Gametes and Embryos Cryopreservation

#### **Chapter 1**

## Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality Controls for Commercial Applications

*Aygül Ekici, Güneş Yamaner and Menekşe Didem Demircan*

#### **Abstract**

In this section, cryopreservation of fish genetic resources, which is one of the important applications to ensure the sustainability of genetic resources of freshwater fish species, is discussed. At the same time, information is provided about the possible sources of contamination that may be encountered during cryopreservation applications. In this context, the results of sperm, egg, and embryo cryopreservation studies of fish and their success and failure in applications were evaluated in addition to the process from past to present. Information is given about the contamination that may develop depending on the applications in the process of cryopreservation and dissolving processes, as well as the studies carried out to eliminate extracellular disease agents. In the section, in addition to the evaluation of the results of scientific studies, commercial companies that commercially carry out gamete cryopreservation applications are also included. The contamination that may develop depending on the applications in the process of cryopreservation and thawing processes, as well as the studies carried out to eliminate extracellular disease agents are mentioned.

**Keywords:** sperm, egg, germ cell, storage, contamination

#### **1. Introduction**

Developing gamete conservation programs will be invaluable in future commercial fish reproductive studies. Gamete conservation is an important tool for fish reproduction and is very important for aquaculture. It may be widely used in breeding laboratories. The growing interest in improving technology has led to an increase in the number of studies on this subject. In particular, sperm has an important place in cryopreservation studies and is still performed both in laboratory studies and in culture applications [1, 2]. Although the freezing of male sperm dates back to the 1600s, the successful method of artificial insemination at the end of the 1950s, with the need for long-term storage of sperm. In 1953, once the method of freezing sperm was successful with the

herring (*Clupea harengus*) that achieved approximately 80% cellular motility upon thawing [3]. After that, the growing interest in improving preservation technology has increased the number of studies on sperm cryopreservation. Therefore, cryopreserved sperm can now be used in routine fish reproduction and aquaculture practices [4].

Before achieving standardization in the sperm cryopreservation procedure, aquaculture conditions, broodstock management, standardization of the feeding regime, and the production of disease-free lineages are essential for the success of standardization. In the next stage, providing appropriate biosecurity conditions in the laboratory where sperm cryopreservation process will be performed will be an important step that increases the success and brings it closer to standardization. Once a standard procedure has been established, for example, the kits used in DNA and RNA isolation, standardization can be achieved by creating fish species-specific sperm cryopreservation kits.

Although sperm cryopreservation studies have focused on Cyprinid, Sturgeon Salmonids, and Catfish species that are intensively cultured, there are many cases of successful freezing studies with other various fish species [5–7]. Since a review on cryopreservation of fish sperm was carried out before [8], the work in this present section will be focused on more recent improvements in fish (carp, sturgeon, eel, Salmonid, and catfish) sperm cryopreservation with the extender (+cryoprotectant)/ additive material and freezing/thawing procedure, mostly.

#### **2. Cryopreservation of freshwater fish sperm**

Populations of aquatic species are threatened by anthropogenic influences, overfishing and poaching, destruction of spawning habitats, as well as an increase in water temperature caused by climate change. Today, fish farms carry out production activities by preserving live fish. However; in addition, because of problem with water resources, gametes, genetic factors, disease-related factors, operational problems, system failure, environmental problems, and the survival chances of these creatures become difficult.

High volume and high-quality sperm quantity must be obtained for the commercialization of sperm cryopreservation applications. The successful use of cryopreserved sperm in gene banks in fertilization studies requires that the preserved material is of an acceptable quality/viability and quantity [9]. A company based in Norway provides the aquaculture industry with cryopreserved sperm of 16 fish species, including 8 species belonging to the Salmonidae family [10]. In addition, sperm preservation, maturation, and cryopreservation solutions specific to aquatic species are commercially available on the market [11]. There are also some solutions developed by various commercial companies for use in tissue and cell freezing.

In sperm cryopreservation studies, the goal is to achieve the highest fertilization rate with frozen sperm. However, the failure to achieve similar results in repeated experiments in sperm cryopreservation studies, using of this application in aquaculture studies is limited [12]. Standardization is the biggest problem in sperm cryopreservation, but it will be useful to understand the molecular mechanisms by using new-generation technologies to ensure standardization.

#### **2.1 Carp sperm cryopreservation**

The common carp (*Cyprinus carpio*) is one of the largest farmed freshwater fish in the world, with its production reaching 4363.3 thousand tons in 2020 [13]. In addition

#### *Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

to being of commercial interest, it is also a model organism within Teleostei, and is one of the first cultured fish [14]. The sperm cryopreservation of carp was developed several decades ago, and cryopreservation of sperm has often been investigated in carp due to its economic (commercial) and recreational value. However, Carp have undergone selective breeding, resulting in different strains. Cryopreservation can be used to secure sperm from the desired common carp strains in sperm banks and for sperm transport [8, 15]. Therefore, there is a growing interest in methodological and practical innovation in the cryopreservation of carp sperm. Even though the protocols for cryopreservation of carp sperm have been developed [16–19], reduction in sperm viability and motility is still observed in cryopreserved sperm. Therefore, before the cryopreservation of sperm, a thorough evaluation of different extender solutions with cryoprotectant, additives material (cyclodextrin, ext.,) and cooling or thawing rates are essential to develop optimum cryopreservation protocols for the carp.

An extender is a chemical compound used in sperm cryopreservation studies and includes inorganic chemicals. The extender has to be similar to blood and sperm seminal plasma in terms of the inorganic chemicals it contains in order for sperm cells to maintain their viability in vitro preservation. Also, the extender functions as nutrients, pH regulators, and seminal plasm osmotic pressure. Therefore, the extender used to freeze the sperm of freshwater fish should be specific to the species. Since the sperm activation mechanism of each species is different, the extenders used vary. Cryoprotectants are added for protection and stored in a cryogen that can produce very low temperature due to its varied state (e.g., liquid nitrogen at −196°C) [8].

A number of efficient cryo-medium context has been developed for carp sperm. One of the most important steps in the successful cryopreservation of carp sperm is selecting the cryoprotectant in the extender during the process. Various cryoprotectants such as glycerol, DMSO, methanol, and DMA have been used as cryoprotectants for fish sperm. Some studies show that DMSO works best as cryoprotectant in carp sperm compared to glycerol and methanol. In another study conducted with carp, it was emphasized that the use of DMSO at a rate of 5–20% was effective in the sperm of carp [16–19]. Another cryoprotectant used to freeze carp sperm is the yolk of an egg. It has been reported that duck egg yolk, which is used as an extracellular cryoprotectant in cryopreservation sperm of carp, increases motility after thawing and the fertilization rate compared to chicken egg yolk [20].

In addition to the studies investigating the effectiveness and success of cryoprotectants in the freezing process of carp sperm, there are studies that revealing the oxidative stress caused by dilution with cryoprotectants during/before the cryopreservation process. Seminal plasma protects sperm cells against oxidative stress. Dilution of sperm cells during cryopreservation reduces the seminal plasma content, which makes sperm cells more sensitive to oxidative stress [21]. Therefore studies have focused on reducing the effect of oxidative stress in the cell. The fact that amino acids have antioxidant properties and are present in high concentrations in seminal plasma has made amino acids an important component in sperm cryopreservation studies [21–24]. It has been shown that the use of L-cysteine in the process of sperm freezing in carp has a positive effect on motility and viability of sperm cells [25]. It has been stated that the use of Cysteine at 20 mM in the freezing medium of carp sperm process makes a significant difference in motility and motility time [26]. Another additive used is cholesterol-laden cyclodextrin (CLC) to reduce cell damage during the freezing process. In the freezing of carp sperm, the use of 1.5 mg of CLC in the extender was found to have the best cryoprotective effect in maintaining sperm motility, duration, and viability of sperm cells [27].

In addition to the cryoprotectant studies mentioned above, one of the factors affecting the freezing process and is the density of the sperm before freezing. It has been reported that sperm concentration is often ignored before freezing, and the sperm and extender are diluted in a certain volume-to-volume ratio [28, 29]. On the other hand, it has been shown that optimization of sperm concentration plays a crucial role in a number of aquaculture species, in particular pikeperch (*Sander lucioperca*) [30], European perch (*Perca fluviatilis*) [9], and Salmonid [31]. However, it was observed that there was no significant difference between sperm samples' motility and velocity after thawing when different sperm concentrations were specially adjusted before freezing in carp sperm [32].

Besides studies on the effect of chemical and non-chemical components used in freezing sperm, studies on the freezing process are varied. In adding to studies on the freezing procedure with liquid nitrogen and liquid nitrogen vapor used for a long time, studies are also performed with programmable freezing devices. In studies using liquid nitrogen vapor and liquid nitrogen, the heights of straws to liquid nitrogen vapor and straws volumes were studied. In Ref. [33], tested different freezing rates by modifying the height (2–6 cm) above the surface of liquid nitrogen, where the straws were placed. The highest fertilization was observed when samples were frozen at 2 cm above the level of liquid nitrogen and 10 min freezing time (74 ± 7%).

#### **2.2 Sturgeon sperm cryopreservation**

Sturgeons are the oldest freshwater fishes, having evolved around 200–250 million years ago [34]. The high economic value of sturgeon, mainly because of their caviar, the failure to manage the caviar trade, and unsustainable fishing (in the seas and river), along with serious habitat fragmentation have led to a significant decline of wild sturgeon populations [35, 36]. Due to this decrease in natural stocks, all sturgeon species have been included in the list of endangered species under CITES (Convention on International Trade in Endangered Species) since 1997 for population restoration; however, thanks to aquaculture, the sturgeon still maintains its place in the valuable product category in fish markets worldwide [37]. Sturgeon culture is developing and increasing in order to meet the need for products obtained from sturgeon and, in addition, support natural stocks. Another method of protection of natural stocks, as already described, is the cryopreservation of gametes or embryos. Especially fish sperm cryobanking is considered a potentially powerful tool in aquaculture for endangered species [38].

Gamete cryoperservation in sturgeon has been given more importance because the species is one of the extinct species. And in sturgeon species, especially sperm freezing, biological, mechanical, and biochemical factors affecting sperm cryopreservation have been studied extensively in order to increase post-thawing motility. Since there is literature [8] on this subject before, current research topics of recent years are given in this section.

The most commonly used cryoprotectant for cryopreservation sperm of sturgeon is methanol. In various species of sturgeon, the lowest fertilization rate obtained using this cryoprotectant 6% (Siberian sturgeon, *Acipenser baeri*); the highest was reported as 40% (Shortnose sturgeon, *A. brevirostrum*) [39–41]. In Russian sturgeon (*A. gueldenstaedtii*), the use of 10% methanol in sperm cryopreservation causes a decrease in acrosin activity and an increase in DNA damage; however, compared to the solution in which methanol was not used, it was reported that this cryoprotectant protects sperm cells during cryopreservation process [42]. In sperm cryopreservation *Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

in sturgeon, in addition to intracellular cryoprotectant substances, there are studies in which cryoproctants such as sugars, egg yolk, and vegetable oil, which are called extracellular, are tested [43]. In the study conducted with Persian (*A. persicus*) and Beluga (*Huso huso*) sturgeon sperm; the addition of different disaccharides such as maltose, trehalose, lactose, and lactulose along with 9% methanol solution has been reported to have an effect on motile sperm cells, except for lactulose. The authors also reported that the cryopreserved sperm using each of the four disaccharides could be stored for at least 30 min without losing sperm motility [44]. In addition to the studies reporting that the protective effect of disaccharides during sperm freezing is due to the high molecular weight they have [44], there are studies reporting that this protective effect is entirely due to the chemical structure of the disaccharides [45, 46]. When the use of sucrose or trehalose alone and in combination with different concentrations was tested for cryopreservation of Dabry's sturgeon (*Acipenser dabryanus*) sperm, low concentration sucrose plus trehalose (S15T15) solution was reported to be the optimal solution. Also, it was reported that mixing of the extender with sucrose, lactose, or trehalose alone or with pairwise mixtures revealed that a mixture of lactose and trehalose (L15T15) gave the best results for both Chinese sturgeon (*A. sinensis*) and Dabry's sturgeon [47].

Along with cryoprotectants that protect the viability of sperm cells, various dilution agents that provide the dilution of the sperm and reduce the cell damage seen in the freezing process are also used. Another substance added in cryomedium in the freezing of sturgeon sperm is antioxidant substances. These substances, which reduce oxidative stress in the sperm cell during the freezing process and therefore increase sperm quality after thawing, are ascorbic acid, catalase, glutathione, and cysteine [48]. Although the protective effect of using catalase (25 U/mL), glutathione (0.25–0.5 mg/ mL), and ascorbic acid (0.5 mg/mL) in freezing the sperm of three species of sturgeon (*A. dabryanus*, *A. sinensis*, and *Acipenser baerii*) on sperm cells have been mentioned; it has been reported that the three antioxidants should not be used together [49].

In order to protect sperm cells from cryodamage in sperm cryopreservation in many fish species, including sturgeon, it is recommended to use various proteins, enzymatic or non-enzymatic antioxidants, and antifreeze proteins in the cryopreservation procedure [50]. However, the effect of antifreezing protein on sperm cells during freezing in sterlet sturgeon was examined and it was found that a significant decrease in motility rate and velocity of curvilinear (VCL) was observed in cryopreserved spermatozoa with/without supplementation of 10 g AFPI compared to fresh spermatozoa. And also the results showed that in partial changes in the ultrastructural compartments, weakening of the midpiece and rupture of the plasma membrane of the flagellum were seen. The author believes that this damage is not due to oxidative stress that can occur in cryopreserved sperm; expressed that there is physical damage that occurs during the formation of ice crystals during freezing process [51].

It is a known fact that after cryopreservation of the sperm, the motility in the sperm cells decreases and if these sperm samples are used in the fertilization study, a low fertilization rate will be obtained [52]. All the studies carried out so far have been aimed at increasing sperm viability/motility, that is, the fertility of sperm, after thawing. However, in the sperm cryopreservation study using methanol in Russian sturgeon, the motility value obtained after thawing sperm samples was found low (18–25%); in the fertilization study conducted with the same sperm samples, fertilization percentage was obtained as 72.67% [53].

Compared to other species, sturgeon sperm is one of the species with low sperm density due to the mixing of sperm with urine and the originality of the maturation process of sperm. This low concentration of sperm in sturgeon fish is especially important for the optimum dilution rate in sperm cryopreservation studies where the dilution rate is important. It has been reported that the percentage postthawed sperm motility in Sterlet sturgeon (*A. ruthenus*) depends on the sperm concentration in the samples. While the highest motility after thawing in the study was found in the frozen sperm samples at concentrations of 0.2–1 × 109 spz/mL; the sperm concentration of 3 × 109 spz/mL, which is higher than the natural sperm concentration in the sterlet, has been reported as suitable for use in cryopreservation procedures as sperm fertilization ability remains at a high level despite a significant decrease in sperm motility percentage. And these findings support the conclusion that high utilization of sperm concentration before freezing may be useful for reducing the volume of sperm retained during freezing and reducing the sample volume required for artificial insemination [54].

In addition to sperm concentration, there are studies investigating the effect of various sperm volumes during freezing. From this point of view, some research was carried out to examine the effect of various volumes (0.5, 0.75, 1.5, and 2 ml) and also the possibility of using the method of vitrification of sperm under deep low-temperature cooling in Russian and Siberian sturgeon. In this study, it was observed that the highest percentage of motility and motility duration was in samples frozen in 0.5 ml Eppendorf tubes. Also, in the study, the following was reported, when cryopreservation of seminal fluid in larger test tubes (0.75, 1.5, and 2 ml), the results were slightly worse [55].

One of the newest issues being investigated in the freezing of sturgeon sperm is the use of ultrasonic waves, which allows the creation of optimum conditions so that the sperm can be preserved at a low temperature. The new methodological approach to low-temperature preservation of fish germ cells using acoustic-mechanical effect offers great opportunities to create new effective deep-freezing methods. The report on the acoustic-mechanical effect on sterlet sperm, it was showed that different parameters of time, frequency, and wavelength can have both positive and negative effects on the reproductive qualities of thawed sperm. It was observed that an increase in the exposure time above 2 min and a frequency up to 5 kHz and a change in the wavelength lead to severe cell damage after defrosting [56].

One of the other current issues studied in sperm cryopreservation in sturgeon is the effect of organotin components (OTs) on fresh and frozen sperm. It has been reported that the accumulation of OTs in the gonad in Russian sturgeon is a stress factor affecting the cells in the cryopreservation process and also this buildup may cause in vitro oxidative stress in sturgeon sperm, reduce gamete quality, and affect fertilization success [57].

#### **2.3 Eel sperm cryopreservation**

Eels are species that contain species of economic importance for fisheries and aquaculture, and have reduced natural stocks such as sturgeons. Since the 1980s, natural stocks have been reported to have decreased by 90% for the populations of European eel (*Anguilla anguilla*) and Japanese eel (*A. japonica*) due to climate change, habitat degradation, pollution, parasite infection, and overfishing [58]. All two temperate eel species have been included in the Red List of the International Union for Conservation of Nature (IUCN) as threatened due to population decline, with *A. japonica* categorized as "Endangered" [59] and *A. anguilla* included as "Critically Endangered" [60], which is the highest category before extinction rating. As mentioned earlier, one of the most common methods of protection in endangered species and species where there are difficulties in the reproductive cycle, such as eels, *Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

is cryopreservation. Sperm freezing in eels was first tried in Japanese eel in the 2000s [61]. And after this study, a lot of studies have been done on freezing the sperm of eels, including the sperm of European eels, with the development of protocols being prioritized [62].

The protocol first developed for sperm freezing of eels was the protocol in which DMSO was used as a cryoprotectant in both species. In cryopreservation study using DMSO, post-thawed motility was 45% and above in Japanese eel; in European eels, it has been reported as 35% and above [61]. Although the post-hawed motility value is considered high; because sperm cells frozen using DMSO gives low rates of fertilization rate, and at the same time, with recent studies, DMSO causes epigenetic changes in eel sperm; the use of methanol instead of DMSO as a cryoprotectant substance in the freezing of eel sperm has become widespread. And studies have shown that the use of methanol as a cryoprotectant in the freezing of both Japanese and European eel sperm has led to an increase in motility values after thawed [61, 63]. In another study conducted on European eel; with the use of 5% egg yolk with methanol (10%) as cryoprotectant, it was reported that the motility after thawing was higher than 50%. And this value was found to be significantly higher than the values obtained by using 10% methanol used as a control group in the study [64].

In a study with Japanese eel; artificial seminal plasma (in mM; 149.3 NaCl, 15.2 KCl, 1.3 CaCl2, 1.6 MgCl2, and 20 NaHCO3, buffered with 20 mM TAPS-NaOH at pH 8.1) and methanol (in 1:100 ratio) and 10% methanol in v/v final concentration were tested as extenders and cryoprotectants for sperm freezing. As a result of the fertilization study, although the embryos obtained with cryopreserved sperm had a low survival rate and a high malformation rate; it has been reported that this freezing procedure can be used successfully [65]. In another study conducted on a Japanese eel, three different cryoprotectants were tried to freeze sperm cells stored by creating artificial seminal plasma. At the end of the study, the most successful result was obtained by using 10% and 15% MeOH, in addition to the combination of 5% MeOH and 5% DMA; however, DMSO in artificial seminal plasma has been reported to have no cryopreservation properties and is toxic to sperm [66].

The latest protocols for sperm cryopreservation of European and Japanese eel use methanol as cryoprotectant and they have been adapted to large volumes. In the case of the protocol for Japanese eel sperm, successful fertilization has been achieved and with similar survival rates as with fresh sperm. Moreover, the morphology of the larvae produced with cryopreserved sperm was similar to larvae produced from fresh sperm. In the case of the protocol for European eel sperm, the latest protocol has not been tested for fertilization trials yet, but the motility of frozen–thawed sperm obtained was over 50%, which is the highest ever obtained in this species [67].

#### **2.4 Salmonid sperm cryopreservation**

The Salmonidae family consists of important species produced in the world, and total Salmonidae production accounts for <1.8% of the total share of global production [68]. There is an increase in water temperature due to global climate change. Salmonid populations distributed in cold waters are the most studied taxonomic group due to their low tolerance to fluctuations in water temperature. These temperature fluctuations are also thought to affect their reproductive performance [69]. In order to support natural stocks and be used in aquaculture, sperm cryopreservation studies are carried out intensively on Salmonid species. Moreover, the wide distribution of fish species belongs to the Salmonidae family in the world and the fact that

they have been produced for many years has made the species of this family suitable for cryopreservation studies. In the sperm cryopreservation studies conducted to date, post-thawing motility parameters and fertilization rates may vary due to reasons such as differences in freezing procedures, genetic differences in species, and differences in culture conditions. For these reasons, the inability to achieve standardization is one of the most important problems in this field.

The issues summarized below about cryopreservation studies; it will help to understand why standardization on fish-species-specific basis cannot be achieved.

Sperm motility parameters after thawing straws containing cryopreserved sperm; fertilization and hatching rate, straws volume, chemicals, cryoprotectants, spermatozoa density, and reproductive season can be affected by factors. One of the first steps in starting cryopreservation is the choice of materials to be used. In cryopreservation of fish sperm, 0.25 or 0.5 mL straws are usually used. In the selection of the straws to be used; it should be ensured that it is in a volume that will not reduce the motility rate after thawing and will allow the fertilization process to be done easily. When using straws of 0.5 mL; it is preferred because sperm motility parameters after thawing are high and save time during fertilization (*Salmo salar*, [70]; *Salmo trutta* m. *trutta*, *S. salar*, *Salvelinus fontinalis*, S.t. m. *fario*, [31]). In addition to the straws volume change, the glucose rate used in the extender (as it changes the osmotic pressure value) affects sperm motility [31]. In the extender of sperm cryopreservation of salmonid species, sucrose, trehalose, and glucose [71–73] are used, however, mostly glucose is preferred [22, 31, 70, 71, 74]. Sperm concentration in the straws also affects sperm motility parameters. However, this sperm concentration may even differ between species belong to the Salmonidae family. In rainbow trout (0.5–1.0 × 109 spz/mL) [75]; sperm concentration in straw, where the survival rate after thawing is the highest, is significantly lower than in other Salmonid species (2.0, 3.0, 4.0 × 109 spz/mL; *S. fontinalis,* S. *trutta*, *S. salar*, respectively) [31]. Due to cryopreservation process, cells are subjected to stress due to imbalances in low temperature and osmotic pressure. In order to reduce this stress on the cell and to protect sperm cells from freezing effects, various cryoprotectant (can/cannot penetrate into the cell) agents are used. In 2017, in ref. [76] listed cryoprotectant agents under the headings "Alcohols and Derivatives; Sugars and Sugar alcohols; Polymers, Sulfoxides, and Amides; Amines".

Although the success in fertilization and motility rates with cryopreserved fish sperm to date has been achieved with 10% DMSO [38]; in *S. salar*, the motility and fertilization success rate is higher in 10% methanol than 10% DMSO [77]. Although the egg yolk used in the extender creates difficulty in use due to its viscous structure after thawing, it is a popular cryoprotectant (not penetrate into the cell) used for freezing and storing sperm of various species. The addition of egg yolk and sucrose to the extender together with cryoprotectant agents with penetrating properties into the cell significantly improves sperm quality [78]. Various antioxidants are used in the extender to prevent lipid peroxidation and Reactive Oxygen Species (ROS) activity that may occur during cryopreservation [22, 79, 80]. The addition of α-tocopherol and ascorbic acid to extender lead to a decrease in membrane lipoperoxidation and O2 − i production of *S. salar* spermatozoa, thereby increasing the fertilization capacity [80]. The addition of ascorbic acid to the extender in *S. salar* increases the cell integrity and sperm function of spermatozoa [80]. A decrease in sperm motility after thawing can be associated with membrane permeability and DNA damage. ROS, which affects sperm motility in cryopreservation studies, may also induce lipid peroxidation in the membrane. This can lead to the induction of cell apoptosis [81]. In salmonids, there is a positive correlation between the mitochondrial membrane permeability of

*Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

cryopreserved sperm and fertilization [70]. The initiation of sperm motility and the duration of motility depend on the ATP provided by mitochondria in most fish [82]. Therefore, any damage to the mitochondria has a negative effect on motility [70] and can limit the motility and fertilization potential of spermatozoa [83].

Potassium ion has an important place in cryopreservation studies because it has the ability to inhibit sperm motility of Salmonid species based on this inhibitory property, the addition of potassium ions to the diluent has a species-specific effect on Salmonid sperm motility after thawing. Although potassium ion negatively affects the percentage of motility after thawing on the species *O. mykiss*, *S. trutta*, *S. fontinalis*; it showed a positive effect on sperm *Coregonus lavaretus*. This effect of the potassium ion is thought to be due to the osmotic pressure, not the concentration used [84]. This has created a similar situation, such as glucose's ability to change osmotic pressure [31]. In sperm during reproductive season and outside the reproductive season, sperm parameters may differ after thawing in sex-reversed rainbow trout [85], however, it is also stated that sperm collection season does not affect post-thawing motility [71]. These different results in the studies depend on the difference in the extender contents used in cryopreservation, the differences in fish strains [71], and the interaction between the extender and the cryoprotectant substance [86].

One of the goals of cryopreservation applications is that this application is commercialized. For this reason, the creation of a cheap and easily prepared cryomedium will be one of the most important factors in the spread of the application. In addition, starting from the studies aimed at spreading the use of natural products in many areas today, non-chemical methods that have the opportunity to be standardized in sperm cryopreservation can be turned to. This allows the use of minimal chemicals in cryomedium to be emphasized. A prototype of the magnetic field that already exists in nature can be created in the laboratory with the help of a magnet, and the motility parameter values of sperm cells can be increased [87]. It has been suggested that the magnetic field may have an effect on the permeability of the sperm cell [88]. Magnetized sperm or water can be used in cryopreservation trials.

#### **2.5 Catfish sperm cryopreservation**

Catfish, which are accepted to have more than 3000 species in the world, show a rapid development among cultured species. However, due to the increase in feed and fuel prices, the production of these fish is also adversely affected. In order to increase the production of these species, hybrid species (with specialty fast growth, disease resistance, and efficient growth rate) are obtained. Since the small number of male fish is asynchronous and the killing of fish is mandatory for sperm collection, sperm cryopreservation provides an important opportunity for these species [89]. Studies have also been carried out to increase sperm motility values and fertilization capacity after cryopreservation in catfish. The rate and temperature of thawing straws after cryopreservation are also parameters that affect sperm motility. In *Ictalurus furcatus*, it is the thawing temperatures that give positive results in motility parameters of 7 and 20 s at 40°C and 40 s at 20°C when using 0.5 mL straws [89, 90]. When straws were thawed for 20 s at 20°C or 40°C, it was observed that the sperm motility results after thawing were similar [89]. In addition to the use of small volumes of straw, catfish sperm cryopreservation was performed in large volumes (1 L) bags using dairy cryopreservation technology, and this method was successfully adapted to catfish [90]. Like studies with salmonid species, egg yolk has been used for sperm cryopreservation in catfish. In *Clarias gariepinus*, 10% egg yolk prevented sperm cell

damage during cryopreservation and thawing processes. It also showed protection against adverse environmental conditions such as temperature, pH and osmotic pressure changes, and against the accumulation of harmful substances caused by the toxicity of cryoprotectant substances. Although fertilization and hatching rates can be achieved in *C. garipionus* without the use of intracellular cryoprotectant agents, this rate is low compared to the use of intracellular cryoprotectant agents such as DMSO [91]. In the extender (365–385 mOsm/kg) in which 10% egg yolk was used with glucose or NaCl in *Silurus triostegus*, cryopreservation was successful in the evaluation of motility parameters after thawing. Necrotic cells were observed in the use of glucose-containing extenders (325 mOsm/kg) with low osmotic pressure [92]. In order to develop standardized protocols for sperm cryopreservation, knowing the sperm concentration is important for the viability rate to be obtained after thawing [89]. This, in turn, can greatly improve the effectiveness of cryopreservation sperm use during the fertilization process. In most of the cryopreservation studies of catfish sperm, motility after dissolution was similar to 1 × 108 spz/mL if 1 × 109 cell/mL was used. It was observed that sperm solutions became viscous at a concentration of 1.7 × 109 spz/mL [89]. Cryopreservation; in the processes of cooling, freezing, and thawing, some biophysical and chemical events occur, such as osmotic changes, dehydration and rehydration, changes in cell volume, formation of ice crystals, and toxicity from cryoprotectant. Sperm cells, which have different characteristics specific to the species, are sensitive to these changes. Therefore, consensus should be achieved between species-specific cryopreservation protocols. An increasing number of studies explaining methods of cryopreservation of sperm in many species are proving this diversity [12]. Cryopreservation has been shown to have detrimental effects on the plasma membrane, mitochondria, chromatin structure, osmotic control, and spermatozoa motility [93, 94]. The cryopreservation process can lead to apoptosis and mitochondrial dysfunction [95], and studies have been carried out at the molecular level in recent years to determine the effect of cryoinjury [96, 97]. After thawing, sperm motility parameters in most species, including *I. furcatus*, show a decrease. This reduction in motility parameters can reduce fertilization potentials and three times higher oxidative stress level has been determined. It indicates that sperm quality may deteriorate after cryopreservation due to a 4-fold increase in the DNA fragmentation level of sperm after thawing [96]. One of the effects of cryopreservation on sperm cells is the increase of apoptotic cells [98, 99]. In order to reduce these effects, "amide" has been used in recent years as a cryoprotector for the protection of sperm [100].

Cryopreservation increases the oxidative stress level and DNA fragmentation of the sperm and thus decreases the sperm kinematic parameter values. Transcriptome analyses are also performed to determine the cryodamage caused by cryopreservation in sperm cells. In these analyses, upregulated genes were identified in sperm samples after thawing and an increase in oxidative phosphorylation activities leading to excessive production of ROS associated with cell death was detected. Despite these negative results, the presence of the potential of sperm to fertilize eggs after thawing is expressed in the fact that compensatory processes occur in the gene expression of sperm cells after thawing to offset these harmful effects (MnSOD, induction to control ROS production; correction of misfolded proteins; apoptosis, functions related to amide biosynthesis) [96]. Sperm cryopreservation can affect several biological processes, including apoptosis, spermatogenesis, mitochondrial activity, ROS production, amide biosynthesis, protein folding, and degradation [96]. Therefore, the effect of cryopreservation is quite complex, with both harmful and compensatory effects on *Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

sperm quality. In addition to the level of gene expression, cryopreservation can also affect DNA methylation, which has been identified as 1266 differentially methylated genes in sperm methyloma [97].

#### **3. Germ cell cryopreservation**

Freezing and storage of gametes is used to protect endangered species as well as to ensure sustainability in aquaculture applications. While only sperm cryopreservation is performed in fish, germ cell cryopreservation, which has the feature of differentiation to both gamete types, can be successfully performed. Although cryopreservation and long-term storage of sperm is a technique that has been practiced for a long time, it is impossible to ensure the continuity of generation by using sperm alone. Therefore, germ cell cryopreservation offers an important opportunity in achieving the above-mentioned goals [101].

Germline stem cells isolated from immature gametes can be intraperitoneal transplantation of immunologically immature newly hatched larvae into the body cavity [102]. In addition to the larvae, germline stem cells can be transferred to broodstock fish and embryos [103]. Germline stem cells, transferred to the larvae, migrate to the genital ridge, multiply and initiate spermatogenesis or oogenesis. Cryopreservation of germ cells is performed for transplantation. For this purpose, in cryopreservation germ cells; slow freezing and vitrification methods are used. After transplantation of germ cells, frozen by both methods, to the larvae, there is no significant difference in the rate of migration to the genital ridge and their reproduction compared to the control group [104]. In addition to cryopreservation of all testis tissue, germ cell isolation can be performed from immature gonads after dissection of fish. Freezing of all testicular tissue; while it is made using immature gonads that are frozen in a freezer and stored without the help of exogenous cryoprotectant [105], it can also be done using cryoprotectant [106].

Germ cell isolation in fish can be performed from all cryopreserved testicular tissue as well as from immature gonads. The differentiation of spermatogonia to the ova after the transfer of spermatogonia isolated from cryopreservation testis tissue to the female recipient fish provides a definitive solution to continuity of the species. As a result of isolation from rainbow trout testis tissue in the absence of cryopreservation and the presence of dead fish, transplantation efficiency was found to be 90.61 ± 5.26%, 82.22% ± 11.76%, 73.33% ± 3.33%, and 6.68 ± 6.66%, respectively [107].

In the cryopreservation of oogonia; DMSO, methanol, glycerol, ethylene glycol, and egg yolk are used as cryoprotectants. Between these, the use of DMSO, which is a cryoprotectant that has the ability to penetrate into the cell, gives the best rate on both motility and fertilization rate. Although egg yolk does not have penetrating properties, success has been observed in its use with lactose [108]. DMSO; has been identified as the cryoprotectant substance with the most successful results for cryopreservation of spermatogonial stem cells of *Oncorhynchus mykiss*, type A spermatogonia of *I. furcatus* [109], ovary of *C. carpio* [110], and oogonia [108].

In sperm cryopreservation, evaluation of motility parameters without determining the post-thawing observation and hatching rate will be incomplete in terms of determining the success of the experiment. In addition, the development of germ cell cryopreservation procedures without germ cell transplantation does not yield results. Since spermatogonia/oogonia has sexual plasticity (the ability to produce both eggs and sperm), these mitotic germ cells can be stored by freezing. This application will

be an alternative to sperm cryopreservation as well as freezing and storing fish eggs or embryos [111]. Reducing the complicated steps in germ cell cryopreservation as much as possible (reducing the use of chemicals, applying some of the experimental stages in fish farms, and easy transferability of samples) will accelerate the conservation of species, which is an urgent global problem.

#### **4. Cryopreservation of fish embryo**

It is an important issue to use gametes obtained by aquaculture to support natural stocks and, to make cryopreservation techniques that provide long-term protection of these gametes available in all species and reproductive cells of species. Sperm cells, due to its small size and greater durability during freezing, have given more successful results than other reproductive cells, and these features have made sperm the most researched cell in the cryopreservation of fish gamete. Freezing sperm cells is successfully practiced in many fish species, and a protocol has been established for almost cultured fish [38]. However, still, egg freezing has not been successful because of its features such as dehydration problems, large volume, and different membrane permeability. For this reason, many studies have focused on freezing fish oocytes and ovarian follicle. The reasons for the increase in oocytes and ovarian follicle cryopreservation studies are that these cells have a small volume, high membrane permeability, membrane systems are simpler and less sensitive to freezing [112, 113].

The cryopreservation of embryo, which allows the storage of both the female and male genomes, has been a challenging subject of cryopreservation studies for many years. In terms of aquaculture, successful fish embryo cryopreservation will significantly facilitate the establishment and management of genetic selection programs in fish farms [38]. Fish embryos have a low surface-to-volume ratio. It also has a large volume of yolk and a low rate of membrane permeability. In addition to these features of fish embryos, their high sensitivity to low temperatures has made it difficult to use and achieve success in cryopreservation studies [113, 114]. However, despite all these difficulties, there are studies on embryo freezing in fish. Studies have been carried out on chilling and cryopreservation of embryos in 20 different teleost fish [115–117]. The first study on cryopreservation fish embryos (slow-freezing method) was tried and successfully recorded in carp fish embryos in 1989 [118], however, a complete standard has not been established yet.

One of the important issues in the cryopreservation of fish embryos is the toxicity and penetration of the cryoprotectant to be used. In addition to the freezing procedure in the cryopreservation of fish embryos, there are also studies on the selection of cryoprotectants to be used [38]. In an embryo-freezing study with zebrafish, known as model fish, methanol was found to be more effective cryoprotectant compared to DMSO and Ethanediol [119]. In another embryo freezing study using vitrification in zebrafish, it was reported that the embryos survived for 3 h after thawing [120]. In Ref. [121], it was observed that continuity in the development seen only 2.96% seven-band grouper (*Epinephelus septemfasciatus*) embryos, postvitrification. In a recent review study on fish embryo freezing, the issues that need to be developed in relation to the vitrification method that is widely used and tried in embryo freezing are systematically given. In this review, the issues that need to be considered for the development of vitrification protocol are listed as cryoprotectant toxicity, developmental stage of the embryo, and the conditions at the time the embryo to be frozen is treated with cryoprotectant and vitrification [122]. Most of the studies focused

*Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

on fish embryo vitrification within the area of toxicity of vitrification solutions. In 2006, it was reported that Japanese flounder (*Paralichthys olivaceus*) embryos were successfully cryopreserved by using vitrification method [123]. Fish embryos show different sensitivity to cryoprotectant permeability at each developmental stage. With this feature that embryos have, some studies have been conducted using various storage methods to determine cryoprotectant flux or concentration at different stages during embryo development [122]. In study conducted on carp, it was reported that the hatching rate was 41% if sucrose was used to protect carp embryos in −4°C. In the same study, it was reported that the use of sucrose and methanol together gave the best results [124]. Recently, in the embryo freezing study, where two different cryoprotectants (DMSO and Methanol) were tested in carp, it was stated that the use of two cryoprotectant substances together had a protective effect by keeping the embryos at −2°C for 1 day and the average larval survival rate was 12.38% [125]. Another study showed that 9.7% of the embryos continued their lives for 2 h by freezing the embryos in liquid nitrogen (−196°C) in *Epinephelus moara* [126]. In a study with carp, using Modified Haga's solution; the toxicity of DMSO and glycerol and its effect on the survival rate of embryos were examined. In the same study where the effect of embryo freezing on the survival rate of the application of different developmental stages was investigated, it was stated that carp embryos were successfully frozen (−196°C) [127].

In cryopreservation of fish embryo studies, the highest success achieved so far has been seen in Persian sturgeon (*A. persicus*) with a hatching rate of 45.45%. In this study, where the vitrification technique was applied, DMSO was selected as a cryoprotectant [128]. Another study whose results were successfully stated was the one which propylene glycol was injected into the zebrafish embryos in freezing and the samples were frozen in liquid nitrogen with a rate of 90,000°C/min. In this study, the survival rate of the thawing embryos was found to be 10% after 24 h post-thawing [129].

Despite all the studies and efforts made on the freezing of fish embryos, unlike fish sperms and germ cells, the work done to prevent crystallization and biological damage during freezing/thawing of fish embryos is still a challenging topic. However, recent studies with cryopreservation primordial germ cells look promising when storing both female and male genomes. Such studies can be used in cryopreservation studies until an undisputed result is obtained in terms of embryo freezing in fish and serve the purpose of cryopreservation.

