet.  The PI will determine 
subject eligibility based upon the following inclusion / exclusion criteria.  
 
4.1 General Inclusion Criteria:  
• Diagnosis of PCOS based on the presence of hyperandrogenism (skin manifestations of 
androgen excess such as hirsutism, acne or temporal balding ‒ or ‒elevation of at least one 
serum androgen [i.e. total testosterone, free testosterone, androstenedione or DHEA-S] 
using predetermined local laboratory cutoffs), oligo/amenorrhea and evidence of withdrawal 
bleeding after progestin administration. 
• 18-40 years of age. 
• Good health as evidenced by medical history, physical examination and gynecologic 
examination within 30 days prior to starting the study.   
• Willingness to provide informed consent according to the guidelines of the University of 
Illinois at Chicago (UIC) Institutional Review Board (IRB).   
• Willingness to use double-barrier contraception such as condoms and topical spermicide 
(foam, cream or gel), condom and diaphragm, diaphragm and topical spermicide or sponge 
with topical spermicide if sexually active. Use of a non-hormonal intrauterine device (IUD), or 
permanent sterilization of the subject or her partner (i.e. tubal ligation or vasectomy) is also 
acceptable in all instances.   
 
4.2 General Exclusion Criteria: 
• Hyperprolactinemia. 
• Uncontrolled thyroid disease. 
• Evidence of Cushing’s syndrome, nonclassic congenital adrenal hyperplasia or a hormone 
producing tumor based on physical findings and serum androgen levels on initial screening. 
• Known or suspected pregnancy. 
• Regular vigorous physical activity during previous 6 months.  
• Use of any medications known to affect carbohydrate or sex hormone metabolism such as 
oral contraceptives, progestins, glucocorticoids or insulin sensitizing agents within 30 days 
of beginning the study.  


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• Acute or chronic inflammatory illnesses (e.g. URI, asthma, rheumatoid arthritis or systemic 
lupus erythematosus). 
• Type 1 or type 2 diabetes mellitus defined as having a fasting glucose >126 mg/dl and/or a 
2-hour postprandial glucose >200 mg/dl.  
• Regular smoking defined as more than 2 cigarettes a month, or any smoking within 30 days 
of beginning the study. 
• History of any illness exacerbated by salicylate use (e.g. peptic ulcer hepatic or renal 
disease, anemia, thrombosis, coagulopathy, congestive heart failure, hypertension or gout). 
• Allergy to salicylate or dairy products. 
• Medication use interacting with salicylates such as anti-platelet drugs (e.g. cilostazol, 
clopidogrel), anticoagulants (e.g. enoxaparin, heparin, warfarin), corticosteroids (e.g., 
prednisone), certain diabetes drugs (e.g. sulfonylureas such as glyburide), certain anti-
seizure drugs (e.g. phenytoin, valproic acid), cidofovir, cyclosporine, drugs for gout (e.g. 
probenecid, sulfinpyrazone), anti-hypertensives (e.g. ACE inhibitors such as captopril, 
angiotensin II receptor antagonists such as losartan, and beta blockers such as metoprolol), 
drugs that affect the acidity of urine (e.g. ammonium chloride, acetazolamide), lithium, 
methotrexate, oral bisphosphonates (e.g. alendronate), pemetrexed, SSRI antidepressants 
(e.g. fluoxetine, sertraline), tenofovir, and diuretics (furosemide, hydrochlorothiazide, 
spironolactone).   
 
4.3 Excluded or Vulnerable Populations 
• Although not the primary source of recruitment, eligible UIC students and employees are 
welcomed to participate with every effort taken to avoid any semblance of coercion (see 
section 5.0).   
• Eligible volunteers from all racial or ethnic backgrounds are also welcomed to participate. 
• Minors and pregnant women will be excluded by virtue of the scientific design.  Moreover, 
the study question is not directed towards minors or pregnant women. 
 
5.0 
Subject Enrollment 
 
     Volunteers with PCOS will primarily be recruited from the principal investigator’s (PI) 
Reproductive Endocrine referral practice located in the University of Illinois Hospital and Health 
System (UI Health) Outpatient Care Center (OCC), Suite 4A. The prevalence of PCOS in the 
general female population is ~15% but is as high as 80% in the referral based patient population 
cared for by the PI. Volunteers with PCOS will also be recruited through physician referral and 
from advertisements on UIC bulletin boards, electronic bulletin boards, websites such as 
Google.com, local newspapers and on social media. The advertisements will include a phone 
number and e-mail address to contact the PI or the Ob/Gyn Research Nurse Study Coordinator 
who will explain the study and administer a screening questionnaire using an approved eligibility 
checklist administered either over the phone, or via email correspondence using a secure UIC 
e-mail server containing a link to a confidential web-based screening questionnaire (Screening 
Questionnaire – Treating Inflammation in Polycystic Ovary Syndrome to Ameliorate Ovarian 
Dysfunction) through UIC’s Qualitrics survey tool (http://accc.uic.edu/service/surveys). 
Advertisements, that correspond with IRB approved flyers or images, may be used on 
Facebook, Twitter, Instagram or the study website (http://www.doyouhavepcos.org).  
Prospective volunteers who view the images will be directed to contact the study phone or email 
address for more information.  No direct contact with prospective volunteers will be made on 
these platforms.  No data will be collected from these platforms.  In addition, other care 
providers who become aware of the study may refer potential subjects for participation.   
 


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     HealthMatch https://healthmatch.io/ and CenterWatch https://www.centerwatch.com/ are two 
online platforms that will also be used to assist with study recruitment. Prospective volunteers 
access these platforms to learn about clinical trials with open enrollment from publicly available 
information gathered by these platforms (e.g. study protocols descriptions listed on 
ClinicalTrials.gov).  If a prospective volunteer expresses interest in study participation, the 
platform sends the investigator an email notification, but only provides the prospective 
volunteer’s contact information at the investigator’s request.  The study team can subsequently 
use an appropriate IRB-approved recruitment tool to confirm interest in study participation and 
study eligibility such as email correspondence containing a link to our confidential web-based 
screening questionnaire as described above.  No direct contact with prospective volunteers will 
be made on these platforms.  No data will be collected from these platforms. 
 
     A targeted recruitment campaign will also be launched with the assistance of PCOS 
Challenge, the largest nonprofit support organization globally for women with PCOS serving 
over 45,000 members (2,542 in Illinois).  PCOS Challenge membership grows at over 20% per 
year, and 92% of PCOS Challenge members have been diagnosed with PCOS. This will be a 
statewide recruitment campaign that includes: 1) One month of bi-weekly emails to members in 
Illinois sent by the PCOS Challenge Marketing and Public Relations Department; thus, the 
research team will not have direct access to any email addresses; 2) A study promotional page 
on the PCOS Challenge website (http://www.pcoschallenge.org/) with additional information 
about the study (i.e. title, description, goals, eligibility criteria, exclusions, etc.); 3) A 120mm x 
240mm banner ad promoting the study on every page of PCOSChallenge.com (>40,000 
pages).  The email message to members, the promotional page PowerPoint presentation and 
the 120mm x 240mm banner ad are included for review. 
 
     The CCTS will further assist in identifying potential study participants using the CRDW–UIC 
CIRCLE as the data source. The data extracted through REDCAP will include age, height, 
weight, laboratory test results, diagnoses associated with PCOS, and contact information (see 
Data Request Authorization Form). The extracted data will subsequently be transferred to an 
Excel spreadsheet that will be stored by the PI in his password protected office computer under 
lock and key.  A letter of introduction to the study will be sent to potentially eligible reproductive-
age women whose data meet the profile for having PCOS, but only after obtaining permission 
from the UIC physicians who care for these women. The letter will indicate that the 
communication was sent with permission from their physician, and will contain study contact 
information to inquire about study participation or to request no further contact.  The data from a 
given individual will initially be identified by medical record number (MRN), and then coded upon 
study enrollment when these individuals are assigned a study number.  The link between an 
individual and the coded data will only be known to the PI.  The coded data set without direct 
identifiers will be stored by the PI in his password protected office computer under lock and key.  
Only the PI and Co-investigators will have access to the coded data set. 
 
     Each volunteer will meet the PI (or his designee) in the UIC Center for Clinical and 
Translational Science’s (CCTS) Clinical Research Center (CRC) to explain the scientific 
rationale, procedures and potential risks of the study, and the rights of the study participant. 
Informed consent will be obtained from each volunteer prior to participation in the study using a 
consent form approved by the UIC IRB.  An electronic note will be written in each participant’s 
medical record regarding the consent, and a copy of the consent form will be given to the 
participant. The original consent form will be kept with the investigator’s study records. 
 
     Eligible volunteers will be informed that participation in the study is entirely voluntary, and 
that they may withdraw their consent at any time. They will also be assured that withdrawal from 


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the study will not affect their on-going care or their relationship with their physician in any way. 
Volunteers who fail the screening process will be promptly notified by telephone that they are 
not eligible to participate in the study.  Data from screen failures will not be included in the 
study. 
 
     An honest and open recruitment process is employed to protect against the possibility of 
coercion or undue influence.  Volunteers are encouraged at all stages to discuss their potential 
participation with trusted advisors including friends, family members, or medical personnel not 
connected with the study.  The aim is to remain objective in the recruitment process to the point 
that volunteers have been excluded in the past on the basis of a perceived failure to reach a 
fully informed state regarding the study.  There will not be any threat of harm or adverse 
consequences if the subject does not agree to participate in the study.  For students or 
employees in particular, they will be reassured that their decision to participate or not to 
participate will not affect their grades (students) or standing in the University or Hospital.  
Furthermore, the information provided during the consent process will be presented in a 
balanced way with equal emphasis on all elements of consent (e.g. there will not be over-
emphasis of benefits and under-emphasis of risks). 
 
