Sucrose concentration influences the rate of human oocytes with normal spindle and chromosome configurations after slow-cooling cryopreservation

BACKGROUND: Recently described slow-cooling cryopreservation protocols involving elevated sucrose concentration have improved survival frequencies of human oocytes, potentially overcoming a major hurdle that has limited the adoption of oocyte storage. Because implantation rates of embryos from frozen oocytes remain generally low, it is still debated whether, irrespective of survival rates, this form of cryopreservation leads inevitably to the disruption or complete loss of the metaphase II (MII) spindle. METHODS: Human oocytes with an extruded polar body I (PBI) were cryopreserved using a slow-cooling method including 1.5 mol/l propane-1,2-diol (PrOH) and alternative sucrose concentrations (either 0.1 or 0.3 mol/l) in the freezing solution. Fresh control and frozen-thawed survived oocytes were analysed by confocal microscopy to evaluate MII spindle and chromosome organizations. RESULTS: Of the 104 oocytes included in the unfrozen group, 76 (73.1%) displayed normal bipolar spindles with equatorially aligned chromosomes. Spindle and chromatin organizations were significantly affected (50.8%) after cryopreservation involving lower sucrose concentration (61 oocytes), whereas these parameters were unchanged (69.7%) using the 0.3 mol/l sucrose protocol (152 oocytes). CONCLUSIONS: Partial disruption of the MII spindle and associated chromosomes accompanies inadequate cryopreservation during slow cooling. However, protocols adopting higher sucrose concentration in the freezing solution promote the retention of an intact chromosome segregation apparatus comparable in incidence to freshly collected oocytes.     

Ultrastructure of human mature oocytes after slow cooling cryopreservation using different sucrose concentrations

BACKGROUND: We studied the ultrastructural characteristics of human mature oocytes frozen/thawed (F/T) using different concentrations of sucrose. Fresh human mature oocytes were used as controls. METHODS: The oocytes (n = 48) were fixed in 1.5% glutaraldehyde at sampling (n = 16) or after freeze/thawing performed using a slow cooling method with propane-1,2-diol 1.5 mol/l and sucrose at either 0.1 mol/l (n = 16) or 0.3 mol/l (n = 16) in the freezing solution. The oocytes were then processed for electron microscopy observations. RESULTS: Fresh and F/T oocytes belonging to both study groups were regularly rounded in sections, with a homogeneous cytoplasm and an intact zona pellucida (ZP). Organelles (mainly mitochondria-smooth endoplasmic reticulum aggregates and mitochondria-vesicle complexes) were abundant and uniformly dispersed in the ooplasm. The amount and density of cortical granules appeared to be abnormally reduced in some F/T samples, independently of the sucrose concentration in the freezing solution: this feature was frequently associated with an increased density of the inner ZP, possibly related to the occurrence of zona 'hardening'. Furthermore, slight to moderate microvacuolization was revealed in the ooplasm of some F/T oocytes, particularly in those treated with sucrose 0.3 mol/l. CONCLUSIONS: Freeze/thawing procedures are associated with ultrastructural alterations in specific oocyte microdomains, presumably linked to the reduced developmental potential of mature cryopreserved oocytes. Further work is needed to determine whether or not a high concentration of sucrose plays a role, at least in part, in producing the above alterations.     

Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation

The present study was conducted to determine if the cryopreservationof immature human oocytes has a deleterious effect on the meioticspindle following maturation in vitro. Oocytes were obtainedin excess from in-vitro fertilization patients and divided intofour groups. Groups 1 (n = 98) and 2 (n = 80) consisted of immatureoocytes cryopreserved before or after maturation in vitro respectively.Groups 3 (n = 37) and 4 (n = 9) served as non-frozen controlsand included oocytes matured in vitro and in vivo respectively.The meiotic spindle was identified after incubation in anti-tubulinmonoclonal antibody (1 h, 37°C) and fluorescein-conjugatedgoat anti-mouse immunoglobulin G (IgG) (1 h, RT). Chromosomeswere counterstained with 4‘, 6’-diamidino-2-phenylindole.Following cryopreservation, group 1 oocytes demonstrated a 63%survival rate and 68% maturation rate in vitro. In all, 58%of the oocytes in group 2 survived the thaw. The number of oocyteswith normal spindles in group 1 (81.0%) was not significantlydifferent from control groups 3 (83.8%) and 4 (88.9%), whilethe number of group 2 oocytes with normal structures (43.5%)was significantly lower than groups 1 (P = 0.0004), 3 (P = 0.0002),and 4 (P = 0.025). These results suggest that cryopreservationof the prophase I human oocyte does not significantly increaseabnormalities in the resulting meiotic spindle.     

Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration

BACKGROUND: Recently, interest in oocyte cryopreservation has steadily increased. Newly developed protocols have dramatically improved survival rates, removing perhaps the major hurdle that has prevented this approach from becoming a fully established form of treatment. However, the clinical efficiency of these protocols has not been exhaustively explored and therefore remains controversial. METHODS: Morphologically normal oocytes displaying the first polar body were frozen-thawed with a slow cooling protocol that utilized 1.5 mol/l propane-1,2-diol (PrOH) and 0.3 mol/l sucrose. RESULTS: A total of 927 oocytes from 146 patients were frozen-thawed, achieving a 74.1% survival rate. Over 76% of microinjected oocytes displayed two pronuclei 16 h post-insemination, while the proportion of embryos at 44-46 h post-insemination was 90.2%. At this time point, the majority (68.3%) of embryos were at the two-cell stage, showing in most cases (78.7%) minimal or moderate fragmentation. Eighteen clinical pregnancies, three of which were twin, were observed, giving rise to rates of 12.3 and 9.7%, calculated per patient and per embryo transfer, respectively.The implantation rate was 5.2%. To date, four children have been born and three pregnancies resulted in spontaneous abortions, while the remaining pregnancies are ongoing. CONCLUSIONS: Our data indicate that although the combination of slow cooling and high sucrose concentration ensures high rates of oocyte survival, it is not sufficient to guarantee a high standard of clinical efficiency.     

The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation

BACKGROUND: Knowing osmotic tolerance limits is important in the design of optimal cryopreservation procedures for cells. METHODS: Mature human oocytes were exposed to anisosmotic sucrose solutions at concentrations of 35, 75, 150, 600, 1200, or 2400 (+/-5) milliosmolal (mOsm) at 37 degrees C. A control treatment at 290 mOsm was also utilized. Oocytes were randomly allocated to each experimental treatment. After the treatment, the oocytes were cultured for 1 h, then fixed in cold methanol. Immunocytochemical staining and fluorescence microscopy were used to assess the morphology of the metaphase II (MII) spindle. Logistic regression was used to determine if media osmolality had a significant effect on spindle structure. RESULTS: Osmolality was a significant predictor of spindle morphology. Hyposmotic effects at 35, 75, and 150 mOsm resulted in 100, 67, and 56% of oocytes having abnormal spindles, respectively. Hyperosmotic effects at 600, 1200, and 2400 mOsm resulted in 44, 44, and 100% of the spindles with abnormal structure, respectively. CONCLUSIONS: Anisosmotic conditions lead to disruption of the MII spindle in human oocytes. Applying this fundamental knowledge to human oocyte cryopreservation should result in increased numbers of cells maintaining viability.     

Fertilization and embryonic development of human oocytes after cooling.

Injury to living cells resulting from rapid cooling to temperatures at or near 0 degrees C has long been recognized, and the phenomenon, which is termed 'cold shock', has been known to occur in some mammalian gametes. Although human embryos have been successfully stored at low temperatures, cryopreservation of the human oocyte is proving to be more difficult. Whether or not this lack of success is a direct result of cellular injury brought about by 'cold shock' is the purpose of the current investigation. Human oocytes were cooled, in the absence of cryoprotectants, at two different cooling rates (-3 degrees C/min and -1000+ degrees C/min) to a temperature of 0 degrees C and rewarmed prior to insemination. In both cases fertilization after cooling was similar to the rates achieved in a routine in-vitro fertilization and embryo transfer procedure. After cooling at -3 degrees C/min, the rate of fertilization was 19/22 (86%) and after cooling at -1000+ degrees C/min, 9/9 (100%), with non-cooled control rates of 62/87 (71%) and 35/50 (70%) respectively. Fertilized oocytes from both groups were successfully cultured for a further 24 h before termination of the experiment.     

