R Fabbri1, R Vicenti1, M Macciocca2, N A Martino3, M E Dell'Aquila4, G Pasquinelli5, A M Morselli-Labate1, R Seracchioli1, R Paradisi1. 1. Gynecology and Pathophysiology of Human Reproductive Unit, Department of Medical and Surgical Sciences, University of Bologna, S. Orsola-Malpighi Hospital, 40138 Bologna, Italy. 2. Gynecology and Pathophysiology of Human Reproductive Unit, Department of Medical and Surgical Sciences, University of Bologna, S. Orsola-Malpighi Hospital, 40138 Bologna, Italy maria.macciocca2@unibo.it. 3. Department of Biosciences, Biotechnologies and Biopharmaceutics (DBBB), University of Bari Aldo Moro, Str. Prov. Casamassima Km 3, 70010 Valenzano, Bari, Italy Experimental Zooprophylactic Institute of Puglia and Basilicata, Via Manfredonia 20, 71121 Foggia, Italy. 4. Department of Biosciences, Biotechnologies and Biopharmaceutics (DBBB), University of Bari Aldo Moro, Str. Prov. Casamassima Km 3, 70010 Valenzano, Bari, Italy. 5. Surgical Pathology, Department of Experimental, Diagnostic and Speciality Medicine, University of Bologna, S. Orsola-Malpighi Hospital, 40138 Bologna, Italy.
Abstract
STUDY QUESTION: Which is the best method for human ovarian tissue cryopreservation: slow freezing/rapid thawing (SF/RT) or vitrification/warming (V/W)? SUMMARY ANSWER: The conventional SF/RT protocol used in this study seems to better preserve the morpho-functional status of human cryopreserved ovarian tissue than the used open carrier V/W protocol. WHAT IS KNOWN ALREADY: Cryopreservation of human ovarian tissue is generally performed using the SF/RT method. However, reduction in the follicular pool and stroma damage are often observed. An emerging alternative procedure is represented by V/W which seems to allow the maintenance of the morphological integrity of the stroma. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study including six patients affected by oncological diseases and enrolled from January to December 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissue was laparoscopically harvested from the right and left ovaries and was cryopreserved using a routinary SF/RT protocol or a V/W method, involving tissue incubation in two solutions (containing propylene glycol, ethylene glycol and sucrose at different concentrations) and vitrification in an open system. For each patient, three pieces from each ovary were collected at the time of laparoscopy (fresh tissue) and after storage (SF/RT or V/W) and processed for light microscopy (LM) and transmission electron microscopy (TEM), to assess the morphological and ultrastructural features of follicles and stroma, and for laser scanning confocal microscopy (LSCM), to determine the functional energetic/redox stroma status. The preservation status of SF/RT and V/W ovarian tissues was compared with that of fresh ones, as well as between them. MAIN RESULTS AND THE ROLE OF CHANCE: By LM and TEM, SF/RT and V/W samples showed cryodamage of small entity. Interstitial oedema and increased stromal cell vacuolization and chromatin clumping were observed in SF/RT samples; in contrast, V/W samples showed oocyte nuclei with slightly thickened chromatin and irregular shapes. The functional imaging analysis by LSCM revealed that the mitochondrial activity and intracellular reactive oxygen species levels were reduced both in SF/RT and in V/W samples compared with fresh samples. The study also showed progressive dysfunction of the mitochondrial activity going from the outer to the inner serial section of the ovarian cortex. The reduction of mitochondrial activity of V/W samples compared with fresh samples was significantly higher in the inner section than in the outer section. LIMITATIONS, REASONS FOR CAUTION: The results report the bioenergetic and oxidative status assessment of fresh and cryopreserved human ovarian tissue by LSCM, a technique recently applied to tissue samples. The use of LSCM on human ovarian tissues after SF/RT or V/W is a new application that requires validation. The procedures for mitochondrial staining with functional probes and fixing are not yet standardized. Xenografting of the cryopreserved ovarian tissue in severe combined immunodeficient mice and in vitro culture have not yet been performed. WIDER IMPLICATIONS OF THE FINDINGS: The identification of a cryopreservation method able to maintain the morpho-functional integrity of the ovarian tissue and a number of follicles comparable with those observed in fresh tissue might optimize results in clinical practice, in terms of recovery, duration of ovarian function and increased delivery outcomes after replanting. The SF/RT protocol allowed better morpho-functional tissue integrity than the V/W procedure. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Fondazione del Monte di Bologna e Ravenna, Italy. Dr N.A.M. was granted by the project ONEV MIUR PONa3 00134-n.254/R&C 18 5 2011 and the project GR-2011-02351396 (Ministry of Health, Young Researchers Grant 2011/2012). There are no competing interests. TRIAL REGISTRATION NUMBER: Clinical trial 74/2001/0 (approved:13 2 2002): 'Pilot study on cryopreservation of human ovarian tissue: morphological and immunohistochemical analysis before and after cryopreservation'.
