Sperm cryopreservation

Sperm cryopreservation was introduced to the human fertility clinic in the 1950s by Bunge and Sherman as a fertility preservation method for adolescent boys and adult men who face a high risk of infertility due to their medical treatments [65]. Moreover, Szell et al. [66] reported that sperms cryopreserved for 40 years retained fertilization function after thawing to produce a healthy offspring. A study showed that 76% of adolescents and young adults (14–40 years old) expressed the desire to have children in the future [67]. The ability to have biological children is part of the recovery path to normalcy, health, and life fulfillment for childhood cancer survivors [9, 11, 13, 68]. However, a study in the United States reported that only 51% of young cancer patients (14–40 years) were offered sperm cryopreservation, and only 24% of them actually froze sperms prior to initiation of their gonadotoxic therapies [67]. An improvement was seen in a 2005 UK study of 13- to 21-year-old adolescents and young adults, in which they were all offered sperm banking; 67% of them successfully banked their sperms [69]. The most common reason cited for failing to preserve sperms was the lack of information among both patients and medical staff [67, 69]. A study in China showed that more than 70% of cancer patients did not know about the existence of sperm banks, and more than 80% of medical staff were not educated about fertility preservation [70]. Moreover, adolescent patients have lower semen volume and sperm motility compared with adult patients [71]. These differences may or may not hinder the success of ARTs in future fertility restoration. Therefore, education on fertility preservation regarding patients who face a significant infertility risk should be broadly provided to medical staff. This allows medical practitioners to thoroughly inform patients at an early stage of their treatments, such that an appropriate plan for preserving the chance of parenthood can be clearly established.

Worldwide testicular tissue cryopreservation programs

Fertility centers around the world are cryopreserving testicular tissues for prepubertal boys with the anticipation that new reproductive technologies will be available to them in the future [22, 51–64, 72, 73]. Table 1 summarizes the efforts of 15 centers that perform testicular tissue or cell cryopreservation for patients who were facing the risk of infertility due to their diseases, medical treatments or other circumstances. Three centers reported samples that included adult patients (older than 18 years old) [22, 52, 58]. The rest included patients with the age range between 0 and 18 years old with oncology as the most common diagnosis [53–56, 59, 60, 62, 74–78]. Eleven out of 15 centers reported using dimethyl sulfoxide (DMSO) as their choice of cryoprotectant [22, 54–56, 60, 62, 74–78] and controlled slow freezing as their preferred freezing method [22, 53–56, 59, 62, 74, 76–78]. Most sites in Table 1 are cryopreserving intact pieces of testicular tissue because this preserves the options for both cell- and tissue-based therapies in the future.

Figure 1.

Spermatogenesis occurs within the ST of the testis that are connected to a common collecting reservoir in the testis space where sperms are deposited before flowing to the epididymis. The intratubular space within the ST includes the basal, adluminal, and lumen compartments. Sertoli cells (yellow cells) are the only somatic cell type that directly interacts with germ cells within the intratubular space. SSCs account for a small proportion of undifferentiated spermatogonia located on the basement membrane within the basal compartment. SSCs are responsible for self-renewing and differentiating divisions to maintain the stem cell pool and continuous sperm production process throughout a man's life. When SSCs differentiate, they undergo several transit amplifying mitotic divisions, giving rise sequentially to differentiating type A spermatogonia (dark purple cells) and differentiated type B spermatogonia (light purple cells). Differentiated type B spermatogonia then give rise to primary spermatocytes (light green cells). During meiosis I, primary spermatocytes lift off the basement membrane and pass through the blood–testis–barrier formed between Sertoli cells to enter the adluminal compartment to produce secondary spermatocytes (dark green cells). Secondary spermatocytes complete meiosis II to form round spermatids (magenta cells). Round spermatids undergo spermiogenesis to form elongating spermatids, elongated spermatids, and finally spermatozoa (gray cells). Spermatozoa are then released into the lumen. Peritubular myoid cells and peritubular macrophages are located on the outside of the basement membrane. The interstitial space is the area between STs where Leydig cells, blood vessels, and interstitial macrophages are located.

