title: Long-term storage does not impact the quality of cryopreserved human ovarian tissue.
abstract: BACKGROUND
  Ovarian tissue cryopreservation is an emerging technique, also addressed to very young cancer patients, for whom it is not possible to perform an ovarian stimulation for oocytes freezing, before gonadotoxic treatment. In this cases, ovarian tissue must be cryopreserved for a long period of time and it is very important to know if it maintains fertility function after a long period of storage. Here we aimed to assess the effect of long-term storage on preservation and viability of cryopreserved human ovarian tissue.
  
  
  METHODS
  Descriptive study of three cases of cancer patients whose cryopreserved ovarian tissue remained stored for 18 years. Long-term stored tissue was examined by histological and immunohistochemical analysis, transmission electron microscopy, TUNEL assay and LIVE/DEAD viability/citotoxicity test.
  
  
  RESULTS
  Ovarian tissue stored for 18 years showed a good morphology. Follicles presented negative staining for estrogen and progesterone receptors, positive staining for ki67 in granulosa cells and/or oocytes and for bcl2 in granulosa cells. Regarding stroma, patch/focal positive expression was found for estrogen receptor and ki67, diffusely positive expression for progesterone receptor and bcl2. After long-term storage, ultrastructural examination showed sub-cellular integrity of follicles and interstitial oedema foci. No apoptosis was observable by TUNEL assay. Stromal cell viability remained >97 % during the culture period.
  
  
  CONCLUSIONS
  The evaluation of different aspects of the tissue provides evidence that the storage time does not impact on tissue quality and gives hope especially to cancer girls, whose tissues could remain cryopreserved for a very long time.
authors.affiliation: Gynecology and Physiopathology of Human Reproductive Unit, Department of Medical and Surgical Sciences, University of Bologna, S. Orsola-Malpighi Hospital of Bologna, via Massarenti 13, 40138, Bologna, Italy.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Fabbri
authors.affiliation: Gynecology and Physiopathology of Human Reproductive Unit, Department of Medical and Surgical Sciences, University of Bologna, S. Orsola-Malpighi Hospital of Bologna, via Massarenti 13, 40138, Bologna, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Macciocca
authors.affiliation: Gynecology and Physiopathology of Human Reproductive Unit, Department of Medical and Surgical Sciences, University of Bologna, S. Orsola-Malpighi Hospital of Bologna, via Massarenti 13, 40138, Bologna, Italy. rossella.vicenti@unibo.it.
authors.forename: Rossella
authors.initials: R
authors.lastname: Vicenti
authors.affiliation: Surgical Pathology, Department of Experimental, Diagnostic and Speciality Medicine, University of Bologna, S. Orsola-Malpighi Hospital, Bologna, Italy.
authors.forename: Gianandrea
authors.initials: G
authors.lastname: Pasquinelli
authors.affiliation: Histopathological and Molecular Diagnostic Unit of Solid Organ and Transplant, S. Orsola-Malpighi Hospital, Bologna, Italy.
authors.forename: Giacomo
authors.initials: G
authors.lastname: Caprara
authors.affiliation: Surgical Pathology, Department of Experimental, Diagnostic and Speciality Medicine, University of Bologna, S. Orsola-Malpighi Hospital, Bologna, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Valente
authors.affiliation: Gynecology and Physiopathology of Human Reproductive Unit, Department of Medical and Surgical Sciences, University of Bologna, S. Orsola-Malpighi Hospital of Bologna, via Massarenti 13, 40138, Bologna, Italy.
authors.forename: Renato
authors.initials: R
authors.lastname: Seracchioli
authors.affiliation: Gynecology and Physiopathology of Human Reproductive Unit, Department of Medical and Surgical Sciences, University of Bologna, S. Orsola-Malpighi Hospital of Bologna, via Massarenti 13, 40138, Bologna, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Paradisi
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods* / Q000592:standards; D003451:Cryoprotective Agents / Q000494:pharmacology; D005260:Female; D006801:Humans; D046529:Microscopy, Electron, Transmission; D009369:Neoplasms / Q000628:therapy; D010053:Ovary / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D013997:Time Factors
chemical_list: D015415:Biomarkers; D003451:Cryoprotective Agents
keywords: Human ovarian tissue cryopreservation; Long-term storage; Tissue quality