Vitrification of Embryos: Main Issues. Dilemmas. Breakthroughs. Perspectives

Abstract: Vitrification of Embryos: Main Issues. Dilemmas. Breakthroughs. Perspectives.

The article was designed as a retrospective overview of the scientific studies for distinguishing the peculiarities of an attractive Ultrarapid Cryopreservation Technique which has gained support as an alternative promising substitute for slow Cryopreservation whereby the embryo is transitioned from 37°C to −196°C in <1 s, resulting in extremely fast rates of cooling (>10 000°C/min) – Vitrification, which is implemented in IVF clinics all over the world. The article focuses on representing, analyzing and comparing the basic overview of main issues, dilemmas, breakthroughs and perspectives in Vitrification’s dimension. The advantages and disadvantages of this technique were distinguished and discussed; the comparison of morulae (day 4) and blastocysts (day 5) using a Vitrification procedure was transparently and inclusively represented; the alternative blastocoele collapse preventive Pre–vitrification method was outlined and postulated: the artificial shrinkage technique is the most effective Vitrification–related embryo damage preventive measure: it was revealed that blastocoele collapse by micropipetting prior to Vitrification gives excellent survival outcomes for Day 5 and Day 6 expanded blastocysts; independent factors that are linked to the Vitrification process are accurately presented.

Key words: Cryoprotectants, Embryo, Slow Freezing, Ultrarapid Cryopreservation Technique, Vitrification

Meta Key Words: Artificial Shrinkage, Blastocoele, Blastocoele Collapse, Blastocoelic Cavity, Blastocyst Stage Embryos, Blastomeres Multicellular Structure, Cell Loss, Cleavage Stage Embryos, Compacted Morulae, Cryoloop, Cryopreservation, Cryoprotectants, Cryoprotectant Chemical Toxicity, Cryotop, Blastocysts, Blastomere survival, Cell Loss, Chromosomal Abnormalities, Chromosomal Mosaicism, Dehydration, Embryo, Embryologic Development, Embryo Transfer, Embryo Toxic Compounds, Expanded Blastocyst, Embryo Cryopreservation, Frozen Embryo Transfer, Inner Cell Mass, Intracellular Physiology, Liquid Nitrogen, Micropipetting, Needle Device, Slow Freezing, Toxic Injury, Trophectoderm Cells, Ultrarapid Cryopreservation Technique, Vitrification, Water–Filled Blastocoele, Zona Fractures Injury

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Introduction

Embryo Cryopreservation plays a significant role in Assisted Reproduction. Two basic techniques have been employed for the Cryopreservation of cells: controlled slow–rate freezing and Vitrification. Although slow freezing remains the most commonly used method of Cryopreservation in IVF laboratories, recent studies have reported increasingly successful clinical results with Vitrification [Mukaida et al., 2003a, b; Kuwayama et al., 2005a, b; Raju et al., 2005; Desai et al., 2007].

Vitrification is an attractive Ultrarapid Cryopreservation Technique which has gained support as an alternative promising substitute for slow Cryopreservation whereby the embryo is transitioned from 37°C to −196°C in <1 s, resulting in extremely fast rates of cooling (>10 000°C/min). Vitrification of blastocysts is being used increasingly to cryopreserve supernumerary embryos following In Vitro Fertilization (IVF) treatment cycle. It uses extremely high concentrations of cryoprotectants and allows the solidification of a solution below the glass transition temperature, without ice crystal formation [Liebermann, 2009; Vajta et al., 2009]. Vitrification has been successfully applied to both cleavage and blastocyst stage embryos and clinical trials have shown high survival rates and promising implantation rates following transfer of thawed embryos at all stages [Liebermann and Tucker, 2006; Mukaida et al., 2006; Hong et al., 2009; Vanderzwalmen et al., 2009, 2003; Desai et al., 2010; Wikland et al., 2010]. The data on the safety of Vitrification in terms of obstetric and perinatal outcomes are also reassuring [Mukaida et al., 2008; Liebermann, 2009; Noyes et al., 2009]. However, blastocysts represent a unique challenge because of the difficulty in accomplishing the required level of dehydration and high viscosity evenly in all blastomeres,
due to their multicellular structure and presence of the water–filled blastocoele [Vanderzwalmen et al., 2002]. High concentrations of cryoprotectants together with rapid cooling rates are essential to cryopreserve embryos in a vitrified, glass–like state [Vajta and Kuwayama, 2006]. To facilitate rapid heat transfer, minimal volumes are used in Vitrification, facilitated through the use of minute tools as carriers. The carrier systems that have been developed for the Vitrification procedure include the electron microscope grid [Park et al., 2000; Son et al., 2002], pulledand hemi–straws [El–Danasouri and Selman, 2001; Vanderzwalmen et al., 2002, 2003], flexipipet [Liebermann and Tucker, 2002], cryotop and cryotip [Kuwayama, 2007] and the cryoloop [Lane et al., 1999; Mukaida et al., 2001, 2003a, b; Reed et al., 2002; Rama Raju et al., 2005]. The use of each technique has recently been reviewed [Chen and Yang, 2007].

No studies have looked into the dividing cells and the effects Vitrification may have on spindle structure, chromosome alignment and ability of spindles to form and continue normal cell division. Therefore, it was postulated as essential issue to investigate the effects of Vitrification on Day 5 on the cytoskeleton and development of blastocysts, by analyzing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy.

The literature on Vitrification of embryos has mainly focused on the blastocyst stage [Lane et al., 1999; Mukaida et al., 2001, 2003a, b; Reed et al., 2002; Vanderzwalmen et al., 2002, 2003; Hiraoka et al., 2004; Liebermann and Tucker, 2004, 2006; Huang et al., 2005; Kuwayama et al., 2005a, b; Stehlik et al., 2005; Takahashi et al., 2005]. There have been fewer publications on the clinical application of cleavage–stage embryo vitrification [Mukaida et al., 1998; Saito et al., 2000; El–Danasouri and Selman, 2001; Kuwayama et al., 2005a, b; Rama Raju et al., 2005; Desai et al., 2007; Kuwayama, 2007]. To date, only Desai et al. (2007) have reported clinically acceptable implantation and pregnancy rates. This study confirms that in terms of embryo metabolism and progression to the blastocyst stage, Vitrification of Day 3 human embryos with the cryoloop is superior to slow freezing. The cryosurvival rate with Vitrification (∼95%) is similar to that reported by Rama Raju et al., and higher than that published by Desai et al. [Rama Raju et al., 2005; Desai et al., 2007].

Vitrification is performed by suspending the embryo(s) in a solution containing a high concentration (5–8 mol/l) of cryoprotectants and then directly plunging the embryo(s) into Liquid Nitrogen (LN) (−196°C). The concentration of the cryoprotectant is so high that during the ultra–rapid freezing until –196°C, the viscosity of the solution increases and forms a glass–like solid. This procedure allows the embryo to be plunged directly into the Liquid Nitrogen, avoiding crystallization during the freezing and warming steps [Vanderzwalmen et al., 2002]. As a consequence, the physical injuries caused by the formation of extracellular as well as intracellular ice crystals during the controlled slow–freezing procedure are eliminated. The advantage of this technique is the prevention of ice crystal formation made possible through increased speed of temperature conduction, reducing associated chilling injuries within the embryo tissue or outside the cytoplasm. However, the toxicity caused by the high concentration of cryoprotectant is a drawback of this technique [Fahy et al., 1984; Rall et al., 1987]. Additionally, the osmotic stress and toxic effect from high cryoprotectant concentrations may constitute an obstacle to using this method. Several new techniques and applications were developed recently that improve the survival rate for blastocysts following Vitrification. These procedures include the use of an Electron Microscope (EM) grid [Martino et al., 1996; Cho et al., 2002], cryoloops [Lane et al., 1999; Mukaida et al., 2001], an open pulled straw (OPS) [Yokota et al., 2000, 2001] and closed plastic straws [Vanderzwalmen et al., 2002], as well as to compacted morulae using closed plastic straws [Vanderzwalmen et al., 2002]. Grids and cryoloops have the highest cooling rate due to the extremely low volume of Vitrification medium and immersion contact with Liquid Nitrogen (LN 2). However, they require laborious embryo handling and carry the risk of Liquid Nitrogen (LN 2) contamination. Vitrification of expanded blastocysts may be further improved by reducing the volume of the blastocoelic cavity [Vanderzwalmen et al., 2002; Son et al., 2003], or by using a sucrose six–step dilution after warming [Cho et al., 2002].

The Open Oulled Straw method (OPS) has been applied to oocytes and early embryos [Liebermann et al., 2002a], but not to compacted morulae and early blastocysts. In the Open Pulled Straw (OPS) technique, the low vessel inner diameter (0.8 mm versus 1.7 mm in French straws) and holding volume (1–2 µl versus up to 250 µl in French straws) increase the cooling rate and allow lower cryoprotectant concentrations to be used, thereby reducing toxic injury; direct contact with LN 2 can be avoided by having extra air and cryoprotectant interfaces on either side of the bead of cryoprotectant solution containing the embryos [Chen et al., 2001; López–Béjar and López–Gatius, 2002]. The cooling and warming rates can be further modified by using glass–pulled micropipettes, due to the higher heat conductivity of glass, reduced capillary size (0.33 mm inner diameter) and reduced loading volume (1–2 µl) [Kong et al., 2000].

