Thermally conductive graphene-based nanofluids, a novel class of cryosolutions for mouse blastocysts vitrification.

Limited heating and cooling rates have long been recognized as bottlenecks in improving embryo cryopreservation. As a result, efforts to achieve higher heat transfer rates gave rise to milestones like open cryodevices and minimal media loading. A crucial but commonly ignored variable is heat conduction by cryosolutions. The low heat conductivity of the aqueous media surrounding embryos slows down cooling and heating rates of the embryo, imposing the risk of preventable damages. In this study, we introduce a novel thermally conductive cryosolution based on graphene oxide nanoparticles and test its performance against conventional sucrose-based solutions for vitrification of mouse blastocysts. Replacing sucrose with graphene oxide brought about similar re-expansion, hatching, and implantation rates of post-vitrification embryos while also preventing an array of cellular and molecular stresses. Our results showed significantly reduced oxidative stress, characterized by control-level expression of Sod1 and significant downregulation of Sod2 transcription when graphene oxide was used instead of sucrose. This molecular response was in agreement with the reduced level of reactive oxygen species produced in vitrified/warmed embryos using graphene-based solutions. The downstream impacts of this stress reduction manifested in significant downregulation of two major pro-apoptotic genes, Bax and Trp53, down to the same level as fresh embryos. Interestingly, embryos maintained their spherical shape during dehydration in graphene-based solutions and did not "collapse" when shrinking, like in sucrose-based solutions. These results provide new insights into the benefits of thermally conductive cryosolutions and showcase the potential of graphene oxide as a cryoprotectant in embryo vitrification.

Using natural honey as an anti-oxidant and thermodynamically efficient cryoprotectant in embryo vitrification.

Embryo cryopreservation is a common practice in reproductive biology and infertility treatments. Despite major improvements over years, the cryoprotectant solutions are still a major source of concern, mostly due to their chemical toxicity and suboptimal protection against cryoinjuries. In this work, we introduced natural honey as a non-permeating cryoprotectant to replace traditionally used sucrose in embryo vitrification. The proposed media were compared with conventional ones by evaluating vitrified/warmed mouse embryos based on their re-expansion, hatching rate and transcription pattern of selected genes involved in heat-shock response, apoptosis and oxidative stress. Despite the similar high re-expansion rate, molecular fingerprint of the cryopreservation is remarkably reduced when honey is used instead of sucrose. The biological response of the proposed media was explained from a fundamental point of view using antioxidant analysis, DSC and GC techniques. It was found that the proposed honey-based medium is less thermodynamically prone to ice formation, which along with its antioxidant capacity can control the production of oxygen radicals and minimize the stress-induced transcriptional response. Furthermore, this work tries to correlate the physico-chemical properties of the vitrification solutions with the cellular and molecular aspects of the cryopreservation and proposes the application of natural cryoprotectants in cryobiology.