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Evgenia Isachenko - One of the best experts on this subject based on the ideXlab platform.
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Technologies of Cryoprotectant-free vitrification of human spermatozoa: asepticity as criterion of effectiveness.
Andrology, 2017Co-Authors: Vladimir Isachenko, Gohar Rahimi, Peter Mallmann, Raúl Sánchez, Evgenia IsachenkoAbstract:This review describes the 120-year history of technology for Cryoprotectant-free cryopreservation of human spermatozoa by direct plunging into liquid nitrogen (vitrification). It explains why Cryoprotectant-free vitrification of some human ejaculate samples is better than conventional freezing and vitrification with the presence of Cryoprotectants. Special attention is given to the extremely high viability of viruses, bacteria, and mycoplasmas after Cryoprotectant-free cryopreservation in culture medium and even in distilled water. This increases the potential risk of disease transmission through liquid nitrogen. The concept of asepticity is concretized as an obvious parameter for any medical assisted reproduction technology that includes cooling of cells in liquid nitrogen. The roles of nonpermeating compounds in media for cytoprotectant-free vitrification—carbohydrates, proteins, lipoproteins, antioxidants—are described. This review summarizes relevant data regarding two groups of different current technologies for Cryoprotectant-free vitrification of human spermatozoa: those involving direct contact of spermatozoa with liquid nitrogen and those involving full isolation of these cells from liquid nitrogen (aseptic technologies).
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Cryoprotectant free vitrification of human spermatozoa in large up to 0 5 ml volume a novel technology
Clinical Laboratory, 2011Co-Authors: Vladimir Isachenko, Gohar Rahimi, P Mallmann, R Maettner, Anna M Petrunkina, K Sterzik, R Sanchez, J Risopatron, Ilija Damjanoski, Evgenia IsachenkoAbstract:BACKGROUND The aim of this study was to develop and to test the aseptic technology of Cryoprotectant-free vitrification of human spermatozoa in large volume (for intrauterine insemination). Spermatozoa, vitrified by this technology, are free of permeant Cryoprotectants and are ready for further use immediately after warming without any additional treatment (centrifugation or separation in the gradient for removal of Cryoprotectant). METHODS Each of 52 swim up-prepared ejaculates were divided into three aliquots and distributed into three treatment groups: Group 1: non-treated control; Group 2: spermatozoa cryopreserved by slow conventional freezing with glycerol-containing medium, and Group 3: spermatozoa vitrified in 0.5 mL insemination "French" straws in culture medium with 0.25 M sucrose. Sperm motility 1, 24 and 48 hours after warming, plasma membrane integrity and capacitation-like changes (spontaneous "cryo-capacitation" and acrosome reaction) were assessed after freezing-thawing. RESULTS In contrast to conventional freezing, spermatozoa vitrified with aseptic Cryoprotectant-free technology displayed superior functional characteristics. The motility rate, integrity rates of cytoplasmic, and acrosomal membranes were significantly higher after vitrification than after conventional freezing (76% vs 52%, 54% vs 28% and 44% vs 30%, respectively) (p 0.1). CONCLUSIONS A basic protection from cryo-injury can be achieved for human spermatozoa using the novel technology of aseptic Cryoprotectant-free vitrification in large volumes.
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dna integrity and motility of human spermatozoa after standard slow freezing versus Cryoprotectant free vitrification
Human Reproduction, 2004Co-Authors: Evgenia Isachenko, Vladimir Isachenko, Igor I Katkov, Gohar Rahimi, T Schondorf, P Mallmann, S Dessole, Frank NawrothAbstract:BACKGROUND: In contrast to the technique of conventional freezing, the vitrification of spermatozoa requires high cooling rates (720 000°K/min), which could be damaging for spermatozoa. The aim of our study was to compare slowly frozen and vitrified spermatozoa in terms of their post-thaw DNA integrity and motility. METHODS: Semen samples were prepared according to the routine swim-up technique and divided into aliquots for comparison of fresh, conventionally frozen and vitrified spermatozoa from the same ejaculate in the presence or absence of Cryoprotectants. Spermatozoa motility and DNA integrity were determined. RESULTS: The motility of spermatozoa conventionally (slowly) frozen with a Cryoprotectant was similar to that recorded for spermatozoa vitrified in the absence of Cryoprotectant (47 versus 52%). The DNA integrity was unaffected by the cryopreservation method or presence of Cryoprotectants. CONCLUSION: The vitrification of human spermatozoa in the absence of conventional Cryoprotectants is indeed feasible. The DNA integrity of vitrified sperm is comparable with that shown by standard slow-frozen/thawed spermatozoa, yet the method is quick and simple and does not require special cryobiological equipment.
