Hum. Reprod. Advance Access originally published online on September 18, 2006
Human Reproduction 2006 21(11):2794-2800; doi:10.1093/humrep/del210
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Novel direct cover vitrification for cryopreservation of ovarian tissues increases follicle viability and pregnancy capability in mice
1 Department of Obstetrics and Gynecology 2 Department of Anatomy and Cell Biology and 3 Department of Pathology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei, Taiwan. E-mail: ysyang{at}ha.mc.ntu.edu.tw
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Abstract |
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BACKGROUND: Cryopreservation of ovarian tissue is valuable for fertility preservation. We develop an innovative vitrification method using less concentrated cryoprotectants and direct application of liquid nitrogen to the ovarian tissue (direct cover vitrification, DCV) to improve its efficiency. METHODS: Ovaries of 5- to 6-week-old C57BL/6J mice were randomly allocated to four groups: DCV, conventional vitrification, slow-freezing and non-frozen controls. Experiment 1: observing the follicle morphology. Experiment 2: assessing viability. Experiment 3: investigating the ultrastructure. Experiment 4: examining the follicle number after grafting. Experiment 5: ascertaining pregnancy potential by allogeneic orthotopic transplantation. RESULTS: The percentages of morphologically normal or viable follicles from DCV were significantly greater than those achieved from conventional vitrification and slow freezing (P < 0.01). The ultrastructure of primordial follicles from DCV appeared better than that achieved from conventional vitrification and slow freezing. After grafting, the follicle number from DCV was greater than conventional vitrification (P = 0.001) and slow freezing (P = 0.021). The pregnancy rate of DCV was higher than conventional vitrification (P < 0.01). The litter size from DCV was comparable with that from non-frozen graft and was significantly greater than that achieved from conventional vitrification and slow freezing (P < 0.01). CONCLUSIONS: DCV is highly efficient for cryopreservation of ovarian tissue. Using less concentrated cryoprotectants appears to reduce toxicity. Direct cover by liquid nitrogen maximizes cooling that could facilitate vitrification and prevent ice crystal injury.
Key words: cryopreservation/ovarian tissue/transplantation/ultrastructure
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Introduction |
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The progress of chemotherapy and radiotherapy has much improved the survival rate of young cancer patients. Yet these treatments are toxic to the gonads. In recent years, cryopreservation technology for mature oocytes has advanced significantly, resulting in better survival, fertilization and pregnancy potential (Porcu et al., 2000
; Fabbri et al., 2001; Chen et al., 2005). While cryopreservation is used to preserve the fertility of female cancer patients (Porcu et al., 2004; Chen et al., 2005), mature oocytes are collected following a period of gonadotrophin stimulation, requiring a delay in the treatment of cancer. Furthermore, mature oocytes are available in limited numbers.
Ovarian tissue cryopreservation has several potential advantages over mature oocyte cryopreservation (Amorim et al., 2003). It preserves both steroidogenic and gametogenic functions. Primordial follicles are present in large numbers in ovarian tissue. The collection of ovarian tissue can be performed independent of the stage of the menstrual cycle, without postponement of cancer treatment. It is also suitable for pre-pubertal girls.
The slow-freezing method using a programmed cryo-machine is traditionally employed for the cryopreservation of embryos, oocytes or ovarian tissues (Trounson and Mohr, 1983; Gosden et al., 1994; Porcu et al., 2000; Donnez et al., 2004; Oktay et al., 2004). These procedures usually take several hours. Vitrification is an alternative method. With high concentrations of cryoprotectants and a faster cooling rate, it transforms cells into an amorphous glassy state, instead of ice crystal formation (Rall and Fahy, 1985; Mazur et al., 1992; Chen et al., 2000a,b). Vitrification is time saving and does not require special equipment. Recently, vitrification has also been applied to ovarian tissues (Sugimoto et al., 2000; Salehnia et al., 2002; Migishima et al., 2003; Yeoman et al., 2005).
