Overview of Embryo Cryopreservation Consequences: Zona Pellucida Damage, Blastomere Damage and Spontaneous Blastomere Fusion

Abstract: Overview of Embryo Cryopreservation Consequences: Zona Pellucida Damage, Blastomere Damage And Spontaneous Blastomere Fusion. 

The article focuses on representing the basic overview of Embryo Cryopreservation Consequences. Undisputedly, Cryopreservation represents an attractive option to the range of Infertility Treatments available at present. There are two important issues, however, remain to be resolved before the role of Cryopreservation technology in current practice can be fully elucidated. Firstly, it would be preferable if continuing scientific research could determine whether modifications to all the Cryopreservation methods could improve the final outcome. Secondly, a tendency to adopt modernized Cryopreservation techniques widely only after they have shown high–quality results, concerning prevention of the negative performance, which is probably related to the breach created in the Zona Pellucida that might alter the diffusion of the cryoprotectant solution compared with zona–intact embryos. Usually, two or three embryos that are considered to have the best chance of implanting are selected for further transfer. After thawing embryos, fully intact embryos co–exist with partially damaged ones and embryos showing some Zona Pellucida damage, blastomere damage, spontaneous blastomere fusion which leads to polyploidy and chromosomal mosaicism, which must be quantified for further theoretical prognosis of the consequences of this damage for the viability of the embryo in vitro. Furthermore, embryos after blastomere biopsy should be cryopreserved using the most accurate Cryopreservation techniques and the most delicate methods to prevent blastocoele collapse. What is vital to emphasize is that the main question for the experts is to distinguish excellent to good quality embryos which probably would be able to implant after Cryopreservation, therefore after selection of two to three excellent to good quality embryos for further transfer in the Oocyte–Collection cycle, the remaining embryos should be cryopreserved. Subsequently, supernumerary embryos with a good chance of implanting are selected for Cryopreservation and possible transfer in the future while remaining embryos are discarded.

Key words: Blastomere Damage, Spontaneous Blastomere Fusion, Zona Pellucida Damage

Meta Key Words: Blastomere Biopsy, Blastocoele Collapse, Blastomere Damage, Blastomere Survival, Chromosomal Mosaicism, Embryo Cryopreservation, Micropipetting, Polyploidy, Spontaneous Blastomere Fusion, Thawing Process, Zona Pellucida Damage, Vitrification

Introduction

Cryopreservation of supernumerary embryos is nowadays a well–accepted procedure in Assisted Reproduction programmes. Within the general framework of Assisted Reproductive Techniques (ART) safety, the definition of criteria that predicates the selection of healthy, viable embryos for further Cryopreservation has become increasingly important in reproductive medicine. Freezing and storing of surplus embryos also allows the number of replaced embryos in both fresh and Frozen Embryo Transfers to be reduced, thereby diminishing the risk of multiple pregnancies [57; 66]. Cryostorage of embryos has offered a valuable complement to this strategy and aims to reduce the number of embryos transferred per one procedure. Additionally, it provides many clinical benefits, including the possibility of increasing the cumulative pregnancy rate from a given Treatment cycle while reducing the risk of multiple pregnancy and avoiding the risk of developing Ovarian Hyperstimulation Syndrome. However, careful consideration of all clinical and embryological factors influencing the outcome of Frozen Embryo Transfer (FET) is a basic condition for a successful Assisted Reproduction programme.

Dimension of Cryopreservation is highly debated theme among the representatives of scientific elite, respected experts and distinguished researchers. There are many contradictions about the cryopreservation methods, techniques, technologies, procedures and decisions regarding Embryo Freezing. Undisputedly, Cryopreservation represents an attractive option to the range of Infertility Treatments available at present. There are two important issues, however, remain to be resolved before the role of Cryopreservation technology in current practice can be fully elucidated. Firstly, it would be preferable if continuing scientific research could determine whether modifications to all the Cryopreservation methods could improve the final outcome. Secondly, a tendency to adopt modernized Cryopreservation techniques widely only after they have shown high–quality results, concerning prevention of the negative performance, which is probably related to the breach created in the Zona Pellucida that might alter the diffusion of the cryoprotectant solution compared with zona–intact embryos. It can be postulated that the Zona Pellucida has an important role in preserving embryo integrity, especially through the steps of dehydration, shrinking and rehydration occurring during freezing and thawing, therefore what is vital to emphasize is that at the present stage, perfecting the existing systems of identifying high–quality embryo during embryo selection process and developing new higher–precision ones for further Cryopreservation and thawing procedure are among priorities in the theory and practice of improving expert systems for special purposes of morphological evaluation, which is a considerable challenge for embryologists.

Usually, two or three embryos that are considered to have the best chance of implanting are selected for further transfer. After selection of two to three excellent to good quality embryos for further transfer in the Oocyte–Collection cycle, the remaining embryos should be cryopreserved. Subsequently, supernumerary embryos with a good chance of implanting are selected for Cryopreservation and possible transfer in the future while remaining embryos are discarded. However, it has been shown that not all embryos survive the Cryopreservation procedure [83]. Embryos for Cryopreservation are supernumerary embryos obtained after In–Vitro Fertilization (IVF) with or without Intracytoplasmic Sperm Injection (ICSI). After selection of two to three excellent to good quality embryos for transfer in the Oocyte–Collection cycle, the remaining embryos should be frozen [63; 71]. Usually, there are two types of embryos that should be frozen: excellent quality embryos containing regular or irregular blastomeres, and good quality embryos containing regular or irregular blastomeres and also containing up to 20% of their volume filled with anucleate fragments. The embryos should be frozen irrespective of their developmental stage [15].

