Beneficial effect of microinjected trehalose on the cryosurvival of human oocytes

Objective: To determine the effectiveness of trehalose as an intracellular cryoprotectant for the cryopreservation of human oocytes. Design: In vitro comparative study. Setting: Clinical and academic research environment at a medical school teaching hospital. Patient(s): Women undergoing in vitro fertilization (IVF). Intervention(s): Discarded human oocytes, obtained from IVF patients, were randomly distributed into three groups: control group (no trehalose), extracellular trehalose group (0.5 M extracellular trehalose), and intracellular trehalose group (0.15 M intra- and 0.5 M extracellular trehalose). Trehalose was introduced into oocytes by microinjection. The oocytes in each group were cooled to different temperatures (i.e., -15°C, -30°C, and -60°C) at rate of 1°C/minute and thawed at ambient air temperature. Survival was examined after overnight culture. Main Outcome Measure(s): Survival of human oocytes cryopreserved in the presence and absence of trehalose. Result(s): The majority of oocytes in the intracellular trehalose group survived cooling to -15°C (63%), -30°C (53%), and -60°C (66%). In contrast, only a small number of oocytes in both the control (13%) and extracellular trehalose group (22%) survived cooling to -15°C, while all oocytes degenerated when cooled to -30°C and -60°C. Conclusion(s): Small amounts of intracellular trehalose in the absence of any other cryoprotectant provide a significant protection against freeze-associated stresses. Our results suggest that sugars such as trehalose should be considered as intracellular cryoprotectants for cryopreservation of human oocytes.