#### **5. Prevention of disease transmission**

#### **5.1 Contamination during the fish gamete cryopreservation and thawing process**

The cryopreservation method is one of the assisted reproductive techniques (ART) and has been in use for many years to serve needs such as infertility treatment or genetic improvement and preservation or transportation in living things. Performing this method under aseptic conditions is one of the important factors in the preservation of gametes without microbial contamination and resulting in successful fertilization [130, 131].

The main sources of contamination encountered in cryopreservation studies are (1) Dewar, (2) Cell, (3) Liquid Nitrogen (LN), and (4) Handling (extender, supply, etc.) [131–134]. When the LN container is opened, the upper part begins to cool, and the water in the air turns into ice crystals with a high electrostatic charge, catching the microorganisms in the air. It is known that these ice crystals fall into the LN container and cause the accumulation of microorganisms in masses at the bottom and can combine with cell residues in the environment. In a study conducted on LN container that was used continuously for 7 and 12 years, it was shown that the contamination intensity did not depend on the year in use, but the microbial diversity was different in each of them [134].

Bacteria are single-celled organisms that have been cryopreserved for extensive research since 1950 [135]. Bacteria are not affected by cold when stored in suitable conditions specific to the species and can reproduce when thawed. For long-term storage at −20°C causes the death of perishable bacteria [136], while bacteria are usually stored in a −80°C freezer with little loss, longer and more appropriate storage is provided with liquid nitrogen and vapor phase (140–196°C) [137]. Cryopreservation is also used for other microorganisms such as viruses and fungi for research purposes [138]. In gamete cryopreservation studies, cryoprotectant agents are added to the extender to protect the cell from freezing. The cryoprotectants and nutritional elements used to store microorganisms at −196°C are very similar to the solutions used to store sperm cells. Therefore, while the gamete cells are frozen, microorganisms can be frozen together with the cells unintentionally [139–141].

Also, infected gamete samples can contaminate other pathogen-free samples when stored in the same LN container. Especially in human sperm storage conditions, cross-contamination poses a major problem. Storing the sperm of an individual carrying the disease and the sperm samples of an individual who is not diseased in the same LN container causes a healthy parent to have a diseased child. It has been shown in studies that liquid nitrogen can contaminate the samples to be placed in contaminated with bacteria and viruses [142].

As we mentioned above, during the transfer of LN used in cryopreservation, microorganisms present in atmospheric air can contaminate liquid nitrogen [134, 143]. Although the fact that many microorganisms in the air are not fish pathogens does not seem to be an important problem in terms of disease transmission, the presence of bacteria in the environment where sperm is present is an important factor that impairs its quality [144]. In other words, microorganisms use the nutrients and oxygen in the environment, which are necessary for the survival of the sperm, as well as changing the pH of the environment [8], and this affects the quality of the sperm and changes its fertilization capacity [145]. In addition, the most important problem will be the use of gamete cells contaminated with the microorganism in fertilization, the transmission of pathogens to the embryo, and the emergence of diseased offspring [146].

#### **5.2 Disease transmission via gametes to embryo**

Research that has been conducted on sperm and embryo has found a relationship with pathogens such as viral, bacterial, fungal, and parasitic organisms [147]. It has been reported that some of them are in the seminal plasma, attach to the sperm cells, but do not enter the cell, and attach to the egg from the zona pellucida [142, 148].

To summarize briefly, the literature on vertical transmission of fish pathogens; It has been proved that the infected pancreatic necrosis (IPN) virus is present in the ovarian fluid and can be attached to the salmonid sperm and transmitted vertically to the embryo [149–152]. Viral hemorrhagic septicemia virus (VHSV) and Infectious hematopoietic necrosis virus (IHNV) have been isolated from the seminal and ovarian fluid of salmonid species [153, 154]. In addition, some bacteria have been shown by staining sperm samples, and *Aeromonas* sp., *Pseudomonas*

*Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

sp., and *Flavobacterium* spp. have been detected in the seminal plasma of trout using culture methods [144, 155, 156]. *Renibacterium salmoninarum* is bind to tail of sperm and has been found intra-ovum of rainbow trout [146]. In a study showing that *Piscirickettsia salmonis* can be found in the seminal fluid of trout, it was shown that vertical transmission is also possible [157].

#### **5.3 Eliminate non-cellular disease agents from gametes**

It has long been known that semen can contain a variety of viruses, and good safety protection systems and methods have been established in many laboratories to reduce virus particles from semen obtained from humans with viral diseases [158]. Viral Infection is recognized as a possible cause of male infertility in humans [159]. Among HIV-infected men, vertical transmission to infants has been reported when in vitro fertilization is performed after sperm washing [160].

However, as far as we know, there is no study on the inhibition of the transmission of viruses from sperm to embryo in fish. It is known as general information that the viral titer decreases after the freezing–thawing process [146], so the viral load in the sperm can be lowered by cryopreservation.

#### **5.4 Antibiotics**

Although antibiotics are used to eliminate bacterial contamination in sperm freezing, it should also be considered that they are harmful to sperm [8, 161]. Although there are studies in which antimicrobial biomolecules (resveratrol, curcumin, etc.) are used instead of antibiotics, these have not yet taken the place of antibiotics [162].

#### **5.5 Sperm washing and cell separating methods**

Density gradient centrifuge method is a method that allows cells to be separated according to their size. Although this method is frequently used for sperm washing in mammals, that is, for removing microorganisms, its applications in fish are generally based on separating quality sperm cells from all other foreign substances [163]. Although the "swim-up" method, which is one of the sperm washing methods, is also used in mammalian sperm cells, this method does not work in fish since motile sperm must be used in this method [155].

During the process of sperm washing, freezing, and thawing, in order to obtain a high concentration of quality spermatozoa, it is critical that we reduce and remove any risk or chance of pathogen contamination during the preservation of frozen sperm in tanks. While sperm washing is usually performed before cryopreservation of mammalian sperm [164], in sperm washing studies applied in fish, since 2010, the washing process is performed after thawing [165–167]. In some species, it is used to separate seminal plasma from cells by centrifugation before freezing [168]. In the study in Ref. [168], firstly, seminal plasma was separated by centrifugation, and then live sperm cells were separated from the dead by magnetic selective. As a result of this study, it was possible to obtain better quality and functional sperm.

Studies using sperm washing method in fish have increased, especially in recent years. In the first study on this subject, in which frozen sperm from carp were used, it was determined that the sperm washed using the percoll gradient method had high motility, and the spermatozoa, which were immobile and whose membrane was damaged during the freezing–thawing process, were also removed. As a result, it is stated

that the use of this technique will allow easier and higher quality spermatozoa to be obtained compared to other biotechnological cell separation methods [169]. Another study conducted in sturgeon includes the evaluation of sperm motility parameters and sperm characteristics after gradient centrifugation method of control and frozen sperm samples [165]. In a freeze–thaw washing study performed with testicular sperm, the fertilization rate was found to be higher than the control [170]. In the only study in which sperm washing was used to remove bacterial load, it was observed that gradient centrifugation method reduced bacterial load, although it caused a decrease in motility rates in rainbow trout sperm [155]. Again, according to the results of the study by the same authors on the relationship between newly completed sperm washing and fertilization success, low motility after washing did not significantly change the fertilization success and survival rate (unpublished data).

In studies using the density gradient centrifugation method, percoll was the most widely used gradient-forming chemical [169, 170]. Sil-Select Plus TM [155] and AllGrad® 90% [167], which are commercial solutions produced in humans, have been tried in fish and successful results have been obtained.

#### **5.6 Disinfection of gametes and embryo**

When the sperm are treated with a disinfectant, the sperm cell loses its vitality. However, iodine and anti-fungal treatment of eggs with formaldehyde is routinely and repeatedly used during the eyed stage of salmonids [146, 171]. Disinfection of eggs with iodophor prevents some viruses (VHSV, etc.) and bacteria (*Flavobacterium psychrophilum*, *Yersinia ruckeri*, etc.) from passing into the embryo, while it cannot provide protection for others (IPNV, *R. salmoninarum*, etc.) [146]. According to the literature, no studies were found regarding the presence or solution of contamination in frozen embryos or eggs (perhaps because embryo and egg cryopreservation is a new subject).

#### **5.7 Suggestions**

In cryopreservation facilities, it has been reported that the microbial load should be controlled before freezing sperm in order to prevent cross-contamination in liquid nitrogen [172]. Freezing sperm from brood stocks that are not infected or have the least pathogen in their sperm will be the best solution. This can be possible with continuous health checks and monitoring of fish. The use of vaccinated brood stocks to prevent the spread of the disease with high mortality would be excellent for sustainability. Sperm cells of brood stocks infected with a possible pathogen can be separated from the pathogen with the sperm washing method and then frozen. This is also promising technique in obtaining quality sperm. In addition, continuous disinfection and use of tanks, UV sterilization of liquid nitrogen used in small volumes, sterilization of all materials used, and sealing the ends of the straws by burning will prevent possible contamination [133].

Establishing special sperm collection laboratories for cryopreservation would also be a good solution to prevent contamination. The use of a common laboratory with professional staff, where special Biosafety measures are taken and hygiene rules are followed, will be beneficial for every fish farm [131]. There are cryobanks where gametes are stored in different countries, but since the number is not sufficient, it would be beneficial to create an easily accessible cryobank for each country [38].

#### *Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

Cryostorage facilities are expensive and may not be affordable or readily available due to their constant need for electrical power and liquid nitrogen production [173]. Additionally, monitoring liquid nitrogen levels is a time-consuming and expensive process that requires constant visual inspection. Therefore, cryostorage systems have recently been said to be prone to failures leading to undesired sample losses [174]. Liquid nitrogen is seen as a pathogenic transmissible agent, and its production is also a high carbon footprint procedure [173]. The most convenient way to prevent LN contamination is to use filtered (sterile) LN. However, this application is not suitable for use in practice due to its high cost. It is recommended to find a cheaper, simpler, greener alternative [173]. Another alternative is to use ultra-low freezers at −150°C, which has increased production today, instead of liquid nitrogen tanks, and it is thought that the risk of nitrogen vapor contamination can be reduced [175]. And even in a recent study in zebra fish, an ultrafreezer of −150°C was used for sperm cryopreservation and successful result was achieved [176].

It has also been reported that freeze-dry technology can eliminate the difficulties and contamination risk of cryo-storage using liquid nitrogen in today's technology [173, 174]. In fact, some mammalian spermatozoa have been studied with this method [173, 174, 177]. In these studies, sperm motility is not important and efforts are made to prevent DNA damage of sperm. Therefore, this method can be used for large mammals in the intracytoplasmic sperm injection (ICSI) method, but it does not seem possible to apply it to animals with multiple eggs such as fish [178]. In the literature, there has not yet been a study in which this technology is used in fish species.

Although the OIE has a "Health code" for terrestrial animals on the prevention of the risk of transmission of cryopreserved sperm-borne disease, there is no such information in the "Aquatic Animal Health Code" [171]. In the future, it will be useful to come up with standard methods for aquatic organisms.

#### **6. Conclusions**

Cryopreservation applications are one of the important assisted reproductive techniques that can be used to maintain the vitality of an organism. When the subject is to ensure the sustainability of a living thing, external factors such as its own biological, physiological, genetic, and epigenetic characteristics as well as its feeding regime, living environment, and presence of pollutants are also effective. For this reason, standardization of all these differences while conducting research is very difficult (perhaps impossible with current practices) and requires long time-consuming studies. However, in order to eliminate the negative effects of cryopreservation on the cell, the standardization of the factors mentioned above, as well as the laboratory environment, ensures the quality of the cells to be obtained. In reproductive biotechnology studies, laboratory studies should be carried out under sterile conditions after obtaining eggs and sperm from the fish broodstock.

Ensuring the importance shown in aseptic laboratory environment also allows the cells obtained to increase the viability rate of the offspring, as well as to be healthy. Fish farms carrying out these practices should be certified and sample entry from noncertified farms should not be allowed. A living organism is in constant contact with its environment and is constantly under this influence. Breeding and fish health practices are subjects that cannot be considered independently of each other. In this context, if it is desired to ensure the continuity of high quality, fast growing, and healthy generations, it is not possible for these two fields to be independent from each other.

#### *Cryopreservation - Applications and Challenges*

Although the concern and necessity of this issue have been stated in the studies, the use of sterile environments has not been actively provided yet. Informing the people working in fish production farms on this subject and organizing meetings/ seminars that emphasize the importance of the subject and organizing trainings/ courses would be beneficial. In addition, these practices may be made compulsory by various regulations.

### **Author details**

Aygül Ekici\*, Güneş Yamaner and Menekşe Didem Demircan İstanbul University, Faculty of Aquatic Sciences, Department of Aquaculture and Fish Diseases, İstanbul, Turkey

\*Address all correspondence to: aekici@istanbul.edu.tr

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

#### **References**

[1] Carolsfeld J, Harvey B, Godinho HP, Zaniboni-Filho E. Cryopreservation of sperm in Brazilian migratory fish conservation. Journal of Fish Biology. 2003;**63**:472-481. DOI: 10.1046/j.1095-8649.2003.00170.x

[2] Hu E, Tiersch T.R. Development of high-throughput cryopreservation for aquatic species. In: Tiersch T.R, Green C.C, editors. Cryopreservation in Aquatic Species. 2nd ed. World Aquaculture Society, Baton Rouge, LO; 2011. p. 995-1003

[3] Blaxter JHS. Sperm storage and cross-fertilization of spring and autumn spawning herring. Nature. 1953;**172**:1189- 1190. DOI: 10.1038/1721189b0

[4] Labbé C, Robles V, Herráez MP. Cryopreservation of gametes for aquaculture and alternative cell sources for genome preservation. In: Allan G, Burnell G, editors. Advances in Aquaculture Hatchery Technology. Woodhead Publishing; Sawston, Cambridge, UK. 2013. pp. 76-116. DOI: 10.1533/9780857097460.1.76

[5] Cabrita E, Robles V, Herráez M.P, editors. Methods in Reproductive Aquaculture: Marine and Freshwater Species. CRC Press, Boca Raton, FL, USA 2008. 549 p. ISBN: 13: 978-0-8493-8053-2

[6] Tiersch TR. Process pathways for sperm cryopreservation research, application, and commercialization. In: Tiersch TR, Green C, editors. Cryopreservation in Aquatic Species. 2nd ed. Baton Rouge, LO: World Aquaculture Society; 2011. pp. 646-671

[7] Kopeika E, Kopeika J, Zhang T. Cryopreservation of fish sperm. Methods in Molecular Biology. 2007;**368**:203-217. DOI: 10.1007/978-1-59745-362-2\_14

[8] Jawahar KTP, Betsy J. Cryopreservation of fish gametes: An overview. In: Betsy J, Kumar S, editors. Cryopreservation of Fish Gametes; Springer. 2020. pp. 151-175. DOI: 10.1007/978-981-15-4025-7

[9] Judycka S, Żarski D, Dietrich M.A, Palińska-Żarska K, Karol H, Ciereszko A. Standardized cryopreservation protocol of European perch (*Perca fluviatilis*) semen allows to obtain high fertilization rates with the use of frozen/thawed semen. Aquaculture2019;498:208-216. DOI: 10.1016/j.aquaculture.2018.08.059

[10] www.cryogenetics.com [Accessed: September 7, 2022]

[11] www.imv-technologies.com [Accessed: September 7, 2022]

[12] Asturiano JF, Cabrita E, Horváth Á. Progress, challenges and perspectives on fish gamete cryopreservation: A mini-review. General and Comparative Endocrinology. 2017;**245**:69-76. DOI: 10.1016/j.ygcen.2016.06.019

[13] The State of World Fisheries and Aquaculture.2022. Available from: https://www.fao.org/fishery/en/ culturedspecies/cyprinus\_carpio/en [Accessed: September 6, 2022]

[14] Balon EK. Origin and domestication of the wild carp, *Cyprinus carpio*: From Roman gourmets to the swimming flowers. Aquaculture. 1995;**129**:3-48. DOI: 10.1016/0044-8486(94)00227-F

[15] Lubzens E, Rothbard S, Hadani A. Cryopreservation and viability of spermatozoa from the ornamental Japanese carp. Israeli Journal of Aquaculture. 1993;**45**:169-174

[16] Gwo JC, Kurokura H, Hirano R. Cryopreservation of spermatozoa from rainbow trout, common carp and marine puffer. Bulletin of the Japanese Society of Scientific Fisheries. 1993;**59**:777-782. DOI: 10.2331/suisan.59.777

[17] Linhart O, Rodina M, Cosson J. Cryopreservation of sperm common carp *Cyprinus carpio*: Sperm motility and hatching success of embryos. Cryobiology. 2000;**2000**(41):241-250. DOI: 10.1006/cryo.2000.2284

[18] Horváth Á, Miskolczi E, Urbányi B. Cryopreservation of common carp sperm. Aquatic Living Resources. 2003;**16**:457-460. DOI: 10.1016/ S0990-7440(03)00084-6

[19] Warnecke D, Pluta HJ. Motility and fertilizing capacity of frozen/thawed common carp (*Cyprinus carpio* L.) sperm using dimethyl-acetamide as the main cryoprotectant. Aquaculture. 2003;**215**:167-185. DOI: 10.1016/ S0044-8486(02)00371-X

[20] Havlar E, Bozkurt Y. Protective effects of different egg yolk sources on cryopreservation of scaly carp (*Cyprinus carpio*) sperm. Acta Aquatica Turcica. 2022;**18**(3):393-402. DOI: 10.22392/ actaquatr.1085283

[21] Cabrita E, Ma S, Diogo P, Martínez-Páramo S, Sarasquete C, Dinis MT. The influence of certain amino acids and vitamins on post-thaw fish sperm motility, viability and DNA fragmentation. Animal Reproduction Science. 2011;**125**:189-195. DOI: 10.1016/j.anireprosci.2011.03.003

[22] Ekici A, Baran A, Yamaner G, Özdas ÖB, Sandal AI, Güven E. Effects of different doses of taurine in the glucosebased extender during cryopreservation of rainbow trout (*Oncorhynchus mykiss*) semen. Biotechnology & Biotechnological Equipment.

2012;**26**:3113-3115. DOI: 10.5504/ BBEQ.2012.0041

[23] Rani KU, Munuswamy N. Effect of DNA damage caused by cryopreservation of spermatozoa using a modified single cell gel electrophoresis in the freshwater catfish *Pangasianodon hypophthalmus* (Fowler, 1936). Journal of Coastal Life Medicine. 2014;**2**:515-519. DOI: 10.12980/ JCLM.2.2014J15

[24] Valdebenito I, Moreno C, Lozano C, Ubilla A. Effect of L-glutamate and glycine incorporated in activation media, on sperm motility and fertilization rate of rainbow trout (*Oncorhynchus mykiss*) spermatozoa. Journal of Applied Ichthyology. 2010;**26**:702-706. DOI: 10.1111/j.1439-0426.2010.01555.x

[25] Kledmanee K, Taweedet S, Thaijongruk P, Chanapiwat P, Kaeoket K. Effect of L-cysteine on chilled carp (*Cyprinus carpio*) semen qualities. The Thai Journal of Veterinary Medicine. 2013;**43**:91-97

[26] Öğretmen F, İnanan BE, Kutluyer F, Kayim M. Effect of semen extender supplementation with cysteine on postthaw sperm quality, DNA damage, and fertilizing ability in the common carp (*Cyprinus carpio*). Theriogenology. 2015;**83**(9):1548-1552. DOI: 10.1016/j. theriogenology.2015.02.001

[27] Yıldız C, Yavaş I, Bozkurt Y, Aksoy M. Effect of cholesterol-loaded cyclodextrin on cryosurvival and fertility of cryopreserved carp (*Cyprinus carpi*o) sperm. Cryobiology. 2015;**70**(2):90-194. DOI: 10.1016/j.cryobiol.2015.01.009

[28] Horvath A, Bokor Z, Bernath G, Csenki Z, Gorjan A, Herraez MP, et al. Very low sperm–egg ratios result in successful fertilization using cryopreserved sperm in the Adriatic grayling (*Thymallus thymallus*).

*Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

Aquaculture. 2015;**435**:75-77. DOI: 10.1016/j.aquaculture.2014.09.032

[29] Molnár J, Bokor Z, Várkonyi L, Izsák T, Füzes-Solymosi E, Láng ZL, et al. The systematic development and optimization of large-scale sperm cryopreservation in northern pike (*Esox lucius*). Cryobiology. 2020;**94**:26-31. DOI: 10.1016/j.cryobiol.2020.05.003

[30] Judycka S, Dietrich MA, Żarski D, Karol H, Palińska-Żarska K, Błażejewski M, et al. Towards standardization of the cryopreservation procedure of cultured pikeperch (*Sander lucioperca*) semen. Aquaculture. 2021;**538**:736-539. DOI: 10.1016/j. aquaculture.2021.736539

[31] Judycka S, Nynca J, Liszewska E, Dobosz S, Grudniewska J, Ciereszko A. Optimal sperm concentration in straws and final glucose concentration in extender are crucial for improving the cryopreservation protocol of salmonid spermatozoa. Aquaculture. 2018;**486**:90-97. DOI: 10.1016/j. aquaculture.2017.12.019

[32] Pataki B, Horváth Á, Mészáros G, Kitanović N, Ács A, Hegyi Á, et al. Adjustment of common carp sperm concentration prior to cryopreservation: Does it matter? Aquaculture Reports. 2022;**24**:101-109. DOI: 10.1016/j. aqrep.2022.101109

[33] Irawan H, Vuthiphandchai V, Nimrat S. The effect of extenders, cryoprotectants and cryopreservation methods on common carp (*Cyprinus carpio*) sperm. Animal Reproduction Science. 2010;**122**(3-4):236-243. DOI: 10.1016/j.anireprosci.2010.08.017

[34] Chebanov MS, Galich EV. Sturgeon hatchery manual. FAO Fisheries and Aquaculture Technical Paper. No. 558. Ankara, FAO. 2011; pp 303

[35] Scarnecchia DL, Lim Y, Ryckman LF, Backes KM, Miller SE, Gangl RS, et al. Virtual population analysis, episodic recruitment, and harvest management of Paddlefish with applications to other Acipenseriform fishes. Reviews in Fisheries Science & Aquaculture. 2014;**221**:16-35. DOI: 10.1080/ 10641262.2013.830592

[36] Wu H, Chen J, Xu J, Zeng G, Sang L, Liu Q, et al. Effects of dam construction on biodiversity: A review. Journal of Cleaner Production. 2019;**221**:480-489. DOI: 10.1016/j.jclepro.2019.03.001

[37] Bronzi P, Rosenthal H. Present and future sturgeon and caviar production and marketing: A global market overview. Journal of Applied Ichthyology. 2014;**30**(6):1536-1546. DOI: 10.1111/ jai.12628

[38] Martínez-Páramo S, Horváth Á, Labbé C, Zhang T, Robles V, Herráez P, et al. Cryobanking of aquatic species. Aquaculture. 2017;**472**:156-177. DOI: 10.1016/j.aquaculture.2016.05.042

[39] Glogowski J, Kolman R, Szczepkowski M, Horváth Á, Urbányi B, Sieczyński P, et al. Fertilization rate of Siberian sturgeon (*Acipenser baeri*, Brandt) milt cryopreserved with methanol. Aquaculture. 2002;**211**(1-4):367-373. DOI: 10.1016/ S0044-8486(02)00003-0

[40] Horváth Á, Wayman WR, Urbányi B, Ware KM, Dean JC, Tiersch TR. The relationship of the cryoprotectants methanol and dimethyl sulfoxide and hyperosmotic extenders on sperm cryopreservation of two North-American sturgeon species. Aquaculture. 2005;**247**(1-4):243-251. DOI: 10.1016/j. aquaculture.2005.02.007

[41] Horváth Á, Wayman WR, Dean JC, Urbányi B, Tiersch TR, Mims SD, et al.

Viability and fertilizing capacity of cryopreserved sperm from three North American acipenseriform species: A retrospective study. Journal of Applied Ichthyology. 2008;**24**(4):443-449. DOI: 10.1111/j.1439-0426.2008.01134.x

[42] Huang X, Zhang T, Zhao F, Feng G, Liu J, Yang G, et al. Effects of cryopreservation on acrosin activity and DNA damage of Russian sturgeon (*Acipenser gueldenstaedtii*) semen. CryoLetters. 2021;**42**(3):129-136

[43] Tiersch TR, Green CC, editors. Cryopreservation in Aquatic Species. 2nd ed. Baton Rouge, LO: World Aquaculture Society; 2011. pp. 1-17

[44] Golshahi K, Aramli MS, Nazari RM, Habibi E. Disaccharide supplementation of extenders is an effective means of improving the cryopreservation of semen in sturgeon. Aquaculture. 2018;**486**:261-265. DOI: 10.1016/j. aquaculture.2017.12.045

[45] Malo C, Gil L, Gonzalez N, Cano R, de Blas I, Espinosa E. Comparing sugar type supplementation for cryopreservation of boar semen in egg yolk based extender. Cryobiology. 2010;**61**:17-21. DOI: 10.1016/j. cryobiol.2010.03.008

[46] Gómez-Fernández J, Gómez-Izquierdo E, Tomás C, Mocé E, de Mercado E. Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation. Animal Reproduction Science. 2012;**133**(1-2):109-116. DOI: 10.1016/j.anireprosci.2012.06.010

[47] Xi MD, Li P, Du H, Qiao XM, Liu ZG, Wei QW. Disaccharide combinations and the expression of enolase3 and plasma membrane Ca2+ ATPase isoform in sturgeon sperm cryopreservation. Reproduction in Domestic Animals.

2018;**53**(2):472-483. DOI: 10.1111/ rda.13134

[48] Ubilla A, Valdebenito I. Use of antioxidants on rainbow trout *Oncorhynchus mykis*s (Walbaum, 1792) sperm diluent: Effects on motility and fertilizing capability. Latin American Journal of Aquatic Research. 2011;**39**(2):338-343. DOI: 10.3856/vol39-issue2-fulltext-1

[49] Li P, Xi M.D, Du H, Qiao X.M, Liu Z.G, Wei Q.W. Antioxidant supplementation, effect on post-thaw spermatozoan function in three sturgeon species. Reproduction in Domestic Animals 2018;53:2:287-295. DOI:10.1111/ rda.13103

[50] Figueroa E, Lee-Estevez M, Valdebenito I, Watanabe I, Oliveira RPS, Romero J. Effects of cryopreservation on mitochondrial function and sperm quality in fish. Aquaculture. 2019;**511**:634190. DOI: 10.1016/j. aquaculture.2019.06.004

[51] Dadras H, Golpour A, Rahi D, Lieskovská J, Dzyuba V, Gazo I, et al. Cryopreservation of Sterlet, *Acipenser ruthenus* spermatozoa: Evaluation of quality parameters and fine ultrastructure. Frontiers in Marine Science. 2022;**78**(32):78. DOI: 10.3389/ fmars.2022.783278

[52] Boryshpolets S, Dzyuba B, Rodina M, Alavi SMH, Gela D, Linhart O. Cryopreservation of Sterlet (*Acipenser ruthenus*) Spermatozoa Using Different Cryoprotectants. Journal of Applied Ichthyology. 2011;**27**:1147-1149. DOI: 10.1111/j.1439-0426.2011.01866.x

[53] Igna V, Telea A, Florea T, Popp R, Grozea A. Evaluation of Russian sturgeon (*Acipenser gueldenstaedtii*) semen quality and semen cryopreservation. Animals. 2022;**12**(16):2153. DOI: 10.3390/ ani12162153

*Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

[54] Nascimento J.P, Horokhovatskyi Y, Kholodnyy V, Rodina M, Dzyuba V, Stechkina T, Dzyuba B. Optimization of sterlet sperm concentration for cryopreservation. Aquaculture2021;540:736682. DOI:10.1016/j.aquaculture.2021.736682.

[55] Krasilnikova A, Ponomareva E, Shvedova S, Magomedov M, Rudoy D, Olshevskaya A. The volume of the sample as a factor of survival of sturgeon spermatozoa after cryopreservation. E3S Web of Conferences. 2020;**210**:07010. DOI: 10.1051/e3sconf/202021007010

[56] Ponomareva EN, Belaya MM, Firsova AV, Krasilnikova AA. Influence of acoustic-mechanical impact on the reproductive qualities of sturgeon sperm during cryopreservation. Doklady. Biochemistry and Biophysics. 2022;**505**(1):170-172. DOI: 10.1134/ S1607672922040081

[57] Kolyada M, Osipova V, Berberova N, Pimenov Y. The effect of tin compounds on the lipid peroxidation level of Russian sturgeon fresh and cryopreserved sperm. Environmental Research, Engineering and Management. 2020;**76**(2):34-42. DOI: 10.5755/j01.erem.76.2.23407

[58] Eels IWGo, Report of the 2011 session of the joint EIFAAC/ICES working group on eels. 2011:24

[59] Jacoby D, Gollock, M. Anguilla japonica. The IUCN Red list of threatened species. 2014:Version 2014.3.a

[60] Jacoby D, Gollock, M. Anguilla anguilla. The IUCN red list of threatened species, 2014.Version 2014.3.b

[61] Tanaka S, Zhang H, Horie N, Yamada Y, Okamura A, Utoh T, et al. Long-term cryopreservation of sperm of Japanese eel. Journal of Fish Biology. 2002;**60**(1):139-146. DOI: 10.1111/j.1095- 8649.2002.tb02393.x

[62] Herranz-Jusdado JG, Gallego V, Morini M, Rozenfeld C, Pérez L, Müller T, et al. Eel sperm cryopreservation: An overview. Theriogenology. 2019;**133**: 210215. DOI: 10.1016/j.theriogenology. 019.03.033a

[63] Herranz-Jusdado JG, Gallego V, Marina M, Rozenfeld C, Pérez L, Kása E, et al. Comparison of European eel sperm cryopreservation protocols with standardization as a target. Aquaculture. 2019;**498**:539-544. DOI: 10.1016/j. aquaculture.2018.09.006b

[64] Herranz-Jusdado JG, Gallego V, Rozenfeld C, Marína M, Pérez L, Asturiano JF. European eel sperm storage: Optimization of short-term protocols and cryopreservation of large volumes. Aquaculture. 2019;**506**:42-50. DOI: 10.1016/j.aquaculture.2019.03.019c

[65] Müller T, Matsubara H, Kubara Y, Horváth Á, Asturiano JF, Urbányi B. Japanese eel (*Anguilla japonica* Temminck & Schlegel, 1846) propagation using cryopreserved sperm samples. Journal of Applied Ichthyology. 2017;**33**(3):550-552. DOI: 10.1111/jai.13316

[66] ICC K, Hamada D, Tsuji YA, Okuda D, Nomura K, Tanaka H, et al. Sperm cryopreservation of Japanese eel, *Anguilla japonica*. Aquaculture. 2017;**473**:487-492. DOI: 10.1016/j. aquaculture.2017.03.011

[67] Nomura K, ICC K, Iio R, Okuda D, Kazeto Y, Tanaka H, et al. Sperm cryopreservation protocols for the large-scale fertilization of Japanese eel using a combination of largevolume straws and low sperm dilution ratio. Aquaculture. 2018;**496**:203-210. DOI: 10.1016/j.aquaculture.2018.07.007 [68] FAO. The State of World Fisheries and Aquaculture 2020. Sustainability in Action. Italy: Food and Agriculture Organization of the United Nations; Rome, Italy. 2020. p. 244 Available from: www.fao.org [Accessed: 2022-09-07]

[69] Huang M, Ding L, Wang J, Ding C, Tao J. The impacts of climate change on fish growth: A summary of conducted studies and current knowledge. Ecological Indicators. 2021;**121**:106976. DOI: 10.1016/j.ecolind.2020.106976

[70] Figueroa E, Valdebenito I, Merino O, Ubilla A, Risopatrón J, Farias JG. Cryopreservation of Atlantic salmon *Salmo salar* sperm: Effects on sperm physiology. Journal of Fish Biology. 2016;**89**(3):1537-1550. DOI: 10.1111/jfb.13052

[71] Ciereszko A, Dietrich GJ, Nynca J, Krom J, Dobosz S. Semen from sexreversed rainbow trout of spring strain can be successfully cryopreserved and used for fertilization of elevated number of eggs. Aquaculture. 2015;**448**:564-568. DOI: 10.1016/j.aquaculture.2015.06.039

[72] Nynca J, Judycka S, Liszewska E, Dobosz S, Grudniewska J, Fujimoto T, et al. Utility of different sugar extenders for cryopreservation and post-thaw storage of sperm from Salmonidae species. Aquaculture. 2016;**464**:340-348. DOI: 10.1016/j.aquaculture.2016.07.014

[73] Di Iorio M, Esposito S, Rusco G, et al. Semen cryopreservation for the Mediterranean brown trout of the Biferno River (Molise-Italy): Comparative study on the effects of basic extenders and cryoprotectants. Scientific Reports. 2019;**9**:9703. DOI: 10.1038/ s41598-019-45006-4

[74] Bozkurt Y, Seçer N, Tekin E, Akçay E. Cryopreservation of rainbow trout (*Oncorhynchus mykiss*) and mirror carp (*Cyprinus carpio*) sperm with glucose based extender. Süleyman Demirel Üniversitesi Eğirdir Su Ürünleri Fakültesi Dergisi. 2005;**1**:21-25

[75] Nynca J, Judycka S, Liszewska E, Dobosz S, Ciereszko A. Standardization of spermatozoa concentration for cryopreservation of rainbow trout semen using a glucose methanol extender. Aquaculture. 2017;**477**:23-27. DOI: 10.1016/j.aquaculture.2017.04.036

[76] Elliott GD, Wang S, Fuller BJ. Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology. 2017;**76**:74-91. DOI: 10.1016/j. cryobiol.2017.04.004

[77] Yang H, Hu E, Buchanan JT, Tiersch TR. A strategy for sperm cryopreservation of Atlantic salmon, *Salmo salar*, for remote commercial-scale high-throughput processing. Journal of the World Aquaculture Society. 2018;**49**(1):96-112. DOI: 10.1111/ jwas.12431

[78] Lahnsteiner F, Berger B, Weismann T, Patzner R. The influence of various cryoprotectants on semen quality of the rainbow trout (*Oncorhynchus mykiss*) before and after cryopreservation. Journal of Applied Ichtyology. 1996;**12**(2):99-106. DOI: 10.1111/j.1439- 0426.1996.tb00070.x

[79] Kutluyer F, Kayim M, Öğretmen F, Büyükleblebici S, Tuncer PB. Cryopreservation of rainbow trout *Oncorhynchus mykiss* spermatozoa: Effects of extender supplemented with different antioxidants on sperm motility, velocity and fertility. Cryobiology. 2014;**69**:462- 466. DOI: 10.1016/j.cryobiol.2014.10.005

[80] Figueroa E, Elías-Farias JG, Lee Estevez M, Valdebenito I, Iván-Risopatron J, *Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

Magnotti C, et al. Sperm cryopreservation with supplementation of a-tocopherol and ascorbic acid in freezing media increase sperm function and fertility rate in Atlantic salmon (*Salmo salar*). Aquaculture. 2018;**493**:1-8. DOI: 10.1016/j.aquaculture.2018.04.046

[81] Wang B, Van Veldhoven PP, Brees C, Rubio N, Nordgren M, Apanasets O, et al. Mitochondria are targets for peroxisomederived oxidative stress in cultured mammalian cells. Free Radical Biology & Medicine. 2013;**5**:882-894. DOI: 10.1016/j. freeradbiomed.2013.08.173

[82] Cosson MP, Cosson J, André F, Billard R. cAMP/ATP relationship in the activation of trout sperm motility: Their interaction in membrane-deprived models and in live spermatozoa. Cell Motility and the Cytoskeleton. 1995;**31**(2):159-176. DOI: 10.1002/cm.970310208

[83] Gallego V, Pérez L, Asturianoa JF, Yoshida M. Relationship between spermatozoa motility parameters, sperm/ egg ratio and fertilization and hatching rates in pufferfish (*Takifugu niphobles*). Aquaculture. 2013;**416**(417):238-243. DOI: 10.1016/j.aquaculture.2013.08.035

[84] Judycka S, Nynca J, Liszewska E, Dobosz S, Zalewski T, Ciereszko A. Potassium ions in extender differentially influence the post-thaw sperm motility of salmonid fish. Cryobiology. 2016;**73**:248-256. DOI: 0.1016/j. cryobiol.2016.07.002

[85] Robles V, Cabrita E, Cuñado S, Herraez MP. Sperm cryopreservation of sex-reversed rainbow trout (*Oncorhynchus mykiss*): Parameters that affect its ability for freezing. Aquaculture. 2003;**224**:203-212. DOI: 10.1016/S0044-8486(03)00221-7

[86] Tekin N, Secer S, Akcay E, Bozkurt Y, Kayam S. Effects of glycerol additions on post-thaw fertility of frozen rainbow trout sperm, with an emphasis on interaction between extender and cryoprotectant. Journal of Applied Ichthyology. 2007;**23**(1):60-63. DOI: 10.1111/j.1439-0426.2006.00792.x

[87] Ahmed MH, Keskin İ, Özkorucuklu S, Ekici A. A study on effect of using magnetized water in dilution the milt of Black Sea Trout (*Salmo trutta labrax*) on sperm motility. Aquaculture Research. **53**(17):6324-6332. DOI: 10.1111/are.16104

[88] Formicki K, Szulc J, Korzelecka-Orkisz A, Tański A, Kurzydłowski JK, Grzonka J, et al. The effect of a magnetic field on trout (*Salmo trutta* Linnaeus, 1758) sperm motility parameters and fertilisation rate. Journal of Applied Ichthyology. 2015;**31**:136-146. DOI: 10.1111/jai.12737

[89] Hu E, Yang H, Tiersch TR. Highthroughput cryopreservation of spermatozoa of blue catfish (*Ictalurus furcatus*): Establishment of an approach for commercial-scale processing. Cryobiology. 2011;**62**(1):74-82. DOI: 10.1016/j.cryobiol.2010.12.006

[90] Lang RP, Riley KL, Chandler JE, Tiersch TR. The use of dairy protocols for sperm cryopreservation of blue catfish (*Ictalurus furcatus*). Journal of the World Aquaculture Society. 2003;**34**:66- 75. DOI: 10.1111/j.1749-7345.2003. tb00040.x

[91] Muchlisin ZA, Nadiah WN, Nadiya N, Fadli N, Hendri A, Khalil M, et al. Exploration of natural cryoprotectants for cryopreservation of African catfish, *Clarias gariepinus*, Burchell 1822 (Pisces: Clariidae) spermatozoa. Czech Journal of Animal Science. 2015;**60**:10-15. DOI: 10.17221/7906-CJAS

[92] Şahinöz E, Aral F, Doğu Z, Koyuncu İ, Yüksekdağ Ö.

