Human Reproduction, Vol. 16, No. 11, 2350-2356,
November 2001
© 2001 European Society of Human Reproduction and Embryology
Vitrification of mouse oocytes using closed pulled straws (CPS) achieves a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws (OPS) and grids
Department of Obstetrics and Gynecology, College of Medicine and The Hospital, National Taiwan University, Taipei, Taiwan
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Abstract |
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BACKGROUND: We modified the loading of pulled straws into a new closed system, called closed pulled straws (CPS) for holding oocytes for vitrification. The morphological survival, dynamics of meiotic spindles, and fertilization in vitro of vitrified oocytes using CPS were compared with conventional straws, open pulled straws (OPS), and grids. METHODS: Surviving oocytes were stained for spindles and chromosomes after 1, 2 and 3 h incubations, and compared with controls. The capacity of fertilization and embryonic cleavage were examined in vitro. RESULTS: The survival rates of the CPS (79%) and straw (77%) groups were significantly higher (P < 0.05) than the OPS (63%) and grid (39%) groups. At a 1h incubation, vitrified oocytes of four groups had significantly fewer normal spindles than controls (P < 0.05). The straw group was inferior to the others in spindle morphology (P < 0.05). After a 3 h incubation, recovery of vitrified oocytes with normal spindles was significantly improved in all groups (P < 0.05). The percentages of fertilization and blastocyst formation of vitrified oocytes after a 1 h incubation was significantly lower than controls (P < 0.05), but they were improved after 2 or 3 h incubations (P < 0.05). CONCLUSIONS: Oocytes vitrified using CPS, OPS or grids could lessen spindle injuries and expedite recuperation. The survival using OPS or grids is lower. Sufficient culture time for recovery of meiotic spindle would be imperative for fertilization events of vitrified oocytes. CPS has the advantages of achieving a high survival and preserving good spindles.
Key words: closed pulled straws/meiotic spindle/mouse oocytes/oocyte vitrification
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The cryopreservation of human oocytes is an important part of infertility treatment (Gook et al., 1994
; Porcu et al., 1997). Embryo cryopreservation is now a successful procedure, but oocyte cryopreservation achieves poorer results (Mandelbaum et al., 1998; Porcu et al., 1998; Emiliani et al., 1999). This has primarily been ascribed to low rates of survival, fertilization, and development of cryopreserved oocytes (Trounson and Kirby, 1989; Gook et al., 1995). The vitrification method of cryopreservation of oocytes, as shown in mammalian experiments (Martino et al., 1996; Vajta et al., 1998), seems to have great potential. Otoi and co-workers vitrified bovine oocytes in conventional straws, and achieved results better than those with the slow freezing method (Otoi et al., 1998). Nevertheless, the value of vitrification for human oocytes remains unclear (Hunter et al., 1995; Hong et al., 1999).
We had examined the effect of vitrification with ethylene glycol (EG)-based cryoprotectants on human oocytes in conventional straws, and found a high survival rate after dilution (Chen et al., 2000a). The morphological survival rate appeared to be greater than that of the slow freezing method reported in the literature (Tucker et al., 1998). The rates of normal fertilization and early cleavage of vitrified oocytes were comparable with those of the controls (Chen et al., 2000a). However, the subsequent development and blastocyst formation seemed impaired. Even so, considering the high rate of survival and the early development of vitrified oocytes, further studies were warranted to look for ways to improve developmental capacity.
Martino and colleagues found that chilling damage occurred very quickly with bovine oocytes and that this could affect the developmental potential. To obtain more rapid cooling and warming rates, they put oocytes on electron microscope grids for vitrification, and found a higher percentage of blastocyst formation than that achieved using conventional straws (Martino et al., 1996). Vajta and co-workers developed open pulled straws (OPS) to hold bovine oocytes in a very small amount of vitrification solution (Vajta et al., 1998). They reported that the pregnancy potential of vitrified oocytes was improved, compared with those using conventional straws. The advantages and disadvantages of conventional straws, OPS, or grids for oocyte vitrification deserve further study.
