**1. Introduction**

During the last couple of decades, assisted reproductive technologies (ART) have become one of the fastest developing branches of medicine. Since they are the main method of fertility treatment, much research has been done in this area. Enormous amount of work has been done to elucidate the benefits of cryopreservation of stem cells, embryos, gametes, tissues, and organs. The idea of maintaining the viability of living reproductive cells and tissues of various species after long-term storage provides a chance for animal and human reproductive applications [1, 2]. Due to the constantly improving cryopreservation techniques, we are now able to preserve cells and tissues by cooling them to extremely low temperatures, such as −195.79°C (the boiling point of the liquid nitrogen). Cooling down biological objects to such degrees prevents any biological activity, including all the biochemical reactions involved in cell death.

Among the biggest scientific achievements, cryopreservation of embryos came into prominence more than 45 years ago [3, 4]. In 1972, Whittingham and associates

**Figure 1.** *Mechanism of hormesis (License: 4371720640523).*

and Wilmut succeeded in cryopreservation of eight-cell mouse embryos [3, 4]. Since that time, vast number of embryos from various mammalian species have been frozen, thawed, and eventually transferred successfully, thus proving the benefits from this ART procedure. In 1983, Trounson and Mohr achieved the first human pregnancy from frozen embryo with the same procedure used successfully for cryopreservation of mouse and cow embryos [5].

Without doubt, the successful cryopreservation of embryos has greatly improved the chances for a successful outcome after a single cycle of ovarian stimulation and in vitro fertilization (IVF). There is also a theory that describes the "treatment" effect of freezing an embryo, which may explain the higher success rates of frozen embryo transfer (FET) compared to fresh embryo transfer (ET). The authors of the "Theory about the Embryo-Cryo treatment" believe that freezing and thawing could activate endogenous survival and repair mechanisms in preimplantation embryos [6]. The idea is that the thawing process induces low levels of stress, which leads to hormesis. This controlled stress could lead to the repair of mitochondrial damage and protein misfolding. This theory may explain the higher success rate of FET compared to fresh ET in women of advanced reproductive age, the higher miscarriage rate after thawed blastocyst transfers compared to thawed early cleavage embryos transfers and the higher perinatal parameters of children born after FET (**Figure 1**).

While much has been discovered about embryo cryopreservation, there are still many things that have to be defined more accurately, such as the freezing medium composition or the stage of the embryo during freezing. Embryo freezing methods are constantly being improved, but they, as well as the freezing equipment, require improvements. Scientists are looking for answers to these and many more questions while the final goal remains clear—successful cryopreservation followed by as high as possible pregnancy rates. In this chapter, we will try to present the most important aspects of cryopreservation. In the end, we hope that the potential of cryobiology in reproductive medicine will have been acknowledged.

#### **2. Cryobiology and reproductive medicine**

Cryobiology represents a branch of biology which studies the effects of low temperatures on organisms, biological systems, or biological materials. Those low temperatures range from hypothermic to cryogenic (−150°C or lower).

**105**

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

tial to affect everyone's lives in the future.

and is a routine procedure, performed worldwide.

oocyte freezing is now also a routine procedure.

or by physical changes induced by ice formation.

**3. Embryo cryopreservation**

While cryobiology is mainly focused on the "living world," in the last decades, it has been expanded to involve treatment of nonliving things, as well. With the development of highly sophisticated cryobiological techniques, like cryosurgery, embryo and gamete preservation and others, this biological branch has the poten-

The idea of freezing human gametes for their future use encouraged scientists to incorporate cryobiology in the field of reproductive medicine. Polge et al. in 1949 have been recognized as the first researchers who cryopreserved spermatozoa while using glycerol as a cryoprotectant [7], although Bernstein and Petropavlovski 12 years earlier demonstrated that glycerol has a cryoprotective role in the cryopreservation of spermatozoa [8]. Encouraged by those findings and driven by the potential benefits of freezing human gametes and embryos, scientists soon began to study much more in the biology of cryopreservation. Rapid progress was made, and the first birth from the use of human frozen spermatozoa was achieved in 1954 [9]. In those days, scientists used spermatozoa for their cryopreservation studies, since they have motility, which was useful when assessing the vitality of the frozen/ thawed probe. Nowadays, cryopreservation of spermatozoa could be achieved easily