These techniques attempt to accelerate the blastocyst cooling rate and decrease the cryosolution volume needed for this procedure. A practical advantage is that the speed of the Vitrification process minimizes the period the embryo is outside of the incubator. Vitrification requires minimal set up time, being performed as needed during the course of the day. Furthermore, there is no need for expensive programmable Cryopreservation equipment. One potential disadvantage of Vitrification is that it is considered technically more challenging than slow freezing and requires more hands–on time per Cryopreserved embryo.

Successful slow freezing and thawing of blastocysts with high survival rates (from 76 up to 94%), originating from both Day 5 and Day 6 good–quality blastocysts, has been reported in several studies [Langley et al., 2001; Behr et al., 2002; Anderson et al., 2003; Veeck et al., 2004; Desai and Goldfarb, 2005; Liebermann and Tucker, 2006]. Slow Freezing was shown to be successful also when unselected blastocysts at all developmental stages, i.e. compacted, expanded and hatching blastocysts, were cryopreserved [Virant–Klun et al., 2003]. Results of slow–freezing programme for blastocyst Cryopreservation have been reported by Van den Abbeel et al. (2005). In this study, two–blastocyst slow–freezing strategies were evaluated in a Day 2/3 fresh Embryo Transfer programme. It was found that early blastocysts showed a better morphological survival than advanced or hatching blastocysts, but this benefit was lost by a lower in vitro developmental capacity. An overall morphological survival rate of 71.9% was obtained. In the case of blastocyst Cryopreservation, one is confronted with a cohort of blastocysts at different developmental stages to be frozen, ranging from early cavitating to expanded and hatching blastocysts. Because of the complex structure of blastocysts containing Inner Cell Mass (ICM) cells and a fluid–filled cavity that is surrounded by a barrier of trophectoderm cells, the Cryopreservation of expanded blastocysts remains a challenge. Therefore, in order to optimize blastocyst Cryopreservation outcome at IVF clinics, slow freezing is often replaced by Vitrification. This method has been modified towards using minimal volume methods [Lane et al., 1999; Kuwayama et al., 2005a,b] and has as such become an interesting alternative to slow freezing for Cryopreservation of embryos, as Vitrification has many advantages: it does not require ice–seeding, slow cooling rates or a programmable freezer, and thus cooling is instant. Vitrification is expected to achieve a high rate
of survival because of the absence of ice. This is crucial for the Cryopreservation of full and expanded blastocysts which are less permeable to water and have a fluid–filled cavity very sensitive to lethal ice crystal formation
[Tucker and Liebermann, 2007]. Therefore, in the literature, Vitrification has become the method of choice to cryopreserve blastocysts [Cho et al., 2002; Son et al., 2003; Mukaida et al., 2003, 2006; Vanderzwalmen et al., 2003; Kuwayama et al., 2005a,b; Liebermann and Tucker, 2006].

Although so far there are no proven viral transmissions between embryos or to the patient at Embryo Transfer, the use of an open system for Vitrification potentially introduces a risk of cross–contamination during Liquid Nitrogen storage [Bielanski and Vajta, 2009]. Therefore, a feasible closed method Vitrification system should be preferentially introduced in IVF laboratories. It was reported in the review by Bielanski and Vajta (2009) that regarding plastic straws, it appears that the CBS High Security Straws which are made from ionomeric resin are the most suitable storage containers for Cryopreservation and Storage of germplasm. These straws are heat sealed on both ends and are impermeable to pathogens. Ionomeric resin straws appeared to be safe in contrast with polyvinylchloride and polyethylene terephthalate glycol straws which could probably be attributed to splashing during ultrasonic sealing. Therefore and because of its validation in a clinical study, the closed Vitrification system using CBS straws, with Dimethylsulphoxide (DMSO) and Ethylene Glycol as the cryoprotectants, can be chosen as the Vitrification system for routine clinical use. In order to optimize blastocyst Cryopreservation, Van Landuyt L. et al. (2011)
through performing the investigation and analyzing its results assessed the efficiency of closed blastocyst Vitrification in relation to embryo development at the time of Cryopreservation and clinical transfer practice
in a large series of consecutive Vitrification cycles. The survival after warming and the transfer rates of Day 5 and 6 vitrified blastocysts of different morphological quality and at different developmental stages were also analyzed by the scientists [Van Landuyt L. et al., 2011].

1. Main issues and dilemmas which should be discussed about Embryo Vitrification procedure

1.1. Overview of the study “Outcome of closed blastocyst Vitrification in relation to blastocyst quality: evaluation of 759 warming cycles in a single–Embryo Transfer policy” written by Van Landuyt L., Stoop D., Verheyen G., Verpoest W., Camus M., Van de Velde H., Devroey P. and Van den Abbeel E.

Pre–vitrification procedures in close scientific focus: In Vitro Fertilization (IVF)/ Intracytoplasmic Sperm Injection (ICSI) Treatment, embryo culture and Embryo Selection

For in vitro culture of oocytes and embryos, sequential media formulations should be used and embryos should be cultured at 37°C in an atmosphere of 6% CO2, 5% O2 and 89% N2. Oocytes and embryos should be cultured individually in 25 µl medium droplets covered with mineral oil, from Oocyte Retrieval (Day 0) until transfer or Cryopreservation (Day 5/6). All embryos should be evaluated daily [Van Landuyt L. et al., 2011].

Embryo Selection for transfer or Cryopreservation should be done in the morning of Day 5. Blastocysts should be scored according to the grading system of Gardner and Schoolcraft (1999). Embryos should be considered for fresh transfer on Day 5 if they reached the stage of full compaction, early blastocyst (Bl1 or Bl2), full (Bl3), expanded (Bl4) or hatching (Bl5–6) blastocyst. Cavitated blastocysts fulfill the criteria for transfer if they have at least an Inner Cell Mass (ICM) type C and trophectoderm quality type B. On Day 5, early or full, expanded and hatching blastocysts with ICM and trophectoderm type A or B (Gardner’s grading system) are considered eligible for Cryopreservation. On Day 6, only full, expanded or hatching blastocysts with ICM type A and trophectoderm quality A or B should be cryopreserved [Van Landuyt L. et al., 2011].

Vitrification and warming Protocols

Vitrification should be performed using closed CBS–VIT High Security (HS) straws in combination with the cryoprotectants. Blastocysts should be vitrified one by one. The Vitrification procedure should be carried out at room temperature (between 22 and 27°C). The blastocyst should be first incubated for 2 minutes in a 50 µl droplet of HEPES–buffered culture medium. Then, the blastocyst should be brought in a 50 µl droplet of equilibration solution containing 7.5% (v/v) DMSO and 7.5% (v/v) Ethylene Glycol and incubated for 10 minutes. The blastocyst should be then transferred consecutively into four 25–µl droplets with Vitrification solution containing 15% (v/v) DMSO and 15% (v/v) Ethylene Glycol. The blastocyst should be incubated for 5 seconds in droplets 1 and 2 and for 10 seconds in the third droplet. The blastocyst should be then transferred to the fourth droplet and immediately loaded onto the CBS–HS straw. The straw should be heat sealed and plunged into Liquid Nitrogen. The total time need to vitrify the blastocyst starting from the first Vitrification droplet to the loading of the straw and plunging into Liquid Nitrogen do not exceed 90 seconds [Van Landuyt L. et al., 2011].

On the day of transfer, blastocysts should be warmed one by one until one or two blastocysts would be suitable for transfer. The choice to transfer one or two embryos in the frozen cycle is decided by the clinician at consultation mainly depending on the patient’s age and the number of embryos replaced in the previous Treatment cycles. Blastocysts should be warmed randomly, independently of the blastocyst stage or quality prior to Vitrification. A Petri dish containing two 25 µl droplets with thawing solution [TS; 1 M sucrose in HEPES–buffered Human Tubal Fluid (HTF) medium supplemented with 20% DSS] should be kept at 37°C. For warming the straw, the straw should be transferred from the Liquid Nitrogen (LN2) storage container to a transport dewar filled with Liquid Nitrogen. After cutting the straw and pulling the capillary from the straw, the gutter should be placed in the first droplet with TS and the blastocyst should be released from the gutter. The blastocyst should be incubated for two times 1 minutes at room temperature in the two TS droplets. The blastocyst should be then transferred to the first of two–Dilution Solution (DS) droplets of 25 µl (0.5 M sucrose in HEPES–buffered HTF medium supplemented with 20% DSS) and after that incubated for 2 minutes in a second DS droplet. Finally, the blastocyst should be washed in three droplets (25 µl) of washing solution (HEPES–buffered HTF medium supplemented with 20% DSS), each for 3 minutes. After warming, the blastocyst should be transferred to a culture dish with blastocyst medium to assess its morphological survival. If the blastocyst is severely or completely damaged, a new one should be warmed immediately. If the blastocyst is fully intact or showed moderate damage, expansion and re–expansion should be assessed 1–2 hours later. If the morphological quality of the blastocyst is regressing or no signs of re–expansion are present, an additional blastocyst should be warmed until one blastocyst is suitable for transfer, for instance,
with good survival (less than half of the blastocyst showing signs of damage) and expansion/re–expansion
of the blastocyst [Van Landuyt et al., 2011].

