Granulocyte-macrophage colony stimulating factor (GM-CSF) and co-culture can affect post-thaw development and apoptosis in cryopreserved embryos

Purpose: The objective of this study was to evaluate the effects of growth factor supplementation and Vero cell co-culture on apoptosis and development of frozen thawed one-cell mouse embryos. Methods: The following treatment regimens were assessed: (a) control medium (b) Vero cell co-culture and (c) growth factor supplemented medium. The individual growth factors tested were: GM-CSF, IGF-I, IGF-II, TNF-alpha, FGF-4. LIF, TGF-alpha, TGF-beta, IL-6, PDGF and EGF. Blastocyst development and differentiation were monitored. At termination of the experiments, overall blastomere number and apoptosis were assessed using the TUNEL assay. Results: No differences were observed in blastulation and hatching rates. lCM differentiation in thawed embryos was notably improved with either co-culture or growth factor supplementation. The only growth factor significantly modulating apoptosis in thawed embryos was granulocytemacrophage colony stimulating factor (GM-CSF). GM-CSF enhanced continued cell survival and prevented apoptosis but did not influence overall cell number in developing blastocysts. Vero cell co-culture significantly increased cell number in blastocysts (124 +/- 42 vs 100 +/- 44 in control; P < 0.05). Embryonic apoptosis was higher in the co-cultured embryos. The increased presence of apoptotic cells in blastocysts of hich cell number may reflect the regulatory role of apoptosis in balancing ICM: TE ratios. Conclusion: These data indicate that culture conditions can modulate post-thaw embryonic development and apoptosis.