The microtubules of oocytes are vulnerable to cryoprotectants and to temperature change (Pickering et al., 1990; Gook et al., 1993; Van Blerkom and Davis, 1994). Damage to the meiotic spindle may impair fertilization and the development of embryos (Eroglu et al., 1998). In mouse oocytes, we found that vitrified oocytes exhibited serious disturbances of the microtubules immediately after dilution (Chen et al., 2000b). After a 1 h incubation, the microtubules could repolymerize so that oocytes vitrified using OPS recovered a significantly higher percentage of normal spindles than those using conventional straws. However, the morphological survival rate with OPS was lower. More research should be done to improve both the survival rate and the microtubules of vitrified oocytes.
Accordingly, we attempted to modify the loading of pulled straws into a closed system, called closed pulled straws (CPS). CPS has the characteristics of OPS as a rapid thermal change method, and of conventional straws as being a non-contact mode. The process of repolymerization of microtubules for cryopreserved oocytes may be time-dependent (Aigner et al., 1992). Whether more spindles of vitrified oocytes can be recovered if they incubate longer post-dilution deserves further investigation. In this study, we explored the effects of vitrification of mature mouse oocytes using CPS, conventional straws, OPS or grids on the morphological survival post-dilution and the meiotic spindles after 1, 2 and 3 h incubations. The capacity of fertilization and embryonic development were examined in vitro.
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Materials and Methods |
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Preparation of oocytes
Female ICR mice were injected with gonadotrophins to induce them to superovulate (Chen et al., 2000b
). The oviducts were excised, and the cumulus-oocyte-complexes were collected in human tubal fluid (HTF) medium. The granulosa cells were removed by pipetting in HTF medium containing 80 IU/ml hyaluronidase (Sigma, St Louis, MO, USA). Mature oocytes with the first polar body were collected for the experiments. They were cultured with HTF medium containing 0.5% bovine serum albumin (BSA; Sigma) in an atmosphere of 5% CO2 in air at 37°C. The oocytes were randomly allocated to five groups: vitrification in CPS, in conventional straws, in OPS, on grids, and untreated controls.
Preparation of pretreatment, vitrification and dilution solutions
The solutions for pretreatment, vitrification, and dilution were prepared using Dulbecco's phosphate-buffered saline (DPBS) (Gibco, Grand Island, NY, USA) plus 20% fetal cord serum. The fetal cord serum was made of heat-inactivated serum of umbilical cord blood that was collected after delivery with the mother's permission. The pretreatment solution contained 1.5 mol/l EG (Sigma). The vitrification solution consisted of 5.5 mol/l EG and 1.0 mol/l sucrose (EG5.5) (Ali and Shelton, 1993). The solutions for dilution contained 0.5, 0.25, and 0.125 mol/l sucrose.
Manufacture of the pulled straws
The 0.25-ml plastic straws (I.V.M., l'Aigle, France) were heat-softened over a hot plate and pulled manually. The pulled straws were cut at the tapered end with a blade. The inner diameter of the tip was 0.8 mm with a wall thickness of ~0.07 mm (Vajta et al., 1998).
Vitrification of oocytes in CPS
The oocytes (4–6 at a time) were pretreated with 1.5 mol/l EG for 5 min. They were transferred into a drop (200 µl) of EG5.5 on a dish and mixed for equilibration. Then they were transferred to another drop (200 µl). The tip of the pulled straw was loaded with 2 mm of vitrification medium, 2 mm of air, 2 mm of vitrification medium containing oocytes, 2 mm of air, and 2 mm of vitrification medium using a syringe (Figure 1A,B). The vitrification medium containing oocytes was isolated by two small segments of air and medium. Through this closed loading system of CPS, the oocytes will not directly contact with liquid nitrogen, which may occur with OPS. The procedures were performed at a room temperature of 22–24°C. The total exposure to EG5.5 lasted for 1 min. They were then plunged into liquid nitrogen for cooling and storage.
After storage from 1 h to 5 days, the CPS was removed from the liquid nitrogen for warming. The opposite end of the pulled straw was sealed using the index finger (Figure 1C). The content was then expelled into a drop of 0.5 mol/l sucrose (400 µl) by using the increase in air pressure in the tube caused by the thermal change. Next, the oocytes were transferred into 0.5, 0.25 and 0.125 mol/l sucrose solutions in a 4-well dish. They spent 2.5 min in each solution. Considering the adverse effect of room temperature on oocytes (Pickering et al., 1990), we operated the extraction steps in a modified incubator at 37°C. The oocytes were then washed, transferred into the culture medium, and incubated.
