Current status of IVM/IVF and embryo culture in humans and farm animals

Human oocytes are usually recovered from preovulatory follicles of women treated with gonadotropins and gonadotropin releasing hormone analogue. The oocytes are usually mature and are inseminated within 4 to 6 hours of aspiration from follicles. High rates of fertilization occur without any particular treatment of washed sperm. Failure of fertilization due to poor quality sperm is very effectively treated by microinjection of sperm directly into the cytoplasm of the mature oocyte. Fertilized oocytes may be cultured in a wide range of media and the cleaving embryos transferred within 48 to 72 hours to the uterus. Coculture methods have been reported for culture of human embryos including human and bovine oviduct epithelial cells and Vero cells. Implantation rates reported for human embryos in individual publications in the literature range from 8 to 28% after culture in simple balanced salt solutions and tissue culture media, and from 18 to 53% after coculture with feeder layer cells. However, the factors that control this variation are not known, nor is it at all certain that coculture significantly increases human embryo development or viability because of the lack of adequate controls or even appropriate comparisons in most studies. Ruminant oocytes may be successfully matured in vitro and cleavage initiated at high rates after insemination in vitro. The production of morulae and blastocysts can be achieved in a wide range of media and coculture cell types. It is most common to use bovine oviduct epithelial cells for coculture and either TCM 199 or Menezo's B2 medium. Around 43 to 53% of 2-cell IVM/IVF bovine embryos develop to blastocysts in coculture for 6 days with bovine granulosa or oviduct epithelial cells in TCM 199 + 10% FCS with mean cell numbers of 112 to 116. Cell numbers of embryos cultured in conditioned media were lower, showing there was a direct benefit of the cocultured cells for embryo development in this study. We have achieved a further improvement in the rate of development to blastocysts and cell number was achieved by culture in simple SOF medium with amino acids and 8 mg/ml BSA. A total of 39% of oocytes matured and developed to blastocysts with mean cell numbers of 87 to 151 in 6 days of culture, depending on the blastocyst stage reached. Fifty percent of blastocysts selected from those produced after 6 days of culture developed to fetuses after transfer to recipients. These results demonstrate that bovine embryos can be produced very successfully in simple culture medium without any supportive cocultured cells. This may reflect improvements in embryo culture methods as well as more suitable ingredients in culture solutions. Further research is required to determine the significant interacting factors for embryo production by IVM/IVF and culture. It is of interest that periodic changing of medium during culture, avoids ammonium toxicity and that the benefit of culturing a number of embryos together in confined volumes to improve the rate of development and cell number of embryos may be due to the production of autocrine factors. It will be useful to apply these culture methods for human embryos and to explore the micromanipulation techniques developed for human gametes and embryos, for fertilization and assisted hatching for embryos of farm animals. .