D. Cell-based GnRHR assay

GnRHR-AAb activity in serum IgG was measured with a calcium flux assay using a Ready-to-Assay GnRHR-expressing Chem-1 cell line (Eurofins Bioanalytics, St Charles, MO), according to the manufacturer’s protocol. Briefly, Chem-1 cells were dispensed into a 96-well microplate and incubated for 24 hours. After the plate was washed with Hanks balanced salt solution supplemented with 20 mM HEPES and 2.5 mM probenecid at pH 7.4, Fluo-8 NW (AAT Bioquest, Sunnyvale, CA), Ca²⁺ dye-loading solution was added to each well and incubated for 1 hour. Serum IgG, GnRHR agonist GnRH, or leuprolide (Sigma-Aldrich) was then added in constant volumes. Calcium flux response was recorded every 20 seconds for 180 seconds on a Hidex Sense microplate reader (Hidex, Turku, Finland). All samples were tested in triplicate. Data are expressed as a percentage of buffer baseline fluorescence signal to normalize the individual values. A value of 100% of basal activity represents the activity associated with the presence of buffer alone. A value of 100% of basal activity represents the activity associated with the presence of buffer alone. The intra-assay coefficient of variation is 8.2% (N = 58) and the interassay coefficient of variation is 8.0% (N = 8). Serum IgG (10-150 μg/mL) was used to assess activity dosage responses. IgG was tested at 4 different concentrations (0, 50, 100, and 150 μg/mL) to determine an optimal concentration for study. In separate experiments, IgG (100 μg/mL) was added in the absence and presence of the GnRHR antagonist cetrorelix (10^–7 M) (Thermo Fisher Scientific) to measure specific receptor activity for individual subjects. GnRH (10^–9-10^–6 M) in the absence and presence of serum IgG (100 μg/mL) was also tested to examine a possible allosteric effect of the PCOS IgG on the established GnRHR orthosteric ligand response.