What clinical practices, patient management strategies and experimental methods are currently being used to preserve and restore the fertility of prepubertal boys and adolescent males? Based on a review of the clinical literature and research evidence for sperm freezing and testicular tissue cryopreservation, and after consideration of the relevant ethical and legal challenges, an algorithm for the cryopreservation of sperm and testicular tissue is proposed for prepubertal boys and adolescent males at high risk of fertility loss. A known late effect of the chemotherapy agents and radiation exposure regimes used to treat childhood cancers and other non-malignant conditions in males is the damage and/or loss of the proliferating spermatogonial stem cells in the testis. Cryopreservation of spermatozoa is the first line treatment for fertility preservation in adolescent males. Where sperm retrieval is impossible, such as in prepubertal boys, or it is unfeasible in adolescents prior to the onset of ablative therapies, alternative experimental treatments such as testicular tissue cryopreservation and the harvesting and banking of isolated spermatogonial stem cells can now be proposed as viable means of preserving fertility. Advances in clinical treatments, patient management strategies and the research methods used to preserve sperm and testicular tissue for prepubertal boys and adolescents were reviewed. A snapshot of the up-take of testis cryopreservation as a means to preserve the fertility of young males prior to December 2012 was provided using a questionnaire. A comprehensive literature review was conducted. In addition, survey results of testis freezing practices in young patients were collated from 24 European centres and Israeli University Hospitals. There is increasing evidence of the use of testicular tissue cryopreservation as a means to preserve the fertility of pre- and peri-pubertal boys of up to 16 year-old. The survey results indicate that of the 14 respondents, half of the centres were actively offering testis tissue cryobanking as a means of safeguarding the future fertility of boys and adolescents as more than 260 young patients (age range less than 1 year old to 16 years of age), had already undergone testicular tissue retrieval and storage for fertility preservation. The remaining centres were considering the implementation of a tissue-based fertility preservation programme for boys undergoing oncological treatments. The data collected were limited by the scope of the questionnaire, the geographical range of the survey area, and the small number of respondents. The clinical and research questions identified and the ethical and legal issues raised are highly relevant to the multi-disciplinary teams developing treatment strategies to preserve the fertility of prepubertal and adolescent boys who have a high risk of fertility loss due to ablative interventions, trauma or genetic pre-disposition. The work was funded by the European Society of Human Reproduction and Embryology (ESHRE). There were no conflicts of interest. Not applicable.

BACKGROUND: Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility.OBJECTIVE AND RATIONALE: With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss.SEARCH METHODS: Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity.OUTCOMES: Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation.LIMITATIONS, REASONS FOR CAUTION: The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention.WIDER IMPLICATIONS: The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss.STUDY FUNDING/COMPETING INTEREST(S): The work was funded by ESHRE. None of the authors has a conflict of interest.

Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful. This review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed. An extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy. The feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23.8%. Yet, functional proof of fertility restoration in the human is lacking. In addition, few to no data are available on the safety aspects inherent to offspring generation with gametes derived from frozen-thawed TT or TCSs. Moreover, clarification is needed on whether it is better to cryopreserve TT or TCS. Fertility restoration techniques are very promising and expected to be implemented in the clinic in the near future. However, inter-center variability needs to be overcome and the gametes produced for reproduction purposes need to be subjected to safety studies. With the perspective of a future clinical application, there is a dire need to optimize and standardize cryopreservation and safety testing before using frozen-thawed TT of TCSs for fertility restoration.

So far, successful de novo formation of testicular tissue followed by complete spermatogenesis in vitro has been achieved only in rodents. Our findings reveal that primary human testicular cells are able to self-organize into human testicular organoids (TOs), i.e., multi-cellular tissue surrogates, either with or without support of a biological scaffold. Despite lacking testis-specific topography, these mini-tissues harbored spermatogonia and their important niche cells, which retained specific functionalities during long-term culture. These observations indicate the posibility of in vitro re-engineering of a human testicular microenvironment from primary cells. Human TOs might help in the development of a biomimetic testicular model that would exert a tremendous impact on research and development, clinical treatment of infertility, and screening in connection with drug discovery and toxicology.

BACKGROUND: Before clinical application, the feasibility and safety of autologous testicular stem cell transplantation should be explored. Apart from limitations in their numbers, spermatogonial stem cells may also be contaminated by malignant cells. Therefore, both enrichment and decontamination before transplantation may be necessary. This study aimed at evaluating the decontaminating potential of magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) for both murine and human testicular cell suspensions. In the mouse, the effectiveness of the transplantation technique after cell sorting was also assessed. METHODS: Murine testicular cells were contaminated with 5% EL4 cells. Fresh and frozen–thawed suspensions were sorted using MACS (CD49f ⁺) and FACS (CD49f⁺, H-2Kb⁻) and evaluated by FACS, cell culture and transplantation into W/W^v mice. Human testicular cells were contaminated with 5 or 0.05% CCRF-SB (SB) cells. Frozen–thawed suspensions were sorted using FACS (HLA class I⁻) and evaluated by FACS, cell culture and PCR for the B-cell receptor. RESULTS: In the mouse, the sorted fractions contained 0.39% H-2K^b-positive and 76.55% CD49f-positive cells. After transplantation, 1 in 20 recipient mice developed a malignancy. In the human experiments, an average of 0.58% SB cells was detected after sorting. In only 1 of 11 samples, there were no SB cells observed. CONCLUSION: MACS and/or FACS are insufficient for completely depleting testicular tissue of malignant cells. Although more research on alternative decontamination techniques is necessary, developing a reliable method to screen a priori testicular tissue for malignant cells may be equally important.

What issues remain to be solved before fertility preservation and transplantation can be offered to prepubertal boys? The main issues that need further investigation are malignant cell decontamination, improvement of in vivo fertility restoration and in vitro maturation. Prepubertal boys who need gonadotoxic treatment might render sterile for the rest of their life. As these boys do not yet produce sperm cells, they cannot benefit from sperm banking. Spermatogonial stem cell (SSC) banking followed by autologous transplantation has been proposed as a fertility preservation strategy. But before this technique can be applied in the clinic, some important issues have to be resolved. Original articles as well as review articles published in English were included in a search of the literature. Relevant studies were selected by an extensive Medline search. Search terms were fertility preservation, cryopreservation, prepubertal, SSC, testis tissue, transplantation, grafting and in vitro spermatogenesis. The final number of studies selected for this review was 102. Cryopreservation protocols for testicular tissue have been developed and are already being used in the clinic. Since the efficiency and safety of SSC transplantation have been reported in mice, transplantation methods are now being adapted to the human testes. Very recently, a few publications reported on in vitro spermatogenesis in mice, but this technique is still far from being applied in a clinical setting. Using tissue from cancer patients holds a potential risk for contamination of the collected testicular tissue. Therefore, it is of immense importance to separate malignant cells from the cell suspension before transplantation. Because biopsies obtained from young boys are small and contain only few SSCs, propagation of these cells in vitro will be necessary. The ultimate use of the banked tissue will depend on the patient's disease. If the patient was suffering from a non-malignant disease, tissue grafting might be offered. In cancer patients, decontaminated cell suspensions will be injected in the testis. For patients with Klinefelter syndrome, the only option would be in vitro spermatogenesis. However, at present, restoring fertility in cancer and Klinefelter patients is not yet possible. 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.