Cryopreservation of Mesopotamian catfish (*Silurus triostegus* H., 1843) spermatozoa: Effects of diluents and osmotic pressure on spermatozoa DNA damage, rate and duration of motility. Iranian Journal of Fisheries Sciences. 2020;**19**(5):2293-2307

[93] Watson P, Holt W. Principles of Cryopreservation. Cryobanking the Genetic Resource: Wildlife Conservation for the Future. Boca Raton, FL: CRC Press; 2001. pp. 23-46

[94] Watson PF, Morris GJ. Cold shock injury in animal cells. Symposia of the Society for Experimental Biology. 1987;**41**:311-340

[95] Cabrita E, Martínez-Páramo S, Gavaia PJ, Riesco MF, Valcarce DG, Sarasquete C, et al. Factors enhancing fish sperm quality and emerging tools for sperm analysis. Aquaculture. 2014;**432**:389-401. DOI: 10.1016/j. aquaculture.2014.04.034

[96] Wang H, Montague HR, Hess HN, Zhang Y, Aguilar GL, Dunham RA, et al. Transcriptome analysis reveals key gene expression changes in Blue Catfish sperm in response to cryopreservation. International Journal of Molecular Sciences. 2022;**23**(14):7618. DOI: 10.3390/ijms23147618

[97] Niu J, Wang X, Liu P, Liu H, Li R, Li Z, et al. Effects of cryopreservation on sperm with cryodiluent in viviparous Black Rockfish (*Sebastes schlegelii*). International Journal of Molecular Sciences. 2022;**23**(6):3392. DOI: 10.3390/ ijms23063392

[98] Martin G, Sabido O, Durand P, Levy R. Cryopreservation induces an apoptosis-like mechanism in bull sperm. Biology of Reproduction. 2004;**71**:28-37. DOI: 10.1095/biolreprod.103.024281

[99] Riesco MF, Oliveira C, Soares F, Gavaia PJ, Dinis MT, Cabrita E. *Solea senegalensis* sperm cryopreservation: New insights on sperm quality. PLoS One. 2017;**12**:e0186542. DOI: 10.1371/journal. pone.0186542

[100] Acosta IB, Corcini CD, Gheller SMM, Brito CR, Goulart TLS, Varela Junior AS. Effect of amide on semen cryopreservation of curimba (*Prochilodus lineatus*). CryoLetters. 2020;**41**:1-5

[101] Yoshizaki G, Yazawa R. Application of surrogate broodstock technology in aquaculture. Fisheries Science. 2019;**85**:429-437. DOI: 10.1007/ s12562-019-01299-y

[102] Okutsu T, Shikina S, Kanno M, Takeuchi Y, Yoshizaki G. Production of trout offspring from triploid salmon parents. Science (New York, N.Y.). 2007;**317**(5844):1517. DOI: 10.1126/ science.1145626

[103] Lacerda SMSN, Costa GMJ, Campos-Junior PHA, et al. Germ cell transplantation as a potential biotechnological approach to fish reproduction. Fish Physiology and Biochemistry. 2013;**39**:3-11. DOI: 10.1007/s10695-012-9606-4

[104] Octavera A, Yoshizaki G. Production of Chinese rosy bitterling offspring derived from frozen and vitrified whole testis by spermatogonial transplantation. Fish Physiology and Biochemistry. 2020;**46**(4):1431-1442. DOI: 10.1007/ s10695-020-00802-y

[105] Lee S, Seki S, Katayama N. Production of viable trout offspring derived from frozen whole fish. Scientific Reports. 2015;**5**:16045. DOI: 10.1038/ srep16045

[106] Lee S, Bang WY, Yang HS, Lee DS, Song HY. Production of juvenile masu

*Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

salmon (*Oncorhynchus masou*) from spermatogonia-derived sperm and oogonia-derived eggs via intraperitoneal transplantation of immature germ cells. Biochemical and Biophysical Research Communications. 2021;**535**:6-11. DOI: 10.1016/j.bbrc.2020.12.021

[107] Yang F, Ichida K, Yoshizaki G. Gametogenesis commencement in recipient gonads using germ cells retrieved from dead fish. Aquaculture. 2022;**552**:737952 DOI: 10.1016/j. aquaculture.2022.737952

[108] Abualreesh M, Myers JN, Gurbatow J, Johnson A, Xing D, Wang J, et al. Development of an effective cryopreservation protocol for Blue Catfish Oogonia. North American Journal of Aquaculture. 2021;**83**:336-345. DOI: 10.1002/naaq.10203

[109] Abualreesh MJN, Myers J, Gurbatow A, Johnson D, Xing J, Wang S, et al. Development of a spermatogonial stem cell cryopreservation protocol for Blue Catfish. Cryobiology. 2020;**97**:46- 52. DOI: 10.1016/j.cryobiol.2020.10.010

[110] Franěk R, Tichopád T, Steinbach C, Xie X, Lujić J, Marinović Z, et al. Preservation of female genetic resources of common carp through oogonial stem cell manipulation. Cryobiology. 2019;**87**:78-85. DOI: 10.1016/j.cryobiol.2019.01.016

[111] Yoshizaki G, Ichikawa M, Hayashi M, Iwasaki Y, Miwa M, Shikina S, et al. Sexual plasticity of ovarian germ cells in rainbow trout. Development. 2010;**137**:1227-1230. DOI: 10.1242/dev.044982

[112] Isayeva A, Zhang T, Rawson DM. Studies on chilling sensitivity of zebrafish (*Danio rerio*) oocytes. Cryobiology. 2004;**49**:114-122. DOI: 10.1016/j.cryobiol.2004.05.005

[113] Zhang T, Rawson DM. Studies on chilling sensitivity of zebrafish (*Brachydanio rerio*) embryos. Cryobiology. 1995;**32**:239-246. DOI: 10.1006/cryo.1995.1023

[114] Hagedorn M, Kleinhans FW, Wildt DE, Rall WF. Chill sensitivity and cryoprotectant permeability of dechorionated zebrafish embryos, *Brachydanio rerio*. Cryobiology. 1997;**34**:251-263. DOI: 10.1006/ cryo.1997.2002

[115] Fornari DC, Ribeiro RP, Streit DJ, Godoy LC, Neves PR, de Oliveira D, et al. Effect of cryoprotectants on the survival of cascudo preto (*Rhinelepis aspera*) embryos stored at −8°C. Zygote. 2014;**22**:58-63. DOI: 10.1017/ S0967199411000517

[116] Liu XH, Zhang T, Rawson DM. Effect of cooling rate and partial removal of yolk on the chilling injury in zebrafish (*Danio rerio*) embryos. Theriogenology. 2001;**55**:1719-1731. DOI: 10.1016/ S0093-691X(01)00515-5

[117] Pessoa NO, Galvão JAS, de Souza Filho FGM, de Sousa MLNM, Sampaio CMS. Cooling of pirapitinga (*Piaractus brachypomus*) embryos stored at −10°C. Zygote. 2015;**23**(3):453-459. DOI: 10.1017/S0967199414000057

[118] Zhang XS, Zhao L, Hua TC, Chen XH, Zhu HY. A study on the cryopreservation of common carp, *Cyprinus carpio* embryos. Cryo-Letters. 1989;**10**:271-278

[119] Zhang T, Rawson DM, Morris BJ. Cryopreservation of prehatch embryo of zebrafish. Aquatic Living Resources. 1993;**6**:145-153. DOI: 10.1051/alr:1993014

[120] Connolly MH, Paredes E, Mazur P. A preliminary study of osmotic dehydration in zebrafish embryos:

Implications for vitrification and ultra-fast laser warming. Cryobiology. 2017;**78**:106-109. DOI: 10.1016/j. cryobiol.2017.08.004

[121] Tian YS, Jiang J, Song LN, Chen ZF, Zhai JM, Liu JC, et al. Effects of cryopreservation on the survival rate of the seven-band grouper (*Epinephelus septemfasciatus*) embryos. Cryobiology. 2015;**71**(3):499-506. DOI: 10.1016/j. cryobiol.2015.10.147

[122] de Carvalho AFS, Ramos SE, de Carvalho TSG, de Souza YCP, Zangeronimo MG, Pereira LJ, et al. Efficacy of fish embryo vitrification protocols in terms of embryo morphology – A systematic review. CryoLetters. 2014;**35**:361-370

[123] Edashige K, Valdez DM Jr, Hara T, Saida N, Seki S, Kasai M. Japanese flounder (*Paralichthys olivaceus*) embryos are difficult to cryopreserve by vitrification. Cryobiology. 2006;**53**:96- 106. DOI: 10.1016/j.cryobiol.2006.04.002

[124] Ahammad MM, Bhattacharyya D, Jana BB. The hatching of common carp (*Cyprinus carpio* L.) embryos in response to exposure to different concentrations of cryoprotectant at low temperatures. Cryobiology. 2003;**44**:114-121. DOI: 10.1016/S0011-2240(02)00012-3

[125] Keivanloo S, Sudagar M, Mazandarani M. Evaluating the suitability of cryopreservation solutions for common carp (*Cyprinus carpio*) embryos stored at −2°C. Iranian Journal of Fisheries Sciences. 2019;**18**(4):1036-1045

[126] Tian T, Chen Z, Tang J, Duan H, Zhai J, Li B, et al. Effects of cryopreservation at various temperatures on the survival of kelp grouper (*Epinephelus moara*) embryos from fertilization with cryopreserved

sperm. Cryobiology. 2017;**75**:37-44. DOI: 10.1016/j. cryobiol.2017.02.007

[127] Serhat E, Ozgur A. Cryopreservation of common carp (*Cyprinus carpio*) embryos using different cryoprotectant. Survey in Fisheries Sciences. 2022;**8**(2):1- 10. DOI: 10.18331/SFS2022.8.2.1

[128] Keivanloo S, Sudagar M. Cryopreservation of Persian sturgeon (*Acipenser persicus*) embryos by DMSO-based vitrificated solutions. Theriogenology. 2016;**85**(5):1013-1018. DOI: 10.1016/j. theriogenology.2015.11.012

[129] Khosla K, Wang Y, Hagedorn M, Qin Z, Bischof J. Gold nanorod induced warming of embryos from the cryogenic state enhances viability. ACS Nano. 2017;**11**(8):7869-7878. DOI: 10.1021/ acsnano.7b02216

[130] Bielanski A. Disinfection procedures for controlling microorganisms in the semen and embryos of humans and farm animals. Theriogenology. 2007;**68**:1-22. DOI: 10.1016/j. theriogenology.2007.03.025

[131] Thibier M, Guerin B. Hygienic aspects of storage and use of semen for artificial insemination. Animal Reproduction Science. 2000;**62**:233-251. DOI: 10.1016/S0378-4320(00)00161 5

[132] Bielanski A, Bergeron H, Lau PCK, Devenish J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology. 2003;**46**:146-152. DOI: 10.1016/ S0011-2240(03)00020-8

[133] Joaquim DC, Borges ED, Viana IGR, Navarro PA, Vireque AA. Risk of contamination of gametes and embryos during cryopreservation and measures to prevent crosscontamination. BioMed Research

*Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

International. 2017;**2017**:1840417. DOI: 10.1155/2017/1840417

[134] Morris GJ. The origin, ultrastructure, and microbiology of the sediment accumulating in liquid nitrogen storage vessels. Cryobiology. 2005;**50**:231-238. DOI: 10.1016/j. cryobiol.2005.01.005

[135] Steel KJ, Ross HE. Survival of freeze dried bacterial cultures. The Journal of Applied Bacteriology. 1963;**26**:370-375. DOI: 10.1111/j.1365-2672.1963.tb04787.x

[136] Pandya SP, Doshi H, Codipilly CN, Fireizen Y, Potak D, Schanler RJ. Bacterial stability with freezer storage of human milk. Journal of Perinatal Medicine. 2021;**49**:225-228. DOI: 10.1515/ jpm-2020-0131

[137] Perry SF. Freeze-drying and cryopreservation of bacteria. In: Day JG, Pennington MW. (eds) Cryopreservation and Freeze-Drying Protocols. Methods in Molecular Biology™, Humana Press, Totowa, NJ. 1995;**38**:21-30. DOI: 10.1385/0-89603-296-5:21

[138] Yamashita H, Mori K, Nakai T. Protection conferred against viral nervous necrosis by simultaneous inoculation of aquabirnavirus and inactivated betanodavirus in the sevenband grouper, *Epinephelus septemfasciatus* (Thunberg). Journal of Fish Diseases. 2009;**32**:201-210. DOI: 10.1111/J.1365-2761.2008.01017.X

[139] Bielanski A, Vajta G. Risk of contamination of germplasm during cryopreservation and cryobanking in IVF units. Human Reproduction. 2009;**24**:2457-2467. DOI: 10.1093/ HUMREP/DEP117

[140] Garcia A, Sierra MF, Friberg J. Survival of bacteria after freezing of human semen in liquid nitrogen.

Fertility and Sterility. 1981;**35**:549-551. DOI: 10.1016/S0015-0282(16)45499-3

[141] Smith D, Ryan M. Implementing best practices and validation of cryopreservation techniques for microorganisms. Scientific World Journal. 2012;**2012**:805659. DOI: 10.1100/2012/805659

[142] Bielanski A, Nadin-Davis S, Sapp T, Lutze-Wallace C. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology. 2000;**40**:110-116. DOI: 10.1006/cryo.1999.2227

[143] Fountain D, Ralston M, Higgins N, Gorlin J, Uhl L, Wheeler C, et al. Liquid nitrogen freezers: A potential source of microbial contamination of hematopoietic stem cell components. Transfusion. 1997;**37**:585-591. DOI: 10.1046/j.1537-2995.1997. 37697335152.x

[144] Ciereszko A, Własow T, Dobosz S, Goryczko K, Glogowski J. Blood cells in rainbow trout *Oncorhynchus mykiss* milt: Relation to milt collection method and sampling period. Theriogenology. 2004;**62**:1353-1364. DOI: 10.1016/j. theriogenology.2004.02.003

[145] Rurangwa E, Kime D, Ollevier F, Nash J. The measurement of sperm motility and factors affecting sperm quality in cultured fish. Aquaculture. 2004;**234**:1-28. DOI: 10.1016/j.aquaculture.2003.12.006

[146] VKM, Rimstad E, Basic D, Brun E, Colquhoun D, Olesen NJ, Bøe KE, Gjøen T, Godfroid J, Janczak AM, Madslien K, Olsen RE, Øverli Ø, Agdestein A. The risk of transmission of infectious disease through trade of cryopreserved milt. Scientific Opinion of the Panel on Animal Health and Welfare of the Norwegian Scientific Committee for Food Safety. VKM report 2019:02, ISBN:

978-82-8259-318-2, ISSN: 2535-4019. 2019. Norwegian Scientific Committee for Food and Environment (VKM), Oslo, Norway.

[147] Hare WCD. Diseases transmissible by semen and embryo transfer techniques. OIE Technical Bulletin. 1985;**4**:1-117

[148] Brackett BG, Baranska W, Sawicki W, Koprowski H. Uptake of heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proceedings of the National Academy of Sciences. 1971;**68**:353-357. DOI: 10.1073/ pnas.68.2.353

[149] Mulcahy D, Pascho RJ. Adsorption to fish sperm of vertically transmitted fish viruses. Science. 1984;**225**:333-335

[150] Mulcahy D, Pascho RJ. Vertical transmission of infectious haematopoietic virus in sockeye salmon, *Oncorhynchus nerka* (Walbaum): Isolation of virus from dead eggs and fry. Journal of Fish Diseases. 1985;**8**:393-396

[151] Ahne W. Presence of infectious pancreatic necrosis virus in the seminal fluid of rainbow trout, *Salmo gairdneri* Richardson. Journal of Fish Diseases. 1983;**6**(4):377-377

[152] Smail DA, Munro ES. Isolation and quantification of infectious pancreatic necrosis virus from ovarian and seminal fluids of Atlantic salmon, *Salmo salar* L. Journal of Fish Diseases. 2008;**31**(1):49-58. DOI: 10.1111/j.1365-2761.2007.00866.x

[153] Eaton WD, Hulett J, Brunson R, True K. The first isolation in North America of infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) in coho salmon from the

same watershed. Journal of Aquatic Animal Health. 1991;**3**:114-117. DOI: 10.1577/1548-8667(1991)003

[154] Meyers TR, Thomas JB, Follett JE, Saft RR. Infectious hematopoietic necrosis virus: Trends in prevalence and the risk management approach in Alaskan sockeye salmon culture. Journal of Aquatic Animal Health. 1990;**2**:85-98. DOI: 10.1577/1548-8667(1990)002

[155] Ercan MD, Ekici A. Reducing bacterial density in the semen of rainbow trout (*Oncorhynchus mykiss*) by applying gradient centrifugation and swim-up washing methods. Aquaculture Research. 2016;**47**:3845-3851. DOI: 10.1111/ are.12835

[156] Yildirim S, Özer S. The existence of Flavobacterium spp. at rainbow trout (*Oncorhynchus mykiss*, Walbaum, 1792) hatcheries in Caglarca village, Mersin-Turkey. Journal of Fisheries. 2010;**4**:112- 122. DOI: 10.3153/jfscom.2010010

[157] Larenas JJ, Bartholomew J, Troncoso O, Fernández S, Ledezma H, Sandoval N, et al. Experimental vertical transmission of *Piscirickettsia salmonis* and in vitro study of attachment and mode of entrance into the fish ovum. Diseases of Aquatic Organisms. 2003;**56**:25-30. DOI: 10.3354/ DAO056025

[158] Wu Y, Zhang X, Wang Z, Xia X. Can we cryopreserve the sperm of COVID-19 patients during the pandemic? Frontiers in Endocrinology (Lausanne). 2022;**13**:753267. DOI: 10.3389/ FENDO.2022.753267

[159] Batiha O, Al-Deeb T, Al-zoubi E, Alsharu E. Impact of COVID-19 and other viruses on reproductive health. Andrologia. 2020;**52**:e13791. DOI: 10.1111/AND.13791

*Cryopreservation Studies in Aquaculture from Past to Present: Scientific Techniques and Quality… DOI: http://dx.doi.org/10.5772/intechopen.108566*

[160] Zafer M, Horvath H, Mmeje O, Van Der Poel S, Semprini AE, Rutherford G, et al. Effectiveness of semen washing to prevent human immunodeficiency virus (HIV) transmission and assist pregnancy in HIV-discordant couples: A systematic review and meta-analysis. Fertility and Sterility. 2016;**105**:645-655. e2. DOI: 10.1016/j.fertnstert.2015.11.028

[161] Ekici A, Baran A, Ozdas OB, Sandal AI, Yamaner G, Guven E. The effect of streptomycin on freezing rainbow trout (*Oncorhynchus mykiss*) sperm. Israeli Journal of Aquaculture – Bamidgeh. 2014;**66**:1-6. DOI: 10.46989/001c.20749

[162] Duracka M, Lukac N, Kacaniova M, Kantor A, Hleba L, Ondruska L, et al. Antibiotics versus natural biomolecules: The case of in vitro induced bacteriospermia by *Enterococcus faecalis* in rabbit semen. Molecules. 2019;**24**:4329. DOI: 10.3390/MOLECULES24234329

[163] Rivers N, Daly J, Daly J, Temple-Smith P. New directions in assisted breeding techniques for fish conservation. Reproduction, Fertility, and Development. 2020;**32**:807-821. DOI: 10.1071/RD19457

[164] Meacham RB. Practical considerations in the preparation of sperm prior to cryopreservation. Journal of Andrology. 2005;**26**(4):451. DOI: 10.2164/jandrol.05061

[165] Horokhovatskyi Y, Dietrich MA, Lebeda I, Fedorov P, Rodina M, Dzyuba B. Cryopreservation effects on a viable sperm sterlet (*Acipenser ruthenus*) subpopulation obtained by a Percoll density gradient method. Rutherford S, editor. PLoS One. 2018;13:e0202514. DOI: 10.1371/journal.pone.0202514

[166] Marinović Z, Šćekić I, Lujić J, Urbányi B, Horváth Á. The effects of cryopreservation and cold storage

on sperm subpopulation structure of common carp (*Cyprinus carpio* L.). Cryobiology. 2021;**99**:88-94. DOI: 10.1016/J.CRYOBIOL.2021.01.007

[167] Pérez-Atehortúa M, Giannotti Galuppo A, Batista Rodrigues R, de Souza FT, dos Santos TN, Rodrigues de Freitas T, et al. The use of differential separation and density gradient with AllGrad 90% after thawing improves the sperm quality of South American catfish (*Rhamdia quelen*). Aquaculture. 2022;**553**:738072. DOI: 10.1016/j. aquaculture.2022.738072

[168] Valcarce DG, Herráez MP, Chereguini O, Rodríguez C, Robles V. Selection of nonapoptotic sperm by magnetic-activated cell sorting in Senegalese sole (*Solea senegalensis*). Theriogenology. 2016;**86**:1195-1202. DOI: 10.1016/j. theriogenology.2016.04.010

[169] Li P, Dzyuba B, Hulak M, Rodina M, Boryshpolets S, Li Z-H, et al. Percoll gradient separation of cryopreserved common carp spermatozoa to obtain a fraction with higher motility, velocity and membrane integrity. Theriogenology. 2010;**74**:1356-1361. DOI: 10.1016/j. theriogenology.2010.06.005

[170] Bravo W, Dumorné K, Beltrán Lissabet J, Jara-Seguel P, Romero J, Farías JG, et al. Effects of selection by the Percoll density gradient method on motility, mitochondrial membrane potential and fertility in a subpopulation of Atlantic salmon (*Salmo salar*) testicular spermatozoa. Animal Reproduction Science. 2020;**216**:106344. DOI: 10.1016/j. anireprosci.2020.106344

[171] OIE. Aquatic Animal Health Code, 22nd edition. 2019. ISBN 978-92-95108- 96-7. World Organisation For Animal Health, Paris, France

[172] Uhrig M, Ezquer F, Ezquer M. Improving cell recovery: Freezing and thawing optimization of induced pluripotent stem cells. Cell. 2022;**11**:799. DOI: 10.3390/CELLS11050799/S1

[173] Saragusty J, Anzalone DA, Palazzese L, Arav A, Patrizio P, Gosálvez J, et al. Dry biobanking as a conservation tool in the Anthropocene. Theriogenology. 2020;**150**:130-138. DOI: 10.1016/J. THERIOGENOLOGY.2020.01.022

[174] Comizzoli P, Loi P, Patrizio P, Hubel A. Long-term storage of gametes and gonadal tissues at room temperatures: The end of the ice age? Journal of Assisted Reproduction and Genetics. 2022;**39**:321-325. DOI: 10.1007/ S10815-021-02392-X

[175] Bajerski F, Nagel M, Overmann J. Microbial occurrence in liquid nitrogen storage tanks: A challenge for cryobanking? Applied Microbiology and Biotechnology. 2021;**105**:7635-7650. DOI: 10.1007/S00253-021-11531-4

[176] Diogo P, Martins G, Quinzico I, Nogueira R, Gavaia PJ, Cabrita E. Electric ultrafreezer (−150°C) as an alternative for zebrafish sperm cryopreservation and storage. Fish Physiology and Biochemistry. 2018;**44**:1443-1455. DOI: 10.1007/S10695-018-0500-6

[177] Keskintepe L, Pacholczyk G, Machnicka A, Norris K, Curuk MA, Khan I, et al. Bovine blastocyst development from oocytes injected with freeze-dried spermatozoa. Biology of Reproduction. 2002;**67**:409-415. DOI: 10.1095/BIOLREPROD67.2.409

[178] Mayer I. The role of reproductive sciences in the preservation and breeding of commercial and threatened teleost fishes. In: Comizzoli P, Brown JL, Holt WV, editors. Reproductive Science in Animal Conservation. Cham: Springer International Publishing; 2019. pp. 187-224

#### **Chapter 2**

## The Current Status of Semen and Oocytes Cryopreservation

*Masindi Mphaphathi, Mahlatsana Ledwaba and Mamonene Thema*

#### **Abstract**

Assisted reproductive technologies are critical in the preservation of gametes from endangered species. As a result, cryobanking is critical in reproduction facilities for the gametes conservation of endangered species for future use. Furthermore, cryobanking allows for the preservation of genetic variability through biotechnological reproduction programs. If oocyte cryopreservation is successful, the timing of *in vitro* maturation and subsequent to *in vitro* fertilization (IVF) will be possible. Cattle oocytes are very sensitive to cryopreservation due to their complex structure, and they are also very sensitive to chilling, which can harm their viability. During the cryopreservation process, sperm membrane proteins and carbohydrate composition change, sperm membrane structure is disrupted, and sperm viability is reduced. Extenders are frequently required during cryopreservation, for improving sperm cryopreservation technologies and is therefore necessary to have a thorough understanding of the properties of the extenders. Extenders have been enriched with antioxidants such as Glutathione to protect sperm motility and integrity from oxidative damage and the reactive oxygen species produced during cryopreservation can be neutralized using antioxidants.

**Keywords:** semen, oocytes, extender, cryopreservation, slow freezing, vitrification

#### **1. Introduction**

Cryopreservation is the expertise of freezing and cryogenic storage of biological materials at extremely low temperatures, occasionally utilizing solid carbon dioxide at −80°C or more often liquid nitrogen at −196°C [1]. This procedure is essential for preserving gametes and genetic diversity in both known and endangered species. Cryopreservation of gametes and genetic diversity provides numerous advantages as it paves the way for the successful application of current biotechnologies like cloning, transgenesis, and long-term storage/conservation of animal genetic resources [2]. Oocyte cryopreservation is beneficial for the treatment of infertility and has broader clinical implications than embryo cryopreservation [3]. However, for the quality of the gametes or tissue not to deteriorate during long-term storage, a good and reliable methodology is required. Many reproductive centers have established

cryopreservation methodologies for many species. However, the survival rate of the gametes or tissues has declined over time due to the lower temperature and metabolic reactions of the gametes which are impaired during cryopreservation. Cryopreservation causes the formation of intracellular ice crystals and osmotic stress, which causes cell damage, oocyte quality degradation due to their susceptibility to chilling, and a reduction in sperm survival rate [4]. Cryopreservation is one method of preserving sperm and oocytes *ex-situ*. Furthermore, the cryopreservation process has proven to can reduce the number of cases of extinction. Moreover, the use of semen extenders, medium, antioxidants and cryoprotectants (CPAs) proved beneficial in preserving the gametes. The semen extenders, mediums, antioxidants, and CPAs are nutrients and antibiotics that increase the quality and survival rate of the gametes. It was discovered that successful gametes cryopreservation, requires the use of semen extenders, mediums, antioxidants, and CPAs. Typically, the pace and susceptibility of sperm to subzero temperatures relate to the content of cryoprotective chemicals and membrane-stabilizing additives. Therefore, cryopreservation may be an effective method of preserving fertility as the frozen– thawed sperm may be utilized for intrauterine insemination, IVF, or intracytoplasmic sperm injection [5].

#### **2. History of cryopreservation**

This chapter will discuss the current state of oocytes and sperm cryopreservation, as well as their prospects. Mammalian sperm cryopreservation has previously proven to be difficult due to the lack of methodologies that can help sperm and oocytes withstand extremely low temperatures. Whereas cryopreservation has been determined to be an ineffective reproductive medicine preservation treatment since 1970 [6]. To date, the sperm cryopreservation in ovine and bovine species has improved over time. Whereas sperm cryopreservation remains a challenge in mammalian species such as porcine and humans. Furthermore, cryopreservation of oocytes has proven to be difficult in all mammalian species. Several methodologies have been tested on mammalian oocytes, in the past, beginning with the first cryopreservation in the year and continuing to the present [7]. Cryopreservation of oocytes in many mammalian species, such as porcine and horses, remains a major challenge.

To achieve the best results during cryopreservation, a thorough grasp of sperm physiology is essential [8]. The fact that sperm are tiny cells with a vast surface area is a crucial aspect of sperm cryobiology [9]. Sperm are less vulnerable to potential harm because of these traits, which impact the intracellular cytosol's viscosity and glass transition temperature [10]. Organelles in the sperm may be destroyed in the absence of cryoprotective substances due to cold shock and the stimulation of ice crystal formation [11]. More research is needed to determine the methodologies that can successfully improve the quality of oocytes after cryopreservation. Cryopreservation of semen and oocytes will aid in the preservation of domestic and wild species' genetic diversity, as well as the dissemination of superior genetics, gene banking, and the extinction of superior and endangered species. Semen cryopreservation is also used in artificial insemination (AI) and IVF procedures. However, previous research has shown that using post-thawed semen for AI results in a lower pregnancy rate. Offspring have also been reported to be born from frozen–thawed oocytes in bovine, ovine, and horse.

#### **3. Fundamentals of the cryopreservation**

#### **3.1 Extenders**

Semen extenders are liquid diluents added to sperm to preserve its ability to fertilize, and they contain protective ingredients that allow sperm to survive outside the reproductive tract of the male animals [12, 13]. Furthermore, semen extenders protect sperm by stabilizing the plasma lemma, providing energy substrates, and preventing the harmful effects of pH and osmolarity changes over time during *in vitro* storage [14]. When a proper semen extender is added to the sample before evaluation, the accuracy of sperm motility determination may improve [15].

During the chilling and shipping processes, semen extenders act as a buffer to protect sperm cells from their own harmful byproducts as well as from cold shock and osmotic shock [16]. Semen extenders has the ability to prolong sperm storage and transportation, allowing it to be used during AI, IVF and other research studies. Currently, the semen extenders are categorized either as short (approximately 3 days; *in vitro* liquid storage) or long term (approximately 5 days; *in vitro* liquid storage or cryopreservation for years) [17]. For the past decades, some commercial vendors have made around 80% of the semen extenders for porcine sperm readily available [18].

#### **3.2 Cryoprotectants**

Cryopreservation methods aim to preserve the viability of tissues and cells by focusing on the mechanisms of harm and protection in living cells and tissues at low temperatures [19]. The impact of subzero temperatures on normally healthy tissue should be recognized to properly comprehend the role of cryoprotective agents. Since water makes up around 80% of tissue mass, both intracellularly and extracellularly, has the greatest impact on the detrimental biochemical and structural changes that are hypothesized to cause cryopreservation injury [20]. Due to the presence of salts and organic molecules in the cells, the freezing point of cell water is substantially lower (even −68°C) than the freezing point of pure water (about 0°C).

Cryoprotectants are divided into two groups: permeable and non-permeable CPAs. Non-permeable and permeable CPAs improve cell survival while decreasing cellular water content to help prevent intracellular ice crystal formation. Permeable CPAs are macromolecules that pass through the sperm plasma membrane. A cryopreservation diluent's functions include providing the sperm with energy sources, shielding them from temperature related harm, and maintaining an environment that allows the sperm to survive for a while. Glycerol, Propylene glycol, and Ethylene glycol (EG) are three examples of permeable CPAs that are commonly used (**Figure 1**). To enhance post-thawed sperm viability and fertility, each of the several media components was studied alone and in combination [21]. The gametes exposed to those penetrating

#### **Figure 1.**

*The penetration CPAs that are widely used: Glycerol (GLY), dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propylene glycol (PG). Adapted from Whaley et al. [20].*

solutes undergo intense initial dehydration, then rehydration, resulting in a chance of gross cellular swelling [22]. Glycerol at 3% has shown to maintain the cryo survival rate of sperm from different species; thus, larger amounts of permeable CPAs concentrations have shown to lead to more cellular damage [23], while the higher concentrated CPAs are more toxic to oocytes. Glycerol reduces intracellular water freezing while adjusting sperm osmolality via invasive thermal protection [24]. The discovery of Glycerol's effectiveness in preventing various phase transitions while freezing via increased water permeability and fluidity of the sperm membranes resulted from research to understand the mechanisms of CPAs [25]. Using minimum volume methods, a higher cooling rate can facilitate vitrification with less concentrated CPAs, and a higher warming rate will prevent it [26]. Non-permeable CPAs such as sucrose can facilitate dehydration and vitrification, which reduce the required concentration of permeable CPAs [27].

#### **3.3 Antioxidant**

During semen cryopreservation and thawing, increased reactive oxygen species (ROS) generation and decreased antioxidant levels were observed. As a result, oxidative stress may have a role in sperm injury during cryopreservation. Oxidative stress is the imbalance between the formation of ROS and antioxidant defenses, resulting in considerable loss of sperm function. Therefore, the ROS generated during oxidative stress can be neutralized by the use of antioxidants. Antioxidants are chemicals and reactions that dispose of scavenging, suppressing, or resisting the creation of ROS. Antioxidants have been shown to inhibit or reduce the lipid peroxidation reaction, resulting in less oxidative stress and damage [28]. Antioxidants and antioxidant enzymes that are found in the seminal plasma of the semen protect sperm from oxidative damage. These antioxidant mechanisms protect the sperm, including enzymatic antioxidants [Catalase, Superoxide Dismutase (SOD), reduced Glutathione & Glutathione Peroxidase (GPx)], [29, 30] and non-enzymatic antioxidant systems [GPx, Vitamin C, E, Cysteine & Glutathione (GSH)]. These antioxidants protect sperm from cryo injuries caused by reactive oxygen species [31, 32]. However, due to the addition of extenders to the seminal plasma during cryopreservation, the quantities of these antioxidants decrease [33]. Therefore, finding low cytotoxic antioxidants at a suitable concentration is very important in improving the frozen–thawed sperm quality [34].

Recent studies suggest that supplementing cryopreservation extenders with antioxidants improves sperm quality in bovine, ovine, porcine, canine, and human, enhancing sperm motility and membrane integrity following thawing [35]. A range of antioxidants is active in the body including enzymatic (endogenous) and nonenzymatic (mainly brought by food) antioxidants. All of them can be intracellular or extracellular antioxidants and can be used during the cryopreservation of semen. To reduce cryodamage, numerous exogenous, non-enzymatic antioxidants were introduced to maturation, vitrification media, and extenders for mammalian sperm, oocytes, and embryos.

#### **4. Methods of cryopreservation**

There are numerous cryopreservation methods for cryopreserving semen and oocytes of different species; methods include slow freezing (programmable freezer), rapid freezing, and ultra-rapid freezing (also known as kinetic vitrification). These methods represent a particular drawback in determining the most appropriate method for cryopreservation [36]. Various processes have been developed for semen and oocytes cryopreservation technology in recent years [37].

#### **4.1 Conventional slow freezing**

Slow cryopreservation of semen has been a useful method and is still used during cryopreserving semen and oocytes [38, 39]. A programmable freezer with significant control over the ideal freezing rate is necessary for conventional slow freezing. The temperature progressively drops below the freezing point during cooling, whereas both extracellular and intracellular spaces can generate ice [40]. In order to ensure fine control over numerous elements (such as thermal shock) that lead to cell damage, slow freezing primarily calls for a relatively low concentration of CPA agent, combined with sufficiently slow cooling/freezing rates [41]. In a summary, slow cooling is mixing low concentrations of a penetrating agent like DMSO (usually ≤1.5 M) as well as a non-permeating agent (often sucrose or trehalose, ≤ 0.3 M) with controlled slow chilling rates to gradually dehydrate sperm and oocytes. The sperm and oocytes are kept in liquid nitrogen at −196°C till they are required for usage after cooling to about ≤150°C [20]. One disadvantage of this procedure is the inability to freeze extremely small amounts of semen, as in the case of surgical testicular sperm retrieval [37]. Slow freezing methods using a programmed freezer is traditionally used for oocyte cryopreservation, and these procedures typically take several hours [3], oocytes are progressively chilled over 2 to 3 hours in two or more steps, either manually or automatically with the aid of a programmable freezer [42]. Semen straws can be frozen in huge quantities with the use of programmable freezers, which also allow for regulating the freezing pace. By cryopreserving the semen straws at −80°C for 7 to 15 minutes and then submerging them in liquid nitrogen, these freezers can be used to stimulate pellet freezing [43]. The advantage of several programmable freezers is the ability


#### **Table 1.**

*Comparison of the characteristics between vitrification and slow freezing methods.*

to personalize the freezing curve, for example, 4 to −5°C at 4°C/min, −5 to 110°C at 25°C/min, and − 110 to 140°C at 35°C/min, before submerging the semen straws in liquid nitrogen [44]. According to some theories, the formation of ice crystals during slow freezing raises the electrolyte concentrations inside cells and could harm the sperm and oocytes chemically and physically. Therefore, slow freezing appears to have a lower survival rate than vitrification [3].

#### **4.2 Conventional straw vitrification**

Vitrification is another important method for improving the survival rate of cryopreserved oocytes that is both time saving and does not require any special equipment [45]. The most typical process for vitrification includes adding CPAs step by step in cryomedia [20]. Although vitrification's rapid freezing defends cells from the majority of chilling-related harm, including membrane damage, it necessitates the use of hazardous CPAs solutions in higher concentrations [46]. Oocytes are introduced to a solution that contains 7.5% v/v EG and 7.5% v/v DMSO for 5 to 15 minutes during the initial equilibrium phase. The oocytes are subsequently subjected to a vitrification solution containing 0.5 M sucrose, 15% v/v DMSO, and 15% v/v EG. The oocytes are then kept in liquid nitrogen at −196°C after a brief incubation (≤ 1 minute). After gradually removing the CPA, the oocytes are promptly warmed to prevent the development of ice crystals, and then cultured in a culture medium until use [4, 47, 48]. Vitrification solution for embryos must be treated at a low temperature of 4°C [47]. One method for increasing the cooling rate and vitrification is to use liquid nitrogen vapor instead of liquid nitrogen only [49]. The study discovered that the vitrification method involving the use of only non-permeable CPAs for cryopreservation of abnormal sperm samples was an effective alternative to the vitrification method [44]. The differences between the vitrification and slow freezing method are shown in **Table 1**.

#### **4.3 Liquid nitrogen vapor**

Semen is poured into 0.25 or 0.5 ml straws, placed on a rack, and frozen in liquid nitrogen vapor. The temperature of which should be determined by the height above the liquid nitrogen after dilution and cooling of the semen samples [21]. When utilizing a styrofoam box, the samples are placed on a rack that is suspended 3 to 4 cm above the liquid for 7 to 8 minutes before the straws are submerged into liquid nitrogen for storage [43]. Alternatively, the freezing height above the liquid nitrogen should be determined by the reported straw size [50]. For storage, it was recommended that 0.5 ml straws be frozen 4 cm over liquid nitrogen for 5 minutes, whereas 0.25 ml straws must be placed 16 cm over liquid nitrogen for 2 minutes, lowered to 4 cm for 3 minutes, and then submerged in liquid nitrogen [21].