However, Cryopreservation and thawing significantly reduces embryo viability and the number of embryos available for uterine transfers [58]. Some embryos do not survive freezing and thawing (17–70%), and in some cases the majority of blastomeres undergo degeneration [83]. The adverse effects of Cryopreservation may also lead to the formation of cracks in the Zona Pellucida, or injuries to the cell membranes and intracellular components [12; 51]. Cryopreservation damage to embryos can be induced by several factors, including intracellular ice formation, solution effects, osmotic effects, or physical damage by growing ice crystals at the advancing ice front. When cryopreserved embryos are recovered from Liquid Nitrogen, therefore, embryos with damaged blastomeres and/or with a cracked Zona Pellucida (ZP) are found. These conditions depend on the speed of cooling and warming, the type of storage container, and the cryoprotectant used [15].

1. In close focus Zona Pellucida damage to embryos after Cryopreservation and the consequences for their blastomere survival and in-vitro viability [based on the conceptions represented in the article “Zona Pellucida damage to human embryos after Cryopreservation and the consequences for their blastomere survival and in-vitro viability” written by the scientists Etienne Van den Abbeel and André Van Steirteghem]

Previously, it had been reported [17; 70] that cryopreserved multicellular embryos with damaged Zona Pellucida (ZP) are better not transferred. However, the incidence of Zona Pellucida (ZP) damage and the relationship between such damage and blastomere damage is poorly described in Embryo Cryopreservation programmes. Zona Pellucida (ZP) damage after freezing–thawing was carefully quantified on a limited number of whole embryos and isolated zonae in four publications [7; 8; 12; 31], two of which indicated that blastomere survival and embryo viability were much lower in embryos with Zona Pellucida (ZP) damage [7; 8].

Embryos can be frozen in polypropylene cryovials or in plastic mini–straws. To find out the most effective preventive measures for excluding blastomere damage and Zona Pellucida (ZP) damage of Сryopreservation process, these both kinds of damage should be carefully evaluated under an inverted microscope at ×400 magnification. Therefore, it is essential to quantify the blastomere damage and Zona Pellucida (ZP) damage in cryopreserved embryos; and to evaluated the viability of the embryos after Cryopreservation.

The scientific results indicate that embryos obtained after In Vitro Fertilization (IVF) in association with Intracytoplasmic Sperm Injection (ICSI) survive the Сryopreservation procedure equally well as do embryos resulting from In Vitro Fertilization (IVF) alone. The hyaluronidase exposure followed by mechanical removal of cumulus cells by aspirating the oocyte–cumulus complexes in and out of a finely pulled pipette and the mechanical piercing of the Zona Pellucida (ZP) and oolemma in Intracytoplasmic Sperm Injection (ICSI) do not make the embryo more susceptible to blastomere damage and do not alter their capacity to cleave further. These results are in agreement with previously published reports [1; 32; 36; 43; 72].

Since the first pregnancy after Vitrification of a blastocyst was reported using cryostraws [84], the scientific 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 blastocysts using the cryotop [37], the cryoloop [46; 47; 55], electron microscope grids [6; 64] and the hemi–straw [69]. However, expanded blastocysts exhibit relatively poor survival rates after Vitrification [5]. Expanded blastocysts have more blastocoelic fluid than early blastocysts, in which ice crystals may form during cooling. This negative performance is probably related to the breach created in the Zona Pellucida that might alter the diffusion of the cryoprotectant solution compared with zona–intact embryos. It can be postulated that the Zona Pellucida has an important role in preserving embryo integrity, especially through the steps of dehydration, shrinking and rehydration occurring during freezing and thawing. According to a recent report, blastomeres near to the breach in the zona are more prone to lysis after thawing [85].

At the present stage, perfecting the existing systems of identifying high–quality embryo during embryo selection process and developing new higher–precision ones for further Cryopreservation and thawing procedures through performing the scientific investigations, distinguished the most transparent correlation of the occurrence of Zona Pellucida (ZP) damage after Cryopreservation significantly influenced by the freezing container, which was used for Embryo Cryostorage. 

It is intriguing that the incidence of Zona Pellucida (ZP) damage was different according to whether the embryos were frozen–thawed in polypropylene cryovials or in plastic mini–straws (16.6% versus 2.3%; P <0.0001). The percentage of embryos with 100% blastomere survival was higher using plastic mini–straws (59.8% versus 34.9%; P <0.0001). Subsequently the percentage of embryos suitable for transfer that cleaved further after a 24 h culture period was higher when using plastic mini-straws (81.0% versus 61.0%; P <0.0001) [15].

Thorough retrospective analysis shown that the incidence of Zona Pellucida (ZP) damage was significantly higher when a freezing procedure with cryovials as storage containers was used, indicating that the material in which the embryos are stored has important consequences for the outcome after thawing procedure. Cracks in the Zona Pellucida (ZP) are considered to be caused by mechanical stress produced from non–uniform volume changes of the freezing medium during phase changes, and are termed fracture damage. Furthermore, it was concluded that the differences observed between plastic mini–straws and glass or plastic cryovials were associated with thermally induced fracturing of the freezing suspensions during rapid changes of temperature. The scientific investigation indicated that the percentage of embryos with 100% blastomere survival was significantly higher when a freezing procedure using plastic mini–straws was used, and that a clear relationship was found between the incidence of Zona Pellucida (ZP) and blastomere damage. Zona Pellucida damage was correlated with low blastomere survival [15]. The same observations were made in another analysis [8]. It has long been considered that the Zona Pellucida (ZP) may play a significant preventive role in the Cryopreservation of embryos by acting either as a barrier to the diffusion of water and cryoprotectants or as a physical barrier to the growth of extracellular ice. Blastomere damage has been described as the result of intracellular ice crystal formation, osmotic stress during addition and removal of concentrated solutions of cryoprotective agents, gas bubble formation, or fracture planes which sometimes pass through the cytoplasm [3]. Compared with embryos without Zona Pellucida (ZP) damage, Etienne Van den Abbeel and André Van Steirteghem’s study indicates that in thawed embryos with Zona Pellucida (ZP) damage after Cryopreservation, blastomere damage is probably also the result of fracture planes passing through the cytoplasm [15].