Freezing oocytes was much harder to achieve, and it took scientists some time. Chen in 1986 reported the first pregnancy, resulting from slow freezing and rapid thawing of human oocytes using DMSO (dimethyl sulfoxide) as a cryoprotectant [10]. However, earlier concerns were raised regarding damage to the meiotic spindle, loss of cortical granules, and the low success rates as compared to the relative success of embryo cryopreservation, which led to little interest in oocyte freezing until 1990s [11]. During those times, Bernard et al. and other researchers demonstrated that reasonable oocyte thaw survival [12] and subsequent fertilization could be achieved [13]. Gradually, interest was raising, and through hard work,

One of the very important attainments that cryobiology has achieved is the ability to successfully freeze and thaw human embryos. Scientists first discovered how to successfully freeze an embryo, and only after that, they achieved successful oocyte cryopreservation. Whittingham et al. achieved the first successful embryo cryopreservation when the research group froze mouse embryos in polyvinylpyrrolidone (PVP) [3]. The first baby born after a frozen/thawed blastocyst transfer was reported by Cohen et al. in 1985 [14]. Embryo cryopreservation is now a routine procedure and there is sufficient published data supporting its effectiveness.

Cryopreservation of embryos is a very delicate procedure, which also hides some risks to the things that are frozen. During the freezing process, embryos are exposed to physical stress, caused either by the direct effects of the low temperature

The direct effects of the low temperature may induce damage to cell structure and function. The mitotic spindle is especially sensitive to cold shock injuries. The extent of the damage, caused by the freezing procedure, depends on various factors, like the shape and size of the cells, the membrane composition, and its permeability. All these factors are variable and are species specific. Embryos and oocytes have

the ability to repair some damage in order to survive and develop properly. The formation of ice crystals is detrimental to cells. The damage that the cells suffer is not due to the crystallization of ice but rather due to the high concentration of solutes occurring when water is removed in order to form ice [15].

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

*Embryology - Theory and Practice*

**Figure 1.**

and Wilmut succeeded in cryopreservation of eight-cell mouse embryos [3, 4]. Since that time, vast number of embryos from various mammalian species have been frozen, thawed, and eventually transferred successfully, thus proving the benefits from this ART procedure. In 1983, Trounson and Mohr achieved the first human pregnancy from frozen embryo with the same procedure used successfully

Without doubt, the successful cryopreservation of embryos has greatly improved the chances for a successful outcome after a single cycle of ovarian stimulation and in vitro fertilization (IVF). There is also a theory that describes the "treatment" effect of freezing an embryo, which may explain the higher success rates of frozen embryo transfer (FET) compared to fresh embryo transfer (ET). The authors of the "Theory about the Embryo-Cryo treatment" believe that freezing and thawing could activate endogenous survival and repair mechanisms in preimplantation embryos [6]. The idea is that the thawing process induces low levels of stress, which leads to hormesis. This controlled stress could lead to the repair of mitochondrial damage and protein misfolding. This theory may explain the higher success rate of FET compared to fresh ET in women of advanced reproductive age, the higher miscarriage rate after thawed blastocyst transfers compared to thawed early cleavage embryos transfers and the higher perinatal parameters of children

While much has been discovered about embryo cryopreservation, there are still many things that have to be defined more accurately, such as the freezing medium composition or the stage of the embryo during freezing. Embryo freezing methods are constantly being improved, but they, as well as the freezing equipment, require improvements. Scientists are looking for answers to these and many more questions while the final goal remains clear—successful cryopreservation followed by as high as possible pregnancy rates. In this chapter, we will try to present the most important aspects of cryopreservation. In the end, we hope that the potential of cryobiol-

Cryobiology represents a branch of biology which studies the effects of low temperatures on organisms, biological systems, or biological materials. Those low temperatures range from hypothermic to cryogenic (−150°C or lower).

for cryopreservation of mouse and cow embryos [5].

