Abstract
OBJECTIVE: Ovarian tissue freezing & transplantation can preserve fertility. VEGF may increase neovascularization of grafts & improve function. The optimal method of delivery & dose of VEGF is unknown. Here we 1) assess if VEGF administered to sham autografts elevates systemic VEGF 2) compare two methods of delivering VEGF, microspheres and hydrogel 3) develop an in vitro assay to estimate an ideal VEGF dose. DESIGN: Descriptive, non-human primate and in vitro assay. MATERIALS AND METHODS: Two female Rhesus had sham, subcutaneous transplants using gel foam as substitute ovary. Both monkeys had 32 implants in the arm & abdomen. Grafts on the left half of the body received 4.5 ug of VEGF; those on the right did not. One monkey had VEGF delivered via slow-release microspheres, & one via hydrogel. VEGF was measured in tissue & blood collected pre and post surgery. A human umbilical vein endothelial cell (HUVEC) invasion assay was used to define the dose-response curve for VEGF concentration & endothelial cell migration. (HUVEC) were cultured in 96 well plates with a membrane separating plain media above, & varying concentrations of VEGF media below. After 24 hrs, the number of cells invading through the membrane was determined by measuring fluorescence. RESULTS: Serum VEGF was minimal & peaked at 7 hrs (hydrogel) & 24 hrs (microspheres). Peak serum VEGF was two-fold lower when microspheres were utilized (microspheres= 24pg/mL, hydrogel= 52pg/mL). In tissue, VEGF concentration at treated sites on day 4 was 3 fold greater at sites with VEGF microspheres (15,000 pg/mg) than with hydrogel (5,000 pg/mg). The HUVEC assay showed maximal endothelial cell migration at 10 ng/ml. CONCLUSION: 1) Serum levels of VEGF are minimal when VEGF is delivered with sham ovarian transplants 2) Microspheres deliver VEGF with lower serum concentrations, & higher tissue levels compared with hydrogel, & therefore, may theoretically be a better mode of VEGF delivery for induction of angiogenesis 3) 10 ng/ml may be the optimal concentration of VEGF.
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