Review Open Access
Factors affecting the outcome of human blastocyst vitrification
Amr A Kader1,2,3, Audrey Choi1,4, Yasser Orief1,3 and Ashok Agarwal*1
Address: 1Center for Reproductive Medicine, Glickman Urological and Kidney Institute, Ob/Gyn and Women's Health Institute, Cleveland Clinic,
Cleveland, Ohio, USA, 2Department of Obstetrics and Gynecology, University of Alexandria, Alexandria, Egypt, 3Center of Surgical Innovation,
Technology and Education, Cleveland Clinic, Cleveland, Ohio, USA and 4Case Western Reserve University School of Medicine, Cleveland, Ohio,
With single blastocyst transfer practice becoming more common in ART, there is a greater demand for a convenient and reliable cryostorage of surplus blastocysts. Vitrification has emerged in the last decade as an alternative promising substitute for slow freezing. Blastocysts represent a unique challenge in cryostorage due to their size, multicellular structure and presence of blastocoele. The continuous acquisition of experience and introduction of many different technological developments has led to the improvement of vitrification as a technology and improved the results of its application in blastocyst cryostorage. The current information concerning safety and efficacy of the vitrification of blastocysts will be reviewed along with the variables that can impact the outcome of the procedure.
With the refinement of extended culture systems, it is becoming more reliable to obtain blastocysts in vitro . Due their high implantation rates, it is becoming a common practice to limit transfer to one or two blastocysts at a time. Therefore, surplus blastocysts require an efficient cryopreservation method [2,3]. Slow freezing was the main method of cryopreservation , but vitrification is now on the rise. Vitrification is the glass-like solidification of a solution at a low temperature without ice crystal formation, which is made possible by extreme elevation in viscosity during freezing. This can be achieved by increasing the freezing and warming rates and/or increasing the concentration of the cryoprotectants . Unlike slow freezing, vitrification results in the total elimination of ice crystal formation, both within the cells being vitrified and outside the cells in the surrounding solution . Although high concentrations of cryoprotectants can be toxic, and the vitrified solution is prone to glass fractures,these effects can be controlled by adjusting the vitrification protocol and technique. With vitrification, the blastocyst is combined with cryoprotectants that maximize cytoplasmic viscosity while exerting a strong dehydrating effect. Vitrification is more convenient and is possibly superior because it avoids ice crystal formation. Over the last decade, vitrification techniques have been standardized, tested and improved via controlled experiments designed to elucidate the optimal conditions under which vitrification should be performed. This review will discuss the most commonly used loading devices, vitrification safety in terms of perinatal outcomes, and the factors that can affect the success of human blastocyst vitrification.
Human blastocysts vitrified using different loading devices
During vitrification, the blastocyst is placed in a loading device surrounded by vitrification media. The device is then placed into liquid nitrogen, where it is stored. There are a variety of loading devices available today: the Cryoloop, Cryotop, Cryoptip, Cut Standard Straws, Cryoleaf
™ and High Security Straws™. The Cryoloop is a nylon loop, whereas the Cryotop is a plastic container. These are considered open systems because the blastocysts come into direct contact with the liquid nitrogen. Cryotips are plastic straws with protective metal sleeves and is heat
sealed from both ends after loading, thus constituting a closed system. The cut standard straw is a system that can
be used as an open method (by direct contact with liquid nitrogen) or closed if placed inside a sealed standard straw (straw within straw). The Cryo-leaf™ is a plastic carrier open system, vitrifying the specimen by direct contact. High security straws are plastic straws sealed after loading, and are thus considered a closed system. Table 1 summarizes
the survival, implantation and pregnancy rates of human blastocysts vitrified using different loading devices.
In 1999, Lane et al  reported that human blastocysts vitrified by cryoloop had hatching rates similar to those of fresh blastocysts. Mukaida et al [8,9] and Reed et al  vitrified blastocysts using the Cryoloop, producing survival rates ranging from 63% to 100% and pregnancy rates ranging from 31% to 37%. In 2001, Mukaida et al reported the first successful delivery of three healthy newborns who had been conceived via blastocyst vitrification using the Cryoloop .
Obstetric and perinatal outcomes
Multiple pregnancy is the main source of obstetric and
perinatal morbidity associated with assisted reproduction.
The transfer of blastocysts allowed one or two blastocysts
to be transferred with high implantation potential, while
minimizing the risks of multiple pregnancies. Single blastocyst
transfer completely avoids dizigotic twin pregnancy
Factors that can affect the outcome
There are a number of variables that can determine the
outcomes of vitrification
•Pre-vitrification blastocyst selection
•Post-thaw blastocyst selection
•Assisted hatching (Figure 1)
•Blastocoele collapse (assisted shrinkage) (Figure 1)