*3.2.1 Slow freezing*

Slow freezing is a conventional cryopreservation method, which is based on a slow cooling rate and use of a low concentration of CPAs. This leads to less toxicity to cells/tissues; however, it does not eliminate ice formation. In 1972, two scientific groups published the first survival of murine embryos after slow freezing [3, 4] and live offspring [3]. Nowadays, after the introduction of the vitrification method, slow freezing is gradually being replaced.

Protocols based on the slow freezing method include an equilibrating step, during which the cells or tissues are placed in an aqueous solution containing PM CPAs in low concentrations (1.0–1.5 M) and sucrose (0.1 M) before which they are placed in ampules or straws. After the exposure to CPAs, initial cellular dehydration is observed followed by a return to isotonic volume with the permeation of CPA and water. After loading the specimen in the straw/ampulla, the temperature is being lowered down slowly with the aid of a controlled rate freezing machine which allows samples to be cooled at different rates, and finally, the frozen objects are placed in liquid nitrogen for storage. The slow cooling is performed to ensure that the cells/tissues are dehydrated before intracellular ice formation occurs. However, the optimal rate of cooling varies greatly among cells and tissue types [22]. A crucial step during the slow freezing protocol is the so-called ice crystal seeding which can be performed either manually or automatically. It takes place after the ampules/straws, preloaded with the embryos, are cooled below the melting point of the solution which is around −5 to −7°C. At these temperatures, solutions remain unfrozen due to the supercooling (lowering the temperature of a solution below its freezing point without extracellular ice formation). Supercooling leads to improper cell dehydration and to avoid such fate, most commonly manual ice nucleation is performed by touching the ampules/straws with a prechilled with liquid nitrogen cold object like forceps which leads to ice crystal formation. In this way, the remaining water in the cells is driven away due to the osmotic imbalance, caused by the formation of ice crystals. After ice crystal seeding, the process of slow freezing continues at various cooling rates. When the temperature has reached values ranging from −30 to −80°C depending on the protocol, the ampules/straws are plunged into liquid nitrogen.

In conclusion, we must say that despite the acceptable results achieved by slow freezing, it also has its negatives, for example, it is time consuming, as freezing an embryo usually takes between 2 and 3 hours depending on the cooling rate. Furthermore, it requires expensive controlled-rate freezers.

### *3.2.2 Vitrification*

Vitrification is an alternative approach to the slow freezing method for the cryopreservation of embryos/gametes. Vitrification differs from slow freezing in that it avoids the formation of ice crystals both intracellularly and extracellularly [23]. This method is easier to conduct, does not require expensive equipment like programmable freezers, and is not that time consuming when compared to the conventional slow freezing.

Physically speaking, vitrification is the solidification of a solution at low temperatures by elevation in viscosity during cooling and not by ice crystallization [24, 25]. The first successful vitrification of embryos was published in 1985 by Rall and Fahy, who froze mouse embryos using DMSO, PEG, and acetamide [23]. Commonly used freezing solutions for vitrifications are composed of permeating CPA (EG, DMSO, G, acetamide, PG, with a concentration of over 4 M) and nonpermeating CPA (most commonly sucrose, >0.5 M). After numerous experiments and further improvements of the vitrification technique, like replacement of DMSO with EG and mixture of several CPAs [26], vitrification was applied to human embryos and live births were achieved with both blastocyst and cleavage stage embryos [27, 28]. Assisted hatching (AH) was added to the freezing/thawing procedure and is performed before the transfer of vitrified embryos. It was reported that AH is beneficial in vitrification cycles by increasing pregnancy and implantation rates [29]. Although several methods of AH had been developed—mechanical, piezo, chemical, and laser, the latter turns out to be the most used one with one of its main advantages lies in minimizing the exposure of embryos outside the incubator. A recent meta-analysis conducted in 2016 encompassing 36 randomized controlled trials and 6459 participants reported increased clinical pregnancy rate and multiple pregnancy rate in couples after AH and nonsignificant difference in miscarriage rates between the AH group and the control one [30]. Despite fears about the safety of AH and the increased chance of multiple pregnancies, many IVF facilities apply the procedure on every thawed embryo. The idea behind this is to improve the implantation and clinical pregnancy rates especially in women with history of repeated IVF failure. Embryos with thicker zona pellucida could benefit the most after AH. A large study by Knudtson et al. with more than 150,000 FET cycles reported a slightly decreased live birth rate in the first autologous FET cycle after AH [31]. Therefore, the application of AH should be carefully considered, and prospective studies should be carried out in order to elucidate its benefits and negatives.

