Table 1.
Characterization of the cohorts of patients studied in this analysis.
Fig 1.
Generation of stable clones and linearity of the assay for GnRH-R-aAb quantification.
A) Stable cultures of HEK293 cells expressing the recombinant fusion protein were established and ten productive clones were compared for luciferase (Luc) activity, which is indicative of the relative concentration of the desired GnRH-R-Luc fusion protein. B) A commercial polyclonal rabbit antiserum to human GnRH-R (anti-GnRH-R Ab) was diluted and tested in the newly generated assay. The signals obtained were related to the anti-GnRH-R Ab, and a proportional response of signal intensity to the antiserum concentration was recorded. Measurements were conducted in duplicates, control samples without human serum typically yield luminescence signals of <100 RLU.
Fig 2.
Prevalence of GnRH-R-aAb in serum samples of controls and PCOS patients.
A) The full set of samples (n = 1051) was analyzed for GnRH-R-aAb concentrations by measuring GnRH-R-Luc activity in the immunoprecipitates. Binding indices (BI) were calculated based on the lower half of all samples. The mean value plus three standard deviations (mean+3SD) was used as criterion for outliers, indicating positive presence of GnRH-R-aAb. Three samples were strongly positive, and 12 samples were slightly above the threshold (dashed line). B) The signals for GnRH-R-aAb were not normally distributed in the cohorts of samples analyzed. The prevalence of highly positive samples is similar in the cohorts of PCOS patients and control subjects (2 out of 651, i.e., 0.31%, vs. 1 out of 400, i.e., 0.25%).
Fig 3.
Test for matrix effects and signal reproducibility.
Three different control samples were equally mixed with one of the three highly positive GnRH-R-aAb samples and analyzed for GnRH-R-Ab concentrations. The signals measured correspond to the theoretical arithmetic mean of the respective serum samples indicating the absence of matrix effects. Measurements were conducted as duplicates.
Fig 4.
Steroid hormone levels in the two patients with high GnRH-R-aAb concentrations in comparison to healthy controls and other PCOS patients.
Two adult female PCOS patients were identified as highly positive for GnRH-R-aAb (PCOS #175; red cross, #225; blue cross). Both patients displayed elevated androgen concentrations in comparison to the reference range of healthy adult women (Ref. Range, green bar), and also in comparison to the other samples from the same cohort of PCOS patients (PCOS-A) (mean +/-SD; black symbols with error bars).