Abstract
Human sperm cryopreservation is a highly desirable technique for preserving fertility potential and future use in couples desiring to have a biological child. Slow cryopreservation of sperm has been the mainstay technique. The drawback of this technique is the inability to freeze extremely small numbers of sperm as in the case of surgical retrieval of testicular sperm. These sperm are extremely few in number and it is difficult to retrieve motile, viable sperm post-thaw. In the past decade, sperm vitrification has been introduced along with both biological and non-biological carriers to freeze extremely low numbers of sperm. Vitrification also allows the ability to freeze single spermatozoa. These techniques are in many ways more efficient and better than the older techniques. This chapter aims to provide a detailed introduction to various approaches used for preserving spermatozoa. It also discusses indications of sperm cryopreservation based on semen quality and summarizes the advantages and shortcomings of these techniques.
12.1 Introduction
Approximately 30–40 percent of infertility cases represent male factor infertility. In the majority of these cases, patients may opt to freeze their sperm for later use for procreation. Spermatozoa are ideal to cryopreserve because (1) they are the smallest human cells; (2) they have a relatively small volume; (3) a large surface area; (4) they contain very little cytoplasm; (5) less total intracellular water compared to other cells; and (6) they exist individually, allowing effective dehydration. Cryopreservation of spermatozoa is the best option available for having a biological child for these patients [1]. It involves freezing of sperm at extremely low temperatures using liquid nitrogen (LN2). This process is carried out in the presence of cryoprotectants. Cryprotectants play a critical role in protecting sperm during freezing [2]. In the absence of cryoprotectant spermatozoa can be damaged because of the swelling of the plasma membrane as water expands during freezing, causing acrosomal leakage and breakdown [3]. Glycerol has been used as an effective primary cryoprotectant for freezing spermatozoa [2]. The function of glycerol is to remove or reduce the water content and help minimize intracellular ice formation during freezing. Osmotic equilibrium is reached as the cryoprotectant penetrates the cell and displaces the intracellular water and the sperm returns to almost its original volume. There are a number of factors that may influence and induce abnormal changes in spermatozoa as a result of cryopreservation. These include failure to maintain the optimum temperature and use of diluents that might have negative effects on sperm quality, including reduced semen parameters such as vitality and motility and increased sperm DNA fragmentation [4].
Physicians recommend preservation of gametes especially for adults and young adolescents diagnosed with cancer before they start treatment for cancer. These treatments interfere with the normal functioning of the gametes as well as the process of gametogenesis [5]. To ensure high success rates of the assisted reproduction via sperm cryopreservation, it is necessary to understand the physiology and cryobiology of the sperm. Unlike embryos, sperm cells are smaller in size and have a larger surface area. These characteristics help them to maintain viscosity and the glass transition temperature prevents their cytosol from cryodamage. The role of cytosol is to provide protection from lipid peroxidation and DNA fragmentation [3]. Cooling of sperm cells at lower temperatures ceases all physiological processes and extends their life span [6].
12.2 Sperm Cryopreservation
Cryopreservation is the process of stabilizing the cells at low temperatures (cryogenic temperature) by applying the principles of cryobiology [5]. Freezing of sperm cells for extended periods is possible by arresting their metabolic processes. This can be achieved by storing these cells at –196 °C in LN2 [7]. At such a low temperature, the cells dehydrate as a result of water loss due to the formation of ice crystals. The energy of the cells to carry out physiological cycles is reduced. Hence, it becomes easier to store them for longer periods of time [8]. The addition of a cryoprotectant is usually accompanied by a reduction in temperature. As the cooling process continues and the temperature reaches –5 °C to –15 °C, extracellular ice formation occurs. This induces the development of the extracellular solid phase. The freezing point of water can be reduced to –42 °C by preventing the process of nucleation – that is, interaction between water droplets to form ice crystals. During the process of cryopreservation, the solutions are cooled below their freezing point without a change in their phase from liquid to solid. This phenomenon is termed supercooling. The formation of ice crystals during this process can be avoided by rapidly reducing the temperature [6]. This starts with forming an ice nucleus in the extracellular space. Solutes are excluded from the ice formed, which increases the concentration of solutes outside the cell. This causes solutes to enter the cell through the plasma membrane and causes them to lose intracellular water, resulting in cell dehydration. During the freezing process, sufficient time should be allowed for sperm cell dehydration and osmotic equilibration. Very slow cooling causes dehydration of the cell while very fast cooling might result in intracellular ice formation. Hence, temperature plays a crucial role here and needs to be controlled accurately [9].
