DOES LEVEL OF EDUCATIONAL ATTAINMENT INFLUENCE THE PURSUIT OF ELECTIVE FERTILITY PRESERVATION AND USE OF CRYOPRESERVED OOCYTES?
Alexandra CampedelliMarja BrolinsonDavid BoedekerRona YuSorana RaiciulescuKathleen DevineMicah J. HillAlan H. DeCherneyTrimble Spitzer
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Educational Attainment
For all patients affected by a disease that could impair fertility before or during the reproductive lifespan, strategies to preserve their fertility and the ability to bear their own children is likely to be of utmost importance. While fertility preservation is a promising option, most of the technologies currently used are far from being well-established or are still experimental. Patients should be aware that no method guarantees success. Psychological and ethical impacts of fertility preservation are major concerns and should be included in the multidisciplinary approach to the patients.
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Research questionWhich cryopreservation method better protects reproductive potential: the cryopreservation of a testicular cell suspension (TCS) or the cryopreservation of testicular tissue (TET)?DesignTwo cryopreservation strategies for spermatogonial stem cells (SSCs) were compared in a mouse model: cryopreservation as TET or as TCS. Evaluated outcomes were number of viable cells after thawing, number and length of donor-derived colonies after spermatogonial stem cell transplantation (SSCT), number of litters, litter size and number of donor-derived pups after mating.ResultsCompared with cryopreserving TCS, cryopreservation of TET resulted in significantly higher numbers of viable cells after thawing (TET: 13.4 × 104 ± 7.2 × 104 versus TCS: 8.2 × 104 ± 2.7 × 104; P = 0.0002), more (TET: 47.6 ± 19.2 versus TCS: 18.5 ± 13.0; P = 0.0039) and longer (TET: 5.2 ± 1.0 mm versus TCS: 2.7 ± 1.5 mm; P = 0.0016) donor-derived colonies, and more donor-derived pups per litter (TET: 2.2 ± 0.2 versus TCS: 0.5 ± 0.1; P = 0.0008).ConclusionsCryopreservation of TET is the preferred method to cryopreserve SSCs prior to SSCT in a mouse model.
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Cryopreservation of preimplantation embryos represents a major challenge due to their shape and relatively large cells. Embryo source and cryopreservation method are key factors to cryotolerance efficiency and few reports have investigated more promising protocols for goat embryos. The study was aimed to compare different cryopreservation methods for goat in vitro produced (IVP) embryos. Goat blastocysts were subjected to conventional freezing (CF), Dimethyl sulfoxide vitrification (DMSO-V) and Dimethylformamide vitrification (DMF-V). Cryopreserved blastocysts were assessed for re-expansion, cell viability and in vivo development rates. Blastocyst re-expansion after cryopreservation was similar between groups, but cell viability was lower for DMF-V (32%) than CF (68%) and DMSO-V (60%). Pregnancy and delivery rates were similar for CF (60% and 50%) and DMSO-V (50% and 45%) and higher then DMF-V (20% and 15%), respectively. Finally, kidding rates were also indistinguishable for CF (40%) and DMSO-V (35%), but higher then DMF-V (12.5%). In conclusion, conventional freezing and vitrification using DMSO have similar efficiencies for cryopreservation of goat IVP embryos and cryoprotectant for vitrification affects its outcome.
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Embryo cryopreservation
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e20672 Background: Loss of fertility is a major problem for childhood cancer survivors treated with gonadotoxic therapy. Cryopreservation of testicular tissue is a promising approach to preserve fertility for prepubertal boys, as sperm banking cannot be considered for these patients. Slow freezing (SF) and vitrification are cryopreservation techniques that were successfully applied in several animal models but need further exploration for clinical application. Methods: Fragments of imamature testicular tissue from 6-day-old mice were assigned to one of the following cryopreservation procedures: SF using dimethyl sulfoxide (DMSO) as cryoprotectants, SF using DMSO free cryoprotectant, vitrification using DMSO, vitrification using DMSO free cryoprotectant. Histological and immunohistochemical analyses were performed to evaluate cell viability, intratubular proliferation (Ki-67), apoptosis (caspase-3) after freezing and thawing. Thawed testicular tissue were cultured for in vitro production of mature sperm using previously described organ culture method. The fertility of induced spermatozoa was tested by intracytoplasmic sperm injection (ICSI). Results: Seminiferous tubules showed good integrity after cryopreservation and thawing in SF with DMSO group and vitrification with DMSO free cryoprotectant group. In these groups, cell viability and their ability to proliferate were observed by immunohistochemistry, and mature spermatozoa were induced after 35 days of organ culture. ICSI showed fertility of induced spermatozoa only in vitrification using DMSO free cryoprotectant group. Conclusions: An optimal cryopreservation protocol enhances the chances for successful fertility restoration.The findings of the present study have potential implications for cryobanking of immature testicular tissue and fertility preservation. Vitrification using DMSO free cryoprotectant would be a preferable cryopreservation method for later induction of functional spermatozoa.