The vitrification protocols for embryo freezing consist of several steps. In the first place, embryos are exposed to high concentrations of CPAs, after that, they are loaded into carriers, most commonly straws, and finally those straws are cooled as fast as possible, reaching a cooling rate of thousands of degrees per minute. To achieve vitrification of solutions, there must be an increase in both the cooling rates and the concentration of CPAs. Those two factors are inverse proportionally connected since, the higher the cooling rate, the lower the required CPA. It is important to mention that there are some concerns regarding the use of high concentrations of CPAs, because they could harm the cells during vitrification. That is why a mixture of CPAs is used during vitrification in order to reduce this toxicity.

Vitrification techniques include the so-called "open" and "closed" systems or carriers. The idea behind them is unambiguous, and with the open carriers, the embryos are directly exposed to the liquid nitrogen, which increases the cooling rate, but hides a potential risk of cross contamination of the probe during the storage in liquid nitrogen. On the other hand, the closed systems isolate the sample from the liquid nitrogen which lowers substantially the risk of contamination; however, the cooling rate is inferior compared to the open carriers. There are dozens of different carrier devices available commercially, but there are not many comprehensive studies that compare the carriers and their efficiency. When comparing open and closed systems (VitriSafe carrier, open and closed variation) for blastocyst freezing, a prospective study by Panagiotidis et al. documented no significant difference between the two carrier systems [32], which highlights the importance of the thawing process. Kuwayama et al. compared open (Cryotop) and closed

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*The Present and Future of Embryo Cryopreservation DOI: http://dx.doi.org/10.5772/intechopen.80587*

type, and others would be answered in due time.

*3.2.3 Method comparison*

cryopreserved [24, 37].

vation in the field of ART.

**3.3 Consequences of freezing an embryo**

(CryoTip) systems for the vitrification of blastocysts and also found no significant difference (survival rate 97 and 93% for Cryotop and CryoTip, respectively, deliveries 51 and 48%). These observations support the thesis that closed systems are comparable to open ones, because they also reduce the risk of cross contamination. We hope that vitrification will be optimized in the near future and questions regarding the composition of the vitrification solution, the most appropriate carrier

In the pool of studies that compare slow freezing of embryos and vitrification,

In 2014, a population-based cohort study in Australia and New Zealand included

All of these results highlight the advantages of vitrification and the drawbacks of slow freezing. Overall, vitrification turns out to be the better method of cryopreser-

There are two major concerns regarding embryo cryopreservation. One of them is about the survival rate after embryo freezing. The second major concern is that the freezing process may induce cryodamage to the embryo. Cryodamage is a collective term which includes various types of injuries that a biological object could experience during the freezing and thawing, like formation of ice crystals, physical stress, and also other types of damage we have mentioned earlier. Assessing survival rate after thawing is the most used technique to evaluate the effects of the

11,644 slow frozen and thawed blastocysts and 19,978 vitrified and thawed blastocysts. A higher clinical pregnancy rate per embryo transfer cycle was reported for the vitrification group (32.7%) than in the slow frozen one (23.8%). The mean maternal age for the slow frozen group was 33.6, while for the vitrification group— 34.2. This is one of the largest known studies in this field; however, a possible drawback could be the lack of information available on clinic-specific cryopreservation protocols and processes for slow freezing-thawing and vitrification-warming

of blastocysts and the potential impact on outcomes [38].

Kuwayama et al. reported that vitrification of 5881 human PN stage embryos resulted in 100% survival, 93% cleavage, and 52% blastocyst rates. In contrast, after slow freezing of 1944 PN stage embryos, the results were 89% survival, 90% cleavage, and 41% blastocyst rates [33]. When freezing cleavage-stage embryos on day 2 with the slow freezing method, the survival rate of 77.0% was reported in a study by Xue et al.; however, when using vitrification, the authors reported 96.6% survival rate when P < 0.05 [34]. A study in 2015 documented the survival rate of 96.95% after vitrification of day 3 embryos, in contrast to the 69.06% of the embryos survived after slow freezing, post-warmed excellent morphology embryos: 94.17 vs. 60.8% [35]. The study included 592 frozen/thawed embryos. Regarding blastocyst cryopreservation, a large retrospective study by Richter et al. in 2016 included 4862 slow frozen blastocysts and 2735 vitrified blastocysts, with no statistical difference between patients BMI and age [36], and reported interesting findings. A survival rate (authors define survival as having >50% of cells intact after thawing) of 95.6% was achieved in the vitrification group versus 91.9% in the slow frozen group, when P < .0001. They also found that the percentage of intact cells was more after vitrification/warming compared to slow freezing and thawing, 95.3 vs. 88.7%, P < .0001. It is important to mention that currently, there is much debate as to the developmental stage at which human embryos are best to be

(CryoTip) systems for the vitrification of blastocysts and also found no significant difference (survival rate 97 and 93% for Cryotop and CryoTip, respectively, deliveries 51 and 48%). These observations support the thesis that closed systems are comparable to open ones, because they also reduce the risk of cross contamination.