6.0 
Study Design and Procedures 
 
     This research will involve the prospective study of 90 reproductive age women (18-40 years) 
with PCOS using a randomized double-blind placebo-controlled study design. The 
randomization schedule will be stratified by body mass index (BMI) (lean: BMI ≥18 but ≤25 
kg/m2; obese: BMI ≥30 but ≤40 kg/m2) and IR status study groups to ensure a balance of 
patients in the two study arms. IR will be defined by the insulin area under the curve from 0-120 
min (insulin AUC0-120) of an oral glucose tolerance test (OGTT) >7,000 which is highly predictive 
and thus, recommended for population assessment of IR in PCOS.35 Forty five (45) of the 
women with PCOS will receive salsalate of which 30 will be lean (15 without IR; 15 with IR) and 
15 will be obese. The other 45 women with PCOS with a similar frequency distribution of BMI 
and IR status will receive placebo. Thus, the study will be comprised of the following 6 groups: 
 
1. Salsalate-treated lean PCOS without IR (n=15)  
       – BMI ≤25 kg/m2 ‒and‒ 2-hour oral glucose tolerance test (OGTT) insulin 
area under the curve (AUC) ≤7,000 
2. Placebo-treated lean PCOS without IR (n=15)   
            – BMI ≤25 kg/m2 ‒ and‒ 2-hour OGTT insulin AUC ≤7,000 
3. Salsalate-treated lean PCOS with IR (n=15)   
            – BMI ≤25 kg/m2 ‒and‒ 2-hour OGTT insulin AUC >7,000  
4. Placebo-treated lean PCOS with IR (n=15)   
            – BMI ≤25 kg/m2 ‒and‒ 2-hour OGTT insulin AUC >7,000                                                                          
5. Salsalate-treated Obese PCOS – BMI>30 kg/m2‒≤40 kg/m2 (n=15) 
6. Placebo-treated Obese PCOS – BMI>30 kg/m2‒≤40 kg/m2 (n=15) 
 
     After the initial pre-screening, volunteers will be asked to come to the CCTS CRC at 912 S. 
Wood Street for a screening visit at ~ 8:00 AM. The screening visit will occur within 1 to 4 weeks 
of beginning the study protocol and will last ~4 hours.  At the start of the screening visit, 
volunteers will undergo informed consent followed by a complete history and physical, vaginal 
sonography, and blood draws for a hormonal evaluation if not performed previously, a complete 
blood count (CBC), a comprehensive metabolic panel (CMP) and an OGTT.  Based on the 
screening results, volunteers deemed eligible for study participation will subsequently enter the 
study protocol beginning between days 3 and 13 following the onset of either a spontaneous or 
progestin-induced withdrawal bleed, or without regard to the onset of menses if vaginal 


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Page 11 of 37                                                                                                                                                  5/9/2022 
sonography within 21 days of study start reveals no evidence of follicular development in the 
face of a negative pregnancy test.   
 
     Visits 1 through 5 will occur during the 
first week of the study protocol.  During 
Visits 1 through 4, subjects will undergo an 
HCG stimulation test (HCG-ST) in the CRC 
consisting of a single IM HCG injection 
during Visit 1 after documenting a negative 
urine pregnancy test, along with a single 
blood draw during all 4 visits at 0, 24, 48 
and 96 hours after HCG administration to 
measure 
serum 
androgens 
and 
17-
hydroxyprogesterone.  Visits 1 through 3 will 
be short lasting ~30 minutes.  After one of 
these short visits, subjects will also undergo 
a body composition assessment consisting 
of 
a 
whole-body 
dual-emission 
X-ray 
absorptiometry 
(DEXA) 
scan 
in 
the 
Integrative Physiology Laboratory (IPL) at 
1640 W. Roosevelt Road, Suite 187, and a 
magnetic resonance image of the abdomen 
in the Center for Magnetic Resonance 
Research at 2242 W. Harrison Street, Suite 
103.  Visit 4 will last ~8 hours during which a 
cream challenge test (CCT) will also be 
performed in the CRC consisting of dairy cream ingestion and serial blood draws at 0, 2, 3 and 
5 hours after cream ingestion to measure inflammation markers.  Visit 5 will last ~7 hours and 
occur 2 days after the CCT during which subjects will undergo a FS-IVGTT in the CRC to 
assess insulin sensitivity and glucose-stimulated inflammation.      
 
     A predefined computer-generated randomization schedule set up by a statistician will have 
centralized allocation and codes secured by a research pharmacist. This schedule will be used 
to assign recruited subjects to receive salsalate (Amneal Pharmaceuticals, Bridgewater, NJ) 3 
grams/day (lean subjects) or 4 grams/day (obese subjects) in 2 divided doses or identical 
placebo for ~12 weeks. Study medication will actually be administered over a range of 11 to 13 
weeks to accommodate scheduling glitches without impacting any anticipated treatment effects. 
Subjects and study personnel will be blinded to the treatment assignment. Serum salicylate 
levels will be measured to assess compliance from a fasting blood sample drawn at baseline 
(Visit 1) and after 4 weeks (Visit 6), 8 weeks (Visit 7) and 12 weeks (Visit 8) of treatment.  
Compliance will be defined as salicylate levels being in the therapeutic range for rheumatology 
practice (10-30 mg/dl) during treatment. A Data and Safety Monitoring Board (DSMB) will keep 
track of salicylate levels to maintain blinding (see Section 6.0).  A fasting lipid profile will also be 
drawn at baseline (Visit 1) and after 12 weeks (Visit 8) of treatment. 
 
     During treatment, all subjects will undergo ovulation monitoring consisting of serum 
progesterone measurements timed by basal body temperatures along with serial follicular 
ultrasonography as indicated.  After the ~12 weeks of treatment, subjects will undergo repeat 
testing consisting of an HCG-ST during Visit 8 through 11, a CCT during Visit 11 and a FS-
Table 1.         PRE-STUDY SCREENING 
Entry 
H&P 
& 
vaginal 
sonography 
for 
ovarian 
morphology; Prolactin, TSH, LH, FSH, total / free T, 
DHEA-S & 17OHP (only if not obtained in previous 6 
months); CBC, CMP & OGTT. 
STUDY PROTOCOL             WEEKS → 
0 4 8 12 
Salicylate Level 
X X X 
X 
Sex Hormone Binding Globulin 
X  
 
X 
Body Composition: MRI & DEXA 
X  
 
 
HCG Stimulation Test: T, A & 17OHP 
@ 0, 24, 48 & 96 hours. 
X  
 
X 
Cream Challenge Test: MNC-derived 
ROS, NFκB, RNA & protein inflammat’n 
markers @ 0, 2, 3 & 5 hours. 
X  
 
X 
Frequently 
Sampled 
Intravenous 
Glucose Tolerance Test: glucose, 
insulin, C-peptide & FFA q 1-30 minutes 
x 34 samples from -20-240 minutes. 
X  
 
X 
Post-Study Safety Tests: CBC & CMP. 
  
 
 
X 
Ovulation Monitoring: BBT from 0-12 weeks; P on 7th 
day of BBT rise; If P >2 ng/dl documented, vaginal 
sonography on day of & day after LH surge. 


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Page 13 of 37                                                                                                                                                  5/9/2022 
                  
• 
A complete medical History and Physical Examination will be performed in the in the CCTS 
CRC. A Pap smear will be performed at the same time only if a volunteer is due for one in 
accordance with ACOG guidelines.  Otherwise, a copy of the most recent Pap smear will be 
requested after obtaining a signed release of medical information from the volunteer.    
 
• 
Vaginal Sonography to assess ovarian morphology will be performed during the initial 
screening in the CCTS CRC on the day of the entry history and physical examination. 
Polycystic ovaries will be defined by published criteria established by Dewailly et al;36 
namely, the presence of 25 or more subcapsular follicles measuring 2-9 mm in diameter or 
increased ovarian volume (>10 cm3). 
 
• 
Body Composition will be assessed at the beginning of the study using 2 modalities: 
 
o Dual Energy X-ray Absorptiometry (DEXA, Model LUNAR iDXA, General Electric 
Healthcare, Wood Dale, IL) will be performed in the IPL at which time anthropometric 
measures of height and weight will be taken using standard techniques. These measures 
are required for the DEXA scan.  All female participants will perform a spot pregnancy 
test prior to DEXA scan. If pregnancy test is positive the participant will be excluded. The 
instrument emits photons at 2 different energy levels as a detection scanner traverses the 
body.  The detected energy passes through the body with differential attenuation that is 
computer analyzed to calculate body fat.  The total fat quantity, and truncal fat quantity 
will be measured, the latter of which is defined as the area between the dome of the 
diaphragm (cephalad limit) and the top of the greater trochanter (caudal limit).37  Fat 
quantity will also be measured in the central abdominal area (R1 area) which is an area 
of 50 cm2 around the central point of the midline between the lateral iliac crests and the 
lowest rib margins at the end of normal expiration (the same midline used for waist 
circumference).38  Study subjects will wear light clothing and lie on their backs during the 
procedure which takes 10-15 minutes to complete.  Subjects will be exposed to a 
radiation dose of ≤0.3 mrem based upon manufacturer’s specifications and calculations 
from Standford Dosimetry, LLC RADAR Medical Procedure Radiation Dose Calculator.  A 
Radiation Safety Committee review is not required for this amount of research protocol 
radiation exposure given the estimated effective equivalent dose is below 100 mrem (1 
mSv) per year, a limit set by the Nuclear Regulatory Commission for “general public” 
exposure. 
  
o Magnetic Resonance Imaging (MRI, Siemens MAGNETOM Trio 3T, Malvern, PA) will be 
performed at the Center for Magnetic Resonance Research, 2242 W. Harrison Street, 
Suite 103. The MRI scanner performs an axial scan in the supine position to measure 
abdominal adiposity as previously described.39 A gradient echo pulse sequence with 
water suppression to obtain a fat-only image is followed by lipid suppression to obtain a 
water-only image and no suppression to serve as an anatomic reference.  Five 4 mm 
slices centered at L3 are obtained and the fat signal intensity ratio [If/(If+Iw)] is calculated 
from the fat-only (If) and water-only (Iw) images on a pixel-by-pixel basis to produce a 
map.  The calibration curve obtained from a previous phantom study is used to convert 
this ratio to a fat percentage value that is used to determine visceral and subcutaneous 
fat volumes with AnalyzeDirect, Inc. software (Stilwell, KS).39    
 
• 
HCG Stimulation Test (HCG-ST):  The test will be performed in the CRC as described by 
Piltonen et al.40 over three (3) short CRC visits.  A baseline blood sample (16 ml) will be 


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drawn after an overnight fast of ~12 hours followed by administration of a single 
intramuscular injection of 5,000 IU HCG (Pregnyl; Schering-Plough, Kenilworth, NJ). Fasting 
blood samples (16 ml each) will again be drawn at 24, 48, and 96 hours after HCG 
administration (please refer to the Risks - Venipuncture / Venous Catheter Blood Sampling 
subsection of Section 7.0 for the approach and alternatives to venipuncture).  Every attempt 
will be made to obtain blood samples on time.  However, the baseline sample may be 
obtained within a fifteen minute window and the post-HCG samples may be obtained within 
a sixty minute window after the scheduled time due to any unforeseen technical difficulty.  
Gathering of vital signs during the single blood draw visits will be considered if there is any 
clinical evidence subject instability.  Plasma will be isolated from the blood samples 
collected and stored at –80o C until assayed for testosterone, androstenedione and 17OHP.  
 