For warming, the cryoloop should be removed from the cryovial and dipped into Warming Solution 1. Embryos fall off the cryoloop and should be moved through 1 ml volumes of a serial sucrose dilution in G–MOPS supplemented with 12 mg/ml HSA: Warming Solution 1 (0.65 M sucrose) for 30 seconds; Warming Solution 2 (0.325 M sucrose) for 1 minute; Warming Solution 3 (0.125 M sucrose) for 2 minutes and Warming Solution 4 (0 M sucrose) for 5 minutes. For subsequent embryo development, embryos should be moved into G2.3 (Vitrolife) for 24 hours (Embryo Transfer) or 48 hours (blastocyst assessment) [Balaban et al., 2008].

The embryological outcome parameters

The embryological outcome measures are the immediate morphological survival after warming (percentage of fully intact and moderately damaged blastocysts/number of warmed blastocysts) and the transfer rate (percentage of blastocysts transferred/number of warmed blastocysts). Immediate morphological survival should be evaluated according to the day of Vitrification, according to the blastocyst stage prior to Vitrification [early (Bl1–2) or advanced blastocysts (Bl3–4)] and according to the blastocyst quality on Day 5 (Inner Cell Mass (ICM) type A or B). The following clinical outcome measures should be also analyzed: clinical pregnancy rate per transfer (exemplification: a pregnancy with an intrauterine gestational sac seen at transvaginal ultrasound scan at least 5 weeks after embryo transfer [Zegers–Hochschild et al., 2009], ongoing pregnancy rate per transfer (exemplification: a clinical pregnancy with a fetal heartbeat (FHB) at ≥12 weeks [Bonduelle et al., 2002] and implantation rate (with FHB) per transferred embryo.

Morphological survival of the warmed blastocysts after warming

According to the study of Van Landuyt L. et al. (2011), the overall morphological survival of the 1185 warmed blastocysts was 77.8%, regardless of their quality or Day of Cryopreservation. In our Vitrification policy, advanced blastocysts with Inner Cell Mass (ICM) A and B as well as good–quality early blastocysts on Day 5 of in vitro culture were vitrified. On Day 6, only blastocysts with Inner Cell Mass (ICM) type A were selected for Vitrification. However, the Day 6 vitrified blastocysts always originated from early blastocysts that were not selected for Vitrification on Day 5 because of their doubtful quality. Before warming, there was no selection concerning the blastocyst quality, i.e. always the first blastocyst that was vitrified was chosen for warming. Hence, different types of blastocyst qualities could be included in this observational analysis [Van Landuyt L. et al., 2011]. Early blastocysts survived better than advanced Day 5 blastocysts as was demonstrated in other studies on blastocyst Vitrification [Cho et al., 2002; Vanderzwalmen et al., 2002; Mukaida et al., 2003, 2006; Zech et al., 2005; Ebner et al., 2009].

The risk of detrimental ice crystal formation is higher in expanded blastocysts compared with early blastocysts due to the large fluid–filled cavity.

Furthermore, it has been observed that later blastocyst stages are less permeable to the cryoprotectant [Cho et al., 2002; Vanderzwalmen et al., 2002], with a higher risk of intracellular ice formation. Several groups perform artificial shrinkage to collapse the fluid–filled blastocoelic cavity before Vitrification in order to obtain higher survival rates in advanced blastocysts [Vanderzwalmen et al., 2002, 2003; Son et al., 2003; Hiraoka et al., 2004; Mukaida et al., 2006]. A survival rate of 78.7 and 72.7% was represented in the Van Landuyt L. et al. (2011) study for Day 5 full and expanded blastocysts in a closed Vitrification system without artificial shrinkage is acceptable. However, the scientists proposed that improvements should be made to make the procedure more robust, for instance increasing the volume of the droplets with warming solution may optimize the warming rate [Van Landuyt L. et al., 2011].

1.2. Overview of the study “A randomized controlled study of human Day 3 Embryo Cryopreservation by slow freezing or Vitrification: Vitrification is associated with higher survival, metabolism and blastocyst formation” written by Balaban B., Urman B., Ata B., Isiklar A., Larman M.G., Hamilton R. and Gardner D.K.

The aim of Balaban et al. (2008) study was therefore to assess the efficacy of slow freezing and Vitrification on the survival, metabolism and subsequent development of cleavage–stage embryos. It was hypothesized that Vitrification is associated with less cellular trauma than slow freezing and should be considered as the primary method of Embryo Cryopreservation. Furthermore, the scientists reported additional clinical data regarding the outcome of transfer of Day 3 human embryos vitrified using the cryoloop [with Ethylene Glycol (EG) and 1,2–Propanediol (PROH) as cryoprotectants] to a cohort of women.

As noted in the discussion overview by Balaban et al. (2008), the results presented there provide the most extensive analysis and comparison between slow freezing and Vitrification of Day 3: there were 466 embryos from 120 patients used in the initial laboratory study to give an indicator of survival, including how many embryos survived with all blastomeres intact, and viability through to subsequent embryo development. As a way of analyzing the cellular effect of the Cryopreservation method, pyruvate uptake was measured in 82 individual embryos post–thawing/warming. The early embryo predominantly uses pyruvate for metabolism, so measuring the amount of pyruvate consumed by the embryo gives an indication of embryonic health. Pyruvate uptake by embryos after Vitrification was significantly higher than that after slow freezing. Pyruvate uptake by embryos after Vitrification was significantly higher than that after slow freezing [Balaban et al., 2008].

Previously published protocols using the cryoloop predominantly used EG and Dimethyl Sulfoxide (DMSO) as the cryoprotectants [Mukaida et al., 2001; Reed et al., 2002; Sheehan et al., 2006; Desai et al., 2007]. However, concerns with regard to the safety of Dimethyl Sulfoxide (DMSO) have been raised. One of the major potential problems with Dimethyl Sulfoxide (DMSO) is that it is a very potent solvent. Embryo toxic compounds present within the system may be easily introduced into the embryo. Furthermore, Dimethyl Sulfoxide (DMSO) has some untoward effects on intracellular physiology; the cryoprotectants Ethylene Glycol (EG), 1,2–Propanediol (PROH) and Dimethyl Sulfoxide (DMSO) have all been shown to be associated with an increase in intracellular calcium in oocytes. Significantly, however, EG and PROH induce an influx of calcium from the extracellular medium. In contrast, DMSO induces an increase in intracellular calcium through a release of intracellular calcium, most likely through disruption of intracellular organelles. Therefore, although the effects of Ethylene Glycol (EG) and 1,2–propanediol (PROH) can be alleviated through the use of a calcium–free medium, Dimethyl Sulfoxide (DMSO) induces intracellular aberrations independent of external calcium, making its presence in Cryopreservation solutions of greater concern. Dimethyl Sulfoxide (DMSO)has also been shown to cause cellular differentiation and affect DNA methylation in other cell types [Morley and Whitfield, 1993; Iwatani et al., 2006].

A potential concern regarding the cryoloop is that it is an open system employing direct contact with Liquid Nitrogen. The possibility of viral contamination of Liquid Nitrogen has been suggested following the spiking of Liquid Nitrogen storage vessels with high viral titers [Bielanski et al., 2000].

1.3. Overview of the study “Influence of cell loss after Vitrification or slow-freezing on further in vitro development and implantation of human Day 3 embryos” written by Van Landuyt L., Van de Velde H., De Vos A., Haentjens P., Blockeel C., Tournaye H. and Verheyen G.

The developmental potential of cryopreserved cleavage–stage embryos has been investigated by several authors and was initially based on slow–freezing and thawing results. Higher implantation rates have been obtained for fully intact Day 2 embryos than that for damaged ones [Van den Abbeel et al., 1997; El–Toukhy et al., 2003]. Blastocyst formation rate was higher for intact Day 2 embryos than that for damaged ones [Archer et al., 2003]. Later on, similar implantation rates were reported for intact 4–cell embryos and 4–cell embryos with 1 cell damaged after thawing [Gabrielson et al., 2006; Edgar et al., 2007]. For day 3 embryos, lower implantation rates were observed when at least 25% of cells were damaged [Tang et al., 2006]. However, other studies reported a similar implantation potential for 7– to 8–cell embryos with 0, 1 or 2 cells damaged [Zheng et al., 2008]. Several studies showed higher survival rates and a higher number of fully intact embryos after Vitrification than after slow–freezing [Kuwayama et al., 2005; Raju et al., 2005; Balaban et al., 2008; Valojerdi et al., 2009; Wang et al., 2012]. Balaban et al. (2008) reported that the development to the blastocyst stage was higher after Vitrification than after slow–freezing. However, overnight cleavage rate and implantation potential of intact versus damaged embryos of different cell stages after Vitrification have not been analyzed so far, therefore, the scientists Van Landuyt L., Van de Velde H., De Vos A., Haentjens P., Blockeel C., Tournaye H., Verheyen G. investigated the survival, further cleavage and transfer rate of different cell stages on Day 3 of development after Cryopreservation using either slow–freezing or Vitrification to reveal the influence of cell loss after Vitrification or slow–freezing procedures. In other words, the scientists aimed to analyze the post–thaw survival and the overnight cleavage rate of Day 3 embryos in relation to their cell stage before Cryopreservation and cell loss post–thawing/warming for both slow–freezing and Vitrification. The impact of cell loss on the implantation potential was analyzed in single Frozen–Embryo Transfers (SFETs) for both Cryopreservation methods. Survived vitrified embryos developed better overnight than slowly frozen embryos, irrespective of the number of cells damaged and the cell stage at Cryopreservation [Van Landuyt et al., 2013].