We hope that vitrification will be optimized in the near future and questions regarding the composition of the vitrification solution, the most appropriate carrier type, and others would be answered in due time.

## *3.2.3 Method comparison*

*Embryology - Theory and Practice*

conventional slow freezing.

programmable freezers, and is not that time consuming when compared to the

Physically speaking, vitrification is the solidification of a solution at low temperatures by elevation in viscosity during cooling and not by ice crystallization [24, 25]. The first successful vitrification of embryos was published in 1985 by Rall and Fahy, who froze mouse embryos using DMSO, PEG, and acetamide [23]. Commonly used freezing solutions for vitrifications are composed of permeating CPA (EG, DMSO, G, acetamide, PG, with a concentration of over 4 M) and nonpermeating CPA (most commonly sucrose, >0.5 M). After numerous experiments and further improvements of the vitrification technique, like replacement of DMSO with EG and mixture of several CPAs [26], vitrification was applied to human embryos and live births were achieved with both blastocyst and cleavage stage embryos [27, 28]. Assisted hatching (AH) was added to the freezing/thawing procedure and is performed before the transfer of vitrified embryos. It was reported that AH is beneficial in vitrification cycles by increasing pregnancy and implantation rates [29]. Although several methods of AH had been developed—mechanical, piezo, chemical, and laser, the latter turns out to be the most used one with one of its main advantages lies in minimizing the exposure of embryos outside the incubator. A recent meta-analysis conducted in 2016 encompassing 36 randomized controlled trials and 6459 participants reported increased clinical pregnancy rate and multiple pregnancy rate in couples after AH and nonsignificant difference in miscarriage rates between the AH group and the control one [30]. Despite fears about the safety of AH and the increased chance of multiple pregnancies, many IVF facilities apply the procedure on every thawed embryo. The idea behind this is to improve the implantation and clinical pregnancy rates especially in women with history of repeated IVF failure. Embryos with thicker zona pellucida could benefit the most after AH. A large study by Knudtson et al. with more than 150,000 FET cycles reported a slightly decreased live birth rate in the first autologous FET cycle after AH [31]. Therefore, the application of AH should be carefully considered, and prospective studies should be carried out in order to elucidate its benefits

The vitrification protocols for embryo freezing consist of several steps. In the first place, embryos are exposed to high concentrations of CPAs, after that, they are loaded into carriers, most commonly straws, and finally those straws are cooled as fast as possible, reaching a cooling rate of thousands of degrees per minute. To achieve vitrification of solutions, there must be an increase in both the cooling rates and the concentration of CPAs. Those two factors are inverse proportionally connected since, the higher the cooling rate, the lower the required CPA. It is important to mention that there are some concerns regarding the use of high concentrations of CPAs, because they could harm the cells during vitrification. That is why a mixture

Vitrification techniques include the so-called "open" and "closed" systems or carriers. The idea behind them is unambiguous, and with the open carriers, the embryos are directly exposed to the liquid nitrogen, which increases the cooling rate, but hides a potential risk of cross contamination of the probe during the storage in liquid nitrogen. On the other hand, the closed systems isolate the sample from the liquid nitrogen which lowers substantially the risk of contamination; however, the cooling rate is inferior compared to the open carriers. There are dozens of different carrier devices available commercially, but there are not many comprehensive studies that compare the carriers and their efficiency. When comparing open and closed systems (VitriSafe carrier, open and closed variation) for blastocyst freezing, a prospective study by Panagiotidis et al. documented no significant difference between the two carrier systems [32], which highlights the importance of the thawing process. Kuwayama et al. compared open (Cryotop) and closed

of CPAs is used during vitrification in order to reduce this toxicity.

**108**

and negatives.

