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Vitrification of human germinal vesicle oocytes with new cut standard straw technology, aseptic mode and reduction of cryoprotectants
RBMOnline - Abstracts - WARM: In-vitro embryology: new trends in reproductive medicine
Course and workshop Vitrification methods of cryopreservation of human oocytes and embryos The preservation of fertility in humans: ultrastructural analysis of different approaches Nottola SA1, Camboni A1,2, Macchiarelli G3, Van Langendonckt A2, Demylle D2, Dolmans MM2, MartinezMadrid B2, Correr S1, Donnez J2 1Department of Anatomy, University of Rome ‘La Sapienza’, Rome 00161, Italy; 2Department of Gynecology, Université Catholique de Louvain, 1200, Brussels, Belgium; 3Department of Experimental Medicine, University of L’Aquila, 67100 L’Aquila, Italy Introduction: Cryostorage and subsequent grafting of frozen– thawed isolated ovarian follicles, ovarian tissue fragments or whole ovaries with an intact vascular pedicle are all possible therapeutic approaches now available to preserve and restore fertility in young patients undergoing cytotoxic treatments for severe pathologies such as neoplastic or other systemic diseases. The aim is to evaluate by transmission electron microscopy (TEM) the impact of enzymatic isolation, cryopreservation and grafting on ovarian tissue integrity. Materials and methods: Ovarian tissue was analysed by TEM after the following treatments: (i) Treatment 1: Enzymatic isolation. Eight human ovarian biopsies were processed for follicle isolation by collagenase or liberase enzymatic digestion. 109 isolated human primordial or primary follicles were fixed and processed for TEM. (ii) Treatment 2: Xenografting of cryopreserved ovarian cortical strips. Sixteen human ovarian cortical biopsies were freeze-thawed. Eight freeze-thawed biopsies were grafted in the peritoneum of nude mice and removed after 3 weeks. At three different steps, (fresh, freeze– thawed and grafted tissue), samples were taken and processed for TEM. (iii) Treatment 3: Intact ovary cryopreservation. Three human ovaries were frozen with their vascular pedicle using a passive cooling device. At three different steps (freshly removed ovary, after perfusion with cryoprotectant, and after thawing), samples were taken and processed for TEM. Results: In treatment 1, the majority of isolated follicles were well preserved but surrounded by a discontinuous basement membrane. Some follicles showed ultrastructural signs of atresia, such as patches of heterochromatin in follicular cell nuclei, focal discontinuities at the oolemma–follicular cell interface, irregularities in the nuclear profile of the oocyte, clusters of lipid droplets or small vacuoles and myelin-like structures in the ooplasm. In treatment 2, healthy-looking and altered primordial and primary follicles were found in the three groups. Altered follicles showed degeneration of follicular cells, increased thickness of the basement membrane and oocyte vacuolization or fragmentation. A few growing preantral follicles observed after xenografting were formed by several layers of follicular cells surrounding intact oocytes, in which organelles showed a perinuclear distribution typical of earlier developmental stages. The degree of maturity of neo-vessels in xenografts was also evaluated. In treatment 3, a well-preserved ultrastructure of follicular, stromal and vascular compartments was observed after freeze-thawing. Occasionally, ultrastructural signs of atresia were observed in
some follicles (as described above). Conclusions: TEM allows enhanced evaluation of actual cell structure, providing some accurate information, hardly obtained by histology or other analysis. For example, there was evidence of early signs of atresia in all cellular microdomains, including alterations in follicular basement membrane and follicular celloocyte interactions, vital for follicular maturation. Preservation and maturity of vessels could also be evaluated. Thus, morphofunctional analysis by TEM allows a marked improvement in the evaluation of integrity of human ovarian tissue after different protocols of sampling, cryostorage and grafting. Vitrification of human germinal vesicle oocytes with new cut standard straw technology, aseptic mode and reduction of cryoprotectants Isachenko V Department of