#### **4.4 Solid surface vitrification**

Solid surface vitrification (SSV) is a cryopreservation technique applied to the preservation of embryos and oocytes. In this technique, which combines many others, oocytes are combined with CPAs and partially submerging a metal surface in liquid nitrogen to pre-cool it to −180°C, SSV employs the metal surface as a cooling template for microdrops of vitrification solution containing oocytes or embryos [51]. The SSV maximizes cooling rates, leaves ample room for tissue, and prevents the formation of gas phase liquid nitrogen bubbles [52]. This technique was initially created for use

with mammalian oocytes [51]. To store the vitrified droplets in liquid nitrogen, they are put into a cryovial. Sperm can be stuffed into tiny capillaries in this experiment. The capillaries can be permitted to be exposed to the cryo-chilly chamber's (−180°C) surface. The benefits of this approach include lessened DNA damage and lessened sperm tail damage [53].

#### **5. Cryo survival rate**

The sperm quality after thawing is believed to be influenced by its pre-freezing qualities. The cryo survival rate of post-thawed sperm can also be impacted by pre-freezing semen quality factors, such as sperm motility and the abstinence time of sperm donors [54]. Moreover, sperm with aberrant motility characteristics (asthenozoospermia and oligoasthenozoospermia) are especially vulnerable to cryo-damage, which could lower their fertility [55]. The sperm plasma membrane's lipid content has a significant impact on the sperm's cryotolerance and cold sensitivity. The size, shape, and lipid composition of sperm from various species may vary, which could have an impact on how resistant they are to cryo-injuries [56–58].

Two competing theories attempt to explain why cryopreserving the gametes is harmful. (i) The creation of intracellular ice crystals during cryopreservation causes severe osmotic and physical damage to the sperm, which later reduced sperm functioning [34], and (ii) during chilling, ice crystals form inside the cells, and fatal increases in solute concentration occur in the remaining liquid phase. The cryopreservation and thawing procedure during cryopreservation creates oxidative, osmotic, and thermal pressures, which might compromise sperm quality and lead to a low fertility rate [33]. Despite advancements in cryopreservation technology, the rate of functional post-thawed sperm recovery remains low [59].

The cryo survival rate of oocytes cryopreserve with the use of the slow freezing method ranged from 74–90% [60]. Oocytes are prone to ice recrystallization episodes during storage and thawing [54, 61, 62]. During the thawing process, sperm and oocytes are vulnerable to metabolic damage caused by oxidative stress [63]. The cytoskeleton, lipid droplets, membrane system, and microtubules are the parts of the cell that are most impacted [49]. Oocyte cell membranes resemble female mammalian gametes visually and are much more vulnerable to the effects of cooling than embryonic and even zygotic membranes [63]. Oocyte membranes have a high melting point, making it easier for the lipids to be damaged by a drop in temperature and lose their ability to function as a membrane.

Transzonal processes, tiny microfilaments that keep the meiotic spindle in the proper position during maturation, are damaged by cryopreservation, which has an impact on communication between oocytes and the cumulus cells around them [64]. It was recently discovered that the gamete's lipid and adenosine triphosphate content is influenced by the number of cumulus cells adhering to the oocyte [65]. The decreased survival and development rates of cryopreserved immature and *in vitro* matured oocytes are believed to be mostly caused by this [64].

#### **6. Conclusion**

The maintenance of fertility through semen/sperm and oocytes cryopreservation is a crucial component of assisted reproductive techniques, although sperm and oocytes functions may be negatively impacted by cryodamage to cellular constituents. However, the effectiveness of freezing sperm and oocytes can be improved by comprehending the cellular and molecular alterations. To optimize cryopreservation and thawing methodologies, increasing pregnancy rates in IVF cycles, better understand the role of oxidative stress in the lower developmental competency of cryopreserved gametes, and additional studies are required.

The extenders, mediums, kind of molecules, and concentration utilized for each species samples are all related to the varied effects of each antioxidant supplementation, which improves distinct measures of sperm quality. Antioxidants are increasingly common when semen is being cryopreserved because they may lessen oxidation. Certain antioxidants have been shown to have superior efficacy, while others have less encouraging outcomes. Antioxidant combination, extender concentration, and quality are just a few of the variables that might make adding antioxidants to semen extender during cryopreservation successful or unsuccessful. The development of more effective methods for cryopreservation of cells and the expansion of their clinical applications may be made possible by understanding the underlying chemistry and biology of freezing and thawing processes.

#### **Acknowledgements**

The Agricultural Research Council (ARC) is acknowledged for funding the running costs and providing the facility for the project, the Department of Agriculture Land Reform and Rural Development (DALRRD), South Africa is acknowledged for funding the running costs of this research. Agricultural Research Council, Animal Production, Germplasm Conservation and Reproductive Biotechnologies colleagues for their support.

### **Conflict of interest**

There are no conflicts of interest.

*The Current Status of Semen and Oocytes Cryopreservation DOI: http://dx.doi.org/10.5772/intechopen.107404*

#### **Author details**

Masindi Mphaphathi1 \*, Mahlatsana Ledwaba1,2 and Mamonene Thema1,2

1 Agricultural Research Council, Animal Production, Germplasm, Conservation and Reproduction Biotechnologies, Pretoria, South Africa

2 Faculty of Science, Department of Animal Sciences, Tshwane University of Technology, Pretoria, South Africa

\*Address all correspondence to: masindim@arc.agric.za

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Yang H, Tiersch TR. Concepts, history, principles, and application of germplasm cryopreservation technology. Fisheries and Aquatic Sciences. 2020;**5**:1- 10. DOI: 10.32473/edis-fa223-2020

[2] Panyaboriban S, Tharasanit T, Chankitisakul V, Swangchan-Uthai T, Techakumphu M. Treatment with chemical delipidation forskolin prior to cryopreservation improves the survival rates of swamp buffalo (Bubalus bubalis) and bovine (Bos indicus) in vitro produced embryos. Cryobiology. 2018;**84**:46-51. DOI: 10.1016/j.cryobiol. 2018.08.003

[3] Chen SU, Yang YS. Slow freezing or vitrification of oocytes: Their effects on survival and meiotic spindles, and the time schedule for clinical practice. Taiwanese Journal of Obstetrics and Gynecology. 2009;**48**(1):15-22. DOI: 10.1016/S1028-4559(09)60030-9

[4] Prentice JR, Anzar M. Cryopreservation of mammalian oocyte for conservation of animal genetics. Veterinary medicine international. 2011;**2011**:146405. DOI: 10.4061/2011/ 146405

[5] Dohle GR. Male infertility in cancer patients: Review of the literature. International journal of urology. 2010;**17**(4):327-331. DOI: 10.1111/j. 1442-2042.2010.02484.x

[6] Aljaser FS. Cryopreservation methods and Frontiers in the art of freezing life in animal models. Animal Reproduction. 2022;**5**:13

[7] Amorim CA, Curaba M, Van Langendonckt A, Dolmans MM, Donnez J. Vitrification as an alternative means of cryopreserving ovarian tissue. Reproductive biomedicine online. 2011;**23**(2):160-186. DOI: 10.1016/j. rbmo.2011.04.005

[8] Morris GJ. Rapidly cooled human sperm: no evidence of intracellular ice formation. Human Reproduction. 2006;**21**(8):2075-2083. DOI: 10.1093/ humrep/del116

[9] Morris GJ, Acton E, Murray BJ, Fonseca F. Freezing injury: The special case of the sperm cell. Cryobiology. 2012;**64**(2):71-80. DOI: 10.1016/j. cryobiol.2011.12.002

[10] Isachenko E, Isachenko V, Katkov II, Dessole S, Nawroth F. Vitrification of mammalian spermatozoa in the absence of cryoprotectants: From past practical difficulties to present success. Reproductive biomedicine online. 2003;**6**(2):191-200. DOI: 10.1016/ S1472-6483(10)61710-5

[11] AbdelHafez F, Bedaiwy M, El-Nashar SA, Sabanegh E, Desai N. Techniques for cryopreservation of individual or small numbers of human spermatozoa: A systematic review. Human Reproduction Update. 2009;**15**(2):153-164. DOI: 10.1093/ humupd/dmn061

[12] Blanchard TL, Varner DD, Schumacher J, Love CC, Brinsko SP, Rigby SL. Semen preservation. In: Manual of Equine Reproduction. Second ed. Philadelphia: Elsevier Health Sciences; 2003. pp. 165-177. DOI: 10.1016/B0-32-301713-4/50015-7

[13] Zamora R, Hidalgo FJ. Lipoproteins. In: Encyclopedia of Food and Health. Cambridge, Massachusetts: Academic Press; 2016. pp. 544-549. DOI: 10.1016/ B978-0-12-384947-2.00422-0

*The Current Status of Semen and Oocytes Cryopreservation DOI: http://dx.doi.org/10.5772/intechopen.107404*

[14] Bustani GS, Baiee FH. Semen extenders: An evaluative overview of preservative mechanisms of semen and semen extenders. Veterinary World. 2021;**14**(5):1220-1233. DOI: 10.14202/ vetworld.2021.1220-1233

[15] Estrada AJ, Samper JC. Evaluation of raw semen. In: Current Therapy in Equine Reproduction. Philadelphia: Elsevier; 2007. pp. 253-257. DOI: 10.1016/ B978-0-7216-0252-3.50044-8

[16] Fichtner T, Kotarski F, Hermosilla C, Taubert A, Wrenzycki C. Semen extender and seminal plasma alter the extent of neutrophil extracellular traps (NET) formation in cattle. Theriogenology. 2021;**160**(Complete):72-80. DOI: 10.1016/j.theriogenology.2020.10.032

[17] Johnston SD, Zee YP, López-Fernández C, Gosálvez J. The effect of chilled storage and cryopreservation on the sperm DNA fragmentation dynamics of a captive population of koalas. Journal of Andrology. 2012;**33**(5):1007-1015. DOI: 10.2164/ jandrol.111.015248

[18] Althouse GC. Artificial Insemination in Swine: Boar Stud Management. In: Current Therapy in Large Animal Theriogenology. Second ed. Philadelphia: Elsevier; 2007. pp. 731-738. DOI: 10.1016/ B978-072169323-1.50100-8

[19] Jungare KA, Radha R, Sreekanth D. Cryopreservation of biological samples–a short review. Materials Today: Proceedings. 2021;**15**:1637-1641

[20] Whaley D, Damyar K, Witek RP, Mendoza A, Alexander M, Lakey JR. Cryopreservation: An overview of principles and cell-specific considerations. Cell Transplantation. 2021;**30**:0963689721999617. DOI: 10.1177%2F0963689721999617

[21] Purdy PH. A review on goat sperm cryopreservation. Small ruminant research. 2006;**63**(3):215-225. DOI: 10.1016/j.smallrumres.2005.02.015

[22] Sharma Y, Sharma M. Sperm cryopreservation: Principles and biology. Journal of Infertility and Reproductive Biology. 2020;**8**(3):43-48. DOI: 10.47277/ JIRB/8(3)/43

[23] Prien S, Iacovides S. Cryoprotectants & cryopreservation of equine semen: A review of industry cryoprotectants and the effects of cryopreservation on equine semen membranes. Journal of Dairy, Veterinary and Animal Research. 2016;**3**(1):1-8

[24] Bai C, Wang X, Lu G, Wei L, Liu K, Gao H, et al. Cooling rate optimization for zebrafish sperm cryopreservation using a cryomicroscope coupled with SYBR14/PI dual staining. Cryobiology. 2013;**67**(2):117-123. DOI: 10.1016/j. cryobiol.2013.05.011

[25] Vishwanath R, Shannon P. Storage of bovine semen in liquid and frozen state. Animal Reproduction Science. 2000;**62**(1-3):23-53. DOI: 10.1016/ S0378-4320(00)00153-6

[26] Pruß D, Yang H, Luo X, Liu D, Hegermann J, Wolkers WF, et al. Highthroughput droplet vitrification of stallion sperm using permeating cryoprotective agents. Cryobiology. 2021;**101**:67-77. DOI: 10.1016/j. cryobiol.2021.05.007

[27] Sarıözkan S, Bucak MN, Tuncer PB, Ulutaş PA, Bilgen A. The influence of cysteine and taurine on microscopic–oxidative stress parameters and fertilizing ability of bull semen following cryopreservation. Cryobiology. 2009;**58**(2):34-38. DOI: 10.1016/j. cryobiol.2008.11.006

[28] Winn E, Whitaker BD. Quercetin supplementation to the thawing and incubation media of boar sperm improves post-thaw sperm characteristics and the in vitro production of pig embryos. Reproductive Biology. 2020;**20**(3):315-320. DOI: 10.1016/j. repbio.2020.06.002

[29] Aitken RJ, Baker MA. Oxidative stress and male reproductive biology. Reproduction, Fertility and development. 2004;**16**(5):581-588. DOI: 10.1071/RD03089

[30] Gadea J, Sellés E, Marco MA, Coy P, Matás C, Romar R, et al. Decrease in glutathione content in boar sperm after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Theriogenology. 2004;**62**(3-4):690-701. DOI: 10.1016/j. theriogenology.2003.11.013

[31] Andrabi SM, Ansari MS, Ullah N, Afzal M. Effect of nonenzymatic antioxidants in extender on post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa. Pakistan veterinary journal. 2008;**28**(4):159-162

[32] Garg A, Kumaresan A, Ansari MR. Effects of hydrogen peroxide (H2O2) on fresh and cryopreserved buffalo sperm functions during incubation at 37 oC in vitro. Reproduction in domestic animals. 2009;**44**(6):907-912. DOI: 10.1111/j. 1439-0531.2008.01115.x

[33] Tariq M, Khan MS, Shah MG, Nisha AR, Umer M, Hasan SM, et al. Exogenous antioxidants inclusion during semen cryopreservation of farm animals. Journal of Chemical and Pharmaceutical Research. 2015;**7**(3):2273-2280

[34] Ledwaba MR, Mphaphathi ML, Thema MA, Pilane CM, Nedambale TL. Investigation of the efficacy of Dithiothreitol and glutathione on In vitro fertilization of cryopreserved large white

boar semen. Animals. 2022;**12**(9):1137. DOI: 10.3390/ani12091137

[35] Bucak MN, Sarıözkan S, Tuncer PB, Sakin F, Ateşşahin A, Kulaksız R, et al. The effect of antioxidants on postthawed angora goat (Capra hircus ancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small Ruminant Research. 2010;**89**(1):24-30. DOI: 10.1016/j. smallrumres.2009.11.015

[36] Wong KM, Mastenbroek S, Repping S. Cryopreservation of human embryos and its contribution to in vitro fertilization success rates. Fertility and sterility. 2014;**102**(1):19-26. DOI: 10.1016/j.fertnstert.2014.05.027

[37] Sharma R, Master K, Agarwal A. Sperm cryopreservation. In: Manual of Sperm Retrieval and Preparation in Human Assisted Reproduction. Vol. 10. New York: Cambridge University Press; 2021. p. 99

[38] Li XH, Chen SU, Zhang X, Tang M, Kui YR, Wu X, et al. Cryopreserved oocytes of infertile couples undergoing assisted reproductive technology could be an important source of oocyte donation: A clinical report of successful pregnancies. Human Reproduction. 2005;**20**(12):3390-3394. DOI: 10.1093/ humrep/dei262

[39] Parmegiani L, Cognigni GE, Bernardi S, Ciampaglia W, Infante F, Pocognoli P, et al. Freezing within 2 h from oocyte retrieval increases the efficiency of human oocyte cryopreservation when using a slow freezing/rapid thawing protocol with high sucrose concentration. Human Reproduction. 2008;**23**(8):1771-1777. DOI: 10.1093/humrep/den119

[40] Tharasanit T, Thuwanut P. Oocyte cryopreservation in domestic animals

*The Current Status of Semen and Oocytes Cryopreservation DOI: http://dx.doi.org/10.5772/intechopen.107404*

and humans: Principles, techniques and updated outcomes. Animals. 2021;**11**(10):2949

[41] De Santis L, Coticchio G. Reprint of: Theoretical and experimental basis of slow freezing. Reproductive biomedicine online. 2011;**23**(3):290-297

[42] Di Santo M, Tarozzi N, Nadalini M, Borini A. Human sperm cryopreservation: Update on techniques, effect on DNA integrity, and implications for ART. Advances in urology. 2011;**2012**:854837. DOI: 10.1155/2012/854837

[43] Evans G, Maxwell WM. Frozen storage of semen. In: Salamon's Artificial Insemination of Sheep and Goats. Wellington: Butterworths; 1987. pp. 122-141

[44] Karthikeyan M, Arakkal D, Mangalaraj AM, Kamath MS. Comparison of conventional slow freeze versus permeable cryoprotectant-free vitrification of abnormal semen sample: A randomized controlled trial. Journal of Human Reproductive Sciences. 2019;**12**(2):150

[45] Lucena E, Bernal DP, Lucena C, Rojas A, Moran A, Lucena A. Successful ongoing pregnancies after vitrification of oocytes. Fertility and sterility. 2006;**85**(1):108-111. DOI: 10.1016/j. fertnstert.2005.09.013

[46] Borges ED, Vireque AA. Updating the impact of lipid metabolism modulation and lipidomic profiling on oocyte cryopreservation. EMJ. 2019;**4**(1):79-87

[47] Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reproductive biomedicine online. 2005;**11**(3):300-308. DOI: 10.1016/S1472-6483(10)60837-1

[48] Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method. Theriogenology. 2007;**67**(1):73-80. DOI: 10.1016/j. theriogenology.2006.09.014

[49] Vajta G, Nagy ZP. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reproductive biomedicine online. 2006;**12**(6):779-796. DOI: 10.1016/ S1472-6483(10)61091-7

[50] Chemineau P, Guérin Y, Orgeur P, Vallet JC. Training manual on artificial insemination in sheep and goats. FAO. 1991;**83**:222

[51] Dinnyés A, Dai Y, Jiang S, Yang X. High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Biology of Reproduction. 2000;**63**(2):513-518. DOI: 10.1095/ biolreprod63.2.513

[52] Bagis H, Sagirkaya H, Mercan HO, Dinnyes A. Vitrification of pronuclearstage mouse embryos on solid surface (SSV) versus in cryotube: Comparison of the effect of equilibration time and different sugars in the vitrification solution. Molecular Reproduction and Development: Incorporating Gamete Research. 2004;**67**(2):186-192

[53] Satirapod C, Treetampinich C, Weerakiet S, Wongkularb A, Rattanasiri S, Choktanasiri W. Comparison of cryopreserved human sperm from solid surface vitrification and standard vapor freezing method: On motility, morphology, vitality and DNA integrity. Andrologia. 2012;**44**:786-790. DOI: 10.1111/j.1439-0272.2011.01267.x

[54] Munck ND, Vajta G, Rienzi L. Oocyte cryopreservation technique. In: Preventing Age Related Fertility Loss. Cham: Springer; 2018. pp. 87-101

[55] Borges E Jr, Rossi LM, de Freitas CV, Guilherme P, Bonetti TC, Iaconelli A, et al. Fertilization and pregnancy outcome after intracytoplasmic injection with fresh or cryopreserved ejaculated spermatozoa. Fertility and sterility. 2007;**87**(2):316-320. DOI: 10.1016/j. fertnstert.2006.06.032

[56] Maldjian A, Pizzi F, Gliozzi T, Cerolini S, Penny P, Noble R. Changes in sperm quality and lipid composition during cryopreservation of boar semen. Theriogenology. 2005;**63**(2):411-421. DOI: 10.1016/j. theriogenology.2004.09.021

[57] Esmaeili V, Shahverdi AH, Moghadasian MH, Alizadeh AR. Dietary fatty acids affect semen quality: A review. Andrology. 2015;**3**(3):450-461. DOI: 10.1111/andr.12024

[58] Fattah A, Sharafi M, Masoudi R, Shahverdi A, Esmaeili V, Najafi A. L-carnitine in rooster semen cryopreservation: Flow cytometric, biochemical and motion findings for frozen-thawed sperm. Cryobiology. 2017;**74**:148-153. DOI: 10.1016/j. cryobiol.2016.10.009

[59] Majzoub A, Agarwal A. Antioxidants in sperm cryopreservation. In: Male Infertility. Cham: Springer; 2020. pp. 671-678

[60] Chen SU, Lien YR, Chao KH, Lu HF, Ho HN, Yang YS. Cryopreservation of mature human oocytes by vitrification with ethylene glycol in straws. Fertility and sterility. 2000;**74**(4):804-808. DOI: 10.1016/S0015-0282(00)01516-8

[61] Seki S, Mazur P. Stability of mouse oocytes at −80 C: The role of the recrystallization of intracellular ice. Reproduction (Cambridge, England).

2011;**141**(4):407. DOI: 10.1530% 2FREP-10-0438

[62] Baust JM, Corwin W, Snyder KK, Buskirk RV, Baust JG. Cryopreservation: Evolution of molecular based strategies. In: Biobanking and cryopreservation of stem cells. Vol. 951. Cham: Springer; 2016. pp. 13-29. DOI: 10.1007/ 978-3-319-45457-32

[63] Ghetler Y, Yavin S, Shalgi R, Arav A. The effect of chilling on membrane lipid phase transition in human oocytes and zygotes. Human Reproduction. 2005;**20**(12):3385-3389. DOI: 10.1093/ humrep/dei236

[64] Brambillasca F, Guglielmo MC, Coticchio G, Mignini Renzini M, Dal Canto M, Fadini R. The current challenges to efficient immature oocyte cryopreservation. Journal of assisted reproduction and genetics. 2013;**30**(12):1531-1539. DOI: 10.1007/ s10815-013-0112-0

[65] Auclair S, Uzbekov R, Elis S, Sanchez L, Kireev I, Lardic L, et al. Absence of cumulus cells during in vitro maturation affects lipid metabolism in bovine oocytes. American Journal of Physiology-Endocrinology and Metabolism. 2013;**304**(6):E599-E613. DOI: 10.1152/ajpendo.00469.2012

#### **Chapter 3**

## Female Fertility Preservation: Different Interventions and Procedures

*Amor Houda, Peter Michael Jankowski, Micu Romeo and Hammadeh Mohamad Eid*

#### **Abstract**

A human being is made up of two living cells: the egg and the sperm, which pass the torch of life to the next generation. After zygote, the fertilized egg undergoes a series of mitotic divisions. First division into two cells is called blastomeres, and then four cells to 64 cells are called the morula stage. Five days after fertilization, the embryo reaches the blastocyst stage. This blastocyst is attaching itself to the uterine wall for implantation. Implantation is complete when the blastocyst is fully embedded in the endometrium a few days later. Cryopreservation of ovarian tissue, oocytes, embryos, and blastocysts has become an integral part of improving the success of infertility treatment and fertility preservation. Various cryopreservation strategies have been proposed to enhance cell survival and preserve cellular function. It also increases the efficiency of assisted reproductive technology (ART) procedures, enables biodiversity conservation, and provides protection to a valuable biological material. However, successful cryopreservation requires the use of cryoprotectants. The chemical and physical effects of these reagents/processes cause extensive cryogenic damage to the plasma membrane, leading to changes in its normal function. In this chapter, we will discuss different interventions to preserve fertility, including cryopreservation methods and cryoprotectants used.

**Keywords:** zygotes, embryos, freezing, slow freezing, vitrification

#### **1. Introduction**

The unexpected discovery of the cryoprotective properties of glycerol revolutionized the field of cryopreservation and sparked a great deal of further research [1]. However, cryoprotectant toxicity, a fundamental barrier to realizing the full potential of artificial cryoprotection, generally remains a little-known phenomenon.

Successful cryopreservation of sensitive cells, tissues, and organs requires the use of cryoprotectants [2]. Cryoprotectants work by increasing the concentration of solutes in cells. However, in order to be biologically viable, they must be easily permeable and non-toxic to cells. The toxicity of CPA depends on the inherent properties of the chemical itself. Therefore, the toxicity of cryoprotectants limits the concentration available, thereby limiting the cryoprotective efficiency of these agents [2–5].

Of course, longer CPA exposure time increases toxicity, which may be non-specific toxicity due to the interference of water molecules with cell membranes or specific toxicity due to CPA type and concentration [6, 7].

Gook identified the delicate balance between protection and toxicity associated with the use of glycerol and other cryoprotectants such as propylene glycol and ethylene glycol [8]. However, some cryo-damage is inevitable regardless of the use of cryoprotectants, as each cell type responds differently to cryoprotectants and cryopreservation.

There are two main categories of cryoprotectants: osmotic cryoprotectants and non-permeable cryoprotectants [9, 10].

#### **1.1 Permeable cryoprotective agents (CPAs)**

Permeable cryoprotectants include glycerol, methanol, dimethyl sulfoxide (DMSO), dimethylacetaldehyde, ethylene glycol, and propylene glycol. They are low molecular weight chemicals that penetrate the plasma membrane and displace water in cells [11]. However, high concentrations of osmotic cryoprotectants can prevent ice formation during cryopreservation of cells, tissues, and organs at low temperatures. However, CPA becomes more and more toxic as the concentration increases [6].

Cryoprotectants work by increasing the concentration of solutes in cells. However, in order to be biologically viable, they must be easily permeable and non-toxic to cells. The toxicity of CPA depends on the inherent properties of the chemical itself. Exposure time and temperature. Of course, longer CPA exposure time increases toxicity [6].

#### *1.1.1 Glycerol*

Glycerol is a non-electrolyte compound, so it can reduce the electrolyte concentration in the remaining unfrozen solution in and around the battery at any given temperature. It has been used for many years in cryobiology as a cryoprotectant for blood cells, animal sperm, and bacteria, which can be stored in liquid nitrogen at low temperatures [12].

In 1937, glycerol was used as a freezing medium for semen from bulls, rams, stallions, boars, and rabbits during the freezing stage (−21°C). About 10 years later, Polge et al. demonstrated the positive effect of glycerol on frozen poultry sperm [1]. However, glycerol is toxic to spermatozoa through protein denaturation, modification of actin interactions, and induction of plasma membrane fragility during cryopreservation [13, 14]. Good cryoprotective effects were obtained when the glycerol concentration was in the range of 0.5–2 M [15].

Therefore, mixtures of cryoprotectants have been shown to be less toxic and more effective than using a single CPA [6].

#### *1.1.2 Dimethyl sulfoxide (DMSO)*

DMSO and glycerol are probably the two most widely studied CPAs, although the relative success rates of each generally vary by species [16]. Since the early history of

cryopreservation, DMSO has been the cryoprotectant of choice for most animal cell systems.

Lovelock and Bishop were the first to document the ability of DMSO to attenuate freezing-induced cellular damage during slow cooling of bull semen [12]. DMSO also protects against certain aspects of biological damage and radiation damage suffered during cryopreservation. It is estimated that the hydrogen bond between DMSO and water is about 30% stronger than that between two water molecules [17].

DMSO crosses biofilms with great ease, with minimal evidence of damage, and has extensive solvation properties [18]. In 1988, Friedler et al. showed that DMSO was more effective than glycerol [19].

Nonetheless, its impact on cell biology and apparent toxicity to patients has been an ongoing topic of discussion, driving research into less cytotoxic CPAs. Cell membrane toxicity is the most common type of specific toxicity associated with DMSO.

Shu et al. also reviewed the effects of DMSO on a variety of organisms and biological systems [20]. However, DNA methylation and histone alterations have been reported to reduce survival and induce cell differentiation [21, 22].

#### *1.1.3 Ethylene glycol (EG) and propylene glycol (PG)*

Ethylene glycol (EG) is approximately half as permeable to human egg cells as propylene glycol (1,2-propanediol) (PG) or dimethyl sulfoxide (DMSO), thus increasing membrane damage through osmotic stress. However, EG is the cryoprotectant of choice because it is less toxic than other drugs [23].

It is important to note that using a relatively high concentration of EG (15%) was prepared in an equimolar mixture with DMSO. This suggests that post-vitrified blastocyst transfer has no negative impact on perinatal outcomes compared to postvitrification post-transfer [24].

Most embryos are more permeable to PG than to glycerol. PG has few systemic toxic effects and is used safely in many foods. PG has been used as an antidote for EG poisoning [25]. Nevertheless, PG (1,2-propanediol) is often toxic when used as a CPA agent. For example, 1,2-propanediol above 2.5 M has been shown to impair the developmental potential of mouse zygotes by lowering the intracellular pH [26].

#### **1.2 Non-permeable cryoprotectants**

Impermeable cryoprotectants are a special class of high molecular weight, impermeable molecules. These include starches such as hydroxyethyl starch and polyethylene oxide, sugars such as trehalose and sucrose, and polyvinylpyrrolidone. They cannot enter cells and therefore remain extracellular during cryopreservation by exerting osmotic effects to support rapid cell dehydration, lower freezing temperature of the medium, and reduce extracellular ice formation [27, 28]. They are used to protect cells from rapid cooling [9–11].

Due to their low toxicity, they are commonly used as extracellular CPAs. Typically, these are not used alone, but together with permeable intracellular standard CPA [29]. However, glucose has specific toxicities such as binding to proteins [30] and causing glycation as a reducing sugar [31].

Many studies report that trehalose is superior to other sugars such as trehalose. In maintaining membrane stability, liposome stability is maintained during drying and preservation of biomaterials [32].

Sucrose is considered a cosmic [33]. Sucrose is used as an extracellular CPA for embryo and oocyte vitrification [34]. However, under acidic conditions, sucrose is more easily hydrolyzed to reducing sugar monosaccharides than the disaccharide trehalose [32].

#### **2. Cryopreservation methods**

#### **2.1 Controlled slow-rate freezing**

Slow freezing or slow programmable freezing technology was introduced in 1966 [35]. This cryopreservation technique was introduced in the 1980s. As the name suggests, it involves slow freezing of eggs or embryos. Treat the cells first with antifreeze (antifreeze) to protect them in the process. Then gradually cool; at a rate of 1–2°C per minute: from +24°C to −7°C, then sowing, 10 minutes after sowing the temperature drops to −30°C, and finally immersion to −196°C for storage of liquid nitrogen [36]. However, optimal cooling rates vary widely between cell and tissue types [37].

#### **2.2 Vitrification**

The word vitrification comes from the Latin vitrum, meaning glass [38]. Vitrification has replaced traditional slow freezing as the primary method for gamete and embryo cryopreservation, while reproductive cryopreservation is slowly shifting research focus to vitrification, a cheaper, faster, and simpler technique [39–41].

Vitrification differs from slow freezing in that it avoids the formation of ice crystals in the intracellular and extracellular spaces [38]. So many laboratories around the world have completely replaced slow freezing with vitrification in order to improve cryogenic survival outcomes [42, 43].

Vitrification is an alternative freezing method based on the solidification of solutions at low temperatures, not by ice crystallization, but by a large increase in viscosity during cooling [44]. It is achieved by briefly exposing the embryos to high concentrations of cryoprotectant (~7–8 M), followed by direct immersion of the embryos in liquid nitrogen, resulting in ultra-rapid cooling at approximately 20,000°C/min. With this technique, a glassy amorphous state can be achieved, and the formation of intracellular and extracellular ice crystals is prevented [45].

This technique can be used to freeze sperm, oocytes, fertilized oocyte (zygotic) embryos, umbilical cord blood, and reproductive tissue in testes or ovaries [46].

#### **3. Fertility preservation interventions**

Fertility preservation may be indicated for the following indications: Women diagnosed with cancer, women with a disease, surgery, or treatment that may affect future fertility (including lupus, endometriosis, and Turner syndrome) Fertility declines with age in women, transgender men, and women worried about aging.

#### **3.1 Oocyte cryopreservation**

Oocytes are cells about 120 microns in diameter with a thick membrane called the zona pellucida. Egg cells are often referred to as the largest cells in the human body.

Surface and volume play important roles in the outcome of cryobiological processes. Therefore, freezing and thawing of unfertilized oocytes require extensive empirical and theoretical knowledge [45, 47].

Oocyte cryopreservation has become an important method for preserving female fertility in medical and non-medical indications [48, 49].

For women with age-related selective fertility, without a male partner, or without donor sperm, oocyte cryopreservation may offer another experimental option under stringent institutional review board (IRB) protocols, early data show promising results [50].

Unfortunately, oocyte cryopreservation is technically more complex than embryo cryopreservation, and unfertilized oocytes are more susceptible to damage during cryopreservation, so these procedures may have lower rates of unsuccessful pregnancies [51]. Cryopreservation of unfertilized oocytes is more technically challenging than embryo cryopreservation but has less ethical and legal implications.

Cryopreservation of human oocytes can be performed by conventional slow freezing or vitrification [46, 52]. Cryopreservation of immature oocytes in prophase I (follicle stage) has been proposed as an alternative to standard oocyte cryopreservation, as these oocytes are thought to be less susceptible to cryo-damage due to the absence of a spindle and different Membrane permeability [53].

ICSI is recommended for insemination of frozen and thawed oocytes because this method offers a reasonable chance of fertilization compared to in vitro fertilization [54].

Chen in 1986 reported the first pregnancy resulting from the slow freezing and rapid thawing of human eggs using DMSO (dimethyl sulfoxide) as a cryoprotectant [55].

Van Uem reported the second litter after cryopreservation of oocytes using a cryopreservation technique different from that described by Chen [56]. Several pregnancies have been reported after oocytes were cryopreserved—thawed and received intracytoplasmic sperm injection (ICSI) [57].

Kuleshova announced the birth of the first child from vitrified oocytes. The newborns were normal and healthy [58]. Other authors successfully used vitrification and found another 10 pregnancies [59].

The total number of children born after fertilization of frozen and thawed oocytes worldwide exceeds 1500 [51, 60]. Furthermore, no intellectual and/or developmental deficits have been found in children born from frozen oocytes [54, 61, 62].

Slow freezing is one of the methods of cryopreservation of oocytes. Compared with the fresh cycle, it has some limitations, such as low oocyte survival [63–65], increased risk of oocyte senescence [63, 66, 67], and reduced embryonic development [63].

Cao et al. conducted a randomized study to compare survival, fertilization, early embryonic development, and meiotic spindles in slowly frozen and vitrified and thawed human oocytes (n = 605) Assembly and Chromosome Arrangement. The vitrification group had significantly higher oocyte survival rate, fertilized egg and developing embryo division rate, and blastocyst development percentage than the slow freezing group (91.8%, 78.0%, 24.0%, 12.0% vs. 61.0%), 54.4%, 42.3%, and 33.1%, respectively). They also noted that vitrification was superior to slow freezing methods, resulting in improved oocyte survival, fertilization, and embryonic development in vitro [68].

Konc et al. used Polscope to determine the presence, position, and spindle dynamics/displacement in each oocyte. They examined frozen and thawed human oocytes

before and after thawing and for 3 h in culture and found that the spindle did not always return to its original position within the oocyte [69].

After thawing and culturing, they were able to see spindles in 84.3% of the oocytes. However, vitrified oocytes tend to reassemble their spindles more efficiently and faster than slowly cooled oocytes [70]. Cobo et al. found comparable spindle recovery after vitrification and slow freezing for 3 hours [62].

Cobo et al. in an oocyte donation program published the results of a randomized controlled trial of more than 3000 fresh oocytes and 3000 vitrified oocytes (92.5% survival rate). Randomized controlled trials demonstrated no adverse effects of vitrification on subsequent fertilization, development, or implantation [71]. Nagy et al. have also reported similar results in an oocyte donation program and Herrero et al. use the same vitrification protocol [72, 73].

#### **3.2 Pronuclear stage (2PN) cryopreservation**

Until recently, our laboratory and others in Germany have focused on cryopreservation of embryos at the prokaryotic stage (PN). PN freezing was performed because of the reported clinical success rates and if embryo selection and thawing techniques improve over time, it ensures that patients have access to a larger cohort of potential embryos [74]. However, at the PN stage, there is evidence that cryopreserved embryos may suffer from damage to prokaryotic integrity and thus their developmental potential may be significantly impaired [75].

Veeck et al. to improve the overall pregnancy rate per search cycle [76], have described cryopreservation of excess prokaryotic stage embryos. They reported that if cryopreserved prokaryotic oocytes survive freezing, thawing has similar implantation and pregnancy potential compared to fresh conception. However, a limitation noted by these researchers is the low rate of embryo survival after thawing (68%) [76].

In another study of more than 300 single frozen embryo transfers of day 2 4-cell stage embryos and embryos that lost only one blastomere (25%), a similar transfer was performed on fully intact frozen embryos and an efficient operation, and fresh embryos were also obtained [77].

As a result, many centers have completely phased out the use of slow freezing and, after long-term adoption of traditional slow freezing, have been replaced by conventional vitrification procedures.

Schroeder et al. reported a pregnancy rate of 10.2% using slow cryopreservation of cryopreserved human fertilized eggs [78].

Sang Shan et al. compared slow freezing and vitrification methods in cryopreservation of 2PN zygotes and reported a 100% survival rate of 5881 vitrified zygotes using cryotop as a carrier [79].

Among 340 vitrified embryos, the zygotic PN stage after vitrification was reported to have a 100% survival rate, a high pregnancy rate (36.9%), and a low miscarriage rate (17.42%). In addition, vitrification of 2-PN fertilized eggs has a high pregnancy rate of 46.2% and a survival rate of 97% [80].

#### **3.3 Embryo cryopreservation**

Embryo freezing and thawing are considered to have a higher survival rate than oocyte cryopreservation. The first successful embryo cryopreservation was achieved when the research team froze mouse embryos in polyvinylpyrrolidone

*Female Fertility Preservation: Different Interventions and Procedures DOI: http://dx.doi.org/10.5772/intechopen.109052*

(PVP) [81] and the earliest pregnancy in frozen-thawed human embryos was reported in 1983 [82].

Rall and Fahy successfully vitrified embryos using high concentrations of cryoprotectant (CPA) and relatively low cooling and heating rates [38].

Embryo cryopreservation is a critical procedure for embryo transfer (ET) discontinuation due to the risk of ovarian hyperstimulation, endometrial bleeding, elevated serum progesterone levels on the day of ejection, or other unexpected events. There is still much debate about optimal tiers, protocols/procedures, and the use of cryoprotective additives (CPA).

Successful pregnancies and live births by thawing frozen human embryos were first reported in the 1980s. Ferraretti et al. showed that the pregnancy rate (PR) and live birth rate (LBR) of patients who subsequently received cryopreserved embryo thaw were like those of patients who received fresh transfer [83].

The average potential of cryopreserved embryos to become live is about 4%, and babies born from cryopreserved embryos do not exceed 8–10% of the total number of babies born with AR [84].

The results of a retrospective study of 11,768 cryopreserved human embryos that underwent at least one thaw cycle between 1986 and 2007 showed that the length of storage, whether by in vitro fertilization or oocytes, had a significant effect on clinical pregnancy, miscarriage, implantation, or survival. Yield did not significantly affect the donation cycle [85].