• 
Cream Challenge Test (CCT):  All subjects will consume a standard weight maintenance diet 
consisting of 50% carbohydrate, 35% fat and 15% protein for 3 consecutive days (days 1 – 
3) before the test through meals prepared by the CRC dietician.  The test will be performed 
in the CRC on the day of the 96 hour HCG-ST blood draw after an overnight fast of ~12 
hours as described by Deopurkar et al.41 A polyethylene intravenous catheter will be 
inserted in an arm vein for blood sampling at ~8:30 AM.  The size of the catheter (i.e. 18-23-
guage) will be of suitable caliber for the size of the vein used to gain access (please refer to 
the Risks - Venipuncture / Venous Catheter Blood Sampling subsection of Section 7.0 for 
the approach and alternatives to intravenous catheter placement).  A baseline blood sample 
(90 ml each) will be drawn in the fasting state at ~9:00 AM.  One hundred (100) ml of dairy 
cream (gourmet heavy whipping cream; Land O Lakes Inc, Arden Hills, MN) will then be 
ingested over 10 minutes.  This dairy cream preparation has 33 gm of fat and 1257 kJ (300 
kcal)/100 ml cream.  The saturated fat content of the cream preparation is 70% (28% 
unsaturated fat), the protein content is <2%, and the glucose content is 0%.  Blood samples 
(80 ml each) will again be drawn at 120, 180 and 300 minutes after cream ingestion.  Every 
attempt will be made to insert the intravenous catheter and obtain blood samples on time.  
However, a sample may be obtained within a fifteen minute window after the scheduled time 
due to any unforeseen technical difficulty.  Upon completion of the test, the catheter will be 
removed, and subjects will be fed a study meal for lunch as described above.  Subjects will 
also be discharged with weight maintenance study meals to consume that evening and up 
until they begin the FS-IVGTT.  MNC will be isolated from the blood samples collected and 
used to measure ROS, or placed in culture, or processed for storage at –80oC until used for 
RT-PCR, Western blotting and electrophoretic mobility shift assay (EMSA).  Plasma will also 
be isolated from the same blood samples and stored at –80oC until assayed for cytokine 
levels.   
 
• 
Diet Instruction and Monitoring:  All subjects will receive detailed diet instructions from a UIC 
Department of Medicine registered dietician on the day of the pre-treatment CCT aimed at 
maintaining a similar weight throughout the study. The instructions will focus on having 
subjects consume their habitual pre-study food intake throughout the ~12 weeks of 
treatment.  Subjects will complete a 24 hour food record just before entering the study to 
serve as a guide for the diet instructions.  Subjects will subsequently weigh themselves at 
home on a weekly basis using the same scale to alert the study team if they have lost or 
gained more than 4 pounds compared with their weight at study entry. The registered 
dietician will provide additional counseling to these subjects as needed to determine if any 
diet adjustments are required for weight maintenance.  Subjects will also keep a 24 hour 
food record on a monthly basis that will be reviewed by trained staff from the Nutrition 


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service to assess compliance. Subjects will return the 24 hour food records to the CRC 
when they report for each monthly salicylate level blood draw. 
 
• 
Insulin-Modified FS-IVGTT:  The test will be performed in the CRC two (2) days after the 
CCT after an overnight fast of ~12 hours as described by Polidori et al.32  An intravenous 
polyethylene catheter will be inserted in an forearm vein at ~7:10 AM (-50 minutes) for 
infusing glucose and insulin.  Another intravenous catheter will be inserted in a contralateral 
forearm vein for blood sampling.  The size of the catheter (i.e. 18-23-guage) will be of 
suitable caliber for the size of the vein used to gain access (please refer to the Risks - 
Venipuncture / Venous Catheter Blood Sampling subsection of Section 7.0 for the approach 
and alternatives to intravenous catheter placement). Baseline blood samples will be 
obtained at ~7:40 AM (-20 minutes), ~7:50 AM (−10 minutes), ~7:59 AM (−1 minute) and ~8 
AM (0 minutes).  The average of the four samples is considered the basal level. A glucose 
solution (0.3 gm/kg) will be administered intravenously beginning immediately after the 0 
minute blood draw.  Insulin (20 mU/kg Humulin; Eli Lilly, Indianapolis, IN) delivered in 1 
unit/ml infused over 60 seconds starting at ~8:20 AM (20 minutes).  Blood samples will be 
obtained at -20, −10, −1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 19, 22, 23, 24, 25, 27, 30, 
40, 50, 60, 70, 80, 90, 100, 120, 150, 180 and 240 minutes.  Every attempt will be made to 
insert the intravenous catheters and obtain blood samples on time.  However, all samples 
with the exception of those used to measure glucose may be obtained within a fifteen 
minute window after the scheduled time due to any unforeseen technical difficulty.  A total of 
272 ml of blood (8 ml/sample) will be collected during the entire procedure.  All infusions will 
be stopped at 12 PM.  Upon completion of the test, both catheters will be removed, and 
subjects will be fed a standard lunch.  Plasma will be isolated from the blood samples 
collected and stored at –80o C until assayed for glucose, insulin, C-peptide and FFA.  
Preparation of glucose and insulin for infusion during the FS-IVGTT procedure will be 
performed by the UI Hospital Pharmacy Investigational Drug Services.  
 
• 
Mathematical Model to Assess Hepatic and Extrahepatic Insulin Clearance.  Hepatic and 
peripheral insulin clearances were estimated using a model for insulin kinetics during the 
FS-IVGTT as described in Fig. 3.  
 
 
The model is based on the following main assumptions: 
1. Endogenously secreted insulin enters the portal circulation where it travels to the liver before 
reaching the systemic circulation.  The insulin secretion rate (ISR) will be calculated by 
deconvolution using C-peptide kinetic parameters.42  The calculated ISR and the known 
insulin infusion rate will be used as inputs to the model. 


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2. The rate of delivery of insulin from the systemic circulation to the liver reported in 
pmoles/minute is equal to the plasma insulin concentration reported in pmoles/liter times the 
assumed hepatic plasma flow (HPF) rate (0.576 L/min/m2).43 
3. Insulin clearance occurs in both the liver and in extrahepatic (peripheral) tissues, which 
includes kidney, muscle, and adipose tissue.  Extrahepatic clearance is assumed to be 
proportional to the plasma concentration. 
4. Hepatic clearance will be modeled using either a linear or a saturable function.  Both 
functions will be tested in each subject, and the one providing the best fit will be retained.  
Both functions will be considered because some previous studies have suggested linear 
clearance, whereas others have suggested nonlinear and/or time-dependent clearance.30,43-
45   No time-dependent changes in parameter values will be modeled. 
 
Given these assumptions, the resulting model requires estimating either three (linear model) 
or four (saturable) parameters from the measured plasma insulin profiles and calculated 
ISR. 
 
The following equations and parameters correspond with these main assumptions.  The 
HPF rate used in the equations is the combined plasma flow to the liver from the portal vein 
and the hepatic artery. 
 
 
 
 
 
 
 
 
 
 
 
 
 
P is plasma insulin reported in pmoles/liter, ISR is reported in pmoles/minute, HPF and CLP 
are reported in liters/minute, FEL is hepatic fractional extraction (dimensionless), Vmax is the 
maximal hepatic degradation rate reported in pmoles/minute, and Km is the hepatic insulin 
delivery rate at which 50% of maximal degradation occurs reported in pmoles/minute. 
 
The differential equations for the linear (Equation 1) and saturable (Equation 2) assumptions 
are as follows:  
  
 
 
 
VP is the extrahepatic distribution volume for insulin reported in liters.  Model-identified 
parameters will be normalized by body weight (BW) for comparison across subjects.  For the 
saturable clearance model, fractional hepatic extraction varies with changes in ISR and P.  
FEL values will be calculated in the basal state when compiling parameter summaries. 
 
To provide measures of clearance that are related to analogous measures obtained by other 
experimental methods, Equations 1 and 2 will be used to calculate measures that would be 


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estimated from steady-state insulin infusions administered either intravenously as in 
hyperinsulinemic-euglycemic clamps or in the portal vein as in endogenous secretion. The 
corresponding clearance values from the model for an intravenous infusion (CLIV) or for a 
portal infusion (CLportal) will be calculated by dividing the insulin infusion rate by the steady-
state plasma insulin concentration.  These linear model calculations are summarized as 
follows: 
 
 
 
 
 
For the saturable clearance model, the effective clearance parameters vary with the insulin 
infusion rate, and values will be calculated assuming an infusion rate of 240 pmol/min/m2 as 
commonly used in hyperinsulinemic-euglycemic clamps.  The proportion of total insulin 
degradation occurring in hepatic versus extrahepatic tissues will be calculated by integrating 
the degradation rates over the 240 minute interval. The insulin sensitivity index (SI) will be 
determined from the FS-IVGTT using MINMOD Millenium version 6.02. 
 
• 
Ovulation Monitoring:  All subjects will maintain a basal body temperature (BBT) chart 
throughout the ~12 weeks of treatment after appropriate instructions.  A blood sample (5 ml) 
will be drawn to measure serum progesterone to detect ovulation on the 7th day of 
temperature elevation of at least ¾oF from baseline indicative of a biphasic BBT response.  
Subjects will be instructed to alert the team regarding the potential need to schedule a 
progesterone measurement when 3 consecutive days of temperature elevation is evident.  
The length of the cycle in which a luteal range progesterone (>2 ng/ml) is documented will 
be used to time initiation of midcycle urine luteinizing hormone (LH) monitoring with a 
commercially available kit.  Vaginal sonography will be performed in the CRC on the day of 
the color change signifying the LH surge and the following day to document follicular 
development and collapse, respectively, as the definitive evidence of ovulation.     
 
