Comparison of human blastulation rates and total cell number in sequential culture media with and without co-culture

Recent interest in delayed embryo transfers necessitated the evaluation of two improved in-vitro systems that could generate viable blastocysts. A total of 178 two-pronucleated embryos (entire cohorts) from 19 patients was cultured in IVF50 medium (100 microl) under oil for 24 h until day 2. Each patient's day 2 embryos were then equally allotted to two in-vitro systems. Embryos in system A were grown until the morning of day 3 on Vero cells covered with IVF50 medium (100 microl) under oil. The medium was then replaced on day 3 with a 1:1 mixture (100 microl) of IVF50:S2 medium and on day 4 with S2 medium only. The same culture protocol was used for system B without Vero cells. Throughout the 5 days all dishes were housed in sealed humidified modular chambers containing a triple gas atmosphere. Separately, 175 spare embryos from 80 patients were grown in system A and B up to days 6 and 7 for total cell number (TCN) analysis. Blastulation rates were not significantly different between system A and B (67.4 versus 68.5%; P > 0.01) although co-cultured embryos cleaved slightly faster by day 4. The overall pregnancy and implantation rates were 52.0% and 32.1% for the 19 patients each of whom received a mixed cohort of three day 5 embryos from both systems. TCN values for the day 6 and 7 blastocysts from both systems were high and increased steadily from days 6-7 and from expanded to hatching stages. There were no significant differences in TCN for day 6 expanded blastocysts between the two systems although day 6 hatching and hatched co-cultured blastocysts had greater values than non-co-cultured blastocysts (246.0 +/- 18.5 and 236.7 +/- 17.8 versus 173.0 +/- 13.5 and 166.5 +/- 16.0; P < 0.01). The results demonstrated that the culture protocol using the sequential IVF50-S2 media combination was a good substitute for Vero cell co-culture for the transfer of viable day 3-6 embryos.