There exists ample evidence on the possibility of cryopreservation negatively affecting sperm DNA integrity; however, its precise underlying mechanism is still much debated. Now, a recent study published in the online issue of the journal Human Reproduction, reports that cryopreservation-induced injury to sperm DNA is mediated primarily through oxidative stress rather than apoptosis.

Laura Kelly Thomson, from the Fertility First-Centre for Reproductive Health, Hurstville, Australia, and co-workers, conducted the study to investigate the cause of sperm DNA damage during cryopreservation in semen samples of 60 patients using the following:
• Oxidative stress biomarker: 8-oxo-7, 8-dihydro-2′deoxyguanosine (8OHdG)
• Apoptosis indicator: percentage caspase positive cells
• Antioxidant: genistein
• Caspase inhibitor: Z-VAD(OMe)-FMK

The researchers noted that cryopreservation considerably increased the percentage of DNA fragmentation, 8OHdG, and caspase positive cells (P<0.001). There was a positive correlation between the percentage of DNA fragmentation and 8OHdG, before and after cryopreservation (P<0.001 vs. P=0.017). It was also reported that incorporation of 50 µM and 100 µM of genistein to the cryoprotectant, significantly protected sperm DNA, while no such effect was observed with the addition of Z-VAD(OMe)-FMK. Based on the findings, the researchers concluded that sperm DNA fragmentation is linked to elevated oxidative stress rather than caspase activation and apoptosis stress, during cryopreservation. It was also suggested that genistein could aid in reducing oxidative stress and thereby DNA fragmentation.

Previously, Zribi et al (Fertility and Sterility, 2008) conducted a study to assess the effect of cryopreservation on sperm DNA integrity, and sperm motility and viability, in 15 semen samples of men undergoing infertility examination. The samples were cryopreserved in liquid nitrogen using SpermFreeze™ freezing medium and then thawed at room temperature for analysis. Sperm DNA fragmentation and oxidation rates were determined using terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick-end labeling and fluorescent assay (to detect 8-oxoguanine), respectively, and then evaluated using flow cytometry, before and after cryopreservation.

The researchers noted a considerable decline in sperm motility and viability after cryopreservation and an increase in oxidative DNA damage and DNA fragmentation following cryostorage/thaw. The findings proposed that the induction of DNA fragmentation and oxidation during cryopreservation exerts deleterious effects to sperm DNA.

In order to investigate if antioxidant (ascorbate, catalase [CAT], or superoxide dismutase) supplementation during cryopreservation would offer protection against oxidative sperm DNA damage, Li et al (Chinese Journal of Medical Genetics, 2007) conducted a study on semen samples from 30 fertile men. The samples were analyzed for nuclear DNA integrity, sperm motility, viable recovery rate, and reactive oxygen species (ROS) before and/or after the process of freeze-thawing. The findings suggested that ascorbate or CAT supplementation during freeze-thawing could reduce ROS levels that trigger sperm nuclear DNA damage, and improve sperm quality.

Cryopreservation has been attributed to trigger and increase the production of ROS in sperms, resulting in oxidative stress. Generation of high levels of ROS in turn lead to a high incidence of membrane lipid peroxidation, DNA strand breaks (base modification, chromatin cross-link, and DNA fragmentation), and protein damage, thereby affecting sperm motility, function and viability.

Despite advancements in cryopreservation techniques, the number of functional spermatozoa available following the procedure is still unsatisfactory. Several studies have reported that incorporation of antioxidants into the freezing media could be one of the means to effectively inhibit cryopreservation-associated sperm DNA oxidative damage. An earlier review study by Chen et al (National Journal of Andrology, 2007) suggested that improving semen quality prior to cryopreservation, selection of optimal spermatozoa, incorporation of suitable cryoprotectants, and following appropriate thawing procedures could remarkably reduce the damage of cryopreserved sperms and improve ART success rate.

References

1. Thomson LK, Fleming SD, Aitken RJ, De Iuliis GN, Zieschang JA, Clark AM. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Hum Reprod. 2009 Jun 12. [Epub ahead of print]

2. Zribi N, Chakroun NF, El Euch H, Gargouri J, Bahloul A, Keskes LA. Effects of cryopreservation on human sperm deoxyribonucleic acid integrity. Fertil Steril. 2008 Nov 20.

3. Li ZL, Lin QL, Liu RJ, Xie WY, Xiao WF. Reducing oxidative DNA damage by adding antioxidants in human semen samples undergoing cryopreservation procedure. Chinese Journal of Medical Genetics. 2007 Dec 4;87(45):3174-7.

4. Chen Y, Liu RZ. Cryopreservation of spermatozoa. National Journal of Andrology. 2007 Aug;13(8):734-8.