6.2 Specimen Collection and Storage  
 
• 
Blood specimens will be collected by venipuncture or from a venous catheter placed by a 
trained medical person who will remain at the bedside to monitor the subject continuously.  
 
• 
For screening, a maximum of 22 mL (4½ tsp) will be drawn to measure hormones if not 
performed previously, a complete blood count and a chemistry panel, and 24 mL (5 tsp) will 
be drawn during the screening OGTT.  During the study, we will draw 64 mL (12¾ tsp) 
during each HCG-ST (Visits 1-4 and 8-11), 330 mL (66 tsp) during each CCT (Visits 4 and 
11), and 272 mL (54 tsp) during each FS-IVGTT (Visits 5 and 12).  We will also draw an 
additional 20 mL (4 tsp) to measure salicylate levels (Visits 1, 6, 7 and 8) and fasting lipid 
profiles, and an additional 5 mL (1 tsp) to measure progesterone for ovulation detection as 
needed only if warranted by the BBT response.  
 
• 
MNC Isolation and Processing:  Blood samples are collected in tubes containing Na-EDTA 
as an anticoagulant. A 12 ml sample will be layered over 12 ml of polymorphonuclear cell 
isolation medium (Polymorphprep™, Accurate Chemical and Scientific Corporation, 
Westbury, NY).  Samples will be centrifuged at 450 x g for 30 minutes at 18oC resulting in 
separation of two bands above the red blood cell pellet.  MNC present in the top band will be 
harvested with a Pasteur pipette and washed repeatedly with Hank’s buffered saline solution 
(HBSS) yielding greater than 95% pure MNC.46  A fresh portion of MNC will be reconstituted 


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in HBSS for ROS generation or suspended in RPMI 1640 medium for culture.  A second 
MNC portion will be suspended in RNAlater reagent (Quiagen, Germantown, MD) for 24 
hours at 4oC to stabilize RNA, centrifuged at 6,000 x g for 5 minutes at 4oC to yield a pellet 
that will be stored at –80oC until processing for real time-PCR (RT-PCR).  A third MNC 
portion will be suspended in phosphate buffered saline (PBS – pH 7.4), centrifuged at 
13,000 x g for 2 minutes at 18oC to yield a pellet that will be stored at –80oC until processing 
for Western blotting.  A fourth portion of MNC will be used to prepare nuclear extracts of 
DNA-binding protein with a method described by Andrews et al., and will subsequently be 
stored at –80oC until processing for electrophoretic mobility shift assays.47     
 
• 
Plasma and/or serum will be separated from whole blood through centrifugation and 
aliquoted into 1.5 ml polyproplyene microtubes for storage.  
 
• 
Individual aliquoted MNC, plasma or serum samples will be placed in a box labeled with the 
study ID and stored in a -80°C freezer.  
 
• 
Samples will be labeled with a numbering system for identification of the test result.  The 
numbering system will give each subject a unique identifier, and indicate which study visit 
each sample is from designated as V1, V2, V3, etc. immediately after the subject number. 
• 
Remaining samples will be stored indefinitely future study as needed. 
 
6.3 Study Medication Preparation and Dispensing  
 
• 
A research pharmacist from the Investigational Drug Service (IDS) will prepare the study 
medication consisting of 500 mg capsules of salsalate along with an identical placebo.  The 
PI currently holds an IND for salsalate administration (IND #113845). 
 
• 
A research pharmacist will also prepare all of the clamp infusates.  
 
• 
The study medication, HCG and clamp infusates will all be stored in the IDS and dispensed 
by an IDS research pharmacist when needed for use in the study. 
 
6.4 Protected Health Information (PHI) and Other Data Collected 
 
• 
PHI data will be collected at the time of study enrollment  
 
• 
Subject demographics will be used for descriptive purposes. 
 
• 
Anthropometric data (height and weight) and body composition data from the DEXA scan 
and abdominal MRI will also be used for descriptive purposes. 
 
• 
Diet logs will be collected. 
 
6.5 Data Storage and Management 
 
• 
All research data will be coded using unique identifiers.  Once the subject has been 
assigned a unique identifier, all data will carry this label.  Only the PI will have the original 
identifier information and this information will be kept in a password-protected spreadsheet 
on his encrypted and password protected personal computer or secure departmental server 
in his locked office at UIC located in CMRB 1111. 
 


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• 
All subject visit data will be transcribed from a written data sheet to password protected files 
on an encrypted university computer and the original data sheet will be kept in a subject 
binder.  Subject binders will be kept in a locked cabinet in the PIs locked office. 
 
• 
Study personnel and the PI will have access to the study information. 
 
• 
Information will be kept indefinitely. 
        
6.6 Specimen Analyses – Laboratory Assays  
 
• 
ROS Generation Assay:  Respiratory burst activity of MNC (4 x 105 cells/ml) will be 
measured by detection of superoxide radical via chemiluminescence. A 500μl aliquot of 
MNC (400 cells/μl) is added to duplicate cuvettes containing a spin bar.  The cuvettes are 
placed into a two-channel lumi-aggregometer (Chronolog Corporation, Haverton, PA). 15μl 
of 10mM luminol followed by 1μl of 10mM formylmethionyl leucinyl phenylalenine are added 
to each cuvette. Chemilumunescence is recorded in mV by computer software (Chronolog 
Aggrolink). The gain is adjusted to visualize the curves within range of the displayed graph.  
The peak responses of the duplicate samples recorded to calculate the mean peak.  This 
method was developed by Thusu and Dandona,48 and is similar to that published by Tosi 
and Hamedani.49 In this assay system, superoxide radical release measured by 
chemiluminescence correlates linearly with that measured by the ferricytochrome C method.  
There is also a dose-dependent chemiluminescence inhibition by superoxide dismutase and 
catalase, and diphenylene, a specific inhibitor of NADPH oxidase, the superoxide producing 
enzyme.  The specific inhibitory effect of diphenylene iodonium on NADPH oxidase has 
been established by Hancock and Jones.50  As validated in our laboratory, the variation of 
ROS generation by MNC in humans using this method varies by less than 8% over a 2 week 
period.12,51  
 
• 
NADPH Oxidase p47phox Subunit, p65 NFκB Subunit, IκB, TNFα, IL-6 and IL-1β Real-Time 
PCR (RT-PCR):  MNC are suspended in RNAlater reagent (Quiagen, Germantown, MD) for 
24 hours at 4oC to stabilize RNA, centrifuged at 6,000 x g for 5 minutes at 4oC to yield a 
pellet that is stored at –80o C until processing for RT-PCR.  Total RNA is isolated from MNC 
(~20 mg) using an RNAeasy kit (Quiagen, Germantown, MD).  The isolated total RNA (1 µg) 
is treated with DNase (Life Technologies, Gaithersburg, MD), and reverse-transcribed using 
TaqMan reverse transcription reagents (PE Biosystems, Foster City, CA) and oligo(dT) 
primers according to the manufacturer’s instruction.  Primers and probes for the individual 
inflammation marker genes are selected using PRIMER EXPRESS software (PE 
Biosystems, Foster City, CA).  The internal probes and a 28S rRNA probe are constructed 
as previously described.52 Internal probes are labeled at the 5’-ends with a reporter dye (6’-
carboxyfluorescein), and at the 3’-end with a non-fluorescent quencher; and subsequently 
phosphate-blocked at the 3’-end to prevent extension.  The 5’-end of the 28S rRNA probe is 
labeled with VIC, a proprietary fluorescent dye (PE Biosystems, Foster City, CA).  Each set 
of target primers and probe are co-amplified with 28S rRNA primers and probe.  
Fluorescence is measured at each amplification cycle in an ABI Prism 7700 Sequence 
Detection System (Perkin Elmer/Applied Biosystems, Foster City, CA).  The signal for the 
28S rRNA is used to normalize against differences in RNA isolation and RNA degradation, 
and in the efficiencies of the reverse transcription and PCRs. All samples are run in triplicate 
and quantified by normalizing the inflammation marker mRNA signal with the 28S signal.  
The final quantification is achieved with a relative standard curve.52    
      


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• 
NADPH Oxidase p47phox Subunit, p65 NFκB Subunit and IκB Western Blotting:  As 
previously described,53 MNC cell lysates are prepared by adding 1 ml of boiling lysis buffer 
(1% SDS), 1mM sodium ortho-vanadate, and 10 mM Tris (pH 7.4) to MNC pellets.  Total 
protein concentrations are determined using BCA protein assay (Pierce Chemical Co., 
Rockville, IL).  Total cell lysate (60 mcg) are electrophoresed on 10% gels for p47phox subunit 
and IκB. The proteins are transferred to a polyvinylidine diflouride membrane and incubated 
with a monoclonal antibody against p47phox subunit (Transduction Laboratories Inc., San 
Diego, CA) or a polyclonal antibody against IκB (Rockland, Gilbertsville, PA). The 
membrane is washed and developed using super signal chemiluminescence reagent (Pierce 
Chemical Co.).  Densitometry is performed using Carestream Molecular Imaging software 
version 5.0.2.30 (Rochester, NY).    
 
• 
NFκB Electrophoretic Mobility Shift Assay (EMSA):  As previously described,46,47 DNA-
binding protein extracts are prepared from MNC pellets, and total protein concentrations are 
determined using BCA protein assay (Pierce Chemical Co., Rockville, IL).  The NFκB gel 
retardation assay is performed using an NFκB-binding protein detection kit (Life 
Technologies, Inc. Long Island, NY).  The double-stranded oligonucleotide containing a 
tandem repeat of the consensus sequence for the NFκB-binding site is radiolabeled with γ-
P32 by T4 kinase.  The nuclear extract (5 μg) is mixed with incubation buffer and 
preincubated at 4oC for 15 minutes.  Labeled oligonucleotide (60,000 cpm) is added, and the 
mixture is incubated at room temperature for 20 minutes.  Samples are electrophoresed on 
a 6% nondenaturing polyacrylamide gel that is dried under vacuum and exposed to x-ray 
film.  Densitometry is performed as described above. 
 
