title: Vitrification preserves proliferation capacity in human spermatogonia.
abstract: OBJECTIVE
  Does vitrification of human immature testicular tissue (ITT) have potential benefits for future fertility preservation? Does vitrification of human ITT have potential benefits in an in vivo murine xenotransplantation model?
  
  
  CONCLUSIONS
  Vitrification is able to maintain proliferation capacity in spermatogonial cells after 6 months of xenografting.
  
  
  BACKGROUND
  Controlled slow-freezing is the procedure currently applied for ITT cryobanking in clinical practice. Vitrification has been proposed as a promising technique for long-term storage of ITT, with a view to preserving spermatogonial stem cells (SSCs) for future fertility restoration in young boys suffering from cancer. After vitrification of ITT, in vitro survival of SSCs was demonstrated, but their functionality was not evaluated.
  
  
  METHODS
  Ten ITT pieces issuing from 10 patients aged 2-12 years were used. Fragments of fresh tissue (serving as controls) and fresh, frozen-thawed and vitrified-warmed testicular pieces xenografted to the scrotum of nude mice for 6 months were compared.
  
  
  METHODS
  Upon graft removal, histological and immunohistochemical analyses were performed to evaluate spermatogonia (SG) (MAGE-A4), intratubular proliferation (Ki67), proliferating SG and Leydig cells (3β-HSD). The entire piece of grafted tissue was assessed in each case.
  
  
  RESULTS
  Seminiferous tubules showed good integrity after cryopreservation and xenografting for 6 months in all three groups. Survival of SG and their ability to proliferate was observed by immunohistochemistry in all grafted groups. SG were able to initiate spermatogenesis, but blockage at the pachytene stage was observed. The recovery rate of SG was 3.4 ± 3.8, 4.1 ± 7.3 and 7.3 ± 6.3%, respectively, for fresh, slow-frozen and vitrified-warmed tissue after 6 months of xenografting.
  
  
  CONCLUSIONS
  The study is limited by the low availability of ITT samples of human origin. The mouse xenotransplantation model needs to be refined to study human spermatogenesis.
  
  
  CONCLUSIONS
  The findings of the present study have potential implications for cryobanking of ITT and fertility preservation. Spermatogonial loss recorded after fresh ITT transplantation indicates that the avascular grafting technique needs to be optimized. There are so far no convincing data justifying modification of current clinical practice for ITT storage with slow-freezing, but this study demonstrates that it is worth pursuing optimization of ITT vitrification as an alternative for preservation of SSCs.
  
  
  BACKGROUND
  The present study was supported by a grant from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie N° 7. 4.572.09.F). The authors declare that there is no conflict of interest.
authors: Gynecology Unit,  Medical School, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier, 52, 1200 Brussels,  Belgium.
authors: Jonathan
authors: J
authors: Poels
authors: Anne
authors: A
authors: Van Langendonckt
authors: Marie-Christine
authors: MC
authors: Many
authors: François-Xavier
authors: FX
authors: Wese
authors: Christine
authors: C
authors: Wyns
mesh_terms: D000755:Anemia, Sickle Cell / Q000378:metabolism / Q000473:pathology; D000818:Animals; D002648:Child; D002675:Child, Preschool; D015925:Cryopreservation; D059247:Fertility Preservation / Q000379:methods*; D006085:Graft Survival; D006801:Humans; D007985:Leydig Cells / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000378:metabolism / Q000473:pathology; D049471:Pachytene Stage; D013091:Spermatogenesis; D013093:Spermatogonia / Q000378:metabolism / Q000473:pathology*; D013737:Testis / Q000378:metabolism / Q000473:pathology / Q000637:transplantation; D014021:Tissue Preservation; D014183:Transplantation, Heterologous; D058989:Vitrification