study question: Is there a better alternative to the conventional cryopreservation protocols for human testicular tissue banking? summary answer: Uncontrolled slow freezing (USF) using 1.5 M dimethylsulphoxide (DMSO) and 0.15 M sucrose as cryoprotectants appears to be a user-friendly and efficient method for the cryopreservation of human testicular tissue.what is known already: Currently, time-consuming controlled slow freezing (CSF) protocols that need expensive equipment are commonly used for human testicular tissue banking. USF and vitrification are cryopreservation techniques that were successfully applied in several animal models but need further exploration with human tissue. study design, size, duration: Fragments (n ¼ 160) of testicular tissue from 14 patients undergoing vasectomy reversal were assigned to a fresh control group or one of the following cryopreservation procedures: CSF using DMSO at a concentration of 0.7 or 1.5 M in the presence (+S) or absence of sucrose (2S), USF using either 0.7 or 1.5 M DMSO combined with sucrose, solid-surface vitrification (SSV) or direct cover vitrification (DCV). materials, setting, methods: Light microscopic evaluations were performed to study apoptosis, germ cell proliferation ability, spermatogonial survival, coherence of the seminiferous epithelium and integrity of the interstitial compartment after cryopreservation. Ultrastructural alterations were studied by scoring cryodamage to four relevant testicular cell types.main results and the role of chance: The USF 1.5 M DMSO + S protocol proved not solely to prevent cell death and to preserve seminiferous epithelial coherence, interstitial compartment integrity, SG and their potential to divide but also protected the testicular cell ultrastructure. A significant reduction in the number of SG per tubule from 21.4 + 5.6 in control tissue to 4.9 + 2.1, 8.2 + 5.4, 11.6 + 5.1, 8.8 + 3.9, 12.6 + 4.4 and 11.7 + 5.7 was observed after cryopreservation combined with at least one other form of cryoinjury when using CSF 0.7 M DMSO 2S, CSF 0.7 M DMSO + S, CSF 1.5 M DMSO + S, USF 0.7 M DMSO + S, SSV and direct cover vitrification (DCV), respectively (P , 0.001).limitations, reasons for caution: Supplementary research is required to investigate the effect on tissue functionality and to confirm this study's findings using prepubertal tissue.wider implications of the findings: An optimal cryopreservation protocol enhances the chances for successful fertility restoration. USF, being an easy and cost-effective alternative to CSF, would be preferable for laboratories in developing countries or whenever tissue is to be procured from a diseased child at a site distant from the banking facility.

Research Foundation, Flanders (G.0385.08 to H.T.), the Institute for the Agency for Innovation, Belgium (IWT/SB/111245 to E.G.), the Flemish League against Cancer (to E.G.), Kom op tegen kanker (G.0547.11 to H.T.) and the Fund Willy Gepts (to HT). E.G. is a Postdoctoral Fellow of the FWO, Research Foundation, Flanders. There are no conflicts of interest.

This project was funded by the scientific Fund Willy Gepts from the UZ Brussel (D.V.S., J.D.S.), grants from the Vrije Universiteit Brussel (E.G.) and a Methusalem grant (K.S.). D.V.S is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

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This xenografting study shows that intratesticular grafting results in high germ cell survival. In grafts derived from the older boys, meiotic activity was maintained in the xenografts for at least 9 months. Although difficult to conduct due to the scarcity of the tissue, more comparative research is needed to elucidate an optimal grafting strategy.

No spermatogenesis was documented in non-mosaic 47,XXY adolescents either by spermaturia, electroejaculation or testicular biopsy. Neither clinical nor hormonal parameters were of value in determining the timing for optimal spermatogonial stem cell retrieval. More data are needed to elucidate the potential role of testicular tissue cryopreservation in adolescents with KS. Therefore, at present, the cryopreservation of testes tissue for clinical reasons should not be recommended.

Intratesticular tissue grafting might be the better choice for fertility restoration. Disrupting the stem cell niche might influence epigenetic patterns. Since the function of H4K5ac and H4K8ac in spermatogonia and spermatocytes still has to be explored, in-depth epigenetical analyses are warranted.

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.