The present data confirmed that damaged Day 3 embryos have a lower overnight developmental potential than intact embryos, both after Vitrification and slow–freezing. Furthermore, overnight cleavage significantly decreases with increasing number of cells lost. In the slow–freezing group, the impact of cell damage on overnight cleavage was more detrimental in 6– and 7–cell embryos than in embryos with at least eight cells. This finding was not observed after Vitrification, probably due to the lower number of damaged embryos. Regardless of the Cryopreservation method, the scientific data confirmed the expectations that the impact of the number of cells lost is more detrimental in 6 and 7–cell embryos than in embryos with ≥8 cells, as they lose a higher percentage of cell volume compared with an 8–cell embryo. However, even when the data were analyzed with respect to the percentage of cell damage (and not according to the number of cells lost), higher cleavage rates were found
for 8–cell embryos. This was also reflected in the significantly higher transfer rates per thawed/warmed embryo obtained with embryos with at least eight cells. This implies a better intrinsic quality of normal developing embryos. In this view, the decision to warm an extra embryo before overnight depends on the cell stage at Cryopreservation and the post–warming cell damage. For 8–cell embryos, up to two cells can be damaged compared with only one cell for 6– to 7–cell embryos, to be considered sufficient for overnight culture without warming an additional embryo [Van Landuyt et al., 2013].

The study also showed that once a survived embryo shows resumption of mitosis after thawing, there was neither an impact of the cell stage at Cryopreservation, nor of the cell damage on its implantation capacity. Edgar et al. (2007) also observed for Day 2 slowly frozen 4–cell embryos that resumption of mitosis of at least two blastomeres, independent of blastomere survival, was indicative for significantly higher implantation rates. Guérif et al. (2002) concluded that the most predictive factor for the implantation potential of a Day 2 thawed embryo is the final number of blastomeres present at transfer, irrespective whether these cells resulted from blastomere survival or from resumption of mitosis.

The integrative question whether the higher cleavage rate of vitrified embryos also results in better implantation rates and whether a vitrified embryo per se is a more viable embryo than a slowly frozen embryo can only be answered by performing a randomized controlled trial [Van Landuyt et al., 2013]. The systematic review and meta–analysis of Loutradi et al. (2008) and Kolibianakis et al. (2009) concluded that further properly designed trials are needed to evaluate the two Cryopreservation techniques in terms of pregnancy rates. The meta–analysis of Abdelhafez et al. (2010) suggested superiority of Vitrification to slow–freezing regarding clinical outcomes. However, only one randomized trial was included for cleavage stage embryos. Taking into account the large difference in post–thawing/warming survival between slow–freezing and Vitrification, it would now be unethical to start a randomized controlled trial. Since vitrified Day 3 embryos survive significantly better, more frozen embryo cycles originating from one oocyte collection cycle can be offered to the patient. Thus, it can be hypothesized that the clinical advantage of Vitrification will be reflected in elevated cumulative pregnancy rates. Expressed per embryo transferred, the retrospective data did not reveal superior implantation rates of vitrified embryos upon slowly frozen ones, taking into account that only survived embryos with overnight cleavage were transferred. Recent retrospective data of Wang et al. (2012) showed higher implantation rates after Vitrification using Cryoleaf compared with slow–freezing (19.8 versus 14.4% and P = 0.01). However, warming and transfer of embryos were planned on the same day, and thus results were analyzed irrespective of overnight cleavage. Therefore, comparing those implantation rates with the results of the present study might not be justified [Van Landuyt et al., 2013]. In the randomized study of Wilding et al. (2010), implantation rates were not significantly different between Vitrification and slow–freezing (14.3 versus 13.5%), even though all embryos that contained at least one surviving blastomere were transferred and more intact embryos were transferred in the vitrified embryo group.

2. Breakthroughs in Embryo Vitrification technique (Ultrarapid Cryopreservation Technique)

2.1. Overview of the study “Experimental Vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes” written by Cremades N., Sousa M., Silva J., Viana P., Sousa S., Oliveira C., Teixeira da Silva J. and Barros A.

Vitrification procedure and warming procedure

Embryos were incubated at 37°C in sperm preparation medium as holding medium (SPM, Medicult: HEPES‐buffered IVF medium, with 10% synthetic serum substitute and 7% human synthetic albumin) for 1 minute, then in SPM + 7.5% Ethylene Glycol (EG) + 7.5% Dimethylsulphoxide (DMSO) for 3 minutes and finally in SPM + 16.5% EG + 16.5% DMSO + 0.67 mol/l sucrose for 25 seconds. They were then aspirated (one or two embryos) with an automatic 0.1–10 µl micropipette (Gilson) using a plastic micropipette tip with a long and soft extremity (0.36 mm inner diameter; Sorenson BioScience, Inc., USA; MiniFlex Round Tips, RNase/DNase–free, Sterile, 0.1–10 µl, Ref: 15110). The tip was then exposed to Liquid Nitrogen (LN 2) vapour for 2 minutes (nearly in contact with LN 2), first almost horizontal and then vertical, before being removed from the automatic micropipette, and then closed inside a pre–cooled 3.6 ml cryotube and plunged into Liquid Nitrogen (LN 2). Embryos were thawed 1 month later: the tip was held with thumb and middle finger for 3 seconds and then immersed in SPM + 0.33 mol/l sucrose (37°C), at a 30–45° angle (from horizontal), taking care that all the vitrified liquid column was immersed.
As the solution softened, the outer medium started to enter the tip. At this moment, the open end of the tip was closed with the index finger and the solution flew out from the tip as the result of the increased pressure
of the warming air inside the tip. After 1 minute, embryos were transferred to SPM + 0.2 mol/l sucrose
for 5 minutes, then to SPM for 2×5 minutes, and finally individually cultured for 24 hours in 50 µl drops
of Blast Assist System Medium–2 [Cremades et al., 2004].

After–Vitrification results

A total of 63 embryos were vitrified. In the first experiment, the overall survival rate was 27/36 (75%), being 14/19 (73.7%) for compacted morulae and 13/17 (76.5%) for early blastocysts. In the next 24 hours, all live morulae evolved to early blastocysts and all but one live early blastocyst evolved to late blastocysts, with 2/13 (15.4%) attaining the hatching stage. After warming, embryos shrunk but then re–expanded, except when degenerated. Degenerated embryos showed darkening of most of the cells, followed by the disappearance of the nuclei and cytoplasmic swelling and lysis. No zona fracture was noticed in this set of experiments. In the case of morula degeneration, blastomere darkening and lysis began at the periphery, with decompaction of the remaining blastomeres being observed only at a late stage. Focal blastomere degeneration did not compromise further evolution of morulae. In the case of blastocyst degeneration, total cell lysis was rare and in most of the cases the trophectoderm was more severely affected than the inner cell mass. When only a few cells in the trophectoderm and inner cell mass showed evidence of degeneration these did not compromise embryo development. In the second set of experiments, the overall survival rate was 22/27 (81.5%), being 8/11 (72.7%) for compacted morulae and 14/16 (87.5%) for early blastocysts. After 24 hours culture, 22 embryos developed further into expanded blastocysts. One of the 16 (6.3%) early blastocysts showed a zona pellucida fracture, but did not degenerate. Two of the 14 (14.3%) early blastocysts that survived hatched. Thus, in total, 49/63 (77.7%) of the vitrified embryos survived, 22/30 (73.3%) from compacted morulae and 27/33 (81.8%) from early blastocysts, with 4/27 (14.8%) of the surviving blastocysts reaching the hatching stage [Cremades et al., 2004].

The scientists have assessed the survival rate of excess compacted morulae and early blastocysts after Vitrification in fine pipette tips. The method differs from those described by other studies [Choi et al., 2000; Yokota et al., 2000, 2001; Mukaida et al., 2001, 2003; Cho et al., 2002; Reed et al., 2002; Vanderzwalmen et al., 2002; Son et al., 2003] blastocysts in that:

The holding solution was adapted to embryos using an IVF medium buffered with HEPES that contained 10% synthetic serum substitute and 7% human synthetic albumin, thus avoiding sera;
The OPS plastic straw (0.8 mm inner diameter, 0.07 mm wall thickness) was replaced by a plastic Emicropipette tip with a reduced size (0.36 mm inner diameter, 0.077 mm wall thickness);
Embryos were vitrified in a smaller volume (0.5 µl versus 1–2 µl in OPS); and
The tip was exposed to LN 2 vapour before plunging it into LN 2 inside a cryotube.