Cryovials or tubes are commonly used to hold ovarian tissues for slow freezing or vitrification. They have the advantage of keeping a larger amount of ovarian tissues in physically isolated containers. However, for achieving vitrification of a larger volume of vitrification solution and tissues by plunging the container into liquid nitrogen, the concentration of cryoprotectants must be higher. Thus, toxicity of the vitrification solution also increases. Sugimoto et al. (2000) used 10-mm (outer diameter) glass tubes as containers for vitrification of rat ovarian tissues. They found that the grafts from vitrified ovarian tissues had significantly fewer follicles than did non-frozen grafts. Migishima et al. (2003) used 1-ml cryotube for vitrification of mouse ovarian tissue. They achieved lower litter size for vitrified grafts than that achieved for non-frozen grafts.
To improve the efficiency and efficacy of cryopreservation, we developed a new vitrification method using less concentrated cryoprotectants and direct application of liquid nitrogen to the ovarian tissue (direct cover vitrification, DCV). We hypothesized that less concentrated cryoprotectants would reduce toxicity and that applying liquid nitrogen directly to the ovarian tissue would maximize cooling to facilitate vitrification and prevent ice crystal injury. We systematically examined the effects of DCV on mouse ovarian tissue by histological examination, viability tests, transmission electron microscopy (TEM), heterotopic autograft and orthotopic allograft. We compared these parameters with conventional vitrification and slow freezing.
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Materials and methods |
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Animals
Five- to six-week-old female C57BL/6J mice (black fur) were obtained from the Animal Center of National Taiwan University Hospital. All mice were housed under a 14-h light/10-h dark regime at 22–24°C, with food and water supplied without restriction. After anaesthesia by inhalation of ethyl ether (Nihon Shiyaku Industries, Osaka, Japan), the ovaries were removed through small dorsolateral skin incisions and placed in Dulbecco’s phosphate-buffered solution (DPBS) (Gibco, Grand Island, NY, USA) with 20% fetal bovine serum (FBS) (Sigma, St Louis, MO, USA). The size of an ovary was approximately 1.2 x 1.5 x 1.5 mm. The skin was closed with 9-mm Michel clips (Becton-Dickinson, Lincoin Park, NJ, USA). The Animal Care and Use Committee of our institute approved this study.
DCV
DPBS medium was traditionally used as base media for the preparation of cryoprotectants in our laboratory (Chen et al., 2000a,b, 2005). HEPES–human tubal fluid (HTF) medium or tissue culture medium-199 can also be used as base media (Isachenko et al., 2004; Huang et al., 2005; Takahashi et al., 2005). The ovary was pretreated with an equilibration solution (0.8 ml) consisting of 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethylsulphoxide (DMSO) in DPBS with 20% FBS for 10 min at room temperature. For example, 10 ml of equilibration medium comprised 0.75 ml of EG, 0.75 ml of DMSO and 8.5 ml of DPBS plus 20% FBS medium. It was then transferred to a vitrification solution (0.8 ml) consisting of 15% EG, 15% DMSO and 0.5 M sucrose for 2 min. This vitrification solution was used very successfully in recent publications on blastocyst and oocyte vitrification (Chian et al., 2004; Hiraoka et al., 2004a,b).
The ovary was then placed on a piece of gauze to remove the surrounding vitrification medium. The ovary was put in a 1.8-ml plastic standard cryovial (Nunc, Roskilde, Denmark). Liquid nitrogen was directly applied onto the ovary for vitrification (Figure 1). The cap of the cryovial was closed. The lid did not have a hole. The vial was then placed into a liquid nitrogen tank.
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For thawing, the vial was taken out of liquid nitrogen, and the cap was opened. It took
20 s after opening the vial for all the liquid nitrogen to disappear. Then, the ovary quickly was moved into 1 ml of 1 M sucrose and kept for 5 min. It was serially transferred into 0.5 M, 0.25 M sucrose and DPBS medium for 5 min each. The procedures were carried out in a sterile hood. The ovary was subjected to subsequent examination or transplantation.
Conventional vitrification
The vitrification solution was prepared on the basis of formula used by Salehnia et al. (2002). It was made of DPBS plus 20% FBS containing 30% (w/v) Ficoll 70 (Sigma), 0.5 M sucrose, 10.7% (w/v) acetamide and 40% (v/v) EG. The ovary was transferred to the vitrification solution at room temperature and held for 5 min (Salehnia et al., 2002). It was loaded into a 0.5-ml standard straw (IMV, L’Aigle, France). The straw was placed directly into liquid nitrogen.
