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How Does Cryopreservation Damage Embryos and Eggs?

Cryopreservation involves cooling cells in special culture media, adding a cryo-protectant (molecules that protect the cell from damage) and ultimately bringing the cells down to a temperature where all cell processes stop and the cells are stored in liquid nitrogen long term. The temperature is -196 ° C. When the cells are to be thawed, the cells are taken from liquid nitrogen, thawed, cryoprotectants removed and cell cultured. Several factors are associated with cooling and cryopreservation that lead to cell injury and compromise post-thaw function and development. The basic mechanisms of direct cell injury is due to, in part and depending on the cooling rate, 1; intracellular ice formation secondary to rapid cooling of intracellular water which nucleates and forms intracellular ice and 2; extra-cellular ice formation leading to increased electrolyte in unfrozen sections and cell dehydration (1). Dehydration and re-hydration induces mechanical stresses on lipid membranes which have been shown to cause physical deformities and changes in the phase behavior using in vitro models of membrane solute interactions (2). These physical alterations may affect the cell at many levels, with some cells being more sensitive. The cell membrane and cellular organelles may be altered. The spindle apparatus and hence chromosome separation may be damaged. Recently, certain genes have been shown to be induced by the freezing and thawing process, suggesting that the cell is actively responding to an insult.

Slow freezing combined with cryoprotectants allows cells to be protected from the detrimental mechanisms noted above by decreasing intracellular ice and effects of increasing solute concentrations. The extracellular ice drives the dehydration of the cell through an equilibrium process. A more rapid technique, vitrification, uses high levels of cryoprotectants and very rapid rates of cooling to minimize the formation of intracellular ice. It is a non-equilibrium technique.

Freezing and ice formation induce dehydration of the bi-lamellar (i.e. two-layered) lipid membrane. The liquid crystallization to gel phase transition with freezing results in an ordering of the phospholipid hydrocarbon tails into a more packed and parallel array which may lead to increased permeability to water and cations, segregation of integral membrane proteins, decreased activities of membrane bound enzymes, and decreased lateral diffusion of proteins (3). It is likely that spermatozoa and oocytes experience similar effects, though their lipid constitution is quite different. Mature oocytes have microtubules located at the meiotic spindle. Reducing the temperature to 25° C for 10 minutes disrupts the microtubules and complete dissolution occurs at 0° C. Re-warming in the mouse leads to reconstitution of the microtubules, however this may not occur in humans due to deficiency of pericentriolar material needed to nucleate tubule polymerization (3). The resultant oocyte may be prone to aneuploidy (i.e. abnormal numbers of chromosomes) as a result of spindle disorganization. Freezing may also affect fertilization by decreasing cortical granules and decreasing the chymotripsin sensitivity of the zona pellucida. The shell may be, in effect, too ‘hard.’ Linford and Meyers (4) showed that sperm DNA may be damaged using single cell gel electrophoresis. Mitochondria are also susceptible to cryopreservation damage (5). The degeneration of oocyte DNA during cryopreservation and subsequent culture appears to involve the process of apoptosis with classic markers and caspases detected (6). Thus the cells are undergoing programmed cell death. It is of interest that genes involved with stress responses including heat shock proteins, oxidative stress scavengers, and enzymes involved with glucose metabolism are also activated(7).

In summary, cryopreservation is not a benign process and many of the functions of the cell may be affected. It is helpful to understand all of these factors so that we may improve this technique to offer patients more with their IVF cycles.

References:

1. Han B, Bischoff JC. Direct cell injury associated with eutectic crystallization during freezing. 2004. Cryobiology 48: 8-21.

2. Wolfe J, Bryant G. Freezing, drying, and/or vitrification of membrane-solute-water systems. 1999. Cryobiology 39:103-129.

3. Parks JE. Hypothermia and mammalian gametes. In Reproductive Tissue Banking. Eds. Karow AM, Critser JK. 1997. Academic Press. San Diego. Pp. 229-261.

4. Linford JJ, Meyers SA. Detection of DNA damage in response to cooling injury in equine spermatozoa using single-cell gel electrophoresis. 2002. J. Androl. 23(1):107-113.

5. O’Connell M, McClure N, Lewis SE. The effects of cryopreservation on sperm morphology, motility and mitochondrial function. 2002 Hum. Rep. 2002 17(3):704-709.

6. Men H, Monson RL, Parrish JJ, Rutlege JJ. Degeneration of cryopreserved bovine oocytes via apoptosis during subsequent culture. 2003. Cryobiology. 47: 73-81.

7. Odani M, Komatsu Y, Oka S, Iwahashi H. Screening of genes that respond to cryopreservation stress using yeast DNA microarray. 2003. Cryobiology. 47:155-164.
 

 

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