Semen cryopreservation (commonly called sperm banking or sperm freezing) is a procedure to preserve sperm cells.

Semen Semen, also known as seminal fluid, is a bodily fluid that contains spermatozoon, spermatozoa which is secreted by the male gonads (sexual glands) and other sexual organs of male or hermaphrodite, hermaphroditic animals. In humans and placen ...

can be used successfully indefinitely after

cryopreservation Cryopreservation or cryoconservation is a process where biological material - cells, tissues, or organs - are frozen to preserve the material for an extended period of time. At low temperatures (typically or using liquid nitrogen) any cell ...

. It can be used for

sperm donation Sperm donation is the provision by a man of his sperm with the intention that it be used in the artificial insemination or other "fertility treatment" of one or more women who are not his sexual partners in order that they may become pregnant by h ...

where the recipient wants the treatment in a different time or place, or as a means of preserving fertility for men undergoing

vasectomy Vasectomy is an elective surgical procedure that results in male sterilization, often as a means of permanent contraception. During the procedure, the male vasa deferentia are cut and tied or sealed so as to prevent sperm from entering into ...

or treatments that may compromise their fertility, such as

chemotherapy Chemotherapy (often abbreviated chemo, sometimes CTX and CTx) is the type of cancer treatment that uses one or more anti-cancer drugs (list of chemotherapeutic agents, chemotherapeutic agents or alkylating agents) in a standard chemotherapy re ...

radiation therapy Radiation therapy or radiotherapy (RT, RTx, or XRT) is a therapy, treatment using ionizing radiation, generally provided as part of treatment of cancer, cancer therapy to either kill or control the growth of malignancy, malignant cell (biology), ...

or surgery. It is also often used by

trans women A trans woman or transgender woman is a woman who was assigned male at birth. Trans women have a female gender identity and may experience gender dysphoria (distress brought upon by the discrepancy between a person's gender identity and their ...

prior to medically transitioning in ways that affect fertility, such as

feminizing hormone therapy Feminizing hormone therapy, also known as transfeminine hormone therapy, is a form of Transgender_health_care#Gender-affirming_care , gender-affirming care and a gender-affirming hormone therapy , gender-affirming hormone therapy to change t ...

and orchiectomies.

Freezing

The most common

cryoprotectant A cryoprotectant is a substance used to protect biological tissue from freezing damage (i.e. that due to ice formation). Arctic and Antarctic insects, fish and amphibians create cryoprotectants ( antifreeze compounds and antifreeze proteins) in th ...

used for semen is

glycerol Glycerol () is a simple triol compound. It is a colorless, odorless, sweet-tasting, viscous liquid. The glycerol backbone is found in lipids known as glycerides. It is also widely used as a sweetener in the food industry and as a humectant in pha ...

(10% in culture medium). Often

sucrose Sucrose, a disaccharide, is a sugar composed of glucose and fructose subunits. It is produced naturally in plants and is the main constituent of white sugar. It has the molecular formula . For human consumption, sucrose is extracted and refined ...

or other di-,

trisaccharide ''Trisaccharides'' are oligosaccharides composed of three monosaccharides with two glycosidic bonds connecting them. Similar to the disaccharides, each glycosidic bond can be formed between any hydroxyl group on the component monosaccharides. Even ...

