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Beneficial effect of directional freezing on in vitro viability of cryopreserved sheep whole ovaries and ovarian cortical slices
No full textSTUDY QUESTION: Does directional freezing improve the structural and functional integrity of ovarian fragments compared with conventional slow freezing and to whole ovary cryopreservation? SUMMARY ANSWER: Compared with slow freezing, the use of directional freezing significantly improves all structural and functional parameters of ovarian fragments assessed in vitro and, overall, whole ovaries were better preserved than ovarian fragments. WHAT IS KNOWN ALREADY: Directional freezing has been developed to provide an alternative way to cryopreserve large biological samples and it is known to improve the structural and functional integrity of whole ovaries. Conventional slow freezing of ovarian fragments is the procedure more widely used in clinical settings but it causes substantial structural damage that limits the functional period after transfer back into the patient. STUDY DESIGN, SIZE, DURATION: We performed a 2 × 2 factorial design experiment on a total of 40 sheep ovaries, divided into four groups (n = 10 ovaries per group): (i) directional freezing of whole ovary (DFwo); (ii) directional freezing of ovarian fragments (DFof); (iii) conventional freezing of whole ovary (CFwo); (iv) conventional freezing of ovarian fragments (CFof). An additional eight ovaries were used as fresh controls. PARTICIPANTS/ MATERIALS, SETTING, METHODS: Ewe ovaries were randomly assigned to one of the experimental groups and frozen accordingly. Upon thawing, ovarian tissue was examined morphologically and cultured in vitro for 7 days. Samples were analyzed for cell proliferation and apoptosis, for DNA damage and repair activity, and for the presence of a panel of heat shock proteins (HSPs) by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Most studied parameters were significantly improved (P < 0.05) in all samples cryopreserved with directional compared with slow freezing. The proportion of primordial follicles, which developed to the primary stage in whole ovaries (53 ± 1.7%) and in ovarian fragments (44 ± 1.8%) cryopreserved with directional freezing, was greater than with slow frozen whole ovaries (6 ± 0.5%, P = 0.001) or fragments (32 ± 1.5%, P = 0.004). After 7 days of culture, cell proliferation in DFwo (28 ± 0.73%) was the highest of all groups (P < 0.05) followed by DFof (23 ± 0.81%), CFof (20 ± 0.79%) and CFwo (9 ± 0.85%). Directional freezing also resulted in a better preservation of the cell capacity to repair DNA damage compared with slow freezing both in whole ovaries and ovarian fragments. Apoptosis and HSP protein levels were significantly increased only in the CFwo group. Direct comparison demonstrated that, overall, DFwo had better parameters than DFof and was no different from the fresh controls. LIMITATIONS, REASONS FOR CAUTION: The study is limited to an in vitro evaluation and uses sheep ovaries, which are smaller than human ovaries and therefore may withstand the procedures better. WIDER IMPLICATIONS OF THE FINDINGS: Improved integrity of ovarian morphology may translate to improved outcomes after transplantation. Alternatively, the particularly good preservation of whole ovaries suggests they could provide a source of ovarian follicles for in vitro culture in those cases when the presence of malignant cells poses a substantial risk for the patient. STUDY FUNDING/COMPETING INTEREST(S): Supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC) IG 10376, Carraresi Foundation and by Legge 7 Regione Autonoma Sardegna (R.A.S). There are no conflicts of interest. © The Author 2013
Multi-thermal gradient freezing allows the cryopreservation of sheep whole ovaries with the same efficiency of ovarian fragments
No full textOvarian tissue cryobanking is proposed as an effective option for preserving female fertility in cancer patients. At present 2 options are available: cryopreservation of ovarian cortical fragments or of the whole ovary. The use of whole ovary reduces ischemic insult. However, the larger the sample volume, the more difficult it is to introduce the cryoprotective agents and to ensure an adequate cooling rate that minimizes tissue damage. For this reason, we used the multi-thermal gradient method, based on running the sample through a temperature gradient. This allows a homogeneous cooling rate through the whole sample independently from its volume. The aim of the study was to determine whether multi-thermal gradient freezing allows a substantial reduction of the damages induced by cryopreservation of large samples by comparing the viability of cortical fragments versus whole ovaries after thawing and grafting in nude mice. Sheep ovaries were collected at the local abattoir and randomly divided into 3 groups: A) ovaries frozen as cortical fragments, B) ovaries frozen as whole organs, and C) fresh ovaries immediately processed for further analysis (control). Ovarian fragments (10×5×1mm) were sliced from the cortical region and immersed into cryoprotectant solution (Leibovitz L-15 medium, 10% FCS, and 1.5M dimethyl sulfoxide), while whole ovaries were perfused with the same solution. Samples were placed into glass freezing tubes 