A bioassay to evaluate oocyte vitrification procedures

Abstract

OBJECTIVE: Vitrified human oocytes display a high potential for survival, fertilization and cleavage, but their developmental potential to blastocyst is low. In contrast, similarly vitrified mouse oocytes develop to blastocyst at a comparable rate to fresh oocytes. In this study we assess the viability of these mouse blastocysts by monitoring their re-expansion and morphology following vitrification-thaw. DESIGN: Blastocysts developing from fresh or vitrified mouse oocytes were evaluated after vitrification to assess their ability for re-expansion and morphology following thaw. MATERIALS AND METHODS: Metaphase II oocytes were collected from PMSG+hCG-treated CB6F1 mice and denuded with hyaluronidase. Mature oocytes were equilibrated and vitrified using a DMSO+ethylene glycol+sucrose procedure. After thaw and cryoprotectant removal in sucrose solutions, the surviving oocytes were inseminated by capacitated sperm. The resultant zygotes were cultured for 4 days at 37C in Quinn's Advantage sequential cleavage/blastocyst media (Sage) supplemented with 6% PlasmanateR. All good quality blastocysts (a well-defined inner cell mass and trophectoderm) were punctured with a micro glass needle to release the blastocoel fluid and vitrified. They were examined for survival immediately post-thaw and for blastocoels re-expansion and morphology 3h later. RESULTS: In response to vitrification-thaw, 98% of 90 mouse oocytes survived; their cleavage and blastocyst formation rates were comparable to those of non-vitrified eggs (78% vs 80%; 71% vs 66%, respectively) as was the formation rate of good quality blastocysts (60% vs 63%). All blastocysts in both groups survived the vitrification-thaw procedure. However, when examined at 3h post-thaw, the number of re-expanded blastocysts derived from vitrified oocytes was significantly lower (62% vs 98%, p<0.001). Moreover, good quality morphology was observed in fewer of the expanded blastocysts that developed from vitrified eggs (51% vs 73%, p<0.05). CONCLUSIONS: The decreased ability of blastocysts derived from vitrified eggs to re-expand and display good quality morphology post-vitrification suggests that they are less viable. However, not all blastocysts are similarly affected. Such results suggest that monitoring blastocyst recovery post-vitrification is a reasonable bioassay to evaluate oocyte vitrification or cryopreservation protocols. Such bioassays will optimize these procedures for fertility preservation.