**1. Introduction**

208 Current Frontiers in Cryobiology

Scott RT Jr, Hofmann GE. Prognostic assessment of ovarian reserve. *Fertility and Sterility*

Shaw JM, Bowles S, Koopman P, Wood EC, Trounson OA. Fresh and cryopreserved ovarian

Singer T, Barad DH, Weghofer A, Gleicher N. Correlation of antimüllerian hormone and

Smith GD, Serafini PC, Fioravanti J, Yadid I, Coslovsky M, Hassun P, et al. Prospective

Sowers MR, Eyvazzadeh AD, McConnell D, Yosef M, Jannausch ML, Zhang D, et al. Anti-

Stachecki JJ, Cohen J, Willadsen SM. Cryopreservation of unfertilized mouse oocytes: the

Tan SL, Doyle P, Campbell S, Beral V, Rizk B, Brinsden P, et al. Obstetric outcome of in vitro

Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer

Wallace WHB, Kelsey TW. Human ovarian reserve from conception to the menopause. *PLoS* 

Wennerholm UB, Söderström-Anttila V, Bergh C, Aittomäki K, Hazekamp J, Nygren KG, et

Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to −196 degrees

Willett LL, Wellons MF, Hartig JR, Roenigk L, Panda M, Dearinger AT, et al. Do women

Zou K, Yuan Z, Yang Z, Luo H, Sun K, Zhou L, et al. Production of offspring from a

tissue samples from donors with lymphoma transmit the cancer to graft recipients.

baseline follicle-stimulating hormone levels. *Fertility and Sterility* 2009; 91(6):2616-9.

randomized comparison of human oocyte cryopreservation with slow-rate freezing

Mullerian hormone and inhibin-B in the definition of ovarian aging and the menopause transition. *Journal of Clinical Endocrinology and Metabolism* 2008;

effect of replacing sodium with choline in the freezing medium. *Cryobiology* 1997;

fertilization pregnancies compared with normally conceived pregnancies. *American* 

al. Children born after cryopreservation of embryos or oocytes: a systematic review

residents delay childbearing due to perceived career threats? *Academic Medicine*

germline stem cell line derived from neonatal ovaries. *Nature Cell Biology* 2009;

https://www.sartcorsonline.com/rptCSR\_PublicMultYear.aspx?ClinicPKID=0.

1995; 63(1):1-11.

*Human Reproduction* 1996; 11(8):1668 – 73.

Society for Assisted Reproductive Technology

Accessed August 2011.

*ONE* 2010; 5(1):e8772.

2010; 85(4):640-6.

11(5):631-6.

93(9):3478-83.

37:346-54.

or vitrification. *Fertility and Sterility* 2010; 94(6):2088-95.

*Journal of Obstetrics and Gynecology* 1992; 167(3):778-84.

of an eight-cell embryo. *Nature* 1983; 305(5936):707-9.

of outcome data. *Human Reproduction* 2009; 24(9):2158-72.

and −269 degrees C. *Science* 1972; 178(59):411–4.

Immediate use of freshly collected testis tissue in diagnosis or in reproductive technologies is not always possible or desirable. Therefore, the ability to properly preserve the tissue for varying intervals is an essential step for maximizing the use of the source tissue. Preservation of gametes and gonads is a topic of interest in reproductive biomedicine. Other chapters in this book have elegantly covered current knowledge on the cryopreservation of sperm, oocytes, and early embryos as well as ovarian tissue, among other cells and tissues. However, the main objective of this chapter is to provide a focused discussion of the importance, methodology, potential applications, and limitations for applying cryopreservation to testicular tissue.

Cryopreservation of human testis tissue obtained by biopsy can be used as a potential future source of sperm. For adult cancer survivors whose only source of sperm is the testis parenchyma, cryopreservation of testis biopsies may be the only option remaining if they prefer to father their own biological progeny. This will require detection of sperm in frozenthawed cell suspensions of testis tissues for use in intra-cytoplasmic sperm injection (ICSI). More importantly, cryopreservation of immature testis biopsies can offer a unique alternative for prepubertal boys undergoing gonadotoxic cancer treatments, whose only future source of spermatogenesis (*i.e.,* spermatogonial stem cells) is at risk. These strategies can also be applied to genetic preservation of endangered species/breeds through the cryopreservation of testis tissue from young animals that die prior to reaching maturity. Restoring the developmental potential of testis tissue after cryopreservation may also provide insight into proper banking of other immature tissues.

