Fertility Preservation and Restoration
About 1 in every 600 children develops cancer before the age of 15 years. Thanks to the remarkable progress that has been made in the treatment of cancer in infants and children, today >80% of them can be cured. It has been estimated that, by now, 1 in 250 adults in the age group of 20 to 30 years is a childhood cancer survivor.[76,77] Besides cancer, other diseases requiring gonadotoxic treatments (e.g., sickle cell disease) or genetic diseases (e.g., Klinefelter syndrome, AZF deletions) may lead to spermatogonial stem cell loss.[78,79] It is unquestionable that the prevention of sterility needs special attention in both oncology and reproductive medicine. The inability to father his genetically own children can have a significant impact on the psychological well-being of the patient in later adulthood. Whereas adult patients can be offered sperm banking before SSC loss, no such option exists to preserve the fertility in prepubertal boys. The cryopreservation of spermatogonial stem cells before gonadotoxic therapy followed by autologous intratesticular transplantation of these stem cells after cure is possibly the only option (Fig. 5).
Spermatogonial stem cell transplantation as a method for fertility restoration. Testicular tissue is removed and cryopreserved before the onset of the cancer treatment. After the patient has been cured, the thawed tissue can be transplanted into the remaining testis. When the boy reaches puberty, spermatogenesis may be established.
Cryopreservation of Stem Cells
To safeguard the reproductive potential of young cancer patients, cryopreservation of testicular tissue containing SSCs is preferred above cryopreservation of SSC suspensions. Indeed, the presence of the extracellular matrix and supporting cells is critical to germ cell survival and germ cell function. Any cryopreservation protocol should thus aim at preserving both the stem cells and their niche cells. Undoubtedly, cryopreservation of testicular tissue is a challenging task. The complexity of the tissue architecture demands optimal conditions for each cellular type. Controlled slow freezing with dimethyl sulphoxide is routinely used to cryopreserve immature testicular tissue.[81–84] In rodents, controlled slow freezing of prepubertal testicular tissue fragments has already led to the birth of healthy offspring. Two teams have published freezing protocols for human testicular tissue using controlled rate freezing. Kvist et al reported the cryopreservation of testicular tissue in boys with cryptorchidism. Later, Keros et al proposed a protocol for prepubertal testicular tissue. Nevertheless, the drawback of controlled slow freezing is the need for expensive computerized equipment. Moreover, this freezing process consumes a lot of time and resources. Therefore, uncontrolled slow freezing has been explored. Like controlled freezing, uncontrolled freezing of prepubertal testicular tissue has been successfully used in different animal species, and it has been fully validated in mice as a means to preserve reproductive potential.[88–91] Recently, in piglets and mice, vitrification was shown to yield similar results compared with slow freezing.[92–94] Because both uncontrolled freezing and vitrification are inexpensive, convenient, and fast executable protocols, these methods might be considered for human testicular tissue too.
Spermatogonial Stem Cell Transplantation
The technique of spermatogonial stem cell transplantation was first reported by Brinster and Zimmermann in 1994. It involves the introduction of a germ cell suspension from a fertile donor testis into the seminiferous tubules of an infertile recipient mouse. Transplanted spermatogonial stem cells were able to relocate onto the basement membrane and colonize the tubules during the first month after transplantation. From that moment on, SSCs started to proliferate and initiated spermatogenesis. The first meiotic germ cells appear after 1 month, and their number gradually increases thereafter. It has been shown that the recipient mice could reproduce in vivo after transplantation and produce transgenic offspring. Similar experiments were performed using SSCs that had been frozen and thawed. Shortly after, this technology was performed in other mammalian species including primates.[98–102] Even the transplantation between different species with close phylogeny was proven successful.[103,104] These encouraging results, especially those from primate studies, suggest a possibility of banking and subsequently transplanting human spermatogonial stem cells to prevent sterility caused by SSC loss.
