Factors affecting the outcome of human blastocyst vitrification

With the refinement of extended culture systems, it is becoming more reliable to obtain blastocysts in vitro [1]. 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 [4], 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 [5]. 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 [6]. 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.

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, Cryo-leaf™ 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 [7] 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 [10] 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 [8].

In 2003, Osada et al [11] studied the vitrification of blastocysts using the Cryotop™ and reported 99% survival rate and 56% pregnancy rate, which was even higher than the 31% pregnancy rate in their fresh blastocyst transfer group. Stehlik et al [12] and Liebermann and Tucker [13] compared vitrification by Cryotop™ with conventional slow freezing methods. Liebermann and Tucker [13] did not find a statistically significant difference in survival and pregnancy rates between blastocysts vitrified by the Cryotop™ and those cryopreserved by slow freezing. On the other hand, Stehlik et al [12] reported that survival and pregnancy rates of blastocysts vitrified by the Cryotop™ significantly exceeded the rates of blastocyst survival after slow cryopreservation.

Despite the wide use and successful vitrification of human and animal oocytes and embryos using open pulled straws (OPS) [14, 15], only modified OPS were used by Cremades et al [16] and resulted in survival rate of 82% in a small sample of 33 human blastocysts.

In 2005, Kuwayama et al [17] performed a study that validated the use of the Cryotip™ for the first time, reporting that the Cryotip™ produced results that were comparable to those of the Cryotop™ carrier. The Cyrotip™ demonstrated 93% blastocyst survival rate and 51% pregnancy rate with no statistical difference when compared with the rates of the Cryotop™ [18].

In 2005, Takahashi et al [19] reported the clinical outcomes of a 4-year study on 1129 vitrified human blastocysts using the cryoloop. This large sample size demonstrated that the pregnancy rate and implantation rates using vitrified blastocysts were comparable to those associated with use of fresh blastocysts.

In a recent report by Liebermann et al [20], of 8,449 blastocysts from 2,453 patients that were vitrified, 1398 vitrified blastocysts were transferred with a survival rate of 96.3%, an implantation rate of 29.4%, and a clinical pregnancy rate per frozen embryo transfer of 42.9%.

Blastocysts can also be vitrified on an electronic microscope (EM) copper grid. Cho et al [21] reported vitrifying human blastocysts in this manner with a survival rate of 83% and a pregnancy rate of 34%.

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 [1, 22‐25].

Vitrification has been in clinical use for more than 15 years. And while multiple studies have reported excellent cryosurvival and pregnancy rates using vitrified oocytes or embryos, there are still concerns regarding the overall safety of vitrification and whether it can cause or lead to chromosomal abnormalities, congenital malformation, and/or developmental abnormalities in the offspring [26, 27]. As a result, no general recommendation in favor of its regular clinical use has been issued.

Part of the problem is a lack of well-controlled clinical trials. Noyes et al [28] reviewed a total of 58 reports (1986-2008) on 900 cryopreserved oocytes looking for data on congenital anomalies in 609 live born babies (308 from slow-freezing, 289 from vitrification and 12 from both methods). Twelve newborns (1.3%) had birth anomalies, which is comparable to the number of congenital anomalies that occur in naturally conceived infants. Analyzing the obstetric and perinatal outcomes following transfer of vitrified blastocysts would be even more challenging due to the limited number of reports, though this number is rapidly rising.

Takahashi et al [19] reported congenital birth defects of 1.4% using vitrified blastocysts which was similar to fresh blastocysts. In a preliminary report on the effect of blastocyst vitrification on perinatal outcomes, Mukaida et al [29] analyzed 560 deliveries of 691 healthy babies following the transfer of vitrified blastocysts. The congenital and neonatal complication rate was 3%, which was comparable to that in their fresh blastocysts transfer group (2.3%). No perinatal abnormalities were reported in Liebermann's report on 348 deliveries of 431 babies following transfer of vitrified blastocysts [20].

