Meiotic spindle imaging in human oocytes frozen with a slow freezing procedure involving high sucrose concentration

V. Bianchi1, G. Coticchio1, L. Fava1, C. Flamigni2 and A. Borini1,3

1 Tecnobios Procreazione, Via Dante 15, 40125 Bologna and 2 University of Bologna, 40125 Bologna, Italy

3 To whom correspondence should be addressed. Email: borini{at}tecnobios.it


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: One of the major concerns derived from the cryopreservation of meiotically mature oocytes is possible damage to the cytoskeletal apparatus, and in particular the meiotic spindle. METHODS: One hundred fresh oocytes showing the polar body I and high meiotic spindle birefringence (maximum retardance±1.5 mol/l SD=2.58±0.1 nm), assessed through analysis, were included in this study. Oocytes were cryopreserved with a 1.5mol/l 1,2-propanediol +0.3 mol/l sucrose solution. After thawing, spindles were imaged at 0, 3 and 5 h. Spindle birefringence was quantified by measuring microtubule maximum retardance. Signals of thawed oocytes were classified as absent (non-detectable), weak (1.55±0.3 nm) or high (2.50±0.2 nm). RESULTS: Immediately after thawing, only 22.9% of oocytes showed a weak birefringence signal, while only 1.2% of oocytes displayed a high signal. Three hours after thawing, the proportion of oocytes exhibiting a weak or high intensity signal was 49.4% and 18.1%, respectively. Finally, after culture for 5 h following thawing, a weak birefringence signal was detected in 51.8% of oocytes, while 24.1% showed a high signal. There was a statistically significant increase in signal restoration after 3 h of culture (P<0.001). CONCLUSIONS: These results suggest that in mature oocytes stored via slow freezing, the meiotic spindle undergoes transient disappearance immediately after thawing but is reorganized in the majority of oocytes, at least to some extent, after 3–5 h of culture.

Key words: human oocyte/meiotic spindle/oocyte cryopreservation/Polscope/thawing


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In human IVF, oocyte cryopreservation so far has been applied with limited success. Various studies (Porcu et al., 2000Go; Borini et al., 2004Go; Chen et al., 2004Go) have shown that conventional slow freezing methods are inadequate to ensure high post-thaw survival, a situation that has prevented the accumulation of data on a large scale and the assessment of clinical efficiency. Recently, modified protocols have been suggested to improve survival rates, as a result of changes involving increase in sucrose concentration (Fabbri et al., 2001Go) or the replacement of sodium with choline in the freezing mixtures (Stachecki et al., 1998Go; Quintans et al., 2002Go).

It is obvious that the achievement of high post-thaw survival is an essential requisite in the attempt to make oocyte freezing competitive with other forms of fertility preservation. On the other hand, it is known that while freezing can cause overt oocyte degeneration immediately after thawing, nevertheless post-thaw survival does not guarantee unaltered viability. In fact, sublethal cell damage that cannot be detected by routine microscopic assessment of the oocyte status may emerge at various developmental stages (Hunter et al., 1995Go), jeopardizing the establishment of a viable pregnancy.

Oocyte cryopreservation has been associated with hardening (Carroll et al., 1990Go) or fracturing (Fuku et al., 1995Go) of the zona pellucida, effects that could interfere with sperm–egg interaction or generate polyspermic fertilization, respectively. Other experiments have shown that frozen mouse oocytes may be affected by disturbances in intracellular free calcium regulation (Litkouhi et al., 1999Go). Intracellular calcium regulation has also been shown to be influenced in frozen human oocytes, in which the increase in the level of this ion, in response to treatment with calcium ionophore A23187, is lower compared with fresh oocytes (Jones et al., 2004Go). Alterations in mitochondrial function in cryopreserved human oocytes have also been documented (Jones et al., 2004Go). Because of its sensitivity to low temperatures (Pickering et al., 1990Go; Zenzes et al., 2001Go), the meiotic spindle is generally believed to be affected by cryopreservation. Alterations of this structure would have major consequences on oocyte viability, consisting of an increase in chromosome segregation errors during meiosis II or, in the most extreme cases, fertilization failure. However, in this respect evidence is insufficient and controversial. In fact, while some authors have reported that freezing can alter meiotic spindle organization (Boiso et al., 2002Go), other studies have suggested that this structure can survive freezing–thawing without consequences (Gook et al., 1993Go; Stachecki et al., 2004Go) in the absence of chromosome dispersal in the oocyte or an increase in aneuploidies in the resulting embryo (Cobo et al., 2001Go).