Before this application can be introduced in a clinical setting, it is important to evaluate the efficiency and the safety of the procedure. In the mouse, it was shown that sperm cells obtained after spermatogonial stem cell transplantation were able to fertilize and produce normal embryos after assisted reproduction, although litter sizes were smaller compared with normal fertile control mice. A detailed analysis of the motility kinematics and concentrations of spermatozoa showed a lower sperm concentration and sperm motility after transplantation. In contrast, when donor and recipient were genetically related, the offspring showed normal genetic and epigenetic characteristics for most of the investigated modifications (Figs. 6 and 7).[107–109] Only histone 4 lysines 5 and 8 acetylation, which is important in spermatids for the histone-to-protamine exchange, was impaired in spermatogonia and spermatocytes. The function of H4K5ac and H4K8ac in these cell types still has to be explored.
Array comparative genomic hybridization on offspring after spermatogonial stem cell transplantation. A detailed graph of chromosome 17 of first-generation offspring. Every genomic gain or loss, detected in the offspring (green box), were found to be polymorphisms.
Methylation status of six cytosine phosphate guanine (CpG) sites of (A) Peg1, (B) Igf2, and (C) α-Actin in the liver of posttransplantation offspring. The X-axis represents the analyzed CpG sites (1 to 6); the Y-axis shows the methylation percentage. Light violet boxes represent the data obtained from first-generation offspring; dark violet boxes show the data obtained from second-generation offspring, and light yellow boxes show the control data. The methylation status of control and first- or second-generation offspring did not show significant differences.
Testicular Tissue Grafting
Testicular tissue grafting has been suggested as an alternative to SSC transplantation. Testis tissue has been grafted under the back skin, in the scrotum, or in the testis. Mature spermatozoa could be obtained from ectopic grafts, and progeny were born using intracytoplasmic sperm injection. Full spermatogenesis was obtained in grafts using immature testis tissue from different species.[110,112–114] Ectopic grafting was also performed using human testicular tissue. The first reports using adult testicular tissue showed only limited spermatogonial survival, with most of the tubules completely regressed.[115,116] Using prepubertal and neonatal tissue, spermatogonial survival and differentiation up to primary spermatocytes were reported in ectopic grafts. In an attempt to improve the results after grafting, immature testicular tissue was placed in the peritoneal bursa inside the scrotum. Long-term survival of spermatogonia and differentiation up to pachytene spermatocytes was observed.
Intratesticular tissue transplantation was first reported in 2002. Sperm was produced after grafting cryopreserved immature testicular tissue from mice and rabbits into the testicular parenchyma. Progeny were born when rabbit sperm from the xenograft was used for microinsemination. A comparison of intratesticular grafting and SSC transplantation (SSCT) in a mouse model showed a better reestablishment of spermatogenesis after grafting. Whereas some epigenetic modifications were found to be altered after SSCT, this was not the case after grafting.
Cryopreservation of the grafts did not adversely affect the colonization efficiency and restoration of spermatogenesis. Although intratesticular grafting seems to be more efficient, it can only be offered to patients with nonmalignant diseases or nonmetastasizing tumors.
Removal of Contaminating Malignant Cells
Many pediatric malignancies are capable of metastasizing through the blood, causing a potential risk of contamination of the collected testicular tissue. The transplantation of as few as 20 leukemic cells could cause malignant recurrence in rats. In the human, the threshold number of malignant cells able to cause malignant relapse when transplanted to the testis is unknown. Therefore, it is of utmost importance to detect even the slightest contamination of the testicular tissue. In the case of contamination, the isolation of SSCs from malignant cells before transplantation is necessary. Apart from ours, another research group studied the use of magnetic-activated cell sorting and/or FACS for depleting cancer cells from mouse and human testicular cell suspensions. However, both reported insufficient depletion.[122–124] Also cell selection by selective matrix adhesion was not efficient enough.
Semin Reprod Med. 2013;31(1):39-48. © 2013 Thieme Medical Publishers