The University of Pittsburgh Medical Center (UPMC) Fertility Preservation Program and coordinated centers have cryopreserved testicular tissues for 517 patients between January 2011 and March 2022 (University of Pittsburgh IRB STUDY19020220; Coordinating Centers IRB STUDY19110083). These include patients at risk of infertility due to medical treatments for cancer, myeloablative conditioning prior to bone marrow transplantation for benign diseases (e.g., sickle cell disease, thalassemia, etc.), hormone modulating treatments for gender dysphoria, and gonadectomy for patients with differences in sexual development (Figure 3). The average age of patients was 7.6 years (standard error of the mean (SEM) = 0.22) and ranged from 3 months to 34 years old. The amount of tissue obtained by biopsy is at the discretion of the surgeons who typically recover as much tissue as possible (to maximize options for downstream use in reproduction) without compromising the function of the remaining testis (usually 10–20% of testicular volume). The average amount of tissue collected was 472.9 mg (SEM = 45.5) and ranged 10.1–9824.8 mg. Our center freezes intact pieces of testicular tissue (9–25 mm³, ∼15 mg) in a medium containing 5% DMSO and 5% serum substitute supplement (84% human serum albumin and 16% human globulins) using the method of controlled slow rate freezing described by Keros and colleagues [74]. We have found that the recovery of transplantable stem cells from cryopreserved testicular tissue pieces is better than the recovery of transplantable stem cells from cryopreserved testicular cell suspensions (data not shown). Moreover, tissue pieces of this size (9–25 mm³) are large enough to suture under the skin for testicular tissue grafting, as previously described [40].

Figure 2.

The schematic diagram of standard and experimental male fertility restoration technologies that have produced offspring in at least one mammalian species (Abbreviations: TESE, testicular sperm extraction; IUI, intrauterine insemination; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; SSC, spermatogonial stem cells; iPSCs, induced pluripotent stem cells; PGCLCs, primordial stem cell-like cells).

Table 1.

Published reports of testicular tissue cryopreservation.

Figure 3.

Testicular tissue cryopreservation of patients at the UPMC Fertility Preservation Program (March 2011–March 2022) (Abbreviation: CNS, central nervous system; BMT, bone marrow transplantation).

Counseling young patients and their families about the reproductive side effects of medical treatments and options for fertility preservation can be challenging and requires a multidisciplinary effort. However, the effort is justified because adult survivors of childhood cancers report that fertility is important to them and patients are willing to undergo experimental therapies to preserve the possibility of having a biological child [9, 12, 13, 79–82].

Spermatogonial stem cell transplantation

Mouse SSC transplantation was first reported by Brinster and colleagues in 1994 [30, 83]. These studies demonstrated that mouse SSCs were able to regenerate complete spermatogenesis when transplanted into the testes of infertile males [30, 83]. The efficiency of the regenerated spermatogenesis depends on the physiology of the recipient testes. Engraftment and initiation of spermatogenesis in neonatal testes is more robust than in adult testes [23]. Ablation of endogenous spermatogenesis by gonadotoxic treatment [25] or genetic mutation [23] reduces competition from endogenous stem cells, thus enhancing donor-derived spermatogenesis.

In addition, functional SSCs can be recovered from frozen and thawed mouse testicular cell suspension after 14 years of storage [84]. Indeed, transplantable spermatogonia have been recovered from frozen and thawed cell suspensions of several species [23–30, 83, 85–91]. Therefore, it is reasonable that testicular tissue frozen from prepubertal patients prior to gonadotoxic therapies can be thawed in the future to obtain functional SSCs for transplantation to regenerate spermatogenesis. These studies have built a strong foundation for translational medicine, so infertile adult survivors of childhood cancers who have cryopreserved testicular biopsies may have a chance to obtain biological children in the future. Autologous and homologous testicular cell transplantation was originally described over 25 years ago in mice [30, 83]. This is a robust technology that has been replicated in many mammalian species, including rats [26, 27], pigs [85, 86], goats [28, 87], bulls [88], sheep [29], dogs [89], and monkeys [24, 90–92] (Table 2). Embryos or offspring born from this technology were reported in mice [23, 25, 30, 93], rats [27], goats [28], and monkeys [24, 92] (Table 2).

Radford and colleagues reported the first autologous testicular cell transplantation in human patients in 1999 [94] and in 2003 [52]. Briefly, testicular cell suspensions were cryopreserved for a total of 12 patients with Hodgkin's disease. Seven of those patients returned to have their frozen and thawed testis cells transplanted back into their testes. The outcomes of those transplants were not reported, but the study provides insights into the motivation of men who were willing to undergo an early-stage experimental procedure for the possibility of having a biologically related child. Homologous species SSC transplantation had only been performed in mice and rats when Radford and colleagues reported the first autologous human testicular cell transplantations in 1999. The technique has now been replicated in numerous mammalian species, demonstrating safety and feasibility that may provide stronger support for translation to the human clinic (Table 2). Human studies must proceed with caution and be performed with regulatory approval in experimental human trials.