This would avoid LN 2 contamination and may have a further beneficial effect, in that the heat transfer would have been more uniform than if the tips had been immersed directly into LN 2 [Arav et al., 2002]. This, together with the low tip inner diameter and volume of the medium may also reduce the probability of zona fractures [Cremades et al., 2004]. To reduce the likelihood of fracture injury even further, the tips were warmed by holding them in the hand for 3, before immersing them into the warm dilution solution. Immediate contact with the dilution solution might cause unequal expansion of the tip and its contents [Arav et al., 2002] as well as causing the dilution solution to freeze around the tip. These factors may thus explain why this simple procedure gave an overall survival rate of 73% for compacted morulae and of 82% for early blastocysts, as well as a low incidence (1/63, 1.6%) of zona fractures.

Embryo degeneration was found in 14/63 (22.2%) of the cases, 8/30 (26.7%) for morulae and 6/33 (18.2%) for early blastocysts. When this occurred, it was characterized by absence of expansion followed by rapid darkening of the cytoplasm and cell lysis, with decompaction having only been found in late stages of degeneration [Cremades et al., 2004].

Although chemical toxicity was theoretically reduced by using a mixture of cryoprotectants and sucrose and by decreasing the time of exposure due to the very low diameter of the tip and volume of the solution [Arav et al., 2002; Liebermann et al., 2002a], the most likely mechanism for the observed degeneration of compacted morulae and early blastocysts seems to be the chemical toxicity of the cryoprotectant. In Cremades et al. (2004) study’s practical performance embryos were equilibrated at 37°C before and after Vitrification. It was postulated that equilibration at room temperature or 4°C would decrease evaporation and cryoprotectant toxicity, but also cryoprotectant diffusion [Cremades et al., 2004]. On the other hand, equilibration and dilution at a higher temperature can increase cell permeability and thus protect against osmotic swelling and osmotic shrinkage [Kasai et al., 2002]. To prevent excess swelling occurring when the cryoprotectant is removed after warming, the embryos are usually placed in a solution with a high concentration of sugar (thought of as non–permeating) [Cremades et al., 2004]. There is still a highly debated dilemma concerning the question which dilution strategy is best for embryos, as some data suggest that six–step sucrose dilution is better [Choi et al., 2000; Cho et al., 2002; Son et al., 2003], while other experts report acceptable survival rates with two [Yokota et al ., 2000 , 2001 ; Cho et al ., 2002] or three–step dilution [Mukaida et al., 2001, 2003; Reed et al., 2002; Vanderzwalmen et al., 2002; Cremades et al., 2004].

The practical performance of the procedure represented by Cremades et al. (2004) has several advantages over previously published Protocols. The tips are sterile, commercially available, very small, simple to load with an exact, very small amount of cryoprotectant and easy to handle as they are attached to a Gilson pipette throughout most of the procedure. This is important, as complex embryo handling increases the time of exposure to the Vitrification solution before cooling [Kasai et al., 2002]. A perspective extension of their research is seen in practical verification and establishment whether this technique will give clinical pregnancy rates as high as previously reported slow–cooled and vitrified embryos.

2.2. The artificial shrinkage technique as the most effective Vitrification–related embryo damage preventive measure: Blastocoele collapse by micropipetting prior to Vitrification [in accordance with the information represented in the study by Kenichiro Hiraoka, Kaori Hiraoka, Masayuki Kinutani, Kazuo Kinutani “Blastocoele collapse by micropipetting prior to Vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts”]

Since the first pregnancy after Vitrification of a blastocyst was reported using cryostraws [Yokota et al., 2000], attention has focused mainly on using very small volumes of cryoprotectant. This greatly increases the cooling and warming rate, while reducing chilling injuries and ice crystal formation. The efficacy of Vitrification in small volumes is demonstrated by good survival rates of early human blastocysts using the cryotop [Kuwayama and Kato, 2000], the cryoloop [Mukaida et al., 2001, 2003; Reed et al., 2002], electron microscope grids [Choi et al., 2000; Son et al., 2003] and the hemi–straw [Vanderzwalmen et al., 2003]. However, expanded blastocysts exhibit relatively poor survival rates after Vitrification [Cho et al., 2002]. Expanded blastocysts have more blastocoelic fluid than early blastocysts, in which ice crystals may form during cooling. Recently, Vanderzwalmen et al. (2002) and Son et al. (2003) reported an increase in the survival rate of blastocysts when the volume of the blastocoele was artificially reduced with a needle. However, this method is invasive because it makes a hole in the zona and trophectoderm. On the other hand, Motoishi (2000) reported that the blastocoele of expanded blastocysts could be artificially collapsed by mechanical pipetting with a glass pipette slightly smaller in diameter than the blastocyst. By using this artificial shrinkage method before Vitrification of expanded blastocysts, Motoishi (2000) reported a survival rate of 91% after warming, a clinical pregnancy rate of 65% and an implantation rate of 61% after the transfer of vitrified blastocysts. This artificial shrinkage method is thought to be non–invasive and useful for Vitrification of expanded blastocysts, but the usefulness of this method has not been well documented. The usefulness of this non–invasive artificial shrinkage method by pipetting before Vitrification was investigated by the scientific group Kenichiro Hiraoka, Kaori Hiraoka, Masayuki Kinutani and Kazuo Kinutani.

Controlled Ovarian Stimulation, Oocytes Retrieval and In Vitro Fertilization (IVF) process

Controlled ovarian stimulation was carried out with Human Menopausal Gonadotropin (hMG). Follicular development was monitored with serial vaginal ultrasound examinations and serum E2 measurements. Human Chorionic Gonadotropin (hCG) was administered when dominant follicles reached a diameter of 18 mm. Oocytes were collected 35 hours after hCG administration using an ultrasound–guided procedure and were incubated in HTF medium containing 10% (v/v) serum substitute supplement at 37°C in an atmosphere of 6% CO2, 5% O2 and 89% N2. Sperm preparation was carried out using discontinuous Isolate™ gradient. Mature oocytes were either inseminated with sperm 5–7 hours after Oocyte Retrieval at a concentration of 100 000 to 200 000 motile sperm per ml for 5–10 oocytes, or microinjected with a single spermatozoon. Fertilization was confirmed at 15–18 hours after Insemination by the presence of two pronuclei [Kenichiro Hiraoka et al., 2004].

Embryo Culture

Fertilized oocytes were washed well and cultured in P–1™ Medium or Blast Assist Medium 1 until day 3, and then placed in Blastocyst medium or Blast Assist Medium 2 until day 6 [Kenichiro Hiraoka et al., 2004]. In all cases, consecutive Embryo Transfer was performed, in which one or two cleaved embryos were transferred on day 2 or day 3 (first step of Embryo Transfer) and one or two blastocysts were transferred on day 5 or day 6 (second step of Embryo Transfer) [Goto et al., 2003]. During this period, two blastocyst culture media systems were used in order to evaluate and compare the results of consecutive embryo transfer between two sequential media at the same gas phase (37°C in an atmosphere of 6% CO2, 5% O2 and 89% N2) in each case. However, blastocyst culture media systems had no effect on the percentage of expanded blastocysts on days 5 and 6, or on the pregnancy and implantation rates. After a consecutive Embryo Transfer, surplus embryos that developed to the expanded blastocyst stage (diameter≥160 μm) were cryopreserved on day 5 or day 6 [Kenichiro Hiraoka et al., 2004].

On day 5, the percentage of blastocysts formation was recorded and classified under an inverted microscope (magnification ×200) according to the degree of expansion of the blastocoele, the quality of the Inner Cell Mass (ICM) and the trophectoderm. The blastocysts on Day 5 were classified into three different categories according to the degree of expansion of the blastocoele: the early blastocyst with a blastocoele being less than half the volume of the embryo, the blastocyst with a blastocoele being greater than half of the volume of the embryo, the expanded blastocyst with a blastocoele larger than the volume of the blastocyst and with a thin zona pellucida [Schoolcraft et al., 1999].

Preparation of Vitrification solutions

The expanded blastocysts were vitrified by the method developed by Kuwayama (2001) using a cryotop that consists of polyethylene laminate film (20 mm × 0.7 mm × 0.1 mm, L × W × T). As the base medium, Dulbecco’s phosphate–buffered saline solution (PBS 1×) plus 20% (v/v) SSS was used. The equilibration solution contained 7.5% (v/v) Ethylene Glycol (EG) and 7.5% (v/v) Dimethylsulphoxide (DMSO). The Vitrification solution was composed of 15% (v/v) EG, 15% (v/v) DMSO and 0.5 mol/l of sucrose (Nacalai Tesque, Inc., Japan). Both cryoprotectant solutions were warmed ∼10 minutes in a heated incubator at 37°C in an atmosphere of 100% air, which was long enough for the temperature to reach ∼35°C, and blastocysts were handled on the stage warmer of a dissecting microscope at 38°C [Kenichiro Hiraoka et al., 2004].

