Introduction
In cases where there are low numbers of spermatozoa, the conventional method for sperm cryopreservation is not suitable. Single sperm cryopreservation has addressed this issue by enabling the freezing of small quantities of spermatozoa using various cryo-devices, particularly the cryotop. Although this technique is a promising strategy for freezing limited numbers of spermatozoa, several challenges remain unresolved. Addressing these challenges could allow clinicians to utilize this method in clinical settings.
‘Cryopreservation’ originates from the Greek word κρύος (krýos), meaning ‘icy cold,’ ‘chill,’ or ‘frost.’ This technique is used to preserve the structure of cells and tissues at low temperatures, keeping them intact and healthy. In 1776, Spallanzani [1] froze sperm cells by placing them in snow and later reported their motility rates upon thawing. Mantegazza [2] later showed that human spermatozoa could survive after being frozen at –17 °C for more than 4 days. The discovery of glycerol as a cryoprotectant in 1949 made it possible to cryopreserve viable spermatozoa for extended periods [3]. In 1957, the first piglet was born using frozen-thawed porcine sperm [4]. The introduction of freezing techniques using dry ice and liquid nitrogen (LN2) vapor in the 1970s significantly improved cryopreservation procedures [5]. The development of controlled rate freezing methods further advanced the field of cryopreservation.
Cryopreservation of human spermatozoa was first successfully performed in 1973 [6]. This process, which preserves the viability and fertilizing capability of spermatozoa, is a crucial step in assisted reproductive technology (ART).
One indication for sperm cryopreservation is fertility preservation and the treatment of male infertility. Various factors, including cancer, can lead to subfertility or infertility [7]. Malignancy-induced stress, inflammation, fever, and hormonal imbalances can significantly change testicular and sperm function [8]. Treatment of cancer with high-dose chemotherapy or radiotherapy can increase DNA fragmentation in spermatozoa, damage Sertoli and Leydig cells, and cause ejaculatory duct obstruction due to the toxic effects of these therapies on the male reproductive system [9]. Additionally, spermatogenesis is altered in patients with lupus, multiple sclerosis, or ulcerative colitis [7]. Surgical treatments for infertility, such as those addressing varicocele and seminal duct obstruction, are linked to injuries to the testicular artery and transection of the vas deferens [10]. Men with high-risk occupations involving exposure to noxious chemicals and hazardous biological substances should be aware of the potential adverse effects on their reproductive system [11]. Men in any of these conditions may benefit from sperm cryopreservation, which allows them to store their sperm cells for future use.
The second indication for cryopreservation techniques involves individuals experiencing infertility due to severe oligozoospermia, ejaculatory dysfunction, and azoospermia. In such cases, retrieved sperm is cryopreserved to eliminate the need for repeated biopsies and aspirations [7]. However, the sperm cryopreservation methods typically employed are unsuitable for testicular samples with low sperm count and motility. Therefore, alternative cryopreservation techniques are necessary to enhance fertility potential in these instances.
In 2020, Liu and Li [12] published a review article on single sperm cryopreservation, specifically addressing cryo-devices. Recognizing their significance in ART, our comprehensive review initially explored various aspects of all cryo-devices, including their pores and cones, along with the challenges they present. Subsequently, our review addressed additional aspects of this technique, which have been largely overlooked in other studies. These include cryoprotectants, cooling and warming rates, testicular sperm enhancer agents, and clinical outcomes, all discussed up to the year 2023.
Methods
This study is a non-systematic literature review of articles published between 1997 and 2023, sourced from the U.S. National Institutes of Health's CENTRAL, Embase, MEDLINE, PubMed, Scopus, and Web of Science databases. The search terms included ‘single,’ ‘low,’ ‘cryopreservation of small spermatozoa count,’ ‘azoospermia,’ ‘rapid freezing,’ ‘slow freezing,’ and ‘vitrification.’ The inclusion criteria focused on individuals with azoospermia and clinical studies employing these techniques. Studies involving normal samples that were frozen using conventional methods were excluded. Out of the 125 manuscripts initially identified, 75 were included in the review.
Sperm cryopreservation techniques
Three freezing methods—slow freezing, rapid freezing, and vitrification—are employed in the cryopreservation of human spermatozoa.
1. Slow freezing
This technique is based on cell dehydration and combines slow cooling with low concentrations of a cryoprotectant to achieve equilibrium. In this cryopreservation method, ice crystals form at a specific temperature as the temperature is gradually decreased. Slow cooling is achieved by lowering the samples into LN2 vapor using a programmable freezer [13,14]. Samples prepared with a cryoprotectant agent (CPA) buffer are diluted at room temperature (RT) and gently shaken for 5 minutes. CPA is added to the original semen sample in a 1:1 ratio and then stored in cryovials. The cooling procedure can be carried out either manually or automatically in a programmable freezer. In the manual method, cryovials are kept at –20 °C for 8 minutes and then placed in LN2 vapor (–80 °C) for 2 hours. The cryovials are subsequently transferred to LN2 for long-term storage [15]. Manual methods offer advantages in terms of validation and consistency compared to computer-controlled freezers [16]. Cryovials are cooled by cold nitrogen vapor from RT to 5 °C at an optimal initial cooling rate of 0.5–1 °C/min. The sample is then frozen from 5 to –80 °C at a rate of 1–10 °C/min before being plunged into LN2 [17,18].
2. Rapid freezing
In the rapid freezing method developed by Sherman [6], the toxicity of CPAs and osmotic damage to membranes are minimized due to the formation of extracellular ice crystals. This technique has been employed for both ejaculated and testicular sperm. For ejaculated sperm, the procedure begins with an assessment of the sperm count, motility, and morphology. Before the addition of cryoprotectants, the sperm sample is rinsed, transferred to a cryo-device, and initially exposed to LN2 vapor for 15 minutes before being submerged in LN2 for storage [19]. For testicular sperm, the samples are finely chopped using sterile scissors or needles while being observed under a stereomicroscope. An inverted microscope, with magnifications of 200× or 400×, is then used to locate sperm within the suspension. Following centrifugation and removal of the supernatant, the pellet is resuspended according to the volume of the tissue. The CPA is introduced at RT. The sample is subsequently placed in a cryo-device, exposed to LN2 vapor for 15 minutes, and finally, the cryovials are plunged into LN2 for storage [20].
3. Vitrification
Vitrification is defined as the solidification of a solution at low temperatures without the formation of intracytoplasmic ice crystals, achieved by a significant increase in viscosity during cooling. In 1938, Luyet and Hoddap [21] first achieved vitrification of frog sperm using liquid air. Subsequently, Polge [22] employed glycerol to freeze sperm samples from various species. This novel technique involves immersing cells in a medium that includes cryoprotectants, followed by rapid submersion into LN2 at −196 °C, thereby preserving cellular function and metabolism. Historically, vitrification has been predominantly applied to the cryopreservation of oocytes and embryos over several decades. However, it is not commonly used for routine sperm cryopreservation for two main reasons: the high concentration of cryoprotectants required (30%–50%) compared to that used in slow freezing (5%–7%) results in high viscosity, and slow freezing achieves more effective dehydration in sperm due to its smaller size relative to oocytes and embryos. These factors lead to lethal osmotic effects and potential chemical changes [23]. Therefore, it is not appropriate for sperm vitrification, despite its successful use for embryos and oocytes. Isachenko et al. [23] presented a new method tailored to specific features of sperm cells, in which sperm cells are vitrified without permeable cryoprotectants.
Khalili et al. [24] found that vitrification adversely affected sperm parameters and DNA integrity in individuals with both normal and abnormal neat samples.
Agha-Rahimi et al. [25] evaluated the impact of rapid freezing and vitrification, both with and without CPAs, on normo-ejaculated sperm parameters, DNA fragmentation, and hyaluronan binding. They found that vitrification without CPAs effectively froze spermatozoa.
Although vitrification is not commonly used in ART laboratories for freezing sperm, it is introducing a promising technique for the vitrification of single sperm, particularly in cases with low sperm counts.
Cryopreservation of a limited number of sperm (single sperm vitrification concept)
Male factor infertility accounts for approximately 40%–50% of infertility cases worldwide [26]. Azoospermia, defined as the absence of sperm in ejaculated semen after evaluating centrifuged semen at least twice [27], affects about 1% of the male population and up to 20% of men in the infertile population [28]. Sixty percent of men with azoospermia have non-obstructive azoospermia (NOA), characterized by small testes, elevated follicle-stimulating hormone levels, and the absence of sperm. Historically, donor insemination was the sole option for these individuals, but today, surgical sperm retrieval techniques are available for both obstructive and NOA. In cases of obstructive azoospermia (OA), techniques such as percutaneous epididymal sperm aspiration, microsurgical epididymal sperm aspiration (MESA), and testicular sperm aspiration (TESA) are employed [29]. For NOA, TESA and testicular sperm extraction (TESE) are utilized. Since the successful application of intracytoplasmic sperm injection (ICSI) in humans, many couples dealing with male factor infertility have opted for spermatozoa retrieved from the testis using microdissection TESE (Micro-TESE) for in vitro fertilization (IVF) treatments. Consequently, TESE and the ICSI technique are considered the first-line treatment in NOA cases. Table 1 presents sperm retrieval techniques and their indications. In the conventional TESE procedure, a small incision is made in the testes, and one or multiple biopsies are taken blindly. The average retrieval rate for this technique is about 50% in males with NOA. Potential side effects include loss of testicular tissue, inflammatory changes, devascularization, and hematoma formation. The Micro-TESE procedure involves opening the tunica albuginea and examining the testicular tissue under an operating microscope at ×20–25 magnification. TESE is effective in only 30%–50% of NOA cases. Moreover, repeated surgical procedures for diagnostic or therapeutic extraction can damage the blood-testicular barrier and impair sexual function [30].