• 
MNC Culture:  A blood sample will be drawn at 0 and 120 minutes of the CCT for MNC 
isolation. Whole blood will be diluted 1:2 with PBS (pH 7.4) and underlayered with 
histopaque-1077 for density gradient centrifugation (400g for 45 minutes) as previously 
described.54  The pellet is obtained from the last wash and resuspended in RPMI 1640 
culture medium (2mM L-glutamine, 100 U/ml penicillin, 100 ug/ml streptomycin, 90 mg/dl 
glucose) at a final concentration of 5X106 cells/ml. MNC isolated from the pre-CCT fasting 
blood sample will be cultured in medium supplemented with palmitate bound to fatty acid-
free bovine albumin (4%) at varying concentrations spanning the physiological 
postabsorptive to pathophysiological range (0.4, 0.8, 1.2 and 2.0 mM), in the presence and 
absence of 1 ng/ml of lipopolysaccharide (LPS) endotoxin (Sigma, St Louis, MO). MNC 
isolated from the 120 minute CCT blood sample will be cultured without FFA 
supplementation in the presence and absence of 1 ng/ml of LPS.  After a 24 hour incubation 
(5% CO2 at 37o C), MNC supernatants will be collected and stored at –80o C until assayed.  
 
• 
Plasma Measurements:  All assays will be performed in the Reproductive Endocrine and 
Inflammation Laboratory except those for salicylate and progesterone which will be 
measured by the Clinical Chemistry Laboratory at IU Hospital.  Glucose concentrations will 
be measured by the glucose oxidase method (YSI, Yellow Springs, OH) while plasma insulin 
concentrations will be measured by direct double antibody RIA (Linco Research, St. 
Charles, MO).  C-peptide will be measured by a 2-site immunenzymatic sandwich assay 
(Roche Diagnostics, Indianapolis, IN).  FFA will be measured by an enzymatic colorimetric 
assay (Wako Diagnostics, Mountain View, CA).  TNFα, IL-6 and IL-1β levels in plasma and 
in MNC culture supernatants will be measured by ELISA (Quantikine, R&D Systems, 
Minneapolis, MN).  CRP levels will be measured by a high sensitivity ELISA (Alpha 
Diagnostics International, San Antonio, TX).  Levels of LH, androstenedione, testosterone, 
DHEA-S, 17OH-progesterone and progesterone will be measured in duplicate by RIA (MP 


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Biomedicals LLC, Solon, OH).  Sex Hormone Binding Globulin will be measured by 
electrochemiluminescence (Mayo Clinic, Rochester, MN).  To reduce interassay variability, 
all samples from an individual subject will be measured in the same assay at the end of the 
study. 
 
7.0 
Expected Risks/Benefits 
 
Risks 
 
• 
Venipuncture / Venous Catheter Blood Sampling – For screening, we will draw a 
maximum of 22 mL (4½ tsp) to measure hormones if not performed previously, a complete 
blood count and a chemistry panel, and 24 mL (5 tsp) during the screening OGTT.  During 
the study, we will draw 64 mL (12¾ tsp) during each HCG-ST, 330 mL (66 tsp) during each 
CCT, and 272 mL (54 tsp) during each FS-IVGTT. We will also draw an additional 20 mL (4 
tsp) to measure salicylate levels and fasting lipid profiles, and an additional 5 mL (1 tsp) to 
measure progesterone for ovulation detection only if warranted by the BBT response. 
 
     The Study Visit Blood Sampling Plan included as an Appendix provides a detailed 
accounting of the volume of blood that will be drawn from each subject during the entire 
study.  These flow sheets provide a detailed breakdown of the tubes that will be used, the 
volumes that will be drawn, and the types of measurements that will be performed.  The 
Laboratory Flow Charts also included as an Appendix illustrates how the collected blood will 
be utilized for molecular work and cell culture in the PI’s laboratory to effectively generate 
key data to address Aims of the study.   
 
     The total amount of blood that will be drawn on each subject during screening and the 
HCG-ST, CCT and the FS-IVGTT is expected to be <1,500 ml consisting of no more than 
750 ml obtained ~12 weeks apart which again is a longer interval than the typical clinical 
practice standard for blood donations.  All subjects will be screened for blood count.  The 
decision to proceed with study participation will be reviewed on a case-by-case basis after 
taking into consideration the health status of the individual if the pre-study screen reveals a 
hemoglobin <11.6 grams; or in the case of a subject who weighs <70.8 kg, if the pre-study 
hemoglobin is <12.6 grams.  In the latter case, blood sampling will be limited to 10.5 ml/kg 
within an 8 week period.  If the complete blood count (CBC) performed during initial 
screening reveals evidence that low hemoglobin is due to iron deficiency anemia in an 
otherwise eligible volunteer, iron supplementation (ferrous sulphate 325 mg twice daily) will 
be recommended and a CBC will be performed at appropriate intervals until higher 
acceptable hemoglobin meeting the above criteria is documented before the volunteer 
enters the protocol. 
 
     The risk of developing anemia due to blood draws during the study is uncommon but still 
possible in subjects with a pre-study hemoglobin at the lower limit of study eligibility (<12.0 
grams, or <13.0 grams for weight <70.8 kg).  Subjects in this circumstance will be advised to 
take iron during the study (ferrous sulphate 325 mg twice daily) as a precaution  Additional 
iron supplementation may be recommended as appropriate if a complete blood count (CBC) 
performed at the end of the study reveals a low hemoglobin.  
 
     The potential risks to the subjects associated with venipuncture needle sticks or IV 
catheter insertions include discomfort, possible bleeding/hematoma (~1/1000) at the site, 
rarely an infection, and uncommonly feeling faint from the procedure.  These risks will be 
minimized by allowing only experienced nurses or doctors to insert intravenous lines.  From 


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time to time, difficulty may be encountered in performing venipuncture or inserting an 
intravenous catheter even after several attempts.  In this instance, a subject may be 
escorted by a physician or a research nurse from the CRC to the Imaging and Diagnostics 
Services Short Stay Unit located in Suite 2600 on the 2nd floor of UI Heath Hospital at 1740 
W. Taylor Street immediately across the skyway adjacent to the CRC, where a member of 
the IV-PICC Team will perform the venipuncture or insert the intravenous catheter using 
ultrasound guidance at no additional risk.  The subject will subsequently be escorted back to 
the CRC to complete the particular study visit. Use of the IV-PICC Team may extend the 
CRC study visit by up to 2 hours.   
 
     The studies will be performed on the CCTS CRC, which provides staff that assists the 
P.I.  Furthermore, the P.I. has staff trained and dedicated to performing these studies.  All 
catheters will be inserted with meticulous use of sterile technique to avoid infection and 
great care will be taken to minimize any discomfort or complications associated with 
insertion.  The catheters will be kept patent with heparinized saline to prevent clotting.  
 
• 
CCT, OGTT and HCG-ST – The major risk associated with these tests is insertion of an 
intravenous catheter for blood drawing as discussed above.  Glucose ingestion during the 
OGTT may cause abdominal discomfort, nausea, increased blood pressure, flushing or 
sweating.  Dairy cream ingestion may cause urticaria, pruritus and in rare cases anaphylaxis 
in individuals with an unknown dairy allergy; and much less often may cause nausea, 
cramps, flatulence, bloating or diarrhea in individuals with previously unknown lactose 
intolerance. 
 
• 
FS-IVGTT – The major risk of this test is hypoglycemia associated with the insulin infusion 
along with the risk associated with insertion of intravenous catheters for the infusions and 
blood drawing as discussed above.  The frequent glucose measurements will allow prompt 
detection of hypoglycemia, in which case the test will be stopped and the intravenous 
glucose infusion will be increased or glucose will be given orally for resolution.   
 
• 
DEXA scans involve a very small amount of radiation. One of the risks associated with 
radiation exposure is cancer. The natural incidence of fatal cancer in the U.S. is about 1 
chance in 5.  Everyday radiation exposure from natural occurring background radiation (sun, 
radon exposure in the home) is approximately 100 mrem (1 mSv) per year.  Each subject in 
this study will receive a whole body DEXA scan.  The radiation dose of one whole body 
DEXA scan is approximately ≤0.3 mrem.  This amount of radiation is very low as to make an 
accurate risk estimate meaningless. A negative urine pregnancy test must be documented 
prior to undergoing the DEXA scan. 
 
• 
HCG Administration – HCG use can cause the following adverse effects:  edema, 
depression, fatigue, headache, irritability, restlessness, ovarian cyst rupture, reaction or pain 
at the injection site, or a local or systemic hypersensitivity reaction.   
 
     The risk of cyst rupture will be minimized by administering the HCG between days 3 and 
13 following the onset of either a spontaneous or progestin-induced withdrawal bleed, or 
without regard to the onset of menses if vaginal sonography within 21 days of HCG 
administration reveals no evidence of follicle cyst development in the face of a negative 
pregnancy test.   
 


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• 
Salsalate Treatment – Salsalate use can cause the following adverse effects characteristic 
of salicylates in descending order of frequency:  tinnitus, nausea, hearing impairment, rash 
and vertigo.  These effects are transient, and resolve upon cessation of salsalate.  Although 
cause and effect has not been established, the following less common adverse effects have 
also been reported:  abdominal pain, abnormal hepatic function, anaphylactic shock, 
angioedema, bronchospasm, decreased creatinine clearance, diarrhea, gastrointestinal 
irritation, inflammation, bleeding or perforation, hepatitis, hypotension, nephritis, urticaria, 
myocardial infarction or cerebral vascular accident.  
 
     Tinnitus is an expected side effect of salsalate treatment that occurs more frequently at 
doses higher than the one proposed for this study. To minimize this side effect, subjects will 
be advised to begin by taking one capsule (i.e. 500 mg of salsalate in subjects receiving 
salsalate) the first day and increase the dose in one capsule increments every 3 days 
thereafter until the desired dose is achieved.  If tinnitus occurs at any point in the upward 
dose titration or later on during the treatment, an affected subject will be advised to reduce 
the daily study medication dose in stepped one capsule increments every 3 days.  In a 
previous study, this approach effectively resolved symptoms enabling subjects to complete 
the study while still maintaining therapeutic salicylate levels.  The nature of any suspicious 
symptoms reported by a subject will be reviewed to determine whether there is actual 
tinnitus before the salsalate dose is reduced in the manner described.  Vital signs and 
potential side effects will be monitored every 4 weeks during treatment when subjects report 
to the CRC to have their blood draw for a salicylate level.  Liver function tests, BUN and 
creatinine levels will be drawn within 4 weeks after study completion to be certain that no 
lasting untoward hepatic or renal impairment has occurred.   
 
