EVOLUTION OF A CULTURE PROTOCOL FOR SUCCESSFUL BLASTOCYST DEVELOPMENTAND PREGNANCY

A cell-free culture system was designed for human embryo development t o the blastocyst stage by testing a range of culture conditions in a s eries of protocols. The culture system that was evolved has a brief 1 h exposure to spermatozoa and then culture of the pronucleate zygote f or 2 days in IVF-50 medium. Two or three embryos were cultured togethe r in 20 mu l microdrops of medium under oil. Embryos were then regroup ed and two or three at a similar stage were cultured together in 50 mu l microdrops of Gardner's G2 medium under oil from days 3 to 5. Embry os were transferred to fresh G2 medium on day 5 and cultured for a fur ther 1 or 2 days (day 6 or 7). No serum was used in any of the culture s. The embryo transfer medium and G2 medium were supplemented with hum an serum albumin. The zonae of all blastocysts to be transferred to pa tients were completely removed enzymatically. Using this protocol, 52% of zygotes developed to blastocysts and 34 out of 35 patients treated received 82 blastocysts and 11 morulae on day 5 or 6. Twenty-one feta l sacs with positive heartbeats (23% implantation rate) were detected in 13 ongoing pregnancies (38% pregnancy rate/transfer or 37%/patient treated). We anticipate that further improvements in embryo developmen t and the selection of viable embryos can be achieved using this simpl e and effective culture system.