Using cryopreserved sperm or performing TESE before oocyte retrieval offers several advantages: (1) it avoids the unnecessary risks associated with ovulation induction in many women; (2) it allows sperm to be frozen at any time, eliminating the need for synchronous oocyte collection; (3) it reduces the time required for sperm retrieval and minimizes oocyte aging [31]; and (4) it prevents the need for repeated testicular biopsies, which can cause injury [32-35]. Therefore, cryopreservation enables the storage of multiple vials for future use. However, when sperm is surgically retrieved, excess sperm are often cryopreserved using conventional methods in standard containers (cryovials or straws), which are not suitable for samples with a low sperm count [35,36]. The size of the container, the volume of the freezing medium, rough and repeated centrifugation, and the adherence of sperm to the container walls render these methods ineffective for freezing small aliquots containing only 10 to 30 sperm, leading to significant sperm loss [37]. It has been reported that freezing spermatozoa in straws with the conventional procedure results in a mere 1% recovery rate after thawing, which is clearly inadequate [38]. Therefore, for cases with a low sperm count, conventional cryopreservation techniques are problematic. Cryopreservation of individual sperm cells is particularly beneficial for patients with a very limited number of spermatozoa, such as those with sperm surgically isolated from the epididymis and the testis.
The concept of single sperm cryopreservation was first introduced by Cohen et al. [39] in 1997, who used an empty zona pellucida (ZP) as a novel freezing carrier to preserve a few sperm cells. Later, Desai et al. [40] applied a cryoloop device to store a small number of sperm cells. Since then, research has focused on identifying the optimal device, freezing technique, and clinical outcomes. However, there has been limited investigation into other aspects of cryopreservation, such as the appropriate cryoprotectant medium, cooling and warming rates, and sperm selection techniques prior to freezing. Therefore, this study reviews various aspects of single sperm freezing methods over the past 20 years to evaluate the advantages and disadvantages of these techniques and to provide insights into enhancing their future clinical applications.
Cryo-devices in the single sperm cryopreservation technique
Handling small-volume samples necessitates special attention due to the potential for crystallization during storage or thawing. The thermal conductance is determined by the size and composition of the device used, which can affect the rates of cooling and warming. To achieve optimal cooling rates and prevent heterogeneous nucleation, the volume of the vitrification solution should be minimized. Various biological and non-biological devices have been specifically designed to address this issue.
1. Biological carriers
1) Empty ZP
To minimize the loss of spermatozoa during the addition and removal of cryoprotectants, enclosed capsules such as an empty ZP were utilized for inserting spermatozoa. In this technique, cellular components are removed from the oocyte ZP, and an ICSI needle is then employed to inject sperm into the empty ZP. Subsequently, a frozen straw is used to load the empty ZP for cryopreservation.
Cohen et al. [39] utilized empty ZP from various sources: immature human oocytes (at the germinal vesicle or metaphase I stage) prior to fertilization, post-fertilization embryos derived from the ICSI technique that failed to develop or fertilize normally, mouse oocytes at the pre-fertilization stage, and frozen hamster oocytes also at the pre-fertilization stage. They obtained ejaculated sperm from semen samples of six patients diagnosed with oligo-astheno-teratozoospermia (OAT). To perforate the ZP, both chemical methods, such as acidified Tyrode’s solution, and mechanical methods, including partial zona dissection (PZD) using closed micropipettes, were employed. The cytoplasmic components were extracted from the zona using a 15 µm micropipette coupled with a suction device. Prior to sperm injection, the spermatozoa were immersed in a 10% polyvinylpyrrolidone solution. Between one and 15 spermatozoa were injected into each zona, with a single spermatozoon being introduced into the hamster zona. Following injection, an 8% glycerol solution in phosphate-buffered saline supplemented with 3% human serum albumin (HSA) was prepared for incubating the zona. The zona was then frozen in 0.25 mL sterile plastic straws. According to the semen freezing protocol, these straws were exposed to LN2 vapor for 120 minutes before being submerged in LN2 and stored for 48 hours. For the extraction of spermatozoa from the ZP, PZD, as proposed by Cohen et al. [39], was utilized. Post-thawing, sperm recovery and ICSI procedures were conducted. The outcomes were quantified, revealing a recovery rate (number of post-warming sperm/number of cryopreserved sperm×100), a motility rate of 82%, and a fertilization rate of 50% [39].
Similar to the approach taken by Cohen et al. [39], Walmsley et al. [41] in 1998 utilized empty ZP. In their study, the samples contained fewer than 1,000 motile sperm cells sourced from fresh ejaculate, as well as epididymal and testicular sources obtained via TESE and MESA procedures. Both human and hamster zonae were employed. Following the findings of Cohen, the hamster zona demonstrated superior recovery rates compared to the human zona; thus, hamster zonae were used in this study. The protocol established by Cohen et al. [39] was adhered to. Between five and six spermatozoa were injected into each zona using an ICSI pipette. The process included freezing and thawing, sperm recovery and ICSI, culturing, embryo selection, and assisted hatching. The outcomes reported were recovery rate, motility rate, fertilization rate, pregnancy rate, and delivery rate (excluding one ongoing pregnancy) at 74%, 82%, 65%, 60%, and 40%, respectively. The first live birth in this study was achieved using the ICSI technique with frozen testicular sperm and empty ZP as a carrier [41].
Montag et al. [42] employed a laser technique to encapsulate individual sperm within an empty ZP. In this study, the sperm samples were from normal ejaculate, and the carrier was human zonae. An 1.48 pm diode laser system created a hole in the ZP to facilitate the preparation of a free-cell ZP. The cytoplasmic components of the oocyte were removed through this hole using a micropipette. The spermatozoa used in the experiment were both motile and immobilized. The immobilized spermatozoa were collected using a single laser irradiation. Sperm membrane integrity was assessed by a hypo-osmotic swelling test prior to freezing. Five to 10 spermatozoa were inserted into the free-cell ZP through the same hole used for cytoplasmic removal. The recovery and survival rates were 87% and 83%, respectively [42].
In 2000, Hsieh et al. [43] conducted a study using empty human or mouse ZP to cryopreserve human spermatozoa. The sperm samples were obtained from individuals diagnosed with azoospermia, oligoasthenozoospermia, or from those with normal spermatozoa, sourced from epididymal, testicular, and ejaculated origins, respectively. To prepare the carrier, cytoplasmic materials were removed, and spermatozoa were then injected into the ZP. The recovery and motility rates were reported at 89% and 92%, respectively. The study found no significant differences in the recovery and motility rates between the two carriers using human and mouse ZP for sperm storage [43].
The 2000 study by Liu et al. [44] pursued two primary objectives: to explore the impact of various glycerol concentrations (6%, 8%, 10%, 12%, and 14%) combined with 3% and 10% synthetic serum substitute (SSS) on the survival rate of cryopreserved ejaculated spermatozoa, and to compare the survival rates of fresh testicular spermatozoa with those cultured in vitro for 3 days prior to cryopreservation. In their experiment, mouse cell-free zonae pellucidae served as carriers, with 5–10 spermatozoa injected into each evacuated ZP. The study found that after the freezing and thawing processes, there were no significant differences in survival rates between the varying concentrations of glycerol combined with 3% and 10% SSS. Additionally, no significant differences in survival rates were observed between fresh and cultured testicular spermatozoa post-freezing. The recovery, motility, and survival rates were reported as 100%, 58%, and 77%, respectively. The study concluded that pre-freezing culture of testicular spermatozoa did not increase survival rates [44].
In their 2000 study, Borini et al. [38] preserved testicular spermatozoa collected via TESA from azoospermic patients using cell-free human ZP. They employed two distinct freezing techniques based on the spermatozoa count: for counts between 100 and 2,000, spermatozoa were frozen using TEST yolk buffer (TYB) with glycerol (method I); for counts below 100, spermatozoa were first loaded into empty human ZP before adding the cryopreservation medium (method II). Following thawing, the ICSI technique was applied to both immature (germinal vesicle) and metaphase I oocytes, which were matured in vitro to evaluate the fertilization potential of the post-thaw testicular spermatozoa. The outcomes showed recovery, motility, and fertilization rates of 1%, 32.3%, and 13.3% for method I, and 88%, 27%, and 23% for method II, respectively [38].
