Abstract
Objective:To compare cryosurvival rates of human spermatozoa in a prolonged period of cryopreservation.Methods:This retrospective study involved 33 cryopreserved semen samples from patients with cancer, between 2002 and 2011. The semen sample was obtained by masturbation and initial semen analysis was performed. The cryoprotectant solution was added and samples were frozen in liquid nitrogen in a slow step-wise process. For thawing, the samples were incubated at 25.0ºC for 15 min, followed by incubation at 36.7ºC for 15 min. The cryosurvival rate (CS) was calculate by CS= [(% total motile sperm post-thaw) x100/(% total motile sperm/tube)]. Each study sample was divided into three aliquots (Study Group; n=23): (I) official patient sample, which was kept cryopreserved for subsequent Assisted Reproduction procedure, cryopreserved between 2002 and 2011; (II) sample destined to post-thaw tests, performed after the sample had been kept cryopreserved for 24 hours; and (III) study sample. Only in 2014, after 3-12 years of cryopreservation, the study samples were thawed and evaluated. To validate the study design, a Validation Group was created including 10 samples obtained between 2014 and 2016, using the same methodology in the study samples. The data was analyzed using the T-test, with a significant p-value of 5%.Results:The mean age was 29.93±9.57 years in the Study Group and 21.80±6.49 years in the Validation Group. No significant difference between the Validation and Study Groups was found in the initial semen analysis (p>0.05). After 24 hours of cryopreservation, the cryosurvival rate was 26.11±46.36% in the Study Group and 23.71±57.06% in the Validation Group. Aliquots of the same sample preserved from 3-12 years demonstrated 23.71±57.06% of cryosurvival rate. Thus, no significant difference was found vis-à-vis the cryosurvival 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 same cryosurvival rate after an extended period.
Highlights
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 same cryosurvival rate after an extended period
Sperm cryopreservation is the main method for preserving male fertility, which was applied in such situations as artificial insemination with donor sperm, in vitro fertilization (IVF) program with sperm obtained by microsurgery, high-risk professions and patients undergoing chemotherapy, radiotherapy and other immunosuppressive therapies (Hallak et al, 2000a; Li et al, 2010)
The improvement of techniques employed in cryopreservation processes, in sample handling and application of diluents and cryoprotectants, which contain osmotically active ingredients, such as glycerol, which is capable of reducing intracellular water and reduce the damage caused by the formation of ice crystals
Summary
Sperm cryopreservation is the main method for preserving male fertility, which was applied in such situations as artificial insemination with donor sperm, in vitro fertilization (IVF) program with sperm obtained by microsurgery, high-risk professions and patients undergoing chemotherapy, radiotherapy and other immunosuppressive therapies (Hallak et al, 2000a; Li et al, 2010). Damage caused by wide temperature variation, ranging from 36.6°C to -196.0°C, such as thermal shock, formation of ice crystals, dehydration and increased intracellular salt concentration caused by osmotic shock (Stanic et al, 2000) can impair the structural and functional integrity of spermatozoa via reduced motility and vitality due to mitochondrial damages and in plasma and acrosome membranes (Succu et al, 2011), increased DNA fragmentation and generation of reactive oxygen species, lipid peroxidation and apoptosis (Zribi et al, 2010; Wang et al, 1997; Alvarez & Storey, 1992; Paasch et al, 2004) These adverse effects were found in 25 to 75% of spermatozoa undergoing the cryopreservation process (Chian & Quinn, 2010; Hallak et al, 1999a). The standardization of thawing steps may enable a satisfactory survival rate of cells, if it is performed following the same steps of freezing process but in reverse (Hallak et al, 1999a; 2000b)
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