SUN-LB5 GnRHR ECL-2 Epitopes Targeted by Activating Autoantibodies in Polycystic Ovary Syndrome

Abstract

Background: Activating autoantibodies (AAb) are directed to the gonadotropin releasing hormone receptor second extracellular loop (GnRHR-ECL2) and are pathogenic when induced in rats. We previously reported GnRHR-ECL2-AAb were elevated in sera from patients with PCOS (Rotterdam criteria) compared to ovulatory infertile controls (OIC). Methods:Human studies: ELISA detection of GnRHR Abs used a synthetic h-GnRHR-ECL2 28 mer peptide (LifeTein) as the target antigen. We assayed AAb activity in GnRHR transfected cells using a GeneBLAzer FRET assay (Invitrogen). ELISA AAb epitope locations on the ECL2 were identified on a minipin plate (pins 1-11 containing sequential 2 aa offsetting octapeptides, Mimotope, Inc) using sera from 30 PCOS subjects, 33 OIC and 18 normal controls (NC). Results:Human sera: An ELISA assay for GnRHR-ECL2-AAbs in the PCOS group was markedly higher than the NC group (P<0.0001) and the OIC subjects (P<0.003). The minipin data demonstrated one or more positive OD peaks on pins 4 (20%), 5 (47%) and 8 (47%) which shared L-aa sequences FSQC or CSFSQ. OIC had only 5 subjects with peaks at minipins 4 or 5 and NC had only 3 lower peaks and one with higher OD values over all minipins. GnRHR-AAb Specific Activity (SA) was estimated by measuring serum activity before and after suppression of AAb sensitive activity by addition of retro inverso D-aa (RID) peptides. These were specifically designed to mimic and decoy the AAb L-aa epitope sequences of pins 5 and 8). SA was measured in 10 selected PCOS and 10 OIC subjects who had positive ELISA values. The baseline activity in the PCOS group was significantly higher than OIC (P < 0.01) and dropped 50% with preincubation with peak 5 RID and 25% with peak 8 RID. The addition of both peak 5 and peak 8 RID suppressed the PCOS group activity to OIC levels (P > 0.2). There was no significant change in activity in the OIC subjects by the addition of peaks 5 + 8 RID peptides. Conclusion: These PCOS GnRHR-AAb data confirm the presence of significant activation of the GnRHR by AAb targeted to specific epitope(s) proximate to the disulfide GnRHR-ECL2 linkage to the nearby ECL1. These data are compatible with a pathophysiological role for GnRHR-AAb in PCOS and may provide both diagnostic and therapeutic opportunities.