The scientists Etienne Van den Abbeel and André Van Steirteghem (2000) were unable to relate the type of Zona Pellucida (ZP) anomalies and the blastomere damage seen. In a prospective study, it would be interesting to investigate whether less Zona Pellucida (ZP) damage is correlated with higher blastomere survival and improved in–vitro viability.

One study [7] concluded that human embryos with Zona Pellucida (ZP) damage after Cryopreservation were less likely to cleave further in vitro. The scientists Etienne Van den Abbeel and André Van Steirteghem also studied the consequence of Cryopreservation damage to embryos in terms of their capacity to cleave further in vitro. They found that Zona Pellucida (ZP) damage had no effect on the further cleavage in vitro of the embryo. Blastomere damage, however, significantly reduced the capacity of the frozen–thawed embryo to cleave further in vitro. The damage to the embryos was not related to the quality of the embryos before freezing, indicating that the blastomere damage in itself was responsible for their impaired further cleavage [15].

Evaluation of the blastomere and the Zona Pellucida (ZP) damage after thawing

Immediately after thawing and dilution of the cryoprotectants, as well a few hours later, the embryos should be inspected for morphological survival under an inverted microscope (×200). Two types of damage are then evaluated, namely of the blastomere and the Zona Pellucida (ZP).

Blastomeres are considered to be damaged if the cytoplasm appeared granular and degenerative, when they remained contracted after dilution of the cryoprotectant, or if they had smooth membranes [9]. Several types of Zona Pellucida (ZP) damage can be also observed. To be recorded as damaged, the Zona Pellucida (ZP) had to be cracked on at least one spot. Whether there was one crack or more cracks, or whether there was complete absence of the Zona Pellucida (ZP), should be separately recorded. Minor Zona Pellucida (ZP) damage (dark Zona Pellucida (ZP), a thinner but not completely cracked ZP at local spots) should be recorded [15].

After freezing and thawing, embryos are suitable for transfer if they contained at least 50% of the initial number of blastomeres intact. Embryos suitable for transfer should be put into culture for 24 hours. Then they should be inspected for further cleavage, which is defined as the number of embryos suitable for transfer where after 24 hours the number of blastomeres has increased [15].

The quality of the embryos thawed should be analyzed before Cryopreservation according to the presence of anucleate fragments and to the developmental stage. It is highly recommended by the scientists that the embryos should be graded in accordance with anucleate fragments criterion: 

1. Embryos contain no anucleate fragments and 
2. Embryos contain up to 20% anucleate fragments. 

The evaluation of damage to embryos after thawing reveals that damage to the Zona Pellucida (ZP) is visible immediately after thawing, while blastomere damage is visible either immediately, or after dilution of the cryoprotectant or after a few hours in culture [15].

For embryos, it was concluded that after a post–thaw culture period, a cleaved embryo group had a significantly increased number of intact blastomeres [86], while in a previous study the scientists Etienne Van den Abbeel and André Van Steirteghem reported that partially damaged embryos implanted less well than fully intact ones [68]. The scientists Etienne Van den Abbeel and André Van Steirteghem postulated that damaged blastomeres exerted “toxic” effects on intact blastomeres. The present study seems to confirm the hypothesis that damaged blastomeres have some toxic effect on the intact ones [15].

Zona Pellucida damage and (or) blastomere damage after Cryopreservation might be signs of suboptimal Cryopreservation procedures [39]. The introduction of plastic mini–straws as storage containers in practice resulted in a clear improvement of the Cryopreservation outcome. Further improvements might be expected by using polymeric substances in the freezing media or by using Vitrification procedures [2; 59].

In conclusion, the study of Etienne Van den Abbeel and André Van Steirteghem indicated that the morphological survival and further cleavage in vitro of cryopreserved embryos obtained after In Vitro Fertilization (IVF) Treatment in association with Intracytoplasmic Sperm Injection (ICSI) are no different from those obtained after In Vitro Fertilization (IVF) only. Zona Pellucida damage and blastomere damage after Cryopreservation can have dramatic consequences for the viability of the embryo in vitro. The occurrence of Zona Pellucida (ZP) and blastomere damage can be carefully controlled using an optimized freezing procedure. Such a procedure includes the use of plastic mini–straws as storage containers and slow–cooling, slow–thawing rates with 1.5 mol/l DMSO as the cryoprotectant. The general aim of a Cryopreservation programme should be to have as many fully intact embryos as possible after thawing [15].

2. In close focus: spontaneous blastomere fusion after Cryopreservation and thawing of early embryos leads to polyploidy and chromosomal mosaicism [based on the conceptions represented in the article “Spontaneous blastomere fusion after freezing and thawing of early human embryos leads to polyploidy and chromosomal mosaicism” written by the scientists Hanna Balakier, Oliver Cabaca, Derek Bouman, Alan B. Shewchuk, Carl Laskin, Jeremy A. Squire]

It was established that both procedures freezing and thawing significantly reduce embryo viability and the number of embryos available for further transfers [58]. Some embryos do not survive freezing and thawing (17–70%), and in some cases the majority of blastomeres undergo degeneration [83]. The adverse effects of Cryopreservation may also lead to the formation of cracks in the Zona Pellucida, or injuries to the cell membranes and intracellular components [12; 51]. In some reports major chromosomal anomalies such as trisomy of chromosome 13, 18 and 21, as well as major and minor congenital malformations, have been identified in fetuses and babies conceived from frozen embryos [11; 54; 65; 80].

