In recent years, increasing environmental pollution owing to continuous industrialization and psychological stresses [1, 2] have resulted in an increase in infertility issues worldwide. Infertility in men now accounts for approximately 50% of the fertility-related problems [3]. Infertility is frequently observed in patients with cancer and in men with severe oligozoospermia or azoospermia owing to spermatogenic dysfunction. The primary challenge in clinical practice is ensuring that the patient is restored to good health after receiving the relevant treatments. However, specific treatment regimens (chemotherapy, radiotherapy, surgery) lead to adverse side effects such as impaired spermatogenesis, testosterone deficiency, or sexual dysfunction, thereby affecting the reproductive and psychological well-being of the patient [4, 5]. Most patients are concerned about the fertility-related side effects, regardless of the treatments received. Therefore, preserving fertility before treatment is critical to ensure the quality of life. In addition, etiological factors involved in male infertility factors also include congenital factors (anorchia, cryptorchidism, congenital absence of vas deferens, genetic abnormalities), acquired factors (testicular torsion, testis trauma, surgery etc.) and idiopathic forms. The etiology is still unknown in about half of the cases and it is termed “idiopathic infertility” [6]. The complete diagnostic workup is important for patients. In general, cryopreservation of sperm is a way to avoid risks.

Sperm banking was initially used for the cryopreservation of sperm [7] and was first developed to facilitate pregnancy in the 1950s [8]. Cryopreservation technologies are integral for managing male-factor infertility. Sperm donors are properly screened and quarantined before semen cryopreservation to eliminate the risk of transmission of HIV, hepatitis B and C, syphilis, and bacterial infections to the recipient. Advancements in intracytoplasmic sperm injection (ICSI) technology provide the opportunity to overcome fertility issues in patients with azoospermia (when the semen is devoid of sperm) and severe oligozoospermia. The ability to cryopreserve small numbers of spermatozoa in males afflicted with nonobstructive azoospermia using testicular sperm extraction (TESE) avoids the requirement for repeated surgery and promotes the preservation of fertility [9,10,11,12,13]. Conventional sperm preservation techniques can result in sperm loss owing to sperm adherence to the carrier vessel, harsh centrifugation, and washing procedures [14]. Thus, the conventional technique is particularly problematic in cases in which sperm numbers are low [15].

In 1997, Cohen et al. were the first to describe a novel cryopreservation technique for individual sperms using an empty zona pellucida (ZP) [16], thereby providing a theoretical and technical foundation for subsequent techniques. They introduced the concept of single-sperm freezing. Since then, a multitude of cryopreservation techniques have been developed to improve sperm counts. In this article, we review the single-sperm cryopreservation methods practiced in the last 20 years (Table 1), including the types of frozen carriers (Table 2), spermatozoa from different sources, prefreezing treatments, freezing procedures, resuscitation after freezing, and clinical applications. We performed an in-depth analysis of the advantages and disadvantages of these techniques to provide the theoretical basis for improving the single-sperm freezing technology in the future.

Repeated TESE is both costly and invasive, and it can frequently have adverse effects on the testis, including deterioration of spermatogenesis, inflammation, irreversible atrophy, and partial testicular devascularization [12]. Repetition of these procedures can be avoided through the cryopreservation of spermatozoa. Although studies [44, 45] have reported sperm survival and the births of live offspring following the cryopreservation of epididymal and testicular sperm, conventional sperm freezing techniques result in low viability [46]. Therefore, it is crucial to develop new sperm freezing methods. The loss of spermatozoa through conventional addition and removal of cryoprotectants in relatively large volumes of media can be circumvented by the insertion of the spermatozoon into an enclosed porous capsule that can be correctly visualized and handled microscopically prior to and after cryopreservation. The chosen vehicle for such a purpose is the ZP that can be used following the removal of its cellular material. The cytoplasmic components in the oocyte ZP were removed, and individual sperm was injected into the empty ZP using an ICSI needle and loaded onto a frozen straw for cryopreservation (Fig. 1a). After thawing, the recovery and fertilization rates were 73% and 50%, respectively. Spermatozoa recovered from human zonae fertilized the same proportion of oocytes as fresh fertile control spermatozoa. The fertilization rate in human zonae was marginally lower (P 58,59], and Schuster et al. [29] used cryoloops for the cryopreservation of small aliquots of spermatozoa. In this method, spermatozoa were submerged in experimental test yolk buffer supplemented with 12% v/v glycerol, followed by freezing in LN2 vapor for 5 min and snap freezing in LN2. Cryoloop-mediated “ultra-rapid freezing” of the oligo sperm is both simple and fast, and in this study, it resulted in 45% motility rate [29]. Moreover, successful vitrification of human spermatozoa has been achieved on cryoloops in the absence of cryoprotectants [14, 55] and this technique can be easily implemented. The post-thaw viability has been reported to be 52%, and sperm function was retained [14]. The DNA integrity and motility of the vitrified spermatozoa frozen on cryoloops were shown to be comparable with that observed for conventionally frozen spermatozoa [55]. To our knowledge, no pregnancies have been reported using vitrified/warmed sperm.

