The most routinely used strategy for fertility preservation in pubertal and adult patients is semen cryopreservation (Sharma 2011). For adult men, semen cryopreservation before their gonadotoxic treatment has been clinically validated as an efficient method to preserve fertility using ART procedures. Live births have been reported after insemination of stored sperm even after freezing for a period of 28 years (Feldschuh et al. 2005). For semen collection, masturbation is recommended. However, some patients may not be able to collect ejaculate by masturbation due to stress or because of certain medical conditions or treatment. These patients include those who have ejaculatory dysfunction, psychogenic anejaculation, and peri-pubertal adolescents unfamiliar with masturbation. For individuals with ejaculatory dysfunction and anejaculation, penile vibratory stimulation (PVS) or electroejaculation (EEJ) methods can be employed. The optimized environment should be provided by eliminating time constraints for sample production, and the appropriate stimulating materials should be arranged. In case of adolescent patients, before their treatment clinicians are recommended to give information regarding the need for fertility preservation and they should explain all available options as early as possible (Lee et al. 2006). If the patients can produce an ejaculate, semen samples are cryopreserved in case of adolescent boys also (Daudin et al. 2015), and it is recommended that parents should not be allowed to attend the appointment for sample collection.

In addition to semen samples, testicular sperm extraction (TESE) and storage is the only method available for cancer patients (adolescent or adult) with azoospermia. The TESE procedure is a surgical intervention. This procedure is done either with local or general anesthesia. However, higher recovery rates were obtained following microsurgical techniques (Donoso et al. 2007; Colpi et al. 2009). Testicular sperm extraction has been used successfully to obtain sperm in approximately 50 % of cases of persistent azoospermia post-cancer therapy with previous failure of cryopreservation or when cryopreservation strategy has not been considered (Hsiao et al. 2011).

In prepubertal boys, when there is no evidence of presence of spermatozoa, immature testicular tissue cryopreservation method is considered as a strategy of fertility preservation. There is increasing trend in the use of testicular tissue cryopreservation before the cancer treatment as a means to preserve the fertility of prepubertal and peri-pubertal boys to 16 years of age (Wyns et al. 2011). This is an experimental approach. Since testicular tissue recovery is a surgical procedure, in order to minimize the trauma to the patient and also to minimize the risk of anesthesia in children, the surgical recovery of testicular tissue should be combined with other interventions requiring anesthesia, such as bone marrow sampling or implantation of venous ports. In order to minimize the manipulation and trauma, retrieving tissue from only one testis is suggested to minimize manipulation (Wyns et al. 2011), and the size of tissue may vary between 80 and 250 mm³ based on testicular size in the different age groups (Goede et al. 2011).

Till date, there is no optimized freezing protocol for human immature testicular tissue. Different freezing protocols have been used such as slow freezing and vitrification. Studies have shown that vitrification may be as effective as slow freezing in preserving testicular tissue (Curaba et al. 2011; Poels et al. 2013). Different cryoprotectants have been used such as ethylene glycol and sucrose (Kvist et al. 2006), DMSO (Keros et al. 2005, 2007), and also DMSO in combination with sucrose (Wyns et al. 2007, 2008; Poels et al. 2014). In order to maximize the quality and viability of human testicular tissue post-thaw, all aspects of the tissue collection and processing, the type and concentration of cryoprotectant used, as well as the cooling and warming protocols must be fully optimized. Since the reproductive potential of cryopreserved immature testicular tissue has still to be proven in humans, the technique remains experimental, and no one preservation protocol has been shown to be superior over any other published methods (Kvist et al. 2006; Keros et al. 2007; Wyns et al. 2008; Baert et al. 2013; Goossens et al. 2013; Poels et al. 2013).

Male germ cell transplantation technique was originally described in the mouse model (Brinster and Zimmermann 1994). In this method, SSC cell suspensions were infused through the efferent duct into the rete testis of sterile recipients with the successful reinstatement of spermatogenesis and finally the restoration of fertility. However, studies have shown that injection of SSC via the rete testis has proved to be a better treatment site for species such as the bovine, primate, and human because of differences in anatomy and consistency and the larger testis size (Schlatt et al. 1999; Ning et al. 2012). At present, SSC injection is considered the most promising method for fertility restoration in prepubertal cancer patients. For this purpose, SSC propagation has to be done in vitro . Studies have shown the ability of SSC propagation in several species (Schlatt et al. 1999; Honaramooz et al. 2002; Kanatsu-Shinohara et al. 2003; Aponte et al. 2008; Nobrega et al. 2010). However, recent study has demonstrated spermatogenesis in vivo after germ cell transplantation and confirmed fertilizing ability of those spermatozoa by ICSI in primates (Hermann et al. 2012). Though this study is a milestone toward restoring fertility in humans, whether epigenetic programming and stability of SSC are not compromised following cryopreservation, culture, and transplantation in humans is yet to be elucidated (Struijk et al. 2013).

Transplantation of testicular tissue fragments is an alternative strategy to the use of SSC suspensions. This approach maintains the SSCs within their natural niche, thus preserving the interactions between the germ cells and their supporting somatic cells. Nutrients and hormones from the body will reach the graft and induce spermatogenesis, and the resultant sperm can be extracted and used in ICSI procedures. Autologous transplantation of the testicular biopsy back into the testis (Van Saen et al. 2009), scrotum (Wyns et al. 2007), or ectopically under the skin (Jahnukainen et al. 2007) can however only be used to restore spermatogenesis if the presence of malignant cells can be excluded.

Spermatogenesis in a culture system provides the one of the best perspectives of fertility preservation. In other methods described in the above sections, there is a chance of reintroducing malignant cells, which can be avoided by the technique of producing sperm in vitro. Several methods have been employed to produce sperm in vitro. However, very little progress has been made in this regard. Conventional testicular cell culture was not successful in differentiating spermatogonia into mature spermatozoa. Recently three-dimensional culture systems have been developed which could successfully generate morphologically mature spermatozoa (Stukenborg et al. 2008, 2009). Major breakthrough in this regard is the production of live offspring in mouse through organ culture systems (Sato et al. 2011a, b). Nevertheless, effective spermatogenesis in vitro still remains to be established.