These findings may provide preliminary reassurance on the safety of blastocyst vitrification. A final verdict on the actual effect of blastocyst vitrification on congenital and perinatal outcomes may not be possible until large-scale trials or further meta-analysis of rapidly accumulating reports can be performed.

There are a number of variables that can determine the outcomes of vitrification:

Selection focuses on the quality of the original embryo and/or the time at which the blastocyst is vitrified.

Post-warming, viable blastocysts re-expand and are usually allowed four to six hours of incubation to regain their vitality before being transferred. Re-expansion is the sign of viability. An important predictor of the transfer of vitrified-warmed blastocyst is the blastocyst re-expansion timing. The earlier the blastocyst expands, the better it is expected to perform after transfer [38].

Pribenszky et al [39] studied the survival of zona-free mouse blastocysts. There was no difference in survival after thawing between these blastocysts and fresh control blastocysts. This experiment suggested that the intact zona pellucida can potentially negatively impact blastocyst vitrification

In lieu of using zona-free blastocysts, which may not be practical with human blastocysts, assisted hatching can be performed prior to vitrification. With assisted hatching, a small hole is created in the zona pellucida so that the blastocyst can more easily escape or "hatch." It was primarily thought to overcome the post-freezing zonal hardening preventing spontaneous hatching and it proved effective [30].

Assisted hatching has been shown to improve the outcome of vitrification of blastocysts through another mechanism. Applying assisted hatching prior to blastocyst vitrification allows better permeation of the cryoprotectants and better blastocoele dehydration [36, 40]. Zech et al [40] found that vitrified warmed blastocysts that had undergone assisted hatching had significantly better survival, implantation and pregnancy rates than blastocysts with an intact zona. In concordance with Zech's findings, we have demonstrated that assisted or spontaneous hatching both have a significantly positive impact on the post-warming DNA integrity index of mice blastocysts post-warming as compared with zona-intact blastocysts [36]. These two studies show that assisted hatching is a useful and effective pre-vitrification intervention that can reduce DNA damage incurred during the vitrification process and improve clinical outcome parameters.

Table 3 summarizes the results of studies assessing the outcomes of pre-vitrification assisted hatching.

Much attention has been paid to the volume of the blastocoele prior to vitrification and its effect on the overall success of vitrification. A negative correlation between blastocelic volume and outcome measures has been attributed to an increased likelihood of intracellular ice formation in an inadequately dehydrated blastocoele [41, 42]. Consequently, a process called assisted shrinkage was developed to reduce blastocelic volume prior to vitrification. Assisted shrinkage can be performed in a variety of ways, including micro-needle puncture of the zona pellucida [37, 41, 43], laser-pulse opening of the zona pellucida [41], repeated micropipetting of the blastocoele [44], and microsuction of the blastocoelic contents [42, 45]

Mukaida et al [41] reported significant improvements in clinical outcome measures in blastocysts that had undergone assisted shrinkage as compared with a retrospective vitrification control group. There were no statistical differences in survival, implantation and clinical pregnancy rates between blastocysts that had undergone laser pulse opening or micro-needle puncture [41]. Vanderzwalmen et al and Son et al have also reported improved results using micro-needle puncture of blastocysts prior to vitrification [37, 43].

Hiraoka et al, [44] mechanically collapsed blastocysts by repeated micropipetting prior to vitrification. The investigators reported 98% survival rate, 33% implantation rate, and 50% pregnancy rate in a sample of 48 vitrified blastocysts.

Chen et al [42] reported significant improvement in survival rates in blastocysts treated with blastocoelic microsuction prior to vitrification. The non-expanded blastocyst survival rate improved significantly with microsuction, and the survival rate for the expanded blastocysts improved from 59% to 89%. We have previously demonstrated significant improvement in the DNA integrity index by microsuction of mice blastocysts prior to vitrification compared with blastocyst vitrification without any pre-intervention [45]

Table 3 summarizes the results of studies assessing the outcomes of pre-vitrification assisted shrinkage.