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Vitrification of oocytes in conventional straws
The oocytes were pretreated with 1.5 mol/l EG and exposed to EG5.5 as above. Using a syringe, the 0.25-ml straw was filled with 1 cm of vitrification medium, 0.5 cm of air, 2 cm of vitrification medium containing oocytes, 0.5 cm of air, and 3.5 cm of vitrification medium. We did not seal the other end of the straw with powder or a plug (Chen et al., 2000b
). It was plunged into liquid nitrogen for cooling and storage. For warming, the straw was taken out, held in the air for 5 s, and then plunged into 37°C water for 10 s. It was cut with scissors and the contents containing the oocytes were expelled into 0.5 mol/l sucrose. The oocytes were diluted in stepwise sucrose solutions and incubated in culture medium.
Vitrification of oocytes in OPS
After pretreatment with 1.5 mol/l EG and exposure to EG5.5, loading into the tip of the pulled straw was done through the capillary effect by simply touching a microdrop (1–2 µl) of vitrification solution containing oocytes (Vajta et al., 1998). Plunging the OPS into liquid nitrogen performed the cooling and storage. At warming, the tip of OPS was put into 0.5 mol/l sucrose solution, and the oocytes were expelled. They were then diluted and incubated.
Vitrification on the electron microscope copper grids
The oocytes were pretreated with 1.5 mol/l EG, and mixed with EG5.5. They were then transferred onto the grid (Agar Scientific Ltd., Essex, England) in very small amounts (Martino et al., 1996). To reduce the volume of vitrification solution, the underside of the grid was blotted on a millicell membrane (Millipore, Bedford, MA, USA). The grids were plunged into liquid nitrogen using fine forceps, and stored there. At warming, the grids were picked up and put into the stepwise dilution solutions. Finally, the oocytes were transferred to culture medium.
Definition of morphological survival
Oocytes were defined as having morphologically survived if the cells possessed an intact zona pellucida and plasma membrane and refractive cytoplasm. They were counted and recorded.
Fluorescent staining of meiotic spindles and chromosomes
The morphologically surviving oocytes of the four treatment groups were examined for spindles and chromosomes after 1, 2 and 3 h incubations. The control oocytes were also tested for comparisons. The procedures for fixing and staining have been described in detail previously (Chen at al., 2000b). In brief, oocytes were preserved in 2% formaldehyde (Merck, Darmstadt, Germany) with 0.02% Triton X-100 (Merck) at 37°C for 30 min. They were then incubated with anti-
-tubulin monoclonal antibody (Sigma) in DPBS with 0.5% BSA for 45 min and washed in 0.01% Tween-20 (Merck) for 15 min. Tubulin was stained by fluorescein isothiocyanate (FITC) conjugated anti-mouse IgG (Sigma) for 45 min, and Hoechst 33258 (20 µg/ml) (Sigma) stained the chromatin. Excess antibody and dye were washed out in 0.01% Tween-20 for 15 min. The oocytes were transferred into DPBS with 0.5% BSA for 60 min and then wet mounted.
Observation of spindles and chromosomes
Fluorescence was observed using an Optiphot microscope with a magnification of 400x (Nikon, Tokyo, Japan). A Nikon filter of set B-2A for the wavelength of 450–490 nm was employed for the FITC green signal of the spindle. A UV-2A filter for the wavelength of 330–380 nm was used to search for the Hoechst 33258 blue fluorescence of chromosomes. The microscope was equipped with a digital imaging camera, and pictures were acquired and processed on a computer (Chen et al., 2000b).
Assessment of spindle morphology and chromosome arrangement
Normal spindle morphology was barrel-shaped with microtubules traversing between both poles and chromosomes. The metaphase chromosomes aligned regularly in a compact group along the equatorial plane (Figure 2A,C). Abnormal spindle morphology included a reduction in the number of microtubules or the size (Figure 2B), disruption, or the complete absence of a spindle (Frydman et al., 1997). Dispersion of chromosomes was defined as abnormal (Figure 2D).