Outi Hovatta - One of the best experts on this subject based on the ideXlab platform.
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preservation of human ovarian follicles within tissue frozen by vitrification in a xeno free closed system using only ethylene glycol as a permeating Cryoprotectant
Fertility and Sterility, 2013Co-Authors: Mona Sheikhi, Kjell Hultenby, Boel Niklasson, Monalill Lundqvist, Outi HovattaAbstract:Objective To study the preservation of follicles within ovarian tissue vitrified using only one or a combination of three permeating Cryoprotectants. Design Experimental study. Setting University hospital. Donor(s) Ovarian tissue was donated by consenting women undergoing elective cesarean section. Intervention(s) Ovarian tissue was vitrified in closed sealed vials using either a combination of dimethyl sulfoxide, 1,2-propanediol, and ethylene glycol (EG), or only EG as permeating Cryoprotectants. Main Outcome Measure(s) Ovarian tissue was vitrified with the use of two vitrification methods. Tissue from the same donor was used for comparison of two different solutions. The morphology of the follicles was evaluated after vitrification, warming, and culture by light microscopy and transmission electron microscopy. Apoptosis was assessed by immunohistochemistry for active caspase-3 in fresh and vitrified tissue. Result(s) Light and electron microscopic analysis showed equally well preserved morphology of oocytes, granulosa cells, and ovarian stroma when either of the vitrification solutions was used. No apoptosis was observed in primordial and primary follicles. Conclusion(s) Using only EG as a permeating Cryoprotectant in a closed tube gives as good ultrastructural preservation of ovarian follicles as a more complicated system using several Cryoprotectants.
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optimizing cryopreservation of human testicular tissue comparison of protocols with glycerol propanediol and dimethylsulphoxide as Cryoprotectants
Human Reproduction, 2005Co-Authors: Victoria Keros, Kjell Hultenby, Bjorn Rosenlund, Lusine Aghajanova, Lev Levkov, Outi HovattaAbstract:Background Cryopreservation of testicular tissue is an option in fertility preservation for pre-pubertal boys who will lose spermatogenic cells as a result of chemotherapy. We compared three different protocols and Cryoprotectants in cryopreservation of testicular tissue. Methods Testicular tissue obtained from 16 infertile men was evaluated by light microscopy(LM), immunostaining against MAGE-A4, transmission electron microscopy (TEM) and organ culture. Seminiferous tubules (1312) from non-frozen (n = 16) and frozen-thawed samples (n = 34) were studied following cryopreservation using protocols with either 1,2-propanediol (PrOH), glycerol or dimethylsulphoxide (DMSO) as Cryoprotectants. Results Normal structure was seen in 86 +/- 6% (mean +/- SD) of the fresh tissue. After freezing with DMSO, 70 +/- 6% and after PrOH, 37+/-3% of the tubules were judged to be good. When glycerol was used, the structure of the basal compartment of the tubules was severely damaged. The ultrastructure of the cryopreserved samples as revealed by TEM and MAGE-positive spermatogonia confirmed the findings. Cryopreserved Leydig cells maintained their morphology and ability to release testosterone in culture. Conclusion DMSO as a Cryoprotectant (at a 0.7 mol/l concentration) proved to maintain the structure of testicular tissue, especially spermatogonia, after cryopreservation better than PrOH or glycerol.