In 2003, Levi-Setti et al. [45] modified the protocol by incorporating TYB into the empty human ZP, utilizing it as an ideal cryoprotective medium. Fifteen motile spermatozoa, obtained from the ejaculate of 10 infertile cases, were injected into each ZP. Upon thawing, the recovery and motility rates were 59% and 73%, respectively. It was concluded that the use of a cryoprotectant medium such as TYB helps preserve sperm motility and prevents the loss of spermatozoa [45].
The use of empty ZP for freezing sperm with a low sperm count is still considered experimental for several reasons. These include federal regulations that prohibit the interaction of human gametes with animal products, the potential for residual host DNA fragments, the requirement for a biological carrier, limited availability, and the method being both labor-intensive and time-consuming. However, there are significant advantages to this method, including the ability to easily add and remove cryoprotectants without losing or diluting the sperm contained within the zona, and the potential for successful pregnancy.
2) Volvox globator algae
Volvox globator is an affordable spherical alga that is easily cultivated and widely available. Its distinctive green color and size of 200 µm make it easily identifiable and useful. In a 2003 study by Just et al. [46], eight motile spermatozoa with intact morphology from a poor ejaculate were injected into each Volvox sphere, and three of these spheres were subsequently cryopreserved in a straw. Upon thawing, the recovery and motility rates were 100% and 60%, respectively. V. globator represents a promising, straightforward, and cost-effective method for the cryopreservation of functional motile spermatozoa. In countries where the destructive use of oocytes is prohibited, V. globator offers an alternative approach even after fertilization failure. However, the potential for genetic material transfer from the algae to the egg cells is a drawback of this method that requires further investigation. According to recent regulations from the U.S. Food and Drug Administration and the European Tissue Directive, the use of algae as a non-human tissue carrier for preserving human spermatozoa is not approved for clinical use [46]. Therefore, due to the limitations associated with biological carriers, there has been increased consideration of non-biological carriers for freezing small sperm counts.
2. Non-biological carriers
1) Alginic acid capsules
Herrler et al. [47] in 2006 utilized polymerized alginic acid drops to freeze small spermatozoa counts. Alginic acid comprises two sugars: β-D-mannuronic and α-L-guluronic acid. The addition of calcium causes these sugars to polymerize, hence the use of a CaCl solution. Alginate, a non-toxic polysaccharide, is employed in its polymerized form for the cryopreservation of hepatocytes and stem cells due to its gel-like properties and chemical inertness. It also serves as a carrier in single sperm cryopreservation. Capsules containing ejaculated sperm were immersed in a sperm freeze medium, then frozen in straws using a slow freezing technique in a programmable freezer. The thawing process involved dissolving the alginate beads in a trisodium citrate solution. Following the liquefaction of the capsules, an additional 30 seconds was allowed to remove the alginic acid, after which the spermatozoa were washed twice by centrifugation. Prior to the use of alginic acid, sperm motility and viability were recorded at 62.7%±1.2% and 69.2%±1.9%, respectively. Following treatment with alginic acid, the rates of motile and viable sperm dropped to 30.0%±3.3% and 56.4%±2.7%, respectively. Cryopreservation using this method resulted in a roughly 20% decrease in motility compared to conventional and standard methods, attributed to the sperm surface being coated with alginic acid. The clinical application of this carrier is limited due to its defects and the complexity of the procedure [47].
2) Hollow-core agarose capsules
Agarose has a structural similarity to a mesh and can regulate the volume of solution on the sheet, facilitating easier handling. In 2015, Araki et al. [48] utilized hollow-core agarose capsules, comparable in size to mammalian oocytes, for the cryopreservation of single spermatozoa. To create these agarose capsules, 0.5% calcium carbonate was dissolved in 4% alginate acid. The solution was then enhanced with mineral oil containing 3% lecithin and 0.5% acetic acid. This mixture led to the formation of tiny spheres. The calcium carbonate in the solution was dissolved by the acetic acid and subsequently reacted with the alginate acid. This reaction transformed the spheres into gel beads. The beads were then collected using a nylon mesh sheet (MS). The capsules were constructed with a hollow core surrounded by an agarose wall.
Devices were constructed from a polycarbonate sheet (PS) equipped with a plastic straw and a nylon MS featuring a hole. Each capsule was injected with one sperm before being placed into a drop of 4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid (HEPES)-HFF99 (6% glycerol with 0.05% methylcellulose; Wako Chemical Co.). Subsequently, the capsules containing the cryoprotectant solution were transferred to the tips of the two devices. The freezing process involved exposing the samples to LN2 vapor for 10 to 30 seconds before plunging them into LN2. For the thawing process, a medium of HEPES-HFF99 (20-mL) supplemented with 0.3% HSA was utilized. The spermatozoa recovery rates achieved were 91.5% with PS and 96.7% with MS. Sperm motility rates were recorded at 85.3% and 82.8%, respectively. The survival rates of the spermatozoa were 97.3% using PS and 91.4% using MS. The results obtained from the two devices were comparable.
In other research, mammalian cells were cultured in an agarose gel [49]. The components of agarose gel are relatively non-toxic, making it suitable for use as a cell culture medium. However, further research is needed on sperm functional parameters before this device can be applied clinically [48].
Hatakeyama et al. [50] encapsulated 3–4 motile spermatozoa within agarose capsules. These capsules were then frozen on a cryotop using 0.5 µL of sperm maintenance medium as a cryoprotectant. The recovery, motility, and survival rates were 100%, 82%, and 88%, respectively [50].
Isaev et al. [51] utilized microspheres composed of 2% agarose gel, each with a diameter of 100 µm, for the cryopreservation of single spermatozoa. Each microsphere could accommodate between one and 10 spermatozoa. These microspheres were then transferred into sperm preparation medium and sperm freezing medium. Subsequently, one to five microspheres were placed onto straws and subjected to LN2 evaporation for 10 minutes before being fully immersed in LN2. Upon thawing, the recovery, motility, and survival rates were recorded at 98%, 78%, and 81%, respectively [51].
These techniques were not pursued or further developed due to concerns about the use of agarose, which was not explained in terms of its stability during the sperm loading procedure. Additionally, the residual effects of alginic acid on sperm surfaces could reduce sperm motility. Furthermore, sperm loss during the washing steps required to remove alginic acid might compromise the effectiveness of this method for surgically retrieved sperm.
3) Hollow hyaluronan-phenolic hydroxyl microcapsules
Tomita et al. [52] developed a technique for single sperm cryopreservation using hollow hyaluronan-phenolic hydroxyl microcapsules, which did not adversely affect sperm parameters. These hollow hyaluronic acid microcapsules were loaded with three sperm each and placed into a cryotop containing 1 µL of freezing medium with sucrose as the cryoprotectant, before being immersed in LN2. Following the thawing procedure, the recovery and motility rates were 96% and 14%, respectively. The recovery rate was notably higher compared to that achieved with an empty ZP carrier. The low motility rate was attributed to gelatin accumulation during freezing, which led to defects in the sperm plasma membrane. While there are no ethical concerns regarding the use of this non-biological carrier, further research is necessary for its clinical application [52].
4) Cryoloops
Schuster et al. [53] employed a cryoloop device as an effective and convenient method for individual sperm cryopreservation. The study consisted of four experiments, which included evaluating sperm motility across various concentrations of cryoprotectants, assessing sperm motility at different time intervals, examining the effects of four cryoprotectant solutions, and comparing two freezing rates: ultra-rapid and slow. Further details can be found in the cryoprotectant and freezing section [53].
Desai et al. [40] in 2004 proposed the use of nylon cryoloops as a novel carrier for freezing spermatozoa. In their study, 77 spermatozoa were cryopreserved across 10 cryoloops, with each cryoloop containing 5–10 spermatozoa. The cryoprotectants used were human tubal fluid (HTF)-HEPES (50:50) with 6% plasmanate and TYB-glycerol. Following the thawing procedure, the recovery, motility, and fertilization rates were 68%, 73%, and 67%, respectively. The post-thaw motility did not show significant changes when compared to the conventional freezing method using vials. It was concluded that the decondensation of the sperm head and the formation of the pronucleus after injection into a human oocyte could occur using the single sperm cryopreservation technique. Additionally, 2–8 epididymal or testicular spermatozoa were cryopreserved on nylon cryoloops using TYB-glycerol and m-HTF medium supplemented with 6% plasmanate as the cryoprotectant, achieving a recovery rate of 72%. Epididymal and testicular spermatozoa cryopreserved by this method were capable of fertilizing oocytes via the ICSI technique [40]. Commercially available cryoloops do not require extra preparation. The materials used in the production of cryoloops are non-biological and do not involve animal products. Initially, this carrier was used for embryo and oocyte freezing. However, due to its challenging handling, it has become less favored, leading to the introduction of other carriers such as cryotops.
