Abstract
OBJECTIVE: To investigate the cytoskeletal apparatus of MII oocytes, specifically the meiotic spindle, before and after cryopreservation using a patented slow-freeze method called the LANDA Technology. DESIGN: Human oocyte freeze/thaw protocols were designed to minimize the damage from intracellular ice formation and high solute concentration. The present study examined and compared the damage rates to the oocyte meiotic spindles apparatus, chromosomal alignment and mitochondrial distribution between fresh and frozen-thawed oocytes. The study was conducted under IRB approval. MATERIALS AND METHODS: MII oocytes from women age < 37 years with day 3 FSH < 10 were obtained for cryopreservation with PrOH as a cryoprotectant agent with temperature and sucrose modifications using the LANDA Technology. Donated oocytes were imaged by polarization microscopy applying PolScope technology before freezing and after thawing employing the LANDA Technology. Using immunohisotochemistry, we attached fluorophores to the meiotic spindles (green), the chromosomes (blue) and the mitochondria (red) to determine whether any meiotic spindle disruption, chromosome disarray, or mitochondrial dispersion could be documented after eggs were thawed. RESULTS: A preliminary analysis was conducted on 56 human oocytes. Of the fresh oocytes controls, 64.7% (11/17) showed normal meiotic spindles, chromosomal alignment, and mitochondrial distribution; whereas 71.8% (28/39) of the oocytes from the study group showed normal characteristics. CONCLUSION: Human oocyte cryopreservation performed under controlled conditions using a patented protocol does not increase injury to the cytoskeletal structures of human oocytes. These results are encouraging and consistent with clinical data showing excellent survival of thawed oocytes (91%), resulting 52 healthy babies born from 117 embryo transfers. We are presently conducting an ongoing study of a larger patient population to assess the efficacy of a new slow-rate oocyte freezing protocol, the LANDA Technology.
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