In this study, we aimed to establish a specific aspect of oocyte viability following freezing with a protocol that enables high oocyte post-thaw recovery. This was pursued by assessing possible detrimental effects of cryopreservation on the meiotic spindle. To this end, we employed the optical system Polscope®, a recently introduced microscopy apparatus that, using polarized light, allows the observation of highly ordered subcellular structures such as the spindle microtubules (Oldenbourg, 1999Go; Wang et al., 2001aGo). Compared with immunostaining or other microscopy methods, the Polscope is totally non-invasive, and therefore oocyte viability can be preserved (Keefe et al., 2003Go). Repeated oberservations are possible over time, and the Polscope quantifies microtubule architecture better than immunofluorescence, because the latter introduces artifact related to fixation, immunostaining and fluorescence quenching.

It should be noted that this system does not allow a detailed analysis of the spindle organization, as opposed to more conventional techniques. However, despite this limitation, the application of the Polscope has already proven to be a valuable technical support in human IVF by showing that fertilization and cleavage rates (Wang et al., 2001bGo), as well as embryo quality (Moon et al., 2003Go), are to some extent dependent on spindle presence and localization.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Source of oocytes
In our centre, patients with more than 18 oocytes retrieved are given the option to donate their supernumerary gametes for research. Approval for use of the donated oocytes was obtained previously from the local internal review board.

One hundred and ten oocytes exhibiting the polar body I were obtained from 18 consenting patients undergoing ovarian stimulation for an IVF procedure. One hundred of those (91%) presented a birefringence signal with maximum retardance of 2.58±0.1 nm (mean±SD) and were included in this study. The mean (±SD) age of patients was 36.4±3.2 years.

Controlled ovarian hyperstimulation was induced with a long protocol using leuprorelin (Enantone; Takeda, Rome, Italy) and rFSH (Gonal-F; Serono, Rome, Italy). HCG (Profasi HP; Serono) was administered when one or more follicles reached a maximum diameter of >23 mm (Dal Prato et al., 2001Go). Oocyte collection was performed transvaginally under ultrasound guidance, 36 h after HCG injection (Profasi HP; Serono).

After retrieval, surplus oocytes were cultured in fertilization medium (Cook IVF, Brisbane, Australia) for at least 5–6 h. Complete removal of cumulus mass and corona cells was performed enzymatically using hyaluronidase (40 IU/ml; Sigma Aldrich SrL, Milan, Italy), and mechanically by using fine bore glass pipettes.

Spindle examination with the Polscope
For spindle imaging before freezing, each oocyte was placed in a 5 µl drop of the fertilization medium covered with mineral oil (Cook IVF) in a glass-bottomed culture dish (Willco Wells, Amsterdam, The Netherlands). The dishes were maintained at 37 °C during examination and oocytes were manipulated using the holding pipette in order to optimize spindle visualization by observing different focal planes. The meiotic spindle visualization was performed at 200x magnification with LC Polscope optics and controller (SpindleView; CRI, Woburn, MA, USA), combined with a computerized image analysis system (SpindleView software; CRI). The instrument settings were maintained unaltered throughout the experiment. Retardance was measured both in fresh and in frozen–thawed oocytes to define spindle characteristics according to Sato et al. (1975)Go, who demonstrated that microtubules are the sole contributor to spindle birefringence, establishing a relationship between spindle retardance and microtubule density.