In the pool of studies that compare slow freezing of embryos and vitrification, Kuwayama et al. reported that vitrification of 5881 human PN stage embryos resulted in 100% survival, 93% cleavage, and 52% blastocyst rates. In contrast, after slow freezing of 1944 PN stage embryos, the results were 89% survival, 90% cleavage, and 41% blastocyst rates [33]. When freezing cleavage-stage embryos on day 2 with the slow freezing method, the survival rate of 77.0% was reported in a study by Xue et al.; however, when using vitrification, the authors reported 96.6% survival rate when P < 0.05 [34]. A study in 2015 documented the survival rate of 96.95% after vitrification of day 3 embryos, in contrast to the 69.06% of the embryos survived after slow freezing, post-warmed excellent morphology embryos: 94.17 vs. 60.8% [35]. The study included 592 frozen/thawed embryos. Regarding blastocyst cryopreservation, a large retrospective study by Richter et al. in 2016 included 4862 slow frozen blastocysts and 2735 vitrified blastocysts, with no statistical difference between patients BMI and age [36], and reported interesting findings. A survival rate (authors define survival as having >50% of cells intact after thawing) of 95.6% was achieved in the vitrification group versus 91.9% in the slow frozen group, when P < .0001. They also found that the percentage of intact cells was more after vitrification/warming compared to slow freezing and thawing, 95.3 vs. 88.7%, P < .0001. It is important to mention that currently, there is much debate as to the developmental stage at which human embryos are best to be cryopreserved [24, 37].

In 2014, a population-based cohort study in Australia and New Zealand included 11,644 slow frozen and thawed blastocysts and 19,978 vitrified and thawed blastocysts. A higher clinical pregnancy rate per embryo transfer cycle was reported for the vitrification group (32.7%) than in the slow frozen one (23.8%). The mean maternal age for the slow frozen group was 33.6, while for the vitrification group— 34.2. This is one of the largest known studies in this field; however, a possible drawback could be the lack of information available on clinic-specific cryopreservation protocols and processes for slow freezing-thawing and vitrification-warming of blastocysts and the potential impact on outcomes [38].

All of these results highlight the advantages of vitrification and the drawbacks of slow freezing. Overall, vitrification turns out to be the better method of cryopreservation in the field of ART.

#### **3.3 Consequences of freezing an embryo**

There are two major concerns regarding embryo cryopreservation. One of them is about the survival rate after embryo freezing. The second major concern is that the freezing process may induce cryodamage to the embryo. Cryodamage is a collective term which includes various types of injuries that a biological object could experience during the freezing and thawing, like formation of ice crystals, physical stress, and also other types of damage we have mentioned earlier. Assessing survival rate after thawing is the most used technique to evaluate the effects of the

cryopreservation process on the embryos. However, freezing an embryo also does not allow the inspection of other types of damages, which occur at the molecular level—DNA damage, altered gene expression, and protein function. These alterations require specific molecular biology methods in order to be assessed, and their impact on the embryo is not that clear. In contrast, when the survival rate after freezing is being assessed, we must say that this approach is straightforward and yields distinct results.

When talking about the embryo survival rate nowadays, with the constantly improving cryopreservation techniques, a survival rate of more than 90% or even 95% could be observed depending on the vitrification protocol, carrier, embryologist experience, thawing process, and other variables. While this rate is indeed very high, unfortunately there is still a risk that a frozen embryo would not survive after thawing. The survival rate is different for the different stages at which the embryos are frozen. In fact, it is still unknown at which stage of development, the embryos are most suitable for freezing and therefore further research is needed. Moreover, the stage at which the embryo is frozen is connected to different types of cryodamage. At the PN stage, there is evidence that embryos may suffer integrity damage of the pronuclei after cryopreservation [39], and therefore, their developmental potential could be significantly impaired. At the cleavage stage, there is evidence of zona pellucida damage [40] and changes in metabolism [41]. Reduced implantation rates have been observed after the loss of blastomeres in day 2 grade 1 embryos with <10% fragmentation in a study with 363 thawing cycles [42]. Blastocyst cryopreservation represents a demanding task due to its size and the presence of blastocoel. Formation of ice crystals is probably the main factor affecting blastocysts survival rates, since the blastocoel contains large amounts of water. In order to reduce the negative effects of the blastocoel on survival rates, it was proposed that blastocysts should be frozen at the contraction stage or the blastocoel should be collapsed artificially before freezing [43] which can be done, for example, with an ICSI pipette. Despite all these difficulties, blastocyst survival rates seem to be higher compared to early cleavage embryos, as shown in a study by Cobo et al., where 6019 embryos were vitrified using Cryotop as a carrier [44]. In the study, 97.6% day 6 embryos survived compared to 95.7% day 5 embryos, 94.9% day 2, and 94.2% day 3 embryos. As a consequence of the freezing procedure, zona pellucida may become thicker, which could affect the implantation ability of the embryo, and this is why assisted hatching is performed with the idea to overcome this problem.