(a) (Adapted from Schuster et al. [29]) Cryoloops can be magnetically attached to a metal wand for easier manipulation. (b) (Adapted from Sereni et al. [34]) Microdrop in a plastic dish. (c) (Adapted from Endo et al. [35]) Cell Sleeper vial (i) is equipped with an inner tray (ii) and a screw cap (iii). (d) (Adapted from Endo et al. [35]) Cryotop comprises a fine polypropylene strip (i), plastic handle (ii), and cover straw (iii). (e) (Adapted from Peng et al. [39]) A cryoleaf bound to a handle by cotton thread is placed to a protective casing containing cotton wool at the bottom. (f) (Adapted from Sun et al. [40]) Droplets and polypropylene piece of cryopiece system. (g) (Adapted from Ma et al. [41]) The closed slice frozen carrier comprised self-made non-toxic polypropylene flakes and conventional sperm cryovials. (h) (Adapted from Berkovitz et al. [43]) The sperm vitrification device

In 2004, Desai et al. [30, 31] proposed a novel cryoloop method as an alternative to hamster zona for freezing individual sperm. In this study, the authors developed a nylon cryoloop to cryopreserve 5–10 ejaculated spermatozoa, with a total of 77 spermatozoa frozen in 10 cryoloops. After thawing, the recovery and motility rates were 68% and 73%, respectively. No difference in post-thaw motility was observed after cryopreservation on loops compared with conventional vials. These individually cryopreserved sperm were indeed capable of inducing sperm head decondensation and pronuclear formation when injected into human oocytes [30]. Each nylon cryoloop was also developed to cryopreserve 2–8 epididymal or testicular spermatozoa, and they achieved 72% recovery. Sperm motility involved minimal head motion and the cryopreserved epididymal and testicular spermatozoa (including non-motile sperms) could fertilize oocytes by using conventional ICSI methods [31].

Cryoloops can be commercially purchased and they require no additional preparation. Because no animal products are required for cryoloops and the materials are non-biological, bioethical problems are avoided, making it more favorable than the hamster ZP. However, the ventilation ports of the cryovials do act as open carriers, thereby posing a risk of LN2 leakage and cross-contamination. Frozen droplets were carried on the surface film, thereby making the storage system unstable. So far, this method has rarely been used.

Another simple method of freezing involves the replacement of the ZP by a small drop of freezing medium and its placement in a culture dish to be frozen (Fig. 3b) [32,33,34]. In 2000, Quintans et al. [32] cryopreserved 4–6 spermatozoa in a microdroplet with oil overlay in a plastic tissue culture dish. The dishes were sealed and stored in LN2, and the post-thaw recovery rate was 90–100% [32]. In 2003, Bouamama et al. [33] described single-sperm cryopreservation in culture dishes using microdroplet freezing. In this procedure, 1–100 spermatozoa were added to freezing media-microdroplet (volume: 0.5 μL) in paraffin oil, and the culture dish was sealed and stored in LN2. After thawing, complete (100%) recovery rate was observed and sperm movement rate was 50%. Sperm function was not further assessed. In the classical straw technique, no sperms were available following freezing-thawing of 1–10 sperms per straw. The cryopreservation of single sperm in the culture dish achieved high recovery rates compared with the classical straw technique [33]. In 2008, Sereni et al. [34] used a similar technique to freeze individual testicular spermatozoa. The total number of frozen spermatozoa was 431 (2–300 spermatozoa/sample) across six patient samples. Cryoprotectant was added to the culture dish. Before freezing, the sperm motility rate was 3.5%, and after thawing, 67% of motile sperm retained their motility. The sperm recovery rate was 100%. A total of 51 oocytes were treated across the six study samples, and in two out of six cases, motile spermatozoa were injected. The fertilization rate was 18% [34].

This method is simple and easy to control, but polystyrene culture dishes with microdroplets are problematic because their size and shape make them more difficult to store for a long term in liquid nitrogen and they cannot be sealed to create a closed system. Therefore, the risk of potential cross-contamination may increase. To date, the clinical application of the cryopreservation of individual sperm in the culture dish remains limited.

There remains no consensus regarding the ideal carrier for the cryopreservation of individual or small quantities of spermatozoa for clinical purposes [15]. In 2012, Endo et al. [35, 36] cryopreserved small numbers of spermatozoa using the Cell Sleeper, which is a closed system. The Cell Sleeper is a vial-based cell-cryopreservation container equipped with an inner tray (Fig. 3c). Individual sperm were added to a droplet (3.5 μL) on the tray. The tray was put into a vial and sealed with a screw cap. The vial was placed in LN2 vapor (− 120 °C) before exposure to sterilized LN2. The authors applied this technique clinically in one NOA patient, resulting in the birth of a healthy boy. In addition, 12 spermatozoa from testis were vitrified. After thawing, 10 were successfully retrieved and injected individually into six mature oocytes, and the fertilization rate was 83% [35]. The effect of different vitrification volumes (0.5, 1.0, and 3.5 μL) on individually vitrified spermatozoa was measured using the Cell Sleeper. The Cell Sleeper was placed 0.5 cm above the LN2 vapor for 2.5 min before exposure to sterilized LN2. After thawing, all spermatozoa were recovered and DNA integrity was maintained. The viable sperm rate was significantly higher when spermatozoa were frozen in a 3.5 μL droplet rather than in 0.5 μL (P