Male fertility preservation : cryopreservation of immature testicular tissue and autotransplantation
(2013)
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- Abstract
- With increasing effectiveness of childhood cancer treatments, survival rates are on the rise and it is estimated that >80% of children now survive their disease. Unfortunately, improvements in treatment efficacy go hand in hand with increased toxicity, especially gonadotoxicity, severely affecting male germ cells (GCs) and leading to loss of fertility. Fertility preservation methods depend on the age of the patient. While sperm cryopreservation prior to gonadotoxic treatment is a well established method after puberty, it is not applicable in prepubertal boys. The alternative is cryopreservation of immature testicular tissue (ITT) containing GCs, and especially spermatogonial stem cells (SSCs). Slow-freezing of ITT is currently offered to prepubertal boys whose fertility is threatened by gonadotoxic treatments. Data from animal studies show very promising results, with healthy offspring obtained after transplantation of frozen testicular tissue or testicular cell suspensions stored for more than 14 years. In humans, after xenotransplantation of frozen ITT, survival, proliferation and initiation of differentiation of spermatogonia (SG) were achieved after 6 months. However, rapid loss of SG was recorded, with recovery rates of 14.5% after 3 weeks and 3.7% after 6 months, and differentiation apparently limited to the pachytene stage. It was suspected that the slow-freezing technique could have a negative impact on the survival of SG after transplantation. In order to evaluate this hypothesis, we developed an alternative method of cryopreservation for human ITT, namely vitrification, which is an innovative strategy preventing ice crystal formation by use of high concentrations of cryoprotectant and ultrafast cooling speeds, which could minimize cellular damage. We first evaluated a vitrification protocol on non-human primate ITT and demonstrated preservation of tissue integrity, maintenance of proliferating SG and functional Leydig cells (LCs) after xenotransplantation for 3 weeks. A comparative study of slow-frozen, vitrified and fresh human ITT grafts showed similar tissue preservation in terms of spermatogonial survival and differentiation, suggesting involvement of the transplantation procedure in graft outcome. Ways of improving graft development were therefore investigated. To limit spermatogonial loss, we evaluated supplementation of our transplants with protective molecules, i.e. N-acetylcysteine (NAC) and testosterone. No clear benefit was observed. However, SG loss appeared to be due to seminiferous tubule (ST) necrosis rather than cell apoptosis during the 5-day grafting period (Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)+STs: 76.4% versus cleaved caspase-3+STs: 10.12%). Altogether, our results point to progressive SG loss over time, concomitant with increasing numbers of STs devoid of spermatogonial cells, raising the question to what extent the SSC pool is depleted after transplantation. Further studies are thus urgently required to improve the transplantation technique in order to increase survival of SG and especially SSCs, which are the basis of spermatogenesis, before considering clinical application of this strategy.
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Poels, J. (2013). Male fertility preservation : cryopreservation of immature testicular tissue and autotransplantation. https://hdl.handle.net/2078.5/26574