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Procedure
Current clinical practice for pre-pubertal boys undergoing chemotherapy, or any other treatment which may be significantly gonadotoxic, is to offer cryopreservation of testicular tissue (TT). This procedure is ideally done before the commencement of any treatments to avoid mutagenic effects of this on the germ cells being preserved.

Testicular fragments are retrieved during surgery and immediately placed into a transport medium at 4 – 8 °C to reduce contamination. It is possible to freeze either whole tissue or cell suspensions from the TT extracted, although whole tissue preserves the ability to pursue both cell or tissue-based therapies in the future and is therefore more widely used. TT is then placed into cryotubes, most often containing sucrose; a non-penetrating cryoprotective agent (CPA). CPAs are added to increase membrane stability during the dehydration phase and reduce damage to the cell structure. Cryopreservation can either be done by slow freezing or vitrification.

Slow freezing and vitrification
Slow freezing allows the temperature of the cells and surrounding medium to be modified in a controlled way, maximising the dehydration of the cells before temperatures are reached which cause intracellular ice to form – this reduces the likelihood of damage from ice crystals. Vitrification freezes the tissue at an ‘ultrafast’ rate, using a higher concentration of CPAs to stabilise the tissue. This method allows fast cooling of the cells without mechanical disruption to the cell body. These procedures are still experimental and clear guidelines on the restoring of fertility after cryopreservation of TT have yet to be published.