For thawing, the straw was warmed in air for 5 s and then in a water bath for 20 s at room temperature. After warming, the contents of the straw were expelled into a Petri dish, and the ovary was serially transferred into 1 ml of 1 M, 0.5 M, 0.25 M sucrose and DPBS medium for 5 min each.
Slow-freezing method
The ovary was put into 1 ml of 1.5 M 1,2-propanediol (PROH) plus 0.1 M sucrose in DPBS with 20% FBS for 25 min. It was then transferred into a 1.8 ml cryovial containing 1 ml of freezing medium of the same solution. The cryovial was transferred into an automated vertical freezer (Kryo 10 series III) (Planer Products, Middlesex, UK). The starting temperature was 22°C. It was slowly reduced to –7°C at a rate of 2°C/min. Ice nucleation was induced manually at –7°C. After holding for 10 min, the cryovial was cooled slowly to –30°C at a rate of 0.3°C/min and then rapidly to –150°C at a rate of 50°C/min. After 10 min of temperature stabilization, the cryovial was transferred into a liquid nitrogen tank.
After thawing, the ovary was placed in 1.0 M PROH and 0.2 M sucrose for 5 min. Then, it was serially transferred to 0.5 M PROH plus 0.2 M sucrose, 0.2 M sucrose and DPBS medium for 5 min each.
Experiment 1: analysis of follicle morphology
To assess the integrity of follicles after freezing and thawing, we examined the follicle morphology by histology. The ovaries removed from female C57BL/6J mice were randomly allocated to four groups: non-frozen controls, DCV, conventional vitrification and slow freezing. Six samples from six different ovaries in each group were examined. The non-frozen controls and specimens after thawing were fixed in Bouin’s solution. The specimens were embedded in paraffin blocks and serially sectioned into 7-µm slices. They were stained with haematoxylin and eosin (H&E) and observed with light microscopy (x400). Follicles were classified as follows: (i) primordial follicles with one layer of flattened granulosa cells surrounding the oocyte; (ii) primary follicles with one layer of cuboid granulosa cells and (iii) secondary follicles with two or three layers of granulosa cells (Gougeon, 1986). Antral follicles were not included in this study.
To avoid counting follicles more than once, in every third section, only follicles with a visible nucleus were counted (Cleary et al., 2001). The definition of normality of follicles was based on the criteria proposed by Lucci et al. (2004). The follicles classified as morphologically normal were those containing an intact oocyte surrounded by well-organized granulosa cells (Figure 2A and B). In contrast, follicles were classified as degenerate if they had one or more of the following aspects: a pyknotic oocyte nucleus, shrunken ooplasm or disorganized granulosa cells (Figure 2C and D).
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Experiment 2: examination of follicle viability
To evaluate the viability of follicles, we used Trypan Blue stain for follicles isolated from frozen–thawed ovarian tissues and non-frozen controls. Five samples from five different ovaries in each group were evaluated. The ovaries in each group were cut into small pieces. They were transferred to a 5-ml centrifuge tube containing 1 ml of HTF medium with 10% FBS plus 200 IU/ml type I collagenase (Sigma). The ovarian tissues were incubated in 5% CO2 at 37°C for 1 h and mechanically disrupted using a Pasteur pipette every 15 min. The digestion was terminated by adding 4 ml of HTF medium with 50% FBS. After centrifuging at 400 g for 5 min, the pellet was resuspended in 100 µl of HTF medium and mixed with 40 µl of 4% Trypan Blue (Sigma). The suspension was spread thinly in a Petri dish and covered with oil for examination with an inverted microscope (x400). Follicles were scored as being alive if the oocyte and the surrounding granulosa cells were clear (Figure 3A and B). Follicles were scored as being non-viable if the oocyte or the surrounding granulosa cells had blue coloration, demonstrating the inability to exclude the dye (Figure 3C and D).
