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.

In vitro versus In vivo: Development-, Apoptosis-, and Implantation- Related Gene Expression in Mouse Blastocyst.

BACKGROUND: While mammalian embryos can adapt to their environments, their sensitivity overshadows their adaptability in suboptimal in vitro conditions. Therefore, the environment in which the gametes are fertilized or to which the embryo is exposed can greatly affect the quality of the embryo and consequently its implantation potential. OBJECTIVES: Since providing an optimal culture condition needs a deep understanding of the environmental effects, and regarding the fact that normal morphology fails to be a reliable indicator of natural embryo development, the current study aimed at comparing in vivo- and in vitro-derived blastocysts at the molecular level. MATERIALS AND METHODS: In vivo and in vitro mouse blastocysts were obtained by flushing the uterine horns and in vitro fertilization/culture, respectively. Normal blastocysts of both groups were evaluated in terms of hatching rate and expression of three lineage-differentiation-, apoptosis-, and implantation-related genes. RESULTS: The hatching rate was lower in In vitro fertilization (IVF)-produced blastocysts in comparison with that of the in vivo counterparts. More importantly, the study results indicated significant changes in the expression levels of eight out of ten selected genes, especially Mmp-9 (about -10.7-fold). The expression of Mmp-9 in trophoblast cells is required for successful implantation and trophoblast invasion. CONCLUSIONS: The current study, in addition to confirming that the altered gene expression pattern of in vitro-produced embryos resulted in normal morphology, provided a possible reason for lower implantation rate of in vitro-produced blastocysts regarding the Mmp-9 expression.