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cryopreservation of follicles in human ovarian cortical tissue comparison of serum and human serum albumin in the Cryoprotectant solutions
Human Reproduction, 2003Co-Authors: Julius Hreinsson, Kjell Hultenby, Pu Zhang, Marja Liisa Swahn, Outi HovattaAbstract:BACKGROUND: Cryopreservation of follicles in ovarian cortical tissue has been suggested as a method for preserving fertility for young women who need to undergo cytotoxic therapy. Varying compositions of Cryoprotectant solutions have been used to prevent tissue damage during cryopreservation and thawing. We compared human serum [20% (v/v)] and human serum albumin (HSA) (25 mg/ml) in Cryoprotectant solutions containing propanediol and sucrose to evaluate whether serum-free medium could be used for this purpose. METHODS: Biopsies of ovarian cortical tissue were obtained from 23 subjects after informed consent. Fourteen underwent Caesarean section and nine underwent sterilization by laparoscopy. The cortical tissue was cut into pieces of 1-1.5 mm 3 and cryopreserved in Cryoprotectant solutions containing serum or HSA. After thawing, a total of 1318 follicles were analysed using light microscopy, transmission electron microscopy (TEM) or live/dead assay. RESULTS: Viability of the follicles was 99.3% in freshly dissected tissue. After thawing, 65% of the follicles and 75% of the oocytes were viable with serum, and 69 and 74%, respectively, with HSA. No significant differences were observed between results in solutions containing serum versus HSA. TEM showed similar results; however, poor survival of stromal tissue was evident in this analysis. The live/dead assay showed 82% viability after thawing for both groups. No benefits were seen from post-thawing culture for 4 h before histological preparation. CONCLUSIONS: A Cryoprotectant solution containing HSA was equally effective as one containing serum. Good viability of follicles was confirmed when using propanediol and sucrose as Cryoprotectants, with a large number of follicles.
Vladimir Isachenko - One of the best experts on this subject based on the ideXlab platform.
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Technologies of Cryoprotectant-free vitrification of human spermatozoa: asepticity as criterion of effectiveness.
Andrology, 2017Co-Authors: Vladimir Isachenko, Gohar Rahimi, Peter Mallmann, Raúl Sánchez, Evgenia IsachenkoAbstract:This review describes the 120-year history of technology for Cryoprotectant-free cryopreservation of human spermatozoa by direct plunging into liquid nitrogen (vitrification). It explains why Cryoprotectant-free vitrification of some human ejaculate samples is better than conventional freezing and vitrification with the presence of Cryoprotectants. Special attention is given to the extremely high viability of viruses, bacteria, and mycoplasmas after Cryoprotectant-free cryopreservation in culture medium and even in distilled water. This increases the potential risk of disease transmission through liquid nitrogen. The concept of asepticity is concretized as an obvious parameter for any medical assisted reproduction technology that includes cooling of cells in liquid nitrogen. The roles of nonpermeating compounds in media for cytoprotectant-free vitrification—carbohydrates, proteins, lipoproteins, antioxidants—are described. This review summarizes relevant data regarding two groups of different current technologies for Cryoprotectant-free vitrification of human spermatozoa: those involving direct contact of spermatozoa with liquid nitrogen and those involving full isolation of these cells from liquid nitrogen (aseptic technologies).
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Cryoprotectant free vitrification of human spermatozoa in large up to 0 5 ml volume a novel technology
Clinical Laboratory, 2011Co-Authors: Vladimir Isachenko, Gohar Rahimi, P Mallmann, R Maettner, Anna M Petrunkina, K Sterzik, R Sanchez, J Risopatron, Ilija Damjanoski, Evgenia IsachenkoAbstract:BACKGROUND The aim of this study was to develop and to test the aseptic technology of Cryoprotectant-free vitrification of human spermatozoa in large volume (for intrauterine insemination). Spermatozoa, vitrified by this technology, are free of permeant Cryoprotectants and are ready for further use immediately after warming without any additional treatment (centrifugation or separation in the gradient for removal of Cryoprotectant). METHODS Each of 52 swim up-prepared ejaculates were divided into three aliquots and distributed into three treatment groups: Group 1: non-treated control; Group 2: spermatozoa cryopreserved by slow conventional freezing with glycerol-containing medium, and Group 3: spermatozoa vitrified in 0.5 mL insemination "French" straws in culture medium with 0.25 M sucrose. Sperm motility 1, 24 and 48 hours after warming, plasma membrane integrity and capacitation-like changes (spontaneous "cryo-capacitation" and acrosome reaction) were assessed after freezing-thawing. RESULTS In contrast to conventional freezing, spermatozoa vitrified with aseptic Cryoprotectant-free technology displayed superior functional characteristics. The motility rate, integrity rates of cytoplasmic, and acrosomal membranes were significantly higher after vitrification than after conventional freezing (76% vs 52%, 54% vs 28% and 44% vs 30%, respectively) (p 0.1). CONCLUSIONS A basic protection from cryo-injury can be achieved for human spermatozoa using the novel technology of aseptic Cryoprotectant-free vitrification in large volumes.