Gynecological Endocrinology and Reproductive Medicine, University of Bonn, Bonn, Germany The aim of this study was to compare the viability of human germinal vesicle oocytes subjected to vitrification using cooling by direct submerging of cut standard straws (CSS) in liquid nitrogen versus vitrification by cooling of CSS located inside closed 0.5 ml straws (aseptic system). Oocytes were cryopreserved in CSS by vitrification in ethylene glycol with DMSO, 0.3 M sucrose and osmotic active and neutral nonpermeable cryoprotectants with a five-step exposure in 12% (v:v), 25%, 50% and 100% of vitrification solution for 2 min, 1 min, 1 min and 60 to 90 s, respectively and plunging into liquid nitrogen. In experiment 1, oocytes of group 1 were vitrified using vitrification medium with 20% ethylene glycol with 20% DMSO and 0.3 M sucrose. Oocytes of group 2 were vitrified using vitrification medium with 15% ethylene glycol with 15% DMSO and 0.3 M sucrose. In experiment 2, when vitrification medium included 15% ethylene glycol with 15% DMSO and 0.3 M sucrose, oocytes of group 1 were rapidly cooled at a speed of 20000°C/min by direct plunging of CSS into liquid nitrogen. Oocytes of group 2 were first located into 0.5 ml straws, which were closed at both sides and plunged into liquid nitrogen with a cooling speed of 600°C/min. Oocytes in both experiments (four groups) were rapidly thawed at a speed of 20000°C/min using an identical protocol. Oocytes were subsequently expelled into a graded series of sucrose solutions (0.5, 0.25, 0.12, 0.06 and 0.03) at 2 min interval. In experiment 1, oocytes of both groups (20% ethylene glycol with 20% DMSO and 0.3 M sucrose as well as 15% ethylene glycol with 15% DMSO and 0.3 M sucrose) had the same rate of maturation to metaphase II stage after in-vitro culture: 80% and 79% (P > 0.1). In experiment 2, oocytes of both groups (direct plunging of CSS into liquid nitrogen as well as with complete isolation of vitrification medium from liquid nitrogen) had the same rate of maturation after in-vitro culture: 75% and 77% (P > 0.1). The efficiency of CSS vitrification of germinal vesicle oocytes with decreased concentrations of permeable cryoprotectants and complete isolation of embryos from liquid nitrogen was noted.
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Course and workshop Vitrification methods of cryopreservation of human oocytes and embryos The preservation of fertility in humans: ultrastructural analysis of different approaches Nottola SA1, Camboni A1,2, Macchiarelli G3, Van Langendonckt A2, Demylle D2, Dolmans MM2, MartinezMadrid B2, Correr S1, Donnez J2 1Department of Anatomy, University of Rome ‘La Sapienza’, Rome 00161, Italy; 2Department of Gynecology, Université Catholique de Louvain, 1200, Brussels, Belgium; 3Department of Experimental Medicine, University of L’Aquila, 67100 L’Aquila, Italy Introduction: Cryostorage and subsequent grafting of frozen– thawed isolated ovarian follicles, ovarian tissue fragments or whole ovaries with an intact vascular pedicle are all possible therapeutic approaches now available to preserve and restore fertility in young patients undergoing cytotoxic treatments for severe pathologies such as neoplastic or other systemic diseases. The aim is to evaluate by transmission electron microscopy (TEM) the impact of enzymatic isolation, cryopreservation and grafting on ovarian tissue integrity. Materials and methods: Ovarian tissue was analysed by TEM after the following treatments: (i) Treatment 1: Enzymatic isolation. Eight human ovarian biopsies were processed for follicle isolation by collagenase or liberase enzymatic digestion. 109 isolated human primordial or primary follicles were fixed and processed for TEM. (ii) Treatment 2: Xenografting of cryopreserved ovarian cortical strips. Sixteen human ovarian cortical biopsies were freeze-thawed. Eight freeze-thawed biopsies were grafted in the peritoneum of nude mice and removed after 3 weeks. At three different steps, (fresh, freeze– thawed and grafted tissue), samples were taken and processed for TEM. (iii) Treatment 3: Intact ovary cryopreservation. Three human ovaries were frozen with their vascular pedicle using a passive cooling device. At three different steps (freshly removed ovary, after perfusion with cryoprotectant, and after thawing), samples were taken and processed