Table 2.

Stem cell- and tissue-based fertility therapies currently in the research pipeline.

Continued.

Continued.

Continued.

Continued.

Continued.

Spermatogonial stem cell culture

SSC transplantation is a robust technology that may be ready for translation to the human fertility clinic (e.g., for patients who cryopreserved immature testicular tissues with SSCs when they were young). However, based on our experiences in Pittsburgh and coordinated centers and other published reports [22, 74, 95], the amount of tissue obtained by biopsy from prepubertal patients is small (30–400 mg) and may contain a limited number of SSCs. Therefore, in vitro SSC expansion may be needed prior to transplantation to achieve robust engraftment and regeneration of spermatogenesis.

In rodents, SSCs can be maintained in long-term culture with significant expansion in number, and these SSCs retain their potential to restore spermatogenesis and fertility upon transplantation [96–101]. The success of SSC culture in rodents required the development of methods to isolate and enrich SSCs while eliminating testicular somatic cells that could rapidly overwhelm the cultures. In their initial report on mouse SSC culture, Kanatsu-Shinohara and colleagues [99] plated heterogeneous testis cells from newborn mice on gelatin-coated plates. Testicular somatic cells rapidly adhered to the plates while germ cells remained floating and could be sequentially aspirated and replated onto secondary plates to gradually remove somatic cells. In contrast, Hamra and colleagues [96] used a positive selection approach with rat pup testis cells that were plated on laminin. SSCs rapidly adhered to the laminin-coated plates and floating testicular somatic cells could be removed by aspiration. Other studies used fluorescence activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) for the cell surface marker THY1 (CD90) to enrich SSCs and reduce contaminating somatic cells prior to culture [102]. Development of a serum-free, defined medium facilitated the discovery of specific growth factors that were required for SSC maintenance and proliferation in culture. While Kanatsu-Shinohara used glial cell-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), and leukemia inhibitory factor (LIF) [99], others have shown that GDNF is necessary and sufficient to support rodent SSC expansion in culture. The addition of FGF2 and the soluble GDNF family receptor (GFRα1) can increase the rate of SSC expansion [100, 103]. Finally, feeder cells, such as mouse embryonic fibroblasts are frequently needed to maintain SSCs in culture, although feeder-free culture conditions on substrates such as laminin have been described [104]. SSC transplantation provided a definitive bioassay to confirm the presence and number of functional SSCs in culture [105].

A number of laboratories around the world have reported culturing nonhuman primate and human SSCs (hSSCs) [106–128]. Langenstroth and colleagues [128] reported on the isolation, enrichment, and short-term 11-day culture of adult marmoset SSCs. Adult marmoset testis cells were placed in regular tissue culture plates with minimal essential media alpha (MEMα) supplemented with 10% fetal bovine serum (FBS) at 35 °C [128]. No feeder cells or other tissue culture substrates (e.g., laminin) were used and no growth factors were added [128]. Similar to the experience with mouse SSC cultures, they found that when heterogeneous testis cell suspensions were placed in culture, the somatic cells rapidly overwhelmed the culture [128]. Therefore, they established a separation culture system in which floating supernatant cells were aspirated after 24 h and placed in a secondary culture dish [128]. Supernatant cells and attached cells were then cultured separately for 11 days [128]. Then, supernatant cells were collected from the supernatant cultures and attached cells were collected from the attached cultures for colonization potential quantification by primate-to-nude-mouse xenotransplantation [128]. Monkey and hSSCs do not produce complete spermatogenesis after xenotransplantation to recipient mouse testes, but they do recapitulate several functions that are unique to SSCs. They migrate and engraft the basement membrane of recipient mouse ST, proliferate to produce chains and clusters of cells with typical spermatogonial appearance (high nuclear to cytoplasmic ratio and frequently connected by intracytoplasmic bridges), and survive long term [107, 129–134]. Using primate-to -nude-mouse xenotransplantation, Langenstroth and colleagues [128] found that the 11-day supernatant culture retained about 60% of the number of colonizing spermatogonia that were originally placed in culture on day 0, and this was 8-fold higher than the colonizing activity from 11-day attached cultures.