Since the inception of vitrification as a technique, many different media protocols have been tested to achieve proper intracellular cryoprotectant delivery.

A high freezing rate is crucial to achieving proper vitrification and survival. This can be achieved via direct contact between the sample and liquid nitrogen or indirect contact if the sample is contained in a closed carrier.

Proper warming is as important as rapid freezing to achieve proper vitrification-devitrification [54]. This is usually done with the immediate transfer of the sample to a pre-warmed (37°C) environment while making sure this temperature is immediately available to the sample. This can be done in open methods by mixing the sample in pre-warmed media or in closed methods by plunging the sample in its loading device into a warm water bath. The heating rate will be controlled by the same factors that control the freezing rate.

Because dilution of the cryoprotectants and re-expansion of the blastocoele occur during the warming process, it is necessary to perform the process using a series of media with gradually decreasing osmotic pressure in an effort to reduce osmotic shock [21]. One commonly used warming protocol uses three steps, beginning with 0.3 mol/L sucrose in base medium, followed by transfer to 0.2 mol/L sucrose in base medium, and finally to a solution containing only base medium [41].

The vitrification outcome is highly operator dependent, and it requires a totally different skill set than is needed with slow freezing. The embryologist should be rapidly handling the embryos in micro-volumes of highly viscous media. Also, because there are a variety of loading devices available, specific training on the use and storage of a certain device and standardization of quality control procedures is mandatory. The embryologist should be well oriented to the different critical procedural details that can affect the vitrification outcome. Those details can be summarized as follows:

Researchers are currently studying different methods to improve vitrification outcome by manipulating the essential factors (Cryoprotectants concentrations, constituents, freezing rate, warming). The vitrification of embryos has shown to be successful at low cryoprotectant concentration and increased rate of freezing. [63].

Simultaneously, non traditional tools such as the effect of high hydrostatic pressure (HHP) in the pre-treatment of oocytes and embryos, including blastocysts to improve vitrification outcomes is also under investigation. Research has shown that HHP leads to the production of heat shock proteins in mammalian cells [64], which could potentially provide enough cellular protection to maintain homeostasis and even improve cryoprotection [65]. The types and amount of such proteins synthesized in the stressed cells depend on the intensity and type of the heat shock as well as on the stressed cell type and state.

Recent studies have reported promising results when applying HHP prior to vitrification of murine blastocysts, mature porcine oocytes and boar semen [39, 66‐69]. For example, applying hydrostatic pressure of 60 MegaPascals (MPa) for 30 minutes then allowing four to five minutes before vitrification significantly improved the survival and hatching rates of vitrified murine blastocysts [68].

The pressure level, pressure duration, temperature at time of pressurizing, and recovery time before vitrification are important parameters that need to be properly identified for oocytes, embryos, and blastocysts of different species [69]. However, further studies would be required to fully understand and control this phenomenon as well as to standardize its use. The use of high hydrostatic pressure before vitrification is still under investigation.

Vitrification of blastocysts can be successfully carried out using many loading devices. It could eventually replace slow freezing of blastocysts as suggested by various reports in the literature[70, 71] Though effect on perinatal outcome has not been fully investigated due to the novelty of the technique in clinical practice, however, the available data supports its potential safety. Other than the patient clinical parameters, the clinical success of transferring vitrified blastocysts would rely on a multitude of factors. The selection of a good quality embryo on preferably day 5 post fertilization is the 1^st step. The selection of blastocysts that show earlier re-expansion post-thaw for transfer could improve the outcome from transferring vitrified blastocysts. The assisted hatching and induction of blastocoele collapse prior to vitrification have also shown to improve the blastocyst vitrification outcome. Current media protocols and loading devices are capable of achieving proper vitrification attaining high level of viscosity and dehydration of the blastocysts and delivering high freezing and warming rates. Still further developments in vitrification media and devices are possible. Finally, the embryologist training would have a major bearing on the vitrification outcome.