It is intriguing that a recent study of aneuploidy and mosaicism of chromosomes X, Y and 1 in frozen embryos (day 2 and 3 of development) using Fluorescence In–Situ Hybridization (FISH) showed that a large proportion of thawed embryos (57%) exhibiting cleavage arrest during the first 24 hours of in–vitro culture carried numerical chromosomal abnormalities [38]. However, it remains uncertain whether those aberrations were induced by the Cryopreservation technique, or were already present in chromosomally abnormal embryos before thawing.

The basic concepts of spontaneous blastomere fusion after freezing and thawing of early embryos leads to polyploidy and chromosomal mosaicism were postulated by the scientists Hanna Balakier, Oliver Cabaca, Derek Bouman, Alan B. Shewchuk, Carl Laskin, Jeremy A. Squire. It was proved that there is an incidence of blastomere fusion after Cryopreservation of early embryos (Day 2 and Day 3). Fusion of two, and occasionally of several, blastomeres resulted in the formation of multinucleated hybrid cells, which clearly indicated that the ploidy of these newly created cells had been altered. This event, depending on the number of fused cells per embryo, transformed the embryos into either entirely polyploid embryos (complete fusion at 2– or 3–cell stage) or into mosaics being a mixture of polyploid and normal cells. Chromosomal preparations of embryos affected by blastomere fusion indicated the presence of tetraploid mitotic plates. Therefore, the purpose of their study was to determine the incidence of blastomere fusion after freezing and thawing of early human embryos, and to characterize numerical chromosomal changes such as ploidy using a standard cytogenetic technique and the FISH method (FISH interphase analysis was performed to investigate further any numerical chromosomal changes in embryos affected by fusion). The implications of the formation of polyploid and mosaic embryos that may arise from such blastomere fusion are discussed [30].

The accurate scientific investigation revealed the following statistics: the overall survival rate of the embryos frozen on Day 2 (2– to 5–cell stage) and the embryos frozen on Day 3 (5– to 10–cell stage) was 76%, including fully intact embryos (53%) and embryos containing at least 50% live cells (23%). The other embryos were either totally degenerated or had the majority of their cells lysed. Among the embryos that survived the thawing (embryos with 50–100% intact cells), 51 cases of blastomere fusion were detected within the first 2 hours of in–vitro culture. About 70% of those affected embryos were of good quality (regular cells, no or few fragments), while the others exhibited poor morphology (20–30% fragmentation, uneven cells or granular cytoplasm). The process of fusion was observed in all developmental stages (from 2 to 10 cells), and the frequency of this event was 4.6% in Day 2 and 1.5% in Day 3 embryos. An especially low incidence of blastomere fusion was found in 7– to 10–cell embryos frozen on Day 3 of development. In total, a slightly higher incidence of fusion was observed in embryos obtained after In Vitro Fertilization (IVF) than Intracytoplasmic Sperm Injection (ICSI) procedure. However, further conclusions on any relationship between fusion and type of procedure cannot be drawn because the observations were distorted by the fact that on Day 2 the majority of cases were represented by In Vitro Fertilization (IVF) and on day 3 by Intracytoplasmic Sperm Injection (ICSI) procedures [30]. 

To compare if similar rates of cell fusion occurred within control embryos that had not been subjected to Cryopreservation, day 2 and day 3 fresh embryos were examined every 2 hours during a period of 4–6 hours. Among 2315 Day 2 embryos, only one fusion of two blastomeres was observed in a 4–cell stage embryo (0.04%). On Day 3 of development, fusion was not recorded in any 5– to 6–cell or 7– to 10–cell embryos (1795 embryos examined). However, in the group of arrested 3– or 4–cell embryos that did not divide from the previous day (from Day 2 to Day 3), blastomere fusion was recorded in eight embryos (8/323; 2.4%). Except for two embryos that underwent double cell fusion, the other six arrested embryos became mosaics, with one binucleated hybrid cell [30].

The scientific investigation has clearly demonstrated that Cryopreservation of early embryos using the standard propanediol technique may induce blastomere fusion resulting in chromosomal aberrations. Simple observations on the number of fused blastomeres indicated that the ploidy of newly created hybrid cells had been altered. It was apparent that embryos affected by fusion were transformed into either polyploid embryos or into mosaics consisting of a mixture of polyploid and normal cells. Therefore, it can be suggested that tetraploid (4n) or hexaploid (6n) embryos might be formed due to complete fusion of all blastomeres at the 2– or 3–cell stage or after double fusion in 4–cell embryos. The creation of entirely polyploid embryos was less frequent (8/51 embryos; 16%), and the majority of affected embryos were converted into tetraploid–diploid (4n/2n; 40/51, 78%) or other complex mosaics (6n/2n or 8n/2n, 6%) when only some blastomeres of an embryo had fused. To reinforce these conclusions based on scientific observations of live embryos, some mosaics obtained by fusion were examined on fixed preparations. Although a number of logistical factors invariably limit these investigations, the FISH and cytogenetic results confirmed the morphological appearance suggestive of altered ploidy in the embryos affected by fusion. Mitotic tetraploid chromosomal plates as well as a mixture of tetraploid and diploid FISH signals within intact nuclei were found in the fixed mosaics, indicating the presence of polyploid cells [30].