Compared to traditional slow-freezing methods, embryo vitrification is a recently introduced ultra-rapid cryopreservation method that prevents freezing within the suspension, transforming it into a glass-like solid, avoiding damage to cells or tissues [86, 87].

Embryo vitrification (VT) was first clinically introduced in Australia in 2006 and is now used for nearly three-quarters of the autologous thaw cycles for transferring blastocysts [88, 89].

Vitrification has been reported to significantly improve pregnancy, delivery, and implantation rates compared to slow freezing of cleavage-stage embryos and blastocysts [90].

Sifer et al. presented the results of a prospective observational study (58 cycles) where early cleavage stage good quality embryos were vitrified and warmed with the results of a retrospective series (189 cycles) where embryos were thawed after a slow freezing procedure (SF). They concluded that the post-thaw survival of vitrified embryos was significantly better than those of embryos resulting from slow freezing procedure. Then, a better clinical pregnancy rate (CPR) per thawed embryo cycle was observed following vitrification [91].

Debrock et al. compared the live birth rate (LBR) per embryo (day 3 cleavage stage embryos) after freezing and thawing by slow freezing or vitrification. They showed that the survival rate after vitrification was significantly higher than that after slow freezing, and the LBR per embryo was significantly higher after vitrification (16%) than after slow freezing (6%) [92].

Zhu et al. compared a retrospective cohort study of 5613 infertile patients with 7862 frozen and thawed day 3 slow frozen (SF) embryos and 3845 vitrified and heated embryos. Day 3 embryos. The proportion of high-quality embryos after thawing in SF was lower than in VT. In a single frozen embryo transfer (FET) cycle, pregnancy and implantation rates were similar between the two groups (35.0 vs. 40.8% and 34.6 vs. 35.9%, respectively). Also, for dual FET, pregnancy rates per cycle were similar between groups (58.8% vs. 58.4%). The implantation rate per embryo transfer was

significantly higher in SF than in VT (38.8% vs. 34.6%). However, SF protocols for cryopreservation of day 3 embryos should be considered [93].

Pooled data from 7 randomized controlled trials (RCTs) (3615 embryos) showed a significant increase in cryopreservation of embryos after vitrification compared to slow freezing (P < 0.001) [94].

When embryos are placed in a freezing solution containing intracellular cryoprotectants (ethylene glycol, propylene glycol, glycerol, dimethyl sulfoxide (DMSO)), due to the extracellular concentration (osmolarity) of cryoprotectants from naive cells Gradient) is higher, intracellular water will leak from the cell. After reaching equilibrium, it gradually diffuses into the cell by cryoprotectant and shrinks until osmotic equilibrium is reached; the cell returns to its normal appearance [95, 96].

The main problem with using cryopreservation techniques is that embryos may be lost due to cryogenic injury. Possible risks of injury to cryopreserved and thawed embryos include exposure to biochemical intracellular ice formation (ICC), cytotoxicity of cryoprotectants due to hyperosmolarity, physical damage (zona pellucida), and deoxygenation during embryo handling ribonucleic acid (RNA) damage. During embryo storage [97].

The most important known mechanism of damage to the cells that occur during cooling to low sub-zero temperature includes chilling injury, ice crystal formation, and fracture damage. In controlled slow freezing, embryos are osmotically equilibrated by incubating in approximately 1–2 M permeable and impermeable CPA prior to freezing. This protects the embryo from the formation of intracellular ice crystals. The extracellular ice is then seeded to form, and the embryos are then cooled at a controlled rate to −30 to −70°C using a programmable slow-speed freezer at 0.2–2.0°C/ minute (min). Finally, embryos are immersed in liquid nitrogen (LN2) for short- or long-term storage [98, 99].

The only danger to cryopreserved cells is suspected to be DNA damage caused by background radiation. Human gametes can safely withstand 3–4 G radiation. Thus, human cells can safely survive for hundreds of years at typical terrestrial background radiation levels of 0.1 cGy/year. However, cosmic rays may be less harmful to embryos stored in high-quality cryogenic tanks than previously thought [100].

Sang Shan et al. vitrified cleavage stage embryos with EG + DMSO+sucrose and showed a small but significant improvement in survival (98% vs. 91%), but no difference in pregnancy rates relative to slow cooling [79]. In a similar comparative study, slow cooling and vitrification were found to have no differences in survival and implantation rates [101].

Balaban et al. observed a higher survival rate (94.8% vs. 88.7%) and a higher rate of intact embryos (73.9% vs. 45.7%) in the vitrification group using the PG + EG + sucrose solution group compared to the slow solution group). Day 3 embryos were frozen in 1.5 MPG + 0.1 M sucrose [102].

#### **3.4 Blastocyst cryopreservation**

Cryopreservation of blastocysts is a challenging task due to the size of blastocysts and presence of blastocysts. Since blastocysts contain a lot of water, the formation of ice crystals may be a major factor affecting blastocyst survival. Cohen et al. reported the first infant born after frozen/thawed blastocyst transfer [103].

Cryopreservation at the blastocyst stage has mainly been performed using slow methods with acceptable results [104–106]. It has been suggested that vitrification results in less apoptosis in blastocysts compared to slow freezing [107].

Outcomes of blastocyst-stage vitrification have improved significantly since 2001 [108, 109], with survival rates as high as 100% [110, 111] and 53% pregnancy rates reported by various investigators [79, 110–112].

Several studies have reported increased blastocyst survival when vesicle volume is artificially reduced using glass microneedles [113], 29-gauge needles [86], and handdrawn Pasteur pipettes for micropipettes [113, 114].

Liebermann and Tucker reported a survival rate of 80.6%. Therefore, highly reproducible vitrification using the Cryotop method is superior to slow freezing. Furthermore, to date, no other technique has consistently achieved the excellent results obtained using this method [115].

Kuwayama et al. found in a comparative study that vitrified blastocysts had a slightly higher survival rate (90%) than slowly cooled blastocysts (84%). However, pregnancy and live birth rates per transfer were not significantly different [79].

In a study of more than 500 blastocysts per group, Liebermann and Tucker found significant differences in survival (96.5% vs. 92.1%), pregnancy per transfer (46.1% vs. 42.9%) and implantation rate (30.6% vs. 28.9%) between the vitrified and slow freezing groups [116].

Loutradi et al. found that blastocyst survival after vitrification was significantly higher than after slow freezing (odds ratio [OR]: 2.20, 95% confidence interval [CI]: 1.53–3.16) [117]. In addition, Hong et al. found a high pregnancy rate (70.5%) and implantation rate (40.6%) when using the new vitrification technique [118].

Recent studies have reported similar clinical outcomes between vitrified blastocyst transfer and fresh blastocyst transfer cycles when similar quality blastocysts were transferred [93, 119].

Cobo et al. vitrified 6019 embryos with cryogenic glass and showed that 97.6% of embryos survived on day 6, compared to 95.7% on day 5, 94.9% on day 2, and 94.9% on day 3, 94.2% at 6 days [120].

#### **3.5 Ovarian tissue cryopreservation**

The ovary has hundreds of primordial follicles containing immature oocytes that are small, quiescent, less differentiated, and devoid of banded cells. Due to the lack of zona pellucida and cortical granules, this immature oocyte can tolerate cryopreservation [121].

Ovarian tissue cryopreservation (OTC) is an evolving technique, although limited outcome information is available. Ovarian tissue can be cryopreserved for later ovarian tissue transplantation in prepubertal patients or when immediate chemotherapy is required [122].

Ovarian tissue is collected laparoscopically and frozen and can later be thawed and reimplanted in situ (in the pelvis) or ectopic (into the subcutaneous tissue of the forearm or abdomen). The cryopreservation process of ovarian tissue involves freezing thin slices of ovarian cortex, which contain a rich reserve of primordial follicles. This method of investigating fertility preservation requires only ovarian cortical tissue [123].

The first frozen-thawed ovarian transplantation was reported in 2000, and since then, several successful pregnancies due to these procedures have been reported [124]. Studies have reported restoration of ovarian function using both approaches [125, 126].

The potential risk of cancer recurrence in preimplantation tissue not exposed to chemotherapy may limit its use in cancer patients, at least until in vitro maturation of immature oocytes becomes more standard [125, 127, 128].

The advantage of cryopreservation of ovarian tissue compared to mature oocytes is that primordial follicles in the ovarian cortex are more resistant to cryoinjury [129]. However, long-term studies have reported graft function for up to 11 years [127, 130].

Porcu et al. reported the first birth of healthy twins in a patient who underwent bilateral oophorectomy for ovarian cancer and was pregnant with her own cryopreserved oocytes [131]. Besides, 131 pregnancies and 75 live births (expected to exceed 200 by 2020) have been reported after slow freezing and transplantation, whereas only 4 deliveries have been described after vitrification [132].

In addition, many deliveries between identical twins using fresh ovarian transfer have been reported [133]. There are also reports of births from two sisters with HLAcompatible whole-fresh ovarian transplants [134].

There are two methods of OTC: slow freezing and vitrification. Early studies have shown that slowly frozen ovarian cortex preserves better than vitrified ovarian tissue [135]. Slow freezing has been the traditional technique for many years, despite reports of massive follicular pool loss and excessive stromal cell damage [136].

Xiao et al. reported that a new vitrification technique was comparable to slow freezing in preserving primordial follicles in human ovarian tissue. The proportion of morphologically prominent primordial follicles was significantly reduced by vitrification compared with slow freezing [93].

To date, only two live births following vitrification of ovarian tissue have been reported and all other live births were caused by slow freezing of the ovarian cortex [137, 138]. Twelve studies collected data on intact primordial follicles, and an overall pooled analysis showed no difference between vitrification and slow freezing for this endpoint [139].

However, it has recently been suggested that vitrification has beneficial effects on granulosa cells and ovarian stroma, providing equal or better results than slow cooling to protect ovarian tissue [139].

#### **4. Storage in LN2 containers after slow freezing and vitrification**

In the field of assisted reproductive technology, little is known about the risks of long-term storage of cryopreserved cells, because vitrification is the solidification of a liquid without forming a crystalline structure—a physically disorganized and therefore potentially unstable system. This raises the question, if this changes over time, does this significantly affect the cryosurvival and implantation potential of vitrified gametes and embryos?

Subsequently, the possible effects on neonatal health remain largely unknown. A study by Wirleiter et al. showed that storage of vitrified blastocysts under sterile conditions did not affect blastocyst viability. In addition, no significant differences were observed in survival rates after warming between the first year of storage and after 5–6 years of storage (83.0% vs. 83.1%); nor did the pregnancy rate decrease (40.0% vs. 38.5%). Furthermore, no increase in neonatal malformation rates was observed over time [140].

To date, there have been no reports of cross-contamination between germ cells and tissues stored in cryovials. Cobo et al. showed that viral sequences (HIV, HBV, and HCV) were not detected in liquid nitrogen samples from containers containing oocytes and embryos from chronically infected patients [120].

*Female Fertility Preservation: Different Interventions and Procedures DOI: http://dx.doi.org/10.5772/intechopen.109052*

To date, neither open systems nor closed systems have resulted in disease transmission during vitrification. However, to ensure biosafety during cryopreservation, aseptic methods are recommended [43].

Germ cells and tissues must be cryopreserved and stored in accordance with European Organization Directive 2006/17/EC (European Union [EU] Directive 2006/17/EC) to prevent pathogen transmission or cross-contamination of samples. Patients must be screened for blood-borne viruses (BBV), such as HIV, Hepatitis B, and C, before processing and freezing gametes/embryos and storing germ cells and tissues for positive and negative patients, respectively. Periodic cleaning of storage containers is also considered good laboratory practice (GLP) for decontamination of viral and microbial agents [141].

#### **5. Conclusion**

Vitrification is now the method of choice for cryopreservation of oocytes due to better results than slow freezing, but more standardized applications are still needed. Transfers of fresh or cryopreserved embryos still performed statistically better than embryo transfers obtained from cryopreserved oocytes. Only a few centers with extensive experience in cryopreservation are comparable between frozen embryo transfer or oocyte cryopreservation embryo transfer.

#### **Conflict of interest**

The authors declare no conflicts of interest.

#### **Author details**

Amor Houda1 \*, Peter Michael Jankowski1 , Micu Romeo2 and Hammadeh Mohamad Eid1

1 Department of Obstetrics and Gynaecology, Saarland University, Germany

2 Obstetrics and Gynaecology Department, University of Medicine and Pharmacy, Cluj-Napoca, Romania

\*Address all correspondence to: houdaamor86@yahoo.fr

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature. 1949;**164**(4172):666. DOI: 10.1038/164666A0

[2] Zhang T, Rawson D, John Morris G. Cryopreservation of pre-hatch embryos of zebrafish (*Brachydanio rerio*). Aquatic Living Resources. 1993;**6**(2):145-153. DOI: 10.1051/alr:1993014

[3] Arakawa T, Carpenter JF, Kita YA, Crowe JH. The basis for toxicity of certain cryoprotectants: A hypothesis. Cryobiology. 1990;**27**(4):401-415. DOI: 10.1016/0011-2240(90)90017-X

[4] Cabrita E, Robles V, Chereguini O, Wallace JC, Herráez MP. Effect of different cryoprotectants and vitrificant solutions on the hatching rate of turbot embryos (*Scophthalmus maximus*). Cryobiology. 2003;**47**(3):204-213. DOI: 10.1016/j.cryobiol.2003.10.001

[5] Cabrita E, Robles V, Wallace JC, Sarasquete MC, Herráez MP. Preliminary studies on the cryopreservation of gilthead seabream (*Sparus aurata*) embryos. Aquaculture. 2006;**251**(2):245-255. DOI: 10.1016/j. aquaculture.2005.04.077

[6] Best BP. Cryoprotectant toxicity: Facts, issues, and questions. Rejuvenation Research. 2015;**18**(5):422-436. DOI: 10.1089/REJ.2014.1656/ASSET/ IMAGES/LARGE/FIGURE1.JPEG

[7] Fahy GM. Cryoprotectant toxicity neutralization. Cryobiology. 2010;**60**(3):S45-S53. DOI: 10.1016/J. CRYOBIOL.2009.05.005

[8] Gook DA. History of oocyte cryopreservation. Reproductive Biomedicine Online. 2011;**23**(3):281-289. DOI: 10.1016/J.RBMO.2010.10.018

[9] Meryman HT. Cryopreservation of living cells: Principles and practice. Transfusion. 2007;**47**(5):935-945. DOI: 10.1111/J.1537-2995.2007.01212.X

[10] Zhang T, Rawson DM, Pekarsky I, Blais I, Lubzens E. Low-temperature preservation of fish gonad cells and oocytes. In: Babin PJ, Cerdà J, Lubzens E, editors. The Fish Oocyte. Dordrecht: Springer; 2007. pp. 411-436. DOI: 10.1007/ 978-1-4020-6235-3\_14

[11] Di Santo M, Tarozzi N, Nadalini M, Borini A. Human sperm cryopreservation: Update on techniques, effect on DNA integrity, and implications for ART. Advanced Urology. 2012;**2012**:12. DOI: 10.1155/2012/854837

[12] Lovelock JE, Bishop MWH. Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature. 1959;**183**(4672):1394-1395. DOI: 10.1038/1831394a0

[13] Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reproduction, Fertility, and Development. 1995;**7**(4):871-891. DOI: 10.1071/ RD9950871

[14] Curry MR, Redding BJ, Watson PF. Determination of water permeability coefficient and its activation energy for rabbit spermatozoa. Cryobiology. 1995;**32**(2):175-181. DOI: 10.1006/ cryo.1995.1016

[15] Isachenko E, Isachenko V, Katkov II, Dessole S, Nawroth F. Vitrification

*Female Fertility Preservation: Different Interventions and Procedures DOI: http://dx.doi.org/10.5772/intechopen.109052*

of mammalian spermatozoa in the absence of cryoprotectants: From past practical difficulties to present success. Reproductive Biomedicine Online. 2003;**6**(2):191-200. DOI: 10.1016/ s1472-6483(10)61710-5

[16] Comizzoli P, Songsasen N, Hagedorn M, Wildt DE. Comparative cryobiological traits and requirements for gametes and gonadal tissues collected from wildlife species. Theriogenology. 2012;**78**(8):1666-1681. DOI: 10.1016/j. theriogenology.2012.04.008

[17] MacGregor WS. The chemical and physical properties of DMSO. Annals of the New York Academy of Sciences. 1967;**141**(1):3-12. DOI: 10.1111/J.1749- 6632.1967.TB34860.X

[18] Brayton CF. Dimethyl sulfoxide (DMSO): A review. The Cornell Veterinarian. 1986;**76**(1):61-90. Available from: http://europepmc.org/abstract/ MED/3510103

[19] Friedler S, Giudice LC, Lamb EJ. Cryopreservation of embryos and ova. Fertility and Sterility. 1988;**49**(5):743-764. DOI: 10.1016/ s0015-0282(16)59879-3

[20] Shu Z, Heimfeld S, Gao D. Hematopoietic SCT with cryopreserved grafts: Adverse reactions after transplantation and cryoprotectant removal before infusion. Bone Marrow Transplantation. 2014;**49**(4):469-476. DOI: 10.1038/bmt.2013.152

[21] Yong KW et al. Phenotypic and functional characterization of longterm cryopreserved human adiposederived stem cells. Scientific Reports. 2015;**5**(1):9596. DOI: 10.1038/srep09596

[22] Noguchi H et al. Cryopreservation of adipose-derived mesenchymal stem cells. Cell Medicine. 2015;**8**:3-7. DOI: 10.3727/215517915X689100

[23] Mullen SF, Li M, Li Y, Chen ZJ, Critser JK. Human oocyte vitrification: The permeability of metaphase II oocytes to water and ethylene glycol and the appliance toward vitrification. Fertility and Sterility. 2008;**89**(6):1812-1825. DOI: 10.1016/J. FERTNSTERT.2007.06.013

[24] Takahashi K, Mukaida T, Goto T, Oka C. Perinatal outcome of blastocyst transfer with vitrification using cryoloop: A 4-year follow-up study. Fertility and Sterility. 2005;**84**(1):88-92. DOI: 10.1016/J. FERTNSTERT.2004.12.051

[25] Holman NW, Mundy RL, Teague RS. Alkyldiol antidotes to ethylene glycol toxicity in mice. Toxicology and Applied Pharmacology. 1979;**49**(2):385-392. DOI: 10.1016/0041-008X(79)90264-3

[26] Damien M, Luciano AA, Peluso JJ. Propanediol alters intracellular pH and developmental potential of mouse zygotes independently of volume change. Human Reproduction. 1990;**5**(2):212- 216. DOI: 10.1093/OXFORDJOURNALS. HUMREP.A137072

[27] Aisen EG, Medina VH, Venturino A. Cryopreservation and post-thawed fertility of ram semen frozen in different trehalose concentrations. Theriogenology. 2002;**57**(7):1801-1808. DOI: 10.1016/S0093-691X(02)00653-2

[28] Cleland D, Krader P, McCree C, Tang J, Emerson D. Glycine betaine as a cryoprotectant for prokaryotes. Journal of Microbiological Methods. 2004;**58**(1):31-38. DOI: 10.1016/J. MIMET.2004.02.015

[29] Jain JK, Paulson RJ. Oocyte cryopreservation. Fertility and Sterility. 2006;**86**(4 Suppl):1037-1046. DOI: 10.1016/J.FERTNSTERT.2006.07.1478 [30] Clark P, Fahy GM, Karow AM. Factors influencing renal cryopreservation. II. Toxic effects of three cryoprotectants in combination with three vehicle solutions in nonfrozen rabbit cortical slices. Cryobiology. 1984;**21**(3):274-284. DOI: 10.1016/0011-2240(84)90323-7

[31] Leslie RDG, Cohen RM. Biologic variability in plasma glucose, hemoglobin A1c, and advanced glycation end products associated with diabetes complications. Journal of Diabetes Science and Technology. 2009;**3**(4):635- 643. DOI: 10.1177/193229680900300403

[32] Crowe JH, Crowe LM, Oliver AE, Tsvetkova N, Wolkers W, Tablin F. The trehalose myth revisited: Introduction to a symposium on stabilization of cells in the dry state. Cryobiology. 2001;**43**(2):89-105. DOI: 10.1006/ CRYO.2001.2353

[33] Moelbert S, Normand B, De Los Rios P. Kosmotropes and chaotropes: Modelling preferential exclusion, binding and aggregate stability. Biophysical Chemistry. 2004;**112**(1):45- 57. DOI: 10.1016/J.BPC.2004.06.012

[34] Kuleshova LL, MacFarlane DR, Trounson AO, Shaw JM. Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes. Cryobiology. 1999;**38**(2):119-130. DOI: 10.1006/ CRYO.1999.2153

[35] Behrman SJ, Sawada Y. Heterologous and homologous inseminations with human semen frozen and stored in a liquid-nitrogen refrigerator. Fertility and Sterility. 1966;**17**(4):457-466. DOI: 10.1016/S0015-0282(16)36003-4

[36] Saragusty J, Arav A. Reproduction review: Current progress in oocyte and embryo cryopreservation by slow

freezing and vitrification. Reproduction. 2011;**141**(1):1. DOI: 10.1530/REP-10-0236

[37] Fuller BJ, Paynter SJ. Cryopreservation of mammalian embryos. Methods in Molecular Biology. 2007;**368**:325-339. DOI: 10.1007/978-1-59745-362-2\_23

[38] Rall WF, Fahy GM. Icefree cryopreservation of mouse embryos at −196°C by vitrification. Nature. 1985;**313**(6003):573-575. DOI: 10.1038/313573a0

[39] Edgar DH, Gook DA. A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Human Reproduction Update. 2012;**18**(5):536- 554. DOI: 10.1093/humupd/dms016

[40] Keros V et al. Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue. Human Reproduction. 2009;**24**(7):1670-1683. DOI: 10.1093/ HUMREP/DEP079

[41] Abdelhafez FF, Desai N, Abou-Setta AM, Falcone T, Goldfarb J. Slow freezing, vitrification and ultrarapid freezing of human embryos: A systematic review and meta-analysis. Reproductive Biomedicine Online. 2010;**20**(2):209-222. DOI: 10.1016/j. rbmo.2009.11.013

[42] Evans J et al. Fresh versus frozen embryo transfer: Backing clinical decisions with scientific and clinical evidence. Human Reproduction Update. 2014;**20**(6):808-821. DOI: 10.1093/ HUMUPD/DMU027

[43] Argyle CE, Harper JC, Davies MC. Oocyte cryopreservation: Where are we now? Human Reproduction Update. 2016;**22**(4):440-449. DOI: 10.1093/ humupd/dmw007

*Female Fertility Preservation: Different Interventions and Procedures DOI: http://dx.doi.org/10.5772/intechopen.109052*

[44] Fahy GM. Vitrification: A new approach to organ cryopreservation. Progress in Clinical and Biological Research. 1986;**224**:305-335. Available from: https://europepmc.org/article/ med/3540994 [Accessed: October 20, 2022]

[45] Kuwayama M. Vitrification of oocytes: General considerations and the use of the Cryotec method. In: Michael T, Juergen L, editors. Vitrification in Assisted Reproduction. Boca Raton: CRC Press; 2015. pp. 94-103. DOI: 10.1201/B19316-14

[46] Vajta G, Kuwayama M. Improving cryopreservation systems. Theriogenology. 2006;**65**(1):236-244. DOI: 10.1016/J. THERIOGENOLOGY.2005.09.026

[47] Dozortsev D et al. The optimal time for intracytoplasmic sperm injection in the human is from 37 to 41 hours after administration of human chorionic gonadotropin. Fertility and Sterility. 2004;**82**(6):1492-1496. DOI: 10.1016/J. FERTNSTERT.2004.09.002

[48] De Vos M, Smitz J, Woodruff TK. Fertility preservation in women with cancer. The Lancet. 2014;**384**(9950): 1302-1310. DOI: 10.1016/S0140-6736 (14)60834-5

[49] Stoop D, Cobo A, Silber S. Fertility preservation for agerelated fertility decline. Lancet. 2014;**384**(9950):1311-1319. DOI: 10.1016/ S0140-6736(14)61261-7

[50] Noyes N, Porcu E, Borini A. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reproductive Biomedicine Online. 2009;**18**(6):769-776. DOI: 10.1016/S1472-6483(10)60025-9

[51] Rodriguez-Wallberg KA, Oktay K. Recent advances in oocyte and ovarian tissue cryopreservation and transplantation. Best Practice & Research. Clinical Obstetrics & Gynaecology. 2012;**26**(3):391-405. DOI: 10.1016/J.BPOBGYN.2012.01.001

[52] Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biology of Reproduction. 1996;**54**(5):1059-1069. DOI: 10.1095/ BIOLREPROD54.5.1059

[53] Son WY et al. Effects of 1,2-propanediol and freezing-thawing on the in vitro developmental capacity of human immature oocytes. Fertility and Sterility. 1996;**66**(6):995-999. DOI: 10.1016/S0015-0282(16)58696-8

[54] Chian RC et al. Live birth after vitrification of in vitro matured human oocytes. Fertility and Sterility. 2009;**91**(2):372-376. DOI: 10.1016/J. FERTNSTERT.2007.11.088

[55] Chen C. Pregnancy after human oocyte cryopreservation. Lancet. 1986;**327**(8486):884-886. DOI: 10.1016/ S0140-6736(86)90989-X

[56] Van Uem JFHM, Siebzehnrübl ER, Schuh B, Koch R, Trotnow S, Lang N. Birth after cryopreservation of unfertilised oocytes. Lancet. 1987;**329**(8535):752-753. DOI: 10.1016/ S0140-6736(87)90398-9

[57] Porcu E, Ciotti PM, Fabbri R, Magrini O, Seracchioli R, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertility and Sterility. 1997;**68**(4):724-726. DOI: 10.1016/S0015-0282(97)00268-9

[58] Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson A. Birth following vitrification of a small number of human oocytes: Case report. Human

Reproduction. 1999;**14**(12):3077-3079. DOI: 10.1093/HUMREP/14.12.3077

[59] Katayama KP, Stehlik J, Kuwayama M, Kato O, Stehlik E. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertility and Sterility. 2003;**80**(1):223-224. DOI: 10.1016/S0015-0282(03)00551-X

[60] Konc J, Kanyo K, Cseh S. Does oocyte cryopreservation have a future in Hungary? Reproductive Biomedicine Online. 2007;**14**(1):11-13. DOI: 10.1016/ S1472-6483(10)60757-2

[61] Porcu E, Fabbri R, Seracchioli R, De Cesare R, Giunchi S, Caracciolo D. Obstetric, perinatal outcome and follow up of children conceived from cryopreserved oocytes. Fertility and Sterility. 2000;**74**(3):S48. DOI: 10.1016/ s0015-0282(00)00849-9

[62] Cobo A, Kuwayama M, Pérez S, Ruiz A, Pellicer A, Remohí J. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the cryotop method. Fertility and Sterility. 2008;**89**(6):1657-1664. DOI: 10.1016/J. FERTNSTERT.2007.05.050

[63] Rienzi L et al. Embryo development of fresh 'versus' vitrified metaphase II oocytes after ICSI: A prospective randomized sibling-oocyte study. Human Reproduction. 2010;**25**(1):66-73. DOI: 10.1093/HUMREP/DEP346

[64] Hull MGR, North K, Taylorb H, Farrow A, Ford WCL. Delayed conception and active and passive smoking. Fertility and Sterility. 2000;**74**(4):725-733. DOI: 10.1016/ S0015-0282(00)01501-6

[65] Van den Abbeel E, Schneider U, Liu J, Agca Y, Critser JK, Van Steirteghem A. Osmotic responses and tolerance limits

to changes in external osmolalities, and oolemma permeability characteristics, of human in vitro matured MII oocytes. Human Reproduction. 2007;**22**(7):1959- 1972. DOI: 10.1093/HUMREP/DEM083

[66] Parmegiani L et al. Freezing within 2 h from oocyte retrieval increases the efficiency of human oocyte cryopreservation when using a slow freezing/rapid thawing protocol with high sucrose concentration. Human Reproduction. 2008;**23**(8):1771-1777. DOI: 10.1093/HUMREP/DEN119

[67] Parmegiani L, Accorsi A, Cognigni GE, Bernardi S, Troilo E, Filicori M. Sterilization of liquid nitrogen with ultraviolet irradiation for safe vitrification of human oocytes or embryos. Fertility and Sterility. 2010;**94**(4):1525-1528. DOI: 10.1016/J. FERTNSTERT.2009.05.089

[68] Cao YX et al. Comparison of survival and embryonic development in human oocytes cryopreserved by slow-freezing and vitrification. Fertility and Sterility. 2009;**92**(4):1306-1311. DOI: 10.1016/J. FERTNSTERT.2008.08.069

[69] Konc J, Kanyo K, Kriston R, Zeke J, Cseh S. Freezing of oocytes and its effect on the displacement of the meiotic spindle: Short communication. Scientific World Journal. 2012;**2012**:785421. DOI: 10.1100/2012/785421

[70] Martínez-Burgos M et al. Vitrification versus slow freezing of oocytes: Effects on morphologic appearance, meiotic spindle configuration, and DNA damage. Fertility and Sterility. 2011;**95**(1):374-377. DOI: 10.1016/J. FERTNSTERT.2010.07.1089

[71] Cobo A, Meseguer M, Remohí J, Pellicer A. Use of cryo-banked oocytes in an ovum donation programme:

*Female Fertility Preservation: Different Interventions and Procedures DOI: http://dx.doi.org/10.5772/intechopen.109052*

A prospective, randomized, controlled, clinical trial. Human Reproduction. 2010;**25**(9):2239-2246. DOI: 10.1093/ HUMREP/DEQ146

[72] Nagy ZP et al. Clinical evaluation of the efficiency of an oocyte donation program using egg cryobanking. Fertility and Sterility. 2009;**92**(2):520-526. DOI: 10.1016/J. FERTNSTERT.2008.06.005

[73] Herrero L, Martínez M, Garcia-Velasco JA. Current status of human oocyte and embryo cryopreservation. Current Opinion in Obstetrics & Gynecology. 2011;**23**(4):245-250. DOI: 10.1097/GCO.0B013E32834874E2

[74] Borini A, Cattoli M, Bulletti C, Coticchio G. Clinical efficiency of oocyte and embryo cryopreservation. Annals of the New York Academy of Sciences. 2008;**1127**(1):49-58. DOI: 10.1196/ ANNALS.1434.012

[75] Isachenko V, Todorov P, Dimitrov Y, Isachenko E. Integrity rate of pronuclei after cryopreservation of pronuclearzygotes as a criteria for subsequent embryo development and pregnancy. Human Reproduction. 2008;**23**(4):819- 826. DOI: 10.1093/HUMREP/DEN002

[76] Veeck LL et al. Significantly enhanced pregnancy rates per cycle through cryopreservation and thaw of pronuclear stage oocytes. Fertility and Sterility. 1993;**59**(6):1202-1207. DOI: 10.1016/S0015-0282(16)55977-9

[77] Edgar DH, Gook DA. How should the clinical efficiency of oocyte cryopreservation be measured? Reproductive Biomedicine Online. 2007;**14**(4):430-435. DOI: 10.1016/ S1472-6483(10)60889-9

[78] Schröder AK et al. Counselling on cryopreservation of pronucleated oocytes. Reproductive Biomedicine Online. 2003;**6**(1):69-74. DOI: 10.1016/ S1472-6483(10)62058-5

[79] Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reproductive Biomedicine Online. 2005;**11**(3):300-308. DOI: 10.1016/S1472-6483(10)60837-1

[80] Youssry M, Youssry M, Ozmen B, Zohni K, Diedrich K, Al-Hasani S. Current aspects of blastocyst cryopreservation. Biomed. Online. 2008;**16**(2):311-320. DOI: 10.1016/ S1472-6483(10)60591-3

[81] Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to −196° and −269°C. Science (80-. ). 1972;**178**(4059):411-414. DOI: 10.1126/ SCIENCE.178.4059.411

[82] Zeilmaker GH, Alberda AT, van Gent I, Rijkmans CM, Drogendijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertility and Sterility. 1984;**42**(2):293-296. DOI: 10.1016/S0015-0282(16)48029-5

[83] Ferraretti AP, Gianaroli L, Magli C, Fortini D, Selman HA, Feliciani E. Elective cryopreservation of all pronucleate embryos in women at risk of ovarian hyperstimulation syndrome: Efficiency and safety. Human Reproduction. 1999;**14**(6):1457-1460. DOI: 10.1093/HUMREP/14.6.1457

[84] De Jong D, Eijkemans MJC, Beckers NGM, Pruijsten RV, Fauser BCJM, Macklon NS. The added value of embryo cryopreservation to cumulative ongoing pregnancy rates per IVF treatment: Is cryopreservation worth the effort? Journal of Assisted Reproduction and Genetics. 2002;**19**(12):561-568. DOI: 10.1023/A:1021211115337

[85] Riggs R, Mayer J, Dowling-Lacey D, Chi TF, Jones E, Oehninger S. Does storage time influence postthaw survival and pregnancy outcome? An analysis of 11,768 cryopreserved human embryos. Fertility and Sterility. 2010;**93**(1):109-115. DOI: 10.1016/J. FERTNSTERT.2008.09.084

[86] Son WY, Tan SL. Comparison between slow freezing and vitrification for human embryos. Expert Review of Medical Devices. 2009;**6**(1):1-7. DOI: 10.1586/17434440.6.1.1

[87] Zegers-Hochschild F et al. The International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) Revised Glossary on ART Terminology, 2009. Human Reproduction. 2009;**24**(11):2683-2687. DOI: 10.1093/HUMREP/DEP343

[88] Costigan S, Henman M, Stojanov T. Birth outcomes after vitrification and slow freezing of supernumerary blastocysts. The Australian & New Zealand Journal of Obstetrics & Gynaecology. 2007;**47**:A6-A6

[89] Chambers GM et al. Assisted reproductive technology in Australia and New Zealand: Cumulative live birth rates as measures of success. The Medical Journal of Australia. 2017;**207**(3):114- 118. DOI: 10.5694/MJA16.01435

[90] Stanger J, Wong J, Conceicao J, Yovich J. Vitrification of human embryos previously cryostored by either slow freezing or vitrification results in high pregnancy rates. Reproductive Biomedicine Online. 2012;**24**(3):314-320. DOI: 10.1016/J.RBMO.2011.11.013

[91] Sifer C et al. Issue de la vitrification des embryons précoces versus congélation lente. Rapport de la première naissance française. Gynécologie,

Obstétrique & Fertilité. 2012;**40**(3):158- 161. DOI: 10.1016/J.GYOBFE.2011.10.004

[92] Debrock S, Peeraer K, Fernandez Gallardo E, De Neubourg D, Spiessens C, D'Hooghe TM. Vitrification of cleavage stage day 3 embryos results in higher live birth rates than conventional slow freezing: A RCT. Human Reproduction. 2015;**30**(8):1820-1830. DOI: 10.1093/ HUMREP/DEV134

[93] Zhu HY et al. Slow freezing should not be totally substituted by vitrification when applied to day 3 embryo cryopreservation: An analysis of 5613 frozen cycles. Journal of Assisted Reproduction and Genetics. 2015;**32**(9):1371-1377. DOI: 10.1007/ S10815-015-0545-8

[94] Rienzi L et al. Oocyte, embryo and blastocyst cryopreservation in ART: Systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Human Reproduction Update. 2017;**23**(2):139. DOI: 10.1093/HUMUPD/DMW038

[95] Liu WX et al. Comparative study between slow freezing and vitrification of mouse embryos using different cryoprotectants. Reproduction in Domestic Animals. 2009;**44**(5):788-791. DOI: 10.1111/j.1439-0531.2008.01078.x

[96] Nicacio AC et al. Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos. Zygote. 2012;**20**(2):117- 122. DOI: 10.1017/S0967199410000717

[97] Sutcliffe AG, D'souza SW, Cadman J, Richards B, Mckinlay IA, Lieberman B. Minor congenital anomalies, major congenital malformations and development in children conceived from cryopreserved embryos. Human Reproduction. 1995;**10**(12):3332-3337.

