title: Long-term spermatogonial survival in cryopreserved and xenografted immature human testicular tissue.
abstract: 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.
authors.affiliation: Department of Gynecology, Université Catholique de Louvain, Cliniques Universitaires Saint-Luc, Brussels, Belgium.
authors.forename: Christine
authors.initials: C
authors.lastname: Wyns
authors.forename: Anne
authors.initials: A
authors.lastname: Van Langendonckt
authors.forename: François-Xavier
authors.initials: FX
authors.lastname: Wese
authors.forename: Jacques
authors.initials: J
authors.lastname: Donnez
authors.forename: Mara
authors.initials: M
authors.lastname: Curaba
mesh_terms: D000293:Adolescent; D000367:Age Factors; D000951:Antigens, Neoplasm / Q000096:biosynthesis; D017209:Apoptosis; D053148:Caspase 3 / Q000096:biosynthesis; D049109:Cell Proliferation; D002470:Cell Survival; D002648:Child; D015925:Cryopreservation / Q000379:methods*; D006801:Humans; D019394:Ki-67 Antigen / Q000096:biosynthesis; D008297:Male; D009363:Neoplasm Proteins / Q000096:biosynthesis; D013093:Spermatogonia / Q000166:cytology* / Q000637:transplantation; D013737:Testis / Q000473:pathology*; D014183:Transplantation, Heterologous
chemical_list: D000951:Antigens, Neoplasm; D019394:Ki-67 Antigen; C095204:MAGEA4 protein, human; D009363:Neoplasm Proteins; D053148:Caspase 3