Testicular Tissue Harvesting

Although spermatogenesis does not occur in prepuber-tal testes, and prepubertal testes do not produce mature spermatozoa, these do contain the diploid stem germ cells from which haploid spermatozoa can be derived. Therefore, testicular tissue can be harvested from a biopsy and stored either as a tissue section or as isolated germ cells, before cancer therapy. Following cure and on entering adulthood, this tissue can be thawed and used to produce offspring in either of the two ways: the stored germ cells can be re-implanted into the patient's own testes to restore natural fertility, a procedure known as germ cell transplantation, or the stored stem cells can be matured in vitro until they are able to achieve fertilization via ICSI.47 Although these two measures have been the subject of intensive research in the last decade, further refinements in the protocols may still be needed before they can be used routinely in clinical practice.

Germ cell transplantation

Germ cells isolated from the testes of donor male mice can repopulate immunologically compatible testes when injected into the seminiferous tubules of recipient animals; the recipients show normal morphological features characteristic of the donor species.48 Similarly, mouse germ cells transplanted into the testes of infertile mice colonize the recipient seminiferous tubules and initiate donor spermatogenesis in more than 70% of recipients.49 The most striking result of these experiments was that healthy offspring (by mating) were produced from spermatozoa generated within the recipient testes by donor germ cells.

Establishing a successful method for testicular stem cell transplantation of frozen, thawed testicular cells would be of immense benefit to boys with childhood cancer undergoing sterilizing treatment. It is possible to reinitiate spermatogenesis after cryopreservation of testicular germ cell suspensions. Although cell survival is acceptable, current protocols need further improve-ment.50 Male germ cells obtained before chemotherapy can be frozen and, after thawing, can be transplanted into animals to maintain the entire genetic information of the donor for a limited period.

Before stem cell transplantation can be considered for preserving the fertility of pre-pubertal boys, two issues must be carefully examined.51 First, the testis biopsy taken from the cancer patient may contain malignant cells. These cells must be removed from the cell suspension because studies in rats have shown that one single malignant cell can reintroduce the disease. Second, the cell suspension consists of all testicular cells, and the proportion of spermatogonial stem cells is low (estimated at 1/5000).52

In vitro maturation

In vitro germ cell maturation would be particularly useful in patients who have received extremely gonadotoxic therapy and in whom the supporting Sertoli cells would be unable to support spermatogene-sis. Mouse spermatogonial stem cells can survive up to 4 months in culture and retain their ability to commence spermatogenesis following transplantation into a recipient.53 However, it appears that current methods for in vitro maturation of diploid stem cells into hap-loid spermatozoa are not well developed. Ongoing research may improve their feasibility.

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