**2. Cryopreservation: principles and methods**

#### **2.1. Cellular cryotolerance**

Cryopreservation of cells and their storage in liquid nitrogen at −196°C is not physiological process. The freezing process can cause stress and mechanical damage of cells by ice crystal formation [2]. Cell damage can occur at any time during cryopreservation process. Cell lysis can be induced by intracellular ice formation. This major change is easily observed through routine microscopic observations. However, damages can also occur in the cellular structural/ functional levels involving intracellular organelle changes, what is more difficult to diagnose.

Three types of damage during cooling process in oocytes and embryos were described:


The using of cryoprotectants makes a damage of oocytes via cytotoxic and osmotic effect. The addition and the removal of cryoprotectants from the oocyte create an osmotic imbalance across the oocyte membrane, which may result in large volumetric changes and cause damages in the cell morphology, cytoskeletal structures and physiologic function.

Protein structure and function, as well as metabolism, can also be affected. Cells require a period of recovery after thawing, and then, they are able to continue normal intracellular function.

Cryopreservation affects various organelles like intracellular lipids [3], mitochondria (**Figure 1**) cortical granules [4], cytoskeletal structure, zona pellucida and also meiotic spindle [5]. Deleterious effects of meiotic spindle can resulted into chromosome disaggregation. Improper chromosome segregation could lead to aneuploidy and genetic errors, which may cause embryonic and foetal abnormalities. Furthermore, cryopreservation can induce releasing of cortical granule what makes changes of zona pellucida (zona hardening) [4].

**Figure 1.** Mitochondria from primary oocyte after cryopreservation with morphological alterations: swollen mitochon‐ dria (\*) and mitochondria with atypical tubular cristae (black arrow). Scale bar represents 2 μm.

#### **2.2. Cryoprotectants**

amount of liquid nitrogen for freezing. Vitrification of human oocytes and embryos (especially

During last years, the practice of single embryo transfer was a greater demand for reliable cryostorage of surplus embryos. The first reports of successful freezing and thawing of human embryos were in 1983 [1]. There are a growing number of indications for oocyte cryopreser‐ vation as oocyte donation, fertility preservation for cancer patients or social egg freezing. Reproductive behaviour of women has been changed in last years. There is a delay in the age of motherhood due to various reasons like career, live style or education. It is known, that in women older than 35 years, reduction of ovarian reserve is observed. The use of younger cryopreserved oocytes can reduce the risk of foetal loss and aneuploidies associated with ageing oocytes. Oocyte cryopreservation simplifies the logistics of assisted reproductive technology (ART) cycles in donation programme, and there is no need for menstrual cycle

Damage of reproductive function is very frequent and well documented side effect associated with the treatment of malignant tumours. The increasing success of cancer treatment and determined efforts to improve the quality of life after successful treatment has turned attention to the preservation of reproductive function in young women and also in young men. Sperm freezing is largely recommended to preserve fertility prior to the oncology treatment. Cryo‐ preservation of spermatozoa is routinely used in a variety of reasons (sperm bank, donor

For this reasons, cryopreservation of gametes and embryos is more and more important part

Cryopreservation of cells and their storage in liquid nitrogen at −196°C is not physiological process. The freezing process can cause stress and mechanical damage of cells by ice crystal formation [2]. Cell damage can occur at any time during cryopreservation process. Cell lysis can be induced by intracellular ice formation. This major change is easily observed through routine microscopic observations. However, damages can also occur in the cellular structural/ functional levels involving intracellular organelle changes, what is more difficult to diagnose.

Three types of damage during cooling process in oocytes and embryos were described:

**•** damage of zona pellucida or the cytoplasm (temperature from −80 to −150°C).

**•** the damage of microtubules, including meiotic spindle with relative high temperature (from

at early stages) is more effective than slow freezing.

synchronization between donor and recipient.

**2. Cryopreservation: principles and methods**

**•** intracellular crystal formation (temperature from −5 to −80°C);

programme, etc.).

166 Cryopreservation in Eukaryotes

of human‐assisted reproduction.