In contrast, when the cells are thawed, the process is reversed. There is a continuous influx of water in the cell, which causes the cells to swell. After a certain period, the cells bursts because of the disruption of the cell membrane. This damage caused by cryopreservation is called cryodamage [2]. The thawing process is as important as the freezing process. The cells should be allowed to restart their physiological activities. Rapid warming can cause heat shock to the cells and damage the cell membrane. Usually 37 °C is used, and higher temperatures are not recommended due to risks associated with cell damage [5]. Cells that have been frozen slowly should also be warmed slowly during thawing, while cells that were frozen rapidly should be thawed rapidly [8]. Although glycerol is a widely used cryoprotectant, it might have negative effects on sperm when added at higher concentrations. The toxicity of glycerol is noted above the concentration of 6%vol./vol. In addition, glycerol also has direct osmotic effects. It tends to cross the plasma membrane comparatively slower than water. For these reasons, addition and removal of glycerol changes the volume of the cell. If the change in volume crosses the osmotic tolerance level of the cell, it causes shrinking or swelling of the cells [6].
12.3 Indications for Sperm Cryopreservation
There are several indications for sperm cryopreservation, including the following:
1. Cancer: Men diagnosed with malignant cancer, including testicular cancer, Hodgkin’s and non-Hodgkin’s lymphoma, leukemia, prostate cancer, and many other types of cancer, prior to radiation therapy for the treatment of cancer [10,11].
2. Surgery: Physicians recommend freezing of spermatozoa before undergoing surgeries for vasectomy, vasectomy reversal, or ejaculation failure. Patients with other medical conditions, including varicocele, spinal cord injury, and infections from Zika virus, hepatitis B, or HIV, are also recommended to preserve their fertility [12].
3. Obstructive or nonobstructive azoospermia: Sperm are absent in the semen of patients suffering from azoospermia, which may be obstructive or nonobstructive. Sperm cryopreservation is a relevant option for them to preserve fertility [3].
4. Male factor infertility: When the infertility is caused due to problems in the male while the female has no abnormal parameters, it is recommended to preserve a semen sample to avoid sterility in a later phase [3].
5. Autologous or donor sperm cryopreservation: Autologous sperm banking or client depositor refers to the individual who preserves spermatozoa for future use with his partner, while a sperm donor is an individual who is serving as a surrogate father for an infertile couple or single female. He may be a directed or anonymous donor. It is necessary for all donors to undergo clinical assessment before sperm donation to avoid infections [6,10].
6. Occupational: Occupations with higher exposure to chemicals such as phthalates, pesticides, polychlorinated biphenyls, etc. might affect the fertility in men by increasing oxidative stress and reducing semen parameters. It is recommended to preserve fertility in such cases [12].
7. Traveling husband/military assignments: In these cases, it becomes difficult to time intercourse with the process of ovulation. Thus, it is necessary to preserve the semen sample from the husband for future use through assisted reproduction [12].
8. Gender reassignment: Approximately three times more individuals opt for male-to-female gender reassignment as compared to female-to-male. These individuals can preserve their sperm before going through gender reassignment and achieve parenthood in the future [10].
12.4 Techniques of Sperm Cryopreservation
12.4.1 Slow Freezing
The principle for this protocol is the formation of ice crystals from extracellular water [13]. This differentiates the two phases: ice crystals formed from extracellular water and water in the liquid phase. The liquid phase consists of cryoprotectants, salts, and sugars [6]. The sperm cells are cooled progressively in several steps over 2–4 hours. This can be carried out manually or with a semi-programmable freezer [5]. The loss of water during this process increases the osmolality of the solution. This causes the cell membrane to shrink. In the andrology laboratory, the commonly used cryoprotectant is modified TES, TRIS and egg yolk buffer (TEST-yolk buffer). The TEST-yolk buffer is an excellent extender that helps maintain sperm viability. Addition of egg yolk helps maintain sperm viability during cryostorage TES, TRIS improves membrane fluidity. Egg yolk-free buffers have also been introduced to avoid potential allergenic reactions and reduce exposure to animal-derived products. Zwitterion buffers also help in the recovery of motile sperm due to their ability to bind free hydrogen and hydroxyl ions in the surrounding medium and to aid in the dehydration process. TEST-yolk buffer contains glycerol. Glycerol has a role in lowering the concentration of salts at an extracellular level by increasing the level of unfrozen water. This decreases the osmotic effects. TEST-yolk consists of low-density lipoproteins which are responsible for protecting the sperm membrane [6]. Sperm suspensions are diluted 1:1 vol./vol. with TEST-yolk buffer, resulting in a final glycerol concentration in the frozen sample of 6 percent. The freezing medium consist of TEST-yolk, glycerol, and gentamycin [14].