*Female Fertility Preservation: Different Interventions and Procedures DOI: http://dx.doi.org/10.5772/intechopen.109052*

#### DOI: 10.1093/OXFORDJOURNALS. HUMREP.A135915

[98] Palasz AT, Mapletoft RJ. Cryopreservation of mammalian embryos and oocytes: Recent advances. Biotechnology Advances. 1996;**14**(2):127- 149. DOI: 10.1016/0734-9750(96)00005-5

[99] Youngs CR, Leibo SP, Godke RA. Embryo cryopreservation in domestic mammalian livestock species. CAB Reviews Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources. 2010;**5**(60):11

[100] Rall WF. Cryopreservation of mammalian embryos, gametes, and ovarian tissues: Current issues and progress. In: Wolf DP, Zelinski-Wooten M, editors. Assisted Fertilization and Nuclear Transfer in Mammals. Totowa, USA: Humana Press; 2001. pp. 173-187

[101] Wilding MG et al. Human cleavagestage embryo vitrification is comparable to slow-rate cryopreservation in cycles of assisted reproduction. Journal of Assisted Reproduction and Genetics. 2010;**27**(9-10):549-554. DOI: 10.1007/ S10815-010-9452-1/FIGURES/1

[102] Balaban B et al. A randomized controlled study of human day 3 embryo cryopreservation by slow freezing or vitrification: Vitrification is associated with higher survival, metabolism and blastocyst formation. Human Reproduction. 2008;**23**(9):1976-1982. DOI: 10.1093/HUMREP/DEN222

[103] Cohen J, Simons RF, Edwards RG, Fehilly CB, Fishel SB. Pregnancies following the frozen storage of expanding human blastocysts. Journal of In Vitro Fertilization and Embryo Transfer: IVF. 1985;**2**(2):59-64. DOI: 10.1007/BF01139337

[104] Veeck LL et al. High pregnancy rates can be achieved after freezing and thawing human blastocysts. Fertility and Sterility. 2004;**82**(5):1418-1427. DOI: 10.1016/J. FERTNSTERT.2004.03.068

[105] Anderson AR, Wilkinson SS, Price S, Crain JL. Reduction of high order multiples in frozen embryo transfers. Reproductive Biomedicine Online. 2005;**10**(3):402-405. DOI: 10.1016/ S1472-6483(10)61803-2

[106] Martin DC, O'Conner DT. Surgical management of endometriosisassociated pain. Obstetrics and Gynecology Clinics of North America. 2003;**30**(1):151-162

[107] Li L, Zhang X, Zhao L, Xia X, Wang W. Comparison of DNA apoptosis in mouse and human blastocysts after vitrification and slow freezing. Molecular Reproduction and Development. 2012;**79**(3):229-236. DOI: 10.1002/ MRD.22018

[108] Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai M, Takahashi K. Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertility and Sterility. 2001;**76**(3):618-620. DOI: 10.1016/ S0015-0282(01)01968-9

[109] Mukaida T et al. Vitrification of human blastocysts using cryoloops: Clinical outcome of 223 cycles\*. Human Reproduction. 2003;**18**(2):384-391. DOI: 10.1093/HUMREP/DEG047

[110] Reed ML, Lane M, Gardner DK, Jensen NL, Thompson J. Vitrification of human blastocysts using the cryoloop method: Successful clinical application and birth of offspring. Journal of Assisted Reproduction and Genetics. 2002;**19**(6):304-306. DOI: 10.1023/a:1015789532736

[111] Stehlik E et al. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. Reproductive Biomedicine Online. 2005;**11**(1):53-57. DOI: 10.1016/ S1472-6483(10)61298-9

[112] Huang C-C et al. Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Human Reproduction. 2005;**20**(1):122-128

[113] Vanderzwalmen P et al. Births after vitrification at morula and blastocyst stages: Effect of artificial reduction of the blastocoelic cavity before vitrification. Human Reproduction. 2002;**17**(3):744-751

[114] Hiraoka K, Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Human Reproduction. 2004;**19**(12):2884-2888

[115] Liebermann J, Tucker MJ. Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmental potential after vitrification. Reproduction. 2002;**124**(4):483-489. DOI: 10.1530/ rep.0.1240483

[116] Liebermann J, Tucker MJ. Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertility and Sterility. 2006;**86**(1):20-26

[117] Loutradi KE et al. Cryopreservation of human embryos by vitrification or slow freezing: A systematic review and meta-analysis. Fertility and Sterility. 2008;**90**(1):186-193

[118] Hong SW, Sepilian V, Chung HM, Kim TJ. Cryopreserved human

blastocysts after vitrification result in excellent implantation and clinical pregnancy rates. Fertility and Sterility. 2009;**92**(6):2062-2064. DOI: 10.1016/J. FERTNSTERT.2009.06.008

[119] Feng G et al. Comparable clinical outcomes and live births after single vitrified–warmed and fresh blastocyst transfer. Reproductive Biomedicine Online. 2012;**25**(5):466-473

[120] Cobo A, Santos MJD, Castellò D, Gámiz P, Campos P, Remohí J. Outcomes of vitrified early cleavage-stage and blastocyst-stage embryos in a cryopreservation program: Evaluation of 3,150 warming cycles. Fertility and Sterility. 2012;**98**(1138):e1131-e1146

[121] Kim SS. Fertility preservation in female cancer patients: Current developments and future directions. Fertility and Sterility. 2006;**85**(1):1-11. DOI: 10.1016/J. FERTNSTERT.2005.04.071

[122] American Cancer Society. Annual cancer facts & figures. Available from: https://www.cancer.org/research/ cancer-facts-statistics/all-cancer-factsfigures.html [Accessed: October 18, 2022]

[123] Bahroudi Z et al. Review of ovarian tissue cryopreservation techniques for fertility preservation. Journal of Gynecology Obstetrics and Human Reproduction. 2021;**51**(2):102290

[124] Oktay K, Karlikaya G. Ovarian function after transplantation of frozen, banked autologous ovarian tissue. The New England Journal of Medicine. 2000;**342**(25):1919

[125] Donnez J et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet. 2004;**364**(9443):1405-1410

*Female Fertility Preservation: Different Interventions and Procedures DOI: http://dx.doi.org/10.5772/intechopen.109052*

[126] Demeestere I, Simon P, Emiliani S, Delbaere A, Englert Y. Fertility preservation: Successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin's disease. The Oncologist. 2007;**12**(12):1437-1442

[127] Dolmans M-M et al. Transplantation of cryopreserved ovarian tissue in a series of 285 women: A review of five leading European centers. Fertility and Sterility. 2021;**115**(5):1102-1115

[128] De Vos M, Smitz J, Woodruf TK. Erratum: Fertility preservation in women with cancer," (Lancet (2014) 384 (1302- 1310)). Lancet. 2015;**385**(9971):856

[129] Sonmezer M, Shamonki MI, Oktay K. Ovarian tissue cryopreservation: Benefits and risks. Cell and Tissue Research. 2005;**322**(1):125-132

[130] Donnez J, Dolmans M-M. Ovarian cortex transplantation: 60 reported live births brings the success and worldwide expansion of the technique towards routine clinical practice. Journal of Assisted Reproduction and Genetics. 2015;**32**(8):1167-1170

[131] Porcu E et al. Healthy twins delivered after oocyte cryopreservation and bilateral ovariectomy for ovarian cancer. Reproductive Biomedicine Online. 2008;**17**(2):265-267

[132] Arapaki A, Christopoulos P, Kalampokas E, Triantafyllidou O, Matsas A, Vlahos NF. Ovarian tissue cryopreservation in children and adolescents. Children. 2022;**9**(8):1256

[133] Silber SJ et al. A series of monozygotic twins discordant for ovarian failure: Ovary transplantation (cortical versus microvascular) and cryopreservation. Human Reproduction. 2008;**23**(7):1531-1537. DOI: 10.1093/ HUMREP/DEN032

[134] Silber SJ, Grudzinskas G, Gosden RG. Successful pregnancy after microsurgical transplantation of an intact ovary. The New England Journal of Medicine. 2008;**359**(24):2617-2618. DOI: 10.1056/NEJMC0804321

[135] Gandolfi F, Paffoni A, Brambilla EP, Bonetti S, Brevini TAL, Ragni G. Efficiency of equilibrium cooling and vitrification procedures for the cryopreservation of ovarian tissue: Comparative analysis between human and animal models. Fertility and Sterility. 2006;**85**:1150-1156

[136] Fabbri R, Pasquinelli G, Keane D, Magnani V, Paradisi R, Venturoli S. Optimization of protocols for human ovarian tissue cryopreservation with sucrose, 1,2-propanediol and human serum. Reproductive Biomedicine Online. 2010;**21**(6):819-828. DOI: 10.1016/J.RBMO.2010.07.008

[137] Kawamura K et al. Hippo signaling disruption and Akt stimulation of ovarian follicles for infertility treatment. Proceedings of the National Academy of Sciences of the United States of America. 2013;**110**(43):17474-17479. DOI: 10.1073/ PNAS.1312830110/SUPPL\_FILE/SM01. AVI

[138] Suzuki N et al. Successful fertility preservation following ovarian tissue vitrification in patients with primary ovarian insufficiency. Human Reproduction. 2015;**30**(3):608-615. DOI: 10.1093/HUMREP/DEU353

[139] Shi Q, Xie Y, Wang Y, Li S. Vitrification versus slow freezing for human ovarian tissue cryopreservation: A systematic review and meta-anlaysis. Scientific Reports. 2017;**7**(1):1-9. DOI: 10.1038/S41598-017-09005-7

[140] Wirleitner B et al. The time aspect in storing vitrified blastocysts: Its impact on survival rate, implantation potential and babies born. Human Reproduction. 2013;**28**(11):2950-2957. DOI: 10.1093/ HUMREP/DET361

[141] European Medicines Agency. European Directorate for the Quality of Medicines and HealthCare (EDQM) of the Council of Europe. Available from: https://www.ema.europa.eu/en/partnersnetworks/international-activities/ multilateral-coalitions-initiatives/ european-directorate-quality-medicineshealthcare-edqm-council-europe [Accessed: October 28, 2022]

## Ovarian Tissue Cryopreservation Guidelines

*Mahboubeh Vatanparast*

#### **Abstract**

With the increase in the survival rate of cancer patients, there has been a growing interest in the field of fertility preservation. One of the main methods in this aim is ovarian tissue cryopreservation, especially for prepubertal girls. From the early time of introducing this opportunity as a chance to preserve future fertility in cancer patients, following gonadotoxic treatments, many guidelines have been published, to introduce the real indications. The need for these guidelines seemed very urgent, and attracted great interest, because this method was performed as an experimental and no standard clinical option, for many years. So patient selection should have been done with the most standard and highly accurate criteria, which could analyze the cost/benefit of this technique after multidisciplinary evaluation, for each patient, individually. For many years the specialist believed that all caution must be taken in referring patients for this technology. To ensure that cancer patients receive highquality uniform treatment, evidence-based clinical practice guidelines (CPGs) are needed. CPGs are essential to enhance care quality and decrease heterogeneity in practice and costs. The guidelines can provide clear advice on the best practice in the field of female FP, based on the best available evidence.

**Keywords:** ovarian tissue cryopreservation, gonadotoxic treatments, guidelines, female fertility preservation, premature ovarian insufficiency

#### **1. Introduction**

cancer survivors have increased dramatically with the improvements in cancer treatment [1]. However, cancer incidence, in the young age group, has increased, during the past 30 years, the mortality rates have declined and the 5-year survival rate has increased from less than 50% in the 1970s to 80% these days, and the 10-year survival rate is estimated 75% [2].

Cancer in adolescents and young adults (AYAs) occurs between 15 and 39 years old. Cancer-diagnosed AYAs are different from other age groups in many components, such as tumor etiology, biology, prognosis, intrinsic and extrinsic risk factors, cancer types and survivorship as well as the effectiveness of treatment. Besides, AYAs suffer more from long-term effects of treatment compared with those older ages, including sexual dysfunction, infertility, and future cancers [3].

Chemotherapy and radiotherapy may have gonadotoxic effects, and compromise ovarian function [4]. A positive correlation has been found between the risk of

gonadal harmful effects and the patient's age, which may be due to the previous reduction of the follicle numbers in older females. Additionally, the type of chemotherapy regimens and the dose are the other determinants of the degree of cytotoxicity [5, 6].

With the increase in the long-term survival rates of cancer patients who have experienced chemo/radiotherapy, there's been greater attention to the long-term effects of treatments, such as premature ovarian failure (POF) [6]. A mixture of health and quality-of-life problems have been created for the young survivors, many of which were not predicted at the time of cancer diagnosis [4].

It needed to be discussed with the patients the risks of cancer treatments on reproductive health, and the options for fertility preservation, before treatment. Consultation with a reproductive endocrinologist is of great value in providing adequate information regarding the side effect of cancer therapies on reproductive consequences and the chance of success rates for the various fertility preservation strategies. It was shown that almost 30% of cancer patients under 50 years old request more information about premature ovarian insufficiency or the probable risks to their children's health, and a third of them would have tended to have a fertility consultation before treatment started [7].

Gametes, embryo, and gonadal tissue cryopreserving may help to preserve fertility, and avoid damage to reproductive organs. Sperm and embryo freezing are well established (clinical stage), but oocytes and ovarian tissue freezing are still experimental [1].

However, in some parts, we referred to the other ways of fertility preservation (FP) options, but the main aim of this chapter is a review of the existing guidelines regarding ovarian cryopreservation, as a fertility preservation option. A comprehensive study of the available literature was conducted, to find updated and evidencebased guidelines and recommendations.

#### **2. The emergence of oncofertility**

Recently, with the increased survival rate of cancer patients, there have been much attention and interest in the long-term effects of treatment on future fertility [2].

Oncofertility is a domain that connects oncology with fertility and having a comprehensive view of these fields got it capable to introduce a standard of care in many institutions. This field has been developed simultaneously with the other lifepreserving advancements in the oncology care unit, such as earlier diagnostics, targeted cancer therapies, new methods with less radiation dose, and local surgical procedures [8].

It passed more than half a century since the first time the concept of "ovarian tissue cryopreservation" was proposed, but it took 12 years, until achieving (reaching) the first successful human ovarian tissue cryopreservation, and the first live birth, following this technique has been reported in 2004. For many years, ovarian tissue cryopreservation and transplantation were done as experimental, in many fertility centers, until 2019, when this approach was accepted as a clinical technique, for fertility preservation [5].

This success has been achieved with the improvement in the technique, protocols, cryoprotectants agents, devices, as well the exact timing for equilibration, throughout many scientific studies, as it got qualified enough, to be considered as "the clinical approach" [9].

#### **3. Female fertility preservation strategies**

increasing the knowledge of the possibility of fertility loss following cancer treatment has led to huge growth in the fertility preservation field, throughout the last two decades. ESHRE 2020, described fully these options, based on the individual case [10].

There are some options for female fertility preservation before cancer treatment. For female fertility preservation, the most successful standard method is emergency IVF and embryo cryopreservation, as an established part of assisted reproduction, before cancer therapy [7, 10].

However, this method is not suitable for pre-pubertal girls, and young patients when there is no partner or when there is not enough time to delay cancer treatment. Oocyte and ovarian tissue cryopreservation are the other less effective options that are still experimental [10, 11].

Ovarian transposition or fertility-sparing cancer surgery is the other option that helps minimize the destructive effects of cancer treatments [10, 11]. Besides restoration of fertility, ovarian tissue transplantation in the POI can restore endocrine and hormonal function [12], also it can be done at any time during the menstrual cycle [13].

In addition, GnRH agonist co-administration also may provide some fertility protection against gonadotoxicity of the chemotherapy, but still prospective controlled trials are needed to approve this method as an established clinical method [7, 10]. In vitro oocyte maturation (IVM), and oocyte cryopreservation are the other choice methods, especially for women with age-related fertility loss, besides women who seek FP for medical indications [10].

Despite the introduction of these strategies, FP stays a particular challenge in the most common candidates such as hematological cancers (leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma) and breast cancer [14]. The most challenge is the patient's selection criteria, under 35 years old, when the ovarian reserve is still high, a realistic chance of 5 years survival rate, and when there the risk of premature ovarian insufficiency is at least 50% [15].

#### **4. Ovarian tissue cryopreservation as an experimental method for FP**

Nearly 20 years passed since the first human pregnancy report following ovarian tissue cryopreservation (OTC) [16], but until recently this method was being considered an experimental method. Embryo and gametes (sperm, and oocyte) cryopreservation are done as the standard practice worldwide, but OTC is a newly established method, which harbors many challenges. One of the reasons is that OT is a complex of various cell types with different physical structures and water permeability, that need a different process to survive during freezing [17].

The other challenge is the methods of cryopreservation, vitrification vs. slow freezing. There are two main methods for OTC; slow freezing (SF) and vitrification (VF). The conventional method is slow freezing, which is the base protocol for OTC worldwide, and nearly all live births have been reported to follow this method. The other is vitrification which compared to slow freezing is a new approach, with small numbers of live births. Some advantages and disadvantages have been reported regarding these methods [18].

The base for slow freezing is tissue exposure to the cryoprotectant agents (CPA), cooling of the tissue to a special temperature using programmable freezers, and final liquid nitrogen (LN2) immersing (196°C). DMSO (1.5 M) has been used as CPA in this method [19].

Vitrification protocol is done commonly using equilibration and vitrification solution exposure (increasing concentration of CPAs), and final LN2 immersion. Ethylene glycol (EG) and dimethyl sulphoxide (DMSO) can be used as the CPAs in this method (7.5% for ES, and 20% for VS) [20].

However, SF has been used as the standard method, in many centers, but some properties of this method created an interest in vitrification. Besides this method is time-consuming, because of the need for a controlled-rate cooling device which is expensive. Also, the probability of cryoinjury is high in the formation of the intracellular ice crystal. Then vitrification was introduced for this purpose, instead of slowfreezing, because of time-saving, and rapid cooling with no need for expensive equipment [17]. Much research has been done to introduce the best method, but the results are controversial [21], but among numerous studies, rigorous documents have been earned that support VF as an alternative way to the SF method [22–28].

The other issue is the techniques of ovarian cryopreservation; ovarian cortical fragments or whole ovary.

Since most primordial follicles are located within the ovarian cortex, so cryopreservation of the small part of cortical tissue enables the storage of large numbers of oocytes. It mainly is recommended to obtain ovarian tissue before cancer treatment. For this purpose, ovarian tissue is commonly obtained by the laparoscopic approach, also it can be done by mini-laparotomy or during ovarian transposition [29].

For the fragment cryopreservation, the cortical tissue will be transferred to the lab, then thin cortical strips will be prepared, using a scalpel, the optimal size will be <sup>5</sup> <sup>10</sup> 1 mm<sup>3</sup> thick, before freezing [18, 20, 30].

The whole ovarian cryopreservation consisted of ovarian removal with the vascular pedicle, through laparotomy or a laparoscopic approach. One of the big advantages is vascular anastomosis of the thawed ovary, which probably provides a larger follicular reserve and also a longer ovarian life span. But there are no currently rigorous documents to support this hypothesis, mainly because of the created damages by applying freezing procedures for a big sample as a human ovary [31].

In one study on sheep ovary, it was shown that the directional freezing method enhances the ovarian tissue cryopreserved viability, for both whole ovary, and ovarian fragments [31]. The physical concept for directional freezing is a sequence of four heat-conductive units positioned in a line. The blocks are set at different temperatures, which creates a temperature gradient. Freezing tubes will be passed, at 0.01 mm/s speed, along the thermal gradient, starting at +4°, and decreasing to 70° C. This results in a cooling rate of 0.3°C/min, and at the end, samples will be plunged into liquid nitrogen [32, 33].

The other problem is how to use cryopreserved ovarian tissue. However the OTC is now a relatively well-established procedure, but the restoration of fertility using the cryopreserved tissue remains a challenge. The only approach for the restoration of fertility is the re-implantation of ovarian tissue [34]. But the re-implantation process is accompanied by attrition of the non-growing follicles population, something about three-quarters [35]. Now the thawed ovarian tissue transplantation can be done in two ways; orthotopic or heterotopic. The other alternative approaches, such as *in vitro* follicular culture need additional research before accepting as an established practice for humans [34].

until 2017, worldwide over 130 babies have been born after ovarian tissue cryopreservation (OTC), and ovarian tissue transplantation (OTT). It was estimated that OTC resulted in 93% restoration of endocrine function [36], 29% successful pregnancy rates and 23% live birth rates [37].

One another important issue regarding OTC is how long the transplanted tissue will remain functional. However, there are some variations in the ovarian grafts' lifespan after ovarian transplantation, it seems that the endocrine function duration is longer than what is expected. On average, the follicular density of nearly 4–5 years will be preserved well, after transplantation, but up to 7–10 years of ovarian function duration also has been reported [36, 38].

For the OTS two surgical processes are needed; one for the extraction and the other for re-implantation [39]. To date, no standard operative technique has been established for obtaining ovarian cortical tissue. Few publications have dealt with the operative technique and outcomes, and the mentioned methods were as follows; ovarian cortical biopsy, unilateral hemi-oophorectomy, as well bilateral hemioophorectomy, and unilateral salpingo-oophorectomy [40].

All of the mentioned challenges, together, cause us to consider the OTC as an experimental method for female fertility preservation. Besides, there are some information gaps, which need to be addressed.

#### **5. Whom, what, when, which, who information gaps**

After the creation of the new approach (fertility preservation), some important questions arose, but there were no unique answers for them. One of them was; for Whom must it be proposed? What were the indications? When must it be done? Which strategy is useful, for each case? Who must provide fertility preservation consultation?

There are some studies, which tried to answer these questions, and some indications have been introduced, a range from very commonly known cases such as cancer patients [41], the latter as the social reason for pregnancy delay [42, 43], and agerelated fertility loss [10], to the new indications such as transgender (fertility preservation for trans men) [43, 44], or Endometriosis [45]. However many guidelines suggested that fertility preservation consultation should be done before treatment is a great information lack in this area [46].

#### **5.1 The challenge of "for whom?"**

Ovaries or testes will not be affected in many cases, by the cancer treatment and physicians can reassure the patients. But there are also other cases, in which, the impact of treatment on fertility is unknown due to a lack of valid studies. Also sometimes the problem is the medical oncologists when there is a belief that there is no good choice/or chance, so discussing it will not help.

The related risk for premature ovarian insufficiency (POI), after chemotherapy, is influenced by some factors such as age, body mass index (BMI), type of treatment, and duration. For example, in breast cancer, the risk of amenorrhoea, after six cycles of CMF (cyclophosphamide, methotrexate, fluorouracil) was estimated at 33% for patients below 40 years and, 81% for patients ≥40 years old, also the risk for amenorrhoea was estimated 51.4% in women under 30 years old and 95.0% for the women above 30 years, after 8 sessions for dose-escalated of BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone) [47].

#### **5.2 The challenge of "which strategy?"**

Which option is appropriate for each patient is the other gap, which must be exactly described in the guidelines. Some important criteria must be fulfilled in the aim to classify a new technology and treatment as an established technique. They are efficacy, safety, procedure, and effectiveness to assess [48]. The ovarian tissue cryopreservation, as a new field of oncofertility, at first must be set up on nonhuman primate models and then would become appropriate for humans, the necessary step to move from the bench to the bedside [4].

Ovarian tissue cryopreservation is done by obtaining ovarian cortical tissue, the part that is rich in primordial follicles, before gonadotoxic treatment, by laparoscopy or laparotomy. Small fragments are prepared and then cryopreserved by the slow freezing or vitrification technique. After cancer treatment, the tissue will be transplanted in two ways of orthoptic (the pelvis) or heterotropic (outside the pelvisabdominal wall, and forearm) transplantation. Orthotopic transplantation the most successful method has been accompanied by spontaneous pregnancies, but in heterotopic transplantation, IVF is necessary [46].

Because of the nature of being experimental, it is needed to identify the accurate indication for this technique [15].

In this regard, there are some guidelines by the American Society for Reproductive Medicine (ASRM) and American Society of Clinical Oncologists (ASCO) which recommend cancer patients must be referred for fertility preservation counseling, related to the risk of infertility after cancer treatment and must be aware of the appropriate plans [49–51]. But, there is not, always, appropriate consultation regarding the potential side effects of cancer treatment on future fertility, and also fewer patients are aware of the fertility preservation program [52].

The other big gap is the information delivery, while the medical oncologists are not exactly aware of the effect of their treatments on reproductive health and outcomes, clinical cancer patients are not routinely treated by reproductive endocrinologists. Some physicians also believe that it involves ethical issues to enable a patient to bear a child while the parent has a lowered life span or cannot take care of a child [1].

#### **5.3 The challenge of "when"?**

when must ovarian tissue cryopreservation be done? It was mentioned that over 90% of patients undergoing high-dose chemotherapy may suffer from ovarian failure [53] and cryopreservation should always be offered when the risk of POF is high (>30–50%) and the risk of ovarian metastasis is low [54]. It suggested that the best time is before the patient receives any chemotherapy [2, 15, 53].

Among many of the guidelines, ASCO mentioned individual cytotoxic treatment regimens and their effects on fertility. The guidelines mentioned that counsel the patient that this method is experimental [50, 55].

The patients' age was one of the most challenges which have been discussed in the various version. Some believed to refer all the patients of reproductive age [50], but some others considered age limitation, and recommended this to special ages, E.g. below 35, 37, or < 42 [51, 56, 57]. The reason for the age limitation is confirmation of the appropriate ovarian reserve [57].

A framework of guidance is needed for healthcare professionals to provide evidencebased care to the women and girls who are a candidate for fertility preservation [43].

#### **5.4 The challenge of "who"?**

Who must provide the fertility preservation consultation? The oncologists and hematologists may be the first ones, in the health care provider team, who find the need for fertility preservation. After referral, the other healthcare professionals which have an important role as the oncologists, in the management of women with cancer, are obstetrics-gynecologists, endocrinologists, andrologists, nurses, embryologists, reproductive biologists, reproductive medicine specialists, psychologists, counselors, and general practitioners [8, 43, 49].

#### **6. Fertility preservation guidelines**

Fertility preservation, nowadays, is a necessary professional domain. It needs close coordination between teamwork which includes oncologists, reproductive biologists, and reproductive medicine specialists in various fields [8]. Many guidelines had been provided to describe the situations, in which fertility preservation is needed to be considered with the patients. However, there is still no general agreement between different guidelines in introducing criteria for ovarian tissue cryopreservation, such as patients' age limits or having a history of chemotherapy.

since the first time scientists succeeded in mammalian ovarian tissue cryopreservation, in 1994 [58], new indications have been introduced and the circumstances have been determined. It had been tried to introduce true indication, in each version. There are many heterogeneities in the criteria for introducing real candidates, such as patients' age limits, excluding patients with a history of chemotherapy, or specific diagnoses which preclude re-transplantation. Tissue preparation and freezing/thawed procedure are the other diverse worldwide [59].

To ensure that cancer patients receive high-quality uniform treatment, evidencebased clinical practice guidelines (CPGs) are needed. CPGs are essential to enhance care quality and decrease heterogeneity in practice and costs [52]. The guidelines can provide clear advice on the best practice in the field of female FP, based on the best available evidence [60]. Several guidelines have been published worldwide. Some of the guidelines focused on efficacy, and safety, while others have paid attention to the aspects of feasibility, and acceptability, and few had ethical considerations. A comprehensive guideline that encloses all aspects of FP, from patients' consultation to the outcome of techniques, would additionally help clinicians in this field.

These guidelines could introduce the potential candidates for OTC, when a woman is at risk for iatrogenic infertility, due to medical or surgical cancer treatment, for a benign or malignant condition [51].

Guidelines are set to address oncologists, gynecologists hematologists, endocrinologists, as well other healthcare providers, such as nurses, counselors, psychologists, and general practitioners who have a role in giving fertility preservation to cancer patients.

#### **6.1 Edinburgh guideline (2005)**

Edinburgh Criteria is one of them that was firstly published in 1996, and then slightly revised in 2000 [15]. Edinburg criteria once again and after multidisciplinary discussion and the report from the Royal College of Obstetricians and Gynecologists working group, described some criteria especially age limit) for patient selection, this guidance is applicable in a patient-specific manner and must be updated by the emerging new evidence and experience [61]. Besides introducing the patient selection criteria, two issues have been addressed; the probability of the congenitofabnormalities in children following chemotherapy, and the legal and ethical issues.

Regarding the concern for child abnormalities, the guideline reassures that a large study did not show any link between these. The guideline mentioned that this technique (FP) raised some critical ethical and legal issues, which must be considered before use. The costs/benefits of any intervention or decision must be evaluated, and all the advantages and disadvantages, in the short, and long term, must be considered. These proposed opportunities should not create unrealistic expectations, and should not bring adverse effects for the subsequent offspring.

Valid informed consent from the patients must be earned voluntarily, and from a competent person. Legal competence was discussed in this guideline, and also described that obtaining valid consent becomes more complicated according to the patient's age and their level of understanding of the discussed issues [61].

Edinburgh criteria validation for ovarian tissue cryopreservation had been done, in 2014, for young women and girls (younger than 18 years). The results validate the guidelines criteria for patient selection for ovarian tissue cryopreservation and show it can identify accurately the girls and young women at risk for premature ovarian failure [15].

#### **Summarized recommendations of the Edinburgh Criteria for Ovarian Tissue Cryopreservation (2005, 2014):**


But, the later study, again challenged one of Edinburgh's criteria, having no history of previous chemotherapy or radiotherapy, while Vatanparast and her colleagues 2021, presented a young girl with acute lymphocytic leukemia, which had a history of chemotherapy (15 sessions, with 30 mg vincristine and 975 mg Adriamycin) before referring. An anti-Mullerian hormone (AMH) had been requested, to survey the patient's fertility situation. It showed a premenopausal situation when reported within the normal range (3.66 ng/mL). after ovarian biopsy, the histology survey also showed a normal follicular density [62]. In the later guidelines, in 2022, it also was described that the OTC can be done also when the gonadotoxic risk is very high, but before the patients undergo chemotherapy is the ideal situation [39].

However, many studies have addressed fertility preservation before starting chemotherapy, fewer paid attention to the survey of pre-menopausal ovarian function [1, 50, 56, 57]. This gap in the available published guidelines is fully answered in the ESHRE guideline, 2020 [10]. There are some hormonal markers as well as ultrasound parameters that can estimate the ovarian reserve [63], behind the age or a history of chemotherapy! Unfortunately, ovarian biopsy only can be done through surgery or

*Ovarian Tissue Cryopreservation Guidelines DOI: http://dx.doi.org/10.5772/intechopen.108201*

laparoscopy, to obtain ovarian reserve from the histological survey. But, one of the most common indirect signs of ovarian reserve is hormone assay; Anti-Mullerian hormone (AMH). In the studies, AMH is accepted as a marker of ovarian reserve [64]. As it has been mentioned that a history of cancer alone does not provide enough documents to decide on patient sterility [7], and does not exclude the patient from the fertility preservation program. It seems that a big gap in the guidelines is the confirmation of pre-menopausal ovarian function, both before or after starting treatment. Also for the aim of ovarian reserve prediction, the other well-validated biomarkers, such as; FSH, LH, estradiol, Inhibin-B levels, ovarian volume, and the antral follicle count (AFC, by transvaginal ultrasound) also may be helpful. Serum AMH is reliable for the assessment after the age of 5, in addition, in older children, ovarian volume and antral follicle assessment can be recommended [65].

It's believed that small ovarian volumes, maybe a more acute marker in the aim of ovarian reserve estimation compared to abnormal hormonal concentrations,18 of which are indirect. Besides AFC, which shows the size of the existing primordial follicle pool may be a more sensitive marker of the ovarian reserve in comparison to ovarian volume. AMH which is produced by the growing follicles may be the most valuable representation of ovarian reserve [6].

#### **6.2 American Society of Clinical Oncology (ASCO) guideline**

The other guidelines also were prepared by the American Society of Clinical Oncology (ASCO) [49] and the American Society of Reproductive Medicine (ASRM) [1].

The ASCO guideline which was published in 2006, has been updated periodically. It was recommended that oncologists should consult with the patients regarding the possibility of infertility through their cancer treatment, before cancer therapy, and be aware of possible fertility preservation options to refer the patients to reproductive specialists.

Sperm and embryo cryopreservation which were introduced as the standard practices are widely available and the other options which were considered investigational should be performed in specialist centers [49]. This version was updated in 2013 [50]. In the new version, it was stressed that health care providers advise the patients regarding the potential effects of treatment on their fertility and inform them about a wide range of fertility preservation options, as soon as possible, during their treatment, although the patients initially focus on their cancer diagnosis. In this version, oocyte cryopreservation is mentioned as a standard practice. In this version, ovarian tissue cryopreservation is still considered experimental and not an established technique [50].

#### **Summarized recommendations of the ASCO (2013):**


#### 5.Discuss common concerns


#### 6.Time:


#### 7.Costs:

	- reproductive specialists
	- mental health professionals
	- advocacy organizations.

#### **6.3 American Society for Reproductive Medicine Clinical Practice Guideline**

In ASRM (ethic committee) recommendations (2005), also as the other guidelines, consulting the patients regarding fertility preservation has been considered as the patient's right. This guideline, in a new approach, pertained to ethical and legal issues, in detail, for physicians and fertility specialists, as well cancer patients and offspring welfare, and the other issues are experimental vs. established options, the minor children's ability to give consent, the welfare of probably resulting children, and posthumous issues. This guideline described that concerns about the welfare of future offspring should not impede cancer patients from fertility preservation programs, also this guideline emphasizes the need for precise instructions in the case of a patient's death, unavailability, or other contingency after doing fertility preservation (disposition of cryopreserved gametes, embryos, or tissues). The necessity for

Preimplantation genetic diagnosis to prevent the birth of children with a high risk of hereditary cancer is ethically accepted [1].

The Practice Committee of ASRM, in 2014, after searching the existing literature, to evaluate the efficacy and safety of ovarian tissue cryopreservation, published new recommendations [29]. In this version, the indication for OTC has been described in detail, and the technique (cortical strips and whole ovary), methods of cryopreservation (slow freezing and vitrification), and transplantation have been discussed. In this guideline slow freezing has been reported as standard protocol. One of the concepts which were discussed in this version is safety concerns regarding the risk of reimplantation of cancer after transplantation. They suggest an alternative way for transplantation when there is a risk of reintroducing; if it is possible to isolate and recover immature oocytes from the ovarian cortex and use them for subsequent IVF, after doing maturation in vitro (either mature oocytes or embryos cryopreservation).

The guideline described the circumstances which must be realized until an experimental procedure is considered an established medical practice. The evidence published in the medical regarding their risks, overall safety, benefits, and efficacy must be documented from the only valid studies which were appropriately designed and performed by several independent researchers [29].

**Summarized recommendations of ASRM (2005)**


#### **Summarized recommendations of ASRM (2014)**


#### **6.4 Backhus et al. guideline (2007)**

A guideline has been written by Backhus et al. [51] that was retrieved from the ASCO and ASRM's criteria. This version is wider than Edinburg's.

These guidelines help to identify correct candidates for OTC, when a woman with a benign or malignant condition is at risk for fertility loss, following medical or surgical treatment. In this guideline, also three methods of ovarian cryopreservation and transplantation, and follicles in vitro maturation were considered experimental and should not be recommended currently for patients who are candidates for fertility preservation, whenever there is no immediate or iatrogenic threat [51].

**Summarized recommendations of Backhus et al. criteria** [51]**.**


### **6.5 Ferti-PROTEKT network<sup>1</sup> (2011–2019)**

FertiPROTEKT network which first was established in 2006, encompasses the university-based, hospital-based, and private infertility and oncology centers. The main

<sup>1</sup> A specialized network and society (of physicians and biologists) in field of fertility preservation, in Germany, Austria and parts of Switzerland, www.fertiprotekt.eu

#### *Ovarian Tissue Cryopreservation Guidelines DOI: http://dx.doi.org/10.5772/intechopen.108201*

aim was to introduce local fertility preservation programs in Germany, Switzerland, and Austria, the techniques which enhanced the chance of achieving a pregnancy [57, 66].

The main theory of the formation of this network was that "a close coordination is needed between oncologists and reproductive medicine specialists, and reproductive biologists", so fertility-preservation activities should be organized, in a network structure, both as the medical-logistic network and as a professional medical society [8]. The three major networks introduced were as follows: (1) the Danish Network,<sup>2</sup> (2) The German-Austrian-Swiss network FertiPROTEKT,<sup>3</sup> and (3) The Oncofertility Consortium.<sup>4</sup> As these networks'structures are different in goals and logistics, so all the aspects of possible network structures are covered [8]. For example, the Danish network, gives practical implementation in the field of fertility-preserving techniques such as ovarian tissue cryopreservation and transplantation, in a small country. The second, the German-Austrian-Swiss network (FertiPROTEKT) organizes these ( fertility-protective techniques) in a large country, and the Oncofertility Consortium facilities knowledge transfer among its members.

In an attempt to introduce indications, in 2011, the Ferti-PROTEKT network prepared a practically oriented recommendation [57], and it was updated in 2018 [47]. This new version has a process-oriented approach, which stressed three main topics; disease prognosis, disease-specific treatment and risks of infertility, and diseasespecific cryopreservation measures. Besides the risk for ovarian metastasis is also assessed which with the other mentioned topics are critical in deciding if fertility preservation is needed, or not. It could provide a disease-specific recommendation for fertility preservation measures [47].

#### **Summarized some of the recommendations of the Ferti-PROTEKT network (2011):**


<sup>2</sup> www.rigshospitalet.dk

<sup>3</sup> www.fertiprotekt.com

<sup>4</sup> www.oncofertility

5. In acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) ovarian tissue cryopreservation was considered experimental because of the high-risk estimation for ovarian contamination with the malignant cells.

At last, they concluded that answering the challenge of fertility preservation necessity depends on several factors such as prognosis, the risks for fertility loss, and some individual factors such as future family planning.

#### **6.6 Cancer Council Australia (2014)**

This guidance which pertained to many aspects of FP in adolescents and young adults (AYA), in detail, has been published by the Clinical Oncology Society of Australia (COSA), assists health professionals by introducing evidence-based recommendations and 'good practice points', to provide an effective consultation for AYA patients and their families.

Three main objects are as follows:


#### **Summarized recommendations of the Cancer Council Australia (2014):**


Regarding OTC, the guidance considered this technique as an investigational technique, which is appropriate for young women who are at high risk for ovarian failure, or when the other options are not suitable. Consult the patient that this technique is not a routine clinical practice. The ovarian tissue must be monitored for the presence of malignant cells. The guidance recommended managing the pregnancy after cancer treatment as a high-risk pregnancy, preferably in a tertiary center.

The guide also has a recommendation for infertile AYA cancer survivors. They should bedsides providing infertility counseling, and be informed about the assisted reproduction technologies opportunities, such as sperm, egg, or embryo donation, surrogacy, and adoption.

The complete version of this guideline is available via: https://wiki.cancer.org.au/a ustralia/COSA:AYA\_cancer\_fertility\_preservation [67].

#### **6.7 Chinese Society of Gynecological Endocrinology affiliated to the International Society of Gynecological Endocrinology Guideline for Ovarian Tissue Cryopreservation and Transplantation (CSGE-ISGE) (2018)**

Besides some guidelines which have no special target, a guideline has been published by the CSGE-ISGE to introduce the standard application of OCT in China. Based on Chinese specific conditions and after assessing the international guideline, they formulated the selection criteria as follows.


In this guideline, the authors described the good indication as the patients with tumors and nonmalignant diseases, as the aim of OTC is to preserve fertility and ovarian endocrine function. This method is suitable for prepubertal girls, patients who do have not enough time to postpone chemo- or radiation therapy, and women with hormone-sensitive tumors [45].

This guideline described that there is no uniform standard protocol for the timing of transplantation. It depends on the primary disease and can be done when the disease is cured and clinical rehabilitation happens. After full situation consideration, the patient's specific treatment is done.

commonly when the primary condition is cured, and the symptoms of menopause have been revealed due to ovarian function destruction; such as hot flashes and sweating, serum level of follicle-stimulating hormone (FSH) ≥ 25 IU/L, and anti-Mullerian hormone (AMH) <1.1 ng/ml ovarian retransplantation can be done at least 3–6 months after stopping chemotherapy [45].

Every month follow-up is needed to assurance of ovarian function recovery (reproductive and endocrine). After recovery, follow-up can be continued every 3–6 months.

For leukemia cases, there is no ideal fertility preservation program and OTC should be done before hematopoietic stem cell transplantation. Due to the high risk of reintroduction of malignant cells, ovarian transplantation should be performed with caution. This guideline also paid attention to ovarian endometrioma fertility preservation, while the inappropriate endometrioma removal can cause destruction effects on the ovarian reserve, and therefore assessment of ovarian function should be done before endometriosis surgery, since fertility preservation may be needed during the operation.

#### **6.8 British fertility society policy and practice guideline (2018)**

This guideline brought together the evidence literature for fertility preservation techniques and their outcome, as well as the associated risks, for medical reasons; both oncological and non-oncological cases. The guideline recommended consultation with women and girls about the risk of cancer treatment on their fertility and available preservation techniques. The four measures were: embryo, oocyte, and ovarian tissue cryopreservation, GnRH agonist administration, and ovarian transposition. In this version also ovarian tissue cryopreservation was still considered experimental. For benign and malignant conditions, current treatment modalities, which were accompanied by a better fertility-sparing profile, were described.