STUDY QUESTION: Which is the best method for human ovarian tissue cryopreservation: slow freezing/rapid thawing (SF/RT) or vitrification/warming (V/W)? SUMMARY ANSWER: The conventional SF/RT protocol used in this study seems to better preserve the morpho-functional status of human cryopreserved ovarian tissue than the used open carrier V/W protocol. WHAT IS KNOWN ALREADY: Cryopreservation of human ovarian tissue is generally performed using the SF/RT method. However, reduction in the follicular pool and stroma damage are often observed. An emerging alternative procedure is represented by V/W which seems to allow the maintenance of the morphological integrity of the stroma. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study including six patients affected by oncological diseases and enrolled from January to December 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissue was laparoscopically harvested from the right and left ovaries and was cryopreserved using a routinary SF/RT protocol or a V/W method, involving tissue incubation in two solutions (containing propylene glycol, ethylene glycol and sucrose at different concentrations) and vitrification in an open system. For each patient, three pieces from each ovary were collected at the time of laparoscopy (fresh tissue) and after storage (SF/RT or V/W) and processed for light microscopy (LM) and transmission electron microscopy (TEM), to assess the morphological and ultrastructural features of follicles and stroma, and for laser scanning confocal microscopy (LSCM), to determine the functional energetic/redox stroma status. The preservation status of SF/RT and V/W ovarian tissues was compared with that of fresh ones, as well as between them. MAIN RESULTS AND THE ROLE OF CHANCE: By LM and TEM, SF/RT and V/W samples showed cryodamage of small entity. Interstitial oedema and increased stromal cell vacuolization and chromatin clumping were observed in SF/RT samples; in contrast, V/W samples showed oocyte nuclei with slightly thickened chromatin and irregular shapes. The functional imaging analysis by LSCM revealed that the mitochondrial activity and intracellular reactive oxygen species levels were reduced both in SF/RT and in V/W samples compared with fresh samples. The study also showed progressive dysfunction of the mitochondrial activity going from the outer to the inner serial section of the ovarian cortex. The reduction of mitochondrial activity of V/W samples compared with fresh samples was significantly higher in the inner section than in the outer section. LIMITATIONS, REASONS FOR CAUTION: The results report the bioenergetic and oxidative status assessment of fresh and cryopreserved human ovarian tissue by LSCM, a technique recently applied to tissue samples. The use of LSCM on human ovarian tissues after SF/RT or V/W is a new application that requires validation. The procedures for mitochondrial staining with functional probes and fixing are not yet standardized. Xenografting of the cryopreserved ovarian tissue in severe combined immunodeficientmice and in vitro culture have not yet been performed. WIDER IMPLICATIONS OF THE FINDINGS: The identification of a cryopreservation method able to maintain the morpho-functional integrity of the ovarian tissue and a number of follicles comparable with those observed in fresh tissue might optimize results in clinical practice, in terms of recovery, duration of ovarian function and increased delivery outcomes after replanting. The SF/RT protocol allowed better morpho-functional tissue integrity than the V/W procedure. STUDY FUNDING/COMPETING INTERESTS: Funding was provided by Fondazione del Monte di Bologna e Ravenna, Italy. Dr N.A.M. was granted by the project ONEV MIUR PONa3 00134-n.254/R&C 18 5 2011 and the project GR-2011-02351396 (Ministry of Health, Young Researchers Grant 2011/2012). There are no competing interests. TRIAL REGISTRATION NUMBER: Clinical trial 74/2001/0 (approved:13 2 2002): 'Pilot study on cryopreservation of human ovarian tissue: morphological and immunohistochemical analysis before and after cryopreservation'.