In the slow-freezing technique, after complete liquefaction, the freezing medium (TEST-yolk buffer) is added dropwise to the liquefied semen by addition of 25 percent of the semen volume four times, with 5-min interval of slow mixing until a 1:1 sample–medium ratio is achieved [15]. The sample is transferred into the cryovials and frozen at –20 °C for 8 min followed by exposure to the liquid nitrogen vapors at –80 °C (Figures 12.1–12.9). After 2 h of incubation in the vapors, it is preserved in LN2 at –196 °C [14]. For thawing, the sample is removed from the LN2 and warmed either slowly at room temperature for 30–40 min or rapidly at 37 °C for 290 min. The conventional slow-freezing protocol performed manually or automated can cause physical and chemical damage to the sperm cell [13].
Figure 12.1 Incubator set at 37 °C and semen sample collected for liquefaction.
Figure 12.2 Mixing of the semen sample and TEST-yolk on a test tube rocker for 5 min.
Figure 12.3 Step-wise addition of TEST-yolk equal to one-quarter volume of buffer to patient sample to give a final 1:1 volume.
Figure 12.4 Distribution of cryodiluted sample into cryovials using a sterile serological pipette.
Figure 12.5 Correct loading of cryovials into cryocanes.
Figure 12.6 Cryovials placed in a cryocane and covered with cryosleeve placed upright in –20 °C freezer for 8 min.
Figure 12.7 Proper loading of cryocanes with cryovials upright into the cryotank canister.
Figure 12.8 Loading of the sperm counting chamber into the load chamber of CASA.
12.4.2 Rapid Freezing
In the rapid-freezing protocol (less than 15 min), the sample is directly exposed to the nitrogen vapors at –80 °C [5,16]. It is mixed with the cryoprotectant in a dropwise manner and incubated at 4 °C for 10 min. The straws are first placed at a distance of 15–20 cm from the level of the LN2 for 15 min for vapor exposure, and later immersed in liquid nitrogen at –196 °C [5,17]. Rapid freezing is not used widely because of its low success rate and reproducibility [5]. Controlling the variation in temperature is also difficult [5]. This difficulty can be reduced by using automated programmable freezers, which are easy to use while handling the specimens at different temperatures [17]. The samples are loaded on a cryoplate and placed in the freezer. Automated systems frequently use a cooling rate of –0.5 °C /min from room temperature to –5 °C and a freezing rate of –10 °C/min from –5 °C to –80 °C or –90 °C, followed by immersion in LN2 [17].
12.4.3 Home Sperm Banking
Home sperm banking is a novel approach for collecting the semen specimen at home. For many patients, collection of a semen specimen is a challenge because of various factors such as discomfort and stress. Sperm banking facilities are not available in many cities and patients may have to travel from distant places to a sperm banking facility. This may also cause emotional trauma and anxiety. The home sperm banking process is a novel approach for such men that helps overcome the issues related to privacy and anxiety. The NextGen kit is a novel, specially designed kit developed by the Cleveland Clinic Andrology Center for sperm collection and transport (Figure 12.10) [18]. The kit is composed of a collection cup and transportation media, ice sleeves, foam inserts, ice packs, Styrofoam packing box, and the outer box (Figure 12.11). Sample collection and shipping instructions are included in the kit. On receipt of the kit, patients are instructed to place the collection cup, ice packs, freezing sleeve, and refrigeration media in a freezer for at least 12 hours. On the day of collection, before collecting the semen, the refrigeration medium and collection cup are removed from the freezer and allowed to thaw at room temperature for 60 min. The semen sample is deposited by masturbation only, avoiding the use of lubricating gels. After sample collection, the entire content of the refrigeration media (5.0 ml) is added to the collection cup, the cup is sealed securely and gently swirled to mix the contents. The cup is then placed in the kit, along with ice bricks, which are placed on the outside of the foam layers. Finally, the kit is sealed and shipped overnight.
