**Part 3**

**Human Assisted Reproduction Techniques (ART)** 

166 Current Frontiers in Cryobiology

Yu, J., Vodyanik, M.A., Smuga-Otto, K., ntosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J.,

Zhang, X.B., Li, K., Yau, K.H., Tsang, K.S., Fok, T.F., Li, C.K., Lee, S.M., & Yuen, P.M. (2003).

Zhou, C.Q., Mai, Q.Y., Li, T., & Zhuang, G.L. (2004). Cryopreservation of human embryonic stem cells by vitrification. *Chin Med. J. (Engl. )*, Vol. 117, No. 7, pp. (1050-1055)

318, No. 5858, pp. (1917-1920)

Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., & Thomson, J.A. (2007). Induced pluripotent stem cell lines derived from human somatic cells. *Science*, Vol.

Trehalose ameliorates the cryopreservation of cord blood in a preclinical system and increases the recovery of CFUs, long-term culture-initiating cells, and nonobese diabetic-SCID repopulating cells. *Transfusion*, Vol. 43, No. 2, pp. (265-272)

**6** 

*USA* 

Juergen Liebermann

**Vitrification of Oocytes and Embryos** 

Currently, controlled ovarian hyperstimulation protocols commonly provide embryos in excess of those needed for fresh transfer. Therefore, techniques have been developed to store these surplus embryos in liquid nitrogen (referred to as cryopreservation) for an indefinite period of time without significant compromise of their quality. Based on data from the Centers for Disease Control and Prevention (CDC) from 2001 to 2004, about 18% of all IVF cycles in the USA used frozen embryos for transfer. In addition, data from the same registry compared live births per transfer using frozen and fresh embryos (25% versus 34% respectively) clearly showing that cryopreservation is an important adjunct to maximize the efficiency of every single patient's oocyte retrieval. The fundamental objectives for successful cryostorage of cells in liquid nitrogen at -196°C can be summarized as follows: **1)** arresting the metabolism reversibly, **2)** maintaining structural and genetic integrity, **3)** achieving acceptable survival rates after thawing, **4)** maintain of developmental competence

Furthermore, all methods and protocols for cryopreservation should be developed such that ice crystals formation and growth inside the cells or tissues must either be eliminated or massively suppressed. One recent hotly debated topic in the area of reproductive cryobiology is whether slow-cooling or rapid-cooling protocols both satisfy the fundamental cryo-biological principles for reduction of damage by ice crystal formation during cooling and warming, and which approach is better. It is the case nonetheless, that both methods of cryopreservation of biological material include six principal steps: **1)** initial exposure to the cryoprotectant (intracellular water has to be removed by gradual dehydration, **2)** cooling (slow/rapid) to subzero temperatures (-196°C), **3)** storage at low temperature, **4)** thawing/warming by gradual rehydration, **5)** dilution and removal of the cryoprotectant agents and replacement of the cellular and intracellular fluid at precise rate and, **6)** recovery

Although initially reported in 1985 as a successful cryopreservation approach for mouse embryos, vitrification has taken a backseat in human assisted reproduction. However, the practical advantages of this cryopreservation method have more recently caught the attention of many ART laboratories as a feasible alternative to traditional slow freezing methods. Since 1985 more than 2,100 publications can be found referring to the topic of "vitrification", which is further evidence of the burgeoning growth of interest in this cryopreservation technology. One "drawback" considered by embryologists who are not

post thaw and, **5)** the technique has to be reliable and repeatable.

and return to a physiological environment.

**1. Introduction** 

*Director of Laboratory, Fertility Centers of Illinois, Chicago, IL* 
