Co-culture of human embryos with autologous cumulus cell clusters and its beneficial impact of secreted growth factors on preimplantation development as compared to standard embryo culture in assisted reproductive technologies (ART)

Abstract

To analyze the concentration of growth factors secreted by autologous cumulus cell clusters during in vitro human embryo co-culture and investigate the impact of culture supplementation with autologous cumulus cell clusters on embryo development and pregnancy rate as compared to standard culture procedure. Randomized prospective comparative study. Private IVF clinic. 90 IVF patient couples ranging at the age between 35 and 40 (mean age of 36.5) agreed to participate in this study. In this IVF group, 64 first trials, 22 s trials and 4 third trials were included. 48 cases have been terminated at day 3 of culture and 42 of them have been continued up to day 5. Of the 48 cases assigned for day-3 culture, 208 MII-oocytes were randomly assigned for co-culture and 197 MII-oocytes were assigned for standard culture. The selection was done by alternating the oocyte immediately after stripping to co-culture or control, starting always with co-culture. All samples were analyzed with Milliplex factor-specific magnetic-bead panels with phycoerythrin fluorescent factor-specific antibodies using immunofluorescence cytometry technology by Lab Supplies Inc. laboratories in Athens, Greece. The MFI (Mean Fluorescence Intensity) values were calibrated and the readings were translated in pg/ml and calculated against the background values of culture medium alone. The statistical analysis of all data were processed with the Two-tailed T-test form. 36 IVF cases terminated at day 3 were randomly chosen. Supernatants from 17 cases were analyzed for seven different growth factors, namely, epidermal growth factor (EGF), fibroblast growth factor 1 and 2 (FGF-1 and FGF-2), vascular endothelial growth factor A, C and D (VEGF-A, VEGF-C and VEGF-D) and Leptin. Supernatants from 19 cases were analyzed for Insulin-like Growth Factor I and II (IGF-I and IGF-II). VEGF-A and VEGF-C were found at statistically significant levels (p < 0.005) in culture supernatant samples where the autologous cumulus clusters were present but were not detectable in supernatants of embryos cultured alone. IGF-I was detected at considerable levels in supernatant samples where the autologous cumulus clusters were present. IGF-I was not detectable in supernatant samples of embryos cultured alone. IGF-II was detected in significant amounts (p < 0.05) in samples of all groups. The highest concentration level of IGF-II was detected in supernatant samples of embryos in standard culture. The investigated factors, among other substances, may be causally connected to the beneficial effect observed on embryo development. Our findings suggest that co-culture with autologous cumulus cell clusters improves the outcome of embryo culture in IVF programs.