Human oocyte vitrification: the permeability of metaphase II oocytes to water and ethylene glycol and the appliance toward vitrification

Abstract

Objective: To determine the permeability of human metaphase II oocytes to ethylene glycol and water in the presence of ethylene glycol, and to use this information to develop a method to vitrify human oocytes.

Design: An incomplete randomized block design.

Setting: A university-affiliated assisted reproductive center.

Patient(s): Women undergoing assisted reproduction in the Center for Reproductive Medicine at Shandong University.

Intervention(s): Oocytes were exposed to 1.0 molar ethylene glycol in a single step and photographed during subsequent volume excursions.

Main outcome measure(s): A two-parameter model was employed to estimate the permeability to water and ethylene glycol.

Result(s): Water permeability ranged from 0.15 to 1.17 microm/(min.atm), and ethylene glycol permeability ranged from 1.5 to 30 microm/min between 7 degrees C at 36 degrees C. The activation energies for water and ethylene glycol permeability were 14.42 Kcal/mol and 21.20 Kcal/mol, respectively.

Conclusion(s): Despite the lower permeability of human metaphase II oocytes to ethylene glycol compared with previously published values for propylene glycol and dimethylsulfoxide, methods to add and remove human oocytes with a vitrifiable concentration of ethylene glycol can be designed that prevent excessive osmotic stress and minimize exposure to high concentrations of this compound.

Figure 1

The steps in the procedure used for image analysis are shown. Panel A shows an original photograph. Notice that the oolemma is darker than its surroundings. A threshold process was applied to the original image, isolating pixels based upon their grayscale value. The result is shown in Panel B. The next process involved filling in areas that are completely surrounded by black pixels (Panel C). Finally, all objects smaller than a minimum size (chosen as 3000 pixels) and those touching an edge of the image were rejected, leaving only the oocyte (Panel D). The area of this object was determined by summing the total number of pixels and multiplying this value by the area per pixel. From this area, the total volume of the oocyte was determined assuming spherical geometry.

Figure 2

Examples of the volume changes that representative oocytes experienced during equilibration with 1.0 mol/L EG at 4 different temperatures are shown. Notice that lower temperatures are associated with a greater total volume loss and a slower return to isotonic volume.

Figure 3

Arrenhius plots of the relationship between temperature and L_p (left) and P_s (right) are shown along with linear regression lines.

Figure 4

Examples of DSC thermograms during warming for solutions containing 0.3 mol/kg sucrose, EG and saline, with a total solute concentration ranging from 55 to 59 wt % (adjusted by increasing the amount of EG). The primary thermal transitions, including the glass transition, crystallization, and melting are noted on the top graph. Notice how the magnitude of the crystallization and melting transitions diminish as the concentration increases from 55 to 59 wt %.

Figure 5

This plot demonstrates changes in the relative cell volume of MII human oocytes resulting from equilibration in vitrification solutions containing varying compositions of EG and sucrose in saline (black circles) having a total solute concentration necessary to maintain a vitreous state as described in the results from experiment 3. The equilibrium volume was calculated by solving equation 1 and equation 2 using the concentrations of permeable (EG) and non-permeable (sodium chloride and sucrose) solutes in the respective solutions (please see text for further details). As the sucrose concentration is increased, the amount of EG in the solution necessary to achieve and maintain a vitreous state during cooling and warming decreases. However, as the sucrose concentration increases, a greater effect on the cell volume will occur. For our choice of an optimal solution, we wanted a combination of EG and sucrose such that the solution would have the maximum amount of sucrose (reducing the EG concentration) yet would not result in excessive cell shrinkage. The lower osmotic tolerance chosen for this analysis would result in the cell shrinking to 57 % of its isotonic volume (horizontal line). Therefore, the optimal solution will occur where the curve intersects the horizontal line. This occurs when the sucrose concentration is 0.75 mol/kg (0.40 mol/L). The corresponding EG concentration is 12.5 mol/kg (6.72 mol/L).