In Vitro Development of Mouse Pronuclear Embryos to Blastocysts in Sequential Media With and Without Co-Culture of Autologous Cumulus Cells

Abstract

The objective of this study was to use mouse embryos as a model system to investigate the effect of co-culture of cumulus cells in Sydney IVF sequential media (Cook) on embryo development, based on the hypothesis that feeder cells in co-culture with a sequential medium could work synergistically to further improve in vitro culture conditions for mammalian preimplantation embryos. The culture systems described here were evaluated by the ability to consistently produce high blastocyst formation rates and high cell number per blastocyst. The role of embryo-to-cell contact was assessed by using Transwell inserts with transparent microporous membranes. Pronuclear embryos of ICR mice were cultured to blastocysts in Cook sequential media, with and without mouse primary cultures of cumulus cells, and with or without inserts. Blastocyst formation rates and cell numbers of in vitro developing embryos in the different culture systems were compared to each other, and to in vivo derived blastocysts. Blastocyst formation rates for Cook medium only was 27.8% (without inserts) and 32.9% (with inserts), whereas Cook-Cumulus cells in identical culture systems was significantly higher at 45.8% (without inserts, P<0.05) and 55.6% (with inserts, P<0.05). When the embryos are suspended above the bottom of the well, for Cook medium significantly lower blastocyst formation rates were observed at 4.2% compared to Cook-Cumulus cells at 17.5% (P<0.05). Mean cell numbers of blastocysts obtained in all co-culture systems were significantly higher (P<0.05) compared to those developing in culture medium only. Although the putative mechanism is as yet unexplained, the improved blastocyst formation rates and cell numbers in co-culture when there is direct contact between the embryo and the cell monolayer suggest that the close proximity between the feeder cells and embryos is in part responsible for these effects.