How are reproductive tissues cryopreserved?
For sperm, a preliminary analysis is performed on the semen sample to assess sperm count and motility. After the analysis, the semen is mixed with a glycerol-based cryopreservation media, placed into several individual vials and slowly exposed to LN2vapors. Approximately 30 to 60 minutes later, the vials are transferred to cryogenic storage tanks for permanent storage.
Oocytes and embryos are cryopreserved utilizing essentially the same methodology but with special modifications. Previously, oocytes and embryos were placed into dehydrating cryoprotectant solutions that removed water from the cells and then replaced the water to protect from ice damage during cryopreservation process. Next the oocytes or embryos were placed into an automated freezer that slowly cooled down to -30oC. Today, oocytes and embryos are cryopreserved by a technique called “vitrification,” whereby the process of cryopreservation is so rapid that the water molecules inside oocytes and embryos do not have the time to form ice crystals and they instantly solidify into a glass-like structure. Slow freezing and vitrification have several other key differences, including the volume of cryoprotectant used (10- to 100-fold greater with slow freezing), devices used for storage and thermodynamic stability (favoring vitrification). Most labs now perform vitrification exclusively. There are also two options for freezing medium: liquid nitrogen and nitrogen vapor. Most of the publicized tank failures have involved nitrogen vapor but vapor is used almost exclusively for shipment of cryopreserved tissues.
Sperm, oocytes, and embryos can be cryopreserved indefinitely, with several published articles reporting live births from reproductive tissues stored for more than several decades.2,3
How are cryopreserved reproductive tissues typically stored?
Cryogenic storage tanks filled with LN2are considered reliably safe and are the standard for most fertility clinics worldwide. These insulated metal containers have walls with two or more vacuumed layers to allow holding and maintenance of LN2at its basal temperature of -196º C, far below the conditions necessary to maintain the viability of cryopreserved reproductive tissues. These tanks do not require electrical power to maintain temperature and are not affected by power outages.
Two types of cryogenic storage tanks are currently used in fertility clinics. Both can be utilized for both the vapor and total immersion phase of LN2 storage. The first type is a small waist-high aluminum cryogenic tank that resemble a large thermos and is frequently placed on a wheeled base to allow for easy evacuation. This type of tank must be filled manually with LN2. Larger aluminum or steel cryogenic storage tanks that resemble chest-type freezers are also available but are not movable. These can function in both the vapor or total immersion phase of LN2. Larger cryogenic storage tanks can also be attached to LN2supply tanks or manifold that will automatically fill the storage tanks when a sensor detects low levels of LN2or an insufficient temperature level. Inside the cryogenic storage tanks are different storage layout systems to hold the many varied cryopreservation devices.
The authors report no potential conflicts of interest with regard to this article.
- Crawford,S., Boulet, S.L., Kawwass, J.F., Jamieson, D.J., Kissin, D.M., 2017. Cryopreserved oocyte versus fresh oocyte assisted reproductive technology cycles, United States. Fertil Steril. 2013;107:110-118.
- Szell AZ, Bierbaum RC, Hazelrigg WB, Chetkowski RJ. Live births from cryopreserved human semen stored for 40 years. J Assist Reprod Genet. 2013;30(6):743-744.
- Dowling-Lacey D, Mayer JF, Jones E, Bocca S, Stadtmauer L, Oehninger S. Live birth from a frozen-thawed pronuclear stage embryo almost 20 years after its cryopreservation. Fertil Steril. 2011;95:1120.e1-1120.e3.
- Tomlinson M, Morroll D.. Risks associated with cryopreservation: a survey of assisted conception units in the UK and Ireland. Hum Fertil. (Camb) 2008;11:33-42.
- Standards and technical manual reproductive cells and tissues. American Association of Tissue Banks, 2002.