• 
Reproductive Risk – Pregnancy is an exclusion criterion for this study.  The risks listed 
above would apply equally to a developing fetus as well as the primary participant.  Potential 
risks of specific impact to a developing fetus include the small amount of radiation exposure 
received from the DEXA scan (point 4 above).  A negative urine pregnancy test must be 
documented prior to undergoing the DEXA scan.  Salsalate is a Category C drug for 
pregnancy and use of double barrier contraception will be required during the study unless 
either the participant or her partner have already undergone permanent sterilization. 
 
• 
Loss of confidentiality is a risk as with all human subject-based medical research owing to 
disclosure of health information. These disclosures may be mandated by local or national 
regulatory agencies (i.e. IRB, Food and Drug Administration (FDA)) or may be unintentional.  
Safeguards regarding both physical and electronic copies of all subject data are in place, as 
dictated by the current standards of UIC and UI Health. 
 
Except for de-identified research data, all other information relevant to patients collected in 
the study will be kept confidential, including locked storage of all paperwork, and secure 
digital storage on a database to which only members of the research group have access.  
The security and integrity of this database are maintained with the direct assistance and 
oversight of UI Health Information Technology support teams, and the database is served 
centrally from a closely monitored, regularly backed up server. 
 
• 
Unforeseen Risks – There may be risks or side effects related to the study that are 
unknown at this time. 
 


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Benefits 
 
• 
Subjects will receive individual body composition results as well as diabetes risk information 
from the OGTT.  Based on the results of a small pilot study that treated women with PCOS 
with salsalate, it is possible that subjects receiving salsalate may experience a reduction in 
circulating androgens and ovulation.  However, because individuals respond differently to 
therapy, it is not known in advanced if these potential benefits will occur.  There are no 
anticipated benefits to subjects receiving placebo.    
 
The knowledge to be gained from this research may be beneficial to other patients, society 
or science.   The results will guide the design of future studies aimed at determining what 
dietary modifications, medical regimen or both will optimally attenuate inflammation in PCOS 
with the goal of reducing medical disease and enhancing fertility.  In particular, the findings 
may help define the therapeutic molecular inflammation target to develop the most ideal 
anti-inflammatory agent. 
 
 
8.0 
 Data Collection and Management Procedures 
 
• 
Data will be collected by the PI, Co-I or an authorized member of the research team and 
coded to the study number assigned to each subject.  All electronic data will be secured on 
the PIs encrypted and password protected computer until it can be moved to a secure UIC 
data server for long-term storage. Hard copy data reporting forms will be stored in a locked 
file cabinet until it can be scanned to electronic copy and archived onto the UIC data server.  
Once files are converted the original hard copy will be kept until the study is closed, at which 
time the documents will be destroyed. In the event that a discrepency or error is found in the 
source data, the PI will determine if the data can be collected again/procedure repeated in a 
subsequent study visit or alternative visit provided this will not disrupt the integrity of the 
data.  Should this occur, the event will be documented and reported to the IRB.  Access to 
the data will be limited to the PI and IRB-authorized members of his research team.  The PI 
will examine safety and review all data on an ongoing basis. Source data will be collected in 
data reporting forms of both electronic and written form. The PI will review these forms and 
data at the completion of each subject visit to ensure both accuracy and completeness.   
 
9.0 
Data Analysis 
 
• 
Data will be analyzed according to the plan for statistical analysis presented below. 
 
10.0 Quality Control and Quality Assurance 
 
• 
The PI will evaluate the data for adherence with the protocol and for accuracy in relation to 
source documents. 
• 
The PI is responsible for the evaluation of data quality and will review the data in an ongoing 
basis. 
 
11.0 Data and Safety Monitoring 
 
• 
The IRB will be responsible for human subject protection oversight through annual periodic 
review of research involving human subjects. All personnel participating in research 


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activities will meet UIC and OHRP IRB requirements for human subjects research, including 
completion of an approved CITI human subjects training (or institutional equivalent) and 
refresher courses at required intervals. 
• 
Each year, the PI will submit a progress report to the UIC IRB, which will review progress 
and re-approve the protocol. As part of these reviews, the PI will report how many subjects 
have been recruited, the outcomes to date and if there have or have not been any non-
serious adverse reactions reported by the study participants. 
• 
Various aspects of the study will be monitored as follows:  data quality, subject recruitment, 
accrual and retention, adverse events, assessment of scientific reports or therapeutic 
development, results of related studies that impact subject safety, and procedures designed 
to protect the privacy of subjects. 
• 
A Data and Safety Monitoring Board (DSMB) will monitor human subject safety, protocol 
compliance and data integrity. The DSMB will consist of three experienced physicians / 
nurses who as a group have a good clinical and investigative understanding of the study 
protocol based on their backgrounds (Dr. Uzma Syed, Assistant Professor of Clinical 
Medicine, Division of Endocrinology and Metabolism; Dr. William Kobak, Associate 
Professor of Clinical Obstetrics and Gynecology; and Dr. Eileen G. Collins, Professor of 
Nursing). The DSMB will be chaired by Dr. Syed who has an excellent knowledge base of 
endocrine and metabolic research and DSMB oversight. Dr. Syed will also be the medical 
safety monitor.  DSMB members will not be directly involved in the study. 
 
Initial Meeting:  The DSMB will meet prior to initiation of the study to review the protocol, 
resolve queries, and approve the research protocol and informed consent document. 
 
Interim Meetings:  In open session, the DSMB will meet at least twice a year to review the 
ongoing recruitment process, participant withdrawal and protocol deviations.  It will review 
any study protocol amendments and follow the continuing review process. It will review 
participant safety and discuss any serious adverse events that may occur, including the 
relationship of adverse events to study intervention.  It will review the study protocol to 
determine if any protocol revisions are merited as suggested by interim results, adverse 
events, or external events such as new FDA recommendations.  It will review the informed 
consent document and confirm consistency with risks of participation to determine if any 
consent document revisions are merited.  It will discuss any issues raised by the clinical 
study team. At the conclusion of each meeting, one of the following dispositions will be 
recommend: continue the study without changes, continue the study with specified changes, 
place the study on Clinical Hold pending further discussion or investigation, or terminate the 
study. 
 
Key Features to be Assessed:  Monthly salicylate levels measured at Visits 1, 6, 7 and 8 by 
Labcorp Clinical Laboratories will eventually be used to assess compliance after the study is 
unblinded upon its completion to accurately interpret the study data, but will not be used for 
clinical management.   To avoid blinding bias, the monthly salicylate levels will not be posted 
in a subject’s electronic medical record.  Instead, DSMB members under Dr. Syed’s 
direction will prospectively receive copies of these salicylate levels for safekeeping.  
Development of tinnitus during salsalate use will be used to assess salicylate toxicity 
clinically as in a previous large multicenter trial.19  As an additional precaution, salsalate will 
be prepared as 500 mg capsules and identical placebo capsules so the blinding will not be 
jeopardized when changing the dose in one capsule increments to manage tinnitus. 
 


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Operating Procedures:  The DSMB will convene after 5 participants have completed the 
study protocol or 6 months following completion of the first participant (whichever occurs 
first); and thereafter at least twice a year.  If there is a concern about serious adverse 
events, more frequent meetings may be called by vote.  If unexpected and/or serious 
adverse events occur disproportionately to what may be anticipated, the study will be placed 
on clinical hold and the IRB will be consulted for guidance. 
 
Minutes will be taken by the Research Nurse Study Coordinator and forwarded to the DSMB 
chair for review.  The final version of the minutes will be sent to all DSMB members for 
approval.  A formal report from the chair, following approval by the DSMB members will be 
supplied to the IRB within six weeks of each meeting.  Each report will conclude with a 
recommendation selecting one of the following dispositions:  continue the study without 
changes, continue the study with specified changes, place the study on clinical hold pending 
further discussion or investigation, or terminate the study. In the case of clinical hold or 
study termination, the report will be disseminated to the research team as rapidly as 
possible. 
 
Interim Reporting:  The PI will provide Interim data reports to the DSMB. Types of reports for 
each meeting include summaries of the following: baseline clinical and demographic 
characteristics of study participants, the recruitment process, including numbers screened, 
numbers enrolled, withdrawals, adherence and deviation from the study protocol. Any 
adverse events, including serious, study-related adverse events, will be evaluated by the 
DSMB.  In particular, they will be concerned with adverse events that may affect continued 
study participation.  Specific items to be reported will include: tinnitus during study 
medication treatment, hypoglycemia during an OGTT or FS-IVGTT, allergic reactions to 
study medications or any other serious adverse events. 
 
• 
Anticipated risks/adverse events noted in the consent form will be handled as follows: 
 
Risks and/or 
Anticipated 
Adverse Events 
Assessment 
Measures 
Individual 
doing 
Assessmen
t 
Assessment 
Intervals or 
Frequency 
Interventions to 
Decrease or 
Respond to Risks 
Venipuncture 
 
DEXA scans 
Patient 
observation 
CRC Staff 
During the 
procedure 
Verbal 
explanation; 
Provide privacy; 
Performed by 
qualified, trained 
personnel. 
Venous access 
and blood draws  
Patient  
observation 
CRC R.N. 
During the 
procedure 
Performed by M.D. 
or R.N.; Provide 
complete 
explanation and 
expectations 
before the 
procedure, both 
verbally and in the 
consent form.  
Pain 
Patient 
observation 
and 
communication 
CRC R.N. 
During the 
procedure 
Tylenol as 
ordered; Explain 
procedure and 
rationale to patient. 


