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Cryostorage of testicular tissue and retransplantation of spermatogonial stem cells in the infertile male

https://doi.org/10.1016/j.beem.2018.10.003Get rights and content

Transplantation of own cryostored spermatogonial stem cells (SSCs) is a promising technique for fertility restoration when the SSC pool has been depleted.

In this regard, cryopreservation of pre-pubertal testicular tissue or SSCs suspensions before gonadotoxic therapies is ethically accepted and increasingly proposed.

SSC transplantation has also been considered to treat other causes of infertility relying on the possibility of propagating SSCs retrieved in the testes of infertile men before autologous re-transplantation.

Although encouraging results were achieved in animals and in preclinical experiments, clinical perspectives are still limited by a number of unresolved technical and safety issues, such as the risk of cancer cell contamination of cells intended for transplantation and the genetic and epigenetic stability of SCCs when cultured before re-transplantation. Moreover, while genome editing techniques raise the hope of modifying the SSCs genome before re-transplantation, their application for reproductive purposes might be a step too far for the moment.

Introduction

Cryostorage of testicular tissue has initially been considered to preserve Spermatogonial Stem cells (SSCs) aiming at fertility preservation for pre-pubertal boys before undergoing fertility threatening therapies leading to SSCs depletion.

Depletion of the SSC pool is often a consequence of cytotoxic therapies. By contrast with adult men where sperm cryopreservation may be proposed to preserve fertility before gonadotoxic therapies, for pre-pubertal boys about to undergo such treatments, the only promising fertility preservation technique is cryopreservation of either immature testicular tissue (ITT) containing SSCs or of isolated SSC suspensions *[1], [2], *[3], [4], [5].

The successful re-transplantation of cryopreserved testicular cells in mice [6], together with the need for fertility preservation options for pre-pubertal boys [4], [7], [8], stressed on the urgency of developing cryopreservation methods for ITT banking.

Even though still considered as experimental, cryostorage of ITT is increasingly being offered worldwide [4], *[9], [10] and is now considered ethically acceptable. Current research is ongoing for determining optimal protocols for cryopreservation and for developing fertility restoration methods with cryostored ITT i.e. SSC transplantation, ITT transplantation and in vitro maturation [11].

Adult males affected by non-obstructive azoospermia (NOA) after gonadotoxic therapies [9] or other acquired or congenital conditions [12] can undergo surgical testicular retrieval of mature sperm in about half of the cases. However, when no spermatozoa are retrieved, no alternative to father a child with the patient's own gametes is available so far. Therefore, use of own SSCs, if still present in the testicular tissue, has recently been proposed to restore fertility in these patients [13]. Indeed, on the one hand, SSCs recovery and transplantation back to the patient's testis after being expanded in culture could theoretically be performed if there are remaining functional SSC niches in order to improve the spermatogenic efficiency. On the other hand, use of new tools for genome editing like crispr-cas9 that allow better understanding of genes involved in male infertility [14] could prove helpful to correct some SSC anomalies responsible for infertility and achieve SSC renewal and differentiation following their re-transplantation.

This review will focus on the current state of the art on cryopreservation of testicular tissue and perspectives with re-transplantation of adult or pre-pubertal SSCs in the infertile male.

Section snippets

Methods

We used the PubMed electronic database to search for articles using the following query: (cryopreservation of mature or immature testicular tissue) OR (transplantation of spermatogonial stem cells) OR (propagation of spermatogonial stem cells) AND (human), representing the main topic of interest. Studies published in English or in French until 01 March 2018 were included. Any additional references found in original articles or review papers that were found relevant and missing from the primary

Cryostorage of SSCs

Current cryopreservation protocols vary depending on tissue and cell characteristics [15].

Slow-freezing and vitrification protocols are mainly applied and include cryoprotective agents (CPAs) in concentrations that must make the balance between avoiding CPAs toxicity and protecting the cells or tissues from damage while freezing and thawing or vitrifying and warming [16].

Cryopreservation of SSCs can be done as whole with tissue fragments *[1], [2], [17], [18], [19], [20] or as germ cell

Transplantation of SSCs

Auto-transplantation of SSCs is a fertility restoration option that has already been described in animals with encouraging results. The successful transplantation of testicular cells including SSCs with development of mature sperm was first described in mice by Brinster and Zimmerman in 1994 [42]. In 1996, Brinster's team also demonstrated completion of spermatogenesis with cryopreserved SSCs that colonized the recipient's empty niches after transplantation [6]. In 2003, live offspring was

Pre-pubertal boys facing gonadotoxic treatments

Pre-pubertal boys facing gonadotoxic therapies were the first candidates to benefit from cryostorage of testicular tissue with a view to preserve their fertility, and a significant number of European and US hospitals currently offer this option [7], [8], *[9], [37], [38], [39].

Chemotherapy and radiotherapy may lead to the destruction of SSCs, resulting in azoospermia in up to 25% of adult survivors of childhood cancer [62]. Preconditioning chemotherapy before bone marrow transplantation or

Perspectives with genome editing and SSC transplantation in the infertile male

SSCs play an important role in the maintenance of spermatogenesis and an abnormal genome of the SSC may impair the spermatogenic process, as aneuploid spermatogonia do not achieve meiosis [78].

The ability to culture SSCs for long periods of time without inducing genetic/epigenetic modifications [51], [85] while preserving their spermatogenic ability [86] is of interest because this means that genome editing tools could be combined to modify the SSC genome in vitro prior to transplantation [75].

Conclusion

Though we are still far from routine clinical application, substantial advances have been done in ITT cryopreservation offering the prospect of future transplantation of cryostored SSCs. Preclinical studies on the feasibility and risk assessment of transplantation of SSCs as well as achievements in non-human primates with SSC transplantation point to the potential of a near future clinical application to restore fertility after gonadotoxic treatments. However, broadening the applications with

Summary

Significant advances have been made since banking of human immature testicular tissue for fertility preservation has been started. Teams around the world progressively developed clinical protocols based on what was done in rodents and higher order mammals. Eighteen years after the first case report on pre-pubertal testicular tissue cryostorage, fertility restoration from cryopreserved human ITT in pre-pubertal boys has not been achieved yet. This is mainly due to numerous challenges ranging

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