LUTEINIZED HUMAN GRANULOSA CELLS VASCULAR ENDOTHELIAL GROWTH FACTOR PRODUCTION

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Recent evidence suggests that VEGF, a cytokine produced by human granulosa cells, is a causative factor in the development of ovarian hyperstimulation syndrome (OHSS). The objective of this study was to determine if in vitro exposure of luteinized granulosa cells to gonadotropin (hCG) stimulates VEGF production and if progesterone (P) produced by these cells is an autocrine regulator of VEGF expression. Luteinized granulosa cells were collected from women undergoing ovarian stimulation for IVF, 34 hours after a 7500 IU bolus of hCG. In Study 1, cells were cultured for 9 days in DMEM/F12 medium (control) supplemented with hCG (CR123, 100 ng/ml), low density lipoprotein (LDL, 100 fg protein/ml), or hCG + LDL. In Study 2, the P synthesis inhibitor, trilostane (Sanofi Winthrop, Collegeville, PA), and/or a P receptor antagonist, ZK137.316 (Schering AG, Berlin, Germany) were added to selected wells. Medium was assayed for VEGF (ELISA; R&D Systems, Minneapolis, MN). Results of Study 1 were divided in two categories based on control VEGF levels on day 1 "low producers" (n=6; <750 pg VEGF/ml) and "high producers" (n=5; >1000 pg VEGF/ml). VEGF levels in cultures of both low and high producers increased (p<0.01) from day 1 to maximal levels on day 3, when they steadily declined through day 9 of culture. VEGF levels of low producers increased (p<0.05) following treatment with LDL on days 3, 5, 7 and 9 and with hCG on days 5 and 7 of culture. The effects of combined hCG and LDL were similar to those of LDL alone. HCG also increased (p<0.05) VEGF production for the high producers by day 7. In Study 2, trilostane treatment decreased (p<0.05) P production by 91% on day 1 of culture, but had no effect on VEGF production on any day. Moreover, no effect of the combined trilostane/ZK treatment was observed on VEGF synthesis. These results suggest that VEGF production by LGC is directly regulated by gonadotropin (hCG) independent of gonadotropin-stimulated progesterone synthesis. These data are consistent with the hypothesis that the exacerbation of OHSS in early pregnancy is mediated by the CG stimulation of VEGF production.