Comparison of ethylene glycol, 1,2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts

The aim of the study was to analyse the toxicity, the osmolar and cryoprotective activity of ethylene glycol (ETG) in terms of survival rate (SR), cleavage rate (CR) and expanded blastocysts percentage (EBP) of mouse embryos. Early mouse embryos and blastocysts were slowly cooled with ETG, 1,2-propanediol (PROH) or glycerol, and thawed. The Van t'Hoff curve for 1.5 mol/l ETG showed recovery of initial volume within 4 min. No differences were observed in CR and EBP of ETG-exposed compared with non-exposed mouse zygotes. The SR of zygotes frozen with PROH was significantly better than with ETG (92% and 60% respectively; P < 0.01), and a significantly better EBP was achieved for blastocysts frozen with glycerol compared with ETG (75% and 50% respectively; P < 0.05). For 4-cell stage embryos, no differences were observed in SR and EBP between ETG and PROH. Higher EBP was observed for 4-cell stage embryos (53%) frozen with ETG compared with pronucleate stage (19%) and blastocysts (48%). Low toxicity, good SR and EBP were observed for mouse embryos frozen with ETG, the best results being obtained at the 4-cell stage. At other embryonic stages, PROH and glycerol respectively seemed to provide better results.     

Cryopreservation of human embryos using ethylene glycol in controlled slow freezing

BACKGROUND: Ethylene glycol (EG) has been successfully used as a cryoprotectant for vitrification of mammalian formula embryos (including human embryos) due to its low formula weight and high permeation into cells compared with other cryoprotectants, including propylene glycol (PROH). This study was carried out to evaluate the permeation and toxicity of EG and to investigate the effects of its use in a slow-freezing protocol on post-thaw development of mouse embryos and on pregnancy outcome of frozen human embryos. METHODS: Spare human embryos after embryo transfer were cryopreserved using 1.5 mol/l EG or PROH using a slow-freezing protocol which had been tested previously in mouse experiments. RESULTS: The post-thaw survival rate of human embryos in the EG group (80.6%) was significantly higher than that in the PROH group (65.2%, P < 0.05). The implantation and clinical pregnancy rates of human embryos in the EG group (20.3 and 46.9%) were significantly higher than those in the PROH group (7.5 and 24.6%, P < 0.05). CONCLUSIONS: Ethylene glycol may be a good substitute for PROH to cryopreserve human embryos using a slow-freezing protocol.     

A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage

BACKGROUND: Routine oocyte cryopreservation remains an elusive technique in the wide range of assisted reproductive technologies available. This study examines the effect of a cryopreservation protocol on the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle (GV) and metaphase II (MII) stage. METHODS: GV oocytes were randomly assigned to one of three groups: (i) control oocytes matured in vitro to MII stage (n = 156); (ii) oocytes cryopreserved at the GV stage and then matured in vitro (n = 90); (iii) oocytes cryopreserved at the MII stage (n = 147). Following cryopreservation and in-vitro maturation, immunostaining of tubulin and chromatin was performed, before visualization using confocal microscopy. RESULTS: A statistically significant increase was observed in the survival rate in group 2 (73.3%, 66/90) compared to group 3 (55.7%, 82/147) (P < 0.007). Exposure of oocytes to the cryoprotective solutions without freezing had no effect on the structure of their second meiotic spindle. However, statistically significant differences were observed on both spindle and chromosome configurations of oocytes from group 2 (5.2 and 5.2% respectively) and group 3 (16.2 and 18.8% respectively) compared with group 1 oocytes (71.6 and 82.0% respectively) (P < 0.001 in all cases). CONCLUSIONS: The protocol followed results in high rates of survival and potential for in-vitro maturation, but has a deleterious effect on the organization of the meiotic spindle of human oocytes cryopreserved at both the GV and MII stages.     