Artificial shrinkage as an alternative blastocoele collapse preventive Pre–vitrification method of expanded blastocysts

Before starting the Vitrification procedure, artificial shrinkage was performed as soon as the expanded blastocysts were placed in the equilibration solution. First, pipetting of the expanded blastocyst was conducted with a glass pipette slightly smaller in diameter (∼140 μm) than the expanded blastocyst. The pipettes used for artificial shrinkage were manually made from a Pasteur pipette hand–drawn using a spirit lamp. The size of the pipette was assessed using stage micrometer. The pipette opening was not flamed. After confirmation of slight shrinkage of the blastocoele, pipetting was performed with a pipette slightly smaller in diameter than the first one (∼100–120 μm). This procedure was repeated two or three times until the blastocoele collapsed completely. Although the shape of the zona and blastocyst was distorted by being aspirated into the pipette, the zona remained intact and all embryos remained within their zona after the artificial shrinkage. After blastocoele contraction, the blastocysts were equilibrated in the equilibration solution for another 2 minutes before exposure to the Vitrification solution. The blastocysts were then incubated in the Vitrification solution and loaded onto the tip of the cryotop within 45 seconds with ∼1 μl of cryoprotectant solution. Then the cryotop was immediately plunged into Liquid Nitrogen which had been filter–sterilized through a 0.22 μm filter and with the aid of forceps covered in a pre–cooled cover before closing it [Kenichiro Hiraoka et al., 2004].

Warming of blastocysts

Before warming blastocysts, 1.0 mol/l sucrose solution, 0.5 mol/l sucrose solution, and the base medium were warmed ∼10 minutes in a heated incubator at 37°C in an atmosphere of 100% air, which was long enough for the temperature to reach ∼35°C. The warming procedure was done as follows. The cryotop tip with the blastocysts was plunged directly into 1.0 mol/l sucrose solution for 1 minute. The blastocysts were then transferred to 0.5 mol/l sucrose solution for 3 minutes and washed twice in the base medium for 5 minutes. All steps were completed on the stage warmer of a dissecting microscope at 38°C. All of the warmed blastocysts were still within their zonae and then cultured in Blastocyst medium containing 10% SSS for further culture until transfer. The post–warming survival of blastocysts was observed 3 hours after warming under a microscope, and re–expanded blastocysts were judged to have survived. Embryo transfer was scheduled on day 5 after ovulation in the spontaneous cycles irrespective of whether they had been vitrified on day 5 or day 6. The time from warming to transfer ranged
from 3 hours to 5 hours. One to three surviving blastocysts were transferred into the patient’s uterus. Pregnancy was first assessed by urinary hCG 9 days after blastocyst transfer, and then clinical pregnancy was confirmed by the presence of fetal heart activity 30 days after blastocyst transfer [Kenichiro Hiraoka et al., 2004].

Results of freezing–thawing procedures

In all, 10 expanded blastocysts were cryopreserved by a slow freezing method without performing artificial shrinkage and thawed for three patients in four thawing cycles. Six blastocysts (60%) re–expanded after warming. A total of 10 blastocysts was transferred to three patients in four cycles. The implantation rate was 10% (1/10) and the pregnancy rate was 25% (1/4) [Kenichiro Hiraoka et al., 2004].

In a preliminary experiment, the scientists evaluated the effect of artificial shrinkage on blastocyst survival after warming of poor quality vitrified day 6 expanded blastocysts. The rate of survival (90%, 9/10) in the artificial shrinkage group was significantly higher than a group (40%, 4/10) which was vitrified after only a short (2 minutes) equilibration step using a protocol adopted from Lane et al. (1999). Usefulness of the procedure was noted [Kenichiro Hiraoka et al., 2004].

Based on the above result, the scientists applied the artificial shrinkage technique clinically. An average of 8.2±4.4 (range, 3–18) minutes was necessary for the blastocoele of expanded blastocysts to collapse completely before Vitrification using artificial shrinkage by the pipetting method. Since the blastocysts were moved to the Vitrification solution 2 minutes after collapsing, the embryos were in the equilibration solution between 5 minutes and 20 minutes. The results of expanded blastocyst Vitrification after artificial shrinkage: 48 blastocysts (98%) re–expanded after warming. A total of 48 blastocysts was transferred to 27 patients in 28 cycles. The implantation rate was 33% (16/48) and the pregnancy rate was 50% (14/28). Three male and five female infants (one set of triplets and five singletons) from six patients have been born, and all of the other eight pregnancies are ongoing and diagnosed as singletons. The triplets following transfer of three day 6 blastocysts were diagnosed as non–identical by ultrasound at 7 weeks of gestation and resulted in delivery of one male and two female infants.
To date, there have been no spontaneous abortions, and all delivered infants have had normal physical profiles up to the present [Kenichiro Hiraoka et al., 2004].

The Kenichiro Hiraoka et al. (2004) study demonstrates that the artificial shrinkage of the blastocoele by micropipetting prior to Vitrification gives excellent survival and pregnancy outcomes for day 5 and 6 expanded blastocysts [Kenichiro Hiraoka et al., 2004].

Unlike the method of Vanderzwalmen et al. (2002) and Son et al. (2003) using a needle, the scientists collapsed the blastocoele by mechanical pipetting with a fine hand–drawn glass pipette slightly smaller in diameter than the blastocyst. This artificial shrinkage method was simple because the pipetting procedure was much the same as that of removal of cumulus cells of mature oocytes before Intracytoplasmic Sperm Injection (ICSI) [Kenichiro Hiraoka et al., 2004].

The mechanism of collapsing the blastocoele by mechanical pipetting is unclear. The pipetting procedure may cause some damage and rupture of the trophectoderm, possibly at a point of weakness, for example in the region of dividing cells where the intercellular contacts may be less strong than normal. Hence, it is suggested that blastocoelic fluid escapes via some rupture of trophectoderm cells by the pressure of the pipetting procedure on the blastocyst. However, there were two observations that lead the scientists to suggest that the damage of artificial shrinkage by the pipetting method is less compared with that of the method using a needle [Kenichiro Hiraoka et al., 2004].

First, the average time of blastocyst contraction (8.2±4.4 min) using artificial shrinkage by the pipetting method was longer than the method (0.5–2 minutes) of Vanderzwalmen et al. (2002) and Son et al. (2003). If the time taken to collapse is determined by the size of the hole, then pipetting makes smaller holes than needles. The blastocysts vitrified in Kenichiro Hiraoka et al. (2004) study were collapsed artificially in the equilibration solution. In addition, the blastocysts were equilibrated in the equilibration solution for another 2 minutes after blastocoele contraction, and so might be damaged as a result of the chemical toxicity of the cryoprotectant. The lower concentration of the equilibration solution used in the present study might be effective in minimizing the risk of injury caused by chemical toxicity [Kenichiro Hiraoka et al., 2004].

Secondly, warmed re–expanded blastocysts, shrunk artificially by the pipetting method, formed a large blastocoelic cavity ∼3 hours after warming. The re–expansion time after warming of vitrified blastocysts in the present study (∼3 hours) was not so different from that of vitrified blastocysts without performing artificial shrinkage (∼2 hours) [Mukaida et al., 2003], thus confirming rapid repair of the trophectoderm. Therefore, the viability of the post–warming expanded blastocysts could be assessed 3 hours after warming. On the other hand, Vanderzwalmen et al. (2002) and Son et al. (2003) estimated the blastocyst survival post–warming ∼20 hours after warming. The blastocysts shrunk artificially by a needle might take a long time to re–expand because of the hole made in the trophectoderm [Kenichiro Hiraoka et al., 2004].

The survival rate (100%) of day 5 blastocysts that were vitrified in Kenichiro Hiraoka et al. (2004) study was not different from that of the report (87%) of Mukaida et al. (2003). However, the survival rate (96%) of day 6 blastocysts in Kenichiro Hiraoka et al. (2004) study was significantly higher than that of the report (55%). Since both groups use the same equilibration solution, the higher survival in Kenichiro Hiraoka et al. (2004) study may reflect longer exposure time and the artificial shrinkage [Kenichiro Hiraoka et al., 2004]. The day 6 blastocysts tend to be at the expanded stage and may be less permeable to cryoprotectants [Mukaida et al., 2003]. Therefore, the day 6 blastocysts may be more vulnerable to damage by ice formation or osmotic stress compared to the day 5 blastocysts. However, both injuries of vitrified day 6 blastocysts can be avoided by the artificial shrinkage [Kenichiro Hiraoka et al., 2004]. Consequently, it can be confirmed that the artificial shrinkage method is a useful technique for the Vitrification of both day 6 blastocysts and even zona–free hatched blastocysts [Kenichiro Hiraoka et al., 2004].