Figure 12.10 View of the NextGen kit optimized for overnight shipping of semen samples.
After receiving the sperm sample with the kit, the cryopreservation protocol is carried out as per the slow cryopreservation technique [14]. The effects of overnight transport of the semen specimen on sperm motility using the home sperm banking kit have been examined. Prefreeze and postthaw sperm motility, total motile sperm, and percentage cryosurvival rates were compared between samples collected from infertile men on-site at the Andrology Center (n = 10) and samples collected from infertile patients at home (off-site; n = 9), and shipped by NextGen to the laboratory (Figure 12.12). A second group (n = 17) consisted of 10 semen samples from cancer patients collected on-site, which were compared with seven semen samples from cancer patients shipped by the NextGen [18]. In the infertile men, percentage cryosurvival rates were similar with NextGen compared with those of on-site collection (53.14 ± 28.9 vs 61.90 ± 20.46 percent; p = 0.51). Similarly, in the cancer patients, all four parameters were comparable between the on-site and NextGen collections. Cryosurvival rates were also similar between NextGen compared with those of on-site collection (52.71 ± 20.37 vs 58.90 ± 22.68 percent; p = 0.46) (Figure 12.13). Cancer patients can bank sperm as effectively as men banking for infertility reasons using the NextGen kit [18].
Figure 12.12 Semen samples from off-site and on-site groups of infertile patients showing differences between (A) prefreeze and postthaw percent motility; (B) prefreeze and postthaw total motile sperm; and (C) percent cryosurvival (n = 7 off-site and n = 10 on-site collections).
Figure 12.13 Semen samples from off-site and on-site groups of cancer patients showing differences in (A) prefreeze and postthaw percent motility; (B) prefreeze and postthaw total motile sperm; and (C) percent cryosurvival (n = 7 off-site; n = 10 on-site collections).
12.4.4 Sperm Vitrification
Sperm vitrification is an emerging technique for improving reproductive outcomes [4]. The first successful live birth of a human by the vitrification process was documented by researchers at the State University of Iowa in 1953 [19]. Sperm vitrification involves solidification of the specimen at ultralow temperature by elevating its viscosity with a high cooling rate of 15 000–30 000 °C/min [13]. Cooling cells at such ultracool temperatures creates a glass-like appearance without formation of ice crystals [9,13]. The glass formation occurs efficiently when the cryoprotectant composing the vitrification solution includes the combination of dimethyl sulfoxide, which is a strong glass former, and ethylene glycol, acetamide, and formamide, which are weak glass formers [13].
The vitrification process with cryoprotective agent (CPA) requires exposing the cells to high concentrations of cryoprotectants at room temperature. The sperm wash media for vitrification contains 5% HSA (human serum albumin) and sucrose. Vitrification is not effective with permeable cryoprotectants [20] as this increases the osmolarity [21] and causes human sperm cells to suffer from osmotic shock when exposed to the hypertonic environment. This results in morphological defects such as coiled tails [20]. This problem can be solved by using isomolar vitrification media which uses nonpermeable cryoprotectants [21]. The sperm suspension is transferred to the cryovial. The cryovial is then placed at the bottom of the cryocane and is exposed to the liquid nitrogen tank [13]. This technique is efficient in maintaining the motility and DNA integrity. It results in higher fertilization and pregnancy rates during the ART procedure compared to the slow-freezing technique. A study conducted on 33 semen samples from humans showed the same outcomes for slow freezing and vitrification [22]. Vitrification is fast, easier, and cost-effective. It has no deleterious effects on sperm quality because of its low toxicity. Sperm vitrification has been performed successfully with and without CPA, but has failed to demonstrate superiority over the conventional sperm-freezing method [4]. In one study, results of conventional slow freezing and vitrification of 105 human semen samples demonstrated that slow freezing yields better sperm motility and vitality. In contrast to this, vitrification showed better results for sperm morphology [23]. Li et al., in their systematic review, concluded that vitrification is a superior technique compared to conventional slow freezing [24]. They reviewed 2428 studies and showed that total motility and progressive motility of post-thaw semen sample were well preserved by vitrification as compared to conventional slow freezing. However, it is important to note that these results varied on the protocol and the sample size [24].
