Dorien Van Saen

Autologous spermatogonial stem cell transplantation in man: current obstacles for a future clinical application

Fertility preservation is becoming an important issue in the management of the quality of life of prepubertal boys undergoing cancer treatment. At present, the only theoretical option for preservation of fertility in these boys is the preservation of the spermatogonial stem cells for autologous intratesticular stem cell transplantation. In animal models, this technique has shown promising results. However, before translation to the clinic, some major concerns should be evaluated. Improving the efficiency of the technique is one of the first goals for further research, besides evaluation of the safety of the clinical application. Also, the cryopreservation of the spermatogonial stem cells needs extra attention, since this first step will be crucial in the success of any clinical application. Another concern is the risk of malignant contamination of the testicular tissue in childhood cancer patients. Extensive research in this field and especially on the feasibility of decontaminating the testicular tissue will be inevitable. Another important, though overlooked, issue is the prevention of damage to the testicular niche cells. Finally, xenografting and in vitro proliferation/maturation of the spermatogonia should be studied as alternatives for the transplantation technique.

Regeneration of spermatogenesis by grafting testicular tissue or injecting testicular cells into the testes of sterile mice: a comparative study.

OBJECTIVE To make a comparison between two different approaches-spermatogonial stem cell transplantation and intratesticular grafting, for preservation and reintroduction of spermatogonial stem cells. DESIGN Prospective experimental study. SETTING Academic medical center and teaching hospital. PATIENT(S) N/A. INTERVENTION(S) Intratesticular transplantation, histologic evaluation of testes. MAIN OUTCOME MEASURE(S) Testicular weight, amount of green fluorescence in the testis, and immunostaining for green fluorescent protein. RESULT(S) In a first experiment donor-derived spermatogenesis was found in 65% of the injected testes (41.8 +/- 72.2 mm) compared with 75% of the testes (122.1 +/- 45.6 mm) after tissue grafting. In the second series of experiments complete spermatogenesis was found in 75% of the testes after fresh grafting (93.8 +/- 21.8 mm) compared with 88% after frozen-thawed tissue grafting (84.8 +/- 45.6 mm). CONCLUSION(S) Both approaches show that spermatogonial stem cells can successfully be introduced to the testis resulting in spermatogenesis. Tissue grafting produced a larger mean donor colony length and there was no significant difference between colonization efficiency using either fresh or frozen-thawed grafts. In a future clinical setting, grafting would be a simple and efficient way for reintroducing stem cells to the testis.

Orthotopic grafting of cryopreserved prepubertal testicular tissue: in search of a simple yet effective cryopreservation protocol.

OBJECTIVE To investigate whether solid-surface vitrification (SSV) is an effective cryopreservation strategy regarding the integrity and function of prepubertal mouse testicular tissue. DESIGN Prospective experimental study. SETTING Academic research unit. ANIMAL(S) Mice. INTERVENTION(S) Testicular tissue from 5- to 10-day-old GFP(+) mice was cryopreserved with the use of a conventional uncontrolled slow freezing (USF) technique and SSV before intratesticular grafting in busulfan-treated GFP(-) mice. MAIN OUTCOME MEASURE(S) Ultrastructural cryoinjury to spermatogonial stem cells (SSCs) and somatic cells was assessed by electron microscopy. Tubular structure was evaluated by histology, and graft survival and spermatogenic recovery by immunohistochemistry. RESULT(S) The tubular morphology and the proportion of ultrastructural cryodamage were similar between vitrified and slow-frozen testicular fragments. Allografting of tissue after both USF and SSV resulted in a recovery of spermatogenesis similar to fresh samples. CONCLUSION(S) SSV resulted in success rates similar to USF in maintaining testicular cell ultrastructure, tubular morphology, and tissue function. These data provide further evidence that vitrification, being an inexpensive and simple technique, can be considered as an alternative for cryopreservation of prepubertal testicular tissue.

Spermatogonial stem cell preservation in boys with Klinefelter syndrome: to bank or not to bank, that's the question

Although early development of testis appears normal in boys with Klinefelter syndrome (KS), spermatogonial stem cell (SSC) depletion occurs in midpuberty, leading to infertility. Therefore, freezing of semen samples or testicular tissue sampling could be offered to boys with KS at onset of puberty. However, only in about half of patients with KS, adult or prepubertal, spermatozoa or SSCs can be observed, and to date, no clinical parameters are available to detect patients who might benefit from these techniques. Furthermore, strategies for the further use of the cryopreserved material are still under investigation. Retrieval of spermatogonial cells in prepubertal boys with KS should therefore still be viewed as experimental and patients and their parents must be counseled accordingly.

Presence of spermatogonia in 47,XXY men with no spermatozoa recovered after testicular sperm extraction.

