Reconstruction of ooplasm recipient oocytes with frozen-thawed donor microcytoplasts and influence on the microtubular spindle

Objective(s): To cryopreserve micromanipulated ooplast segments (microcytoplasts) from mouse oocytes, compare microcytoplast and parent or recipient oocyte fusion performed within or without the zona pellucida, compare electrofusion of fresh or frozen oocyte with frozen-thawed microcytoplasts, and assess spindle integrity after reconstruction of oocytes. Design: Prospective experimental study. Setting: University-based experimental laboratory. Animal(s): Mouse (MII) oocytes obtained after superovulation (n = 363). Intervention(s): Micromanipulation of oocytes (n = 363) into microcytoplasts (n = 181), cryopreservation of microcytoplasts along with parent and sibling control oocytes (n = 182), reconstruction by electrofusion of microcytoplast and parent or recipient oocyte performed with (group A, n = 35) or without a zona pellucida (group B, n = 32), comparison of electrofusion of fresh oocyte (group C, n = 40) or frozen oocyte (group D, n = 36) with frozen-thawed microcytoplasts fused within zona, and assessment of spindle morphology of reconstructed oocyte. Main Outcome Measure(s): Post-thaw survival, success of fusion, and spindle integrity as assessed by immunostaining. Result(s): Higher success of post-thaw fusion was seen in group A (91.4%) compared with group B (56.2%). The post-thaw fusion of microcytoplasts with either fresh or frozen oocytes was not significantly different. Spindle integrity was 82.5% in group C as compared with 47.2% in group D. Conclusion(s): Microcytoplasts created from oocytes can be successfully cryopreserved, thawed, and used to reconstruct oocytes with intact spindles.