Harman

The cytokine assays tested in this study included traditional solid-phase sandwich enzyme-linked immunosorbent assays (ELISA) utilizing colorimetry or luminometry platforms, immunobead-based assays utilizing the fluidics Luminex fluorometry platform, and a newer class of solid-phase microspot assays based on ultra-low-noise charge-coupled device (CCD) cameras represented by the Meso Scale Discovery (MSD) electrochemiluminescence platform. ELISA assays were obtained from Invitrogen BioSource (Carlsbad, CA), R&D Systems (Minneapolis, MN), and Pierce Endogen (Rockford, IL). Luminex assay kits were either purchased from Millipore Upstate (Charlottesville, VA), or were assembled in-house using reagents from R&D Systems. For comparison purposes in this paper, these two assays are labeled as Beadlyte Luminex and in-house Luminex. MSD assay kits were purchased from Meso Scale Discovery (Gaithersburg, MD). A detailed description of immunoassays is provided in Table 1.

KEEPS was designed as a randomized, placebo-controlled, double-blinded, prospective trial (KEEPS; NCT000154180) to evaluate effects of MHT on progression of atherosclerosis as defined by carotid intima–media thickness (CIMT) [44 (link)] and coronary arterial calcification (CAC) [8 (link), 73 (link)] in women who more closely match the age of initiation of MHT reported by prior observational studies. Women meeting inclusion criteria subsequently were randomized to daily placebo, oral CEE, or transdermal 17β-estradiol with placebo or pulsed progesterone for 12 days/month. The detailed inclusion and exclusion criteria for KEEPS have been published elsewhere [36 (link)]. In brief, women between the ages of 42 and 58 years of age who were at least 6 months and no more than 36 months from their last menses with plasma follicle-stimulating hormone (FSH) level ≥35 ng/mL and/or E₂ levels <40 pg/mL were eligible. A history of clinical CVD including myocardial infarction, angina, congestive heart failure, or thromboembolic disease excluded women from KEEPS. Other major cardiovascular risk factors excluding participation were current heavy smoking (more than ten cigarettes/day by self-report), morbid obesity [body mass index (BMI) >35 mm²/kg], dyslipidemia (LDL cholesterol >190 mg/dL), hypertriglyceridemia (triglycerides >400 mg/dL), and uncontrolled hypertension (systolic blood pressure >150 mm Hg and/or diastolic blood pressure >95 mm Hg) and glucose >126 mg/dL. Complete blood count and chemistry panel, estradiol, and FSH were performed at the clinical laboratories at each recruiting center. Lipid profiles and thyroid-stimulating hormone (TSH) were performed at the Kronos Science Laboratories (Phoenix, AZ, USA). At screening, women were asked to rank their menopausal symptoms (hotflashes, night sweats, vaginal dryness, dyspareunia, palpitations, insomnia, depression, mood swings, and irritability) as either none, mild, moderate, or severe. Finally, all subjects were screened for CAC and women with Agatston score ≥50 U, indicating significant subclinical coronary artery disease, were excluded. All women meeting inclusion criteria underwent baseline measurements of CIMT by B-mode ultrasound [44 (link)]. All imaging results are read centrally by individuals blinded to participant demographics (CAC at the Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, CA, USA under the direction of Dr. M. Budoff and CIMT at the Atherosclerosis Research Unit Core Imaging and Reading Center, University of Southern California, Los Angeles, CA, USA under the direction of Dr. H. Hodis).
Analysis of variance was used to determine statistical significance except where an alternative test is specified. Statistical significance was accepted at P < 0.05.