Freezing solutions
All cryoprotectant solutions were prepared using Dulbecco's phosphate-buffered solution (PBS) (Gibco Life Technologies, Paisley, UK), 1,2-propanediol (PROH) (Fluka Chemika; Sigma Aldrich SrL) and a plasma protein supplement (PPS) (BAXTER AG, Vienna, Austria). The freezing solutions were (i) 1.5 mol/l PROH +30% PPS in PBS (equilibration solution) and (ii) 1.5 mol/l PROH +0.3 mol/l sucrose +30% PPS in PBS (loading solution), as described by Fabbri et al. (2001)Go.

Freezing procedure
Three hours after cumulus removal the oocytes were cryopreserved according to laboratory procedures normally applied in our centre.

Oocytes were washed in PBS solution supplemented with 30% PPS, and put into the equilibration solution for 10 min, then transferred to the loading solution for 5 min. Each step was performed at room temperature (RT) (22±1 °C).

The oocytes were loaded individually in plastic straws (Paillettes Cristal 133 mm; Cryo Bio System, France), transferred into an automated Kryo 10 series III biologic vertical freezer (Planer Kryo 10/1.7 GB) and frozen according to the following conditions: the start chamber temperature was 20 °C then slowly reduced to –7 °C at a rate of –2 °C/min. Ice seeding was induced manually at –7 °C; after a hold ramp at –7 °C for 10 min, the straws were 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. The straws were finally transferred into liquid nitrogen and stored until thawing.

Thawing procedure
The straws were rapidly air-warmed for 30 s and then plunged into a 30 °C water bath for 40 s. The cryoprotectant was removed at RT by step-wise dilution. The oocytes were expelled in the first solution (1.0 mol/l PROH +0.3 mol/l sucrose +30% PPS) (5 min), then equilibrated in 0.5 mol/l PROH +0.3 mol/l sucrose +30% PPS for another 5 min. Finally they were placed in a 0.3 mol/l sucrose +30% PPS for 10 min before final dilution in PBS solution +30% PPS for 20 min (10 min at RT and 10 min at 37 °C). The oocytes were placed in 20 µl drops of cleavage medium (Cook IVF) under warm mineral oil (Cook IVF) at 37 °C in an atmosphere of 5% CO2 in air.

Data collection of spindle imaging after thawing
Each oocyte was observed before freezing and after rewarming. Examinations were conducted at 0, 3 and 5 h after thawing. Birefringence was recorded for each oocyte and classified, depending on signal intensity, as absent (non-detectable), weak (with maximum retardance of 1.55±0.3 nm) or high (with maximum retardance of 2.50±0.2 nm) (Figure 1A, B and C, respectively).



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Figure 1. oocyte displaying (A) non-detectable signal, (B) a weak signal (with maximum retardance of 1.55±0.3 nm and (C) a high signal (with maximum retardance of 2.50±0.2 nm).

 
Confocal microscopy assessment
For immunostaining analysis, oocytes were fixed at 37 °C for 30 min with a buffer containing 3.7% formaldehyde. Afterwards, they were transferred to a solution including 0.2% azide, 0.2% powdered milk, 2% goat serum, 1% bovine serum albumin, 0.1 mol/l glycine and 0.1% Triton X-100. Incubation with anti-tubulin primary antibody diluted 1:150 in the same solution was carried out at 37 °C for 1 h. Treatment with FITC-conjugated secondary antibody diluted 1:50 was performed in the dark at 37 °C for 1 h. DNA staining was obtained by including propidium iodide (10 mg/ml) in the mounting medium. Confocal analysis was performed by using an Olympus IX8 laser confocal imaging system equipped with an argon laser and integrated with an Olympus microscope.