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Vitrification
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This study designed to assess the expression level of CATSPER2 and TEKT2 and to evaluate the levels of CatSper2 and Tektin2 proteins in human spermatozoa before and after cryopreservation. One hundred and twenty semen samples were included in this study. All the samples were subjected to qPCR and Western blot analysis. The results showed a significant reduction in the expression levels of CATSPER2 and TEKT2 in the cryopreserved compared to the fresh samples (P = 0.0039 and P = 0.0166, respectively), and the results showed down-regulation in the expression level of CATSPER2 and TEKT2 genes between the study groups. Moreover, the protein levels of the CatSper2 and Tektin2 were lower in cryopreserved samples compared to fresh samples (P = 0.0001). In conclusion, the reduction in the proteins level and expression level of the CATSPER2 and TEKT2 in cryopreserved samples could be used as an indicator of sperm motility loss.
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Objective:To observe the effect of the cryopreservation of human peripheral blood stem cells(PBSC) by -80℃ non rate-controlled freezing. Method:We mobilized and collected peripheral blood stem in common methods ,froze cells with 120 g/L HES and 10% DMSO and 10% human plasma of AB type without rate-controlled and preserved them at -80℃.Typeran blue viability and recovery rate of mononuclear cells (MNC), CD34+cells, colony-forming unit-granulocyte macrophages(CFU-GM) were detected before and after cryopreservation in different periods.Result:Having been cryopreserved from 15 to 720 days at -80℃, there were no statistically significant differences in trypan blue viability and MNC recovery rate (P0.05). Although recovery rate of CD34+ and CFU-GM decreased obviously after cryopreservation for 240 days (P0.05), the recovery rate was 82.5% and 79.6%, respectively.After corresponding pretreatment, twenty one patients received transfusion with -80℃ cryopreserved PBSC which had been stored for 12 to 46 days (mean 29 days) and hematological reconstitution was obtained successfully in 14 to 27 days (mean 20.5days).Conclusion:-80℃ non rate-controlled freezing method could be suitable to human PBSC short-term cryopreservation with the cryoprotectant composed of 60 g/L HES and 5% DMSO and 5% human plasma of AB type.
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Trypan blue
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This study deals with cryopreservation of spermatogonial stem cells (SSCs) of native goat using different media along with cryoprotectant. Morphological differentiation and immunocytochemistry tests were used to identify the cells. Furthermore anti-vimentin and anti-Oct-4 immuno staining methods were used for identification of sertoli cells and SSCs, respectively. Cryopreservation of SSCs was done with two sets of media. One with 40% dulbecco’s modified eagle’s medium (DMEM), 50% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO). The second medium included 90% FBS and 10% DMSO. Post thaw cryopreserved cells were subjected to viability and colony area formation on 4th, 8th and 12th days of culturing after and before cryopreservation. Results clearly indicated the viability, area and number of colonies in the first medium registered to 61.37%, 0.87 mm2and 246.88 averagely in every 25 cm2 culture flask, respectively. Similarly with second medium, post thaw cryopreserved sperm registered viability, area and number of colonies to 73.87%, 2.74 mm2 and 364.36 in every 25 cm2 culture flask, respectively. After the thawing of spermatogonial cells, the best viability percentage was obtained in the freezing medium containing 90% FBS. Thus the study demonstrated that serum concentration had a distinct positive effect on the maintenance and proliferation of SSCs in culture after cryopreservation.
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Fetal bovine serum
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Increasing numbers of childhood cancer survivors reach adulthood making therapy induced infertility a growing concern. Sperm cryopreservation is not possible prior to puberty. Testicular tissue cryopreservation has been proposed as an alternative fertility preservation method for prepubertal males but no standardised cryopreservation procedure for immature tissue has been agreed to date. Here we review the current literature of cryopreservation protocols to determine which method best preserves the morphology and function of immature testicular tissue; and to examine which tissue intervention, grafting or tissue culture, is mostly likely to restore fertility. Embase, Medline, and Web of Science were systematically searched using relevant MeSH headings and search terms for testis, cryopreservation, and fertility preservation. This systematic search returned 4748 unique entries which were screened for relevance. Eleven studies were found to be eligible and were included in the systematic review. We found that cryopreservation protocols differ in freezing rate and cryoprotectant media, the optimum combination of which for ITT has yet to be determined. Further investigations must be carried out to decipher which method best preserves tissue integrity and function and which application method is most likely to induce spermatogenesis.
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Ovarian Tissue Cryopreservation
Embryo cryopreservation
Semen cryopreservation
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