#239 : Granulocyte-Macrophage-Colony Stimulating Factor Improve Embryo Development, Mitochondrial Function and Spindle Alignment in a Mouse Model of Advanced Maternal Age

Abstract

Background and Aims: We have previously shown that GM-CSF can improve embryo development when added during in vitro oocyte maturation (IVM) as well as increase pregnancy and birth rates following embryo transfer in mice. The aim of the present study was to determine whether GM-CSF during IVM can improve embryo development in a mouse model of advanced maternal age (AMA) Method: 12–14-month-old CBAF1 female mice were injected with 5IU of PMSG and cumulus-oocyte complexes (COCs) aspirated 46–48 h post-injection. Ten COCs were cultured per 50 [Formula: see text]L drop in bicarbonate-buffered [Formula: see text]-MEM containing 3 mg/ml BSA, 1 mg/ml Fetuin and 5 mIU/mL FSH plus 0 or 10 ng/ml of GM-CSF. Zygotes were cultured in Cleave Media and cleavage and blastocyst rates were determined. Blastocysts were differentially stained on Day 5 to determine cell numbers. To determine mitochondrial membrane potential (MMP) COCs were incubated for 15 minutes in HEPES buffered alpha-MEM media with JC-1 dye for 15 minutes. To examine spindle formation and chromosomal alignment, MII oocytes were fixed and then incubated with FITC conjugated mouse anti-[Formula: see text]-tubulin for 40 minutes at 25°C and chromosomes stained using Hoechst. Fluorescence was visualised using Cell Voyager CV1000 Confocal Scanner and Z-stack images were analysed using Fiji Image J software. Data were analysed using a univariate general liner model in IBM SPSS. Results: GM-CSF during IVM increased Day 5 blastocyst rate by 21% (P0.05). Conclusion: We have shown that the addition of GM-CSF during IVM can improve embryo development and mitochondrial activity in a mouse of AMA.