Rescue of zona-free embryos by partial encapsulation with collagen followed by coculture with endometrial cells

Abstract

Zona-free oocytes, a frequent occurrence when micromanipulation techniques are employed in IVF laboratories, are usually discarded. The purpose of this study was to see whether these embryos can be rescued by encapsulation with artificial zona pellucida. Zona-free mouse embryos encapsulated with sodium alginate (AZP, Group A), partially encapsulated with collagen micro-wells (Group B), zona-free embryos (Group C), and zona-intact embryos as control (Group D), were cocultured with endometrial cells. Embryo development of these studied groups were compared. Enzymatically isolated mouse endometrial cells were plated in 4-well plates (2 × 105 cells/well) and cultured with F12/199 medium supplemented with 10% FCS before coculture. Pronuclear-staged embryos were harvested from superovulated female CF1 mice (zona intact, control), treated with pronase to remove the zona pellucida (zona-free). Zona-free embryos were immersed in 1.5% sodium alginate solution, then aspirated with a small amount of sodium alginate and expelled into 1.5% calcium chloride to form microgels (AZP). All zona-intact, zona-free, and AZP embryos were cocultured with endometrial cells plated in 4-well plates directly. For the collagen microwells group, collagen type-I were coated at the surface of the monolayer endometrial cells in the 4-well plates and these micro-wells were formed by introducing small air bubbles in the layer of the collagen gel. The size of the wells is slightly larger then that of the embryos in order to encapsulate the embryos. Zona-free embryos were then transferred to each of the micro-well for coculture (collagen micro-wells group). Embryo development were evaluated every day for 5 days. Zona-free embryos tend to have abnormal early embryo development. They either disassociated into fragments or aggregated to form mosaic embryos. Addition of fully or partially encapsulated zona pellucida (AZP and collagen micro-wells, respectively) did enhance embryo development of these zona-free embryos. Most of the AZP appeared to have abnormal blastomere arrangement and blastocoele collapse and its blastocyst formation rate was compromised. However, collagen group exhibited normal embryo development and resulted in a blastocyst formation rate similar to that of the control. Our data suggests that artificial zona pellucida may rescue some zona-free embryos encountered during IVF. Partial encapsulation of collagen appears to increase cell-cell contacts resulting in normal blastomere polarization and allow space for embryo development. Recently, a new method for zona-free cloning has been demonstrated to increase the efficiency of cloning for stem cell study. Zona-free embryos generated from zona-free cloning can be benefited by culturing in our collagen micro-well culture system to improve embryo quality, thus avoiding monozygotic twinning and preventing adhesion of zona-free embryos to the feeder cells before transfer.