AbstractMale infertility, due to depletion of spermatogonial stem cells (SSC), is a common side effect of gonadotoxic treatments (chemo- and radiotherapy) or can be a symptom of a genetic disorder (eg. Klinefelter syndrome). Pre-pubertal patients cannot benefit from sperm banking before reproductive stem cell loss because of the lack of complete spermatogenesis. As a consequence they are at risk of life-long sterility. The autotransplantation of banked testicular tissue might become a possible fertility preservation strategy. Nevertheless, this technique requires a good cryopreservation protocol capable of preserving the tissue and cell integrity as well as the tissue function. In this study we evaluated a controlled-rate freezing method, noncontrolled-rate freezing and a vitrification procedure to select the most appropriate protocol for human adult testicular tissue cryopreservation.
Tissue integrity: Only noncontrolled-rate freezing didn't led to a significant decrease in either intact seminiferous tubules or in SSC per tubule compared to the control. Freezing with a noncontrolled rate was statistically better in preserving the tubule morphology and SSC than freezing with a controlled rate, although it was not different from vitrification. There was no difference in tubule preservation, but significantly more SSC survived cryopreservation after vitrification compared to controlled-rate freezing. All cryopreservation protocols showed excellent Sertoli cell preservation. The rate of freezing had clearly no effect on Sertoli cell survival. Cell integrity: The distribution of SSC, according to the degree of ultrastructural damage, was statistically different between the fresh group and the cryo groups and between the cryo groups mutually. Controlled freezing led to the highest proportion of damaged SSC. This method damaged nearly all SSC (99%) of which 63% showed severe aberrations. Noncontrolled-rate freezing resulted in 20% undamaged SSC and 80% damaged cells. Vitrification outshines both freezing methods by resulting in 52% unharmed SSC approaching the SSC distribution of the reference sample. Tissue function: Four months after intratesticular xenografting only sclerotised grafts with degenerated tubules were found. Although, 71% of the fresh grafts showed some surviving SSC. Clearly no conclusion could be drawn from the transplantation experiment.
Cryopreservation of human adult testicular tissue with the vitrification procedure as well as with the noncontrolled-rate freezing method led to promising results. Eventually a mouse model was used to study the effect of both cryopreservation protocols on the tissue function. The vitrification technique was introduced at a later time point consequently the analyses of the vitrified grafts are still ongoing. No statistical difference in number of tubules with complete spermatogenesis was seen between the fresh and cryopreserved grafts. So noncontrolled freezing had no effect on the function of pre-pubertal murine tissue. In addition, FACS analyses were performed to detect GFP+ spermatozoa in the acceptor epididymii. The data showed an average GFP+ fraction of 7.8±9.0% and 3.3±3.9% on the side of fresh tissue grafted testes and on the side of the cryopreserved grafts, respectively. These findings need confirmation by PCR. In the future this study needs to be repeated with pre-pubertal human tissue due to the structural and functional differences with adult tissue.
|Date of Award||1 Jul 2010|
|Supervisor||Ellen Goossens (Promotor) & Herman Tournaye (Co-promotor)|