Insulin and Insulin-Like Growth Factors (IGFs) promote Vascular Endothelial Growth Factor (VEGF-A) secretion by human luteinizing granulosa cells following Controlled Ovarian Stimulation (COS)

Abstract

Objective: To investigate the role of insulin-related growth factors and luteinizing hormone (LH) in the physiological regulation of VEGF-A production by luteinized granulosa cells (LGC) from preovulatory follicles in humans. Design: Prospective, comparative, experimental study. Materials and Methods: LGC were obtained from women (n=6), with IRB approval, by follicular aspiration after undergoing COS for in-vitro fertilization. Subjects (33.5 ± 1.2 years, mean age ± SEM) had regular menstrual cycles with no evidence of PCOS or other endocrinopathies. LGC were isolated and plated at 50,000 cells/well on fibronectin in DMEM-Ham’s F-12 medium with 5 μg/ml transferrin, 5 ng/ml selenium, 10 μg/ml aprotinin and 25 μg/ml human LDL. Cells were incubated in the presence and absence of 100 ng/ml recombinant human (r-h) LH (Ares Serono) and 0, 1, 10, 100 ng/ml of r-h IGF-1, IGF-II or insulin (Sigma). Media were collected daily and assayed for free VEGF-A (Quantikine VEGF ELISA, R&D Systems). After 3 days (d), cells were fixed, Crystal Violet stained and analyzed for DNA content to estimate cell numbers. Data were analyzed by two-way repeated ANOVA, using a randomized block design. Results: In the absence of LH or insulin-related growth factors, LGCs secreted low but detectable levels of VEGF-A through d 3 of culture (179–304 pg/ml on d 3). However, exposure to IGF-I or -II resulted in a dose-related increase in VEGF-A levels by d 1 (e.g., 568 vs 183 pg/ml or 453 vs 278 pg/ml,100 ng/ml IGF-I or-II vs controls, respectively; p<0.05), which was sustained through d 3 (1001 vs 179 pg/ml or 701 vs 255 pg/ml, p<0.05). Conversely, the addition of insulin (100 ng/ml) did not increase VEGF-A levels until d 2 (737 vs 436 pg/ml, p<0.05). Although LH alone did not increase VEGF-A levels above controls on d 1–3, the combination of LH and insulin-related growth factors increased VEGF-A levels to a greater extent than IGFs alone at all doses (1181 vs 717 or 800 vs 567 pg/ml, LH + 10 ng/ml IGF-I or -II vs IGF-I or -II alone, respectively, on d 2; p<0.05). The synergistic effect of LH with IGF-I/-II was evident earlier on d 2 and at lower doses (10 ng/ml), compared to insulin and LH, which was not significant until d 3. There was no change in DNA content following incubation for 3 days in the presence of IGF-I, -II, insulin or LH when compared to controls. However, DNA content decreased (p<0.05) in the presence of LH + IGF-II at the maximum dose. Conclusion: Exposure to both insulin-related growth factors and LH is required for optimal VEGF-A secretion by human LGC. Thus, local (IGFs) as well as endocrine (LH, insulin) factors may combine to regulate VEGF-A production, and thus angiogenesis and vascular permeability, in the primate ovulatory follicle and corpus luteum. Evidence of insulin/IGFs, as well as LH, regulation of VEGF-A production supports further studies on their role in regulating angiogenic factors during COS cycles in women with endocrinopathies, such as PCOS. Supported by: NICHD U54 HD18185, RR 00163.