The guideline highlighted the role of psychological support in decision-making. The guideline also highlights that it is needed for the patients to meet the exact criteria to undergo invasive procedures, and FP must be done for curative intent [43].

The summary of its recommendations is as follows:


7.Patients should be aware of the time limitation for storing their oocytes/embryos.


#### **6.9 ESHRE guideline (2020)**

The last version of the guidelines has been published by the European Society of Human Reproduction and Embryology (ESHRE, 2020) [60]. In this guideline fertility preservation has been discussed in women and transgender men concluded assessment before FP, the interventions, and also the post-treatment recommendations. It includes 50 evidence-based recommendations which were approved by the ESHRE Executive Committee and the Guideline Group. They consider this version needs to be updated 4 years after this publication.

This guideline considers intrinsic and extrinsic factors for the patients' assessment and selection. Intrinsic factors such as patient's health status, age, consent obtaining, and ovarian reserve assessment, and extrinsic such as the risk for fertility, and risk for pregnancy, … are discussed. The needs and ways for "ovarian reserve testing" are argued. Some of the important recommendations regarding OTC have been brought below.

In preparing a guideline, it's the first time that OTC methods have been reviewed, and the slow-freezing protocol is considered as standard protocol and offered the vitrification protocol only in the research program. One of the limitations of the present guidelines is the lack of discussion about the best cryopreservation method, although the choice of the method is selected by the embryologist and based on the conditions and facilities of the specialized centers, the freezing method is needed to be discussed in the guidelines.

#### **Summarized recommendations of the ESHRE (2020):**


<sup>5</sup> AMH < 0.5 ng/ml and AFC < 5.


#### **7. Guidelines regarding the concerns of cancer reintroducing**

One of the serious concerns regarding OTC and transplantation is the risk of reimplantation, by the possible existing malignant cells in the harvested ovarian tissue after remission and resulting in the exclusion of ovarian cancer or when there's a high risk for ovarian metastasis [45, 68, 69]. It is of paramount importance in the cases of hematologic malignancies, which are at the most risk of ovarian metastasis, with the transferring of malignant cells via the bloodstream, and the highest risk was found with leukemia [69].

Recently, there is a new idea that ovariectomy after chemotherapy may be resulted in tissue cancer cell-free and increase transplantation safety in hematologic patients [12, 59, 70]. To evaluate the presence of leukemic cells in cryopreserved ovarian tissue histology and immunohistochemistry can be used to identify the ovarian infection by the malignant cell, but the molecular analysis is a better sensitive way. In leukemia patients, polymerase chain reaction (PCR) can reveal malignant cells in up to 75% of cases of thawed ovarian tissue [12]. RT-qPCR, and long-term (6 months) xenografting to immunodeficient mice, also have been used, in this aim [69, 71]. It must be mentioned that a disease-specific molecular marker may not be found in some cases of ALL, and AML [12]. In one study immunohistochemistry showed no evidence of the presence of residual leukemic cells, in the ovarian tissue after freeze and thaw. But the malignant cells were identified in 6 of 8 specimens, by the RT-PCR [72]. In the other study, it was shown that in the ovarian tissue harvested from ALL patients, there was no evidence of malignant cells in the ovarian grafts into immunodeficient mice, or in any of the other tissues, it's despite 4 RT-PCR positive malignant cells of the 7 grafts [73].

However, in a report in 2013, the author strongly discouraged ovarian autotransplantation when there's a risk of malignant cell reimplantation, especially in leukemia cases, and suggested patients consultation before reimplantation, in other cases in which the estimated risk is low [69], in the later publication in 2016, it was suggested that leukemia survivors also may benefit from OTC after providing maximal safety measures. These measures include harvesting after chemotherapy and an exact and intense search for leukemia cells within the graft. These cases must be informed that to date tissue involvement with malignant cells cannot be ruled out entirely with no measures [59]. Studies are needed to reassure the safety of this technique in different types of cancer.

In leukemia survivors, for ovarian tissue transplantation, careful consideration and evaluation must be taken to increase safety. Informed the patients thoroughly regarding the limitations of present investigation modalities, and the risk of cancer re-introducing should be weighed against the opportunity of being a biological parent [12].

#### **8. Is ovarian tissue cryopreservation and transplantation still experimental?**

Until recently, and for many years, ovarian tissue cryopreservation has been considered an experimental method, in many guidelines [15, 49, 50, 74]. One study described that the studies on tissue cryopreservation are limited, and most of them were done with no control group. There's a need for more experience, to accept this technique as a routine technique for fertility preservation. They said the published success of pregnancies may be because of the patient's native ovarian tissue [75]. In one study, in 2018, authors discussed this subject with more caution, and said because a live birth has not been achieved in the woman who had ovarian cryopreservation in the prepubertal stage, the efficacy is not still approved in children, and also in women with leukemia, the safety of this technique is still poor, so they considered it as experimental in children and in adults' patients which the risk for ovarian contamination with malignant cells is high.

For a technique to be defined as experimental or established some clear criteria are needed. European Society of Human Reproduction and Embryology proposed a platform to classify a technique as experimental, innovative, or established. The suggested applying criteria were efficacy, safety, procedure, and effectiveness, which all of them must be fulfilled, to accept a new treatment as established. These criteria also can be applied for the classification of the OTC and OTT in children, adult malignant cases with a high risk of ovarian contamination with malignant cells (e.g., leukemia), or low risk of ovarian involvement (e.g., breast cancer or Hodgkin lymphoma). In final, they classified this technique as established and no longer experimental when the risk of ovarian metastasis is low [48].

The first time, in 2016, in a study which was published by ASRM, it was concluded that there are enough national ethical and professional authorities for considering OTC as a standard modality, in fertility preservation programs [59]. Also, CSGE-ISGE (2018) mentioned that now there is large evidence of data that can confirm the effectiveness of the OTC, and it must be considered as a clinical and standardized procedure that would be promoted as soon as possible. The cryopreservation indications are extensive, and it is the only chance for prepuberty girls and patients who need emergency treatment (radiotherapy or chemotherapy) [45].

After many evidence-based studies and live birth reports, following transplantation, it's time to consider this technique as a clinical approach for medical indications (by the American Society of Reproductive Medicine), which can restore both patients` fertility and ovarian endocrine function [76]. Subsequently to more and more achievements of OTC reports, in the literature, researchers concluded that we passed the experimental phase.

In one study, the pregnancy rate per re-implantation was calculated, in the presence of evidence. In this large series of 111 cases, which the results were collected from Denmark, Belgium, Spain, Germany, and also from Australia, 29% of the women conceived (n ¼ 32). Knowing the exact number of transplantations, the data was highly relevant and evidence-based. This study approved the efficacy of the OTC technique [9].

According to one of these studies on 20 cases, the success rate for restoration of hormone activity was calculated at 94% [77]. Other studies, also referred to the worldwide live birth rate of 30–70% and concluded that this is a rigorous document to consider OTC as a standard protocol, for female fertility preservation options [36, 77, 78]. Many pioneers believe that now enough evidence support OTC and we reach the time to stop considering it as an investigational or experimental procedure [79].

#### **9. Conclusions and recommendations**

While the aim is to gather more evidence of the efficacy of the OTC, it is essential to consult the children and their families about this chance. There is no doubt that OTCP will be accepted as a successful standard method, soon with the fast growth in the technologies. They may suffer by delaying the offer.

We must discuss with all the patients, who may be at risk for premature ovarian insufficiency, following chemo- or radiotherapy, about the FP techniques. Discuss FP using OTCP, to the patients who do have no enough time for ovarian stimulation, just the time the diagnosis is established.

Patients should be well-informed about the different choices to be able to make valuable decisions according to the available evidence, as it may be their only real chance to be genetic parents in their future lives. A multidisciplinary team is needed, that could support all the necessary aspects of the FP, such as clinical, psychological, as well as legal, and ethical issues. After a comprehensive review of the present literature, a flow chart of the guidelines regards to OTC for cancer patients has been provided (**Figure 1**).

Regarding future FP programs, it must be stressed that however, the emerging options are promising, but their efficacy and safety of both established and newer techniques are needed to be confirmed by rigorous clinical trials, maybe with a focus on live birth, before introducing them as international clinical standards, and then can be offered as a medical intervention. Moreover, the exact criteria and relevant indication must be identified, in the aim to achieve the most efficacy of each FP method, also to reach more advancement in this field more research is needed on both established and newer techniques. Recent evidence has approved ovarian tissue cryopreservation as one of the promising options for female FP.

In the aim to give the patients appropriate and good clinical practice medical intervention about FP, widening access to the FP options and technologies is the key aspect (both appropriately patient selection and relevant indication).

However, negligence in referring the true cases to fertility preservation will result in depriving someone to have a family, applying FP to all cases, even with a low risk for gonadotoxicity, creating a large amount of unused stored reproductive cells and tissue, which burden costs for both patients and health services.

However, there are some new treatments in oncology with unknown gonadotoxicity effects, the risk of ovarian gonadotoxicity can be estimated in specific treatments which enough studies were done, and with sufficient detail, also ovarian reserve testing may be helpful, however, they have limited authority for predicting future fertility. So decision-making for many patients requires multidisciplinary discussion to evaluate the risk and benefits of FP interventions exactly. Providing comprehensive information is crucial to support the patients to have true decision-making.

Despite some general limitations in the current review of the published guidelines and the limited provided evidence, there is a hope that this document will help best practices in female FP.

From all guidelines, the following recommendations can be extracted:


#### **Figure 1.**

*Flow chart of a comprehensive guideline for referring cancer patients for fertility preservation.*


*Cryopreservation - Applications and Challenges*

#### **Author details**

Mahboubeh Vatanparast Molecular Medicine, Research Center, Research Institute of Basic Medical Sciences, Rafsanjan University of Medical Sciences, Rafsanjan, Iran

\*Address all correspondence to: mahboob\_vatan@yahoo.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Ovarian Tissue Cryopreservation Guidelines DOI: http://dx.doi.org/10.5772/intechopen.108201*

#### **References**

[1] Ethics Committee of the American Society for Reproductive Medicine. Fertility preservation and reproduction in cancer patients. Fertility and Sterility. 2005;**83**(6):1622-1628

[2] Revel A, Revel-Vilk S. Fertility preservation in young cancer patients. Journal of Human Reproductive Sciences. 2010;**3**(1):2

[3] Miller KD, Fidler-Benaoudia M, Keegan TH, et al. Cancer statistics for adolescents and young adults, 2020. CA: A Cancer Journal for Clinicians. 2020; **70**(6):443-459

[4] Woodruff TK. The emergence of a new interdiscipline: Oncofertility. In: Oncofertility Fertility Preservation for Cancer Survivors. New York: Springer; 2007. pp. 3-11

[5] Ruan X, Cheng J, Du J, et al. Prevention and treatment of iatrogenic premature ovarian insufficiency: Interpretation of the first Chinese guideline on ovarian tissue cryopreservation and transplantation. Global Health Journal. 2021;**5**(2): 70-73

[6] Fleischer RT, Vollenhoven BJ, Weston GC. The effects of chemotherapy and radiotherapy on fertility in premenopausal women. Obstetrical & Gynecological Survey. 2011;**66**(4):248-254

[7] Gracia CR. Reproductive health after cancer. Oncofertility. 2010;**156**:3-9

[8] von Wolff M, Andersen CY, Woodruff TK, et al. Ferti PROTEKT, oncofertility consortium and the danish fertility-preservation networks—What can we learn from their experiences? Clinical Medicine Insights:

Reproductive Health. 2019;**13**: 1179558119845865

[9] Donnez J, Dolmans M-M, Diaz C, et al. Ovarian cortex transplantation: Time to move on from experimental studies to open clinical application. Fertility and Sterility. 2015;**104**(5): 1097-1098

[10] TEGGoFF et al. ESHRE guideline: female fertility preservation†. Human Reproduction Open. 2020;**2020**(4):1-17

[11] Rodriguez-Wallberg KA, Oktay K. Fertility preservation during cancer treatment: Clinical guidelines. Cancer Management and Research. 2014;**6**:105

[12] Shapira M, Raanani H, Barshack I, et al. First delivery in a leukemia survivor after transplantation of cryopreserved ovarian tissue, evaluated for leukemia cells contamination. Fertility and Sterility. 2018;**109**(1):48-53

[13] Demeestere I, Simon P, Emiliani S, et al. Fertility preservation: Successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin's disease. The Oncologist. 2007;**12**(12):1437-1442

[14] Dolmans M-M, Donnez J, Cacciottola L. Fertility preservation: The challenge of freezing and transplanting ovarian tissue. Trends in Molecular Medicine. 2021;**27**(8):777-791

[15] Wallace WHB, Smith AG, Kelsey TW, et al. Fertility preservation for girls and young women with cancer: Population-based validation of criteria for ovarian tissue cryopreservation. The Lancet Oncology. 2014;**15**(10):1129-1136

[16] Anderson RA, Baird DT. The development of ovarian tissue

cryopreservation in Edinburgh: Translation from a rodent model through validation in a large mammal and then into clinical practice. Acta Obstetricia et Gynecologica Scandinavica. 2019;**98**(5):545-549

[17] Abedelahi A, Rezaei-Tavirani M, Mohammadnejad D. Fertility preservation among the cancer patients by ovarian tissue cryopreservation, transplantation, and follicular development. Iranian Journal of Cancer Prevention. 2013;**6**(3):123-132

[18] Sugishita Y, Taylan E, Kawahara T, et al. Comparison of open and a novel closed vitrification system with slow freezing for human ovarian tissue cryopreservation. Journal of Assisted Reproduction and Genetics. 2021;**38**(10): 2723-2733

[19] Isachenko V, Lapidus I, Isachenko E, et al. Human ovarian tissue vitrification versus conventional freezing: Morphological, endocrinological, and molecular biological evaluation. Reproduction. 2009;**138**(2):319-327

[20] Kagawa N, Silber S, Kuwayama M. Successful vitrification of bovine and human ovarian tissue. Reproductive BioMedicine Online. 2009;**18**(4):568-577

[21] Labrune E, Jaeger P, Santamaria C, et al. Cellular and molecular impact of vitrification versus slow freezing on ovarian tissue. Tissue Engineering Part C: Methods. 2020;**26**(5):276-285

[22] Amorim CA, Dolmans M-M, David A, et al. Vitrification and xenografting of human ovarian tissue. Fertility and Sterility. 2012;**98**(5): 1291-1298.e2

[23] Huang L, Mo Y, Wang W, et al. Cryopreservation of human ovarian tissue by solid-surface vitrification. European Journal of Obstetrics &

Gynecology and Reproductive Biology. 2008;**139**(2):193-198

[24] Wang Y, Xiao Z, Li L, et al. Novel needle immersed vitrification: A practical and convenient method with potential advantages in mouse and human ovarian tissue cryopreservation. Human Reproduction. 2008;**23**(10): 2256-2265

[25] Li Y-B, Zhou C-Q, Yang G-F, et al. Modified vitrification method for cryopreservation of human ovarian tissues. Chinese Medical Journal. 2007; **120**(02):110-114

[26] Zhou XH, Zhang D, Shi J, et al. Comparison of vitrification and conventional slow freezing for cryopreservation of ovarian tissue with respect to the number of intact primordial follicles: A meta-analysis. Medicine. 2016;**95**(39):e4095

[27] Campos ALM, Guedes JS, Rodrigues JK, et al. Comparison between slow freezing and vitrification in terms of ovarian tissue viability in a bovine model. Revista Brasileira de Ginecologia e Obstetricia. 2016;**38**: 333-339

[28] Rahimi G, Isachenko E, Isachenko V, et al. Comparison of necrosis in human ovarian tissue after conventional slow freezing or vitrification and transplantation in ovariectomized SCID mice. Reproductive BioMedicine Online. 2004;**9**(2):187-193

[29] Medicine PCotASfR. Ovarian tissue cryopreservation: A committee opinion. Fertility and Sterility. 2014;**101**(5): 1237-1243

[30] Silber S. Ovary cryopreservation and transplantation for fertility preservation. Molecular Human Reproduction. 2012; **18**(2):59-67

*Ovarian Tissue Cryopreservation Guidelines DOI: http://dx.doi.org/10.5772/intechopen.108201*

[31] Arav A, Patrizio P. Techniques of cryopreservation for ovarian tissue and whole ovary. Clinical Medicine Insights: Reproductive Health. 2019;**13**: 1179558119884945

[32] Arav A, Gavish Z, Elami A, et al. Ovarian function 6 years after cryopreservation and transplantation of whole sheep ovaries. Reproductive BioMedicine Online. 2010;**20**(1):48-52

[33] Arav A, Revel A, Nathan Y, et al. Oocyte recovery, embryo development and ovarian function after cryopreservation and transplantation of whole sheep ovary. Human Reproduction. 2005;**20**(12):3554-3559

[34] Demeestere I, Simon P, Emiliani S, et al. Orthotopic and heterotopic ovarian tissue transplantation. Human Reproduction Update. 2009;**15**(6): 649-665

[35] Wallace WHB, Kelsey TW, Anderson RA. Fertility preservation in pre-pubertal girls with cancer: The role of ovarian tissue cryopreservation. Fertility and Sterility. 2016;**105**(1):6-12

[36] Donnez J, Dolmans M-M, Pellicer A, et al. Restoration of ovarian activity and pregnancy after transplantation of cryopreserved ovarian tissue: A review of 60 cases of reimplantation. Fertility and Sterility. 2013;**99**(6):1503-1513

[37] Fertility preservation in patients undergoing gonadotoxic therapy or gonadectomy: A committee opinion. Fertility and Sterility. 2019;**112**(6): 1022-1033

[38] Kometas M, Christman GM, Kramer J, et al. Methods of ovarian tissue cryopreservation: Is vitrification superior to slow freezing?—Ovarian tissue freezing methods. Reproductive Sciences. 2021;**28**(12):3291-3302

[39] Santaballa A, Márquez-Vega C, Rodríguez-Lescure Á, et al. Multidisciplinary consensus on the criteria for fertility preservation in cancer patients. Clinical and Translational Oncology. 2022;**24**(2):227-243

[40] Rowell EE, Corkum KS, Lautz TB, et al. Laparoscopic unilateral oophorectomy for ovarian tissue cryopreservation in children. Journal of Pediatric Surgery. 2019;**54**(3):543-549

[41] Oktay K. Ovarian tissue cryopreservation and transplantation: Preliminary findings and implications for cancer patients. Human Reproduction Update. 2001;**7**(6):526-534

[42] Dolmans M-M, Donnez J. Fertility preservation in women for medical and social reasons: Oocytes vs ovarian tissue. Best Practice & Research Clinical Obstetrics & Gynaecology. 2021;**70**:63-80

[43] Yasmin E, Balachandren N, Davies MC, et al. Fertility preservation for medical reasons in girls and women: British fertility society policy and practice guideline. Human Fertility. 2018;**21**(1):3-26

[44] De Roo C, Tilleman K, T'Sjoen G, et al. Fertility options in transgender people. International Review of Psychiatry. 2016;**28**(1):112-119

[45] Ruan X. Chinese Society of Gynecological Endocrinology affiliated to the International Society of Gynecological Endocrinology Guideline for ovarian tissue cryopreservation and transplantation. Gynecological Endocrinology. 2018;**34**(12):1005-1010

[46] Mahajan N. Fertility preservation in female cancer patients: An overview. Journal of Human Reproductive Sciences. 2015;**8**(1):3

[47] Schüring A, Fehm T, Behringer K, et al. Practical recommendations for fertility preservation in women by the FertiPROTEKT network. Part I: Indications for fertility preservation. Archives of Gynecology and Obstetrics. 2018;**297**(1):241-255

[48] von Wolff M, Sänger N, Liebenthron J. Is ovarian tissue cryopreservation and transplantation still experimental? It is a matter of female age and type of cancer. Journal of Clinical Oncology. 2018;**36**(33): 3340-3341

[49] Lee SJ, Schover LR, Partridge AH, et al. American Society of Clinical Oncology recommendations on fertility preservation in cancer patients. Journal of Clinical Oncology. 2006;**24**(18):2917-2931

[50] Loren AW, Mangu PB, Beck LN, et al. Fertility preservation for patients with cancer: American Society of Clinical Oncology clinical practice guideline update. Journal of Clinical Oncology. 2013;**31**(19):2500-2510

[51] Backhus LE, Kondapalli LA, Chang RJ, et al. Oncofertility consortium consensus statement: Guidelines for ovarian tissue cryopreservation. In: Oncofertility Fertility Preservation for Cancer Survivors. New York: Springer; 2007. pp. 235-239

[52] Font-Gonzalez A, Mulder RL, Loeffen EA, et al. Fertility preservation in children, adolescents, and young adults with cancer: Quality of clinical practice guidelines and variations in recommendations. Cancer. 2016; **122**(14):2216-2223

[53] Lee D. Ovarian tissue cryopreservation and transplantation: Banking reproductive potential for the future. Oncofertility Fertility Preservation for Cancer Survivors. 2007: **138**:110-129

[54] von Wolff M, Donnez J, Hovatta O, et al. Cryopreservation and autotransplantation of human ovarian tissue prior to cytotoxic therapy—a technique in its infancy but already successful in fertility preservation. European Journal of Cancer. 2009;**45**(9): 1547-1553

[55] Levine J, Stern CJ. Fertility preservation in adolescents and young adults with cancer. Journal of Clinical Oncology. 2010;**28**(32):4831-4841

[56] Harada T, Kuji N, Ishihara O, et al. Guideline for cryopreservation of unfertilized eggs and ovarian tissues in Japan Society of Reproductive Medicine: Ethics Committee in Japan Society of Reproductive Medicine. Reproductive Medicine and Biology. 2019;**18**(1):3

[57] von Wolff M, Montag M, Dittrich R, et al. Fertility preservation in women—A practical guide to preservation techniques and therapeutic strategies in breast cancer, Hodgkin's lymphoma and borderline ovarian tumours by the fertility preservation network FertiPROTEKT. Archives of Gynecology and Obstetrics. 2011;**284**(2): 427-435

[58] Gosden RG, Baird D, Wade J, et al. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at-196 C. Human Reproduction. 1994;**9**(4):597-603

[59] Meirow D, Ra'anani H, Shapira M, et al. Transplantations of frozen-thawed ovarian tissue demonstrate high reproductive performance and the need to revise restrictive criteria. Fertility and Sterility. 2016;**106**(2):467-474

[60] Preservation EGGoFF, Anderson RA, Amant F, et al. ESHRE guideline: Female fertility preservation. *Ovarian Tissue Cryopreservation Guidelines DOI: http://dx.doi.org/10.5772/intechopen.108201*

Human Reproduction Open. 2020; **2020**(4):hoaa052

[61] Wallace WHB, Anderson RA, Irvine DS. Fertility preservation for young patients with cancer: Who is at risk and what can be offered? The Lancet Oncology. 2005;**6**(4):209-218

[62] Karimi-Zarchi M, Khalili MA, Binesh F, et al. Ovarian tissue reservation and risk of reimplantation in a young girl with acute lymphocytic Leukemia after 6-month chemotherapy: A case report. South Asian Journal of Cancer. 2021;**10**(02):112-114

[63] Coccia ME, Rizzello F. Ovarian reserve. Annals of the New York Academy of Sciences. 2008;**1127**(1):27-30

[64] Peluso C, Fonseca F, Rodart I, et al. AMH: An ovarian reserve biomarker in assisted reproduction. Clinica Chimica Acta. 2014;**437**:175-182

[65] Oktay K, Bedoschi G, Berkowitz K, et al. Fertility preservation in women with turner syndrome: A comprehensive review and practical guidelines. Journal of Pediatric and Adolescent Gynecology. 2016;**29**(5):409-416

[66] Jones JM, Fitch M, Bongard J, et al. The needs and experiences of posttreatment adolescent and young adult cancer survivors. Journal of Clinical Medicine. 2020;**9**(5):1444

[67] AYA cancer fertility preservation guidance working group. Fertility Preservation for AYAs Diagnosed with Cancer: Guidance for Health Professionals. Sydney: Clinical Oncology Society of Australia; 2014. Available from: https://wiki.cancer.org.au/austra liawiki/index.php?oldid=78825

[68] Kristensen SG, Giorgione V, Humaidan P, et al. Fertility preservation and refreezing of transplanted ovarian tissue—A potential new way of managing patients with low risk of malignant cell recurrence. Fertility and Sterility. 2017;**107**(5): 1206-1213

[69] Dolmans M-M, Luyckx V, Donnez J, et al. Risk of transferring malignant cells with transplanted frozen-thawed ovarian tissue. Fertility and Sterility. 2013;**99**(6):1514-1522

[70] Dolmans M-M. Safety of ovarian autotransplantation. Blood, The Journal of the American Society of Hematology. 2012;**120**(22):4275-4276

[71] Dolmans M-M, Marinescu C, Saussoy P, et al. Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe. Blood, The Journal of the American Society of Hematology. 2010;**116**(16): 2908-2914

[72] Rosendahl M, Andersen MT, Ralfkiær E, et al. Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia. Fertility and Sterility. 2010;**94**(6): 2186-2190

[73] Greve T, Clasen-Linde E, Andersen MT, et al. Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells. Blood, The Journal of the American Society of Hematology. 2012;**120**(22): 4311-4316

[74] Imbert R, Moffa F, Tsepelidis S, et al. Safety and usefulness of cryopreservation of ovarian tissue to preserve fertility: A 12-year retrospective analysis. Human Reproduction. 2014; **29**(9):1931-1940

[75] Forman EJ. Ovarian tissue cryopreservation: Still experimental? Fertility and Sterility. 2018;**109**(3): 443-444

[76] Oktay KH, Marin L, Petrikovsky B, et al. Delaying reproductive aging by ovarian tissue cryopreservation and transplantation: Is it prime time? Trends in Molecular Medicine. 2021;**27**(8): 753-761

[77] Donnez J, Dolmans M-M. Ovarian cortex transplantation: 60 reported live births brings the success and worldwide expansion of the technique towards routine clinical practice. Journal of Assisted Reproduction and Genetics. 2015;**32**(8):1167-1170

[78] Shi Q, Xie Y, Wang Y, et al. Vitrification versus slow freezing for human ovarian tissue cryopreservation: A systematic review and meta-anlaysis. Scientific Reports. 2017;**7**(1):1-9

[79] Zhang X, Niu J, Che T, et al. Fertility preservation in BRCA mutation carriers—Efficacy and safety issues: A review. Reproductive Biology and Endocrinology. 2020;**18**(1):11

#### **Chapter 5**

## Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes

*Peter Kilbride, Julie Meneghel, Mira Manilal Chawda, Susan Ross and Tessa Crompton*

#### **Abstract**

This chapter covers the key physical, biological and practical challenges encountered when developing cryopreservation protocols for larger biological structures and examines areas where cryopreservation has been successful in scaling to larger structures. Results from techniques being used in attempts to overcome these challenges are reviewed together with the indicators for future development that arise from them. The scale-up of cryopreservation to tissues with diverse functions and cell types makes the control of freezing and thawing more challenging. Technology may or may not—be available depending on the size of the material involved. To meet the challenge there must be innovation in technology, techniques and understanding of damage-limiting strategies. Diversity of cell structure, size, shape and expected function means a similarly diverse response to any imposed cryopreservation conditions and interaction with ice crystals. The increasing diffusion distances involved, and diversity of permeability properties, will affect solutes, solvents, heat and cryoprotectant (CPA) transfer and so add to the diversity of response. Constructing a single protocol for cryopreservation of a larger sample (organoids to whole organs) becomes a formidable challenge.

**Keywords:** cryopreservation, tissues, organs, slow cooling, diffusion, cryoprotectants, ice

#### **1. Introduction**

Historically, the predominant application of cryopreservation was in agriculture and reproductive medicine, starting with stored spermatozoa in the 1950s and oocytes being widely cryopreserved beginning in the late 1980s [1–3]. In the past decade, a revolution in tissue engineering has changed the landscape of cryopreservation and there is now a growing and critical need for successful cryopreservation of somatic cells not only as low volumes of cell suspensions but also in larger quantities and, increasingly, as part of a complex cell network. In such a network, different cells may have a range of different functions and structural requirements [3–6]. These larger subjects can contribute directly to a therapeutic treatment or can be

cryopreserved as tissue from which cells can be isolated to begin a manufacturing process [5, 7, 8]. A new demand has, therefore, been created for cryopreservation of larger subjects ranging from cell spheroids and organoids to tissue slices and, eventually, entire organs [3, 4, 6–11]. The potential benefits of cryopreservation of these multicellular and differentiated structures range from facilitating population-wide biopsy studies to supporting large-scale manufacturing and providing economies of scale within organoid preservation. Realising these benefits would support a sizable fraction of the needs of regenerative medicine and would advance progress towards organ cryopreservation, a key and as yet unmet need in transplantation technology.

The first steps towards large-volume cryopreservation must necessarily exploit the knowledge gained from the widespread, successful cryopreservation of cell suspensions [12, 13]. This success stems from the level of control of pre-treatment, cooling, warming and recovery that can be exerted over the cells [14, 15]. Appropriate control is supported by a specific technology, including programmable freezers and mathematical modelling, and benefits significantly from the relative uniformity of cell size, shape and cytoplasmic content of the majority of cell types of interest [15–19]. The important diffusion distances for solutes, solvents and heat are short for these suspended cells with little cell-to-cell differences and so provide relatively uniform responses to imposed conditions. Responses to applied cryoprotectant (CPA), whether physiological, osmotic or related to toxicity are also relatively uniform within a single cell type [20]. Additional complications that are introduced by a relatively large bulk volume of suspension, such as heat transfer across the sample, can be modified by altering the geometry of the sample, e.g., by flattening a cryobag containing suspended cells during cooling and warming [6, 8, 16].

While there is a promise with ice-free techniques, also known as vitrification, these have been covered in other reviews and so will not be examined here [21, 22].

#### **2. The cryobiology of scale up**

#### **2.1 Practical challenges**

Significant challenges that arise when moving up to the cryopreservation of large, coherent cell masses are caused directly by the size and volume of the tissues concerned. As noted above, in a cell suspension the diffusion distances between the cytoplasm and surrounding medium are effectively constant for each cell, ensuring relatively uniform responses to imposed physical and chemical diffusion gradients, such as external cooling and CPA addition. For larger cell masses such as organoids, a much greater range of diffusion distances exists because cells further towards the centre of the structure are increasingly distant from the external medium [6, 8, 17, 18, 23, 24]. Rates of diffusion for these cells are further complicated as diffusion within the overall cell mass will involve transfer across adjacent cells and intracellular spaces, with a range of differing properties before the external medium is reached [4, 24].

For numerous mammalian cell types (including cells derived from blood, liver and ovaries), cryopreservation of suspensions is straightforward with limited loss of cell viability and function [12]. Moving up in size to single cell-type spheroids this success is often continued, as in the case for liver spheroids [8, 25, 26]. As biological structures become more complicated, e.g., cell organoids composed of several different cell types, success is more limited with a strong, negative influence of size [4, 6]. Many smaller, immature organoids, typically consisting of no more than a few hundred


*Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes DOI: http://dx.doi.org/10.5772/intechopen.108254*

#### **Table 1.**

*A summary of some tissue types currently cryopreserved in the presence of ice successfully, and the methods used to achieve results.*

cells can be cryopreserved [27] but for larger, mature organoids in their final state for therapeutic use, cryopreservation success is more limited. **Table 1** summarises some successful strategies for a range of such tissue types.

For even larger tissues and whole organs, success is largely limited to those which can operate as discrete units when dissected, for example, ovarian tissue and thymic slices [31, 32, 34, 35]. These can be removed from the body and cut into smaller functional units, which can each be successfully cryopreserved, thawed and transplanted independently. Mammalian organs lacking this ability such as the heart and kidneys cannot, as yet, be cryopreserved successfully [9, 10]. A famous 1978 paper on the subject started with the line '*Attempts to preserve viable kidneys by freezing in the presence of cryoprotective agents have been notoriously frustrating*'—a statement no less true today than it was 45 years ago! [36]. The ability to cryopreserve elements of structure and function in excised tissues also has clear medical benefits when applied to biopsy samples. For microscopic investigation where the function is not required then

structure/tissue architecture is of primary concern [37]. Conversely, when functional assessment is required, then balanced and optimal cellular performance must take precedence over structure. This indicates an interesting, and valuable, halfway house for cryopreservation where success can be measured in terms of either the structural integrity or function of recovered material [37, 38].

Cell therapies and regenerative medicine treatments require methods that offer successful cryopreservation, are practical and meet regulatory requirements if they are to form parts of medical devices and/or require cGMP manufacture [14, 26, 39, 40]. This can become an issue for larger samples if novel, larger sample containers need to be devised to facilitate effective processing, including CPA treatment, cooling and warming. For example, only cryobags and hermetically sealed cryovials are permissible for cGMP therapies. The latter enables simplified aseptic filling operations and typically has thickened plastic walls to prevent damage at low temperatures. These thick walls limit the heat transfer rates achievable and so may influence the design of the cryopreservation protocol [41]. On the contrary, cryostraws, commonly used in reproductive medicine, have internal diameters in the order of 1–2mm that increase their surface-to-volume ratio for more efficient thermal transfers of the sample and so they are only suitable for the smallest spheroids and organoids [42]. Regulations of course vary between regions, but broadly align when the manufacture and use can take place over multiple jurisdictions, requiring compliance with all regulatory regimes [14, 40, 43].

It is important to accommodate such practical difficulties into the initial design of the cryopreservation protocol as retro-adapting methods for clinical delivery once they have been developed are lengthy and costly and can delay (and in some cases prevent) a treatment gaining widespread use. Other issues such as a need for automation during processing may also have an impact [14, 40].

#### **2.2 CPA loading and unloading**

An early event where the extended diffusion pathways of larger structures are evident is in the loading and unloading of permeating CPAs such as Me2SO [6, 11, 17, 18, 24, 34]. Following the addition of a permeating CPA an initial, cellular response of exposed cells is to shrink due to the osmotic gradient the CPA exerts [34]. As the CPA then permeates into the cell, the gradient is diminished and cell volume recovers to a significant extent [34]. In larger structures, exposure to the gradient, and the responses to it, will be delayed for those cells embedded deeper in the structure [44]. This generates a risk of insufficient CPA protection if cooling proceeds before CPA equilibration is reached in the central regions of the structure. However, an extended incubation time in the CPA to ensure deep equilibration can lead to damaging levels of toxicity for more peripheral cells. The larger and more complex the structure the more challenging this issue becomes, with both extracellular channels, cell membrane parameters, viscosity, temperature and physical distance all playing a role [19, 24, 44]. A similar issue, but reversed in direction, is encountered on warming and subsequent CPA removal [18].

Tissue architecture can provide additional complications for CPA treatments. For example, mature organoids may contain a central cavity devoid of cells, or with a different cellular composition [27, 45] and sufficient time for CPA diffusion into this cavity is necessary to prevent further CPA diffusion from the innermost cells into the cavity following cellular equilibration. This would result in an overall CPA loss from the inner cells, compromising the chances of achieving the required level

#### *Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes DOI: http://dx.doi.org/10.5772/intechopen.108254*

of their post-thaw cell survival to maintain organoid integrity. Chondrocyte and cartilage samples, typically cryopreserved with bone attached, provide a further example. As commonly used CPA cannot pass through bone, this further limits the surface area for diffusion of water and CPA, restricting diffusion pathways and transfer speed [17, 23].

Several methods have been employed to alleviate CPA loading and unloading difficulties that may prove to be applicable if modified for larger structures. One such method involves adding an initial CPA concentration to the external medium that is higher than that considered necessary for successful cryopreservation. As CPA diffusion is driven by concentration gradients, this higher concentration external to the biological sample will increase the CPA diffusion rate and thereby reduce the required incubation time. When the tissue is calculated to be sufficiently protected, the extracellular CPA concentration can be reduced to its equilibrium value [17, 23, 44]. Such methods are more often used with systems preserved through vitrification (ice-free cryopreservation) but are equally useful to overcome CPA loading issues in slow-cooling techniques. However, the high concentration of CPAs, at the relatively high incubation temperatures employed, can cause significant cytotoxic responses in sensitive cells near the outer surfaces of a larger structure. The temperature could be reduced to lower CPA toxicity, but as viscosity is temperature dependent [46, 47], any lowering of temperature would increase incubation times to achieve the required level of diffusion, thereby negating any benefit of the lowered temperatures. Using a mixture of different CPAs can reduce the concentration, and so toxicity, of any one given CPA can also be used to mitigate this problem. Such techniques are common in large-volume vitrification and may help in slow-cooled systems with long incubation times [20, 48, 49].

When working with entire organs in which the circulatory system is intact, the blood vessels can be perfused to reduce CPA distribution time and ensure homogenous CPA loading [35]. Perfusion is an established technique in major surgery and organ analysis [50] and the replacement of blood or stabilising solutions with CPAs can effectively reach areas of tissues difficult to reach by diffusion or surface-induced effects alone [21, 31, 35, 51]. This has shown to be effective in some cases [21, 31, 33, 51], yet most studies focus on the very high CPA concentrations required for vitrification that are currently less applicable to larger structures using slower cooling rates. The systems involved may be susceptible to vasculature cryoinjury, with damage to small blood vessels during cooling, sufficient to prevent effective CPA removal resulting in necrotic areas after thawing due to CPA toxicity. These methods are also limited to tissues with the full circulatory system—immune privileged tissues without vasculature cannot benefit from this technique—and require specific technical skills to perfuse the organs successfully.

Extracellular CPAs, which can help dehydrate cells and protect cell membranes pose particular problems for larger structures as they will only protect the outermost cells of the structure, or ones that can be reached through extracellular liquid channels. Innovative methods to exploit the potential benefits of perfusion to slow cooling techniques are required**.**

#### **2.3 Diffusion of heat and intracellular water**

In a suspension of separated cells undergoing cryopreservation, the diffusion distance for heat, water and solutes between individual cells and the external medium is no larger than the radius of a cell. Additionally, diffusion of water and intracellular

CPAs is influenced by membrane permeability to these compounds and the cell surface area to volume ratio. These factors will vary in differing, but limited ways when small-cell aggregates are present. Having relatively uniform characteristics means that the cellular responses of single cells, and small-cell aggregates, to imposed thermal or chemical gradients will be similarly uniform, providing the level of control needed for successful cryopreservation. As noted above, the consequence of working with larger, multicellular structures is that the diffusion pathways are extended and depend on the dimensions of the cell mass. They will also involve transfer across a number of cells and extracellular space [11, 24, 44]. The location of individual cells within the cell mass and their type—each with their specific membrane permeability coefficients and surface area to volume ratios—will influence their response to any imposed diffusion gradient over time and so the level of overall control of heat and water and diffusion of CPAs will be diminished.