**2.1. Cellular cryotolerance**

+5 to −5°C);

Cryoprotectants are substances with high solubility and cytotoxicity, what is directly propor‐ tional to their concentration and temperature. They aim to protect cells from any damage what is known as cold shock, during freezing‐warming procedure. Cryoprotectants bond water and they reduce the toxic effect of high concentrations of other compounds. At high concentrations,

cryoprotectants minimize the damage caused by ice formation, as they cause the water to form a glass rather than ice crystal. After thawing, the cryoprotectants must be removed from cells to avoid their deleterious effect on further fertilization and embryonic development.

#### *2.2.1. Membrane permeable cryoprotectants*

These solutions displace water via an osmotic gradient and partly occupy the place of the intracellular water. Indeed, increase in the extracellular osmolarity generates an osmotic gradient across the cell membrane‐supporting dehydration of the cell. In this group, com‐ pounds are with relatively low molecular weight (<100 g/mol). The most commonly used cryoprotectants for oocyte and embryos are ethylene glycol, 1,2‐propandiol and dimethyl sulfoxide (DMSO) [6]. Ethylene glycol is widely used during the vitrification of human oocytes and embryos due to its low toxicity and high permeability. In present time, it is standard part of all vitrification protocols. During equilibration step especially in oocyte vitrification, the compounds of very high concentration (>4 M concentration) are used.

#### *2.2.2. Membrane nonpermeable cryoprotectants*

Nonpermeable cryoprotectants are usually large molecules, which remain in extracellular solution. Extracellular saccharides and macromolecules (sucrose, trehalose, Ficoll, PVP) are commonly added to vitrification solutions. They help draw water out of the blastocoel to attain better dehydration and reduce osmotic shock. Very frequent approach is combination of more cryoprotectants for decreasing the individual specific toxicity of each solution. At least, one of these cryoprotectants should be permeable (with higher toxicity) and one or two nonpermeable (lower toxicity) [7].

For example, during vitrification commonly used ethylene glycol or DMSO or propanediol (permeable) are often combined with sucrose or PVP (nonpermeable), which reduce the concentration of permeable cryoprotectants and facilitate dehydration and vitrification.

Cell permeability is an important factor for determining the conditions for cryopreservation. The permeability of mouse embryos increases as development proceeds to the compacted morula. Ethylene glycol is less permeating than propylene glycol at the one cell stage. In morula stage, ethylene glycol is far more permeating than other cryoprotectants. Exchange of water and cryoprotectants in expanded pig blastocyst occurs predominantly by facilitated diffusion but in oocytes predominantly by simple diffusion [8]. This was related to the expression of aquaporin three mRNA, which was abundantly active in expanded blastocyst, but not in oocytes. The common consensus is that rapidly permeating agents are favoured for oocyte cryopreservation, because the exposure time before cooling can be shortened, and because osmotic swelling during removal of the cryoprotectant can be minimized.

#### **2.3. Slow freezing**

This technique involves stepwise programmed decrease in temperature. The procedure is lengthy and requires the using of expensive equipment (**Figure 2**). This process does not exclude ice crystal formation, which can have extremely deleterious effects [9].

Cryopreservation of Human Gametes and Embryos: Current State and Future Perspectives http://dx.doi.org/10.5772/64950 169

**Figure 2.** Programmable cryo freezer Planer Kryo F10.

Slow freezing is a technique with long history but in comparison to vitrification actually does not bring any advantages. Vitrification methods are more efficient and reliable than any version of slow freezing [10]. After a long period of practising was the convention slow freezing method completely stopped in many centres and was replaced by routine vitrification.

