Improved methods for blastocyst formation and culture.

Transfer at the blastocyst stage has been proposed to increase the pregnancy rates after in-vitro fertilization (IVF) and embryo transfer. In the first period of experiments, culture in a single medium, from fertilization to blastocyst led to disappointing results: low blastocyst formation rates and low implantation rates per blastocyst transferred. Then the period of co-culture began (starting with animals in the early 1980s and with humans in the early 1990s). With this technique, using tubal or granulosa cells or layers obtained from established cell lines of transport epithelium origin, blastocyst formation has reached approximately 50% and the overall implantation rate is approximately 25%. The embryos obtained have high numbers of cells (> 200 cells for a day 6 expanded blastocyst). Co-culture with fibroblasts has been found to be useless. This technology has been proven reliable and reproducible: Blastocyst formation is highly dependent on maternal and paternal factors. It has enabled the design of efficient freezing and thawing protocols. Numerous interesting observations have been obtained to reach the third period, i.e. the use of sequential media. A simple medium is used for fertilization, then another one is used from fertilization up to the 4-cell stage (beginning of waves of transcription), then a third medium is used for development up to the blastocyst stage. The results obtained seem very similar to the one obtained with co-culture. Obviously it is now time, in humans, to switch to sequential media.