Formulation of a protein-free medium for human assisted reproduction

The optimal concentrations of individual amino acids, antioxidants, vitamins, osmolytes and energy sources were determined using a 1-cell Swiss outbred (SO) and or F(1) [(CBAxC57BL/6J)xSO] mouse assay in Earle's balanced salt solution containing bovine serum albumin. Based on the findings of these experiments, a number of media were formulated. Of these, the medium showing optimal embryo development and a significantly higher blastocyst hatching rate was investigated further. A protein-free medium (ART-7) was formulated and assessed using 1-, 2- and 4-cell SO mouse embryos. The generation of viable human embryos in the ART-7 series of media in micro- and ultra micro-droplet culture under oil with and without cumulus co-culture following intracytoplasmic sperm injection (ICSI) was investigated. The quality of sibling day 2 human embryos generated in the ART-7 media series was statistically comparable to or better than control embryos. The ART-7 medium was not toxic to human spermatozoa. Fertilization by conventional IVF and subsequent embryo development was not affected. A clinical trial of ICSI-derived embryos generated in the protein-free medium, with and without cumulus co-culture, has resulted in clinical pregnancies (10 of 20 transfers) of which two have proceeded to term, and the remaining patients are in various stages of pregnancy.