#### **2.4. Vitrification**

cryoprotectants minimize the damage caused by ice formation, as they cause the water to form a glass rather than ice crystal. After thawing, the cryoprotectants must be removed from cells

These solutions displace water via an osmotic gradient and partly occupy the place of the intracellular water. Indeed, increase in the extracellular osmolarity generates an osmotic gradient across the cell membrane‐supporting dehydration of the cell. In this group, com‐ pounds are with relatively low molecular weight (<100 g/mol). The most commonly used cryoprotectants for oocyte and embryos are ethylene glycol, 1,2‐propandiol and dimethyl sulfoxide (DMSO) [6]. Ethylene glycol is widely used during the vitrification of human oocytes and embryos due to its low toxicity and high permeability. In present time, it is standard part of all vitrification protocols. During equilibration step especially in oocyte vitrification, the

Nonpermeable cryoprotectants are usually large molecules, which remain in extracellular solution. Extracellular saccharides and macromolecules (sucrose, trehalose, Ficoll, PVP) are commonly added to vitrification solutions. They help draw water out of the blastocoel to attain better dehydration and reduce osmotic shock. Very frequent approach is combination of more cryoprotectants for decreasing the individual specific toxicity of each solution. At least, one of these cryoprotectants should be permeable (with higher toxicity) and one or two nonpermeable

For example, during vitrification commonly used ethylene glycol or DMSO or propanediol (permeable) are often combined with sucrose or PVP (nonpermeable), which reduce the concentration of permeable cryoprotectants and facilitate dehydration and vitrification.

Cell permeability is an important factor for determining the conditions for cryopreservation. The permeability of mouse embryos increases as development proceeds to the compacted morula. Ethylene glycol is less permeating than propylene glycol at the one cell stage. In morula stage, ethylene glycol is far more permeating than other cryoprotectants. Exchange of water and cryoprotectants in expanded pig blastocyst occurs predominantly by facilitated diffusion but in oocytes predominantly by simple diffusion [8]. This was related to the expression of aquaporin three mRNA, which was abundantly active in expanded blastocyst, but not in oocytes. The common consensus is that rapidly permeating agents are favoured for oocyte cryopreservation, because the exposure time before cooling can be shortened, and because

This technique involves stepwise programmed decrease in temperature. The procedure is lengthy and requires the using of expensive equipment (**Figure 2**). This process does not

osmotic swelling during removal of the cryoprotectant can be minimized.

exclude ice crystal formation, which can have extremely deleterious effects [9].

to avoid their deleterious effect on further fertilization and embryonic development.

compounds of very high concentration (>4 M concentration) are used.

*2.2.1. Membrane permeable cryoprotectants*

168 Cryopreservation in Eukaryotes

*2.2.2. Membrane nonpermeable cryoprotectants*

(lower toxicity) [7].

**2.3. Slow freezing**

Limiting factor for all cryopreservation methods is ice crystal formation that drastically reduced survival of embryos and oocytes. Vitrification process produces a glasslike solidifi‐ cation of living cells, which completely avoids ice crystal formation. It is well known that vitrification requires a greater amount of cryoprotectants, what increases the toxicity of their environment. However, it was claimed higher survival rate after using vitrification instead of slow freezing [11]. Vitrification is very simple, cost‐effective process, but the skills to perform require good manual training.

### **3. Gamete cryopreservation**

#### **3.1. Spermatozoa cryopreservation**

Cryopreservation of human semen is well‐established laboratory procedure to maintain the fertilizing potential of spermatozoa during storage in liquid nitrogen. Modern trends in assisted reproduction technologies influenced the indications for human sperm cryopreser‐ vation. Spermatozoa are not so sensitive to cryopreservation damage (in comparison with other cell), because of the high fluidity of the membrane and the low water content (about 50%). The effect of cryopreservation on sperm DNA integrity is still unclear. There is no agreement in literature on whether or not affect cryopreservation sperm chromatin integrity.

When clinicians became aware that azoospermia or very severe oligozoospermia could not be improved by medical treatment, it arises the idea to create sperm banks. Today, cryopreser‐ vation of spermatozoa is routinely used in a variety of reasons:


Semen preservation before the beginning of therapy should be proposed to all adult men and postpubertal boys. To date, no clinically proven methods are available to preserve fertility in prepubertal males. The testicular cancer survivors have a good chance of fathering a child by using sperm cryopreserved prior to the oncology treatment thanks to assisted reproduction methods [12].

In the ICSI era, almost all cryopreserved semen sample, even when it contains only few sperm, could be used for subsequent infertility treatment. Genetic damage is unknown.