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Infection 
Patient 
observation 
and vital signs 
CRC R.N. 
During the 
procedure 
Blood draws 
performed under 
sterile technique; 
Instruct patient 
regarding infection 
signs and 
symptoms and 
importance of 
reporting these to 
the M.D.; Provide 
M.D. and CRC 
telephone number 
in the event of a 
problem. 
Bleeding, bruising 
or anemia 
Patient 
observation 
and 
communication 
CRC R.N. 
During 
procedure 
Careful closure of 
stopcocks 
between blood 
draws; Meet 
hemoglobin IRB 
requirements. 
Thrombosis 
Observe blood 
flow during 
draws 
CRC R.N. 
Every blood 
draw to check 
patency of 
lines. 
O.9% NS infusion 
@ 30cc/hr 
throughout the 
test. 
Hypoglycemia 
during FS-IVGTT 
infusion of insulin. 
Patient 
observation 
and frequent 
glucose 
measurements 
CRC R.N. 
and 
Research 
Nurse Study 
Coordinator 
Every 1-10 
minutes during 
the initial 1½ 
hours of the 
FS-IVGTT 
starting 20 
minutes before 
the test, then 
every 20-30 
minutes for the 
next 1½ hours. 
Cessation of the 
infusions and 
intravenous 
glucose 
administration if 
patient becomes 
symptomatic (i.e. 
headache, 
shakiness, visual 
changes and 
palpitations) or 
glucose level falls 
below 70 mg/dl. 
 
• 
A participant will be monitored continuously during an entire CRC visit to undergo study 
procedures, and the PI will be present within the hospital and available by telephone, pager 
or e-mail throughout each visit. In the situation of an anticipated or an unanticipated Adverse 
Event (AE), a standard IRB Adverse Event Report Form will be sent to the CRC and IRB in 
a timely manner.  This report will include a full description of the event, including the 
relationship of the AE as not related, possibly related, or definitely related to the study 
procedure.  The PI, in consultation with the DSMB, will grade adverse events severity as 
follows (as recommended by the National Cancer Institute): 
               0 – “No adverse event or within normal limits or not clinically significant” 
               1 – “Mild AE, did not require treatment” 
               2 – “Moderate AE, resolved with treatment”     
               3 – “Severe AE, resulted in inability to carry on normal activities and required 


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                     professional medical attention” 
               4 – “Life threatening or disabling AE” 
5 – “Fatal AE” 
 
• 
A participant’s study will be stopped in the event she requests not to continue, or if she has 
a reaction to any procedure, the ingestion of the glucose beverage during the OGTT, the 
HCG injection, dairy cream ingestion during the CCT, difficulty obtaining a peripheral 
intravenous line, administration of salsalate (i.e. allergies, persistent or serious side effects, 
unusual anxiety).  The participant’s treating physician will attend to any serious adverse 
events that may occur until they have resolved or refer the participant to the appropriate 
provider as needed.  A participant will also be asked to stop the study if she is not compliant 
with the requirements of the study protocol.  All participants will be assessed for side effects 
at the end of the study.  Study discussions will be documented within each participant’s 
study binder.  Follow-up with the IRB or CCTS CRC will also be noted, and copies will be 
kept of all correspondence.  
• 
If a participant withdraws from the study, she will be replaced by a different participant to 
achieve accrual of 90 participants to complete the study.  Additional reasons for withdrawal 
may include but are not limited to a family emergency, a new medical diagnosis, participant 
preference or development of any persistent study medication side effects that do not 
resolve with common clinical measures in a timely fashion.  The IRB and CCTS CRC will be 
notified during an annual continuing review of subject accrual to date, and any withdrawals 
or dropouts. 
• 
If a serious adverse event occurs during the study, the PI will promptly report the event to 
the IRB (in accordance with UIC OPRS Prompt Reporting policy) and to the study DSMB for 
review and possible action.  In the unlikely event of any serious adverse reactions occurring 
during this study, the DSMB will also provide appropriate clinical expertise for advice and 
assistance.  The study will be completely suspended in the event of death of a subject.    
• 
The PI will keep abreast of scientific reports or therapeutic development, and results of 
related studies that impact subject safety; and will inform subjects immediately of new 
information from these reports or studies that change the risk/benefit ratio for study 
participation.  This type of information will also be reported to the DSMB and to the IRB 
during an annual continuing review. 
 
12.0 Statistical Considerations 
 
12.1 Data Analysis 
 
• 
OGTT Dynamic Responses:  The glucose and insulin responses during the OGTT is defined 
as the area under the curve (AUC) created by plotting the entire sequence of glucose or 
insulin values and calculated using the trapezoidal rule.55 
 
• 
HCG-stimulated Dynamic Responses: The testosterone, androstenedione and 17-
hydroxtprogesterone responses during the HCG-ST is defined as the area under the curve 
(AUC) created by plotting the entire sequence of each of these hormone values, and 
calculated using the trapezoidal rule.56 
 
• 
Lipid-stimulated Responses:  Responses of the various inflammation markers from MNC or 
plasma will be defined as the difference between the maximum lipid-stimulated value and 
the baseline basal value. If baseline basal values exhibit considerable intersubject 


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variability, the percent (%) change will be calculated defined as follows:  (maximum lipid-
stimulated value – baseline basal value / baseline basal value) x 100. 
 
• 
Insulin Sensitivity: The calculated fractional glucose disappearance per insulin 
concentration unit adjusted for insulin clearance will be determined during a FS-
IVGTT as a measure of peripheral insulin sensitivity (SI). 
 
12.2 Power Analysis   
 
     We do not expect >20% dropout based on previous studies.19,20  Thus, we do not anticipate 
any substantial bias in the results. Based on the high cost of adding a patient, recruitment will 
continue until 90 pre- and post-evaluations are completed up to a maximum of 113 patients 
rather than increasing the overall sample size up front.  
 
Aim 1:  The primary outcome for Aim 1 is HCG-stimulated testosterone AUC. Our salsalate 
treatment preliminary data sample size (n=6) is too small to calculate power for the main 
outcome.  As there are three groups (lean non-IR, lean IR, obese), the three contrasts of 
interest will be comparing the mean difference in HCG-stimulated testosterone AUC between 
the salsalate and placebo arms within each study group (i.e. salsalate-treated lean non-IR vs 
placebo lean non-IR, salsalate-treated lean IR vs placebo lean IR, and salsalate-treated obese 
vs placebo obese).  To keep the familywise type I error at 0.05, we will use a Sidak adjustment 
and set α=0.017 for each contrast for the primary outcome.   
 
     There are no previous studies in women with PCOS that address any of the outcome 
variables related to salsalate treatment.  Thus, there is no pilot data of salsalate vs. placebo 
from which to base our power calculations.  However, our preliminary baseline HCG-ST data 
revealed similar results in women with PCOS or controls regardless of BMI.  The HCG-
stimulated testosterone AUC (ng/dl x 96 hr) in women with PCOS (n=17) compared with 
ovulatory controls (n=16) revealed a mean (±SD) of 7370±2481 vs. 3659±959 (p<0.0001, effect 
size [ES] =1.8).  Furthermore, previous data gathered in women with PCOS (n=12) evaluating 
HCG-stimulated testosterone AUC in response to treatment with an insulin sensitizer revealed a 
significant (p<0.04) decrease in the mean (±SD) from 4395±1294 (pre) to 3288±1259 (post).57 
   
     Our preliminary data presented above revealed an ES of 1.8 for the mean change in the 
PCOS group vs. controls.  We expect that the post-salsalate treatment HCG-stimulated 
testosterone AUC will be similar to controls.  The proposed sample size of 90 patients (15 
salsalate and 15 placebo for each study group), will provide 90% power to detect an ES ≥1.4 
(80% power to detect an ES ≥1.24), based on a two-sided two-sample t-test at 1.7% 
significance to account for multiple comparisons which will yield similar power to our proposed 
GLM model with a Sidak adjustment to control type I error level.  We used the pre- and post-
treatment HCG-stimulated testosterone AUC data above136 to obtain a reasonable estimate for 
common SD of change for each treatment group. A post-treatment decrease of 30% (7672 - 
0.3(7672)) in the salsalate-treated group and a post-placebo decrease of 10% (7672 - 
0.1(7672)) in the placebo group with common standard deviation of 990 (assuming a correlation 
of 0.7 between pre/post measures) will yield an ES of 1.55.  This analysis is indicative of 
adequate power to detect a difference in our primary outcome.  
 
     The secondary outcome for Aim 1 is insulin sensitivity (SI).  Our primary comparison will be 
change in SI between the salsalate and placebo groups in insulin-resistant subjects (lean IR and 
obese) as we do not anticipate a change in lean non-IR subjects.  Previous data gathered in 
another insulin resistant population (n=9) evaluating insulin sensitivity in response to salicylate 


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treatment revealed a significant (p<0.04) increase in the mean (±SD) insulin sensitivity from 
4.0±2.7 (pre) to 5.9±1.8 (post).23  These data indicate that a sample size of 30 subjects per 
group is sufficient to achieve 80% power to detect an ES ≥1.74 for salsalate vs. placebo based 
on a two-sided two-sample t-test at a 0.05 alpha level to account for multiple comparisons again 
yielding similar power to our GLM model.   
 
     This study is not powered to compare ovulation rates among the separate groups.  Thus, 
ovulation rate post-treatment based on detection of luteal range progesterone elevations guided 
by BBTs will be compared between the salsalate and placebo groups overall.  Based on the 
inclusion criteria, patients will be anovulatory pre-treatment, thus only post-treatment rates will 
be compared.  With a sample size of 45 in each treatment group, if we expect approximately 
10% to ovulate in the placebo arm, we will have over 80% power to detect a statistically 
significant difference if ovulation increases by 40% in the salsalate-treated group based on 
Fisher’s exact test with type I error set at 0.05. 
 
Aim 2:  The primary outcome for Aim 2 is lipid-stimulated NFκB EMSA density (i.e. change from 
baseline). The three contrasts of interest will be comparing the mean difference in lipid-
stimulated NFκB EMSA density between the salsalate and placebo group within each study 
group (i.e. salsalate-treated lean IR vs placebo lean IR, salsalate-treated lean non IR vs placebo 
lean non IR, and salsalate-treated obese vs placebo obese). Our preliminary baseline CCT data 
revealed similar results in women with PCOS and obese controls.11 The lipid-stimulated NFκB 
EMSA density in women with PCOS (n=17) compared with lean controls (n=8) revealed a mean 
(±SD) % change of 20.1±10.4 vs. -3.0±9.1 (p<0.0001, ES=1.9). The secondary outcomes for 
Aim 2 are glucose-stimulated NFκB EMSA density and lipid-stimulated ROS generation (i.e. 
change from baseline). For women with PCOS compared with lean controls, our previously 
published baseline OGTT data showed a mean (±SD) % change of 34.6±45.7 vs. -21.6±35.6 
(p<0.04, ES=1.4),13 and our baseline preliminary CCT data showed a mean (±SD) % change of 
92±38 vs. -7±22 in ROS generation (p<0.0001, ES=3.8). For each of these outcomes, we will 
use a similar GLM framework as described in Aim 1 with the same three contrasts of interest. 
We will use a Sidak adjustment to control the type I error for each outcome. We expect post-
salsalate treatment measures in PCOS will be similar to lean controls. Based on a two-sided 
two-sample t-test with a 0.017 α level to account for multiple comparisons which will yield similar 
power to our GLM model, we will have 80% power to detect an ES ≥ 1.24. Thus, our study is 
sufficiently powered for these outcome measures. 
 