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Experiment 3: investigation of ultrastructure of follicles
To ascertain the ultrastructural changes of follicles, we examined the frozen–thawed and non-frozen specimens by TEM using four samples from four different ovaries in each group. The ovarian tissue was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 h at room temperature. The specimen was washed with the same buffer for 10 min and post-fixed in 1% osmium tetroxide for 1 h. After dehydration in an increasing ethanol concentration, the ovarian tissue was embedded in Epon 812 resin (TABB Laboratories, Aldermaston, UK). Semi-thin sections (1 µm) were stained with 1% toluidine blue for locating the follicles. Ultrathin sections (70 nm) were obtained and mounted on a nickel grid and were stained with alcoholic uranyl acetate together with aqueous lead citrate.
They were examined by TEM at magnifications ranging from x3000 to xl5 000. The ultrastructural examination was focused on the primordial follicle with intact cytoplasmic membrane and nuclear envelope. The normal primordial follicle had a round oocyte tightly enclosed by the surrounding elongated granulosa cells (Figure 4A). Mitochondria with normal cristae and other membranous organelles such as Golgi apparatus and endoplasmic reticulum could be found in the cytoplasm of oocyte (Figure 4B and C). The follicular oocyte with a percentage of swollen mitochondria greater than 30% was defined as abnormal (Figure 4D).
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Experiment 4: autologous transplantation of ovaries beneath the renal capsule
To assess the follicles surviving from freezing–thawing and grafting, we transplanted frozen–thawed and non-frozen ovaries autologously beneath renal capsules. A dorsolateral incision was made into the skin and peritoneum to expose the kidney. The kidney was exteriorized, and a small hole was torn in the capsule. The ovary was then inserted under the capsule. The kidney was returned to its normal anatomical position. The contralateral side was performed in a similar manner. The skin was closed with Michel clips. Two weeks after grafting of ovaries, mice were killed by cervical dislocation. The ovaries were removed and placed in Bouin’s fixative and sectioned for H&E staining and follicle counts. Eight samples from eight different ovaries in each group were used for calculating graft follicle number.
Experiment 5: allogeneic orthotopic transplantation of ovaries
We further investigated the pregnancy potential of frozen–thawed and non-frozen ovaries after transplantation. Female C57BL/6J mice were randomly allocated into the four groups. Non-frozen and frozen–thawed ovaries were orthotopically transplanted into allogeneic female recipient mice of C57BL/6J-Tyrc-2J-J albino strain (Behringer, 1994). Fifteen mice with 30 different ovaries in each group were transplanted. The recipient female mice were oophorectomized by removing ovaries from ovarian bursae before transplantation. Three weeks after grafting, recipients were paired with fertile albino male mice of C57BL/6J-Tyrc-2J-J strain. Mating was confirmed by the presence of a vaginal plug, and then the female was housed individually. If no vaginal plug was seen, females were left in continuous pairings with males for 28 days. The pregnant female mice were allowed to litter, and the size of each litter was recorded. The pups have black eyes and fur (Figure 5).
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Statistics
The percentages of follicles with normal morphology or viability were calculated in each experiment. Follicular counts and litter sizes are reported as means ± SD. The Mann–Whitney U-test was used to compare the study and the control groups. The Bonferroni method was used for multiple comparison adjustments. Comparability between the fecundity of the study groups and control recipients was tested with the Fisher’s exact test.
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Results |
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Follicle morphology
Percentages for normal morphology of various developmental follicle stages after treatment with different freezing methods and non-frozen controls are presented in Table I. The percentages of normal morphology for primordial, primary and secondary follicles in the non-frozen controls were significantly higher than those of the groups of DCV, conventional vitrification and slow freezing. The percentages of normal morphology of primordial, primary and secondary follicles in the DCV group were significantly greater than conventional vitrification and slow freezing.
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Follicle viability
Percentages of viable follicles for frozen–thawed ovarian tissues and non-frozen controls are presented in Table II. The percentage of viability of primordial follicles of the DCV group was comparable with that of non-frozen controls but was significantly greater than that achieved from conventional vitrification and slow freezing. For primary and secondary follicles, the percentages of viability in non-frozen controls were significantly higher than those of the groups of DCV, conventional vitrification and slow freezing. There was significantly greater viability in the DCV group than that achieved from conventional vitrification and slow freezing.
