Cryopreservation of Gametes and Embryos and Their Molecular Changes

Abstract

The process of freezing cells or tissues and depositing them in liquid nitrogen at –196 °C is called cryopreservation. Sub-zero temperature is not a physiological condition for cells and water ice crystals represent the main problem since they induce cell death, principally in large cells like oocytes, which have a meiotic spindle that degenerates during this process. Significantly, cryopreservation represents an option for fertility preservation in patients who develop gonadal failure for any condition and those who want to freeze their germ cells for later use. The possibility of freezing sperm, oocytes, and embryos has been available for a long time, and in 1983 the first birth with thawed oocytes was achieved. From the mid-2000s forward, the use of egg vitrification through intracytoplasmic sperm injection has improved pregnancy rates. Births using assisted reproductive technologies (ART) have some adverse conditions and events. These risks could be associated with ART procedures or related to infertility. Cryopreservation generates changes in the epigenome of gametes and embryos, given that ART occurs when the epigenome is most vulnerable. Furthermore, cryoprotective agents induce alterations in the integrity of germ cells and embryos. Notably, cryopreservation extensively affects cell viability, generates proteomic profile changes, compromises crucial cellular functions, and alters sperm motility. This technique has been widely employed since the 1980s and there is a lack of knowledge about molecular changes. The emerging view is that molecular changes are associated with cryopreservation, affecting metabolism, cytoarchitecture, calcium homeostasis, epigenetic state, and cell survival, which compromise the fertilization in ART.

Aquaporins and Animal Gamete Cryopreservation: Advances and Future Challenges

“…In cellular cryopreservation, cells are excluded from the forming ice matrix and the increased solute concentration constitutes a major damaging factor during this process. Gamete cryopreservation leads to several cellular and molecular alterations (reviewed in detail by Estudillo et al [17]). Among them, the most important to highlight are the damage to the cellular and intracellular membranes [18] and ROS formation [19].…”

“…After cooling to a raging temperature of −5 to −7 • C, cells are exposed to a slow temperature decrease of about 0.3 to 0.5 • C per minute until reaching a temperature between −30 and −65 • C. Straws are then directly immersed into liquid nitrogen to rest for as long as cryopreservation is needed [62]. This procedure is unique by its slow cooling rate that, in combination with the adequate concentration of CPAs, ensures that the formation of ice crystals only happens outside the cell [17]. This mechanism happens due to slow and controlled loss of water as the temperature decreases and consequent concentration of the solutes that prevent ice crystals formation in the intracellular medium.…”

“…The first protocol developed to cryopreserve embryos by vitrification was used by Rall et al in 1987 [65]. This protocol is based on the incubation of embryos and oocytes in a solution of multiple CPAs in high concentrations (up to 8 M) [17,63]. Then, embryos or oocytes are divided into thin straws and submitted to cooling rates of −200 • C per minute.…”

“…incubation of embryos and oocytes in a solution of multiple CPAs in high concentrations (up to 8 M) [17,63]. Then, embryos or oocytes are divided into thin straws and submitted to cooling rates of −200 °C per minute.…”

“…Membrane permeability is crucial for preventing intracellular ice crystal formation during the controlled freezing protocol. Despite that, both cryopreservation protocols inflict damage and changes to the cells on multiple fronts (for review see [17]). Controlled-rate slow freezing was found to inflict more structural damage to mitochondria and other organelles, including cytoskeleton, plasmatic and nuclear membrane in bovine embryos [88].…”