As freezing and thawing of human spermatozoa may affect the post-thaw qualities, including the sperm morphology, motility, DNA integrity and viability, optimization of the cryopreservation protocol is vital for the success of assisted reproductive technologies. According to a recent study published in the journal Reproductive BioMedicine Online, the supplementation of the cryopreservation medium with vitamin E may enhance the motility of human spermatozoa post-thaw, especially in patients with abnormal semen parameters.
The research headed by K Taylor, from the Concept Fertility Centre, King Edward Memorial Hospital, Western Australia, analyzed the effect of antioxidant supplementation of cryopreservation medium in augmenting the post-thaw integrity of cryopreserved human spermatozoa. During the study, both normal (n=23) and abnormal semen samples (n=20) were collected by masturbation and were assorted into three aliquots before performing the cryopreservation. The second and third aliquots were added to sperm freezing medium containing 100 µmol or 200 µmol vitamin E analogue, while the first aliquot was mixed with cryopreservation medium only.
The cryopreservation protocol included freezing the samples at –10 °C to –80 °C per minute, and plunging them into liquid nitrogen. After the thawing procedure, vitality, motility and DNA integrity of the samples were evaluated. Further evaluation were carried out using split-plot repeated-measures analysis of variance (ANOVA) to study the difference in vitality staining, post-thaw motility index, and DNA fragmentation within the subject (dose), and between normal and abnormal groups. The study results showed a significant association between vitamin E dose and post-thaw motility (P=0.041). Also, the pattern of response across doses was found comparable in both normal and abnormal groups. Although 200 µmol supplementation of vitamin E did not alter the vitality or sperm DNA fragmentation, it substantially improved the post-thaw motility of cryopreserved human spermatozoa.
In a similar study, Stanic, et al. (International Journal of Andrology, 2002) evaluated the effect of pentoxifylline, a vasodilator indicated to treat intermittent claudication, on sperm motility and membrane integrity prior to and after the cryopreservation-thawing procedures. After dividing the 24 semen samples into 4 equal aliquots, they were incubated at 37 °C for 30 minutes. A slow programmable freezing protocol was used for the cryopreservation of the samples using TEST-yolk freezing medium. The aliquots were thawed, washed twice with Quinn’s Sperm Washing Medium, and incubated at 37 °C for 30 minutes, following two weeks of cryopreservation. Further, the sperm samples were variously treated with 3 mmol/L of pentoxifylline:
• during the period of incubation prior to cryopreservation
• as a supplement to the cryoprotectant before cryopreservation
• after thawing during the period of incubation
The supplementation of fresh samples with 3 mmol/L pentoxifylline during the incubation period before the cryopreservation caused a substantial reduction in the progressive and total motility, when compared to the controls without treatment. However, a significant escalation in the total and progressive motility was observed in the cryopreserved samples added with pentoxifylline after thawing. The comparison between controls and samples, with pentoxifylline as a supplement to cryoprotectant, did not show any significant difference in motion characteristics post-thaw.
Also, the post-thaw hypoosmotic swelling (HOS) test scores did not show substantial improvement with the supplementation of pentoxifylline. The study results concluded that the post-thaw motility of cryopreserved human spermatozoa can be enhanced by the addition of pentoxifylline after thawing.
The addition of various antioxidants, such as vitamin C, vitamin E, and pentoxifylline, to the sperm freezing media may help to augment the fertilization potentials of the cryopreserved sperms, thereby increasing the success rates of ART cycles.
References
1. Taylor K, Roberts P, Sanders K, Burton P. Effect of antioxidant supplementation of cryopreservation medium on post-thaw integrity of human spermatozoa. Reprod Biomed Online. 2009 Feb;18(2):184-9.
2. Stanic P, Sonicki Z, Suchanek E. Effect of pentoxifylline on motility and membrane integrity of cryopreserved human spermatozoa. Int J Androl. 2002 Jun;25(3):186-90.
