Assessment of growth factor effects on post-thaw development of cryopreserved mouse morulae to the blastocyst stage

The objective of this study was to assess the influence of specific factors on post-thaw development of mouse cryopreserved morulae, Thawed morulae (n = 206) were randomly distributed between 10 treatment groups: medium alone control (CT), Vero (VR) cells, leukaemia inhibitory factor (1 ng/ml), inte rleukin-6 (1 ng/ml), transforming growth factor (TGF) alpha (2 ng/ml), epid ermal growth facto; (EGF) (1 ng/ml), platelet-derived growth factor (1 ng/m l), insulin-like growth factor (IGF)-I (30 ng/ml), IGF-II (1 ng/ml) and TGF beta (2 ng/ml), At 4, 8, 20, 30 and 48 h, a digitized image of each thawed embryo was captured and stored for later analysis, The following parameter s were examined: blastocoel formation, blastocyst expansion, zona thickness and hatching. At termination of the experiment, cell number per embryo was determined by bisbenzimide staining, When contrasted to the medium done co ntrol, co-culture consistently accelerated the development of frozen-thawed morulae to the hatched blastocyst stage, allowing embryos to recover rapid ly from any damage sustained during the cryopreservation process. While no single growth factor/cytokine was able to completely mimic the results achi eved with co-culture, all of the growth factors impacted positively on at l east one of the morphological parameters studied. Cell proliferation was si gnificantly stimulated by just 48 h exposure to growth factors, either thro ugh co-culture or by direct media supplementation. Go-culture again yielded the best results with a mean cell count of 217 +/- 76 cells per blastocyst as compared with 131 +/- 36 in control medium alone, Amongst the factors t ested, IGF-I, IGF-II and EGF had the greatest impact. with mean cell counts of 172 +/- 50, 168 +/- 50 and 179 +/- 55 respectively, Whereas only 5% of CT embryos developed to blastocysts Kith > 200 cells, 51% of thawed embryos placed on co-culture monolayers and 25-32% of embryos cultured with IGF-I, IGF-II or EGF had > 200 cells. This study for the first time systematicall y describes the effect of culture regimen and growth factor additives an th e post-thaw development of cryopreserved embryos.