Introduction Cryopreservation Effects on Sperm Fertility

The spermatozoon, like all cells living under aerobic conditions, constantly faces the oxygen paradox; oxygen is required for life, but oxidative metabolism of biological molecules can be potentially toxic due to the formation of reactive oxygen

School of Veterinary Medicine, Anatomy, Physiology, and Cell Biology, University of California, Davis, CA, USA e-mail: [email protected]

A. Agarwal et al. (eds.), Studies on Men's Health and Fertility, Oxidative Stress 41

in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-61779-776-7_3, © Springer Science+Business Media, LLC 2012

species (ROS) that can modify cell functions or viability. Although oxidative stress was suggested as an important factor in disruption of sperm function over 60 years ago [1, 2], it is only within the past 10-15 years that the importance of oxidative stress has gained a wider understanding. Recent studies indicate that ROS play an important role in normal sperm function; however, an imbalance in the production or degradation of ROS may have adverse consequences for sperm function. Excessive production of ROS could be responsible in part for loss of viable, fertilization-competent sperm either directly or indirectly by intensifying the cellular response to other stressors, such as osmotic imbalance.

Long-term storage of numerous mammalian somatic and germ cell types has been accomplished using cryopreservation, typically in liquid nitrogen (cryogenic tanks) or in other subzero environments (-80°C ultra-low-type freezer). Conventional cryopreservation for sperm is termed equilibrium, or slow, freezing of the bulk of water, followed by storage at extremely low temperatures, usually -196°C, although other types of low-temperature freezing such as vitrification and lyophilization have been reported [3-7]. The process of cryopreservation has profound effects on cells, many of which result in sublethal damage and subsequent reduction of function. Many cell types do not tolerate frozen storage above -80°C and undergo severe deterioration with subsequent lethal damage. Some hematopoietic and human embryonic stem cell lines and gametes undergo sublethal and lethal changes associated with the complex interaction of low-temperature cryoprotective agent (CPA) reagents, osmotic and oxidative balance, solute and electrolyte balance, and ice crystallization [8, 9], However, in the presence of CPAs, which can be cytotoxic, many cell types can survive low-temperature storage. As a routine consequence of cryogenic storage, approximately 25-75% of cells stored this way are lost due to necrotic and apoptotic cell death and this is dependent on cell type, freezing rate, and CPA. Cryoprotectants have been broadly classified as penetrating and non-penetrating substances. Penetrating CPAs are small nonionic molecules (glycerol, dimethylsulfoxide, propylene glycol, ethylene glycol, methylformamide) with high water solubility while the nonpenetrating CPAs are long-chain polymers or sugars (methylcellulose, sucrose, raffinose, trehalose). One of the most significant origins of cell damage and death during freezing is that of intracellular and extracellular ice formation. Cryoprotectants act primarily by reducing the speed at which ice forms and the size of ice crystals [10, 11] , With adequate CPA, freezing damage can be minimized. However, high concentrations of CPA can cause osmotic and toxic cell damage. Thus, CPA addition and removal can have long-lasting cellular effects.

Although cryopreservation of ejaculated sperm has been in clinical and agricultural use for decades, it is not completely clear how the damage that sperm incur as a result of cryopreservation contributes to fertilization failure, or embryonic or fetal loss. This problem is multifactorial and has species-specific contributing factors [12], Frozen ejaculated semen has enabled the cattle industry to make significant genetic progress since the 1950s when artificial insemination (AI) was embraced commercially. However, due to species variability and individual male variability, the use of AI with frozen semen has been less successful in the swine, sheep, and horse breeding industries. A considerable issue has been that of male variation, but in swine and horses the relatively poor sperm survivability has magnified the high variability among males intended to be breeding males [10, 13]. In pigs, the use of frozen semen for AI has resulted consistently in decreased farrowing rates and litter sizes [14, 15], thus limiting the usefulness of frozen semen for this economically important industry. In sheep, frozen semen has also resulted in decreased pregnancy rates [10], although insemination practices have recently increased pregnancy rates. In human medicine, sperm cryopreservation is widely used for insemination both in vivo and in vitro, but successful pregnancy rates are highly variable and male dependent. The use of assisted reproductive technologies, such as intracytoplasmic sperm injection, has obviated a portion of the need, in large part, to greatly improve cryopreservation success in humans. The bovine artificial insemination industry has had great success for more than 50 years owing to active selection for bulls with high fertility with frozen sperm. It is no surprise that a considerable amount of sub-lethal and lethal damage occurs as a result of exposure to the extreme temperature and osmolality effects associated with cryopreservation [16-19]. Cryopreservation poses a severe osmotic insult to the cell that leads to generation of ROS; this process can be minimized but not eliminated by using CPA agents. Additionally, there is substantial evidence that cryopreservation results in increased DNA damage, aneu-ploidy, and chromosome fragmentation [5, 20-22]. Lipid peroxidation can also contribute directly to specific sublethal effects, like chromatin cross-linking, base changes, and DNA strand breaks [23-26] , A limited ability to store antioxidant enzymes combined with a membrane rich in unsaturated fatty acids makes spermatozoa particularly susceptible to oxidative stress and peroxidative attack by ROS, specifically superoxide anion and hydrogen peroxide [27].

There is clinical evidence that damage to sperm DNA results in downstream impaired embryo development and pregnancy in mice and humans [28-31]. High levels of ROS have been associated with sperm DNA damage in the semen of 25% of infertile men [32]. Recent evidence indicates that men with high DNA fragmentation indices (DFIs) have significantly higher rates of spontaneous abortion in their partners [33]. Further, apoptotic processes may contribute to DNA damage in sperm [34]. It has been proposed that some sperm with DNA damage may have initiation and possibly escape of apoptosis [35]. There is also evidence for a number of other inducers of DNA damage that are associated with embryonic loss and infertility, such as tobacco use, environmental toxins, chronic orchitis/genital tract inflammation, varicoceles, radiation, chemotherapeutic drugs, and testicular hyperthermia [34],

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