The effects of cryoprotectant concentration and cooling rate are not similar among tissues or species. Therefore, we will discuss the basis for a number of successfully applied strategies and workable protocols that have been used to effectively cryopreserve testis tissue in various species.

In summary, this chapter provides an overview of the current literature and contributions by the author and colleagues on cryopreservation of testicular tissue and its potential applications in experimental and clinical settings in reproduction medicine.

#### **1.1 Developmental changes in the structure of testis tissue**

In mammals at birth, all organs/tissues required for sustaining life display functional competence and histological similarity to those in mature individuals. Reproductive tissues,

Cryopreservation of Testicular Tissue 211

As highlighted in **Figure 1**, the cellular composition of a typical mature testis is quite different from that of an immature testis; for instance, the latter hosts a considerably higher number of differentiating germ cells, known to be more sensitive to manipulations and temperature changes (Franca et al., 2000; Frankenhuis et al., 1981). Consequently, the tissue composition of the testis changes during development and proportionally larger volumes of the mature testis are occupied by the seminiferous tubules. Therefore, the developmental state of the testis affects the tissue composition and has important implications for its

**2. Rationale for preserving testis tissue from human and animal donors** 

for future use in emerging reproductive technologies.

**3. Methodology for cryopreservation of testicular tissue** 

Preservation of testicular tissue could be pursued for multiple reasons. An estimated 1 in 650 children will be diagnosed with malignancies by age 16, of which 80% will be cured (Stiller et al., 2006). However, irreversible gonadotoxic insult of chemo/radio-therapy remains a major concern in the use of these life-saving treatments, which render about 20% of boys sterile in the long term, likely as a result of the loss of spermatogonial stem cells (Apperley & Reddy, 1995; Naysmith et al., 1998). With improved treatments, the proportion of childhood cancer survivors is expected to increase, posing an even greater challenge for reproductive medicine and oncologist practitioners in the decades to come. A routine strategy to offer preservation of future fertility for adult men undergoing sterilizing cytotoxic treatments is to freeze semen samples; however, some men may be azoospermic at the time of cancer diagnosis. More critically, in pre-adolescent boys, collection of sperm is not possible because spermatogenesis has not yet started. In such cases, cryopreservation of testicular biopsies collected prior to the start of the treatment may provide a potential source

In animal conservation, preventing the permanent loss of a male's potential contribution to the genetic variability of a rare or endangered species/breed is feasible through the collection of sperm before or even shortly after death by retrieval from the ejaculate, epididymis, or testes, which is then cryopreserved for future use in assisted reproduction (Gañán et al., 2009; Kishikawa et al., 1999; Martínez et al., 2008; Maksudov et al., 2009). Preservation of sperm, however, is not an option when young offspring die prior to reaching sexual maturity. Cloning has been used for a number of species and especially where the goal has been to produce a genetically exact replica of an individual animal. However, development of cloning for a new species is technically demanding and costly but, more importantly, does not immediately provide the genetic diversity that would otherwise be offered by gametes. In such cases, cryopreservation of testicular tissue can again provide an alternative strategy for *ex situ* generation of sperm from these neonatal/immature animals for use in reproductive technologies (Abbasi & Honaramooz,

A number of cryogenic strategies have been developed to serve as a means to maintain functional properties of the preserved cells and tissues. Apparently, the first successful cryopreservation of cells was carried out by accidental freezing of fowl sperm in diluents containing glycerol (Polge et al., 1949). Later, cryopreservation of bull sperm using glycerol (Polge & Lovelock, 1952; Smith, 1961), set the stage for revolutionizing the bovine artificial

cryopreservation.

2011).

on the other hand, attain maturity much later and only when other bodily requirements of parenthood are also in place. Therefore, in discussion of testis tissue cryopreservation, the developmental stage of the tissue is an important factor to be considered. For instance, for cryopreservation of testis tissue from an immature individual, the differing tissue texture and need for maintaining its future developmental potential are to be taken into account.

Embryonic development of the testis begins when the SRY gene in a genetic male is expressed, driving the transformation of an indifferent early gonad to a testis. This in turn causes differentiation of Sertoli cells to enclose the fetal germ cells, to mark the differentiation of primordial germ cells into gonocytes, and results in the formation of seminiferous cords. In humans, this process begins at 7-9 wk gestation (Wilhelm et al., 2007) and is immediately followed by differentiation of fetal Leydig cells, located in the interstitial spaces between the seminiferous cords, to allow production of testosterone thus causing masculinization of the foetus (Scott et al., 2009).