OBJECTIVE To evaluate the presence of spermatogonia in men diagnosed with Klinefelter syndrome (KS), in whom no testicular spermatozoa were recovered by testicular sperm extraction. DESIGN Retrospective case series. SETTING University hospital. PATIENT(S) Testicular samples from 22 nonmosaic 47,XXY men, aged 24-43 years, with no spermatozoa at multiple biopsies. INTERVENTION(S) Paraffin-embedded testicular tissue was sectioned and stained with hematoxylin-eosin, and immunostainings were performed for both MAGE-A4 and vimentin. MAIN OUTCOME MEASURE(S) The presence of spermatogonia. RESULT(S) Massive fibrosis and hyalinization were observed in all men with KS. Spermatogonia were observed in 4 of 22 men with KS, with differentiation up to the spermatocyte level in 2 of them. CONCLUSION(S) A few men with KS, having no spermatozoa after testicular sperm extraction, still had few spermatogonia. These patients may eventually benefit from in vitro maturation using spermatogonial stem cells. The adult KS population can thus be divided into three subgroups: one subgroup showing focal spermatogenesis, a second having spermatogonia, and a third group in which no germ cells can be recovered. Further research is necessary to unravel the mechanism leading to these different patterns in patients with KS.

Mouse spermatogonial stem cells obtain morphologic and functional characteristics of hematopoietic cells in vivo

BACKGROUND The aim of this study was to explore the plasticity and transdifferentiation potential of murine spermatogonial stem cells (SSCs) into hematopoietic cells. METHODS GFP(+)CD49f(+)H-2K(b-) SSCs of male donor mice were isolated and injected into the bone marrow (BM) of Busulfan-treated GFP(-) female mice. Twelve weeks post-transplantation, the recipients were sacrificed and their BM, peripheral blood (PB) and spleen (SL) cells were collected and evaluated by phenotypical methods, i.e. fluorescence-activated cell sorting, immunohistochemistry and fluorescence in situ hybridization, and functional assays, i.e. colony-forming units assay and intra-BM transplantation. RESULTS Green fluorescent protein (GFP)- and Y chromosome-positive cells were observed in the BM, PB and SL of transplanted female mice. These cells presented phenotypical and functional characteristics of hematopoietic cells in vitro and in vivo. CONCLUSIONS Our results indicate that SSCs have the potential to transdifferentiate into hematopoietic cells in vivo.

Exogenous administration of recombinant human FSH does not improve germ cell survival in human prepubertal xenografts

In a previous study, meiotic activity was observed in human intratesticular xenografts from peripubertal patients. However, full spermatogenesis could not be established. The present study aimed to evaluate whether the administration of recombinant human FSH could improve the spermatogonial survival and the establishment of full spermatogenesis in intratesticular human xenografts. Human testicular tissue was obtained from six boys (aged 2.5-12.5years). The testicular biopsy was fragmented and one fragment of 1.5-3.0mm(3) was transplanted to the testis of immunodeficient nude mice. Transplanted mice were assigned to different experimental groups to enable evaluation of the effects of FSH administration and freezing. The structural integrity of the seminiferous tubules, the spermatogonial survival and the presence of differentiated cells were evaluated by histology and immunohistochemistry. Freezing or administration of FSH did not influence tubule integrity and germ cell survival in human xenografts. Meiotic germ cells were observed in the xenografts. More tubules containing only Sertoli cells were observed in frozen-thawed grafts, and more tubules with meiotic cells were present in fresh grafts. There was no clear influence of FSH treatment on meiotic differentiation. Administration of FSH did not improve the establishment of full spermatogenesis after intratesticular tissue grafting.

Bone marrow stem cells transplanted to the testis of sterile mice do not differentiate into spermatogonial stem cells and have no protective effect on fertility

Four months after transplanting bone marrow cells into the testis, no differentiation to spermatogonial stem cells was observed. Bone marrow transplantation had no protective effect on fertility after chemotherapy.

Does early cell death cause germ cell loss after intratesticular tissue grafting

OBJECTIVE To assess cell death in intratesticular grafts. DESIGN Experimental animal study. SETTING University. ANIMAL(S) F1-hybrids from SV129 X C57BL. INTERVENTION(S) Intratesticular tissue transplantation was performed and cell death in the grafts was evaluated at different time points after transplantation. MAIN OUTCOME MEASURE(S) Apoptotic cell death in spermatogonia was evaluated by flow cytometry with the use of the annexin V assay. Immunohistochemistry was used to evaluate graft development and the global occurrence of cell death. RESULT(S) The highest level of spermatogonia-specific cell death was found on days 4 and 10, although no statistical difference was observed compared with control tissue. Statistically significant reductions in tubule integrity were observed 1 day and 2 months after transplantation. More degenerated tubules were observed in the center of the grafts 1 and 4 days after transplantation, and higher numbers of apoptotic tubules were found 1 day after transplantation. No difference in overall cell death was observed between grafts and controls for any time point except for the frozen grafts 1 day after transplantation. CONCLUSION(S) Spermatogonia-specific apoptosis does not explain the stem cell loss observed after intratesticular tissue grafting; it probably results from degeneration of tubules in the center of the graft owing to hypoxia during the first days after transplantation.

Cryopreservation of Human Testicular Tissue by Isopropyl-Controlled Slow Freezing

Tissue cryopreservation uses very low temperatures to preserve structurally intact living cells in their natural microenvironment. Cell survival is strongly influenced by the biophysical effects of ice during both the freezing and the subsequent thawing. These effects can be controlled by optimizing the fragment size, type of cryoprotectant, and cooling rate. The challenge is to determine cryopreservation parameters that suit all cell types present in the tissue. Here we describe a quick and convenient protocol for the cryopreservation of testicular tissue using an isopropyl-insulated freezing device, which was validated in both a mouse and a human model.