Hanna Balakier’s, Oliver Cabaca’s, Derek Bouman’s, Alan B. Shewchuk’s, Carl Laskin’s, Jeremy A. Squire’s observations may also suggest that early embryos are more susceptible to cryodamage and blastomere fusion when compared with older, more advanced embryos. It is likely that the properties of cell membranes, for instance fluidity, are changing during embryo development, and perhaps for this reason more fusion has been observed in day 2 than in day 3 thawed embryos (4.6% versus 1.5%). Similarly, the frequency of hybrid formation appeared to be higher in 5– to 6–cell than in 7– to 10–cell embryos examined on the third day of development (1.2% and 0.3% respectively). An interesting observation was that in the control group of unfrozen embryos, the only fusion that was found had occurred in early–arrested 2– to 4–cell stage embryos. Alternatively, the occurrence of blastomere fusion could be associated with existing membrane abnormalities that may promote fusion either after freezing and thawing or in unfrozen fresh embryos due to other factors such as pH, temperature, osmotic pressure, etc. [30].

It seems that freezing and thawing is responsible for blastomere fusion, and this may occur regardless of the type of cryoprotectants used [30; 67]. These studies also suggest that blastomere fusion is not only attributed to fair and poor–quality embryos, as was previously thought [67], but it can also affect morphologically good embryos, as was shown by Hanna Balakier’s, Oliver Cabaca’s, Derek Bouman’s, Alan B. Shewchuk’s, Carl Laskin’s, Jeremy A. Squire’s observation (70% of affected embryos were of good quality) [30]. Although the molecular mechanism of cell fusion has not yet been elucidated, the general studies have proven that a defect in the cell membrane is required for initiation of fusion, which can be induced by many membrane–disrupting agents such as a virus, polyethylene glycol or electric field, as well as freezing and thawing [33; 87; 88]. Based on these reports, it seems that cryoprotectants may also contribute to the process of fusion by causing cell dehydration and osmotic swelling which induce other changes that are necessary to induce fusion (for instance, changes in the cytoskeleton, tight contact between cells, etc.) [30]. It can also be assumed that cytoplasmic bridges may play a role in blastomere fusion; however, research on early human embryos did not detect them when embryos were examined
in electron microscope serial sections or after injection of specific dyes into single blastomeres of 2– to 8–cell stage embryos [10; 45].

The transfer of embryos containing numerical chromosomal aberrations due to the process of fusion may potentially have clinical relevance; at present, the developmental result of such transfers remains unclear. Since there is no evidence that Cryopreservation increases the incidence of birth defects [83], this implies that embryos affected by fusion – if transferred to the uterus – either fail to implant or are spontaneously aborted, and for this reason are not seen among the babies born from the frozen cycles. The fact that in Hanna Balakier’s, Oliver Cabaca’s, Derek Bouman’s, Alan B. Shewchuk’s, Carl Laskin’s, Jeremy A. Squire’s study [“Spontaneous blastomere fusion after freezing and thawing of early human embryos leads to polyploidy and chromosomal mosaicism”] cleavage arrest and fragmentation of the nuclei were observed in the hybrid cells also suggests that embryos affected by fusion are either eliminated before clinical recognition, or they are rescued by protective mechanisms that correct embryonic errors [30]. It is possible that abnormal cells    report on a twin pregnancy of two empty tetraploid sacks that had resulted from a transfer of frozen zygotes [21]. It is probable that in such cases mitotic spindles of the zygotes were destroyed, resulting in the formation of tetraploid embryos. Therefore, it cannot be ruled out that similar abnormal abortuses may also arise from frozen thawed embryos by means of blastomere fusion [30]. More extensive studies are required from abortive tissues to determine whether chromosomal abnormalities are associated with Embryo Cryopreservation. It is important to note that in natural conceptions the incidence of tetraploidy (4–8%) has been reported in human abortive tissues as well as in born infants [60; 79]. It is generally assumed that such a chromosomal anomaly is formed due to genome duplication and suppression of the early cleavage divisions [60]. However, it can be suggested that blastomere fusion may be an alternative mechanism by which embryos acquire polyploidy. This concept is supported by the observations that blastomere fusion can be induced by freezing and thawing, or in rare circumstances may occur spontaneously in fresh/unfrozen embryos [30].

3. In close focus: blastomere biopsy and Cryopreservation techniques [based on the conceptions represented in the article “Impact of blastomere biopsy and Cryopreservation techniques on embryo viability” written by the scientists Magli M.C., Gianaroli L., Fortini D., Ferraretti A.P., Munné S.]

The availability of an efficient Cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for Preimplantation Genetic Diagnosis (PGD) of aneuploidy before freezing. The standard Embryo Cryopreservation method is still less than optimal for biopsied embryos. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. The poor survival rate obtained after thawing biopsied embryos is of great concern for the general outcome of a PGD cycle, especially when considering the complexity, both technical and psychological, of the procedure. For this reason, the supernumerary embryos are considered to be particularly valuable having being screened for a genetic abnormality. More recently, the technique of Comparative Genomic Hybridization (CGH) has been proposed for the analysis of all chromosomes in single cells [77; 81]. The time currently needed to complete the diagnosis is not compatible with a fresh embryo transfer and embryos must be cryopreserved after biopsy [82]. This makes the availability of an efficient Cryopreservation Protocol mandatory to minimize the damage related to Embryo Cryopreservation and thawing.

Preimplantation Genetic Diagnosis (PGD) has now been widely used for screening abnormal embryos in couples with a high risk of genetic disease especially for single gene disorders [75]. In some clinics, routine Preimplantation Genetic Diagnosis (PGD) has been used to screen all embryos to be able to transfer only chromosomally normal embryos to improve implantation and pregnancy rates in couples with previous persistent failure of implantation [20]. Thus, Cryopreservation of biopsied embryos will be important for routine Preimplantation Genetic Diagnosis (PGD) since many couples will have some excessive biopsied embryos to be cryopreserved for subsequent transfers.