#### **3.4 Artificial shrinkage**

In a well expanded blastocyst, the large blastocoel may interfere with the permeation of CPAs during the vitrification procedure which in turn would decrease the survival rates after thawing. Mukaida et al. back in 2003 stated that blastocyst survival rate after vitrification/warming correlate negatively with the expansion of the blastocoel [45]. Artificial shrinkage (AS) of the blastocoel by different methods—laser pulse, microneedle, micropipetting, and 29-gauge needle was developed with the idea of overcoming this obstacle. A study by Gala et al. in 2014 encompassing 185 warming cycles reported a higher survival rate after AS (99.0%) compared to 91.8% survival rate in the control group without AS [46]. Darwish and Magdi in 2016 assessed clinical pregnancy rates, implantation rates, and blastocyst survival rates in more than 400 patients, divided into two groups—untreated expanded blastocysts and blastocysts undergone AS by laser pulse [47]. The study group found that after AS, there was significantly increased survival rates (97.3 vs. 74.9%), implantation (39.1 vs. 24.5%), and clinical pregnancy rates (67.2 vs. 41.1%).

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**4. Embryo cryopreservation and ART**

these very important questions.

**4.1 Embryo storage**

observed.

applied practically.

Despite the promising results of AS, this technique is relatively new and not well studied. More studies must be carried out to validate if the procedure is safe and to assess its impact on the developing embryo. Moreover, a high survival rate can be

All of the advances that had been made in the last two decades regarding embryo

cryopreservation would be of no significance if the success rates after FET were minimal. So what is the place of embryo cryopreservation among other basic ART methods? What are the positives and negatives of having embryos frozen/thawed and transferred instead of having a fresh transfer? We will try to give answers to

The main indication of embryo cryopreservation is for storage purposes. We have reviewed the basic cryopreservation methods. Our interpretation of this thought is: however, no matter how much they have improved recently, they could not be successful unless proper storage and thawing of the frozen objects are carried out. After freezing, the embryos are placed in storage tanks which are filled with liquid nitrogen. There is substantial variety of storage tanks and automated storage systems have been recently introduced, which offer optimal storage conditions and safety. It is not known for how long embryos can be stored in liquid nitrogen without affecting their potential, because embryo freezing was developed during the 1980s, which means that the longest time an embryo has been stored is around 35 years, and there is little chance that patients would come back for them after such a long period. There are some differences in the laws regarding embryo cryopreservation, and therefore, embryo storage limit varies between countries, for example, 3 years in Portugal, 5 years in Denmark, Norway, and many other countries, 10 years in Austria and Australia, 55 years in the UK, while in Venezuela, embryo freezing is prohibited [48]. However, a storage time of 5–10 years is most commonly

Keeping embryos in liquid nitrogen raises some concerns about the safety of the procedure. First of all, liquid nitrogen that is used by the IVF laboratories has chemical standards of purity, not biological. That means that there might be some kind of contamination and we should think if there is any way to sterilize this liquid nitrogen. Bielanski et al. describes the potential for viral transmission from experimentally contaminated liquid nitrogen to vitrified embryos, stored in open freezing containers [49]. From a pool of 83 batches, 21% were positive for viral association. In contrast, vitrified embryos in sealed plastic cryovials and straws were free from viral contamination. These data support that sealing of the freezing container might prevent exposure to contaminants; however, that does not mean 100% safety, as the seal can be damaged. This information leads us back to the idea of sterilizing the liquid nitrogen. However, if this is possible, it should be evaluated if it can be

Regarding the thawing process, it is very similar in both vitrification and slow freezing technique. The idea is to submerge the frozen object into a solution prewarmed at 37°C which is the core temperature in human body. Closed systems are usually plunged into water baths, while open systems could be put directly into a prewarmed medium. As mentioned before, CPAs are used for the cryopreservation of embryos and those CPAs must be removed during the thawing process and also

obtained without AS, and therefore, the use of AS is questionable.

Despite the promising results of AS, this technique is relatively new and not well studied. More studies must be carried out to validate if the procedure is safe and to assess its impact on the developing embryo. Moreover, a high survival rate can be obtained without AS, and therefore, the use of AS is questionable.
