Sperm Freezing Procedure

Cryopreservation of spermatozoa is an essential aspect of the work conducted in many semen analysis laboratories, particularly those associated with infertility treatment clinics.

The field of human sperm cryobiology began in the late 1940s when it was found that glycerol could protect spermatozoa from the damaging effects of freezing. This discovery led to storing human spermatozoa on dry ice at approximately -79 °C. Eventually, the adoption of liquid nitrogen further propelled the field of semen cryopreservation forward, resulting in the establishment of commercial sperm banks and organized national services in various countries.

Currently, a variety of cryopreservation methods are utilized, employing different cryoprotectants and freezing techniques. The survival of cells post-freezing and thawing largely hinges on reducing the formation of intracellular ice crystals.

This is achieved by selecting suitable cryoprotectants and controlling the cooling and warming rates to minimize the freezing of intracellular water. If spermatozoa cells are kept at temperatures above -130 °C for extended periods, especially during thawing, recrystallization may occur, leading to the expansion of harmful intracellular ice crystals.


Cryoprotectants play a pivotal role in spermatozoa cryopreservation and can be categorized into two main types: permeable, such as dimethyl sulfoxide (DMSO) and glycerol, and impermeable, including substances like albumins, dextran, and egg yolk citrate. Human spermatozoa cells are adaptable to various cooling and warming rates, showing a remarkable resilience to damage from rapid initial cooling, possibly due to the high membrane fluidity attributed to unsaturated fatty acids in the lipid bilayer. Additionally, spermatozoa may be inherently more resistant to cryopreservation damage because of their low water content, around 50%. Despite this, cryopreservation can adversely affect sperm function, particularly motility, with studies indicating a reduction in the proportion of motile spermatozoa from 50.6% to 30.3% post-cryopreservation, highlighting the importance of optimizing the cryopreservation process to mitigate such effects.

Pregnancy rates following artificial insemination using cryopreserved donor semen significantly depend on the quality of sperm after thawing, the timing of insemination, and crucial recipient factors such as age, history of previous pregnancies using donor insemination, and issues related to ovulation and the uterine tubes. Properly stored semen does not exhibit a notable decrease in sperm quality over time, with successful fertilizations reported using semen stored for more than 28 years. In cases where high levels of leukocytes are present in the semen, selecting highly motile spermatozoa fractions is recommended for better outcomes.

The practice of human semen cryobanking encompasses two primary purposes: autologous use (for the individual’s future use) and homologous use (donor banking). Storage for personal future use varies widely in reasons and is subject to national regulations. Society of Scientific Oncology advises healthcare providers to offer sperm cryopreservation to all post-pubertal males of reproductive age undergoing cancer treatment, as it represents the most reliable method to preserve fertility. Men should also be informed about the increased risk of genetic damage in sperm collected after starting chemotherapy or radiotherapy, thus emphasizing the need for banking sperm prior to treatment. While current evidence does not show significant differences between the use of cryopreserved and fresh sperm in assisted reproductive technologies (ART), there are reports of increased DNA fragmentation following cryopreservation, suggesting that in some instances, adjustments to the cryopreservation protocol may be beneficial.

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