Radioimmunoassay

Mouse cytokine array (R&D Systems, Minneapolis, MN) was performed on isolated protein from aortas from F WT, F WT ovx, and F ArKO ovx mice according to the manufacturer’s instructions (n=4 mice per group). Protein samples from each group were pooled for analysis, and all samples were then run in duplicate.
To determine circulating endogenous hormone levels, serum concentrations of estradiol and testosterone were determined with enzyme-linked immunosorbent assay (ELISA, Calbiotech, Spring Valley, CA) and radioimmunoassay (Siemens Healthcare Diagnostic, Tarrytown, NY), respectively. Tests were performed by the Ligand Assay and Analysis Core Facility at the University of Virginia Center for Research in Reproduction.

The AMIGOS trial was designed to discern if ovarian stimulation with letrozole would result in a significantly lower frequency of multiple gestations when compared to gonadotropins and/or clomiphene citrate (7 (link)–8 ). This IRB approved study enrolled 900 couples with unexplained infertility. The female partner was required to be between 18 and 40 years of age, with regular menstrual cycles, a normal uterus, and at least one patent fallopian tube (8 ). Male partners were required to have a baseline semen analysis with at least 5 million motile sperm, recorded within 1 year of study initiation (7 (link)). This minimum required sperm count was considered sufficient for sperm washing and intrauterine insemination—which was performed in all AMIGOS treatment cycles. While fasting, male partners also provided blood samples, which were processed for TT levels at the University of Virginia Ligand Assay and Analysis Core (Charlottesville, VA). TT was determined by radioimmunoassay (Siemens; functional sensitivity = 10 ng/dL; intra-assay coefficient of variation = 4.3%; inter-assay coefficient of variation = 7.4%). The assay performance was validated with determination of accuracy, linearity, precision, and functional sensitivity (i.e., the lowest concentration with accuracy to a known standard within 20% and intra-assay coefficient of variation <20%).

Fasting blood samples were drawn in the supine position and processed using a standardized protocol, plasma and serum were prepared from samples by sedimentation in a refrigerated centrifuge within two hours of blood collection, stored at −70°C and sent to central laboratories (University of Minnesota) (16 , 17 (link)). Serum aldosterone was measured by radioimmunoassay (Siemens) and the intra-assay coefficients of variation were 8.7% and 6.2% for low and high concentrations. PRA was measured at baseline using immunoradiometric assays of PRA in ng/ml/hr (n=2,252) with intra-assay coefficient of variation of 8.0%.