16mm in diameter filled with cryoprotectant solution. Samples were frozen with the multi-thermal gradient freezing apparatus (Core Dynamics, Ness Ziona, Israel) progressing along the thermal gradient at a rate of 0.01mms(-1), resulting in a cooling rate of 0.3°Cmin(-1). Two weeks later, samples were thawed by plunging the tubes into a 37°C water bath with gentle shaking. Whole ovaries were perfused with 10mL of HEPES-Talp medium, 0.5M sucrose, and 10IUmL(-1) of heparin and their cortical region was cut into fragments. These fragments and those derived from group A were rehydrated in L-15 medium with decreasing sucrose concentrations. Fragments (2×2×1mm) were xenografted in the dorsal region of 6 nude mice for each group. Mice were killed after 8 weeks and grafts were collected for analysis. Cryopreserved samples were compared with each other and fresh controls (group C). Morphologically normal follicles at primordial, primary, and secondary stages were visible in all samples. Cell proliferation was assessed measuring Ki-67 mRNA and counting immunohistochemically positive cells. The FSH receptor and GDF9 gene expression were used to evaluate tissue viability. No significant differences for any of these parameters were measured amongst the groups. We conclude that directional freezing is an effective method for ovarian tissue cryopreservation independently from the sample volume, thus overriding the limitations usually associated with whole-organ banking
In vitro viability of sheep whole ovaries and cortical fragments after cryopreservation with different techniques
No full textCryopreservation of ovarian tissue is a promising technique for preserving fertility in young female cancer patients. At present two options are available: cryopreservation of ovarian cortical fragments or of the whole ovary. We compared ovarian tissue viability, cryopreserved as fragments or whole organs using a conventional (CF) or directional freezing apparatus (DF).
Cortical fragments (10x5x1 mm) were immersed into Leibovitz L-15 medium, 10% FCS and 1,5 M DMSO, while whole ovaries were perfused with the same solution. CF was performed at 0.5°C/min in a Kryo 560M (Planer, UK). DF was performed at 0.01 mm/sec, resulting in cooling rates of 0.3°C/min with a Multi-Thermal-Gradient (IMT, Israel). In both cases freezing was arrested at -40°C, before plunging the samples into liquid nitrogen. After thawing, whole ovaries and cortical fragments were cut in 2x2x1 mm pieces and cultured for 7 days in α-MEM medium supplemented with ITS, glutamine, pyruvate, hypoxantine, BSA, FSH and bFGF. After culture, the percentage of primordial follicles developed to the primary stage in DF whole ovaries, was comparable to controls. A lower rate (P<0.05) of growing follicles was observed in all other groups, and CF whole ovaries were unable to support any development. DNA double-strand breaks formation and repair was analyzed by immunofluorescent expression of γ-H2AX and of RAD51. At the beginning of the culture, DNA damage in frozen samples was higher than in fresh controls. However after 7 days, DNA damage significantly decrease in DF groups and in CF cortex samples concomitantly with an increase of DNA repair. Conversely, in CF whole ovaries DNA repair signal was absent leaving the rate of DNA damage substantially unchanged. We conclude that DF allows a better preservation than CF of whole ovaries and cortical fragments and eliminates any disadvantage related to the bigger volume of whole organs
Ovarian tissue viability in vitro after cryopreservation with different techniques
No full textOvarian tissue cryopreservation, is a viable option for preserving female fertility. Since different options are available, we performed a 2x2 factorial design comparing viability of sheep ovarian tissue cryopreserved as fragments or whole organs using a conventional (CF) or directional freezing apparatus (DF).
Cortical fragments (10x5x1 mm) were immersed into Leibovitz L-15 medium, 10% FCS and 1,5 M DMSO, while whole ovaries were perfused with the same solution. CF was performed at 0.5°C/min in a Kryo 560M (Planer, UK). DF was performed at 0.01 mm/sec, resulting in cooling rates of 0.3°C/min with a Multi-Thermal-Gradient (IMT, Israel). In both cases freezing was arrested at -40°C before plunging the samples into liquid nitrogen. Immediately after thawing, the percentage of morphologically normal follicles found in DF whole ovaries (81%) was similar to that of fresh controls (90%). DF (72%) and CF (63%) cortical fragments showed both a significantly lower rate (P<0.05). Only 23% survived after CF of whole ovaries.
Whole ovaries and cortical fragments were cut in 2x2x1 mm pieces and cultured for 7 days in α-MEM medium supplemented with ITS, glutamine, pyruvate, hypoxantine, BSA, FSH and bFGF. After culture, the percentage of primordial follicles developed to the primary stage in DF whole ovaries, was similar to controls. A lower rate (P<0.05) of growing follicles was observed in all other groups, and CF whole ovaries were unable to support any development. We conclude that DF ensures a better tissue viability than CF and that DF better preserve whole ovaries than cortical fragments.
Supported by AIRC IG 10376 and by Carraresi Foundatio
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