The most common method of Preimplantation Genetic Diagnosis (PGD) entails the biopsy of one or two cells from Day 3 embryos [Preimplantation Genetic Diagnosis (PGD) has been proposed as an early form of prenatal diagnosis that is based on the analysis of a single cell: a blastomere biopsied from morphologically normal Day 3 embryos or the first and second Polar Body retrieved from the oocyte [28; 29; 74]; the procedure does not adversely affect embryo development, since at that stage all cells are undifferentiated and still totipotent. In addition, events of empty zonae occur more frequently compared with the controls. These effects are probably related to the opening in the Zona Pellucida which can cause an altered diffusion of the cryoprotectant solution compared to intact embryos. The Zona Pellucida has an important role in embryo viability and variation in its thickness corresponds to an increased chance of implantation, especially in the case of thawed embryos [8]. This condition may be an indicator of increased membrane transport and production of enzymes capable of digesting the zona itself in a localized area. The following hatching process represents one of the first signals of polarity in the recently compacted embryo, which leads to the formation of two primary cell lines: the inner cell mass and the trophectoderm.

Biopsied embryos have a hole on the Zona Pellucida, and cryoprotectant and frozen medium can freely enter the previtelline space. It is suggested that the increased susceptibility of biopsied embryos could be a consequence of both zona drilling and blastomere removal [35]. However, others found that micromanipulation does not appear to be a major factor for reducing the cryosurvival of embryos since embryos derived from Intracytoplasmic Sperm Injection (ICSI) survive Cryopreservation at similar rates to those derived from conventional In Vitro Fertilization (IVF) [40]. The hole on the Zona Pellucida after Intracytoplasmic Sperm Injection (ICSI) is obviously much smaller than that after biopsy and the small hole tends to close. It was noted that blastomeres near to the hole of the Zona Pellucida were more likely to lyse after the warming of standard slow Сryopreservation. It was hypothesized that it is possibly because water velocity is different among the blastomeres located close to and further away from the hole in the Zona Pellucida during the warming procedure. It is proposed that the Zona Pellucida acts as a partial water barrier to prevent blastomeres from rupturing by excessive high–speed rehydration during the warming procedure. To verify the hypothesis, the Сryopreservation Protocols were revised accordingly. The common component of these successful modified Protocols is that the concentration of sucrose of the warming media was increased to slow down the rehydration velocity. Also, the cells shrink more after freezing with higher osmolality sucrose media. These factors help the cells to remain intact during the warming process. the preliminary results of this study suggest that Vitrification may be a better method for Cryopreservation of biopsied human embryos [85].

Basically, Preimplantation Genetic Diagnosis (PGD) is recommended for the screening of single–gene disorders and the analysis of chromosomal abnormalities, both numerical and structural. Preimplantation Genetic Diagnosis (PGD) for aneuploidy is by far the most common technique as the selection of chromosomally normal embryos for transfer has a positive effect on the clinical outcome [25; 49]. This is probably due to the strong association between chromosomal abnormalities and embryo non–viability, mainly resulting in spontaneous abortions and implantation failure [26; 50; 76].

Numerical chromosomal abnormalities occurring at meiosis, syngamy or during the first cleavage stages of embryo development are frequent, and result in embryos with reduced implantation potential [48]. Preimplantation Genetic Diagnosis (PGD) of aneuploidy is routinely performed in Fertility Clinics by using the multicolour Fluorescence In–Situ Hybridization (FISH). Preimplantation Genetic Diagnosis (PGD) of aneuploidy allows for the identification of these abnormalities in in–vitro generated embryos; the technique is especially advantageous in those patients with a poor prognosis of pregnancy due to their embryos having a high incidence of chromosomal abnormalities [22; 24; 48]. After the multicolour Fluorescence In–Situ Hybridization (FISH) diagnosis, the feasibility of identifying and transferring euploid embryos has an immediate impact on embryo implantation, which is higher after chromosomal analysis compared to the controls [23]. In addition, the number of embryos being cryopreserved is dramatically reduced due to the low number of chromosomally normal embryos which are generally available.

Therefore, it is essential to evaluate the effect of the freezing–thawing procedure on biopsied embryos, in order to estimate the feasibility of their Cryopreservation. 

According to the current data, alteration of the zona integrity, possibly associated with the removal of one blastomere before freezing, is detrimental to embryo viability during Cryopreservation. Physical factors involved in the irreversible damage of embryos during the freezing and thawing procedures have been reported [3]. Consequently, it may be inferred that the glycoprotein coat surrounding the oocyte has a fundamental role in maintaining cellular integrity and shape throughout the steps of dehydration, shrinking and re–hydration involved in freezing and thawing. The combination of blastomere biopsy and Cryopreservation procedures gives rise to results which are not expected. This effect could be associated with an interference with intercellular junction formation, due to the freezing and thawing process, as suggested by the facility of removing the blastomere in comparison to fresh embryos at the same stage. In addition, the ensuing cell biopsy could contribute a mechanical disarrangement in their subsequent organization. In all cases, if damage occurs to the cytoskeleton because of abnormal exposure to Propanediol (PROH) and sucrose, and/or the blastomere biopsy procedure itself, cell shape is not maintained and this causes an alteration in the bilayer associated glycoproteins and transport systems, which results in cellular death [41].