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Ultrastructure of follicles
The results from TEM showed that the 16 primordial follicles observed in non-frozen controls were all ultrastructurally normal. The number of primordial follicles with normal ultrastructure in the DCV group (13/15, 87%) was comparable with non-frozen controls (16/16, 100%) (Figure 4). However, compared with the conventional vitrification (4/15 or 27%) (P = 0.003) and slow-freezing (6/14, 43%) (P = 0.021) groups, the incidence of primordial follicles with normal ultrastructure was higher in the DCV group.
Follicle number after transplantation
The sum of primordial, primary and secondary follicles in each graft represents the follicle number. The mean follicle number of the DCV group (828 ± 141, range 630–1107) was similar to that of non-frozen ovarian grafts (857 ± 156, range 648–1175). The follicle number of the DCV group was significantly greater than that of the conventional vitrification (380 ± 128, range 187–593) (P = 0.001) and slow-freezing (601 ± 162, range 410–879) (P = 0.021) groups.
Pregnancy capability after grafting
The numbers of pregnant recipients and pups derived from grafted non-frozen or frozen–thawed ovaries are summarized in Table III. Overall, 176 pups were delivered, 170 of which had black eyes. Six pups (3.4%) with red eyes were excluded from results. The pregnancy rate of females receiving frozen–thawed ovaries from DCV was similar to that of those receiving non-frozen ovaries. However, the pregnancy rate of females receiving ovaries from the DCV or non-frozen groups was significantly higher than that from conventional vitrification. Pups delivered by recipients in the group of DCV (4.3 ± 2.4) were comparable with the non-frozen group (4.4 ± 2.2). They were significantly greater than those delivered in the groups of conventional vitrification (0.7 ± 1.1) and slow freezing (2.0 ± 1.9).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionAcknowledgementsReferences |
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The DCV cryopreservation method was highly efficient at increasing morphologically normal and viable follicles from cryopreserved ovarian tissue, compared with slow freezing and conventional vitrification. Direct cover of the ovarian tissues with liquid nitrogen maximized the cooling rate, facilitating vitrification and reducing ice crystal formation. When thawing, the vitrified tissues were directly put into a dilution solution that maximized the warming rate to minimize devitrification. After transplantation, the follicular counts and litter size of the DCV group were significantly greater than those of the slow-freezing and conventional vitrification groups. In all experimental groups, a decrease in the percentage of morphologically normal or viable follicles with increased stage of follicular development was observed. The cause for this decrease may be due to the larger size of follicles with increased stage of development that was more difficult for sufficient dehydration.
Using cryoloops instead of straws for vitrification resulted in significant improvement in the cryopreservation of oocytes and blastocysts (Cai et al., 2005; Takahashi et al., 2005). With a small amount of less concentrated cryoprotectants and direct contact with liquid nitrogen, the cryoloop technique achieved high thermal change and reduced the toxicity from the cryoprotectants. However, the cryoloop is not appropriate for handling larger amounts of tissue, such as an ovary. Unlike oocytes or embryos that have to be transferred under a microscope, the ovarian tissues can be manipulated without a microscope. Therefore, we used cryovials for the development of the DCV method that can hold larger amounts of tissue for vitrification, with direct contact by liquid nitrogen. We employed less-concentrated vitrification medium consisting of 15% EG, 15% DMSO and 0.5 M sucrose. This formula of cryoprotectants was used by Chian et al. (2004) for vitrifying bovine oocytes, with a survival rate of 92%. In the conventional vitrification solution, a much higher EG concentration (40%) combined with a polymer like Ficoll was used, which was considered to be more toxic. A clear drop in the outcome using conventional vitrification may be related to the higher concentration of cryoprotectants used in this protocol. We further demonstrated that using the less-concentrated vitrification solution achieved better outcome for ovarian tissues by the DCV method.