for TEM. Results: In treatment 1, the majority of isolated follicles were well preserved but surrounded by a discontinuous basement membrane. Some follicles showed ultrastructural signs of atresia, such as patches of heterochromatin in follicular cell nuclei, focal discontinuities at the oolemma–follicular cell interface, irregularities in the nuclear profile of the oocyte, clusters of lipid droplets or small vacuoles and myelin-like structures in the ooplasm. In treatment 2, healthy-looking and altered primordial and primary follicles were found in the three groups. Altered follicles showed degeneration of follicular cells, increased thickness of the basement membrane and oocyte vacuolization or fragmentation. A few growing preantral follicles observed after xenografting were formed by several layers of follicular cells surrounding intact oocytes, in which organelles showed a perinuclear distribution typical of earlier developmental stages. The degree of maturity of neo-vessels in xenografts was also evaluated. In treatment 3, a well-preserved ultrastructure of follicular, stromal and vascular compartments was observed after freeze-thawing. Occasionally, ultrastructural signs of atresia were observed in
some follicles (as described above). Conclusions: TEM allows enhanced evaluation of actual cell structure, providing some accurate information, hardly obtained by histology or other analysis. For example, there was evidence of early signs of atresia in all cellular microdomains, including alterations in follicular basement membrane and follicular celloocyte interactions, vital for follicular maturation. Preservation and maturity of vessels could also be evaluated. Thus, morphofunctional analysis by TEM allows a marked improvement in the evaluation of integrity of human ovarian tissue after different protocols of sampling, cryostorage and grafting. Vitrification of human germinal vesicle oocytes with new cut standard straw technology, aseptic mode and reduction of cryoprotectants Isachenko V Department of Gynecological Endocrinology and Reproductive Medicine, University of Bonn, Bonn, Germany The aim of this study was to compare the viability of human germinal vesicle oocytes subjected to vitrification using cooling by direct submerging of cut standard straws (CSS) in liquid nitrogen versus vitrification by cooling of CSS located inside closed 0.5 ml straws (aseptic system). Oocytes were cryopreserved in CSS by vitrification in ethylene glycol with DMSO, 0.3 M sucrose and osmotic active and neutral nonpermeable cryoprotectants with a five-step exposure in 12% (v:v), 25%, 50% and 100% of vitrification solution for 2 min, 1 min, 1 min and 60 to 90 s, respectively and plunging into liquid nitrogen. In experiment 1, oocytes of group 1 were vitrified using vitrification medium with 20% ethylene glycol with 20% DMSO and 0.3 M sucrose. Oocytes of group 2 were vitrified using vitrification medium with 15% ethylene glycol with 15% DMSO and 0.3 M sucrose. In experiment 2, when vitrification medium included 15% ethylene glycol with 15% DMSO and 0.3 M sucrose, oocytes of group 1 were rapidly cooled at a speed of 20000°C/min by direct plunging of CSS into liquid nitrogen. Oocytes of group 2 were first located into 0.5 ml straws, which were closed at both sides and plunged into liquid nitrogen with a cooling speed of 600°C/min. Oocytes in both experiments (four groups) were rapidly thawed at a speed of 20000°C/min using an identical protocol. Oocytes were subsequently expelled into a graded series of sucrose solutions (0.5, 0.25, 0.12, 0.06 and 0.03) at 2 min interval. In experiment 1, oocytes of both groups (20% ethylene glycol with 20% DMSO and 0.3 M sucrose as well as 15% ethylene glycol with 15% DMSO and 0.3 M sucrose) had the same rate of maturation to metaphase II stage after in-vitro culture: 80% and 79% (P > 0.1). In experiment 2, oocytes of both groups (direct plunging of CSS into liquid nitrogen as well as with complete isolation of vitrification medium from liquid nitrogen) had the same rate of maturation after in-vitro culture: 75% and 77% (P > 0.1). The efficiency of CSS vitrification of germinal vesicle oocytes with decreased concentrations of permeable cryoprotectants and complete isolation of embryos from liquid nitrogen was noted.
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