In the effort of investigating the Cryopreservation process and designing a Protocol more suitable for biopsied embryos, the role of sucrose, a protein source and freezing medium has been reconsidered. Comparing the standard Cryopreservation with slow programmed procedures and Vitrification, there should have been mentioned that Vitrification could be obtained by combining the high concentration cryoprotectant with high cooling and warming rates. During Vitrification, there will not have been cell damage caused by ice. Jericho et al. (2003) Have proposed an increased concentration of sucrose in the freezing solution that could enable a more complete dehydration of blastomeres, in combination with the use of human serum instead of albumin. Due to their chemical structure (polyhydroxy domains facilitating the interaction with water molecules), the content of globulins in human serum could substantially contribute to an adequate dehydration, improving the recovery of viable embryos after thawing [52]. Increasing sucrose concentrations in the modified method of Jericho et al. (2003) appear to be beneficial to survival of frozen–thawed biopsied embryos and the further development of blastocysts of surviving embryos. Others have also reported that increasing the concentration of sucrose to 0.2 mol/l improves survival of frozen–thawed oocytes [4; 16; 61]. For slow freezing programmes for embryos, the cryoprotectant generally used is PROH (membrane–permeating cryoprotectant) and sucrose (non–membrane–permeating cryoprotectant). Sucrose does not enter the cell and it mainly induces cellular dehydration through changes in osmotic pressure. Therefore, varying the sucrose concentration can change the rate of cellular dehydration to draw water out of the cells sufficiently during freezing and change the speed of cellular rehydration during warming. The use of this Protocol for biopsied embryos significantly improved the results compared with a standard freezing protocol: the survival index was 67 versus 46%, respectively, with a pregnancy rate of 16.7% per transfer (6 clinical pregnancies yielded by 36 transfers and 41 thawing cycles) and an implantation rate of 12% [34].

There also has been proposed the use of choline–based, sodium–free medium to replace the conventional sodium–based medium [62]. The proportion of survived blastomeres was significantly improved compared with a standard freezing Protocol (80 versus 50%, respectively). Unfortunately, no clinical application was reported.

Together with this, further scientific investigations revealed a valid alternative method which could be represented by Vitrification that avoids ice–crystal formation by combining high cryoprotectant concentration and high cooling and warming rates. Recently, encouraging results have been reported using non–transferable biopsied embryos with a survival index of 90% [85]. Neither in this case a clinical application was achieved, whereas in another preliminary study the Vitrification of blastocysts developed from biopsied embryos yielded a survival index of 73% and a clinical pregnancy [18].

Magli M.C., Gianaroli Luca, Grieco N., Cefalù E., Ruvolo G., Ferraretti A.P. in their scientific article “Cryopreservation of biopsied embryos at the blastocyst stage” outlined that basically, two considerations supported the decision of cryopreserving biopsied embryos at the blastocyst stage, as it was done in their current study: 

1. It permits the selection of the most viable embryos for Cryopreservation by discarding those having developmental arrest. In this way, the number of frozen embryos is kept at a minimum. 
2. The thinning of Zona Pellucida in the expanding blastocyst possibly reduces its effect of water barrier, making the presence of a breach due to blastomere biopsy of less effect as far as water permeability and turbulence is concerned. In addition, the structure of the blastocyst itself, with ∼100 tightly connected cells, could possibly make it more resistant to high–speed rehydration [42].

The usage of the alternative method for Cryopreservation – membrane–permeating cryoprotectant – Propanediol (PROH) for blastocyst freezing instead of glycerol was based on its characteristics of permeability and capacity of replacing H₂O molecules to protect against thermal shock or dilution stress [53; 56]. This was considered to be especially important for the presence of the breach in the Zona Pellucida and the consequent relevance of water flow at warming. As the preliminary data derived from the experimental part of Magli M.C., Gianaroli Luca, Grieco N., Cefalù E., Ruvolo G., Ferraretti A.P. study [“Cryopreservation of biopsied embryos at the blastocyst stage”] were promising, the same protocol was also applied to blastocysts originated from non–biopsied embryos for which the traditional use of glycerol as cryoprotectant had not provided satisfactory results in terms of implantation and incidence of miscarriage [42].

According to the data obtained with the use of propanediol (PROH), the survival rate still remains the critical point corresponding to 53% of thawed blastocysts in the Preimplantation Genetic Diagnosis (PGD) group. This figure was not different from that obtained in non–biopsied embryos which were frozen with the same Protocol (58%). However, compared with the data reported in the literature, the survival rate of the IVF/ICSI group is lower than expected by using the glycerol as cryoprotectant [19; 44; 73] suggesting that possibly different strategies could be optimal for the freezing of blastocysts generated from biopsied or intact embryos. The current results suggest that, if the embryo survives the Cryopreservation/thawing process, its chances of implanting are not negatively affected [42].

A number of scientific articles represented that blastomere loss in frozen–thawed early cleavage stage embryos is associated with a reduction in implantation potential. Frozen embryo transfer (FET) cycles in which all embryos transferred remain fully intact at thawing achieve a better outcome than those in which at least one partially damaged embryo is transferred [13; 14; 27].

In conclusion, the combination of blastomere biopsy and Cryopreservation adversely affects embryo viability, irrespective of the sequence in which the two procedures are carried out. Furthermore, the low survival rate in biopsied embryos after freezing and thawing suggests that the implementation of Cryopreservation after Preimplantation Genetic Diagnosis (PGD) of aneuploidy should be restricted until adequate Protocols for freezing, including ultrarapid methods, are designed. Alternatively, less aggressive methods to open the Zona Pellucida should be considered in the hope of alleviating chemical stress to the embryo. At the present time, one of the strategies is adopted to postpone the transfer of biopsied embryos to Day 4. This has two advantages: 

1. In cases of supernumerary euploid embryos it allows for a better selection based on morphological evaluation, and 
2. It gives more time for the screening of additional chromosomes after overnight re–hybridization with specific probes. 