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In-vitro fertilization and culture
Spermatozoa were obtained from mature ICR male mice. The vas deferens and cauda epididymides were dissected, and the spermatozoa were released into 1 ml pre-equilibrated Whittingham's medium (Whittingham, 1971
) with 3% BSA for 15min at 37°C. After dispersion, the concentration was adjusted to a final value of 1–2x106 spermatozoa/ml. The insemination dishes were then incubated for 2 h to capacitate spermatozoa before the addition of oocytes. The control oocytes and surviving oocytes of the four treatment groups after 1, 2 and 3 h incubations were transferred into the individual insemination medium. After 4 h of culture with spermatozoa, the oocytes were washed and then cultured in HTF medium with 0.4% BSA and 0.01 mmol/l ethylenediamine-tetraacetic acid (EDTA, Sigma). Oocytes were judged to be fertilized by the presence of two uniform blastomeres with two definite nuclei and a second polar body at 24 h after insemination (Fraser and Drury, 1975). The resulting embryos were examined daily for the extent of developmental progression to the blastocyst stages for 4 days.
Statistics
The morphological survival rates of oocytes in the four treatment groups were calculated. The patterns of the meiotic spindles and chromosomes after 1, 2 and 3 h incubations were analysed for the five groups. The percentages of fertilization and blastocyst formation were compared. A contingency table analysis was performed with several rows and columns for overall difference prior to comparisons between individual groups. If it was significant, 
2 or Fisher-exact test was then carried out for comparisons of groups two by two. A P value < 0.05 was considered statistically significant.
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Results |
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After dilution, the morphological survival rates of vitrified mouse oocytes using CPS, conventional straws, OPS or grids are shown in Table I
. The oocytes that further degenerated during incubation were included in the non-surviving group. The survival rates of the CPS and conventional straw groups were significantly higher than those of the OPS and grid groups (P < 0.05). There was no obvious difference between the former two groups. Oocytes vitrified using grids (61%) had significantly more morphological damage than those using OPS (37%) (P < 0.001).
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The spindle morphology and chromosomal patterns of vitrified oocytes of the four groups after a 1 h incubation post-dilution and controls are presented in Table II
. The oocytes from the CPS, conventional straw, OPS or grid groups had significantly lower percentages of normal spindles than the control group (P < 0.05). In addition, oocytes vitrified using the conventional straw had a significantly smaller percentage of normal spindles than those using CPS, OPS or grids (P < 0.05). Differences among the CPS, OPS, or grid groups were not apparent. The majority of abnormal patterns consisted of reduced spindles. Compared with the controls, the vitrified oocytes of the four groups had more chromosomal dispersion, but only the difference in the conventional straw group was significant (P < 0.05).
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After a 2 h post-dilution incubation, more vitrified oocytes of the four groups recovered normal spindles than those with the 1 h incubation (Table III
). The percentages of oocytes with normal spindles in the CPS, conventional straw, OPS and grid groups were lower than the controls, however the differences were not significant. The percentage of compact chromosomes of vitrified oocytes increased with a 2 h incubation. The CPS, OPS and grid groups had a higher percentage of compact chromosomes similar to the controls and higher than the conventional straw group, but the difference was not significant.
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With a 3 h post-dilution incubation, significantly more vitrified oocytes with normal spindles were recovered in the four groups than after the one-hour incubation (Table IV
). The percentage of oocytes with normal spindles from the CPS, OPS, and grid groups were comparable with that of the controls. The conventional straw group had more abnormal spindles than other groups, but the difference was not significant. The percentage of compact chromosomes was also increased for the vitrified oocytes following a 3 h incubation period. Although the conventional straw group had a slightly smaller percentage of compact chromosomes than the other groups, this was significantly better than the 1 h incubation. The spindle morphology and chromosomal patterns of control oocytes did not change during the 3 h incubation.