*Mechanism of hormesis (License: 4371720640523).*

ogy in reproductive medicine will have been acknowledged.

**2. Cryobiology and reproductive medicine**

born after FET (**Figure 1**).

**104**

While cryobiology is mainly focused on the "living world," in the last decades, it has been expanded to involve treatment of nonliving things, as well. With the development of highly sophisticated cryobiological techniques, like cryosurgery, embryo and gamete preservation and others, this biological branch has the potential to affect everyone's lives in the future.

The idea of freezing human gametes for their future use encouraged scientists to incorporate cryobiology in the field of reproductive medicine. Polge et al. in 1949 have been recognized as the first researchers who cryopreserved spermatozoa while using glycerol as a cryoprotectant [7], although Bernstein and Petropavlovski 12 years earlier demonstrated that glycerol has a cryoprotective role in the cryopreservation of spermatozoa [8]. Encouraged by those findings and driven by the potential benefits of freezing human gametes and embryos, scientists soon began to study much more in the biology of cryopreservation. Rapid progress was made, and the first birth from the use of human frozen spermatozoa was achieved in 1954 [9]. In those days, scientists used spermatozoa for their cryopreservation studies, since they have motility, which was useful when assessing the vitality of the frozen/ thawed probe. Nowadays, cryopreservation of spermatozoa could be achieved easily and is a routine procedure, performed worldwide.

Freezing oocytes was much harder to achieve, and it took scientists some time. Chen in 1986 reported the first pregnancy, resulting from slow freezing and rapid thawing of human oocytes using DMSO (dimethyl sulfoxide) as a cryoprotectant [10]. However, earlier concerns were raised regarding damage to the meiotic spindle, loss of cortical granules, and the low success rates as compared to the relative success of embryo cryopreservation, which led to little interest in oocyte freezing until 1990s [11]. During those times, Bernard et al. and other researchers demonstrated that reasonable oocyte thaw survival [12] and subsequent fertilization could be achieved [13]. Gradually, interest was raising, and through hard work, oocyte freezing is now also a routine procedure.

One of the very important attainments that cryobiology has achieved is the ability to successfully freeze and thaw human embryos. Scientists first discovered how to successfully freeze an embryo, and only after that, they achieved successful oocyte cryopreservation. Whittingham et al. achieved the first successful embryo cryopreservation when the research group froze mouse embryos in polyvinylpyrrolidone (PVP) [3]. The first baby born after a frozen/thawed blastocyst transfer was reported by Cohen et al. in 1985 [14]. Embryo cryopreservation is now a routine procedure and there is sufficient published data supporting its effectiveness.

### **3. Embryo cryopreservation**

Cryopreservation of embryos is a very delicate procedure, which also hides some risks to the things that are frozen. During the freezing process, embryos are exposed to physical stress, caused either by the direct effects of the low temperature or by physical changes induced by ice formation.

The direct effects of the low temperature may induce damage to cell structure and function. The mitotic spindle is especially sensitive to cold shock injuries. The extent of the damage, caused by the freezing procedure, depends on various factors, like the shape and size of the cells, the membrane composition, and its permeability. All these factors are variable and are species specific. Embryos and oocytes have the ability to repair some damage in order to survive and develop properly.