This expedient not only keeps to a minimum the need of resorting to Embryo Cryopreservation, but also enables the screening of a wider panel of chromosomes, thus offering patients with a poor prognosis increased chances of receiving embryos with the highest implantation potential [42].

Conclusion

1. In conclusion, the retrospective analysis represented that it was established that both procedures freezing and thawing significantly reduce embryo viability and the number of embryos available for further transfers. Some embryos do not survive freezing and thawing (17–70%), and in some cases the majority of blastomeres undergo degeneration. The adverse effects of Cryopreservation may also lead to the formation of cracks in the Zona Pellucida, or injuries to the cell membranes and intracellular components. Cryopreservation damage to embryos can be induced by several factors, including intracellular ice formation, solution effects, osmotic effects, or physical damage by growing ice crystals at the advancing ice front. When cryopreserved embryos are recovered from liquid nitrogen, therefore, embryos with damaged blastomeres and/or with a cracked Zona Pellucida (ZP) are found. These conditions depend on the speed of cooling and warming, the type of storage container, and the cryoprotectant used.

Zona Pellucida damage and (or) blastomere damage after Cryopreservation might be signs of suboptimal Cryopreservation procedures. The study of Etienne Van den Abbeel and André Van Steirteghem indicated that the morphological survival and further cleavage in vitro of cryopreserved embryos obtained after In Vitro Fertilization (IVF) Treatment in association with Intracytoplasmic Sperm Injection (ICSI) are no different from those obtained after In Vitro Fertilization (IVF) only. Zona Pellucida damage and blastomere damage after Cryopreservation can have dramatic consequences for the viability of the embryo in vitro. The occurrence of Zona Pellucida (ZP) and blastomere damage can be carefully controlled using an optimized freezing procedure.

2. It was interesting to explore the link between chromosomal aberrations and blastomere fusion: the scientific investigation established that Cryopreservation of early embryos using the standard propanediol technique may induce blastomere fusion resulting in chromosomal aberrations. Simple observations on the number of fused blastomeres indicated that the ploidy of newly created hybrid cells had been altered. It was apparent that embryos affected by fusion were transformed into either polyploid embryos or into mosaics consisting of a mixture of polyploid and normal cells. Hanna Balakier’s, Oliver Cabaca’s, Derek Bouman’s, Alan B. Shewchuk’s, Carl Laskin’s, Jeremy A. Squire’s observations may also suggest that early embryos are more susceptible to cryodamage and blastomere fusion when compared with older, more advanced embryos. It is likely that the properties of cell membranes, for instance fluidity, are changing during embryo development, and perhaps for this reason more fusion has been observed in day 2 than in day 3 thawed embryos (4.6% versus 1.5%). Similarly, the frequency of hybrid formation appeared to be higher in 5– to 6–cell than in 7– to 10–cell embryos examined on the third day of development (1.2% and 0.3% respectively). An interesting observation was that in the control group of unfrozen embryos, the only fusion that was found had occurred in early–arrested 2– to 4–cell stage embryos. Alternatively, the occurrence of blastomere fusion could be associated with existing membrane abnormalities that may promote fusion either after freezing and thawing or in unfrozen fresh embryos due to other factors such as pH, temperature, osmotic pressure, etc. [30]. It seems that freezing and thawing is responsible for blastomere fusion, and this may occur regardless of the type of cryoprotectants used [30; 67]. These studies also suggest that blastomere fusion is not only attributed to fair and poor–quality embryos, as was previously thought [67], but it can also affect morphologically good embryos, as was shown by Hanna Balakier’s, Oliver Cabaca’s, Derek Bouman’s, Alan B. Shewchuk’s, Carl Laskin’s, Jeremy A. Squire’s observation (70% of affected embryos were of good quality) [30]. Although the molecular mechanism of cell fusion has not yet been elucidated, the general studies have proven that a defect in the cell membrane is required for initiation of fusion, which can be induced by many membrane–disrupting agents such as a virus, polyethylene glycol or electric field, as well as freezing and thawing [33; 87; 88]. Based on these reports, it seems that cryoprotectants may also contribute to the process of fusion by causing cell dehydration and osmotic swelling which induce other changes that are necessary to induce fusion (for instance, changes in the cytoskeleton, tight contact between cells, etc.) [30]. It can also be assumed that cytoplasmic bridges may play a role in blastomere fusion; however, research on early human embryos did not detect them when embryos were examined in electron microscope serial sections or after injection of specific dyes into single blastomeres of 2– to 8–cell stage embryos [10; 45].

3. The availability of an efficient Cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for Preimplantation Genetic Diagnosis (PGD) of aneuploidy before freezing. The standard Embryo Cryopreservation method is still less than optimal for biopsied embryos. The usage of the alternative method for Cryopreservation – membrane–permeating cryoprotectant – Propanediol (PROH) for blastocyst freezing instead of glycerol was based on its characteristics of permeability and capacity of replacing H₂O molecules to protect against thermal shock or dilution stress [53; 56]. This was considered to be especially important for the presence of the breach in the Zona Pellucida and the consequent relevance of water flow at warming. As the preliminary data derived from the experimental part of Magli M.C., Gianaroli Luca, Grieco N., Cefalù E., Ruvolo G., Ferraretti A.P. study [“Cryopreservation of biopsied embryos at the blastocyst stage”] were promising, the same Protocol was also applied to blastocysts originated from non–biopsied embryos for which the traditional use of glycerol as cryoprotectant had not provided satisfactory results in terms of implantation and incidence of miscarriage [42].

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