5) Culture dish
Using microdroplets in a culture dish represents a simple and innovative method for the cryopreservation of small spermatozoa counts. In 2000, Quintans [54] froze 4–6 spermatozoa in a tiny droplet of freezing medium, which they then placed in plastic tissue culture dishes covered with an oil overlay. The dishes were subsequently sealed and stored in LN2. Upon thawing, they achieved a recovery rate of 90% to 100% [54].
In 2003, Bouamama et al. [55] introduced a microdroplet freezing medium for single sperm cryopreservation, utilizing culture dishes. This method involved placing 1–100 spermatozoa in microdroplets composed of a 50:50 mixture of TYB-glycerol and sperm freezing medium, which were then overlaid with paraffin oil. The culture dishes were subsequently sealed and stored in LN2. The post-thaw recovery rate achieved was 100%, with a motility rate of 50%. In contrast, the classical straw technique, which involves freezing 1–10 sperm per straw, resulted in no viable sperm after thawing. Consequently, the microdroplet freezing medium in sealed culture dishes demonstrated a significantly higher recovery rate compared to the straw technique [55].
In 2008, researchers employed a similar carrier to cryopreserve a small quantity of testicular spermatozoa. Testicular fine needle aspiration was used to collect 1,063 spermatozoa, which were then cryopreserved using a culture dish technique, with 10–340 sperm cells per sample. Initially, motility was reported at 13.7%, but this decreased to 3.6% post-thaw. However, the recovery rate of spermatozoa was 100%. In other study groups, 431 spermatozoa (2–300 sperm cells per sample) were cryopreserved. The motility rates before freezing and after thawing were 3.5% and 2.3%, respectively, with a recovery rate of 100%. Motile sperm were injected into the oocytes in two of the six cases, resulting in one biochemical pregnancy. The fertilization rate was recorded at 17.6% [56].
This technique is straightforward and simple; however, polystyrene culture dishes containing microdroplets are challenging to preserve for extended periods in LN2 due to their size and shape. These culture dishes do not create closed systems as they cannot be sealed, which heightens the risk of cross-contamination. Additionally, the substantial volume of oil used to overlay the droplets necessitates a lengthy warming period, potentially disrupting the procedure.
6) ICSI pipettes
Gvakharia and Adamson [57] utilized ICSI micropipette tips as carriers for the cryopreservation of small spermatozoa counts. In their study, samples were collected from ejaculate samples (n=15) and testicular tissues (n=3). Following the collection, spermatozoa were aspirated into ICSI micropipettes, then placed into a cryoprotectant before being aspirated again into the ICSI pipette tip. The ICSI pipettes were then positioned in holders and exposed to LN2 vapor before being submerged in LN2. Upon thawing, the recovery rate of spermatozoa was 92% for both ejaculated and TESA samples, with a motility rate of 52%. These results suggest that glass carriers are effective for the cryopreservation of small spermatozoa counts [57].
In 2003, Sohn et al. [58] conducted a study to evaluate the effectiveness of the ICSI pipette in the cryopreservation of samples from cases of severe oligozoospermia. Prior to freezing, samples from 10 severe oligozoospermia cases were centrifuged, and the resulting pellets were prepared for both ultra-rapid and slow freezing methods. Each type of sperm pellet was then placed into a drop of freezing media. An ICSI micropipette was utilized to collect motile spermatozoa. Following the freezing and thawing processes, the recovery rates were found to be comparable, with slow freezing at 91.1% and ultra-rapid freezing at 79.6%. The motility rates were low in both groups after the procedures. Initially, sperm viability was 57.4%, but it decreased in both the slow and ultra-rapid freezing groups after the freezing and thawing. However, sperm viability was higher in the slow freezing group compared to the ultra-rapid freezing group [58].
Desai et al. [37] conducted a case report study in 2012 using a high security straw (HSV) to freeze epididymal, testicular, and poor ejaculated sperm samples from individuals with azoospermia and OAT. This device comprises an outer straw and a capillary tube. TYB-glycerol served as the cryoprotectant. Sperm were aspirated into the tip of a glass micropipette and then placed into the capillary tube. The sperm, suspended in a medium (1.0 µL), were positioned in the gutter near the open bottom of the capillary tube. The capillary tube was then inserted into the HSV outer straw, and the wide end was sealed. The straw was exposed to LN2 for 1 hour and subsequently immersed in the LN2. Following the thawing procedure, the recovery and motility rates were 70% and 74%, respectively. Spermatozoa with minimal motility, recovered post-thaw, were injected into the oocytes of six cases, resulting in a fertilization rate of 53%. The pregnancy and delivery rates were both 100% [37].
The use of ICSI pipettes as carriers for single sperm freezing is straightforward and convenient; however, they are not suitable for long-term storage due to their susceptibility to breakage with temperature fluctuations. The tip of an ICSI pipette is extremely fragile and prone to breaking. Additionally, storing sperm directly in LN2 exposes it to potential cross-contamination. Consequently, the application of this carrier has become restricted, leading to the introduction of alternative cryo-devices.
7) Straws
Kuznyetsov et al. [59] investigated the effectiveness of the closed straw system in freezing samples from normozoospermic and severely oligozoospermic individuals using nonpermeating CPAs. In this study, spermatozoa were introduced into the open-pulled straw using two methods: spontaneous capillary action and a polar body biopsy pipette. The research involved samples from 15 normozoospermic and 10 severely oligozoospermic participants. The post-thaw recovery rate, viability, and motility rate were reported as 80%, 80%, and 41.5%, respectively. Additionally, it was noted that using the polar body biopsy pipette for loading sperm increased recovery rates in both normal and abnormal samples [59].
In 2017, Liu et al. [60] compared three different types of straws for freezing samples from fertile individuals: LSL straws (50–100 μL), and traditional 0.25 and 0.5 mL straws. The LSL straws featured an outer metal shell and an inner tube. Samples were mixed with a modified glycerin-yolk-sodium citrate (GYC) cryoprotectant in a 1:1 ratio. Following the freeze-thaw process, the LSL straw group exhibited a higher percentage of sperm motility compared to the groups using traditional 0.25 and 0.5 mL straws. Morphology, acrosome, and DNA integrity remained similar across all three straw types. The LSL straw's thin shape and minimal volume contributed to a faster freezing rate. It was concluded that LSL micro-straws are advantageous for freezing samples with low sperm counts in cases undergoing ICSI treatment [59].
However, this method is not recommended for severely low-quality specimens where only a few sperm are present, as sperm can be lost due to adherence to the vessel wall. Additionally, individually selected sperm cannot be easily sequestered within the straw.
8) Cell sleeper
In 2012, Endo et al. [61] utilized a cell sleeper, a closed system, to freeze a small number of spermatozoa. This device comprised a vial container and an inner tray. The sperm samples originated from cases of poor ejaculation and testicular sources in NOA and OAT patients. A 3.5 µL droplet was placed on the tray, to which individual sperm samples were then added. Subsequently, the tray was inserted into a vial and securely sealed with a screw cap. It was positioned above LN2 vapor at –120 °C and rapidly frozen in LN2. Cryoprotection was achieved using a mixture of 0.7 mL sperm freez and 1.0 mL HFF99 with 20% SSS. In one NOA case, the technique led to the birth of a healthy boy. Additionally, 12 sperm cells were cryopreserved from testicular tissue, and upon thawing, a recovery rate of 83% (10/12) was achieved. Each sperm was injected into six mature oocytes, resulting in a fertilization rate of 83% (5/6). The rates of motility, pregnancy, miscarriage, and delivery were 0%, 100%, 0%, and 100%, respectively [61].
In a study by Endo et al. [62], researchers investigated the impact of different vitrification volumes (0.5, 1.0, and 3.5 µL) on vitrified spermatozoa using a cell sleeper carrier. The sperm samples were obtained from normal ejaculate. The cryoprotectant solution consisted of a mixture of cryoprotectants and 20% SSS. During the freezing process, the cell sleeper was first exposed to LN2 vapor before being fully immersed in LN2. The thawing process revealed recovery, motility, and survival rates of 96%, 30%, and 65%, respectively. Apoptotic DNA fragmentation remained unchanged when compared to fresh sperm [62].
In 2016, Coetzee et al. [63] utilized a cell sleeper device to cryopreserve testicular spermatozoa from OA and NOA patients. A 2 µL droplet was placed on the tray, to which 5–27 spermatozoa were added. The spermatozoa were then submerged in a 50:50 mixture of Sperm Freeze solution (Vitrolife) and IVF Gamete Buffer supplemented with HSA, followed by freezing in LN2 vapor and subsequent immersion in LN2. Twenty cell sleepers were employed to freeze 304 spermatozoa, of which 265 were subsequently warmed. The recovery and motility rates post-thawing were 94% and 65%, respectively. The thawed spermatozoa were injected into 179 mature oocytes, resulting in a fertilization rate of 66% and a pregnancy rate of 58%. Although this technique is straightforward and rapid, locating sperm post-thaw takes about 30 minutes, which is longer compared to other methods. In this approach, the sperm remain in the freezing medium for 30 minutes after warming and are not washed in a physiological medium. Additionally, during microsurgery, the ICSI needle must be moved up and down, a motion that may cause the needles to break [63].