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The outcome of IVF and embryonic development of vitrified oocytes after 1, 2 and 3 h incubations and controls are shown in Table V
. The vitrified oocytes from the CPS, conventional straw, OPS and grid groups after a 1 h incubation had significantly lower percentages of fertilization and blastocyst formation than controls. The differences among the CPS, OPS and grid groups were not significant. At 1 h incubation, the percentage of fertilization of the conventional straw group was significantly lower than the CPS group. After 2 or 3 h incubations, the percentages of fertilization of vitrified oocytes were greater than 1 h incubation in that the CPS, conventional straw, and OPS groups showed significant improvements. In comparison with 1 h incubation, the percentages of blastocyst formation of vitrified oocytes after 2 or 3 h incubations were increased in that the conventional straw and OPS groups showed significant improvements.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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In this study, we found that mouse oocytes vitrified using OPS or grids had a smaller chance of morphological survival than those vitrified using conventional straws or CPS. Furthermore, oocytes vitrified on grids had more morphological injuries than those in OPS. It is possible that direct contact with liquid nitrogen on grids or a portion of the oocytes in OPS may have a negative effect on oocyte survival. In addition to damage caused by different ways of holding oocytes, the damage to oocytes during vitrification has been demonstrated to be dependent on the interaction of all the steps, including the rate of increase of exposure of the cells to the cryoprotectants by mixing them repetitively, the temperature and time of exposure, and methods of dilution (O'Neil et al., 1997
). Finding the most appropriate conditions for vitrification of oocytes deserves further study.
Vitrified oocytes revealed severe disturbances of microtubules immediately post-dilution (Chen et al., 2000b). During the incubation period, the microtubules could repolymerize and start to restore normal spindles. In the present study, we verified that vitrified oocytes recovered more normal spindles after 2 or 3 h incubations, a significant improvement over 1 h incubation periods. In addition, we demonstrated that oocytes vitrified in CPS, OPS, or grids had more normal spindles than conventional straws at 1 h after thawing. The former also had more compact chromosomes than the latter did. Appropriate organization of microtubules is indispensable for the alignment of chromosomes, and disorganization of the spindle may result in chromosome dispersion.
Oocytes held in a pulled straw or on a grid for vitrification achieve a faster cooling and warming rate (a theoretical rate of 20 000°C/min and 180 000°C/min respectively) than those in conventional straws (2500°C/min) (Rall and Fahy, 1985; Martino et al., 1996; Vajta et al., 1998). Moreover, oocytes in a small amount of vitrification solution can be directly warmed and immediately diluted into the dilution solution. That reduces exposure to unsuitable temperatures and concentrated cryoprotectants. In contrast, the conventional straw is warmed in water and then cut with scissors. The oocytes in a larger vitrification volume are expelled into the dilution solution and then placed into another dilution solution. This allows more time to pass through the inappropriate conditions (Chen et al., 2000b). That may explain why vitrification of oocytes using CPS, OPS, or grids preserves spindles better than conventional straws. It may also partly explain the findings of others (Martino et al., 1996; Vajta et al., 1998) that the developmental competence of vitrified bovine oocytes could be enhanced using OPS or grids, compared with conventional straws.
Chung and co-workers used grids for vitrifying human oocytes, and found that the culture time before vitrification did not significantly affect embryo development after thawing, but that the best results were obtained in the longest culture period group (Chung et al., 2000). The visual changes in spindles after vitrification were linked with functional effects of oocytes on fertilization and development (O'Neil et al., 1997). It has been found that insemination of oocytes, cryopreserved by a slow freezing method immediately after thawing caused impairment of fertilization and cytoskeletal dynamics post-fertilization that was improved by insemination after a 1 h incubation (Eroglu et al., 1998). A sufficient restoration of cytoskeleton after incubation would be critical for fertilization events (Joly et al., 1992). In our study, the vitrified oocytes at 1 h incubation had significantly lower percentages of fertilization and blastocyst formation than controls. After 2 or 3 h incubations, the fertilization outcome of vitrified oocytes was improved in a way that appeared compatible with the recovery of spindles. The timing of insemination of human cryopreserved oocytes varies in the literature, ranging from 1–4 h post-dilution (Porcu et al., 1997; Tucker et al., 1998; Young et al., 1998; Kuleshova et al., 1999). The most appropriate time for insemination may be related to the method of cryopreservation and the recovery of spindles after dilution. This deserves further study.
