**4.2 Testis tissue (xeno)grafting**

Another potential strategy for the use of cryopreserved testis tissue is represented by testis tissue xenografting. Grafting of both fresh and cryopreserved testis tissue fragments from

Cryopreservation of Testicular Tissue 221

or endangered ungulates can be used for this application, and when allowed to develop in the host mouse, lead to full spermatogenesis (Abbasi & Honaramooz, 2011). Therefore, testis tissue xenografting can be used as unique solution for genetic conservation of immature males by producing sperm from these otherwise resource-less donors in xenografts,

However, xenografting of human gonadal tissues into animals to harvest the resultant gametes for use in IVF for humans is prohibited in Canada, and possibly in other countries, due to the potentially serious risk of animal viral transmission or contamination with animal genetics. Nevertheless, the promising results from animal research suggest a potential hope for future use of cryopreserved testis biopsies from pre-adolescent boys to be grafted back to the individual; whether this technique can be used to produce viable sperm for future use from prepubertal boys undergoing gonadotoxic treatments remains to be determined. However, the same safety risks as for autologous germ cell transplantation exist and require

In theory, cryopreserved testicular tissues can also be used for *in vitro* induction of differentiated germ cells and ideally production of sperm or spermatids to be used for ICSI. If successful, this approach can circumvent the potential risk of reintroducing cancer cells into post-recovery patients. Many labs have experimented with the idea, and some have had success with maturation of later stages of human spermatogenesis (but not from SSCs), including live births (Tesarki et al., 1999). Availability of a culture system to support complete *in vitro* spermatogenesis from the SSC stage was, however, elusive until very recently when it was reported that all spermatogenic lineage cells including fertilizationcompetent sperm could be produced from neonatal mouse testes maintained exclusively in a culture system (Shinohara et al., 2011). This is a very promising step, indicating that

similar results may be achievable in future using immature human testis biopsies.

Since the first reports of successful germ cell transplantation and xenografting of testis tissue raised new interest in this field, several promising cryopreservation protocols have been introduced. Perhaps not surprisingly, the results differed and at times conflicted depending on the tissue donor species/developmental stage. These first reports of cryopreservation of pig and mouse testis tissues were based on DMSO-based slow freezing protocols originally developed for isolated testis cells or for ovarian tissue, respectively (Honaramooz et al., 2002a; Schlatt et al., 2002). Later, other detailed studies comparing multiple protocols showed high cell viability with programmed slow-freezing of immature mouse testis tissue using 1.5M DMSO as a cryoprotectant (Milazzo et al., 2008; Traverse et al., 2011). DMSO has also been found to be a more suitable cryoprotective agent than ethylene glycol for immature mouse and rat testis tissue (Goossens et al., 2008; Jezek, 2001). Shinohara et al. (2002) reported the birth of mouse offspring from sperm retrieved from cryopreserved pre-pubertal testis tissue with DMSO after transplantation under tunica albuginea of the recipient testes (Shinohara et al., 2002). Similar results were obtained using primate testis tissue, where 1.4M (but not 0.7M) DMSO was able to protect some of the developmental potential of grafts from rhesus monkeys (Jahnukainen et al., 2007) but the 0.7M DMSO protocol was successful for cryopreservation of

followed by extraction and cryopreservation of sperm for future use in ICSI (**Fig. 5**).

addressing before such an option can be offered clinically.

**5. Current trends in testis tissue cryopreservation** 

**4.3** *In vitro* **maturation of germ cells** 

donors of different species under the back skin of recipient mice results in the production of functional sperm (Honaramooz et al., 2002a). The approach has especially been successful using neonatal/immature donors (**Fig. 5**), from laboratory animals to domestic animals, primates, and even humans (Honaramooz et al., 2002a, 2004, 2008; Schlatt et al., 2002; Oatley et al., 2004; Snedaker et al., 2004; Rathi et al., 2005, 2006; Arregui et al., 2008; Abrishami et al., 2010b).

