Figure 1. Strategy for microdrop culture of synchronizing preimplantation embryos and the timing of culture media collection.

Embryos were collected from mated superovulated mice at 18 hours post-hCG, and embryos with two pronuclei were discarded. Other embryos were incubated in KSOM and then those with 2PN were selected to synchronize in vitro embryo development (at 24 hours post-hCG). A hundred of zygotes were cultured in 50-µl drops of KSOM under oil at 37°C in an atmosphere of 95% air/5% CO₂. The embryos were then transferred into fresh KSOM at 55 hours post-hCG and further cultured until the blastocyst stage. Embryo-free control drops were incubated alongside the embryo-containing drops. Each of the spent media was analyzed by CE-TOFMS.