The formation of ice crystals is detrimental to cells. The damage that the cells suffer is not due to the crystallization of ice but rather due to the high concentration of solutes occurring when water is removed in order to form ice [15]. Cryoprotectants (CPAs) act to reduce cellular damage by increasing the volume of the unfrozen residual phase. When the first cryopreservation experiments took place, two opposing methods had been developed simultaneously—a method of slow freezing of the cells and vitrification. Since these methods were very different, scientists started to compare them, in order to elucidate their benefits and drawbacks. Vitrification offers the possibility of eliminating the formation of ice crystals [16], and over the years, it has gradually replaced slow freezing as the preferred method of cryopreservation in the field of reproductive medicine. The main reason behind this fact is that vitrification achieves better survival rates, and moreover, it is less time consuming to perform and does not require highly specialized and expensive equipment like in the slow freezing technique.

#### **3.1 Cryoprotectants**

Cryoprotectants (CPAs) are substances that protect cells/tissues from the damage that may occur during the freezing process. In order to achieve successful cryopreservation of any biological material, the freezing protocols must be optimized, starting with the correct choice of CPA and ending with the thawing process and post-thawing handling of the material. The choice of the most appropriate CPA for a certain freezing procedure is difficult to make, because it must take into consideration the CPA's toxicity, permeability, and also its physicochemical properties. CPAs are widely used to improve the cryosurvival rates, although their mechanism of action is not fully understood. One of their properties is to lower the freezing point of a certain solution, while also protecting the cell membrane during the freezing process. CPAs may also act to stabilize intracellular protein structure. As mentioned earlier, freezing an embryo is a very delicate procedure and embryos may be damaged by chilling, fracturing, ice formation (intra and extracellular), the chemical toxicity of CPAs, osmotic swelling, and osmotic shrinkage [17]. Chilling injuries can lead to changes in lipid-rich membranes and can also result in cytoskeletal disorganization. The mechanical effect of a solidified solution may cause fracture damage, especially to embryos. One of the first documented studies that introduced the concept of cryoprotectants was that of Polge et al. [7, 18], which assessed the use of glycerol in sperm freezing, and it also provided the basis of many future investigations concerning CPAs.

Regarding their structure, CPAs are small molecular weight solutes possessing high aqueous solubility and polar groups that interact weakly with water [19]. CPAs are generally divided into two groups based on their ability to penetrate through the cell membrane—permeating (PM) and nonpermeating (NPM). It is important to point out that PM and NPM cryoprotectants are often used together in order to achieve a successful cryopreservation of cells and tissues. In fact, the core of a cryopreservation solution is made of a mixture of those CPAs, and it also includes various components, like some salts, pH buffers, and others. In the PM group are included some of the most studied CPAs like glycerol (G), ethylene glycol (EG), dimethyl sulfoxide (DMSO), formamide, propylene glycol (PG), and others. PM cryoprotectants are the most important component in the vitrification solution. G and EG are the most commonly used PM CPAs. NPM cryoprotectants include saccharides, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), and others. They are large molecules, usually polymers. Sucrose is the most commonly used NPM cryoprotectant. The effect of these NPM agents is the dehydration of the cells by osmosis. They also act to stabilize the cell's membrane [20] and aid the entry of PM cryoprotectants [21]. Moreover, NPM CPAs are added during the thawing process as they act to reduce the osmotic shock.

Nowadays, there are many freezing media produced by biotechnology companies, which are made by mixing various substances to achieve maximum

**107**

into liquid nitrogen.

*3.2.2 Vitrification*

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

discuss their positives and negatives.

slow freezing is gradually being replaced.

**3.2 Methods**

*3.2.1 Slow freezing*

is no "perfect formula" and therefore the search goes on.

cryoprotection. The composition of these freezing media varies greatly, since there

Practically, two methods for embryo cryopreservation have been used—slow freezing and vitrification. Here, we will briefly review both of them and we will

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,

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

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.

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

Furthermore, it requires expensive controlled-rate freezers.

cryoprotection. The composition of these freezing media varies greatly, since there is no "perfect formula" and therefore the search goes on.
