Wyns Christine, Curaba Mara, Martinez-Madrid Belen, Van Langendonckt Anne, François-Xavier Wese, Donnez Jacques
Gynecology Research Unit, Université Catholique de Louvain, 1200 Brussels, Belgium.
Hum Reprod. 2007 Jun;22(6):1603-11. doi: 10.1093/humrep/dem062. Epub 2007 May 3.
Fertility preservation has become an urgent clinical requisite for prepubertal male cancer patients undergoing gonadotoxic treatment. As these patients do not yet produce spermatozoa for freezing, only immature tissue is available for storage. We studied the survival and proliferative activity of spermatogonia and Sertoli cells after cryopreservation of cryptorchid testicular tissue pieces followed by xenografting for 21 days.
Single pieces of tissue from cryptorchid testes (2-9 mm(3)) of young boys (2-12 years) were cryopreserved, thawed and transplanted into the scrotum of mice. Quantitative morphometric and immunohistochemical techniques were used to evaluate the integrity of the tissue, as well as the survival and proliferative capacity of spermatogonia and Sertoli cells before and after freezing/thawing/grafting. Three weeks after grafting, cryopreserved tissue was removed and analysed. Most of the tubules (88.3%) were intact and there was no fibrosis or sclerosis, 14.5% of the initial spermatogonial population remained, as identified by the MAGE A4 antibody, and 32% of these cells showed proliferative activity evidenced by Ki67, compared to 17.8% before cryopreservation and grafting. The number of Sertoli cells was unchanged and 5.1% were Ki67-positive, compared to none at all before freezing and grafting.
Through our orthotopic xenografting model, we have demonstrated the survival and proliferative activity of spermatogonia and Sertoli cells in cryopreserved immature human cryptorchid tissue. Testicular tissue banking may thus prove to be a promising technique for the preservation of fertility in prepubertal boys undergoing oncological treatments. As the stem cell niche is maintained, the cryopreserved tissue can potentially be used for future autotransplantation. In addition, whole tissue freezing does not exclude alternative clinical uses, including isolated cell transplantation after dissociation, selection and enrichment. However, as this work was done on cryptorchid tissue, studies on normal immature testicular tissue, involving longer grafting periods, are needed to demonstrate a differentiation capacity before clinical implementation. Ethical and safety issues should also be addressed.
对于接受性腺毒性治疗的青春期前男性癌症患者而言，生育力保存已成为一项紧迫的临床需求。由于这些患者尚未产生可冷冻保存的精子，因此仅有未成熟组织可供储存。我们研究了隐睾睾丸组织块冷冻保存后进行异种移植21天，精原细胞和支持细胞的存活及增殖活性。
将年轻男孩（2至12岁）隐睾的单个组织块（2至9立方毫米）进行冷冻保存、解冻，然后移植到小鼠阴囊中。运用定量形态计量学和免疫组织化学技术评估组织的完整性，以及冷冻/解冻/移植前后精原细胞和支持细胞的存活及增殖能力。移植三周后，取出冷冻保存的组织并进行分析。大多数小管（88.3%）保持完整，未出现纤维化或硬化，通过MAGE A4抗体鉴定，初始精原细胞群体中有14.5%留存，其中32%的细胞表现出增殖活性，这由Ki67证实，而冷冻保存和移植前这一比例为17.8%。支持细胞数量未变，5.1%的支持细胞Ki67呈阳性，而冷冻和移植前无一例阳性。
通过我们的原位异种移植模型，我们证明了冷冻保存的未成熟人类隐睾组织中精原细胞和支持细胞的存活及增殖活性。睾丸组织库因此可能被证明是一种有前景的技术，用于为接受肿瘤治疗的青春期前男孩保存生育力。由于干细胞生态位得以维持，冷冻保存的组织有可能用于未来的自体移植。此外，全组织冷冻并不排除其他临床用途，包括解离、筛选和富集后进行分离细胞移植。然而，由于这项工作是在隐睾组织上进行的，因此需要对正常未成熟睾丸组织进行研究，涉及更长的移植期，以在临床应用前证明其分化能力。伦理和安全问题也应得到解决。