Fertilization and early embryology: Parthenogenetic activation of human oocytes following cryopreservation using 1, 2-propanediol

Fresh and aged human oocytes were cryopreserved using 1, 2-propanediol(PROH). After thawing, the oocytes were cultured for 20 h andexamined for parthenogenetic activation using light microscopyand an ultraviolet DNA stain. Control fresh or aged oocytesand oocytes exposed to PROH without cryopreservation were alsoexamined for activation. No control oocytes were observed toactivate spontaneously (n = 43) and parthenogenetic activationwas not induced by exposure to PROH alone (n = 26). In bothfresh and aged cryopreserved oocytes, 27 and 29% of the oocytesrespectively were activated, and these proportions were significantlyelevated compared with the controls (P < 0.01). Althougha similar rate of activation was observed for the cryopreservedfresh and aged oocytes, the form of parthenogenetic activationvaried between these two types of oocyte. A single pronucleuswas observed in 18% of the fresh and 5% of the aged cryopreservedoocytes. In contrast, the presence of two or more pronucleiwas observed in 0% of the fresh and 19% of the aged cryopreservedoocytes.     

Cytoskeletal organization and zona sensitivity to digestion by chymotrypsin of frozen--thawed mouse oocytes

Mouse oocyte—cumulus masses were added to 1.5 dimethylsulphoxide (DMSO) + 20% fetal bovine serum (FBS) that had beenprecooled at +4°C, were frozen by slow cooling to an intermediatetemperature of –60°C before being plunged into liquidnitrogen at –196°C, subjected to a controlled thaw,expelled into 1.5 M DMSO + FBS at 4°C, and then washed inmedium + FBS at 37°C. Of 7733 oocytes treated, 78.4% wereviable (controls; no treatment: 94.2% of 2764 oocytes; cryoprotectantonly: 92.2% of 2991 oocytes). The oocyte losses were not dueto complete loss of all oocytes from some straws or mice, sinceanalysis of individual straws containing oocytes from a singlemouse revealed considerable inter-straw/mouse variation. Amongstsurviving oocytes, no significant differences between frozenand control oocytes in spindle, chromosomal or microfilamentorganization were recorded. Two significant differences wereobserved: (i) fewer frozen—thawed oocytes had zonae resistantto chymotrypsin digestion, and (ii) spindle organization incontrol oocytes, but not frozen—thawed oocytes, was improvedby 3 h incubation at 37°C. More of the abnormal than thenormal frozen—thawed and control oocytes were surroundedby zonae which were resistant to digestion by chymotrypsin.     

Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures

BACKGROUND: The clinical efficacy of the current methods used for cryopreservation of metaphase II human oocytes is low. Meiotic spindle disorders are thought to be largely responsible for this situation. METHODS: Supernumerary fresh metaphase II human oocytes were cryopreserved in 1,2-propanediol with 0.1 M sucrose using a slow freezing/rapid thawing programme. Meiotic spindles were analysed in these living metaphase II oocytes at sequential steps of the freezing and thawing procedures with the use of a computer-assisted polarization microscopy system (Polscope). RESULTS: The meiotic spindle was detected in all 56 oocytes (from 16 patients) before freezing and remained visible in all these oocytes throughout the preparation for freezing up to the time that they were loaded into cryopreservation straws. Immediately after thawing, the spindle was visible in 35.7% of oocytes, but it disappeared in all of the thawed oocytes during the subsequent washing steps. However, the spindle reappeared in all surviving thawed oocytes after washing (57.4%), by 3 h of incubation at 37 degrees C in culture medium. CONCLUSIONS: The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post-thaw reconstruction.     