Cryopreservation is known to cause some changes in sperm morphology, including damage to mitochondria, the acrosome and the sperm tail. The sperm motility is particularly sensitive, and it is generally accepted that it can be reduced to 50% after the cryopreservation/thawing procedure. Due to this fact, it is necessary to choose potential donors with an emphasis on this sperm parameter.

#### **3.2. Oocyte cryopreservation**

Cryopreservation of human oocyte can be an alternative to circumvent many of the ethical issues associated with embryo cryopreservation. For oocyte cryopreservation, it is very suitable to use vitrification method. Oocyte cryobanking is a new more efficient approach in oocyte donor‐recipient treatment. On the basis of guideline from the Practice Committees of the American Society for Assisted Reproductive Medicine (from 2013) and, in March 2012, European Society of Human Reproduction and Embryology (ESHRE), it is indicated that mature oocyte vitrification and warming are not experimental and should no longer be considered as experimental procedures. This progress in the field of cryopreservation opens new perspectives in assisted reproduction. Recent effective oocyte vitrification systems have a significant impact on clinical practice. It is a possible way in countries where the law forbids the cryopreservation of embryos. Indeed, efficient oocyte vitrification technology eliminates synchronization between donor and recipient. It enables the establishment of egg banks by eliminating the logistics of coordinating egg donors with their recipients. Progress in oocyte vitrification brings new possibilities mainly for women, who are trying to postpone child‐ bearing from professional or social reasons. The process was originally developed as a way to preserve the fertility of cancer patients undergoing possibly sterilizing chemotherapy, and it is relatively simple.

cell), because of the high fluidity of the membrane and the low water content (about 50%). The effect of cryopreservation on sperm DNA integrity is still unclear. There is no agreement in

When clinicians became aware that azoospermia or very severe oligozoospermia could not be improved by medical treatment, it arises the idea to create sperm banks. Today, cryopreser‐

**2.** Sperm banking for husband sperm for psychological or other reasons (it is not always

**3.** Storage of epididymal or testicular spermatozoa after MESA/TESE, to avoid repeated

**5.** Preservation of semen before surgical, chemical or radiological cancer therapy, which may lead to testicular failure or ejaculatory dysfunction. Also other nonmalignant diseases,

**6.** Male gamete freezing is largely recommended to preserve fertility in those subjects who are exposed to potentially toxic agents, which may interfere with gametogenesis.

Semen preservation before the beginning of therapy should be proposed to all adult men and postpubertal boys. To date, no clinically proven methods are available to preserve fertility in prepubertal males. The testicular cancer survivors have a good chance of fathering a child by using sperm cryopreserved prior to the oncology treatment thanks to assisted reproduction

In the ICSI era, almost all cryopreserved semen sample, even when it contains only few sperm,

Cryopreservation is known to cause some changes in sperm morphology, including damage to mitochondria, the acrosome and the sperm tail. The sperm motility is particularly sensitive, and it is generally accepted that it can be reduced to 50% after the cryopreservation/thawing procedure. Due to this fact, it is necessary to choose potential donors with an emphasis on this

Cryopreservation of human oocyte can be an alternative to circumvent many of the ethical issues associated with embryo cryopreservation. For oocyte cryopreservation, it is very suitable to use vitrification method. Oocyte cryobanking is a new more efficient approach in oocyte donor‐recipient treatment. On the basis of guideline from the Practice Committees of the American Society for Assisted Reproductive Medicine (from 2013) and, in March 2012, European Society of Human Reproduction and Embryology (ESHRE), it is indicated that mature oocyte vitrification and warming are not experimental and should no longer be

could be used for subsequent infertility treatment. Genetic damage is unknown.

literature on whether or not affect cryopreservation sperm chromatin integrity.

possible to produce sperm samples at the appropriate time in the cycle).

such as diabetes or autoimmune disorders, may lead to testicular damage.

vation of spermatozoa is routinely used in a variety of reasons: **1.** Donor or husband semen storage for assisted reproduction.