Salehnia et al. (2002) applied conventional vitrification for mouse ovarian tissue by plunging a cryovial into liquid nitrogen. The tissue was exposed to a more concentrated vitrification solution. They found that the examination of the ultrastructure of the oocytes of primary follicles in vitrified ovarian tissues revealed increased numbers of swollen mitochondria with disappearance of cristae. In our study, the mitochondrial morphology of oocytes of primordial follicles in the DCV group was comparable with that of the non-frozen controls. The incidence of ultrastructurally abnormal oocytes with swollen mitochondria, indicating cells under stress, was higher in the conventional vitrification and slow-freezing groups than in the DCV group.
Yeoman et al. (2005) performed vitrification of cortical pieces of monkey ovarian tissues suspended in cryoprotectants, by dropping these pieces from a serological pipette into a shallow container of liquid nitrogen. The solid drops were then picked up with forceps and sealed in liquid nitrogen-filled cryovials. They achieved survival rates for ovarian follicles similar to slow freezing. However, the volume of the vitrification solution surrounding the tissue pieces could slow the cooling by liquid nitrogen. It could also reduce the warming rate during thawing. In the mouse model, we verified that the follicle viability and the litter size from the DCV method were significantly greater than those from slow freezing.
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionAcknowledgementsReferences |
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We developed DCV and explored the effects of DCV on mouse ovarian tissue in the five different experiments using histology, viability tests, TEM, heterotopic autograft and orthotopic allograft. The DCV method had greater percentages of morphologically normal follicles in the histological examination, compared with conventional vitrification and slow freezing. In vitro viability tests showed larger survival of follicles in the DCV group. The DCV method also preserved better ultrastructure of oocytes of primordial follicles, indicating less cellular stress. In vivo viability of follicles after autotransplantation beneath the renal capsule revealed higher follicular counts in the DCV group. After allogeneic orthotopic transplantation, the in vivo function of frozen–thawed ovaries treated by DCV was comparable with that of non-frozen ovaries regarding the pregnancy capability and the litter size and was better than that achieved with conventional vitrification and slow freezing. Therefore, the DCV method was highly efficient at increasing follicular survival and pregnancy of cryopreserved mouse ovarian tissues. It also resulted in less ultrastructural injury. The principle of the DCV method allows the cryopreservation of many tissue pieces, and the size of containers may also be increased. The effects of DCV on the ovarian tissues of other species, including humans, deserve further investigation. The possibility of vitrifying pieces of other tissues, such as liver, may merit further studies.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionAcknowledgementsReferences |
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This study was supported in part by grants from the National Science Council (NSC 93-2314-B-002-158) and National Taiwan University Hospital (93A12-5, 95A05) Taipei, Taiwan. The authors are grateful for Dr Chi-Kuang Wang for his technical assistance.
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References |
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|---|
Amorim CA, Goncalves PB, Figueiredo JR. (2003) Cryopreservation of oocytes from pre-antral follicles. Hum Reprod Update 9:119–129.
Behringer R. (1994) Transferring ovaries. In Brigid H, Rosa B, Frank C and Elizabeth L (eds). Manipulating the Mouse Embryo 2nd edn. (Cold Spring Harbor Laboratory Press, New York) pp. 185–186.
Cai XY, Chen GA, Lian Y, Zheng XY, Peng HM. (2005) Cryoloop vitrification of rabbit oocytes. Hum Reprod 20:1969–1974.
Chen SU, Lien YL, Chao KH, Lu HF, Ho HN, Yang YS. (2000a) Cryopreservation of mature human oocytes by vitrification with ethylene glycol in straws. Fertil Steril 74:804–848.[CrossRef][Web of Science][Medline]
Chen SU, Lien YL, Chen HF, Chao KH, Ho HN, Yang YS. (2000b) Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod 15:2598–2603.
Chen SU, Lien YL, Chen HF, Tsai YY, Chang LJ, Yang YS. (2005) Observational clinical follow-up of oocyte cryopreservation using a slow-freezing method with 1,2-propanediol plus sucrose followed by ICSI. Hum Reprod 20:1975–1980.
Chian RC, Kuwayama M, Tan L, Tan J, Kato O, Nagai T. (2004) High survival rate of bovine oocytes matured in vitro following vitrification. J Reprod Dev 50:685–696.[CrossRef][Web of Science][Medline]
Cleary M, Snow M, Paris M, Shaw J, Cox SL, Jenkin G. (2001) Cryopreservation of mouse ovarian tissue following prolonged exposure to an ischemic environment. Cryobiology 42:121–133.[CrossRef][Medline]
Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, Martinez-Madrid B, Van Langendonckt A. (2004) Live birth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 364:1–6.[CrossRef][Medline]
Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S, Flamigni C. (2001) Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 16:411–416.
