Treating Inflammation in Polycystic Ovary Syndrome to Ameliorate Ovarian Dysfunction
Treating Inflammation in Polycystic Ovary Syndrome to Ameliorate Ovarian Dysfunction
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.
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 vitro4 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.
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 dehydroepiandrosterone-sulphate] 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.

 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.

 -  Acute or chronic inflammatory illnesses (e.g. upper respiratory infection, 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. angiotensin converting enzyme 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, selective serotonin
 reuptake inhibitor antidepressants (e.g. fluoxetine, sertraline), tenofovir, and
 diuretics (furosemide, hydrochlorothiazide, spironolactone).