3. Perspectives of Embryo Vitrification Technique (Ultrarapid Cryopreservation Technique) [overview of the study “Births after Vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before Vitrification” written by Vanderzwalmen P., Bertin G., Debauche Ch., Standaert V., van Roosendaal E., Vandervorst M., Bollen N., Zech H., Mukaida T., Takahashi K., Schoysman R.]

In 1996 the scientists Vanderzwalmen P., Bertin G., Debauche Ch., Standaert V., van Roosendaal E., Vandervorst M., Bollen N., Zech H., Mukaida T., Takahashi K. and Schoysman R. set up a programme of Cryopreservation of supernumerary morulae (day 4) and blastocysts (day 5) using a Vitrification procedure. Their results showed that the efficiency of the Vitrification method was dependent on the stage of embryo development and was negatively correlated with the expansion of the blastocoele. The percentage of blastocysts that remained intact and produced a pregnancy was much lower compared with those of morula and early blastocyst. The scientists postulated that a large blastocoele might disturb cryopreservative potential due to ice crystal formation during the cooling step. It was analyzed therefore the effectiveness of reducing before Vitrification the volume of the blastocoelic cavity. The scientists postulated that the loss of viability after Vitrification of blastocysts could be attributed to physical damage resulting from ice formation during the cooling procedure. It is probable that inadequate permeation of the cryoprotectants (Ethylene Glycol–Ficoll–sucrose) or a too slow cooling rate leads to intra-blastocoelic ice formation during freezing, damaging the embryos. In order to improve the efficiency of Vitrification of blastocysts and expanded blastocysts, we suggest reducing the blastocoele by removing artificially the fluid in order to decrease ice crystal formation at low temperature. Vitrification technique which was used: Day 4 and day 5 embryos were vitrified in 40% Ethylene Glycol–18% Ficoll and 0.3 mol/l sucrose before plunging the straws directly into Liquid Nitrogen. Artificial shrinkage of the blastocyst was achieved after pushing a needle into the blastocoele cavity until it contracted. The survival rate post–thawing of day 4 and intact day 5 embryos was correlated with the volume of the blastocoele. In the control group only 20.3% blastocysts or expanded blastocysts survived as compared with 54.5 and 58.5% with morulae and early blastocyst respectively. After puncturing the blastocoelic cavity, an increase in the survival rate of up to 70.6% was noted. The pregnancy rates were improved after the artificial shrinkage procedure (20.5%) compared with the control intact blastocyst group (4.5%) (not significant). After reduction of the blastocoelic cavity, a significant increase in the implantation rate per vitrified blastocyst was observed (12.0 versus 1.4% P < 0.01). The results showed that survival rates in cryopreserved expanded blastocysts could be improved by reducing the fluid content. This was presumably because mechanical damage caused by ice crystal formation was avoided. These observations should be considered when establishing a strategy and a protocol for Cryopreservation of day 5 embryos [Vanderzwalmen et al., 2002].

Additionally it was distinguished that in order to apply extended culture in sequential media in a clinical setting, it is essential to investigate causes responsible for the low survival rate of blastocysts after cryopreservation. It is difficult to explain discrepancies in survival rate. The difference in volume of the cells between morulae and the blastocysts and the presence of the blastocoelic cavity are two factors to consider [Vanderzwalmen et al., 2002].

If only the volume of the blastomeres constituting the morula and the blastocyst is considered, the experts can expect a better prevention of ice crystal formation in the more advanced stage. Due to the small volume of the blastomeres forming the blastocysts, the concentration of the cryoprotectant (Ethylene Glycol) normally increases faster inside the cells, allowing a sufficient permeation of the cryoprotectant and a more rapid equilibration before freezing Furthermore, smaller blastomeres are less sensitive to osmotic stress and, consequently, less osmotic injury when the cryoprotectant is removed [Vanderzwalmen et al., 2002]. However, another factor that can affect the survival rate is that the blastocyst consists of a fluid–filled cavity called the blastocoele. The likelihood of ice crystal formation is directly proportional to volume and inversely proportional to viscosity and the cooling rate. A decrease in survival rate after Vitrification was noticed when the volume of the blastocoelic cavity increased. It is suggested that an insufficient permeation of Ethylene Glycol inside the cavity might cause ice crystal formation during the cooling step, reducing the post–warming survival. Intra–blastocoelic water, which is detrimental to Vitrification, may remain in the cavity after a 3 minutes exposure to Ethylene Glycol (EG20) solution [Vanderzwalmen et al., 2002].

The inclusion of a macromolecule such as Ficoll, present outside the trophoblast and Inner Cell Mass (ICM) cells, protects the outer part of the embryo against crystallization. The cytoplasm of the blastomeres contains various intrinsic macromolecules that increase during equilibration and favour the amorphous state. Inside the blastocoele however, there must be few macromolecules. Due to the short exposure time to the Ethylene Glycol (EG20) and EFS solution, a low concentration of permeable cryoprotectant is present inside the cavity, probably not sufficient to protect the blastocysts against formation of ice crystals inside the blastocoele. Early blastocysts can survive after the Vitrification procedure probably because the initial amount of liquid is reduced [Vanderzwalmen et al., 2002].

According to such a hypothesis, Vanderzwalmen et al. (2002) study investigated the effectiveness of reducing artificially the fluid from the blastocoelic cavity when touching the trophectoderm cells with a glass pipette. After the artificial shrinkage of the blastocyst, the post–thaw survival rate increased dramatically, suggesting the negative influence of the cavity using the two–step Vitrification procedure with Ethylene Glycol as the main permeating cryoprotectant. The beneficial effect of removing the blastocoelic fluid is also manifest when blastocysts are artificially shrunk before slow freezing procedure (personal observation). Vanderzwalmen et al. (2002) study showed that decreasing artificially the volume of the blastocoele has a beneficial effect on the post–thaw survival rate of blastocysts vitrified inside a 0.25 ml French straw. It was suggested that the blastocoelic fluid was the source of injury probably due to the presence of ice crystals inducing a mechanical damage [Vanderzwalmen et al., 2002].

If the lower viability of blastocysts was related to an insufficient permeation of cryoprotectant inside the cavity, alternatives other than the artificial shrinkage can be advocated in order to reduce the negative effect of the blastocoele. Increasing either the time of exposure or temperature to EG20 and/or EFS solutions or a stronger dehydration by increasing the sucrose concentration are alternative options. Using a solution of cryoprotectant containing 1 mol/l sucrose, it was suggested that the prior removal of the blastocoelic fluid could be beneficial and could enhance the survival of vitrified blastocysts [Vanderzwalmen et al., 2002].

Immediately after warming and dilution of the cryoprotectant, it is difficult to assess exactly the viabilit of the embryos at the stereomicroscopic level. Under high magnification (×200), if only minor morphological changes are detectable, a prolonged culture period would be necessary to evaluate the re–expansion and the viability of the embryos. The beneficial effect of the artificial shrinkage, assessed by the rate of blastocysts that survived after thawing and that re–expanded, is encouraging. However, considering the satisfactory level of re–expansion and the optimal morphological aspect of the blastocysts before transfer, the scientists found that the pregnancy and implantation rates of blastocyst and expanded blastocyst after artificial shrinkage were below their expectation [Vanderzwalmen et al., 2002].

It was also emphasized that independent factors that are not linked to the Vitrification process, such as the effect of the artificial shrinkage procedure and the in–vitro culture conditions, may also explain the lower effectiveness achieved after Vitrification of artificially shrunk blastocysts [Vanderzwalmen et al., 2002].

Another factor that can affect the viability of blastocysts to implant resides in the in–vitro culture conditions. The introduction of sequential culture media allows the production of blastocysts without the need for co–culture with [Vanderzwalmen et al., 2002] feeder cells. Notwithstanding the fact that fresh blastocysts give good implantation rates, it has been shown [Dumont–Hassan et al., 1999] that cryopreserved IVF embryos obtained in sequential media are less viable than cryopreserved blastocysts after co–culture. It was represented that a high survival rate of cryopreserved, in–vitro cultured, embryos requires improvement in the techniques of maturation and culture, rather than simple changes in Cryopreservation methods [Dumont–Hassan et al., 1999].

In conclusion, in Vanderzwalmen et al. (2002) study it is postulated that this Vitrification procedure using a solution of Ethylene Glycol–sucrose–Ficoll as cryoprotectant is simple and efficient for the Cryopreservation of day 4 morulae or day 5 early blastocysts in sealed 0.25 ml French straws. Current results strongly support the concept outlined above, namely that the results were dependent upon the structural differences between earlier stage embryos and blastocysts. In the case of blastocysts exhibiting a large fluid–filled cavity, artificial reduction of the blastocoele has a beneficial effect on their post–thaw survival rate [Vanderzwalmen et al., 2002].

In order to improve blastocyst culture, efforts have to focus on better in-vitro culture conditions and to adopt specific Cryopreservation methods for each developmental stage of the embryo, especially for expanded blastocysts. Recent attention has been focused on extremely rapid cooling as a more successful method for the cryopreservation of blastocysts. The preliminary results with the extremely hig–speed cooling of blastocysts are encouraging. A combination of this technique associated with the artificial shrinkage of very expanded blastocysts seems promising [Vanderzwalmen et al., 2002].