Statistical analysis
Statistical analysis was performed using the {chi}2-test for qualitative variables. Differences were considered significant when a P-value was <0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Before freezing the meiotic spindle was visualized in 100 metaphase II oocytes with maximum retardance of 2.58±0.1 nm. After thawing, 83 oocytes were recovered with a survival rate of 83%. A statistical difference in spindle birefringence was found when the spindle was imaged immediately after completion of the thawing procedure, before transfer into cleavage medium (time 0): 63 (75.9%) oocytes did not show a detectable signal, 19 (22.9%) presented a weak (maximum retardance of 1.55±0.3 nm) signal while a single oocyte (1.2%) displayed a very marked birefringence (maximum retardance of 2.50±0.2 nm). After 3 h of culture, the proportions of oocytes displaying either no, weak or high spindle birefringence were 27 (32.5%), 41 (49.4%) and 15 (18.1%), while after 5 h they were 20 (24.1%), 43 (51.8%) and 20 (24.1%), respectively (Figure 2).



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Figure 2. Diagram showing different rates of birefringence signal intensity at different time points after thawing.

 
The number of oocytes within each category of spindle birefringence intensity at different times after cryopreservation were compared and highly statistically significant differences (P<0.001) were found between 0 and 3 h, while no differences were found between 3 and 5 h (Table I).


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Table I. Birefringence at different times of observation

 
Frozen–thawed oocytes displaying different intensity of the birefringence signal were subjected to confocal microscopy analysis. Inability to identify the spindle after Polscope assessment coincided with the absence of a recognizable microtubule organization, with only minor tubulin staining associated to chromosomes. Metaphase II configurations were found in oocytes in which Polscope evaluation had revealed the presence of the spindle, irrespective of signal intensity. In addition to typical bipolar spindles with chromosomes organized on the equatorial plane, other tubulin and chromosome distributions were observed, including non-polar spindles and scattered chromosomes. However, these observations were insufficient to ascertain possible statistical differences in the distribution of diverse spindle configurations between the two groups of oocytes. For this reason, a separate study is currently being conducted to establish possible associations between intensity of spindle birefringence and microtubule configurations.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study, using polarized light microscopy, we monitored the presence of the spindle in meiotically mature oocytes that had been frozen–thawed with a modified slow freezing protocol. Our observations indicate that immediately after thawing and cryoprotectant removal, only a minority of oocytes show spindle birefringence. After 3 h, this proportion increases significantly, suggesting that short-term culture after thawing may be a critical requirement to ensure spindle recovery, a factor essential to establish optimal timing for insemination. In addition, the reduced spindle retardance found in the majority of frozen–thawed oocytes even after 5 h of incubation raises concerns that after thawing the spindle microtubular organization may not strictly coincide with the highly ordered structure usually present in fresh oocytes. In effect, the lower retardance suggests a lower microtubular density. The implications of these finding are not entirely clear, and certainly warrant further investigation in this field.

The unique features of the mature oocyte make the cryopreservation of this cell a daunting challenge. In particular, during freezing and thawing the low surface-to-volume ratio hinders the exchange of water and cryoprotective agents (CPA) through the plasmalemma, increasing the risk of cryoinjury caused by intracellular ice formation, osmotic stress and other factors. This is consistent with the fact that most studies have documented low survival rates following the application of conventional slow freezing protocols (Coticchio et al., 2001Go).

Fabbri et al. (2001)Go have achieved improved survival rates (58% and 83%) by applying high sucrose concentrations (0.2 and 0.3 mol/l, respectively) in the freezing solution. This may be explained by the fact that higher concentrations of this CPA cause enhanced cell dehydration, thereby reducing the risk of intracellular ice formation during slow freezing. On the other hand, treatment with elevated cryoprotectant concentrations coincides with fluctuations in cell volume (Paynter et al., 2001Go) that can cause cell injury. Therefore, exposure to CPA needs to respond to a delicate balance, in order to obtain sufficient protection from freezing injury while avoiding damage caused by osmotic stress. Another reason for concern associated with oocyte freezing lies in the susceptibility of the meiotic spindle to low temperatures and the potentially increased risk of errors in chromosome segregation. It is well established that in metaphase II oocytes even supra-zero cooling induces depolymerization of microtubules and disappearance of microtubule organising centres. Incubation of mouse oocytes at 25, 18 or 4 °C causes spindle disassembly and a series of cytoskeletal changes derived by the increase in monomeric tubulin (Pickering and Johnson, 1987Go). To a large extent, however, in most of these oocytes spindle organization can be re-established following re-incubation at 37 °C. The spindle of human oocytes exhibits a different sensitivity to suboptimal temperatures. This is shown by the fact that incubation at RT generates spindle alterations, including total disassembly, that are recovered only by a small minority of oocytes upon rewarming at normal temperature (Pickering et al., 1990Go). More recently, testing human oocytes with the Polscope, Wang et al. (2001c)Go found that a slight temperature reduction to 33 °C results in spindle depolymerization within 10 min, observing a direct correlation between extent of temperature decrease and rapidity of spindle depolymerization. In addition, imaging the spindles 20 min after rewarming, the same authors reported that spindle repolymerization occurs with rates of 100%, 40% and 0% in oocytes cooled at 33, 28 and 25 °C, respectively.