Gosden RG, Baird DT, Wade JC, Webb R. (1994) Restoration of fertility to oophorectomized sheep by ovarian autografts stored at –196 degrees C. Hum Reprod 9:597–603.
Gougeon A. (1986) Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod 1:81–87.
Hiraoka K, Hiraoka K, Kinutani M, Kinutani K. (2004a) Case report: successful pregnancy after vitrification of a human blastocyst that had completely escaped from the zona pellucida on day 6. Hum Reprod 19:988–990.
Hiraoka K, Hiraoka K, Kinutani M, Kinutani K. (2004b) Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod 19:2884–2888.
Huang CC, Lee TH, Chen SU, Chen HH, Cheng TC, Liu CH, Yang YS, Lee MS. (2005) Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Hum Reprod 20:122–128.
Isachenko V, Montag M, Isachenko E, Nawroth F, Dessole S, van der Ven H. (2004) Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration. Hum Reprod 19:660–665.
Lucci CM, Kacinskis MA, Rumpf R, Bao SN. (2004) Effects of lowered temperatures and media on short-term preservation of zebu (Bos indicus) preantral ovarian follicles. Theriogenology 61:461–472.[CrossRef][Web of Science][Medline]
Mazur P, Cole KW, Hall JW, Schreuders PD, Mahowald AP. (1992) Cryobiological preservation of Drosophila embryos. Science 258:1932–1935.
Migishima F, Suzuki-Migishima R, Song SY, Kuramochi T, Azuma S, Nishijima M, Yokoyama M. (2003) Successful cryopreservation of mouse ovaries by vitrification. Biol Reprod 68:881–887.
Oktay K, Buyuk E, Veeck L, Zaninovic N, Xu K, Takeuchi T, Opsahl M, Rosenwaks Z. (2004) Embryo development after heterotopic transplantation of cryopreserved ovarian tissue. Lancet 363:837–840.[CrossRef][Web of Science][Medline]
Porcu E, Fabbri R, Damiano G, Giunchi S, Fartto R, Ciotti PM, Venturoli S, Flamigni C. (2000) Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol 169:33–37.[CrossRef][Web of Science][Medline]
Porcu E, Fabbri R, Damiano G, Fratto R, Giunchi S, Venturoli S. (2004) Oocyte cryopreservation in oncological patients. Eur J Obstet Gynecol Reprod Biol 113:Suppl 1, S14–S16.
Rall WF and Fahy GM. (1985) Ice-free cryopreservation of mouse embryos at –196°C by vitrification. Nature 313:573–575.[CrossRef][Medline]
Salehnia M, Moghadam EA, Velojerdi MR. (2002) Ultrastructure of follicles after vitrification of mouse ovarian tissue. Fertil Steril 78:644–645.[CrossRef][Web of Science][Medline]
Sugimoto M, Maeda S, Manabe N, Miyamoto H. (2000) Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification. Theriogenology 53:1093–1103.[CrossRef][Web of Science][Medline]
Takahashi K, Mukaida T, Goto T, Oka C. (2005) Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study. Fertil Steril 84:88–92.[CrossRef][Web of Science][Medline]
Trounson A and Mohr L. (1983) Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 305:707–709.[CrossRef][Medline]
Yeoman RR, Wolf DP, Lee DM. (2005) Coculture of monkey ovarian tissue increases survival after vitrification and slow-rate freezing. Fertil Steril 83:1248–1254.
Submitted on April 15, 2006; resubmitted on April 29, 2006; accepted on May 10, 2006.
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Y. Wang, Z. Xiao, L. Li, W. Fan, and S.-W. Li Novel needle immersed vitrification: a practical and convenient method with potential advantages in mouse and human ovarian tissue cryopreservation Hum. Reprod., October 1, 2008; 23(10): 2256 - 2265. [Abstract] [Full Text] [PDF]
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