In early postnatal humans and most domestic species, the testis still contains interstitial tissue and seminiferous cords, with gonocytes as the only type of germ cells present (Franca et al., 2000). Initially, gonocytes reside in the centre of the seminiferous cords (**Fig. 1A**), but they gradually migrate toward the periphery of the cords and remain in close contact with Sertoli cells and peritubular myoid cells at the basement membrane to form the stem cell niche (Pelliniemi, 1975; Van Straaten & Wensing, 1977). Gonocytes eventually give rise to spermatogonial stem cells (SSCs), which have the ability to both self-renew and give rise to differentiating germ cells. Postnatal development of the testis also involves proliferation and maturation of Sertoli cells to transform testicular cords into seminiferous tubules (containing a lumen), followed by sequential division and differentiation of germ cells to generate sperm (**Fig. 1B**) (Hughes & Varley, 1980; Ryu et al., 2004). Therefore, SSCs form the foundation of spermatogenesis and are responsible for a lifetime supply of sperm.

Fig. 1. Histological differences between an immature and a mature testis tissue. In the immature testis (**A**), seminiferous cords contain only one type of germ cells - gonocytes (arrow heads). In the mature testis (**B**), on the other hand, seminiferous tubules are much larger in diameter, contain a lumen, and a repertoire of germ cell types. The composition and extent of the interstitial tissue also changes over development. These differences may affect the response of the tissue to a given cryopreservation protocol even within the same donor species. Scale bar = 100 mg. Images modified from Abbasi & Honaramooz (2011).

on the other hand, attain maturity much later and only when other bodily requirements of parenthood are also in place. Therefore, in discussion of testis tissue cryopreservation, the developmental stage of the tissue is an important factor to be considered. For instance, for cryopreservation of testis tissue from an immature individual, the differing tissue texture and need for maintaining its future developmental potential are to be taken into account.

Embryonic development of the testis begins when the SRY gene in a genetic male is expressed, driving the transformation of an indifferent early gonad to a testis. This in turn causes differentiation of Sertoli cells to enclose the fetal germ cells, to mark the differentiation of primordial germ cells into gonocytes, and results in the formation of seminiferous cords. In humans, this process begins at 7-9 wk gestation (Wilhelm et al., 2007) and is immediately followed by differentiation of fetal Leydig cells, located in the interstitial spaces between the seminiferous cords, to allow production of testosterone thus causing

In early postnatal humans and most domestic species, the testis still contains interstitial tissue and seminiferous cords, with gonocytes as the only type of germ cells present (Franca et al., 2000). Initially, gonocytes reside in the centre of the seminiferous cords (**Fig. 1A**), but they gradually migrate toward the periphery of the cords and remain in close contact with Sertoli cells and peritubular myoid cells at the basement membrane to form the stem cell niche (Pelliniemi, 1975; Van Straaten & Wensing, 1977). Gonocytes eventually give rise to spermatogonial stem cells (SSCs), which have the ability to both self-renew and give rise to differentiating germ cells. Postnatal development of the testis also involves proliferation and maturation of Sertoli cells to transform testicular cords into seminiferous tubules (containing a lumen), followed by sequential division and differentiation of germ cells to generate sperm (**Fig. 1B**) (Hughes & Varley, 1980; Ryu et al., 2004). Therefore, SSCs form the

foundation of spermatogenesis and are responsible for a lifetime supply of sperm.

Fig. 1. Histological differences between an immature and a mature testis tissue. In the immature testis (**A**), seminiferous cords contain only one type of germ cells - gonocytes (arrow heads). In the mature testis (**B**), on the other hand, seminiferous tubules are much larger in diameter, contain a lumen, and a repertoire of germ cell types. The composition and extent of the interstitial tissue also changes over development. These differences may affect the response of the tissue to a given cryopreservation protocol even within the same donor species. Scale bar = 100 mg. Images modified from Abbasi & Honaramooz (2011).

masculinization of the foetus (Scott et al., 2009).

A B

As highlighted in **Figure 1**, the cellular composition of a typical mature testis is quite different from that of an immature testis; for instance, the latter hosts a considerably higher number of differentiating germ cells, known to be more sensitive to manipulations and temperature changes (Franca et al., 2000; Frankenhuis et al., 1981). Consequently, the tissue composition of the testis changes during development and proportionally larger volumes of the mature testis are occupied by the seminiferous tubules. Therefore, the developmental state of the testis affects the tissue composition and has important implications for its cryopreservation.