s are added to glycerol solution. Cryoprotectant media may be supplemented with either egg yolk or soy lecithin, with the two having no statistically significant differences compared to each other regarding motility, morphology, ability to bind to hyaluronate in vitro, or DNA integrity after thawing. Additional cryoprotectants can be used to increase sperm viability and fertility rates post-freezing. Treatment of sperm with heparin binding proteins prior to cryopreservation showed decreased cryoinjury and generation of ROS. The addition of nerve growth factor as a cryoprotectant decreases sperm cell death rates and increased motility after thawing. Incorporation of cholesterol into sperm cell membranes with the use of cyclodextrins prior to freezing also increases sperm viability. There are different techniques for freezing semen samples: *Block freezing (slow): First the sample is washed, the cryoprotectant is added. A cryoprotectant solution is added to the sperm to protect them from possible damage during freezing and thawing. The solution helps prevent ice crystals from forming in the sperm, which can damage the cell membrane and affect its viability. The sperm is separated into aliquots and frozen little by little, then immersed in liquid nitrogen and stored in sperm banks. It is a slow procedure. *Freezing in pills (fast): It is the same procedure but now it is stored in the form of CO2 pills. Then the pills are put in aliquots and put into N2 and stored. This is done if the seminal samples are considered valuable, for example if the sample is from a person who wants to have an ICSI or IVF, as this will allow the optimization of the semen, since it allows the sample to be thawed little by little. *Vitrification (ultrafast): is a very expensive procedure. Sperm vitrification is an ultra-fast cryopreservation method that involves direct exposure to liquid nitrogen. In this way, the crystallization of intracellular water and cryodamage is avoided, without using permeable cryoprotectants that can imply mutagenic risk. It prevents ice crystals from forming, the main cause of irreparable deterioration or cell death. Through this practice, we ensure that the samples maintain their fertilizing capacity and a similar behavior to the fresh sample. Vitrification gives superior post-thaw motility and cryosurvival. This current technique, invented by the Japanese, is used in the best centers around the world. It is extremely fast (-23000°C/min), so as a result it avoids the appearance of small ice crystals, preventing the "knife" effect.

Packaging Method

The packaging method is a crucial aspect of cryopreservation processes, as it directly affects thermal stability, storage capacity, and the efficiency of sample thawing. Several packaging techniques are used for sperm cryopreservation, each with its advantages and disadvantages: ;Cryo-tops (Cryovials or Cryotubes) Advantages: *Provide a good surface for sample identification. *Offer greater thermal stability during the process. Disadvantages: *Nitrogen may come into contact with the stored material. *Require more storage space. *Exhibit a less uniform freezing rate. ;Goblets Advantages: *Low cost. *Allow partial thawing of samples. *Ensure uniform freezing. Disadvantages: *Nitrogen comes into contact with the sample. *Pills stored in these devices are more sensitive to temperature changes. ;Straws Advantages: *Require less storage space. *Enable a more uniform freezing rate. *Allow rapid thawing. Disadvantages: *More sensitive to temperature changes. *Offer a smaller surface for identification. *The cost of filling and sealing equipment is high.

Thawing

Thawing at seems to result in optimal sperm motility. On the other hand, the exact thawing temperature seems to have only minor effect on sperm viability, acrosomal status, ATP content, and DNA. As with freezing, various techniques have been developed for the thawing process, both discussed by Di Santo et al.

Refreezing

In terms of the level of sperm

DNA fragmentation DNA fragmentation is the separation or breaking of DNA strands into pieces. It can be done intentionally by laboratory personnel or by cells, or can occur spontaneously. Spontaneous or accidental DNA fragmentation is fragmentation that gradually a ...

, up to three cycles of freezing and thawing can be performed without causing a level of risk significantly higher than following a single cycle of freezing and thawing. This is provided that samples are refrozen in their original

and are not going through sperm washing or other alteration in between, and provided that they are separated by

density gradient centrifugation Density (volumetric mass density or specific mass) is the ratio of a substance's mass to its volume. The symbol most often used for density is ''ρ'' (the lower case Greek letter rho), although the Latin letter ''D'' (or ''d'') can also be use ...

or swim-up before use in assisted reproduction technology.

Effect on quality

Some evidence suggests an increase in

single-strand break DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. A weakened capacity for DNA repair is a risk factor for the development of cancer. DNA is constantly modified ...

condensation Condensation is the change of the state of matter from the gas phase into the liquid phase, and is the reverse of vaporization. The word most often refers to the water cycle. It can also be defined as the change in the state of water vapor ...

and fragmentation of DNA in sperm after cryopreservation. This can potentially increase the risk of

mutation In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, ...

s in offspring DNA.

Antioxidant Antioxidants are Chemical compound, compounds that inhibit Redox, oxidation, a chemical reaction that can produce Radical (chemistry), free radicals. Autoxidation leads to degradation of organic compounds, including living matter. Antioxidants ...

s and the use of well-controlled cooling regimes could potentially improve outcomes. In long-term follow-up studies, no evidence has been found either of an increase in

birth defect A birth defect is an abnormal condition that is present at birth, regardless of its cause. Birth defects may result in disabilities that may be physical, intellectual, or developmental. The disabilities can range from mild to severe. Birth de ...

s or chromosomal abnormalities in people conceived from cryopreserved sperm compared with the general population.

References