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dna integrity and motility of human spermatozoa after standard slow freezing versus Cryoprotectant free vitrification
Human Reproduction, 2004Co-Authors: Evgenia Isachenko, Vladimir Isachenko, Igor I Katkov, Gohar Rahimi, T Schondorf, P Mallmann, S Dessole, Frank NawrothAbstract:BACKGROUND: In contrast to the technique of conventional freezing, the vitrification of spermatozoa requires high cooling rates (720 000°K/min), which could be damaging for spermatozoa. The aim of our study was to compare slowly frozen and vitrified spermatozoa in terms of their post-thaw DNA integrity and motility. METHODS: Semen samples were prepared according to the routine swim-up technique and divided into aliquots for comparison of fresh, conventionally frozen and vitrified spermatozoa from the same ejaculate in the presence or absence of Cryoprotectants. Spermatozoa motility and DNA integrity were determined. RESULTS: The motility of spermatozoa conventionally (slowly) frozen with a Cryoprotectant was similar to that recorded for spermatozoa vitrified in the absence of Cryoprotectant (47 versus 52%). The DNA integrity was unaffected by the cryopreservation method or presence of Cryoprotectants. CONCLUSION: The vitrification of human spermatozoa in the absence of conventional Cryoprotectants is indeed feasible. The DNA integrity of vitrified sperm is comparable with that shown by standard slow-frozen/thawed spermatozoa, yet the method is quick and simple and does not require special cryobiological equipment.
Gohar Rahimi - One of the best experts on this subject based on the ideXlab platform.
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Technologies of Cryoprotectant-free vitrification of human spermatozoa: asepticity as criterion of effectiveness.
Andrology, 2017Co-Authors: Vladimir Isachenko, Gohar Rahimi, Peter Mallmann, Raúl Sánchez, Evgenia IsachenkoAbstract:This review describes the 120-year history of technology for Cryoprotectant-free cryopreservation of human spermatozoa by direct plunging into liquid nitrogen (vitrification). It explains why Cryoprotectant-free vitrification of some human ejaculate samples is better than conventional freezing and vitrification with the presence of Cryoprotectants. Special attention is given to the extremely high viability of viruses, bacteria, and mycoplasmas after Cryoprotectant-free cryopreservation in culture medium and even in distilled water. This increases the potential risk of disease transmission through liquid nitrogen. The concept of asepticity is concretized as an obvious parameter for any medical assisted reproduction technology that includes cooling of cells in liquid nitrogen. The roles of nonpermeating compounds in media for cytoprotectant-free vitrification—carbohydrates, proteins, lipoproteins, antioxidants—are described. This review summarizes relevant data regarding two groups of different current technologies for Cryoprotectant-free vitrification of human spermatozoa: those involving direct contact of spermatozoa with liquid nitrogen and those involving full isolation of these cells from liquid nitrogen (aseptic technologies).