In a 2021 case report, Herbemont et al. [64] utilized a stripper tip and a cell sleeper for the cryopreservation of spermatozoa in a patient with cryptozoospermia. They froze seven spermatozoa in a cell sleeper and 12 in a homemade stripper tip. Following the thawing process, the recovery, survival, and motility rates for the cell sleeper were 100%, 85.7%, and 42.9%, respectively. In contrast, the stripper tip showed recovery, survival, and motility rates of 100%, 66.7%, and 16.7%, respectively. Spermatozoa recovered from the cell sleeper and stripper tip were used to inject six and seven oocytes, respectively. The use of spermatozoa from the stripper tip resulted in four zygotes, achieving a fertilization rate of 57%. Subsequently, two of the selected embryos were transferred, leading to the birth of a healthy girl [64].
The cell sleeper, which is commercially available, is a closed system. The main disadvantage of this carrier is an inner rigid tray and its high edge, which increases the risk of breaking ICSI pipettes due to moving them up and down [62].
9) Cryotop and similar devices (Cryoplus, Cryolock, and Cryopiece)
Cryotop, a non-biological carrier, is made up of polypropylene strips and a plastic handle with a cover, which is commercially available. It is used to cryopreserve a small number of sperm in microdroplets [35]. Additionally, this technique was first applied to the vitrification of oocytes and embryos, achieving a 99% recovery rate after warming [39].
Endo et al. [65] utilized the cryotop method to cryopreserve individual spermatozoa. In their study, they compared two sources of sperm, two different carriers, and cryoprotectants. The carriers used for sperm cryopreservation were cryotop and empty ZP. The ZP sources included germinal vesicles, metaphase I stage, and unfertilized metaphase II stage oocytes. Oocytes were evacuated using an injection micropipette. In the cryotop technique, sperm were transferred to a 1 µL droplet of freezing medium on the cryotop strip. The cryotop was then exposed to LN2 vapor for 2 minutes before being plunged into LN2. The sperm sources included normal ejaculated sperm and testicular sperm from OA and OAT patients. Post-thaw recovery rates were 90% and 95%, respectively. There was no significant change in motility rate between the ejaculated group (44.4%) and the testicular sperm group (42.1%). The average time required to locate sperm was 265 seconds for the ejaculated sample and 286 seconds for the testicular sperm. One hundred sperm were frozen using both devices. After thawing, 98% of the sperm in the cryotop and 88% in the ZP were recovered. There were no significant differences in motility rates between the cryotop (30.6%) and ZP (23.9%) groups. The results of using two different cryoprotectants are discussed in the cryoprotectant section. It can be concluded that the cryotop is an appropriate and ideal device for the cryopreservation of a small number of sperm, and sucrose is an effective cryoprotectant [65]. In another study, spermatozoa from ejaculated samples were frozen using two devices: cryotop and cell sleeper. After the thawing procedure, the sperm recovery rate was high (>96%) in both the cryotop and cell sleeper groups. There were no differences in motility and viability rates between the two carrier groups. Additionally, the cryotop was reported to be an efficient device for the cryopreservation of a limited number of spermatozoa. The cryoprotectant used included 0.7 mL sperm freeze and 1.0 mL HFF99 supplemented with 20% SSS [61]. This technique was applied in two cases of severe oligozoospermia or NOA. The vitrification procedure was performed for 81 spermatozoa from testicular tissue across eight carriers (approximately 10.1 sperm per carrier), and warming was conducted for 10 of these. After complete recovery, they were injected into four mature oocytes, resulting in a 75% fertilization rate [62]. It should be noted that in an open system, spermatozoa are exposed to LN2 using these carriers, which may potentially increase the risk of cross-contamination.
In 2023, Bagheripour et al. [66] developed a novel cryotop vial device system. This study involved categorizing 25 normal samples into four groups: fresh, rapid freezing, and ultra-rapid freezing using the cryotop device and cryotop vial device. Ultra-rapid freezing was performed using sucrose. The biological characteristics of the sperm were evaluated. The results indicated that the new device preserved sperm motility, viability, and DNA integrity more effectively than the other groups. The cryo-solution, consisting of 1 µL supplemented with 0.25 M sucrose, was dissolved in a 10 µL drop of warming medium, resulting in a dilution at least 10 times less concentrated than the sucrose. Consequently, centrifugation is not necessary with this method. This approach serves as an effective alternative to rapid freezing for individuals with sterility and low ejaculate volume, providing more frozen sperm carriers for repeated ART cycles [66].
Cryoplus is similar to cryotop but is designed to load more than 5 µL of a solution. This modified carrier features an enlarged polypropylene strip measuring 32 mm by 1.2 mm, larger than that of the cryotop carrier. Consequently, it can accommodate over 10 µL of solution on both sides of the device [67].
In a 2018 study by Wang et al. [67], Cryoplus, along with two other carriers—0.25-mL straws and 2-mL cryogenic vials—were utilized to cryopreserve samples from oligozoospermia and azoospermia cases to assess clinical outcomes. A 10 µL research plus pipette was employed to load sperm onto the adapted strip. Spermatozoa motility rates showed improvement with Cryoplus when compared to the 2-mL cryogenic vials. DNA fragmentation in sperm was found to be similar across all three carriers. Cryoplus carriers were also used for the cryopreservation of testicular spermatozoa in ICSI cycles. Clinical pregnancy, implantation, miscarriage, live birth rates, and birth weight were comparable between cycles using fresh or cryopreserved testicular spermatozoa for frozen-thawed embryo transfers. The delivery rates for the cryopreserved and fresh sperm groups were 19 and 23, respectively. This open cryopreservation system offers the advantages of requiring less cooling time and fewer thawing procedures compared to other methods [67].
Stein et al. [68] in 2014 utilized a cryolock device, similar to the cryotop, to develop an effective method for freezing individual spermatozoa. The device is square-shaped with four surfaces, allowing for the placement of small-volume samples. This design facilitates high cooling and warming rates. The study was conducted in three phases. In the first phase, sperm samples from individuals with OAT were mixed with two cryoprotectants: TYB-glycerol and HEPES-buffered salt solution with glycerol-glucose (HBGG). The samples were subjected to two freezing methods: exposure to LN2 vapor and direct immersion in LN2. The primary goal was to assess the effectiveness of the cryolock in freezing sperm samples. The results showed that the survival rate was higher when samples were frozen in LN2 vapor. Additionally, the motility rate of spermatozoa was 95% with TYB-glycerol, significantly higher than the 35% achieved with the HEPES-glycerol-glucose solution. In the second phase, groups of 10–50 spermatozoa were cryopreserved using TYB-glycerol as the cryoprotectant and frozen in LN2 vapor. The recovery rate was 100% (sperm retrieved in all 15 samples), and the motility rate was 93%. The third phase involved cryopreserving 10 testicular samples from non-obstructive azoospermic individuals using the cryolock device and the methods described in phases 1 and 2. The recovery rate was 90% (sperm detected in nine samples), and the motility rate was 44%. It was suggested that the cryolock is an effective carrier when used with TYB as the cryoprotectant and LN2 vapor as the freezing method for cryopreserving both OAT and testicular samples [68].
The cryopiece system is a polypropylene piece with sperm droplets on it placed into a cryo-tube. In this procedure, sperm samples and a polypropylene piece with droplets were prepared in a dish covered with an oil overlay. Sperms were selected using an ICSI pipette, and the chosen sperm was then placed onto the droplets on the polypropylene piece. Finally, the cryopiece was inserted into a cryo-tube and stored in an LN2 tank [69].
In 2016, Sun et al. [69] employed the cryopiece technique on four individuals diagnosed with severe oligozoospermia or NOA. The sources of sperm were testicular and normal ejaculate. High-quality spermatozoa, characterized by normal morphology and high motility, were placed on droplets of freezing medium on the cryopiece. The samples were then exposed to LN2 vapor for 15 minutes before being fully immersed in LN2. Upon warming, the recovery and motility rates were recorded at 83% and 43%, respectively. The fertilization, pregnancy, and delivery rates were notably high at 73%, 75%, and 75%, respectively. The study reported the birth of four full-term babies, all in good physical condition [69].
The sperm vitrification device (Sperm VD) is an innovative tool designed for the freezing of a limited number of spermatozoa, proving to be both effective and efficient in fertility preservation. This device functions similarly to a cryopiece. In this technique, spermatozoa are placed into microdroplets ranging from 0.8 to 1 µL of Quinn’s Advantage Sperm Freezing Medium and Quinn’s Sperm Washing Medium on the Sperm VD. Subsequently, cryovials (3.6 mL) containing the Sperm VD are immersed in LN2. This method significantly reduces the time required to search for spermatozoa from hours to minutes. Additionally, sperm recovery and motility rates are high, at 96% and 33%, respectively. Fertilization and pregnancy rates have been reported at 59% and 55%, respectively, while delivery and miscarriage rates stand at 32% and 29%, respectively. Although the Sperm VD is ideal for the cryopreservation of motile spermatozoa and enhances clinical outcomes, it is unable to preserve viable immotile spermatozoa. Therefore, further research is necessary [70].
