Permeability of human oocytes to ethylene glycol and their survival and spindle configurations after slow cooling cryopreservation

Abstract

BACKGROUND: To develop novel cryopreservation methods, we estimated the permeability coefficients Lp (hydraulic conductivity) and P EG (cryoprotectant permeability) of mature human oocytes after exposure to ethylene glycol (EG) and tested the efficiency of a multi-step slow cooling protocol based on this cryoprotectant. METHODS: Oocytes were perfused with 1.5 mol/l EG for 10 min. Oocyte volume at each time point was calculated and normalized to the original volume. Slow cooling was conducted by exposing oocytes to increasing EG concentrations (0.5, 1.0 and 1.5 mol/l n 5 155) or 1.5 mol/l of propane-1,2-diol (PrOH) n 5 102. Oocytes which survived cryopreservation n 5 80 and fresh oocytes n 5 73 were prepared for confocal microscopy analysis of the meiotic spindle. RESULTS: During EG exposure, oocytes underwent an abrupt 50% volume reduction. Complete recovery of the initial volume was not observed. From the values of a best fit plot, the coefficients Lp 5 0.82 + + + + + 0.29 mm min 21 atm 21 (mean + + + + + SD) and P EG 0.10 + + + + + 0.01 mm s 21 were generated. Survival rates after freezing with EG were lower than with PrOH (51.6 versus 71.5%, respectively, P < 0.05). The frequencies of normal spindle configuration were lower in frozen EG and frozen PrOH oocytes compared with fresh oocytes (53.8, 50.9 and 66.7%, respectively, P < 0.05). CONCLUSIONS: The oocyte plasmalemma possesses limited permeability to EG and EG exposure causes considerable osmotic stress. However, post-thaw rates of survival and normal meiotic spindle organization may be preserved by protocols which are designed in order to minimize osmotic stress.

In Vitro Oocyte Maturation and Preantral Follicle Culture from the Luteal-Phase Baboon Ovary Produce Mature Oocytes

“…Spindle morphology and chromosome alignment of in vitro matured oocytes from small antral follicle COCs was assessed using a previously described classification system [36]. Figure 3, A-D, shows representative images from the 1L-COC and ML-COC for each of the four classifications of nuclear status: 1) bipolar spindle/aligned chromosomes show bipolar organization with pointed or flattened poles, microtubules converging at both poles, and all chromosomes present and evenly aligned at the equatorial plate (Fig.…”

Qualitative and morphometric analysis of the ultrastructure of human oocytes cryopreserved by two alternative slow cooling protocols

“…Disagreement also exists on the entity and type of cell damage that the oocyte may suffer as a result of cryopreservation. For example, several lines of evidence have been reported for and against the hypothesis that the metaphase II (MII) spindle can be irreversibly disrupted by low temperature storage [8][9][10][11][12][13]. In effect, during the process of freezing-thawing (or vitrification-warming) the oocyte is exposed to a variety of physical and chemical conditions, such as low or suboptimal temperatures, formation of intracellular ice crystals (IIC), osmotic stress and replacement of intracellular water with cryoprotective agents (CPAs), that may endanger its mere survival [14].…”

Ultrastructural evaluation of human metaphase II oocytes after vitrification: closed versus open devices

“…Until recently, only slow freezing methods were applied, resulting in poor oocyte survival, pregnancy, and implantation rates (3)(4)(5)(6). Despite the implementation of modified protocols (7)(8)(9), alterations in the appearance of cytoplasm, mitochondria, zona pellucida (ZP), meiotic spindle, and cortical granules, along with impaired intracellular Ca 2þ fluxes, have been reported (10)(11)(12)(13)(14)(15)(16)(17)(18)(19).…”