Some dehydration may occur in response to CPA treatment in the initial phase of the preservation protocol but the greater part occurs once the ice has formed in the system [41, 44]. This is referred to as cryodehydration. During controlled, slow cooling the extracellular solution commonly falls below its melting point, entering a supercooled state, before ice forms by spontaneous, or induced nucleation [52–54]. When ice nucleates there will be a temperature discontinuity (an exotherm) within the system related to the release of latent heat of freezing accompanied by a sharp increase in the osmolality of the extracellular medium as water molecules, and only water molecules, become components of ice crystals [55]. Biological material is excluded from the crystal lattice [52]. The nucleation event initiates protective cryodehydration, as described above, but if supercooling is extreme prior to nucleation then the large and immediate osmotic shock delivered once ice forms can be damaging to the sample. The overall size of a sample (tissue mass plus cryomedium) influences ice nucleation and the larger the volume the earlier ice nucleates [52, 56].

Once nucleation has taken place, cells in suspension become entrapped in channels between ice crystals and cellular dehydration is primarily limited by their membrane permeability to water [54, 57, 58]. This protective cryodehydration during cooling is essential as cells retaining a high intracellular water content are more likely to experience lethal intracellular ice formation (IIF) than their more dehydrated counterparts [11, 59, 60]. Cells that have a high membrane permeability to water can survive relatively rapid cooling as water is able to leave the cell quickly enough to prevent IIF. However, at lower temperatures cell permeability decreases, the level of this reduction being cell type dependent. The lower the permeability the slower cooling must proceed to ensure sufficient dehydration occurs, with 1°C/min after ice nucleation being a typical value for somatic mammalian cells in suspension [41, 54].

Larger structures will become embedded in the matrix of ice crystals after nucleation. In a cell spheroid, for example, not all the cells are at the outer surface and so, rather than dehydrating directly into the cryoprotective medium, some cells will transfer water to those in physical contact with them that generate an osmotic gradient, and only those at the outer surface of the sphere will interact directly with the extracellular medium. The overall dehydration rate for the spheroid is, therefore, slower than would be observed for single cells in suspension and, inevitably, the fastest acceptable cooling rate for cryopreservation of the biological sample will also be lower. However, as the slowest cooling rate is essentially defined by the sensitivity of the cell type to CPA toxicity, this remains unaltered, resulting in a narrowing of the range of acceptable cooling rates for successful post-thaw survival [59]. In some

#### *Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes DOI: http://dx.doi.org/10.5772/intechopen.108254*

instances, a lower recovery rate than is seen in suspensions can be the consequence of the slower rate for the complex system—the highest survival after optimisation being lower than the value achieved in suspensions. In HepG2 liver cell spheroids, the optimal cooling rate falls to 0.3°C/min from 1 to 2C°C/min for a cell cluster of a few hundred cells [8]. This problem becomes more pronounced in organoids containing multiple cell types where dehydration will be limited by the cells with the lowest membrane permeability—the maximum cooling rate becoming increasingly slower as the biological structure becomes larger and more complex. The solution to this dehydration problem is likely to be to lower the cooling rate, where this does not impact post-thaw cell functions. Most somatic mammalian cells can tolerate a relatively low cooling rate, down to 0.1–0.3°C/min, which is usually sufficient for dehydration to occur. T cells for example have shown similar optimal survival at rates of 1°C/min and as low as 0.1°C/min [41], and ovarian tissue samples are typically cooled at rates of 0.2–0.3°C/min [31, 61–64]. As can be seen in **Table 1**, most spheroid and organoid cryopreservation methods currently use passive coolers, where control of the cooling rate is limited and producing rates in the vicinity of/of approximately 1°C/min—moving to controlled rate freezers with lower and more precise rates would allow for more precise control over cell dehydration [15].

Ice formation can be physically damaging for cell suspensions when the cells become trapped in channels between crystals [58]. At higher temperatures, the channels are relatively wide, and the cells have minimal direct contact with ice crystals, minimising the potentially damaging effects of distortion, crushing and shear forces. As the temperature falls, more water molecules are locked away as ice and the channels reduce in size [54, 55, 58]. Larger samples are at an increased risk of direct contact with ice under these circumstances, resulting in damage that can impact negatively on recovery. Relatively delicate tissues such as spheroids and organoids can be crushed in this way. Extracellular ice also damages complex tissue structures by disrupting cell-cell contacts, and thereby damaging intercellular communications. Severing these connections is not only damaging to individual cells, it can also reduce the overall function and communication between the surviving cells tissue or organoid.

Different CPAs can be used, perhaps in combination, to help with dehydration difficulties with larger samples. Where lower cooling rates are not practically possible or biologically tolerable with only the permeating CPA Me2SO, then dehydration can be accelerated through the use of extracellular CPAs such as sugars [3, 20, 31, 64]. These CPAs decrease the osmotic potential in the extracellular space, and so can drive more rapid dehydration. This may offset the effect of a lower surface-to-volume ratio of spheroids and organoids relative to individual cells. The addition of different types of CPAs, such as apoptosis inhibitors to the cryopreservation and post-culture medium, has been shown to improve organoid survival in some systems [4]. Altering the size and shape of samples where original structure and integrity are not the priority can also improve the outcome. Ovarian tissue for example is often cryopreserved in strips to maximise the dehydration rates as these tend to be more effective than spheres due to the larger surface area they provide, and an increased surface area can improve biological outcomes [63, 64]. However, in many cell types, when it comes to large, mature organoids containing several cell types the problems faced by dehydration issues cannot easily be overcome. Intracellular ice can still form and be lethal and more research is required to increase dehydration rates, or perhaps lower the possibility of IIF even at relatively high cell hydration levels.

#### **2.4 Ice nucleation and direct ice damage**

A further issue with extracellular ice formation is the increased volume of ice crystals—when ice forms it expands to occupy approx. 12% more volume than the liquid state. In cell systems such as organoids, the formation of ice in the internal, liquid-filled cavity, can generate sufficient mechanical pressure on the cells lining the cavity to cause significant fractures. This can disrupt the organoid structure, yet individual cells may survive the cryopreservation procedure.

Intracellular ice is lethal for the cell in which it forms but in a cell suspension, where the cells have limited direct contact with each other, a frozen cell rarely nucleates others. A proportion of weakened or damaged cells in the suspension will experience intracellular freezing but this poses little risk for the greater cell population. However, in a larger structure where cells can be tightly pressed together and/or physically interconnected, ice that forms in one cell can spread to another [65]. This triggers a chain of intracellular freezing throughout the structure that can cause significant damage and cell mortality. Strong evidence of the damage that can be caused by ice comes from tissues and organs which survive cryopreservation, at least in part, with slow cooling. Excised ovarian and thymus tissues are notable in this regard and are dealt with in more detail below. The impact of this damage has been observed in thymus slices, cryopreserved at 1°C/min in 10% Me2SO [32].

A good example of this chain reaction of cell-to-cell ice growth is seen when considering the studies presented by Ross et al. [32]. In this work, histology was carried out (H&E staining) to detect viable tissue and areas of autolysis (indicating cell death); autolysis was seen over continuous areas with some completely devoid of surviving cells and other areas with almost total survival. Autolysed areas form in different places in different samples and so are not related to location in the tissue or placement in the vial in which it was preserved. In a thawed tissue, mass with limited intercellular connections, living and dead cells would be expected to be distributed relatively uniformly throughout the tissue. The aggregated areas of autolysed cells observed suggest there was a significant intercellular connection (as far as required for ice to spread) within the tissue and that once intracellular ice nucleation occurred in a small number of cells it spread rapidly to conjoined neighbours. When thawed, these slices were transplanted into an athymic mouse model where they were able to support T-cell development, showing preservation of function [32].

In certain circumstances, supercooling techniques have been proposed as an alternative cryopreservation method that avoids ice and its associated lethal impacts. Supercooling involves cooling a sample to high sub-zero temperatures, typically between 0 and − 10°C, under conditions where ice is relatively unlikely to form thermodynamically. At such temperatures, biological activity is reduced and both structure and function can be protected for several days. Whilst such a short timeframe is limiting, this can be sufficient to overcome extreme time constraints associated with, for example, transport, quality checks and organ transplants [11, 62, 66–68].

#### **2.5 Control of ice structure: a way forward?**

Ice damage is generally accepted to be the most severe and the leading cause of cryopreservation-related injury and cell death in large biological tissues [11, 21, 60, 69] and can be considered the most difficult problem to overcome. However, ice crystal structure is not constant [41, 58] and new ways of manipulating ice growth may help reduce the damage it causes.

#### *Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes DOI: http://dx.doi.org/10.5772/intechopen.108254*

One of the simplest ways to change ice structure is by manipulating the cooling rate, especially in the high sub-zero zone where most ice forms (c. −5 to −40°C) [41, 47]. In **Figure 1**, the ice structure of a 10% Me2SO solution is shown for samples experiencing cooling at 10°C/min; cooled at 1°C/min and at 0.1°C/min. These rates were those recorded after ice was nucleated at −4°C. At very low rates of cooling where ice growth rates are also very slow, the ice has time to organise into large crystals—the most thermodynamically favourable state. Research is limited as to how different forms of macroscopic ice structure impact cryopreservation; however, slower rates of ice growth are known to inhibit damaging ice-recrystallisation on thawing and reduce the osmotic pressure on the cells as the rate at which water molecules are locked into any recrystallising ice is reduced [41, 70]. The ice structure is very different at 1°C/min, a typical cooling rate for cell suspensions, compared with cooling at the much slower 0.1°C/min.

There are some indications that by using very low rates of cooling, more structure can be preserved. **Figure 2** shows the whole mouse embryonic kidney, heart and liver cryopreserved at only 0.2°C/min in 12.5% Me2SO. As can be seen in the figure, these organs (2–5 mm max. dimension) had good post-thaw structure.

#### **Figure 1.**

*The structure of ice in a 10% Me2SO saline solution in a cryomicroscope at 10x magnification after controlled cooling at different rates to −100°C. samples were cooled, left to right, at 10°C/min, 1°C/min and 0.1°C/min. The extremely low cooling rate used in C results in a markedly different ice structure.*

#### **Figure 2.**

*Mouse embryonic kidney, heart, and liver after cooling at 0.2°C/min and storage in LN for >30 days. The overall structure of the organ (top), and histology (bottom, H&E stain) of the tissue indicate minimal cell and structural damage on cooling.*

New developments in cryopreservation technology allow ultra-slow cooling rates and long cooling times, and so open the door to new ice structures—mammalian nucleated somatic cells tend to be robust to very slow rates of cooling. Many of the large tissues currently cryopreserved use very slow rates of cooling—ovaries at 0.2 or 0.3°C/ min [31, 63, 64], liver spheroids at 0.3°C/min [8] and uterus at 0.2°C/min [33] —while this will help in dehydration and CPA diffusion as discussed above, the different ice structure in these ultra-slow cooling regimes likely plays a role.

Historically, the manipulation of cooling rates was seen as a key parameter to the successful cryopreservation of whole organs. A 1984 study found that extremely low rates of cooling, as low as 1°C/hr. in this case, resulted in better vascular resistance readings, tissue architecture observations, with ice seeming to have been localised to extracellular zones more at these slower rates of cooling [71]. Microscopic studies using freeze-substitution paint a similar picture [72]. Such slow rates of cooling have been scarer in recent years, partly due to the practical difficulties of applying low cooling rates at the time, and due to fewer needs for larger structure cryopreservation. Applying these exciting but somewhat neglected methods to modern tissue-engineered structures and organs, along with combining them with new cryoprotectant knowledge and technologies offers perhaps the best chance for widespread tissue preservation.

Ice structure can also be effectively manipulated through the introduction of ice nucleation and ice-inhibiting particles, as well as cooling rates and CPA concentration [60]. Higher nucleation temperature tends to cause larger ice crystals as less of the freezable water solidifies at the initial point of nucleation, more supercooling—as is seen in the absence of ice nucleators—causes a smaller, more dendritic and ice structure. More viscous CPAs will slow the rate of ice crystals growth by inhibiting the diffusion of water molecules onto the crystal-liquid interface [19, 70]. In future, adapting parameters such as this may be able to reduce the damage caused by ice enough to allow for the preservation of a larger portion of a larger number of tissues.

#### **3. Cryopreservation of larger structures: the special case of ovaries and thymus**

The ovary consists of follicles at various states of maturity, in which immature oocytes reside. These follicles and slices of the mammalian ovary have been successfully cryopreserved [61, 63, 64, 73]. Ovarian tissue can be cryopreserved before cancer treatments which may damage the ovaries and can be thawed and transplanted when the patient wants to have a baby, allowing for natural conception [63, 64, 74, 75]. In human ovaries, the tissue is often cryopreserved in follicle-containing slices, which in addition to simplifying the physical problems of larger tissue cryopreservation, has the additional advantage that only a single slice has to be transplanted back at any one time, allowing for multiple pregnancies following separate thawing procedures. Ovarian tissue preservation can be particularly beneficial in pre-pubescent girls undergoing treatment where hormonal stimulation to produce mature oocytes for cryopreservation is usually not possible [63, 64, 74].

Carroll et al. [76] first published successful births in mouse ovarian follicles (a liquid membrane containing immature oocytes which are surrounded by layers of granulosa cells) in 1990. The method involved incubating the samples in Me2SO (1.5 M) and serum for 10–12 minutes, then seeding ice at −7°C and followed by a cooling rate of 0.3°C/min [76]. By 2014, Campbell et al. cryopreserved whole sheep

#### *Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes DOI: http://dx.doi.org/10.5772/intechopen.108254*

ovaries, which were able to produce fertile offspring after thaw and re-transplantation [31]. For these larger tissues, the ovary was first perfused [35] using the blood vessel architecture with CPAs (Me2SO, calf serum, and extracellular CPA sucrose, for up to 60 minutes, and cooling proceeded at only 0.2°C/min). The success of these techniques shows that, with sufficient CPA incubation, appropriate cooling rates and controlled ice nucleation, then larger structures can be preserved with widespread success [61–64, 75, 77]. It is observed that tissue that can be physically sliced and still function on transplant can also survive cryopreservation, and tissues that cannot be sliced and survive do not survive cryopreservation. This may indicate that physical damage due to ice disruption within tissues is certainly a central issue in the cryopreservation of larger tissue samples.

Another tissue that can be cryopreserved with success is the paediatric thymus. Thymus transplantation is carried out to treat paediatric diseases such as complete DiGeorge syndrome, in which infants lack a thymus [78]. Thymus is obtained from a donor and sliced into up to 30 pieces, approximately 1 mm thick. These slices are then cultured to deplete the donor thymocytes (large numbers of donor thymocytes could potentially cause an immune reaction in the host), leaving mainly stromal and epithelial cells for transplantation. Transplantation is done in the well-vascularised thigh where circulating recipient progenitor cells are able to populate the transplanted slices and undergo T-cell development, eliminating the need for more complicated chest surgery where the thymus usually resides [32, 78, 79].

Cryopreserving such tissues will allow for the creation of thymic tissue banks, giving a supply of tissue on patient demand and allowing for future recipient tissue or partial tissue matching, surgery at the optimal time and location for the recipient. The authors have found that these samples can be cryopreserved at 1°C/min in 10% Me2SO without the need for ice nucleation [32]. Rapid diffusion of water and solutes is facilitated by the slicing of the tissue pre-cryopreservation. The thymus does not have to be completely intact to fulfil its function of supporting T-cell development, so the areas of tissue that survive the freeze/thaw have sufficient capacity to restore the peripheral T-cell population in the mouse model [32].

#### **4. Biopsies**

An area of cryopreservation that is sometimes overlooked is that of biopsies. These small pieces of tissue, typically of the order of 1–3 mm3 , are cryopreserved for reasons ranging from diagnostics and cell extraction to fundamental research [7, 29, 30, 80, 81]. Typical cryopreservation of these structures involves direct plunging into liquid nitrogen without the use of CPAs [80]—this may allow the recovery of some markers and DNA but living cells and faithful tissue architecture is lost. A particularly promising use of optimising biopsy preservation is their use for population-wide studies where biopsies are taken from many patients over many years and stored in biobanks [11, 82]. For the most effective use of such biobanks, preservation methods should allow tissue architecture to be preserved, together with undamaged DNA and protein content, and for viable cells to be available for regrowth. This would open up the possibility of extracting an increased range of data from the samples as well as future-proofing samples for examination by techniques not developed at the time of preservation.

Using current methods, even with the use of CPAs and some control in cooling, liver biopsies can have recovery of oxygen consumption and mitochondrial functions—something elusive with the whole organ [81]. Cryopreserving as tissue or at least as cell clusters may give better single-cell performance than tissue fully digested prior to cryopreservation [83].

Current preservation techniques can provide high level, tissue architectural preservation in organs as complex as the brain, and success has been reported in heart valves using ice-free methods (either vitrification or non-low temperature preservation) to preserve the structure [11, 21, 84, 85]. However, such methods tend to preserve only architecture and not viable cells. Accepting current technical limitations, the balance between preserving tissue architecture/structure or cellular function can be altered. Typically, the structure is the preferred option for biopsies with samples cryopreserved rapidly sometimes in the absence of CPA, resulting in near-total cell death. However, biopsies can also be used to extract living cells, typically for regenerative medicine and organoid culture [29, 30, 82], and slow cooling methods discussed above could allow for sufficient structural preservation as well as ensuring an acceptable recovery of some viable cells. A cryopreservation method where the structure is preserved but also allows for live cells to be extracted would enable considerably more data to be extracted from population-wide samples, markedly improving scientific efficiency and productivity. Overcoming these challenges with new techniques may require an initial focus on specific applications where known demand exists, for example, in biopsy preservation in cancer patients for extraction of tissue infiltrating lymphocytes. Success here might also provide valuable new knowledge relevant to the development of protocols for larger structure cryopreservation.

#### **5. Conclusions and future direction**

Cryopreservation is a rapidly developing field that is continually adapting to meet the challenges presented by ice and low temperatures when trying to preserve viability in larger tissues and structures. The larger structures become, the more challenging attempts at cryopreservation, using current techniques, becomes. It is possible that some methods, such as optimisation of known CPAs, may be approaching maximally optimised thanks to modelling (although the door to new CPAs and their reactions remains open), and most current knowledge gained from cell suspensions has already been applied. However, many relatively unexplored avenues of research are available and actively being explored to achieve a viable post-thaw outcome combining these new techniques with the manipulation of ice structure from lower cooling rates shown to minimise ice damage [71, 72] is an obvious route forward.

There are also cryopreservation methods exploiting higher temperatures, such as supercooling discussed above. Taking samples below the appropriate glass transition temperature (as in conventional storage in liquid nitrogen) will provide dramatically extended storage time for samples, measured at least in decades. However, at a practical level, many applications may not need such a guarantee e.g. preparations for cell therapy or organ and tissue samples destined for application in the short term. Where storage of several weeks would suffice, for example, then storage at a relatively high temperature, where ice could be avoided or at least occupy a lesser fractional volume was harnessed, may provide significant benefit.

In some special cases, such as the thymus and ovaries, it is already becoming possible to cryopreserve mammalian organs and, in time, the number of these cases will doubtless grow through the development of new CPAs, new loading and unloading methods, and techniques to overcome the damaging effects of ice crystals. *Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes DOI: http://dx.doi.org/10.5772/intechopen.108254*

While ice-free methods offer a promising, but more distant avenue for cryopreservation, slow-cooling methods enjoy current success and will likely form the key to the delivery of many cell therapies, tissue-engineered constructs and other larger tissues in the future.

#### **Acknowledgements**

This work was supported by a grant from Great Ormond Street Hospital Children's Charity; MMC was supported by a PhD studentship from the MRC; research at UCL GOSICH is supported by the NIHR BRC at GOSH and UCL.

#### **Author details**

Peter Kilbride1 \*, Julie Meneghel1 , Mira Manilal Chawda<sup>2</sup> , Susan Ross2 and Tessa Crompton<sup>2</sup>

1 Cytiva, Sovereign House, Histon, Cambridge, UK

2 UCL Great Ormond Street Institute of Child Health, London, UK

\*Address all correspondence to: peter.kilbride@cytiva.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Chen C. Pregnancy after human oocyte cryopreservation. The Lancet. 1986;**327**(8486):884-886

[2] Walters EM, Benson JD, Woods EJ, Critser JK. The History of Sperm Cryopreservation. Sperm Banking: Theory and Practice. Cambridge, UK: Cambridge University Press; 2009. pp. 2-10

[3] Mutsenko V, Knaack S, Lauterboeck L, Tarusin D, Sydykov B, Cabiscol R, et al. Effect of 'in air'freezing on postthaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds. Cryobiology. 2020;**92**:215-230

[4] Han S-H, Shim S, Kim M-J, Shin H-Y, Jang W-S, Lee S-J, et al. Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of rho kinase inhibitor. World Journal of Gastroenterology. 2017;**23**(6):964

[5] Kratochvil MJ, Seymour AJ, Li TL, Paşca SP, Kuo CJ, Heilshorn SC. Engineered materials for organoid systems. Nature Reviews Materials. 2019;**4**(9):606-622

[6] Dolezalova N, Gruszczyk A, Barkan K, Gamble JA, Galvin S, Moreth T, et al. Accelerating cryoprotectant diffusion kinetics improves cryopreservation of pancreatic islets. Scientific Reports. 2021;**11**(1):1-18

[7] Pendergraft SS, Sadri-Ardekani H, Atala A, Bishop CE. Three-dimensional testicular organoid: A novel tool for the study of human spermatogenesis and gonadotoxicity in vitro. Biology of Reproduction. 2017;**96**(3):720-732

[8] Kilbride P, Lamb S, Gibbons S, Bundy J, Erro E, Selden C, et al. Cryopreservation and re-culture of a 2.3 litre biomass for use in a bioartificial liver device. PLoS One. 2017;**12**(8):e0183385

[9] Giwa S, Lewis JK, Alvarez L, Langer R, Roth AE, Church GM, et al. The promise of organ and tissue preservation to transform medicine. Nature Biotechnology. 2017;**35**(6):530

[10] Lewis JK, Bischof JC, Braslavsky I, Brockbank KG, Fahy GM, Fuller BJ, et al. The grand challenges of organ banking: Proceedings from the first global summit on complex tissue cryopreservation. Cryobiology. 2016;**72**(2):169-182

[11] Taylor MJ, Weegman BP, Baicu SC, Giwa SE. New approaches to cryopreservation of cells, tissues, and organs. Transfusion Medicine and Hemotherapy. 2019;**46**(3):197-215

[12] Wolkers WF, Oldenhof H. Principles underlying cryopreservation and freeze-drying of cells and tissues. In: Cryopreservation and Freeze-Drying Protocols. New York: Springer; 2021. pp. 3-25

[13] Fuller BJ, Lane N, Benson EE. Life in the Frozen State. Boca Raton, Florida, United States: CRC Press; 2004

[14] Meneghel J, Kilbride P, Morris GJ. Cryopreservation as a key element in the successful delivery of cell-based therapies—A review. Frontiers in Medicine. 2020:**7**

[15] Kilbride P, Meneghel J. Freezing technology: Control of freezing, thawing, and ice nucleation. In: Cryopreservation and Freeze-Drying Protocols. New York: Springer; 2021. pp. 191-201

*Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes DOI: http://dx.doi.org/10.5772/intechopen.108254*

[16] Xu F, Moon S, Zhang X, Shao L, Song YS, Demirci U. Multi-scale heat and mass transfer modelling of cell and tissue cryopreservation. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences. 1912;**2010**(368):561-583

[17] Abazari A, Thompson RB, Elliott JA, McGann LE. Transport phenomena in articular cartilage cryopreservation as predicted by the modified triphasic model and the effect of natural inhomogeneities. Biophysical Journal. 2012;**102**(6):1284-1293

[18] Lawson A, Mukherjee IN, Sambanis A. Mathematical modeling of cryoprotectant addition and removal for the cryopreservation of engineered or natural tissues. Cryobiology. 2012;**64**(1):1-11

[19] Karlsson J, Cravalho E, Toner M. A model of diffusion-limited ice growth inside biological cells during freezing. Journal of Applied Physics. 1994;**75**(9):4442-4455

[20] Elliott GD, Wang S, Fuller BJ. Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology. 2017;**76**:74-91

[21] Fahy GM, Wowk B. Principles of ice-free cryopreservation by vitrification. In: Cryopreservation and Freeze-Drying Protocols. New York: Springer; 2021. pp. 27-97

[22] Fahy GM, Wowk B, Wu J, Phan J, Rasch C, Chang A, et al. Cryopreservation of organs by vitrification: Perspectives and recent advances. Cryobiology. 2004;**48**(2):157-178

[23] Abazari A, Jomha NM, Elliott JA, McGann LE. Cryopreservation of

articular cartilage. Cryobiology. 2013;**66**(3):201-209

[24] Warner RM, Higgins AZ. Mathematical modeling of protectant transport in tissues. In: Cryopreservation and Freeze-Drying Protocols. New York: Springer; 2021. pp. 173-188

[25] Lee KW, Park JB, Yoon JJ, Lee JH, Kim SY, Jung HJ, et al. The viability and function of cryopreserved hepatocyte spheroids with different cryopreservation solutions. Transplantation proceedings. Elsevier; October 2004;**36**(8):2462-2463

[26] Massie I, Selden C, Hodgson H, Fuller B, Gibbons S, Morris GJ. GMP cryopreservation of large volumes of cells for regenerative medicine: Active control of the freezing process. Tissue Engineering Part C: Methods. 2014;**20**(9):693-702

[27] Reichman S, Slembrouck A, Gagliardi G, Chaffiol A, Terray A, Nanteau C, et al. Generation of storable retinal organoids and retinal pigmented epithelium from adherent human iPS cells in xeno-free and feeder-free conditions. Stem Cells. 2017;**35**(5):1176-1188

[28] Drost J, Karthaus WR, Gao D, Driehuis E, Sawyers CL, Chen Y, et al. Organoid culture systems for prostate epithelial and cancer tissue. Nature Protocols. 2016;**11**(2):347-358

[29] Tsai Y-H, Czerwinski M, Wu A, Dame MK, Attili D, Hill E, et al. A method for cryogenic preservation of human biopsy specimens and subsequent organoid culture. Cellular and Molecular Gastroenterology and Hepatology. 2018;**6**(2):218-22.e7

[30] Bui BN, Boretto M, Kobayashi H, van Hoesel M, Steba GS, van Hoogenhuijze N, et al. Organoids can be established reliably from cryopreserved biopsy catheter-derived endometrial

tissue of infertile women. Reproductive Biomedicine Online. 2020;**41**(3):465-473

[31] Campbell B, Hernandez-Medrano J, Onions V, Pincott-Allen C, Aljaser F, Fisher J, et al. Restoration of ovarian function and natural fertility following the cryopreservation and autotransplantation of whole adult sheep ovaries. Human Reproduction. 2014;**29**(8):1749-1763

[32] Ross S, Cheung M, Lau CI, Sebire N, Burch M, Kilbride P, et al. Transplanted human thymus slices induce and support T-cell development in mice after cryopreservation. European Journal of Immunology. 2018;**48**(4):716-719

[33] Dittrich R, Maltaris T, Mueller A, Dimmler A, Hoffmann I, Kiesewetter F, et al. Successful uterus cryopreservation in an animal model. Hormone and Metabolic Research. 2006;**38**(03):141-145

[34] Han J, Sydykov B, Yang H, Sieme H, Oldenhof H, Wolkers WF. Spectroscopic monitoring of transport processes during loading of ovarian tissue with cryoprotective solutions. Scientific Reports. 2019;**9**(1):1-11

[35] Ding Y, Shao J-l, Li J-w, Zhang Y, Hong K-h, Hua K-q, et al. Successful fertility following optimized perfusion and cryopreservation of whole ovary and allotransplantation in a premature ovarian insufficiency rat model. Journal of ovarian. Research. 2018;**11**(1):1-10

[36] Pegg DE, Green CJ, Walter CA. Attempted canine renal cryopreservation using dimethyl sulphoxide helium perfusion and microwave thawing. Cryobiology. 1978;**15**(6):618-626

[37] Gastal G, Alves B, Alves K, Paiva S, de Tarso S, Ishak G, et al. Effects of cryoprotectant agents on equine ovarian biopsy fragments in preparation for cryopreservation. Journal of Equine Veterinary Science. 2017;**53**:86-93

[38] Thompson RE, Johnson AK, Prado TM, Premanandan C, Brown ME, Whitlock BK, et al. Dimethyl sulfoxide maintains structure and function of cryopreserved equine endometrial explants. Cryobiology. 2019;**91**:90-96

[39] Fuller B, Gonzalez-Molina J, Erro E, De Mendonca J, Chalmers S, Awan M, et al. Applications and optimization of cryopreservation technologies to cellular therapeutics. Cell & Gene Therapy Insights. 2017;**3**(5):359-378

[40] Hunt CJ. Technical considerations in the freezing, low-temperature storage and thawing of stem cells for cellular therapies. Transfusion Medicine and Hemotherapy. 2019;**46**(3):134-150

[41] Baboo J, Kilbride P, Delahaye M, Milne S, Fonseca F, Blanco M, et al. The impact of varying cooling and thawing rates on the quality of cryopreserved human peripheral blood t cells. Scientific Reports. 2019;**9**(1):3417

[42] Heo YT, Lim JK, Xu YN, Jang WI, Jeon SH, Kim N-H. Development of a method of vitrification, thawing, and transfer of mammalian blastocysts using a single closed cryo-straw. CryoLetters. 2014;**35**(2):108-113

[43] Association PD. Standard 02-2021: Cryopreservation of cells for use in cell therapies. Gene Therapies, and Regenerative Medicine Manufacturing. 2022

[44] Warner RM, Shuttleworth R, Benson JD, Eroglu A, Higgins AZ. General tissue mass transfer model for cryopreservation applications. Biophysical Journal. 2021;**120**(22):4980-4991

[45] Alzamil L, Nikolakopoulou K, Turco MY. Organoid systems to study the human female reproductive tract and pregnancy. Cell Death & Differentiation. 2021;**28**(1):35-51

*Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes DOI: http://dx.doi.org/10.5772/intechopen.108254*

[46] Morris GJ, Goodrich M, Acton E, Fonseca F. The high viscosity encountered during freezing in glycerol solutions: Effects on cryopreservation. Cryobiology. 2006;**52**(3):323-334

[47] Kilbride P, Morris G. Viscosities encountered during the cryopreservation of dimethyl sulphoxide systems. Cryobiology. 2017;**76**:92-97

[48] Phatak S, Natesan H, Choi J, Brockbank KG, Bischof JC. Measurement of specific heat and crystallization in VS55, DP6, and M22 Cryoprotectant systems with and without sucrose. Biopreservation and Biobanking. 2018;**16**(4):270-277

[49] Warner RM, Ampo E, Nelson D, Benson JD, Eroglu A, Higgins AZ. Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method. Cryobiology. 2021;**98**:219-232

[50] Van Raemdonck D, Rega F, Rex S, Neyrinck A. Machine perfusion of thoracic organs. Journal of Thoracic Disease. 2018;**10**(Suppl 8):S910

[51] Chiu-Lam A, Staples E, Pepine CJ, Rinaldi C. Perfusion, cryopreservation, and nanowarming of whole hearts using colloidally stable magnetic cryopreservation agent solutions. Science Advances. 2021;**7**(2):eabe3005

[52] Morris GJ, Acton E. Controlled ice nucleation in cryopreservation—A review. Cryobiology. 2013;**66**(2):85-92

[53] Kilbride P, Meneghel J, Fonseca F, Morris J. The transfer temperature from slow cooling to cryogenic storage is critical for optimal recovery of cryopreserved mammalian cells. PLoS One. 2021;**16**(11):e0259571

[54] Meneghel J, Kilbride P, Morris JG, Fonseca F. Physical events occurring during the cryopreservation of

immortalized human T cells. PLoS One. 2019;**14**(5):e0217304

[55] Körber C. Phenomena at the advancing ice–liquid interface: Solutes, particles and biological cells. Quarterly Reviews of Biophysics. 1988;**21**(2):229-298

[56] Daily MI, Whale TF, Partanen R, Harrison AD, Kilbride P, Lamb S, et al. Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation. Cryobiology. 2020;**93**:62-69

[57] Fleck R, Fuller B. 21 Cell Preservation. In: Medicines from Animal Cell Culture. Chichester, UK: Wiley; 2007

[58] Luyet GRB. Microscopic variations on the development of the ice phase in the freezing of blood. Biodynamica. 1960;**8**(166):195-239

[59] Mazur P, Leibo S, Chu E. A twofactor hypothesis of freezing injury: Evidence from Chinese hamster tissueculture cells. Experimental Cell Research. 1972;**71**(2):345-355

[60] Chang T, Zhao G. Ice inhibition for cryopreservation: Materials, strategies, and challenges. Advanced Science. 2021;**8**(6):2002425

[61] Morewood T, Getreu N, Fuller B, Morris J, Hardiman P. The effect of thawing protocols on follicle conservation in human ovarian tissue cryopreservation. CryoLetters. 2017;**38**(2):137-144

[62] Liebenthron J, Montag M, Reinsberg J, Köster M, Isachenko V, van der Ven K, et al. Overnight ovarian tissue transportation for centralized cryobanking: A feasible option. Reproductive Biomedicine Online. 2019;**38**(5):740-749

[63] Hinkle K, Orwig KE, Valli-Pulaski H, Taylor S, van Leeuwen K, Carpentieri D, et al. Cryopreservation of ovarian tissue for pediatric fertility. Biopreservation and Biobanking. 2021;**19**(2):130-135

[64] Silber SJ, DeRosa M, Goldsmith S, Fan Y, Castleman L, Melnick J. Cryopreservation and transplantation of ovarian tissue: Results from one center in the USA. Journal of Assisted Reproduction and Genetics. 2018;**35**(12):2205-2213

[65] Acker J, Larese A, Yang H, Petrenko A, McGann L. Intracellular ice formation is affected by cell interactions. Cryobiology. 1999;**38**(4):363-371

[66] de Vries R, Tessier SN, Banik PD, Ozer S, Crorin SE, Nagpal S, et al. Extending the human liver preservation time for transplantation by supercooling. Transplantation. 2018;**102**:S396

[67] Bruinsma BG, Berendsen TA, Izamis M-L, Yeh H, Yarmush ML, Uygun K. Supercooling preservation and transplantation of the rat liver. Nature Protocols. 2015;**10**(3):484-494

[68] Tessier SN, de Vries RJ, Pendexter CA, Cronin SE, Ozer S, Hafiz EO, et al. Partial freezing of rat livers extends preservation time by 5-fold. Nature Communications. 2022;**13**(1):1-13

[69] Pegg D. The history and principles of cryopreservation. In: Seminars in Reproductive Medicine. New York: Thieme Medical Publishers, Inc; 2002

[70] Morris GJ, Acton E, Murray BJ, Fonseca F. Freezing injury: The special case of the sperm cell. Cryobiology. 2012;**64**(2):71-80

[71] Jacobsen I, Pegg D, Starklint H, Chemnitz J, Hunt C, Barfort P, et al. Effect of cooling and warming rate on glycerolized rabbit kidneys. Cryobiology. 1984;**21**(6):637-653

[72] Hunt C, Taylor M, Pegg D. Freezesubstitution and isothermal freezefixation studies to elucidate the pattern of ice formation in smooth muscle at 252 K (−21°C). Journal of Microscopy. 1982;**125**(2):177-186

[73] Oktay K, Newton H, Aubard Y, Salha O, Gosden RG. Cryopreservation of immature human oocytes and ovarian tissue: An emerging technology? Fertility and Sterility. 1998;**69**(1):1-7

[74] Radford JA, Lieberman B, Brison DR, Smith A, Critchlow J, Russell S, et al. Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin's lymphoma. The Lancet. 2001;**357**(9263):1172-1175

[75] Nahata L, Woodruff TK, Quinn GP, Meacham LR, Chen D, Appiah LC, et al. Ovarian tissue cryopreservation as standard of care: What does this mean for pediatric populations? Journal of Assisted Reproduction and Genetics. 2020;**37**(6):1323-1326

[76] Carroll J, Whittingham D, Wood M, Telfer E, Gosden R. Extraovarian production of mature viable mouse oocytes from frozen primary follicles. Reproduction. 1990;**90**(1):321-327

[77] Campbell LD, Astrin JJ, DeSouza Y, Giri J, Patel AA, Rawley-Payne M, et al. The 2018 revision of the ISBER best practices: Summary of changes and the editorial team's development process. Biopreservation and Biobanking. 2018;**16**(1):3-6

[78] Davies EG, Cheung M, Gilmour K, Maimaris J, Curry J, Furmanski A, et al. Thymus transplantation for complete

*Scaling up Cryopreservation from Cell Suspensions to Tissues: Challenges and Successes DOI: http://dx.doi.org/10.5772/intechopen.108254*

DiGeorge syndrome: European experience. Journal of Allergy and Clinical Immunology. 2017;**140**(6):1660-70.e16

[79] Markert ML, Boeck A, Hale LP, Kloster AL, McLaughlin TM, Batchvarova MN, et al. Transplantation of thymus tissue in complete DiGeorge syndrome. New England Journal of Medicine. 1999;**341**(16):1180-1189

[80] Lee CC, Hoang A, Segovia D, Herbst A, Barthelemy F, Gibbs E, et al. Enhanced methods for needle biopsy and cryopreservation of skeletal muscle in older adults. Journal of Cytology & Histology. 2020;**11**(2):1-13

[81] García-Roche M, Casal A, Carriquiry M, Radi R, Quijano C, Cassina A. Respiratory analysis of coupled mitochondria in cryopreserved liver biopsies. Redox Biology. 2018;**17**:207-212

[82] He A, Powell S, Kyle M, Rose M, Masmila E, Estrada V, et al. Cryopreservation of viable human tissues: Renewable resource for viable tissue, cell lines, and organoid development. Biopreservation and Biobanking. 2020;**18**(3):222-227

[83] Guillaumet-Adkins A, Rodríguez-Esteban G, Mereu E, Mendez-Lago M, Jaitin DA, Villanueva A, et al. Single-cell transcriptome conservation in cryopreserved cells and tissues. Genome Biology. 2017;**18**(1):1-15

[84] Brockbank KG, Schenke-Layland K, Greene ED, Chen Z, Fritze O, Schleicher M, et al. Ice-free cryopreservation of heart valve allografts: Better extracellular matrix preservation in vivo and preclinical results. Cell and Tissue Banking. 2012;**13**(4):663-671

[85] McIntyre RL, Fahy GM. Aldehydestabilized cryopreservation. Cryobiology. 2015;**71**(3):448-458

Section 2