In this study, only motile sperm were cryopreserved. Consequently, further research is necessary to demonstrate the efficacy of this carrier for single sperm cryopreservation. These carriers operate in an open system where spermatozoa are exposed to LN2, thereby increasing the potential risk of cross-contamination.
10) Cryoleaf
In 2011, Peng et al. [71] developed a new technique for the cryopreservation of a limited number of spermatozoa. This method involved using clear polystyrene from a Falcon petri dish to construct a cryoleaf in the laboratory. Small squares were cut from the dish cover, and one square was heated to create a sheet measuring 20.0 mm in length and 3.0 mm in width. The cryoleaf was then attached to a handle and enclosed within a protective cover, which included moist cotton wool at one end. Both the handle and the protective cover were crafted from a human embryo transfer tube package. A specialized chamber was constructed in the laboratory to maintain humidity during the process of injecting spermatozoa into the droplet. The sperm sources for this study included epididymal and normal ejaculate. A droplet (0.2 µL) consisting of 0.1 µL seminal plasma and 0.1 µL cryoprotectant—a mixture of 12% glycerol and 20% egg yolk in 0.1 M citrate buffer (pH 7.2)—was placed on the cryoleaf. Individual spermatozoa were then added to the droplet. The cryoleaf was sealed with its cover and exposed to LN2 vapor for 2 minutes before being fully immersed in LN2. Upon warming, the motility recovery rate for spermatozoa collected via percutaneous epididymal punctures was 92.9%, while the recovery rate for ejaculated spermatozoa was 61.5%. Following the ICSI procedure, there were no significant differences in fertilization and cleavage rates between fresh and frozen-thawed spermatozoa. To prevent droplet evaporation, the cryopreservation procedure must be performed swiftly by skilled operators. No pregnancies were reported using this method. This carrier is homemade and is not available commercially [71].
11) New cryopreservation container
In 2019, Nakata et al. [72] introduced a novel carrier designed for the cryopreservation of small quantities of spermatozoa. This carrier, made from polydimethylsiloxane, features a transparent bottom that facilitates the evaluation of sperm morphology under a microscope. To fit into a 1.8 mL cryo-tube, the dimensions of the carrier were set at 30×10×5 mm for width, depth, and height, respectively. In their research, samples from 44 normal spermatozoa and 16 spermatozoa from OAT patients were collected using a micropipette and placed onto the droplet on the new device under microscopic observation. The container was then frozen at –80 °C for 5 minutes, inserted into the cryo-tube, and subsequently plunged into LN2. Additionally, the study compared the cryotop with the new device. Post-thaw, the sperm recovery rate was 21.2% (11/52) for the cryotop and 96.7% (58/60) for the new container. While other studies have reported recovery rates for the cryotop above 80%, Nakata et al. [72] recorded a rate of 21.2%, suggesting potential issues with their use of the cryotop. Sperm motility rates were 19.2% (10/52) with the cryotop and 35.0% (21/60) with the new device. The study concluded that the new device is suitable for the cryopreservation of a small number of spermatozoa, effectively minimizing sperm loss [72].
Effective agents in the single spermatozoa cryopreservation technique
Several factors and agents influence the cryopreservation of one or a few spermatozoa, including carriers, sperm sources, freezing techniques, and cryoprotectants.
1. The ideal carrier for single sperm cryopreservation
Table 2 summarizes the advantages and disadvantages of various carriers used for storing small numbers of spermatozoa. The key factor in the freezing of single spermatozoa using these carriers is their recovery and survival rates. Among the carriers, agarose gel microcapsules exhibit the highest recovery and survival rates, while the straw has the lowest recovery rate [73]. This variation in recovery rates among the carriers is linked to the volume of freezing; carriers that achieve high recovery rates require smaller volumes for freezing. The recovery rate exceeded 80% for cryotop, cell sleeper, cryopiece, and Sperm VD, reaching 100% for cell sleeper and agarose gel microcapsules. The lowest survival rate was recorded for the cell sleeper [73]. Additionally, the rates of fertilization, pregnancy, miscarriage, and delivery were similar across the different carriers. Specifically, fertilization rates were low in thawed single human spermatozoa following the conventional ICSI procedure. There was no significant difference in fertilization rates among the various carriers [73]. According to published data, cryotop and similar devices appear to be more ideal carriers due to their acceptable recovery and motility rates in single sperm cryopreservation. Future research should therefore concentrate on these carriers.
2. Cooling rate and single sperm cryopreservation
Conventional freezing and vitrification are two methods used for sperm cryopreservation. In research focusing on the cryopreservation of single spermatozoa, vitrification is predominantly employed as the freezing technique. A meta-analysis indicated that vitrification, or ultra-rapid freezing, is more effective than conventional freezing methods at preserving spermatozoa [74]. Additionally, our previous studies have demonstrated that directly submerging the cryotop into LN2 significantly reduces sperm motility rates (unpublished data). While some researchers have cryopreserved sperm using either vapor or direct submersion in LN2, further studies are needed to compare the efficacy of these two cooling methods.
Schuster et al. [53] employed a cryoloop device for the cryopreservation of individual sperm. In the fourth experiment of their study, two freezing techniques were evaluated: ultra-rapid freezing, which involved direct immersion into LN2, and slow rate freezing, which entailed exposure to LN2 vapor for 5 minutes before immersion. The study cryopreserved 10 semen samples using a freezing medium that included TYB with 12% v/v glycerol. Initially, sperm motility was recorded at 55%±2.2%. Post-thaw, motility rates were 45%±3.1% for the slow rate method and 45%±3.4% for the ultra-rapid method. Both methods resulted in a significant decrease in motility after thawing. However, there were no significant differences in motility outcomes between the two methods [53].
Hosseini et al. [75] compared two cryopreservation methods—vapor and direct submersion in LN2—using a cryotech device on samples with a low number of normal spermatozoa. They found that direct submersion in LN2 was superior in preserving motility, viability, and chromatin structure compared to the vapor method [75].
Stein et al. [68] utilized a cryolock device for freezing and compared two phases of the process: exposure to LN2 vapor and immersion in LN2. They found that exposing samples to LN2 vapor was the more effective freezing method, while plunging the cryolock into LN2 proved to be detrimental [68].
Bagheripour et al. [66] modified the previous cryotop device, which consisted of polypropylene strips with a cap inserted into a hard plastic straw. The main disadvantages of this design were the time-consuming process required to seal the straw and the tendency for the drop to attach to the cap's wall. To improve this carrier, the cryotop was placed into a cryo-vial, and a screw cap was used for sealing. Although sperm motility decreased in both devices, the modified carrier preserved motility more effectively. The new carrier features two protective walls with an airspace between them, in contrast to the previous device, which had only one protective wall to reduce temperature conduction. The formation of ice crystals, the primary cause of sperm cryo-injury, is exacerbated by a slower cooling rate in the previous device. Therefore, the new carrier could be considered an ideal device due to its effectiveness in preserving sperm characteristics [66].
3. Warming temperature and single sperm cryopreservation
In the thawing process, temperature plays a critical role in preventing the formation of ice crystals and cellular damage. Increasing the warming temperature is another technique used in cryopreservation; however, no relevant studies have been identified concerning the cryopreservation of a small number of spermatozoa.
For the ejaculated samples, the temperature of the warming medium was increased to 38, 40, and 42 °C for durations of 5 and 10 seconds. The percentage of sperm progressive motility was higher at 42 °C (65.4%±15%) compared to 38 and 40 °C (26.4%±8.4% and 56.6%±16.3%, respectively). The function of the plasma membrane was assessed using the hypo-osmotic swelling test, and the results indicated that 42 °C preserved the plasma membrane function more effectively than the other two temperatures. In the warming process, temperature plays a critical role, and 42 °C has been identified as the ideal temperature for maintaining sperm parameters [76].
Furthermore, warming at both 37 and 42 °C significantly preserved sperm motility, morphology, and results of the hyaluronan-binding assay and acrosome reaction tests [77].
4. Cryoprotectants and single sperm cryopreservation
Several studies have explored the use of various cryoprotectants for freezing small quantities of spermatozoa. Other research has examined the impact of different cryoprotectants when used in conjunction with carriers. Using a cryoloop as the freezing carrier, Schuster et al. [53] assessed sperm motility across various concentrations of cryoprotectants and examined the effects of four different solutions.