Combined impact of the number of pre-ovulatory oocytes and cryopreservation on IVF outcome

Three hundred and twenty-seven consecutive in-vitro fertilization (IVF) cycles in which luteal-phase leuprolide had been given were ranked according to the number of pre-ovulatory oocytes obtained (1-5, 6-10, greater than 10). Excess pre-embryos were cryopreserved at the pronuclear stage and later transferred into monitored natural cycles on the day after ovulation. The results indicate that the retrieval of large numbers of pre-ovulatory oocytes (greater than 10) has a small negative impact on oocyte quality as judged by fertilization rates (4% lower). However, implantation was not impaired compared to lower levels of retrieval in either the original IVF or subsequent cryo-thaw cycles. Overall, despite the small reduction in fertilization rate, the retrieval of many pre-ovulatory oocytes has produced a 'take-home baby rate' per stimulation cycle of 28.3% when 6-10 pre-ovulatory oocytes were retrieved and 41.5% when greater than 10 were retrieved: even higher rates are anticipated when the remaining cryopreserved pre-embryos are ultimately thawed.     

Cryopreservation of human embryos and oocytes

The success rate of human embryo cryopreservatlon depends ontechnical and embryonic parameters. First of all, the cryoprotectantcan affect embryo survival as we found by comparing two freeze-thawprocedures using propanediol (PROH) (1.5 mol) alone or withsucrose (0.1 mol). Embryo survival was significantly enhancedwith sucrose (62 versus 32%). Embryo quality is another majorparameter involved in the success of freezing; the rates ofpositive survival were found to be 67% for morphologically normalembryos versus 49% for embryos with fragments (P < 0.001).The efficiency of embryo cryopreservatlon in an IVF programmecould be estimated in 1986: a woman with extra embryos, storedafter transfer of 3–4 fresh embryos (16% of all cydes),can expect a 22% pregnancy rate per transfer of fresh embryosand a 32% pregnancy rate per collection after transfer of thestored eggs. A comparative study of the cryopreservability ofimmature or mature oocytes was performed in humans. Human oocyteshave a low survival rate (36%) whatever the cryopreservationprotocol or the initial maturation stage. Immature human oocytescould survive freezing and thawing, mature and be fertilizedin vitro, but with a very low efficiency.     

Cryopreservation of human oocytes: a review of current problems and perspectives

It is well recognized that the ability to cryopreserve unfertilizedhuman oocytes would make a significant contribution to infertilitytreatment. However, despite considerable interest, very fewsuccessful pregnancies have arisen from cryopreserved oocytesafter thawing, insemination and transfer of the subsequent embryo.The reasons for this lack of progress may well result from adearth of information on how the various biophysical changesduring a cryopreservation regimen affect human oocyte function.Recently, fundamental studies on the effects of cooling, membranepermeability, cryoprotectant addition and ice formation havebeen performed on human oocytes by a number of groups, and theseform the basis of the current review. It is likely that successfulhuman oocyte cryopreservation will only follow once these factorsare fully understood, but the existing base of knowledge shouldprovide a platform for further improvements in the techniquescurrently employed.     

Cryopreservation of mouse and human oocytes using 1, 2-propanediol and the configuration of the meiotic spindle

Human and mouse oocytes were cryopreserved by a slow freeze,rapid thaw method, using propanediol (PROH) as the cryoprotectant.A simulated cryopreservation was also included in the studyto detect the level of damage attributable to the PROH alone.Comparison of the mouse and human oocytes cryopreserved by thesame method showed opposing results, with a poor morphologicalsurvival rate of 4% observed for mouse oocytes and a subsequentnormal fertilization rate of 0%. In 171 cryopreserved humanoocytes a higher survival rate of 64% was achieved, and thisshowed more similarity to the mouse pronuclear oocytes survivalof 53%. A comparison of human oocytes, cryopreserved withinthe cumulus and denuded of cumulus and corona prior to cryopreservation,demonstrated a higher survival rate in the denuded oocytes of69% compared to 48%. A delay prior to cryopreservation of 1or 2 days had no effect on the immediate survival of oocytes,but culture for a further 24 h after thawing reduced survival,with the day 1 oocytes exhibiting the most dramatic reductionin survival (28%). Using a lectin binding method, abundant corticalgranules were observed in all cryopreserved oocytes analysed.The meiotic spindle and chromosomes were examined in cryopreservedoocytes using fluorescence microscopy and 60% of the survivingoocytes had a normal spindle and chromosome configuration.     

