Received August 17, 2018
Accepted July 19, 2019

Objective: To compare cryo-survival rate of human spermatozoa in a prolonged period of cryopreservation. Methods: This retrospective study involved 33 cryopreserved semen samples from patients with cancer diagnosis between 2002 and 2011. Semen sample was obtained by masturbation and semen analysis was performed. Cryoprotectant solution was added and samples were frozen in liquid nitrogen in slow step-wise process. For thawing, samples were incubated at 25 °C for 15 min, followed by incubation at 36.7 °C for 15 min. Cryo-survival rate (CS) was calculate by CS= [(% total motile sperm post-thaw) x100/(% total motile sperm/tube)]. Each study sample was divided into (Study Group; n = 23): (I) official patient sample, which was kept cryopreserved for Assisted Reproduction procedure, cryopreserved between 2002 and 2011; (II) sample destined to post-thaw tests, performed after 24 hours; and (III) research sample. In 2014, after 3–12 years of cryopreservation, research samples were thawed. Validation Group was included 10 samples obtained between 2014 and 2016, using the same methodology in the study samples. Data were analyzed using T-test and adopted a significant p-value of 5%. Results: Mean age was 29.93 ±9.57 y.o. in Study Group and 21.80 ±6.49 y.o. in Validation Group. No significant difference between Validation and Study Groups was observed in semen analysis (p>0.05). After 24 hours of cryopreservation, cryo-survival rate was 26.11 ±46.36% in Study Group and 23.71 ±57.06% in Validation Group. Aliquots of the same sample preserved to 3-12 years demonstrated 23.71 ±57.06% of cryo-survival rate. No significant difference was observed in cryo-survival rates (p=0.56). Conclusion: We concluded that the method introduced in the late 1990s, which enables the removal of debris, potentially toxic elements and generators of reactive oxygen species from the seminal sample before cryopreservation, exhibited efficiency in maintaining the cryo-survival rate after an extended period.