A new technique called cryoprotectant-free vitrification has been introduced. It is carried out in the absence of cryoprotectants. Vitrification results of 35 human semen samples using the cryoprotectant-free approach showed that high membrane potential and low DNA damage were observed in samples. On the contrary, another study failed to show significant differences in postthaw motility between conventional slow freezing and cryoprotectant-free vitrification [25]. This study was further supported by Aizpurua et al. [26]. This approach yields a greater number of live sperm and maintains the acrosome with reduced DNA fragmentation. Sucrose yields better results in the absence of cryoprotectants. Use of sucrose during vitrification has been correlated with better results in postthaw motility. The plasma membrane and acrosome integrity are also maintained [27].
Sucrose is a sugar and acts like a solute with high viscosity. The increased viscosity during vitrification makes it easy for the cells to achieve a glassy state [13]. Sperm have been shown to be particularly sensitive to exposure to high concentrations of CPAs used routinely in oocyte and embryo vitrification [28,29]. The sperm suspension is directly exposed to the LN2; however, the non-penetrating cryoprotectants inhibit the flow of water outside the sperm cells and thereby it may cause osmotic damage [13]. In addition to freezing, there is a need for extreme care during the process of thawing. The velocity of thawing plays a challenging role in the cryosurvival rate of spermatozoa [6]. Despite high-speed freezing of spermatozoa prefreeze, the thawing velocity should be high as well. The reason for this is that the water in the sperm cells must move from the glassy state to the liquid phase. This process should occur without forming ice crystals. Sperm samples show higher motility when thawed at 42 °C [30]. Vitrification of neat ejaculates is associated with several effects on sperm parameters and DNA integrity, suggesting that seminal plasma does not have the same protective effects during sperm vitrification [31].
12.5 Sperm Storage Techniques
Before proceeding with ART, it is recommended to store the sperm cells. The process of cryopreservation damages sperm health on some levels. As a result, the quality of the sperm is reduced in terms of motility [16]. To reduce the amount of damage caused to spermatozoa, it is recommended to store them for only a short interval [32].
In cases of infertility with obstructive and nonobstructive azoospermia, sperm are surgically retrieved and patients are recommended to cryopreserve their samples [3]. Cryopreserving spermatozoa after surgery through the slow-freezing protocol results in an approximately 1 percent recovery rate. Among all the ART procedures, sperm recovery for intracytoplasmic sperm injection (ICSI) is noted to be difficult [33]. There are several methods to cryopreserve spermatozoa, including biological and nonbiological methods [16].
12.5.1 Sperm Storage Inside Zona Pellucida
When the spermatozoa are few in number, they can be stored in the empty zona of human or animals. The process of storing the sperm in zona is critical and requires the stripping of the oocytes. The sperm are aspirated using the ICSI pipette and then transferred to the empty zona [34]. Small holes are created using laser-assisted techniques in the zona membrane to allow the sperm to enter. In the case of the large-sized holes, there are chances for the sperm to leak out and the DNA of the host might enter the oocyte. Small-sized holes may result in entrapped DNA, which causes it to adhere to the sperm. Following the insertion of sperm into the zona, they are frozen using slow freezing. This process has shown a significant improvement in sperm recovery during postthaw [16].
12.5.2 Volvox Globator Spheres
This method is based on the use of an algae named Volvox globator. These algae have spherical colonies. Sperm cells are mixed with the cryoprotectants and introduced into the spheres. This process is followed by slow freezing of the spermatozoa. The success rate of this process is noted to be 100 percent [16]. However, the Food and Drug Administration (FDA) restricts the use of this method as the sperm are exposed to algae [1].
12.5.3 Straws and Pipette
The sperm sample can be stored using straws. These straws are distinguished into many types, including open, mini, and open pulled. The open pulled straws use capillary action to help protect samples from mechanical damage. They are highly recommended for the process of vitrification. However, there is a high risk of contamination as the system is open [35]. Mini straws, which are smaller in size, are used when the volume of the sample is comparatively lower. The sample is loaded inside with cryprotectants and the ends of this straw are closed. The disadvantage of this type of straws is that the sperm might stick to the walls [15].
12.5.4 Preserving Spermatozoa Using Microdroplets
The sperm sample, along with cryoprotectants, is rapidly cooled in the presence of dry ice. This process forms microdroplets. These microdroplets are exposed to LN2 at –196 °C. This method has resulted in six clinical pregnancies (33 percent) [36].
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
