# Semen cryopreservation

Semen cryopreservation (commonly called sperm banking or sperm freezing) is a procedure to preserve sperm cells. Semen can be used successfully indefinitely after cryopreservation. It can be used for sperm donation where the recipient wants the treatment in a different time or place, or as a means of preserving fertility for men undergoing vasectomy or treatments that may compromise their fertility, such as chemotherapy, radiation therapy or surgery. It is also often used by trans women prior to medically transitioning in ways that affect fertility, such as feminizing hormone therapy and orchiectomies.

## Freezing

The most common cryoprotectant used for semen is glycerol (10% in culture medium). Often sucrose or other di-, trisaccharides 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:
1. 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.
2. 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.
3. 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 40 °C (104 °F) 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, 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 cryoprotectant and are not going through sperm washing or other alteration in between, and provided that they are separated by density gradient centrifugation or swim-up before use in assisted reproduction technology.

## Effect on quality

Some evidence suggests an increase in single-strand breaks, condensation and fragmentation of DNA in sperm after cryopreservation. This can potentially increase the risk of mutations in offspring DNA. Antioxidants 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 defects or chromosomal abnormalities in people conceived from cryopreserved sperm compared with the general population.