Compared with the conventional Vitrification procedure, the artificial shrinkage of blastocysts or expanded blastocysts improves the results after Vitrification, but the efficacy of the method compared with the rate of implantation after fresh blastocysts transfers is still not optimal and needs further investigation. Therefore, in order not to reduce the patients’ chances, the scientists recommend the cryopreservation of some zygotes with conventional freezing procedures. The remaining embryos should be left in culture until the blastocyst stage for transfer and Vitrification [Vanderzwalmen et al., 2002].

Conclusion

Vitrification is an attractive Ultrarapid Cryopreservation Technique which has gained support as an alternative promising substitute for slow Cryopreservation whereby the embryo is transitioned from 37°C to −196°C in <1 s, resulting in extremely fast rates of cooling (>10 000°C/min). Vitrification is performed by suspending the embryo(s) in a solution containing a high concentration (5–8 mol/l) of cryoprotectants and then directly plunging the embryo(s) into Liquid Nitrogen (LN) (−196°C). The advantage of this technique is the prevention of ice crystal formation made possible through increased speed of temperature conduction, reducing associated chilling injuries within the embryo tissue or outside the cytoplasm. However, the osmotic stress and toxic effect from high cryoprotectant concentrations may constitute an obstacle to using this method. Several new techniques and applications were developed recently that improve the survival rate for blastocysts following Vitrification. These procedures include the use of an electron microscope (EM) grid [Martino et al., 1996; Cho et al., 2002], cryoloops [Lane et al., 1999; Mukaida et al., 2001], an pen pulled straw (OPS) [Yokota et al., 2000, 2001] and closed plastic straws [Vanderzwalmen et al., 2002], as well as to compacted morulae using closed plastic straws [Vanderzwalmen et al., 2002]. Grids and cryoloops have the highest cooling rate due to the extremely low volume of Vitrification medium and immersion contact with Liquid Nitrogen (LN 2). However, they require laborious embryo handling and carry the risk of Liquid Nitrogen (LN 2) contamination. Vitrification of expanded blastocysts may be further improved by reducing the volume of the blastocoelic cavity [Vanderzwalmen et al., 2002; Son et al., 2003], or by using a sucrose six–step dilution after warming [Cho et al., 2002].

Among the various hypotheses that can explain the differences between the cryopreservability of the different stages of embryo development, the structural difference between morula and blastocyst is one parameter to consider. Compared morphologically with early stage embryos, blastocysts and expanded blastocysts show a fluid-filled cavity, i.e. the blastocoele. A large proportion of the water content of embryos at the morula stage is present inside the cells, while in the case of blastocysts and expanded blastocysts the blastocoele contains the largest amount of water [Vanderzwalmen et al., 2002].

Vitrification of blastocysts is being used increasingly to cryopreserve supernumerary embryos following In Vitro Fertilization (IVF) Treatment cycle. It uses extremely high concentrations of cryoprotectants and allows the solidification of a solution below the glass transition temperature, without ice crystal formation [Liebermann, 2009; Vajta et al., 2009]. Vitrification has been successfully applied to both cleavage and blastocyst stage embryos and clinical trials have shown high survival rates and promising implantation rates following transfer of thawed embryos at all stages [Liebermann and Tucker, 2006; Mukaida et al., 2006; Hong et al., 2009; Vanderzwalmen et al., 2009, 2003; Desai et al., 2010; Wikland et al., 2010]. The data on the safety of Vitrification in terms of obstetric and perinatal outcomes are also reassuring [Mukaida et al., 2008; Liebermann, 2009; Noyes et al., 2009]. However, blastocysts represent a unique challenge because of the difficulty in accomplishing the required level of dehydration and high viscosity evenly in all blastomeres, due to their multicellular structure and presence of the water–filled blastocoele [Vanderzwalmen et al., 2002]. High concentrations of cryoprotectants together with rapid cooling rates are essential to cryopreserve embryos in a vitrified, glass–like state [Vajta and Kuwayama, 2006]. To facilitate rapid heat transfer, minimal volumes are used in Vitrification, facilitated through the use of minute tools as carriers. The carrier systems that have been developed for the Vitrification procedure include the electron microscope grid [Park et al., 2000; Son et al., 2002], pulled and hemi–straws [El–Danasouri and Selman, 2001; Vanderzwalmen et al., 2002, 2003], flexipipet [Liebermann and Tucker, 2002], cryotop and cryotip [Kuwayama, 2007] and the cryoloop [Lane et al., 1999; Mukaida et al., 2001, 2003a, b; Reed et al., 2002; Rama Raju et al., 2005]. The use of each technique has recently been reviewed [Chen and Yang, 2007].

Principle variables of a Vitrification procedure consist of cooling and warming rates and cryoprotectants, the probability of Vitrification being determined as a correctinterplay between cooling and warming rates and the concentration of cryoprotectants. During Vitrification, water does not form ice crystals but solidifies into a glassy state. Vitrification depends on temperature conduction, the concentration of the cryoprotectant and the volume of the solution [Arav et al., 2002; Liebermann et al., 2002a]. Many of the latest methods used for Vitrification revolve around a technique that utilizes minimal volumes of cryoprotectants in order to maximize heat transfer and thus create a very rapid cooling/warming environment. Among these are Cryotop and Cryoloop methods. These methods although very efficient have been brought into question by the possibility of cross–contamination from direct contact with Liquid Nitrogen. In an experiment to test Liquid Nitrogen as a vector for contamination, a contaminated tank was used to hold samples in both closed and open systems, showing that the open system tested positive for contamination [Bielanski et al., 2000]. Therefore, the preference should be given to use closed devices for the Vitrification of embryos in order to avoid contamination issues, and thereby meet the quality standards. However, a drop of the cooling rate could be expected using a closed method for Vitrification with a thermo–insulating wall between the solution and the Liquid Nitrogen. Whether this lower cooling rate would have an impact on the success of the Vitrification procedure and would be detrimental for the immediate morphological survival was therefore first investigated in a preclinical study on blastocysts from abnormally fertilized oocytes [Guns et al., 2008]. No difference was found in the percentage of fully intact blastocysts after warming when comparing the open CryoTop system (86.4%) with the closed CBS Vitrification straws (90.9%) using DMSO–EG–sucrose as the cryoprotectant solution with 100% expansion/re–expansion of the blastocoelic cavity after 24 hours [Van Landuyt L. et al., 2011]. This reassuring result led to the introduction of the closed system for clinical use. The fact that the lower cooling rate as a consequence of using a closed Vitrification system did not affect the survival rates negatively can probably be explained by the dominance of the warming rate upon the cooling rate in the outcome after Vitrification. Seki and Mazur (2009) studied the relative importance of cooling and warming rates on the survival of mouse oocytes using Vitrification. They found survival rates above 80% at the highest warming rate of 2950°C/minute and this for all cooling rates between 187 and 1827°C/minute and hypothesized that the primary cause of injury after Vitrification is recrystallization during warming instead of failure to vitrify during cooling. Isachenko et al. (2007) published a method for aseptic Vitrification of human zygotes by using Open Pulled Straws hermetically isolated from Liquid Nitrogen before cooling. The isolation resulted in a drop of cooling rate from 15 000 to 600°C/minute but this did not compromise survival and further development. The embryos were warmed ‘open’ resulting in warming rates of 90 000°C/minute which was thought to compensate for the drop–in cooling rate.

Outlining the perspectives of Embryo Vitrification technique in general and the further peculiarities of the Post–vitrification consequences, it was established that it is difficult to explain discrepancies in survival embryo rate. The difference in volume of the cells between morulae and the blastocysts and the presence of the blastocoelic cavity are two factors to consider [Vanderzwalmen et al., 2002].

Immediately after warming and dilution of the cryoprotectant, it is difficult to assess exactly the viability of the embryos at the stereomicroscopic level. Under high magnification (×200), if only minor morphological changes are detectable, a prolonged culture period would be necessary to evaluate the re–expansion and the viability of the embryos. The beneficial effect of the artificial shrinkage, assessed by the rate of blastocysts that survived after thawing and that re–expanded, is encouraging. However, considering the satisfactory level of re–expansion and the optimal morphological aspect of the blastocysts before transfer, the scientists found that the pregnancy and implantation rates of blastocyst and expanded blastocyst after artificial shrinkage were below their expectation [Vanderzwalmen et al., 2002].

Compared with the conventional Vitrification procedure, the artificial shrinkage of blastocysts or expanded blastocysts improves the results after Vitrification, but the efficacy of the method compared with the rate of implantation after fresh blastocysts transfers is still not optimal and needs further investigation. Therefore, in order not to reduce the patients’ chances, the scientists recommend the Cryopreservation of some zygotes with conventional freezing procedures. The remaining embryos should be left in culture until the blastocyst stage for transfer and Vitrification [Vanderzwalmen et al., 2002].

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Vitrification of Embryos: Main Issues. Dilemmas. Breakthroughs. Perspectives

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