In experiment 1, Cryo A consisted of 40% ethylene glycol, 0.5 mol/L trehalose, 5% dextran serum substitute (DSS), and 10% TYB. DSS was composed of 50 mg/mL HSA and 20 mg/mL dextran in a saline solution. The TYB included 20% egg yolk, 1,000 IU/mL penicillin-G, and 1,000 mg/mL streptomycin sulphate. Cryo B contained 25% ethylene glycol, 0.5 mol/L trehalose, 5% DSS, 10% TYB, and 25% glycerol. These cryoprotectants were utilized in 10 samples diluted at a 1:1 ratio. Cryoprotectants were prepared with TYB at dilutions of 1:1, 3:1, and 4:1 to evaluate the impact of varying cryoprotectant concentrations on sperm motility, which was 87%±1.5%. Pure Cryo A and Cryo B resulted in 1% and 0% sperm motility, respectively. Sperm motility in the Cryo A group was 77%±2.4%, 51%±4.3%, and 39%±5.7% for the 1:1, 3:1, and 4:1 dilutions, respectively. In the Cryo B group, the 1:1, 3:1, and 4:1 dilutions showed sperm motility of 41%±6.3%, 11%±3.5%, and 2%±1.7%, respectively. Each solution significantly reduced sperm motility compared to the control group, with notable differences observed between the solutions. Specifically, the Cryo A 1:1, Cryo A 3:1, Cryo A 4:1, and Cryo B 1:1 dilutions had detrimental effects on sperm motility. In experiment 3, the efficacy of four different cryoprotectants was tested during the freezing of 14 samples. Two solutions, cryoprotectants 1 and 2, were selected from the first experiment using Cryo A with 1:1 and 3:1 dilutions. The other two cryoprotectants were a freezing medium composed of TYB with 12% v/v glycerol (cryoprotectant 3) and an unprocessed semen sample with seminal plasma, without any added cryoprotectant. Before freezing, sperm motility and viability were 54%±3.4% and 66%±3.8%, respectively. After thawing, sperm motility for Cryo 1, 2, 3, and 4 were 5%±1.9%, 0%, 40%±3.2%, 16%±3.6%, and 21%±4.7% (seminal plasma and TYB), respectively. Post-thaw sperm viability was 4%±2.0% for Cryo 1, 0% for Cryo 2, not reported for Cryo 3 due to reagent incompatibility, and 17%±3.9% and 21%±4.7% for Cryo 4 resuspended in seminal plasma and TYB, respectively. There were significant decreases in sperm motility and viability in the ultra-rapid freezing groups compared to the control group. Cryo 3 demonstrated better motility compared to the other cryoprotectants [53].
Endo et al. [65] conducted a comparison of two different cryoprotectants and also explored the effectiveness of using a cryotop as a carrier for freezing. In their study, the freezing medium containing sucrose consisted of 0.1 M sucrose in HFF99, supplemented with 20% SSS. The sperm freezing medium was composed of 0.7 mL of Sperm Freeze and 1 mL of HFF99 with 20% SSS. The survival rate in the sucrose group was significantly higher at 65.3%, compared to 37.3% in the sperm freeze group. Sucrose was identified as an effective cryoprotectant for sperm cryopreservation [65].
In 2015, Chen et al. [78] collected 10 spermatozoa from each of 21 normozoospermic individuals and preserved them using a cryotop, either without CPAs or with sucrose added to the freezing medium (0.5 µL). The sperm recovery and motility rates were comparable between the two groups. It was observed that spermatozoa frozen without CPAs exhibited higher viability and less chromatin and DNA damage than those preserved with sucrose [78].
In 2021, Maleki et al. [79] employed a single sperm vitrification method using a cryotech device, a type of cryotop. The primary objective of their study was to evaluate the effectiveness of two cryoprotectants: a sperm freezing medium (Life Global) and a sucrose medium (Sigma-Aldrich). The study involved 20 ejaculated samples from individuals with severe oligozoospermia and 20 testicular samples from individuals with azoospermia. After collecting 25 spermatozoa, they were immersed in droplets of the cryoprotectant on the cryotech device and then plunged into LN2. The findings indicated that the sucrose medium better preserved the motility, viability, mitochondrial membrane potential, and DNA integrity of the spermatozoa compared to the sperm freezing medium in the single sperm vitrification method. Therefore, this technique shows promise as a strategy for the cryopreservation of spermatozoa in infertility clinics [79].
Stein et al. [68] utilized a cryolock device as a carrier in their study, which included examining sperm samples from individuals diagnosed with OAT. These samples were mixed with two cryoprotectants: TYB-glycerol and HBGG. Furthermore, the survival rate of spermatozoa was 95% when using the TYB-glycerol cryoprotectant, significantly higher than the 35% survival rate observed with the HEPES-glycerol-glucose solution [68].
In 2015, a new method called closed slice was developed using polypropylene and sperm cryovials to freeze small quantities of spermatozoa. The study involved vitrifying spermatozoa in two different groups: one group used a combination of sperm cryoprotectant and HEPES buffer with 10% HSA, while the other group used only HEPES buffer with 10% HSA, without any cryoprotectants. In the cryoprotectant group, the effects of different volumes (0.5, 1.0, and 3.5 µL) were examined. Ten progressive spermatozoa were placed in droplets and exposed to the surface of LN2 for 2 minutes before being submerged in LN2. The post-thaw analysis indicated that recovery, motility, and viability rates were consistent across the three volume groups. Additionally, there were no significant differences in recovery, activity, and survival rates between the spermatozoa preserved with cryoprotectants and those preserved without [12].
Studies have shown that media containing sucrose as a cryoprotectant are as effective as those using permeable agents. Consequently, it is necessary to evaluate various sucrose concentrations to determine the optimal level for single sperm cryopreservation. Glycerin is a primary component of cryoprotectants, known for mitigating the negative effects of freezing and enhancing sperm quality. Despite this, there has been little advancement in cryoprotectant formulations. Due to the cryopreservation process and the volume of the droplet, current cryoprotectants have not achieved ideal or optimal freezing results [12]. Therefore, it is important to develop an effective and suitable cryoprotectant to maintain testicular sperm quality in single sperm cryopreservation, as well as to explore different carrier options.
5. Techniques for improving testicular sperm motility
There are strategies that improve survival and fertilization rates by adding chemicals to the spermatozoa samples before performing ICSI. These chemicals, such as pentoxifylline, 2-deoxyadenosine, and theophylline, stimulate in vitro improvements in sperm motility by inducing variations in sperm cyclic adenosine monophosphate contents. However, these chemicals may be toxic to the spermatozoa and the resulting embryos in assisted reproduction techniques. Therefore, in vitro incubation or culture of testicular sperm samples may be an effective method for stimulating motility in hypokinetic spermatozoa in cases of azoospermia [80]. Recent research has demonstrated the beneficial effects of in vitro cultured testicular spermatozoa on the differentiation of spermatogenic cells and the enhancement of motility. Hosseini and Khalili [81] evaluated sperm motion characteristics after culturing TESE samples at various temperatures and time intervals. The samples were cultured at different temperatures, and sperm total motility was assessed at various time intervals post-testicular biopsy. The results indicated that the optimal in vitro culture condition for testicular spermatozoa was at 25 °C for 1 day, which significantly improved sperm motility in azoospermic TESE samples [81]. Another study explored the impact of in vitro culture of testicular spermatozoa on motility and pregnancy outcomes. Twenty testicular biopsy specimens were washed and cultured in HTF+10% SSS for 24 hours, after which the selected spermatozoa were injected. After 24 hours, the motility rate had increased in 18 of the 20 samples. The fertilization, implantation, and pregnancy rates were 58%, 20%, and 45%, respectively. The study concluded that these rates were comparable to those achieved with ejaculated spermatozoa [82].
Although various studies have explored the activation of testicular sperm movement, there is a need for further research focusing on the cryopreservation of individual sperm from a testicular source.
Clinical outcomes of the single sperm cryopreservation technique
Several studies have investigated the method of single sperm cryopreservation and have developed various vehicles for sperm freezing. Many of these studies have documented clinical pregnancies and deliveries achieved through this approach (Table 3). Clinical pregnancies were achieved using seven different sperm carriers, including empty ZP, straw, cell sleeper, cryotop, cryopiece, Sperm VD, and stripper tip.
In 1997, three clinical pregnancies were achieved using an empty ZP for freezing spermatozoa from individuals with azoospermia, extreme oligozoospermia, or oligoasthenozoospermia. Following the thawing procedure, spermatozoa were injected into five oocytes. This approach resulted in two twin deliveries, an ongoing singleton gestation, one negative pregnancy test, and one biochemical pregnancy [39].
In 2012, one clinical pregnancy was achieved using an HSV straw. This method was employed to freeze spermatozoa from eight individuals diagnosed with azoospermia and oligospermia. ICSI was subsequently performed in six cases. Fertilization was successful in all instances, and one of these resulted in a clinical pregnancy. An embryo at the 8-cell stage was transferred on day 3 to a 32-year-old woman, leading to a clinical pregnancy. Ultimately, this resulted in the birth of a healthy girl [37].
Seven clinical pregnancies were achieved using the cell sleeper for cryopreservation of small numbers of spermatozoa. In their research, Endo et al. [62] compared two carriers: the cell sleeper and the cryotop. They cryopreserved spermatozoa from individuals with NOA (two motile and 10 non-motile) in cell sleeper vials. Following oocyte retrieval and thawing, sperm were injected into the oocytes. The injected oocytes were then activated with calcium ionophore A23187 for 15 minutes. The fertilization rate was 83% (five out of six oocytes), and all of the resulting zygotes underwent cleavage. An expanded blastocyst was transferred to a 29-year-old woman on day 5, resulting in the birth of a healthy and normal boy. In contrast, no pregnancies or live births were reported using the cryotop device in this study [62].