**4.** Storage of sperm as a fertility "insurance" for future.

biopsies or aspirations.

170 Cryopreservation in Eukaryotes

methods [12].

sperm parameter.

**3.2. Oocyte cryopreservation**

Oocyte cryopreservation is less successful than embryo cryopreservation for many reasons. Oocytes have small surface to volume ratio, temperature‐sensitive metaphase spindle [13], zona pellucida as very specific structure and susceptibility to parthenogenetic activation. Oocytes are one of the biggest cells with high likelihood of intracellular ice formation [14] Oocytes are very unique cells, because of their developmental capacity to be fertilized and then to support early embryonic development. This capacity derives from maternal legacy of the myriad of transcript, proteins and energetic substrates and also cytoplasmic organelles, which facilitate early mitotic divisions of the embryo until embryonic genome activation occurs [15]. This highly organized structure often incurs serious damage after cryopreservation. The volume of mammalian oocyte is much bigger than that of spermatozoa, thereby substantially decreasing the surface to volume ratio and making them sensitive to chilling and highly susceptible to intracellular ice formation. In fact, in a developing embryo, cleavage division occurs without any increase in volume until blastocyst stage, leading to higher nucleus‐ cytoplasmic ratio of embryo blastomeres compared with the oocyte. Oocytes are substantially more prone to cryo damage than are embryos. Number of blastomeres in early embryos provides great flexibility to compensate for any detrimental effects of cryopreservation, because missed blastomeres can be replaced by the daughter cells of dividing intact ones. Oocytes contain one‐half of the genetic material of the future individual, and so any damage to its chromatin structure may result in deleterious defects in the developmental competence of the resulting embryos. Damage of meiotic spindle can result in chromosomal abnormalities after thawing. The permeability of oocyte plasma membrane to cryoprotective agents is low compared with embryo [6].

Although mature oocytes in metaphase II are sensitive to cryopreservation (detrimental effect on meiotic spindle or premature cortical granule release) and immature oocytes on prophase I (GV oocytes) look that are more suitable for cryopreservation. It is well known that oocytes frozen at GV stage exhibited decreased affectivity of *in vitro* maturation and increased spontaneous parthenogenetic activation [16]. For this reason in case of immature oocytes, it is recommended to use *in vitro* maturation and after that perform their subsequent vitrification.

It was presented that highly organized structure of fresh oocyte changes dramatically (at cellular, ultrastructural, molecular and developmental levels) after cryopreservation. Cryo‐ preserved oocytes have cellular characteristics that differ from those of the fresh oocytes.

#### *3.2.1. Cryopreservation of ovarian tissue*

Fertility preservation has a great importance to many young women with cancer [17]. Cryo‐ preservation of ovarian tissue is a safe, simple and effective option for preserving fertility in young patients facing or undergoing gonadotoxic therapy. Oocytes in primordial follicles are very small and tolerate cryopreservation very well. The removal of ovarian tissue is a simple procedure. Ovarian tissue can be obtained using minimally invasive techniques during laparoscopy, with unilateral ovariectomy or partial ovariectomy. Ovarian tissue can be cryopreserved independently of the menstrual phase.

**Figure 3.** Primary follicle from ovarian cortex before (a) and after (b) cryopreservation with morphological alterations. The oolemma of oocyte after cryopreservation is more undulated and interrupted (E), and the cytoplasm of follicular cells (F) is vacuolated. N, nucleus; M, mitochondria; L, lipid droplets. Scale bar represents 5 μm [19].

In 2004, first live birth after autotransplantation of human ovarian tissue was reported [18]. To date, 60 live births have been reported worldwide following transplantation of cryopreserved ovarian tissue. However, research on the cryopreservation of ovarian tissue as a method of fertility preservation has now been continuing for more than a decade, and considerable successes have recently been achieved.

In centres that offer cryopreservation of ovarian tissue, the procedure can be performed one day after the patient's first visit. After the tissue has been removed, it can be processed immediately or transferred in special transportation containers to a centre specializing in the cryopreservation of ovarian tissue, with an associated cryobank (**Figure 3**).
