An improved medium for long-term culture of human embryos overcomes the in vitro developmental block and increases blastocyst formation

Abstract

To determine if a culture medium, designated CZB after the authors who first described it, which is supplemented with 0.11 mM ethylenediaminetetraacetic acid, 1.0 mM glutamine, 31.30 mM lactate, and 0.27 mM pyruvate and is lacking glucose for the initial stages of culture that overcomes the in vitro two-cell block of mouse embryos, can improve the rate of blastocyst formation of human embryos in long-term cultures and increase the pregnancy rate (PR) when used in a clinical in vitro fertilization (IVF) program. The study is in two parts. Initially, excess oocytes from IVF and gamete intrafallopian transfer patients were fertilized in vitro and then placed in long-term culture of either CZB plus 10% heat-inactivated human serum (32 zygotes) or Earle's balanced salt solution (EBSS) supplemented with 0.45 mM pyruvate plus 10% human serum (28 zygotes) to determine if CZB medium could enhance in vitro development and increase blastocyst formation when compared with EBSS. Subsequently, CZB or EBSS medium was used for short-term cultures of embryos in a clinical IVF program to determine if the use of CZB could increase the clinical IVF PR. Private practice of one author (M.D.). The excess oocytes were donated by couples not wishing to have cryopreservation. In the clinical trial, 49 couples presenting with tubal or male factor infertility and who had three or more fertilized zygotes were randomly assigned to one of the culture media being used. In long-term cultures, embryos were observed at 42, 66, 90, 114, and 138 hours after fertilization and scored for blastomere number, degree of fragmentation, and developmental arrest. When CZB- and EBSS-cultured embryos were evaluated over 138 hours, there was a significant increase in the number of CZB-cultured embryos reaching the blastocyst stage (56% versus 20%; P less than 0.009) and less fragmentation of CZB-cultured embryos (18.8% versus 50%; P less than 0.01). In short-term cultures, pronuclear stage embryos from patients undergoing IVF were cultured in CZB or EBSS for 24 hours, graded, and then used in embryo transfers. Of the 28 patients assigned to EBSS, 6 became pregnant (21.4%), and of the 21 assigned to CZB, 5 attained pregnancy (23.8%). These results were not significantly different. The use of CZB for the long-term culture of human embryos is highly beneficial and increases the rate of blastocyst formation, but its use in an IVF program does not increase the clinical PR.