Achieving faster cooling, warming, and dilution, mouse oocytes vitrified using CPS, OPS, or grids could alleviate the injury of spindles and accelerate their recuperation, compared with conventional straws. The effect on the spindle and chromosome organization for the former three methods appears similar. However, the number of oocytes in each processed group is too low to draw a conclusion. The morphological survival of oocytes using OPS or grids is lower. Therefore, CPS has the advantages of achieving a high survival rate and preserving good spindles. The grid, OPS, CPS, and even the conventional straw for storing the oocytes in liquid nitrogen have a potential risk of infection (Kuleshova and Shaw, 2000). For any clinical application of the method the aseptic condition and avoidance of contamination should be specially planned. Recently, several successful pregnancies from vitrified human oocytes using OPS or grids have been reported in the literature (Hong et al., 1999; Kuleshova et al., 1999). Vitrification of human oocytes using CPS regarding the survival, timing of insemination, and potential of pregnancy may deserve further study. The value of CPS for vitrification or ultra-rapid freezing of embryos may also merit investigations.
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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This study was supported in part by grants from the National Science Council (NSC 89-2314-B-002-263), Taipei, Taiwan. The authors are grateful to Ms Ju-Cheng Chang, Ms I-Ching Chen, Ms Li-Jung Chang, Ms Yi-Yi Tsai, and Dr Ko-Chen Lin for their technical assistance.
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Notes |
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1 Department of Obstetrics and Gynecology, College of Medicine and The Hospital, National Taiwan University, Taipei, Taiwan1To 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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References |
|---|
|
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Aigner, S., Van der Elst, J., Siebzehnrubl, E. et al. (1992) The influence of slow and ultra-rapid freezing on the organization of the meiotic spindle of the mouse oocyte. Hum. Reprod., 7, 857–864.
Ali, J. and Shelton, J.N. (1993) Vitrification of preimplantation stages of mouse embryos. J. Reprod. Fertil., 98, 459–65.
Chen, S.U., Lien, Y.L., Chao, K.H. et al. (2000a) Cryopreservation of mature human oocytes by vitrification with ethylene glycol in straws. Fertil. Steril., 74, 804–808.[Web of Science][Medline]
Chen, S.U., Lien, Y.L., Chen, H.F. et al. (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.
Chung, H.M., Hong, S.W., Lim, J.M. et al. (2000) In-vitro blastocyst formation of human oocytes obtained from unstimulated and stimulated cycles after vitrification at various maturational stages. Fertil. Steril., 73, 545–551.[Web of Science][Medline]
Emiliani, S., Van den Bergh, M., Vannin, A.S. et al. (1999) The outcome of cryopreserved human embryos after intracytoplasmic sperm injection and traditional IVF. J. Assist. Reprod. Genet., 16, 405–409.[Web of Science][Medline]
Eroglu, A., Toth, T.L. and Toner, M. (1998) Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes. Fertil. Steril., 69, 944–957.[Web of Science][Medline]
Fraser, L.R. and Drury, L.M. (1975) The relationship between sperm concentration and fertilization in vitro of mouse eggs. Biol. Reprod., 13, 513–518.[Abstract]
Frydman, N., Selva, J., Bergere, M. et al. (1997) Cryopreserved immature mouse oocytes: a chromosomal and spindle study. J. Assist. Reprod. Genet., 14, 617–623.[Web of Science][Medline]
Gook, D.A., Osborn, S.M. and Johnston, W.I.H. (1993) Cryopreservation of mouse and human oocytes using, 1,2-propanediol and the configuration of the meiotic spindle. Hum. Reprod., 8, 1101–1109.
Gook, D.A., Osborn, S.M., Bourne, H. and Johnston, W.I.H. (1994) Fertilisation of human oocytes following cryopreservation: normal karyotypes and absence of stray chromosomes. Hum. Reprod., 9, 684–691.
Gook, D.A., Schiewe, M.C., Osborn, S.M. et al. (1995) Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol. Hum. Reprod., 10, 2637–2641.