Fig. 5. Schematic representation of testis tissue (xeno)grafting from an immature donor individual into the back skin of a host mouse. The testes are collected from a donor animal (**A**), which could include post-mortem testis recovery from a recently deceased newborn animal of an endangered species. The testis tissue (**B**) could be cryopreserved (**C**) until grafting. At the time of grafting, tissue fragments of ~0.5 mm3 (**D**) are prepared and the fragments are grafted subcutaneously into an immunodeficient host mouse (**E**). When given enough time, the grafts can grow in size (**F**) and undergo development, leading to the production of complete spermatogenesis, including fertilization-competent sperm (**G**). The sperm can then be extracted from the grafts and used in intracytoplasmic sperm injection (ICSI) (**H**), which after embryo transfer can potentially lead to birth of progeny (**I**).

The sperm recovered from such grafts, including those from primates, have been shown to be fertilization competent after ICSI (Honaramooz et al., 2002a, 2004, 2008), leading to the birth of healthy progeny (Schlatt et al., 2003; Nakai et al., 2010). We recently showed that testes recovered post-mortem from newborn bison calves, as a model for closely-related rare

donors of different species under the back skin of recipient mice results in the production of functional sperm (Honaramooz et al., 2002a). The approach has especially been successful using neonatal/immature donors (**Fig. 5**), from laboratory animals to domestic animals, primates, and even humans (Honaramooz et al., 2002a, 2004, 2008; Schlatt et al., 2002; Oatley et al., 2004; Snedaker et al., 2004; Rathi et al., 2005, 2006; Arregui et al., 2008; Abrishami et al.,

**I**

Fig. 5. Schematic representation of testis tissue (xeno)grafting from an immature donor individual into the back skin of a host mouse. The testes are collected from a donor animal (**A**), which could include post-mortem testis recovery from a recently deceased newborn animal of an endangered species. The testis tissue (**B**) could be cryopreserved (**C**) until grafting. At the time of grafting, tissue fragments of ~0.5 mm3 (**D**) are prepared and the fragments are grafted subcutaneously into an immunodeficient host mouse (**E**). When given enough time, the grafts can grow in size (**F**) and undergo development, leading to the production of complete spermatogenesis, including fertilization-competent sperm (**G**). The sperm can then be extracted from the grafts and used in intracytoplasmic sperm injection (ICSI) (**H**), which after embryo transfer can potentially lead to birth of progeny (**I**).

**F**

**E**

**C**

**D**

**A**

**B**

The sperm recovered from such grafts, including those from primates, have been shown to be fertilization competent after ICSI (Honaramooz et al., 2002a, 2004, 2008), leading to the birth of healthy progeny (Schlatt et al., 2003; Nakai et al., 2010). We recently showed that testes recovered post-mortem from newborn bison calves, as a model for closely-related rare

2010b).

**G**

**H**

or endangered ungulates can be used for this application, and when allowed to develop in the host mouse, lead to full spermatogenesis (Abbasi & Honaramooz, 2011). Therefore, testis tissue xenografting can be used as unique solution for genetic conservation of immature males by producing sperm from these otherwise resource-less donors in xenografts, followed by extraction and cryopreservation of sperm for future use in ICSI (**Fig. 5**).

However, xenografting of human gonadal tissues into animals to harvest the resultant gametes for use in IVF for humans is prohibited in Canada, and possibly in other countries, due to the potentially serious risk of animal viral transmission or contamination with animal genetics. Nevertheless, the promising results from animal research suggest a potential hope for future use of cryopreserved testis biopsies from pre-adolescent boys to be grafted back to the individual; whether this technique can be used to produce viable sperm for future use from prepubertal boys undergoing gonadotoxic treatments remains to be determined. However, the same safety risks as for autologous germ cell transplantation exist and require addressing before such an option can be offered clinically.