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Cryoprotectant free vitrification of human spermatozoa in large up to 0 5 ml volume a novel technology
Clinical Laboratory, 2011Co-Authors: Vladimir Isachenko, Gohar Rahimi, P Mallmann, R Maettner, Anna M Petrunkina, K Sterzik, R Sanchez, J Risopatron, Ilija Damjanoski, Evgenia IsachenkoAbstract:BACKGROUND The aim of this study was to develop and to test the aseptic technology of Cryoprotectant-free vitrification of human spermatozoa in large volume (for intrauterine insemination). Spermatozoa, vitrified by this technology, are free of permeant Cryoprotectants and are ready for further use immediately after warming without any additional treatment (centrifugation or separation in the gradient for removal of Cryoprotectant). METHODS Each of 52 swim up-prepared ejaculates were divided into three aliquots and distributed into three treatment groups: Group 1: non-treated control; Group 2: spermatozoa cryopreserved by slow conventional freezing with glycerol-containing medium, and Group 3: spermatozoa vitrified in 0.5 mL insemination "French" straws in culture medium with 0.25 M sucrose. Sperm motility 1, 24 and 48 hours after warming, plasma membrane integrity and capacitation-like changes (spontaneous "cryo-capacitation" and acrosome reaction) were assessed after freezing-thawing. RESULTS In contrast to conventional freezing, spermatozoa vitrified with aseptic Cryoprotectant-free technology displayed superior functional characteristics. The motility rate, integrity rates of cytoplasmic, and acrosomal membranes were significantly higher after vitrification than after conventional freezing (76% vs 52%, 54% vs 28% and 44% vs 30%, respectively) (p 0.1). CONCLUSIONS A basic protection from cryo-injury can be achieved for human spermatozoa using the novel technology of aseptic Cryoprotectant-free vitrification in large volumes.
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dna integrity and motility of human spermatozoa after standard slow freezing versus Cryoprotectant free vitrification
Human Reproduction, 2004Co-Authors: Evgenia Isachenko, Vladimir Isachenko, Igor I Katkov, Gohar Rahimi, T Schondorf, P Mallmann, S Dessole, Frank NawrothAbstract:BACKGROUND: In contrast to the technique of conventional freezing, the vitrification of spermatozoa requires high cooling rates (720 000°K/min), which could be damaging for spermatozoa. The aim of our study was to compare slowly frozen and vitrified spermatozoa in terms of their post-thaw DNA integrity and motility. METHODS: Semen samples were prepared according to the routine swim-up technique and divided into aliquots for comparison of fresh, conventionally frozen and vitrified spermatozoa from the same ejaculate in the presence or absence of Cryoprotectants. Spermatozoa motility and DNA integrity were determined. RESULTS: The motility of spermatozoa conventionally (slowly) frozen with a Cryoprotectant was similar to that recorded for spermatozoa vitrified in the absence of Cryoprotectant (47 versus 52%). The DNA integrity was unaffected by the cryopreservation method or presence of Cryoprotectants. CONCLUSION: The vitrification of human spermatozoa in the absence of conventional Cryoprotectants is indeed feasible. The DNA integrity of vitrified sperm is comparable with that shown by standard slow-frozen/thawed spermatozoa, yet the method is quick and simple and does not require special cryobiological equipment.
Kjell Hultenby - One of the best experts on this subject based on the ideXlab platform.
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preservation of human ovarian follicles within tissue frozen by vitrification in a xeno free closed system using only ethylene glycol as a permeating Cryoprotectant
Fertility and Sterility, 2013Co-Authors: Mona Sheikhi, Kjell Hultenby, Boel Niklasson, Monalill Lundqvist, Outi HovattaAbstract:Objective To study the preservation of follicles within ovarian tissue vitrified using only one or a combination of three permeating Cryoprotectants. Design Experimental study. Setting University hospital. Donor(s) Ovarian tissue was donated by consenting women undergoing elective cesarean section. Intervention(s) Ovarian tissue was vitrified in closed sealed vials using either a combination of dimethyl sulfoxide, 1,2-propanediol, and ethylene glycol (EG), or only EG as permeating Cryoprotectants. Main Outcome Measure(s) Ovarian tissue was vitrified with the use of two vitrification methods. Tissue from the same donor was used for comparison of two different solutions. The morphology of the follicles was evaluated after vitrification, warming, and culture by light microscopy and transmission electron microscopy. Apoptosis was assessed by immunohistochemistry for active caspase-3 in fresh and vitrified tissue. Result(s) Light and electron microscopic analysis showed equally well preserved morphology of oocytes, granulosa cells, and ovarian stroma when either of the vitrification solutions was used. No apoptosis was observed in primordial and primary follicles. Conclusion(s) Using only EG as a permeating Cryoprotectant in a closed tube gives as good ultrastructural preservation of ovarian follicles as a more complicated system using several Cryoprotectants.