More than 20 studies on hSSC culture methods have been published [106–126], including three with fetal or prepubertal testicular cells [76, 108, 109]. Many have used differential plating on plastic, lectin, collagen, or gelatin to enrich SSCs and reduce testicular somatic cell contamination. Others have used positive or negative FACS or MACS selection alone or in combination with differential plating. Positive selection markers have included integrin subunit alpha 6 (ITGA6), CD9, G-protein coupled receptor 125 (GPR125), expression of stage-specific embryonic antigen 4 (SSEA4), and epithelial cell adhesion molecule (EpCAM). Negative selection markers include KIT proto-oncogene (cKIT), protein tyrosine phosphatase receptor type C (PTPRC or CD45), and THY1 (reviewed in [135]). It is noteworthy that THY1 is a positive selection marker for mouse spermatogonia but a negative selection marker for transplantable human spermatogonia [102, 119, 135, 136].

Most hSSC culture studies used culture conditions similar to those originally described by Kanatsu-Shinohara and colleagues in mice, including StemPro-34 medium supplemented with various combinations of GDNF, FGF2, EGF and LIF. Some of those studies reported significant expansion of hSSC numbers in culture [76, 107, 137], while others reported a rapid decline in hSSC numbers using the same conditions [118, 120, 122, 125]. These disparate outcomes may be explained by differences in starting cell populations, culture conditions, and approaches that were employed to identify and quantify hSSCs in culture (ranging from counting total cells in culture to quantifying xenotransplantation colonizing events). Therefore, there is no consensus “best method” for culturing hSSCs that has been independently replicated in different laboratories and no consensus on best methods to identify and quantify bona fide hSSCs in culture. Nonetheless, a number of research groups have reported hSSC survival and expansion for periods ranging from 1 week to 6 months [106–126]. While the results for hSSC culture are promising, they are challenged by the inability to test the full spermatogenic potential of these cells in xenotransplantation; and autologous or homologous transplantation in humans is currently not feasible.

De novo testicular morphogenesis

De novo testicular morphogenesis is defined as the reconstruction of testicular architecture and spermatogenesis from a heterogenous testicular cell suspension to replicate the in vivo cell-to-cell interactions and functions of germ cells and somatic cells. De novo testicular morphogenesis can be performed entirely in culture or by subcutaneous xenotransplantation into animal recipients (Figure 2, Table 2). This approach can be used to dissect, manipulate, or modify the component parts of a complex tissue to learn the impact on testicular cell development or function. The approach may also be used to eliminate unwanted and unnecessary components, such as contaminating malignant cells, which could disrupt development or be unsafe for therapy. Cells from a single testis can be disaggregated to produce many organoids that can be used for high throughput toxicity testing or novel compound screening. De novo testicular morphogenesis has been described in rodents as well as farm animals, nonhuman primates, and humans [33, 138–149].

Methods for de novo testicular morphogenesis, in vitro, were first described in rodents (Figure 2, Table 2). Yokonishi and colleagues [150] pioneered the technique by producing organoids from neonatal mouse testis cells in V-bottomed 96-well plates. After 2 days, organoids were collected and cultured at the gas–liquid interface by placing on agarose gel islands half-soaked in culture medium that was supplemented with knockout serum replacement (KSR). Seminiferous tubule-like structures were apparent by two weeks in culture. Sertoli cells and germ cells were arranged inside ST and Leydig cells were located in the space between the de novo-formed tubules. Spermatocytes and spermatids were observed by 60 days in culture. Zhang and colleagues used a variation of the gas–liquid interface culture system; testicular cell/collagen aggregates were deposited on a nucleopore filter that was then floated on culture medium [151]. Seminiferous tubule-like structures contained spermatogonia and spermatocytes [151]. Sertoli cell differentiation occurred only when KSR was included in the medium. Baert and colleagues [152] seeded neonatal mouse testis cells on an alginate lattice scaffold alone or embedded with testicular interstitial cells. The testicular cells formed organoids within the lattice but did not self-organize into ST [152]. Nonetheless germ cell differentiation to the elongated spermatid stage was reported on both the cell-free and cell-laden scaffold [152]. Donor age is a factor. Alves-Lopes and colleagues [139] reported seminiferous tubule formation from 5- to 8-day-old and 20-day-old rat pup testis cells sandwiched in a three-layer Matrigel culture system but not from 60-day old rat testicular cells. To our knowledge, no offspring have been produced from any mouse in vitro de novo testicular morphogenesis approach.

