O-296: Lower culture temperature during vitrification freezing/thawing improves viability of human and mouse oocytes

Abstract

Human oocytes are sensitive to temperature change. Reduced temperature may cause disruption of meiotic spindle structure, and even irreversibly comprise the development of oocytes. However, on the other hand, high temperature is generally associated with more toxicity of cryoprotectants. Studies of the effect of temperature on viability of oocytes have given mixed results. In this study, we examine the effect of temperature on a vitrification system and its influence on the viability of human and mouse oocytes. Prospective study using mouse oocytes and retrospective data of human oocytes. Metaphase II (MII) mouse oocytes were collected from 6-8 weeks old CB6F1 hyperstimulated by injection of 5IU PMSG and 5IU hCG. Human oocytes were retrieved from Ovarian stimulated FSH, GnRH agonist and hCG. MII oocytes were cultured in vitro for less than 1 for mouse oocytes and less than 2hours for human oocytes before denude by brief exposure to hyaluronidase and expose to DMSO-EG-sucrose based vitrication - devitrification system. Mouse oocytes were divided into two groups, group one, the vitrification - devitrification is carried out under body temperature (37°C), group two, the experiment is under room temperature (23°C). Human oocytes group similar. Oocytes were loaded in CryoTip™ straw and heat-sealed just before plunged directly into liquid nitrogen. After thaw, oocytes were allowed to one hour (mouse) and three hours (human) recovery. Survival mouse oocytes were inseminated by capaticitated sperm, human oocytes were fertilized via ICSI. Embryos were cultured in sequential Quinn’s Media supplement with 6% plasmanate under condition of 37°C and 5%CO2. Human and mouse embryos were graded at day 5, and mice blastocysts were corroborated by double staining of Oct 3/4 (detect functional inner cell mass) and of Hoechst 33342 (detect total embryonic cell number). Decrease media temperature in the process of vitrification and devitrification from 37°C to 23°C, the viability of mouse oocytes increase significantly, more embryos develop to blastocysts (from 43% to 75%) and good blastocyst (from 28% to 64% ≥3BB). Human oocytes benefit similar from the temperature change, significant more oocytes survive (from 74% to 98%) and cleave (from 38% to 70%), and develop to blastocyst (from 11% to 30%). It is noted that, under a similar condition, the rate of blastocyst is lower in human oocytes (30%) than in mouse oocytes (75%). These data suggest a benefit to performing vitrification at room temperature over the current standard of 37 °C. Better embryo quality will hopefully translate to improved pregnancy outcome following oocyte freezing and thawing. The vitrification system developed base on mouse oocytes is required to be further modified for more human oocytes developing into good embryos.