Hong, S.W., Chung, H.M., Lim, J.M. et al. (1999) Improved human oocyte development after vitrification: a comparison of thawing methods. Fertil. Steril., 72, 142–146.[Web of Science][Medline]
Hunter, J.E., Fuller, B.J., Bernard, A. et al. (1995) Vitrification of human oocytes following minimal exposure to cryoprotectants; initial studies on fertilization and embryonic development. Hum. Reprod., 10, 1184–1188.
Joly, C., Bchini, O., Boulekbache, H. et al. (1992) Effects of 1,2-propanediol on the cytoskeletal organization of the mouse oocyte. Hum. Reprod., 7, 374–378.
Kuleshova, L.L. and Shaw J.M. (2000) A strategy for rapid cooling of mouse embryos within a double straw to eliminate the risk of contamination during storage in liquid nitrogen. Hum. Reprod., 15, 2604–2609.
Kuleshova, L., Gianaroli, L., Ferraretti, A. and Trounson, A. (1999) Birth following vitrification of a small number of human oocytes: case report. Hum. Reprod., 14, 3077–3079.
Mandelbaum, J., Belaisch-Allart, J., Junca, A.M. et al. (1998) Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum. Reprod., 13, 161–174.
Martino, A., Songsasen, N. and Leibo, S.P. (1996) Development into blastocysts of bovine oocytes cryopreserved by ultrarapid cooling. Biol. Reprod., 54, 1059–1069.[Abstract]
O'Neil, L., Paynter, S.J., Fuller, B.J. and Shaw, R.W. (1997) Murine oocyte cytoskeletal changes, fertilization and embryonic development following exposure to a vitrification solution. Cryo-Lett., 18, 17–26.
Otoi, T., Yamamoto, K., Koyama, N. et al. (1998) Cryopreservation of mature bovine oocytes by vitrification in straws. Cryobiology, 37, 77–85.[Medline]
Pickering, S., Cant, A., Braude, P.R. et al. (1990) Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil. Steril., 54, 102–108.[Web of Science][Medline]
Porcu, E., Fabbri, R., Seracchioli, R. et al. (1997) Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil. Steril., 68, 724–726.[Web of Science][Medline]
Porcu, E., Fabbri, R., Seracchioli, R. et al. (1998) Birth of six healthy children after intracytoplasmic sperm injection of cryopreserved human oocytes. Hum. Reprod., 13 (Abstract Bk. 1), p.124.
Rall, W.F. and Fahy, G.M. (1985) Ice-free cryopreservation of mouse embryos at –196 °C by vitrification. Nature, 313, 573–575.[Medline]
Trounson, A. and Kirby, C. (1989) Problems in the cryopreservation of unfertilized eggs by slow cooling in dimethyl sulfoxide. Fertil. Steril., 52, 778–786.[Web of Science][Medline]
Tucker, M.J., Morton, P.C., Wright, G. et al. (1998) Clinical application of human egg cryopreservation. Hum. Reprod., 13, 3156–3159.
Vajta, G., Holm, P., Kuwayama, M. et al. (1998) Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol. Reprod. Dev., 51, 53–58.
Van Blerkom, J. and Davis, P.W. (1994) Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microscopy Res. Tech., 27, 165–193.
Whittingham, D. (1971) Culture of mouse ova. J. Reprod. Fertil., 14, 7–21.
Young, E., Kenny, A., Puigdomenech, E., et al. (1998) Triplet pregnancy after intracytoplasmic sperm injection of cryopreserved oocytes: case report. Fertil. Steril., 70, 360–361.[Web of Science][Medline]
Submitted on January 8, 2001; accepted on July 28, 2001.
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M. Lane, J. M. Maybach, and D. K. Gardner Addition of ascorbate during cryopreservation stimulates subsequent embryo development Hum. Reprod., October 1, 2002; 17(10): 2686 - 2693. [Abstract] [Full Text] [PDF]
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S.-U. Chen, Y.-R. Lien, Y.-Y. Tsai, L.-J. Chang, H.-N. Ho, and Y.-S. Yang Successful pregnancy occurred from slowly freezing human oocytes using the regime of 1.5 mol/l 1,2-propanediol with 0.3 mol/l sucrose Hum. Reprod., May 1, 2002; 17(5): 1412 - 1412. [Full Text] [PDF]
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