STUDY QUESTION 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? SUMMARY ANSWER 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. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SIZE, DURATION 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. PARTICIPANTS/MATERIALS, SETTING, METHODS 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. MAIN RESULTS AND THE ROLE OF CHANCE 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. LIMITATIONS, REASONS FOR CAUTION The data collected were limited by the scope of the questionnaire, the geographical range of the survey area, and the small number of respondents. WIDER IMPLICATIONS OF THE FINDINGS 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. STUDY FUNDING/COMPETING INTEREST(S) The work was funded by the European Society of Human Reproduction and Embryology (ESHRE).

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A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys

Distinctive chromatin in human sperm packages genes for embryo development

Fertility in adult life may be severely impaired by gonadotoxic therapies. For young boys who do not yet produce spermatozoa, cryopreservation of immature testicular tissue (ITT) is an option to preserve their fertility, albeit still experimental. This paper covers current options for ITT cryopreservation and fertility restoration. Relevant studies were identified by an extensive Medline search of English and French language articles. Search terms were: gonadotoxicity, cytoprotection, cryopreservation, ITT, spermatogonia, testicular transplantation, testicular grafting and in vitro maturation (IVM). Although no effective gonadoprotective drug is yet available for in vivo spermatogonial stem cell protection in humans, current evidence supports the feasibility of ITT cryopreservation before gonadotoxic treatment with a view to fertility preservation. Controlled slow freezing with dimethyl sulfoxide allows survival and proliferation of human spermatogonia after xenotransplantation, but only partial differentiation. Animal data look promising, since healthy offsprings have been obtained after transplantation of frozen testicular cell suspensions or tissue pieces. However, none of the fertility restoration options from frozen tissue, i.e. cell suspension transplantation, tissue grafting and IVM have proved efficient and safe in humans as yet. While additional evidence is required to define optimal conditions for ITT cryopreservation with a view to transplantation or IVM, the putative indications for such techniques, as well as their limitations according to disease, are outlined.

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Options for fertility preservation in prepubertal boys

BACKGROUND: Preservation of the male germ line in prepubertal boys undergoing gonadotoxic treatment is a crucial consideration in terms of their future quality of life. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival, proliferation and differentiation capacity of spermatogonia after cryopreservation and long-term transplantation of immature testicular tissue pieces. METHODS: Single pieces of testicular tissue (2-8 mm(3)) from prepubertal boys (7-14 years) were cryopreserved, thawed and transplanted into the scrotum of mice for 6 months. Upon removal, histological, immunohistochemical and ultrastructural analyses and testicular sperm extraction (TESE) were used to evaluate the tissue. RESULTS: Histology showed 55 +/- 42% of tubules to be intact. MAGE-A4 immunostaining showed mean spermatogonial recovery to be 3.7 +/- 5.5%, with 35% of these cells expressing Ki67, evidencing proliferation in tissue from boys <14 years of age. No apoptosis was found, as demonstrated by the absence of active caspase-3 and TUNEL staining. Numerous premeiotic spermatocytes, a few spermatocytes at the pachytene stage and spermatid-like cells were observed. No immunostaining was observed for lactate dehydrogenase-C, ACE or proacrosin, indicating that the spermatid-like structures observed by histology did not express the meiotic and post-meiotic markers characteristic of normal spermatids. No ultrastructural alterations of the tissue were encountered. CONCLUSIONS: The present study demonstrates that spermatogonia are able to survive and proliferate after cryopreservation and long-term transplantation. Complete regeneration of normal spermatogenesis was not observed since, beyond the pachytene stage, no adequate characterization of germ cells was obtained. Further studies are thus required to investigate the differentiation potential of cryopreserved germ cells.

Long-term spermatogonial survival in cryopreserved and xenografted immature human testicular tissue

Long-Term Spermatogonial Survival in Cryopreserved and Xenografted Immature Human Testicular Tissue Editorial Comment

BACKGROUND: Spermatozoa with large vacuoles (SLV) may have a negative impact on embryo development. The origin of these vacuoles is unknown. We evaluated acrosome and nucleus alterations in isolated SLV, versus unselected spermatozoa. METHODS: We studied 20 patients with teratozoospermia. Spermatozoa from the native semen sample and spermatozoa presenting a vacuole occupying > 13.0% total head area, isolated under high magnification (× 6600), were assessed. Confocal and transmission electron microscope evaluations were performed on SLV and native sperm, respectively. Acrosome morphology and DNA fragmentation were analysed using proacrosin immunolabelling (monoclonal antibody 4D4) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Chromatin condensation was evaluated with aniline blue staining. Sperm aneuploidy was assessed using fluorescence in situ hybridization. RESULTS: SLV represented 38.0 ± 5.10% of motile spermatozoa obtained after gradient density centrifugation. Vacuoles were mainly in the anterior and median sperm head (45.7 ± 2.90 and 46.1 ± 3.00%, respectively). Abnormal acrosomes were increased in SLV compared with unselected spermatozoa (77.8 ± 2.49 versus 70.6 ± 2.62%; P = 0.014). Microscopic observations showed an exclusively nuclear localization of large vacuoles. Complete DNA fragmentation was higher in native spermatozoa (P < 0.0001) than SLV, while chromatin condensation was altered in SLV (P < 0.0001). Aneuploidy and diploidy rates were increased in SLV (P < 0.0001). CONCLUSIONS: Sperm vacuoles were exclusively nuclear. In our selected teratozoospermic population, aneuploidy and chromatin condensation defects were the main alterations observed in SLV. Based on results from this small sample of spermatozoa, we propose a global impairment of the spermatogenesis process as a common origin of the morphological alterations.

Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles

BACKGROUND: Cryopreservation of immature testicular tissue could be considered as a major step in fertility preservation for young boys with cancer. In the present study, eight different freezing protocols were evaluated in immature mice testis. METHODS: Testis from six-day-old mice were frozen using either 1,2-propanediol (PROH) or dimethylsulphoxide (DMSO: D) at 1.5 M. Different cooling rate curves were tested: (i) controlled slow protocol with seeding (CS+) or (ii) without seeding (CS-), (iii) controlled rapid protocol and (iv) non-controlled protocol. Cryodamage of seminiferous cords was semi-quantitatively determined, establishing a scoring of alterations. Cell viability and apoptosis induction were assessed on testicular cell suspensions immediately after digestion (DO) and after a 20-h culture period (D1). Cells recovered after digestion of 100 mg tissue and the rate of living and non-apoptotic cells were quantified at DO and Dl. A long-term culture (9 days) of testis pieces was carried out for the protocol offering the best survival. Testosterone production, intratubular cell proliferation and tubule growth were assessed. RESULTS: DMSO produced optimal results in the different cooling rate curves tested when compared with PROH. Optimal results were obtained for the DCS - procedure (P < 0.05). Testosterone production, tubule growth and cell proliferation of post-thaw pieces were similar to fresh samples. CONCLUSIONS: Testis freezing with 1.5 M DMSO in a CS -procedure was found to maintain not only immature testicular tissue architecture, but also viability of testicular cells, endocrine and partial exocrine functions of the testis. Semi-quantitative evaluation of seminiferous cord cryodamage can be effectively used to rapidly screen optimal freezing conditions and as a possible quality control in a human application.

Comparison of conditions for cryopreservation of testicular tissue from immature mice

BACKGROUND Sperm banking is a suitable procedure to prevent infertility after cancer therapy in male adolescents. We evaluated the feasibility of semen preservation in 156 adolescents aged between 13 and 20 years and then we assessed fertility outcome after treatment. METHODS Age, urogenital history, indications for cryopreservation, histological diagnosis and semen parameters were recorded. Fertility status after treatment was assessed by a questionnaire addressed to those patients who had utilized sperm storage. Post-treatment semen analysis was performed for 22 patients. RESULTS Cryopreservation was possible in 88.5% of cases. Azoospermia was detected in 2.6% of the patients at the time of diagnosis. Malignant disease accounted for 84% of our male adolescents. In this type of disease, semen parameters were significantly altered only among patients with metastatic malignant bone tumour. After treatment, nine patients presented azoospermia, five patients achieved pregnancy spontaneously, two achieved it after assisted reproductive technique using fresh ejaculated spermatozoa and one following sperm donation. Three failed with cryopreserved sperm. CONCLUSIONS Semen cryopreservation is possible for most adolescents and, regardless of disease type, may be a means of preserving fertility prior to gonadotoxic treatment that might impair the spermatogenesis process.

Fertility preservation in adolescent males: experience over 22 years at Rouen University Hospital

Chromosome meiotic pairing during male meiosis is a major event for chromosome segregation during anaphase I and spermatogenesis normal process. Chromosome non-disjunctions responsible for aneuploidy in male gametes can be observed during the first and the second meiotic divisions. The analysis of sperm nuclei chromosome constitution is a major and indirect tool for assessing male meiotic non-disjunctions and the genesis of chromosomal abnormalities. This evaluation has been performed initially by the human sperm/hamster oocyte fusion assay and more recently by fluorescence in situ hybridisation (FISH). Therefore, male populations with increased risk of aneuploidy for their progeny could be identified before entering an in vitro fertilization procedure, and depending on the potential risk a preimplantation or prenatal genetic diagnosis could be performed. For males with constitutional chromosome abnormalities, a specific genetic counselling could also be proposed.

Anomalies chromosomiques du spermatozoïde

Resume Objectif Malgre les nombreux progres realises dans le domaine de la fecondation in vitro (FIV), les resultats en termes de taux d’implantation embryonnaire restent relativement bas puisqu’ils ne depassent generalement pas les 15 a 20 %. L’eclosion embryonnaire assistee a ete proposee afin de faciliter l’eclosion puis l’implantation de l’embryon. Patient(e)s et methodes Notre travail a consiste a evaluer l’efficacite de l’eclosion embryonnaire assistee, en termes de taux d’implantation, taux de grossesse et taux d’accouchement, dans trois indications principales. Une etude retrospective a ete conduite sur 143 cycles de FIV et ICSI realises chez des femmes d’âge superieur ou egal a 38 ans, 166 cycles de FIV et ICSI concernant les tentatives de rang superieur ou egal a 3 et 180 cycles de transferts d’embryons decongeles. Resultats Le profil des patientes est comparable dans les differents groupes. Aucune difference significative n’a ete observee sur le taux d’implantation, de grossesse et de naissance avec cette technique pour les femmes de plus de 38 ans. Pour les echecs repetes d’implantation, les taux d’implantation, de grossesse et d’accouchement sont superieurs dans le groupe ayant beneficie d’une eclosion embryonnaire assistee ; cependant, les differences observees n’apparaissent pas significatives. Le taux d’implantation est significativement superieur dans le groupe ayant beneficie d’une eclosion embryonnaire assistee (19,14 % contre 8,84 % [ p  = 0,02]) apres transfert d’embryons decongeles. Neanmoins, aucune difference significative n’a ete mise en evidence concernant les taux de grossesse et de naissance. Discussion et conclusion L’eclosion embryonnaire assistee ameliore significativement le taux d’implantation des embryons decongeles.

N. Mousset-Siméon

Papers

Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles

Comparison of conditions for cryopreservation of testicular tissue from immature mice

Fertility preservation in adolescent males: experience over 22 years at Rouen University Hospital

Anomalies chromosomiques du spermatozoïde

Éclosion embryonnaire assistée : évaluation des indications au CHU de Rouen