Coetzee et al. [63] utilized a cell sleeper device to cryopreserve spermatozoa in cases of OA, NOA, and cryptozoospermia. A total of 304 retrieved spermatozoa were cryopreserved using 20 cell sleepers. Upon thawing, the recovered spermatozoa were injected into 179 mature oocytes, achieving a fertilization rate of 65.9% (118 out of 179 oocytes). Embryo transfer procedures were conducted in 10 cases, with three transfers on day 3 and seven on day 5. Of these, three did not result in pregnancy, and one was identified as a chemical pregnancy. Overall, six pregnancies were initially classified as chemical pregnancies, leading to four ongoing pregnancies [63].
Three clinical pregnancies were achieved using the cryopiece device. In a study conducted by Sun et al. [69], 126 spermatozoa from individuals diagnosed with NOA or severe oligozoospermia were cryopreserved using the cryopiece. Following the thawing process, the recovered spermatozoa were injected into mature oocytes. The fertilization rate was 73% (22 out of 30 oocytes), and 76% of the zygotes underwent cleavage. Embryo transfer procedures were performed in four cases, with each woman receiving two fresh embryos (three on day 3 and one on day 2). The outcomes included two single live births and one twin live birth. However, one of the cases did not result in pregnancy [69].
Jiang et al. [83] in 2022 utilized a SpermCD carrier made from RA-Cryopiece and a grooved petri dish. The RA-Cryopiece comprises two sheets: a flat sheet for a droplet of freezing medium and a vertical sheet for labeling and handling the device. Sperm from 35 individuals with NOA, virtual azoospermia, and cryptozoospermia were cryopreserved using a single freezing method. The thawing procedure involved 125 frozen sperm samples, from which 121 spermatozoa were successfully recovered, yielding a recovery rate of 97.1%±4.6%. Following the ICSI procedure, the rates of fertilization and good-quality embryos were 68.0%±33.2% and 24.4%±22.2%, respectively. Nineteen embryos were obtained, and eight of these were transferred to five couples, resulting in four successful deliveries [83].
Chen et al. [78] utilized a cryopiece device to cryopreserve both ejaculated and testicular spermatozoa. The recovery and motility rates were 79.1% and 29.7%, respectively, with the motility rate being higher for ejaculated sperm. The fertilization rate, embryo cleavage rate, and clinical pregnancy rates were 61.9%, 84.6%, and 43.3%, respectively, showing no significant differences between ejaculated and testicular spermatozoa. There were 10 cases of successful delivery [78].
Twenty-four clinical pregnancies were achieved using a Sperm VD for the cryopreservation of sperm from individuals with NOA. This study encompassed 44 cases. Following the thawing procedure, 180 motile spermatozoa were recovered and injected into mature oocytes. The fertilization rate was 59%, and the clinical pregnancy rate was 55% (24/44). The delivery rate was 32% (14/44), and three ongoing pregnancies were reported [70].
Testicular spermatozoa from individuals with azoospermia were cryopreserved using a Cryoplus device. Fertilization rates were evaluated by injecting either fresh or cryopreserved testicular spermatozoa during ICSI cycles. In frozen-thawed embryo transfer cycles, clinical pregnancy, implantation, miscarriage, live birth rates, and birth weights were comparable between groups using fresh and cryopreserved testicular spermatozoa. The delivery rate for the cryopreserved sperm group was 19%, while it was 23% for the fresh sperm group [67].
A novel micro-straw technique was employed for the cryopreservation of samples from individuals with severe oligozoospermia or azoospermia, sourced from testicular, epididymal, and ejaculate origins. The rates of fertilization, cleavage, and high-quality embryo development were 81.7%, 98.8%, and 53.9%, respectively. This approach resulted in 69 clinical pregnancies [84].
In 2021, a clinical pregnancy was achieved using a stripper tip for sperm cryopreservation in a 34-year-old man with cryptozoospermia. Twelve thawed spermatozoa were injected into seven mature oocytes, resulting in four zygotes. A fertilization rate of 57% was reported. Two of these zygotes were selected for fresh transfer, ultimately leading to the birth of a healthy girl [64].
Endo et al. [85] reported clinical and neonatal outcomes using both the cryotop and cell sleeper methods. The study involved 10 individuals with low sperm counts, whose samples were frozen using a single sperm vitrification technique. Following the thawing process, the cryotop group exhibited higher sperm motility and fertilization rates post-ICSI compared to the cell sleeper group. The pregnancy rate in the cryotop group was 15.4%, resulting in the birth of two healthy babies. Meanwhile, the cell sleeper group had a pregnancy rate of 14.3%, with one baby born [85].
Chen et al. [86] conducted a retrospective study using a modified micro cryo-tube for individuals with OA who underwent the TESA technique to retrieve sperm samples. The study encompassed 155 ICSI treatment cycles, with frozen sperm used in 79 cycles and fresh sperm in 76 cycles. The fertilization rate, good-quality embryo rate, and blastocyst rate were comparable between the groups using frozen and fresh sperm. Additionally, there were no significant differences in total clinical pregnancy rate (54.43% vs. 57.89%), implantation rate (46.08% vs. 49.47%), miscarriage rate (13.95% vs. 13.64%), and live birth rate (45.57% vs. 48.68%) between the groups when comparing fresh embryo transfers and the first frozen-thawed embryo transfers.
It was concluded that testicular samples with small numbers of spermatozoa can be cryopreserved in modified microtube without adverse effects on pregnancy and neonatal results in TESA-ICSI cycles [86].
Risks of contamination in the single sperm cryopreservation technique
Cryopreserved sperm samples are susceptible to contamination from various sources, including the samples themselves, which may harbor viruses and bacteria, unsealed cryo-devices, and contaminated LN2. Therefore, it is crucial to screen for infectious agents such as human immunodeficiency virus, hepatitis B and C, syphilis, and bacterial infections in individuals who opt to preserve their sperm. Additionally, to minimize the risk of transmitting infections, cryo-devices used by patients with viruses should be stored in a special tank separate from other samples. The process of preparing and washing spermatozoa using the density gradient centrifuge method, followed by resuspending the samples in an appropriate medium, can significantly reduce the presence of viruses. Utilizing sealed cryo-devices, such as the cryo-vial device mentioned in the previous section, is an effective strategy to prevent external contamination, including from impure LN2. In the single sperm cryopreservation technique, spermatozoa are selected using an ICSI needle and added to sterile cryoprotectant, which diminishes the risk of contamination compared to the conventional sperm freezing method. However, because this technique is not commonly used in clinical settings, there is insufficient data regarding sperm cross-contamination in the single sperm cryopreservation method.
Conclusions and future prospective
Freezing spermatozoa is a technology used to preserve fertility potential under specific conditions. It is crucial to select the most appropriate freezing method tailored to individuals with varying semen qualities. Samples from individuals with severe oligozoospermia and NOA contain only a small number of spermatozoa. Consequently, a suitable method should be recommended for these cases, offering hope for biological offspring in the future. This method has been developed globally for single sperm cryopreservation. In this approach, most studies have concentrated on carriers to enhance the recovery and survival rates of spermatozoa post-freezing and to reduce the time spent searching for spermatozoa. Most research in this area has focused on ejaculated sperm, although ejaculated specimens do not adequately represent the condition of testicular sperm. This technique is particularly necessary for individuals with severe oligozoospermia and those with azoospermia, where sperm are surgically retrieved from testicular tissue. This is because the characteristics of testicular sperm, such as motility, DNA fragmentation level, and plasma membrane quality, differ from those of ejaculated sperm. Therefore, future research should focus on testicular sperm, similar to the studies mentioned, to develop more accurate models. In terms of cryo-device protocols, the cryotop appears to be an effective device for maintaining optimal recovery and motility rates, especially when used in a cryo-vial to create a closed system that ensures aseptic conditions. Importantly, this carrier is commercially available. Moreover, few studies have explored the use of appropriate cryoprotectants that achieve ideal freezing outcomes, optimal cooling and warming rates, and the selection of testicular sperm prior to freezing. Thus, various aspects of this technique need further investigation to establish reliable and practical protocols. Cryopreserved sperm samples are at potential risk of cross-contamination. Contamination can occur for various reasons, including the use of non-sealed carriers, samples contaminated by viruses and bacteria, and non-sterile LN2. Therefore, individuals must be screened for microbial infections, and sealed carriers should be used to prevent contamination by LN2. Implementing this technique in a clinical setting requires more time to address all aspects of the procedure. For example, a skilled operator experienced with testicular samples and microinjection apparatuses is necessary to select viable testicular samples and freeze them using a cryotop. Therefore, more experimental studies are needed before this technique can be routinely used in clinical settings. All in all, more well-designed clinical trial studies are needed to explore clinical approaches to increase fertilization rates and live birth rates in patients with few spermatozoa.
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