Huleihel and coworkers [153] cultured heterogeneous testicular cell suspensions from prepubertal rhesus macaques in 3-dimensional (3D) methylcellulose or soft agar culture systems. Cells aggregated to produce colonies, including CAMP-responsive element modulator-positive (CREM)+ and acrosin (ACR) + spermatids, but did not reconstitute ST [153]. Sakib and colleagues [149] reported a microwell aggregation approach to producing 3D testicular organoids from neonatal or prepubertal testicular cell suspensions of mice, pigs, macaques, and humans. In all species, the organoids exhibited an inverted seminiferous tubule architecture with germ cells and Sertoli cells on the outside and other somatic cells on the inside of the tubules [149].

Two studies reported adult and pubertal human testicular organoids formation in human testicular extracellular matrix (htECM) [146, 147]. Pendergraft and colleagues [146] used a hanging-drop method to induce organoid formation from cultured adult human spermatogonia mixed with immortalized human Sertoli and Leydig cells suspended in a hydro-gel of htECM. Baert and colleagues [147] seeded adult or pubertal heterogeneous human testis cell suspensions onto a 3-dimensional htECM scaffold that retained the tubular architecture. Both approaches led to the production of organoids including germ cells and somatic cells but neither approach produced seminiferous tubule-like structures [146, 147]. Furthermore, Baert and colleagues [147] reported that human testis cell organoids formed with or without the benefit of the scaffold. Similar to results in rodents, age appears to be a factor in the formation of seminiferous tubule-like structures. Oliver and colleagues [154] sandwiched fresh human fetal testicular cells in a three-layer Matrigel gradient system. Seminiferous cord-like structures appeared within 7 days with a clear basement membrane separating Sertoli cells on the inside from peritubular myoid cells and Leydig cells on the outside [154]. However, although DEAD-box helicase 4 (DDX4 or VASA) + prospermatogonia were observed in the preculture 8-week-old fetal testicular tissues, they disappeared over 7 and 14 days of culture [154]. In summary, fetal or prepubertal testis cell suspensions (mouse, rat, or human) can self-organize to produce seminiferous cord- or seminiferous tubule-like structures that do not appear when pubertal or adult cells are used [154]. Meiotic or postmeiotic cells were occasionally observed, and this does not appear to depend on the formation of tubules [154]. To our knowledge, the function of haploid cells produced by in vitro de novo testicular morphogenesis approaches has not been tested [154].

The second de novo morphogenesis approach is to inject the heterogenous testicular cell suspension under the skin of a mouse host (Figure 2, Table 2). Pelleted testicular cells from mice [33, 34], rats [33, 138, 155, 156], pigs [33, 141, 142], sheep [143], and monkeys [144] have been transplanted into mouse hosts and self-organized in vivo to form seminiferous tubule-like structures (Table 2). Studies from Kita et al. [33] and Zhang et al. [151] confirmed that cells from different sources could be mixed to test their importance in tubule formation or spermatogenesis and that haploid mouse cells produced in de novo reconstructed tubules were competent to fertilize and produce live offspring. Similar seminiferous tubule-like structures containing spermatogenesis with haploid germ cells have been reported from pig [141, 142] and sheep [143] by transplanting neonatal testis cells under the skin of immune-compromised mice. Spermatogenic efficiency was improved by addition of Matrigel but not by introduction of an enriched germ cell fraction into a heterogeneous pig testis cell suspension [142]. The fertilization potential of pig and sheep spermatids in de novo-formed tubules was not tested in those studies [141–143]. Neonatal marmoset monkey testis cells self-organized into seminiferous cord-like structures without lumens after subcutaneous xenotransplantation to mouse hosts [144]. The cords contained Sertoli cells and germ cells, but complete spermatogenesis was not observed and there was no evidence of meiotic or postmeiotic cells [144]. Cords were not formed from juvenile or adult marmoset testis cells [144]. To our knowledge, in vivo de novo testicular morphogenesis has not been attempted with human cells. If human testis cells can produce results similar to those reported above for mice, pigs and sheep, de novo-derived haploid germ cells could theoretically be used for clinical applications. This may raise concerns about xenobiotics that could be circumvented if de novo testicular morphogenesis from humans can be achieved in organoid cultures described above. However, this has not occurred yet.