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optimizing cryopreservation of human testicular tissue comparison of protocols with glycerol propanediol and dimethylsulphoxide as Cryoprotectants
Human Reproduction, 2005Co-Authors: Victoria Keros, Kjell Hultenby, Bjorn Rosenlund, Lusine Aghajanova, Lev Levkov, Outi HovattaAbstract:Background Cryopreservation of testicular tissue is an option in fertility preservation for pre-pubertal boys who will lose spermatogenic cells as a result of chemotherapy. We compared three different protocols and Cryoprotectants in cryopreservation of testicular tissue. Methods Testicular tissue obtained from 16 infertile men was evaluated by light microscopy(LM), immunostaining against MAGE-A4, transmission electron microscopy (TEM) and organ culture. Seminiferous tubules (1312) from non-frozen (n = 16) and frozen-thawed samples (n = 34) were studied following cryopreservation using protocols with either 1,2-propanediol (PrOH), glycerol or dimethylsulphoxide (DMSO) as Cryoprotectants. Results Normal structure was seen in 86 +/- 6% (mean +/- SD) of the fresh tissue. After freezing with DMSO, 70 +/- 6% and after PrOH, 37+/-3% of the tubules were judged to be good. When glycerol was used, the structure of the basal compartment of the tubules was severely damaged. The ultrastructure of the cryopreserved samples as revealed by TEM and MAGE-positive spermatogonia confirmed the findings. Cryopreserved Leydig cells maintained their morphology and ability to release testosterone in culture. Conclusion DMSO as a Cryoprotectant (at a 0.7 mol/l concentration) proved to maintain the structure of testicular tissue, especially spermatogonia, after cryopreservation better than PrOH or glycerol.
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cryopreservation of follicles in human ovarian cortical tissue comparison of serum and human serum albumin in the Cryoprotectant solutions
Human Reproduction, 2003Co-Authors: Julius Hreinsson, Kjell Hultenby, Pu Zhang, Marja Liisa Swahn, Outi HovattaAbstract:BACKGROUND: Cryopreservation of follicles in ovarian cortical tissue has been suggested as a method for preserving fertility for young women who need to undergo cytotoxic therapy. Varying compositions of Cryoprotectant solutions have been used to prevent tissue damage during cryopreservation and thawing. We compared human serum [20% (v/v)] and human serum albumin (HSA) (25 mg/ml) in Cryoprotectant solutions containing propanediol and sucrose to evaluate whether serum-free medium could be used for this purpose. METHODS: Biopsies of ovarian cortical tissue were obtained from 23 subjects after informed consent. Fourteen underwent Caesarean section and nine underwent sterilization by laparoscopy. The cortical tissue was cut into pieces of 1-1.5 mm 3 and cryopreserved in Cryoprotectant solutions containing serum or HSA. After thawing, a total of 1318 follicles were analysed using light microscopy, transmission electron microscopy (TEM) or live/dead assay. RESULTS: Viability of the follicles was 99.3% in freshly dissected tissue. After thawing, 65% of the follicles and 75% of the oocytes were viable with serum, and 69 and 74%, respectively, with HSA. No significant differences were observed between results in solutions containing serum versus HSA. TEM showed similar results; however, poor survival of stromal tissue was evident in this analysis. The live/dead assay showed 82% viability after thawing for both groups. No benefits were seen from post-thawing culture for 4 h before histological preparation. CONCLUSIONS: A Cryoprotectant solution containing HSA was equally effective as one containing serum. Good viability of follicles was confirmed when using propanediol and sucrose as Cryoprotectants, with a large number of follicles.