**7. Conclusions**

Human spermatozoa can be successfully cryopreserved and utilized. Cryopreservation now plays an essential role in fertility preservation under the following scenarios:


Normozoospermic semen samples appear to be more tolerant to damage induced by freezing and thawing compared with oligozoospermic or asthenozoospermic samples. Cryopreservation of surgically retrieved epididymal and testicular spermatozoa is challenging, but a valuable component in effective treatment and management of severe male factor infertility. Cryopreservation of low numbers or single spermatozoa has multiple biological and technical aspects yet to be worked out; therefore, further research is required to introduce this technique into clinical practice. During cryopreservation, cells and tissue undergo dramatic transformation in chemical and physical characteristics as temperature drops from +37 to -196°C, thus risking cryoinjury. Velocity of cooling and warming is crucial and inaccurate cooling or thawing rates negatively correlate with sperm survival.

Spermatozoa cannot survive slow freezing without CPAs; CPAs have to be used at low concentrations with minimum exposure as CPAs are toxic and can cause osmotic damage. Gradual, stepwise introduction before freezing and removal of CPAs after thawing is essential. Conventional slow freezing with CPAs can offer cooling rates of 1–10°C/min. Vitrification, currently only an experimental technique, allows for extremely rapid freezing at rates of up to a 1000°C/min. LN2 can offer long-term survival of spermatozoa due to essentially absent metabolic activity and aging of cells and tissues in the frozen state. Rigorous standards of operation and quality control are essential for sperm banks. Social, psychological, legal and ethical issues surrounding sperm banking are very complex and must be considered in each case.

The vitrification method uses no specially developed cooling program; it does not need permeable cryoprotectants; it is much faster, simpler and cheaper; and it can also provide a high recovery of motile spermatozoa after warming as effective protection of spermatozoa against cryodamage and helps to avoid many problems relevant to slow freezing such as ice formation; shifts in pH, extensive rehydration and osmotic damage.

Successful vitrification of human spermatozoa without toxic CPAs has been reported now by two independent groups. Moreover, live births were reported after vitrification of semen utilized for intrauterine insemination and IVF with ICSI procedures, making this freezing technique even more attractive in clinical practice.