Visualization of the metaphase II meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI

BACKGROUND: Meiotic spindles in living human oocytes can be visualized by the Polscope. This study investigated the relationship between the presence/location of the spindle in metaphase II (MII) oocytes and developmental competence of embryos in vitro. METHODS: The spindles in 626 MII oocytes were examined by the Polscope and divided into six groups (A-F) based on the presence or absence of the spindles and the angle between the spindle and the first polar body. After ICSI, the fertilization and embryo development were evaluated. RESULTS: Meiotic spindles were imaged in 523 oocytes (83.5%), while 103 (16.5%) did not have a visible spindle (group F). The majority of oocytes (68.8%) had the spindle directly beneath or adjacent to the first polar body (groups A and B: 48.2 and 20.6%). Oocytes in group C (11.2%) had the spindle located between 60 and 120 degrees angle away from the first polar body, those in group D (2.4%) had the spindle located between 120 and 180 degrees angle and those in group E (1.1%) had the spindle located at 180 degrees angle to the first polar body. The fertilization and embryonic development were similar in the oocytes with spindles regardless of spindle position. However, the rate of high quality embryos was significantly higher in the oocytes (64.2%) with visible spindles than in the oocytes (35.9%) without spindle and multipronuclear proportion showed a slight tendency to increase in oocytes without spindles. (10.7 versus 5.9%, P = 0.12; NS). CONCLUSIONS: the presence of a bi-refringent meiotic spindle in human oocytes by using the Polscope can predict a higher embryonic developmental competence. However, the relative position of the spindle within the oocyte doesn't appear to influence the developmental potential of embryos.     

Fertilization and development of the human oocyte following exposure to cryoprotectants, low temperatures and cryopreservation: a comparison of two techniques.

Both glycerol and dimethyl sulphoxide (DMSO) equilibration prior to cryopreservation produced adequate rates of survival and development to the hatching blastocyst stage for mouse oocytes using the two chosen cooling and warming regimes. Successful fertilization of human oocytes and further development of the embryos produced, was achieved following equilibration with DMSO at 0 degrees C. Although fertilization of fresh human ova previously exposed to glycerol was recorded, all oocytes with two pronuclei failed to continue to undergo cell division by 48 h in culture. Following cryopreservation, human oocytes were successfully inseminated after equilibration with both glycerol and DMSO. However, cell division to the 2-cell stage was recorded in only one oocyte which had undergone exposure to DMSO, freezing and thawing. No cell divisions were recorded after glycerol cryopreservation, or even after simple exposure to glycerol. Therefore it appears that DMSO and slow cooling may be the best protocol, although further evaluation will be necessary.     

Differential effects of repeated ovarian stimulation on cytoplasmic and spindle organization in metaphase II mouse oocytes matured in vivo and in vitro

The effects of four rounds of ovarian stimulation spaced 1-6 weeks apart on the normality of metaphase II (MII) spindle formation, chromosomal alignment and cytoplasmic organization were examined in intact ovulated mouse oocytes and at MII for oocytes obtained at the germinal vesicle stage from the same ovaries and matured in vitro. The terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling assay was used to identify DNA strand breaks in chromosomes, and histological studies of ovaries between and at each round of ovarian stimulation were performed. The results demonstrate a progressive and significant increase in the frequency of spindle defects with each round of ovarian stimulation, including those spaced weeks apart. Oocytes with spindle defects were also characterized by the occurrence of detached chromosomes and cytoplasmic asters. In contrast, in-vitro matured oocytes derived from the same ovaries were normal. No evidence of DNA strand breaks with repeated rounds of ovarian stimulation was detected in ovulated or in-vitro matured oocytes. The development and persistence of nodules of hypertrophied granulosa in regions where follicular growth occurs suggest that a progressively increasing proportion of oocytes in the ovulatory pathway may experience an intrafollicular milieu that has negative consequences for competence. The results are discussed with respect to ovarian and oocyte biological ageing and possible adverse implications for human oocyte competence with repeated hyperstimulation.     

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.

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.