Comparison and Avoidance of Toxicity of Penetrating Cryoprotectants

Abstract

The objective of this study was to elucidate the toxicity of widely used penetrating cryoprotective agents (CPAs) to mammalian oocytes. To this end, mouse metaphase II (M II) oocytes were exposed to 1.5 M solutions of dimethylsulfoxide (DMSO), ethylene glycol (EG), or propanediol (PROH) prepared in phosphate buffered saline (PBS) containing 10% fetal bovine serum. To address the time- and temperature-dependence of the CPA toxicity, M II oocytes were exposed to the aforementioned CPAs at room temperature (RT, ∼23°C) and 37°C for 15 or 30 minutes. Subsequently, the toxicity of each CPA was evaluated by examining post-exposure survival, fertilization, embryonic development, chromosomal abnormalities, and parthenogenetic activation of treated oocytes. Untreated oocytes served as controls. Exposure of MII oocytes to 1.5 M DMSO or 1.5 M EG at RT for 15 min did not adversely affect any of the evaluated criteria. In contrast, 1.5 M PROH induced a significant increase in oocyte degeneration (54.2%) and parthenogenetic activation (16%) under same conditions. When the CPA exposure was performed at 37°C, the toxic effect of PROH further increased, resulting in lower survival (15%) and no fertilization while the toxicity of DMSO and EG was still insignificant. Nevertheless, it was possible to completely avoid the toxicity of PROH by decreasing its concentration to 0.75 M and combining it with 0.75 M DMSO to bring the total CPA concentration to a cryoprotective level. Moreover, combining lower concentrations (i.e., 0.75 M) of PROH and DMSO significantly improved the cryosurvival of MII oocytes compared to the equivalent concentration of DMSO alone. Taken together, our results suggest that from the perspective of CPA toxicity, DMSO and EG are safer to use in slow cooling protocols while a lower concentration of PROH can be combined with another CPA to avoid its toxicity and to improve the cryosurvival as well.

Optimization of cryoprotectant loading into murine and human oocytes

“…Some studies have suggested that cell volume excursions in excess of ±30% should be avoided when loading CPAs into human oocytes [52; 58; 59]. Considering the variability in oocyte quality [67; 72] and difference in toxicity of various CPAs [68], in the present study, we implemented an additional safety margin and limited volume excursions of oocytes to ±25% when optimizing CPA loading protocols. It should be noted that the simplified version of our two-step protocol for room-temperature loading exceeded our preset cell volume constraints and resulted in a volume excursion of 28%, but still delivered excellent results similar to controls, suggesting that mouse oocytes can tolerate volume excursions beyond ±25% in the presence of Me 2 SO.…”

“…It is important to note that 1.5 M Me 2 SO solution was prepared in PBS supplemented with BSA (i.e., in the absence of any appreciable Ca 2+ and Mg 2+ ). In our previous study, when mouse M II oocytes were exposed to 1.5 M Me 2 SO prepared in PBS containing 10% FBS and thus small amounts of extracellular Ca 2+ and Mg 2+ , no significant adverse effect was observed [68]. We have performed similar experiments with PROH (i.e., exposure of mouse oocytes to 1.5 M PROH in the presence and absence of extracellular Ca 2+ /Mg 2+ ) and obtained similar results in our recent study, suggesting that extracellular presence/absence of Ca 2+ /Mg 2+ modulates cell injury induced by CPA loading [69].…”

“…Known manifestations of oocyte cryoinjury include intracellular ice formation [45], cell lysis [6], osmotic stress [1], disruption of cytoskeleton and spindle microtubules [18; 75], premature exocytosis of cortical granules and zona hardening [12; 64], parthenogenetic activation [65; 68; 73], and polyploidy [2; 18; 25]. Only after a decade of additional research and implementation of intracytoplasmic sperm injection (ICSI), it has become possible to mitigate some of these cryoinjury mechanisms, and to reproduce the initial success of human oocyte cryopreservation [40; 60; 71].…”

“…It is known that such insults compromise oocyte viability and developmental capacity [1; 32; 34; 53; 65; 68]. Consequently, optimization of CPA loading procedures is of importance to maximize the probability of success in oocyte cryopreservation.…”

Large-volume vitrification of human biopsied and non-biopsied blastocysts: a simple, robust technique for cryopreservation

“…For example, both DMSO and ethylene glycol were found to induce a transient increase in intra-cellular calcium in mouse oocytes [14] and further, DMSO was responsible for release of calcium from intracellular stores, as opposed to the influx of calcium from extracellular culture media generated by ethylene glycol exposure. For mouse oocyte cryopreservation, the potential for cryoprotectant toxicity was reduced by eliminating or combining cryoprotectants, and exposing oocytes to these agents at room temperature rather than 37°C [15]. In regard to post-fertilization embryonic cells, recent reports demonstrate cellular/genetic perturbations following DMSO exposure for mouse blastocysts [16], mouse embryoid bodies [17], and human induced pluripotent stem cells [18].…”