Spermatogonial stem cells An update on spermatogonial stem cell banking and transplantation.

Introduction:
Approximately one in 600 children will develop cancer before the age of 15 years. Thanks to recent advances in chemo- and radiotherapy, cure rates of childhood cancers are as high as 80%. It has been estimated that by 2010, one in 250 young adults may be long-term survivors of childhood cancer. Unfortunately, however, as a consequence of their disease and treatment, these long-term survivors now face significant adverse effects, which they must deal with for the rest of their lives. One such implication of an aggressive cancer treatment is that patients may become infertile as a result of chemo- and radiotherapy. Chemotherapy will indiscriminately destroy diseased as well as healthy cells, so that spermatogenic failure and infertility may occur. One subgroup of children, especially, who need aggressive treatment including chemo- and radiotherapy and bone marrow transplantation are at risk of life-long sterility. It is obvious that prevention of sterility requires special attention in reproductive medicine. For an adult, sperm banking before treatment may circumvent this adverse effect. However, pre-pubertal patients cannot benefit from this option because of the lack of complete spermatogenesis in their testes1. In female patients, preventive cryopreservation of ovarian tissue has been carried out and has recently led to the first reproductive success2. In analogy with this strategy, the storage of pre-pubertal testicular tissue is a potential solution. At this stage, the testicular stem cell transplantation3 seems the most promising method. However, before such an application can be applied, both the safety and the efficiency of the procedure have to be assured.

Material and Methods:
Donor cells were obtained from six-day-old male C57Bl x WBRej F1 mice by a two-step digestion protocol. Two - 10 µl from a 30 milj/ml testicular cell suspension was injected through the efferent duct in the rete testis of 4-6 week old genetically sterile W/Wv mice (Figure 1). At least five months after transplantation, the histology of the testis tissue as well as the vitality, the concentration, the motility, the individual sperm movement and the hyperactivity of the spermatozoa were evaluated and compared to data obtained from fertile mice.
Transplanted male mice were mated with females in order to evaluate in-vivo conception. Foetuses were evaluated on the 17th gestational day. The length, weight and morphological age were compared to control mouse foetuses. The live born offspring were then investigated for their reproductive potential over three generations.
Functionality of spermatozoa obtained after SSCT was also investigated by performing both in-vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI). Fertilisation rate, blastocyst development rate and blastocyst quality were evaluated.
When SSCT becomes available for clinical use, efficient protocols for the cryopreservation of SSCs and testicular tissue will be of great benefit.

Results:
In the mouse, we have shown that sperm obtained after SSCT were able to fertilize and produce offspring in-vivo and after assisted reproduction4. However, post-transplantation spermatozoa showed a lower motility than spermatozoa from fertile males. It was also observed that the fertilizing potential was lower with transplanted males compared to control mice. Moreover, the litter sizes were smaller and the fetal length and weight were significantly lower in the first-generation offspring from transplanted animals, whereas subsequent generations did not show those abnormalities5. This observation may be suggestive for imprinting disorders.
By using a non-controlled freezing protocol, the survival rate of SSCs was higher compared with other testicular cells, which resulted in an enrichment of SSCs in the final suspension, but an important loss of functionality of spermatogonial stem cells was found after freezing and thawing6. An alternative way to preserve SSCs is to freeze the whole testicular tissue instead of cell suspensions. The structure of the tissue could be well preserved and especially the spermatogonia survived7.

Discussion:
The efficiency of SSCT depends on the number of SSCs injected in the recipient's tubules. Since only the SSCs can relocate to the basement membrane and initiate colonization, enriching the proportion of SSCs may improve tranplantation efficiency. Although, SSCT could prove important for fertility preservation, this technique may not be without any risk. Testicular cell suspensions from cancer patients may be contaminated with cancerous cells. It is obvious that reintroduction of malignant cells into an otherwise cured patient must be omitted. Magnetic Activated Cell Sorting and Fluoresence Activated Cell Sorting are two strategies that can be used to decontaminate the cell suspension from malignant cells or to enrich the cell suspension for SSCs8.
An alternative to cryopreservation could be in-vitro culture of SSCs. Recently, it was reported that mouse SSCs could be expanded in-vitro with maintenance of functionality. When this approach would be feasible with human SSCs, it may improve the efficiency of cryopreservation, either by increasing the number of SSCs before freezing or after thawing.

References:
1 Tournaye H, Goossens E, Verheyen G, Frederickx V, De Block G, Devroey P, Van Steirteghem A. Preserving the reproductive potential of men and boys with cancer: current concepts and future prospects. Hum Reprod Update 2004;10:525.
2 Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, Martinez-Madrid B, van Langendonckt A. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;364:1405-1410.
3 Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 1994;91:11298-302.
4 Goossens E, Frederickx V, De Block G, Van Steirteghem AC, Tournaye H. Reproductive capacity of sperm obtained after germ cell transplantation in a mouse model. Hum Reprod 2003;18:1874-80.
5 Goossens E, Frederickx V, de Block G, van Steirteghem A, Tournaye H. Evaluation of in vivo conception after testicular stem cell transplantation in a mouse model shows altered post-implantation development. Hum Reprod 2006;21:2057-60.
6 Frederickx V, Michiels A, Goossens E, De Block G, Van Steirteghem AC, Tournaye H. Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells. Hum Reprod 2004;19:948-53.
7 Goossens E, Frederickx V, Geens M, De Block G, Tournaye H. Cryosurvival and spermatogenesis after allografting prepubertal mouse tissue: comparison of two cryopreservation protocols. Fertil Steril 2008;89:725-7.
8 Geens M, Van de Velde H, De Block G, Goossens E, Van Steirteghem A, Tournaye H. The efficiency of magnetic-activated cell sorting and fluorescence-activated cell sorting in the decontamination of testicular cell suspensions in cancer patients. Hum Reprod 2007;22:733-42.