Testicular tissue grafting

For testicular tissue grafting, SSCs are maintained within intact pieces of tissue, preserving the original architecture of the ST, extracellular matrix, and testicular somatic cells with associated paracrine signaling. Tissue grafting is performed by implanting testicular tissue pieces at an orthotopic (e.g., scrotum) or ectopic (e.g., skin) site in the recipient animals (autologous, allogenic, or xenogeneic recipients) with or without exogenous matrices or hormones [35–41, 75, 143, 157–188] (Table 2). The recipients are usually castrated to stimulate the gonadotropic hormone secretion to promote maturation of the grafted tissues [35–41, 75, 143, 157–188]. The overall goal is not to restore fertility since grafted testicular tissues are not connected to the excurrent duct system. The goal is to mature the grafted tissue to produce sperm that can be used to fertilize eggs by ICSI and produce offspring. Honaramooz and colleagues [85] reported that immature testicular tissues from mice, pigs, and goats could be transplanted under the back skin of immune-deficient nude mice and matured to produce sperm. Two groups later reported that sperms from fresh [35] or cryopreserved [37] immature mouse testicular tissue grafts were competent to fertilize mouse eggs and produce offspring. Immature testicular tissue grafting (autologous or allogeneic) or xenografting is a robust technology that has been replicated in mice [35–37, 157, 158, 160], hamsters [157], rabbits [37], dogs [165], cats [162–164], horses [168, 176], pigs [36, 38, 166, 167, 169, 170], bulls [166, 171–175], goats [36], sheep [143, 166], and monkeys [39, 41, 168, 177–179] with the production of spermatids or spermatozoa (Table 2). Function of graft-derived sperm has been tested by fertilization, and pre-implantation embryo development up to blastocyst stage and/or production of viable offspring were reported in mice [35–37], rabbits [37], pigs [38], goats [36], and rhesus macaques [39–41] (Table 2). Only immature (fetal, neonatal, pre-pubertal) testicular tissues were able to survive and undergo complete spermatogenesis; whereas adult tissues gradually lost differentiated germ cells and degenerated [75, 158, 161, 163–165, 168, 175, 176] (Table 2).

In addition, the efficiency of inducing complete spermatogenesis varies widely among species. In studies of pigs, goats, sheep, and monkeys, at least 50% of tubules contained complete spermatogenesis post xenografting [36, 40, 166]. However, only less than 10% of tubules contained complete spermatogenesis in horse and bull xenografts [171, 173, 174, 176]. Graft retrieval time also greatly affects spermatogenesis efficiency. In mice, grafted tissues recovered after 120 days yielded about four times more tubules with complete spermatogenesis compared with grafts recovered before 120 days [158]. Another mouse study also showed increasing percentage of tubules containing complete spermatogenesis from ∼20 to ∼55% in grafts retrieved at later timepoints between 4–16 weeks post-grafting [36]. However, the number of surviving spermatogonia in pre-pubertal human testicular tissues xenografted to mice decreased over time and complete spermatogenesis was not observed [158]. Honaramooz et al. [189] reported that the fertilization potential of sperm produced in the pig-to-mouse xenografts decreased over time and suggested that this might be due to lack of excurrent ducts to remove older sperm.

Live birth outcomes from autologous, allogeneic, or xenograft experiments suggest that these approaches may have application for maturing prepubertal testicular tissues that were frozen for patients prior to gonadotoxic therapies (Table 2). Furthermore, xenografting of human tissues to mice or other animal hosts may circumvent the risk of reintroducing malignant cells to patient survivors. Spermatogonia survival from fresh and cryopreserved prepubertal grafts were reported at various timepoints, ranging from days to months [75, 158, 159, 182, 183, 187, 190]. The longest observation time was 9 months with orthotopic xenografts retaining spermatogonia survival better than the ectopic grafts [75, 183]. A few studies have reported the initiation of spermatogenesis up to early spermatocytes from fresh or cryopreserved immature human orthotopic or ectopic xenografts [184–186, 188]. However, complete spermatogenesis from xenografting human testicular tissues into mice has not yet been achieved [75, 158, 159, 180–188]. Recipient choice may be a factor that should be tested in future studies, including porcine or nonhuman primate species, which are more closely related to humans.