     Based on baseline PCOS vs. control data and using the approach described to analyze the 
primary and secondary outcomes, there is also 80% power to detect an ES ≥ 1.24 for lipid-
stimulated p47phox WB density (16.4±10.9 vs. -6.4±6.3 [p<0.0001, ES=2.6]) and lipid-stimulated 
MNC-derived TNFα release (8.4±11.8 vs. -7.4±9.4 [p<0.008, ES=1.5]). These 2 variables and 
those for which we lack preliminary data to estimate power; (i.e. salsalate-treated vs. placebo 
lipid- and glucose-stimulated p47phox, p65 NFκB and TNFα mRNA content, and plasma CRP 
and TNFα) are exploratory outcomes key to providing means and SDs to plan larger studies. 
 
12.3 Statistical Analysis   
 
     Initially, a full descriptive analysis of the data will be performed. Frequency distributions will 
be computed for all raw data and for change from baseline responses to lipid or glucose 
stimulation. Summary statistics will be provided by treatment group (salsalate, placebo) overall 
as well as by treatment group and study group (lean non-IR, lean IR, obese).   
 


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i. 
Baseline data such as age, BMI, androgens, insulin sensitivity, total body fat and visceral fat 
will be compared between groups (salsalate: lean non-IR, lean IR, obese; placebo: lean 
non-IR, lean IR, obese) using analysis of variance (ANOVA) for normally distributed data or 
Kruskal-Wallis test for non-normal data to validate similarity of characteristics within each 
study group across the 2 treatment arms. If there is statistical significance, multiple 
comparisons between groups will be performed using a Sidak adjustment.   
 
ii. The primary outcome for Aim 1 is a difference in HCG-stimulated testosterone AUC between 
salsalate vs. placebo. To test our hypothesis, we will use a GLM framework with a 
dependent variable of post-treatment HCG-stimulated testosterone AUC including 
covariates for pre-treatment HCG-stimulated testosterone AUC, treatment indicator, study 
group (lean non-IR, lean IR, obese) and the treatment by study group interaction. Of primary 
interest will be the three contrasts of difference in mean between salsalate vs. placebo for 
these study groups. A Sidak adjustment will be used to control the Type I error. Analysis for 
the secondary measure of difference in SI between salsalate and placebo for the insulin-
resistant subjects only (lean IR, obese) will be compared using the same GLM framework, 
although only the overall contrast of difference in means between salsalate and placebo is 
of interest. Assumptions and model fit will be checked for all primary and secondary 
outcome analyses. Data will be transformed if necessary.   
 
     Post-treatment ovulation rates based on ultrasound documentation of follicular collapse 
will be compared between the salsalate-treated group and the placebo group using Fisher’s 
exact test with type I error at 0.05.  Also, ovulation rates and 95% exact confidence intervals 
will be estimated by study group (lean with IR, lean without IR, and obese) and treatment 
group.   
 
iii. Aim 2 will evaluate lipid-stimulated NFκB EMSA density (primary outcome), glucose-
stimulated NFκB EMSA density and lipid-stimulated ROS generation (secondary outcomes) 
along with the exploratory variables described in the power analysis. Primary and secondary 
outcomes will be transformed (e.g. logarithmic transformation) if either measure is not 
normally distributed and will be analyzed using a GLM framework as described for Aim 1 to 
compare salsalate vs. placebo for each study group. A Sidak adjustment will be used to 
control type I error. Descriptive statistics will be provided for exploratory variables. 
  
13.0 Regulatory Requirements 
 
13.1 Informed Consent 
• 
Written consent will be obtained in the CCTC CRC during the screening visit after the 
volunteer has successfully completed an initial phone screening.  Each member of the 
research team is designated to administer the informed consent.  Each research team 
member has been trained by the PI in administering informed consent and has completed 
CITI and HIPAA training as required by IRB. 
 
• 
Each volunteer will receive a copy of the signed informed consent document (ICD) for 
his/her records.  The study copy of the signed ICD will be stored in the subject’s study 
binder in a locked file cabinet in the PIs locked office.  Each member of the study team has 
access to these study binders. Because they are stored in the PI’s office, the PI will 
supervise access. 
 


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13.2 Subject Confidentiality  
 
• 
Subject’s confidentiality will be maintained by using unique identifiers and keeping source 
documents with sensitive subject information in protected environments as described above.  
Further, all research team members are Collaborative Institutional Training Initiative (CITI) 
and HIPAA trained. 
 
• 
All research team members have access to the study data that will be stored digitally on a 
secure database.  Study data are kept separate from source documents containing sensitive 
subject information.  Source documents such as initial screening forms, informed consent 
and other data sheets containing dates are kept in subject binders in a locked cabinet in the 
PI’s office.  Security standards will include 2 locked doors for paper records, and 2 electronic 
signatures for electronic data (i.e. main computer, then specific access to data files). The PI 
will oversee who has access to these files.   
 
• 
Upon subject enrollment, the subject is assigned a unique identifier.  This unique identifier 
will be used for all data sheets and data spreadsheet entries.  Only the initial screening 
form, the informed consent and the identifier key will contain direct identifiers.   
 
• 
The research team will request access to participant’s PHI through a patient authorization 
document, which if signed by the subject will give the researcher permission to use PHI 
collected during the research study for defined purposes.  
 
• 
After study completion, we plan to scan all source documents into electronic format for long 
term storage on an Academic Computing and Communications Center (ACCC) protected 
UIC server that only the PI can access.  Upon verification of electronic transfer, original 
source documents will be shredded and disposed of into confidential file disposal bins.  
 
• 
Sensitive subject information such as personal identifiers are required to determine subject 
eligibility and required to contact the individual for study visits.  Once the subject is enrolled, 
the subject is assigned a unique identifier and these initial source documents are no longer 
needed for the successful completion of the study.  The steps above describe the handling 
of these source documents. 
 
• 
A Certificate of Confidentiality is not required, as the information collected if disclosed would 
not significantly harm or damage the participant.   
 
13.3 Unanticipated Problems 
 
• 
Based on the low level of risk during study participation, serious adverse events are unlikely 
to occur. Discomfort during the blood draws will be most likely problem that could occur 
during participation. Every effort will be made to provide comfort to participants whenever 
possible. Should participants experience discomfort or emotional upset from any of the 
research procedures, they will be reminded that they are under no obligation to participate in 
the study, and can withdraw consent and discontinue participation at any time without 
prejudicing their current or future relations with UIC or UI Health, or its physicians.  Students 
of employees will be reminded that participation or nonparticipation in the study will not 
affect current or future medical care at UIC or the UI Health and/or position of employment 
or course of studies in these institutions. 
 


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• 
Additional potential problems such as breach of confidentiality and emotional upset may 
also occur. Every effort will be made to maintain subject confidentially using the data 
management procedures described above. In the event of breaches or suspected breaches 
of data security, including sensitive information, an unanticipated problem will be reported to 
the IRB using the Event Requiring Prompt Reporting form within 5 working days of 
becoming aware of the event. Possible data breaches include, but are not limited to, the 
following: lost or misplaced files/folders, improper disposal of paper containing sensitive 
information, loss/violation of the integrity of decryption key or process. 
   
• 
A participant’s study will be stopped in the event she requests not to continue, or if she has 
a reaction to any procedure, the ingestion of the glucose beverage during the OGTT, the 
HCG injection, dairy cream ingestion during the CCT, difficulty obtaining a peripheral 
intravenous line, administration of salsalate (i.e. allergies, persistent or serious side effects, 
unusual anxiety).  In the unlikely event that a serious side effect occurs, the participant will 
immediately be referred for further evaluation and treatment.  All participants will be 
assessed for side effects at the end of the study and any discussions will be documented 
within each participant’s study binder.  Follow-up with the IRB or CRC will also be noted, 
and copies will be kept of all correspondence.  
 
• 
A participant will be withdrawn from the study if she fails to comply with any of the research 
procedure, if the study is stopped for an unforeseen reason, or in the event of physical injury 
or illness resulting from the research procedures.  Failure to take the appropriate dose of 
medication not related to downward dose titration for expected symptom control can result in 
withdrawal of a subject from the study.  The degree of noncompliance with medication use 
that can have a significant adverse impact on the study may vary from subject to subject 
and the decision to withdraw a subject for medication use noncompliance will be left to the 
discretion of the investigator.  
 
• 
The study will be completely suspended in the extremely unlikely event of death of a 
subject, or at the moment it becomes apparent that any particular serious side effect is 
occurring at an unanticipated increased frequency.    
 
• 
The PI will keep abreast of scientific reports or therapeutic development, and results of 
related studies that impact subject safety; and will inform subjects immediately of new 
information from these reports or studies that change the risk/benefit ratio that might cause 
them to change their mind about continued participation.  This type of information will also 
be reported to the DSMB and to the IRB during an annual continuing review.  Participants 
will be instructed to notify the principal investigator or his designee as soon as the desire to 
withdraw is apparent. The circumstances related to the request to withdraw will be reviewed 
to determine the necessity of performing a post-study evaluation to insure safety.     
  
14.0 References 
 
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3. Dunaif A, Segal KR, Futterweit W, Dobrjansky A. Profound insulin resistance, independent 
of obesity in polycystic ovary syndrome. Diabetes 1989, 38:165-1174. 


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APPENDICES 
 
Telephone Eligibility Assessment Form 
Electronic Eligibility Assessment Form 
Clamp Infusions Preparation 
Study Visit Blood Sampling Plan 
González Laboratory Flowsheet 
ChooseMyPlate Diet Instructions  
3-Day Food Record  
Basal Body Temperature Chart  
Advertisement (Flyer or Newsprint) 
PCOS Challenge Promotional E-Mail 
PCOS Challenge Promotional Presentation and Script