Clinical data are insufficient and inadequate to answer the question as to whether frozen oocytes are exposed to an increase in aneuploidy risk. Abortion rates, often a reflection of the incidence of aneuploidy, associated with pregnancies from frozen oocytes are either not available or insufficient in terms of sample size (Quintans et al., 2002Go). On the other hand, microscopy studies on the spindle configuration in frozen oocytes are not entirely consistent. For example, it has been described that the proportion of metaphase II oocytes with a morphologically normal spindle is comparable in fresh and frozen oocytes (Gook et al., 1993Go; Stachecki et al., 2004Go). In another study, it has been described that cryopreservation prior to in-vitro maturation does not compromise spindle organization, with >81.0% of oocytes with normal morphology in frozen, as well as control, groups (Baka et al., 1995Go). In contrast, Boiso et al. (2002)Go have reported that spindle configuration is significantly perturbed by freezing in oocytes frozen after maturation in vivo as well as in oocytes stored at the germinal vesicle stage and matured in vitro. In mouse oocytes it has been established that mature oocytes can be stored without a rise in the incidence of aneuploidy or digyny provided that improved protocols based on either slow cooling (Bos-Mikich and Whittingham, 1995Go) or vitrification (Bos-Mikich et al., 1995Go) are applied. In the human, cytogenetic evidence is scarce. Nevertheless, it appears that slow freezing does not affect the oocyte rate of aneuploidy (Van Blerkom and Davis, 1994Go). Moreover, the occurrence of aneuploidy associated with chromosomes 13, 18, 21, X and Y is very similar (28% and 26%, respectively) in embryos obtained from fresh or frozen oocytes (Cobo et al., 2001Go).

To contribute to the elucidation of possible effects of cryopreservation on the meiotic spindle, in this study we aimed to analyse human oocytes treated with a slow freezing protocol involving the use of high sucrose concentration. This treatment, while generating higher survival rates and giving rise to several live births (Porcu, 2001Go), to our knowledge has not been tested so far in terms of objective criteria of oocyte quality following thawing. We applied the non-invasive approach offered by the Polscope in order to monitor in a dynamic fashion spindle stability before freezing and after thawing, and therefore gain clinically relevant information.

Before freezing, Polscope analysis revealed the presence of the spindle in the large majority of oocytes, confirming previous data suggesting that absence of the spindle is a rather sporadic condition (Rienzi et al., 2003Go). Some authors have reported lower incidence of spindle visualization in fresh oocytes (Wang et al., 2001bGo), but this may reflect technical limitations of the early Polscope prototype, as well as patient-specific factors affecting spindle resiliance. Our results indicate that the meiotic spindle disappears at some point during the freezing–thawing procedure, being visible immediately after thawing only in ~24% of oocytes. This is essentially in agreement with the study by Rienzi et al. (2004)Go, who failed to detect the spindle in all the surviving oocytes following thawing and before culture under standard conditions. The same authors suggested that the spindle disappears during the actual thawing process, since this structure is not affected by cryoprotectant exposure at RT. This is in apparent contrast to data on the loss of spindle organization in human oocytes exposed to RT. However, it should be noticed that there is evidence that cryoprotectants, apart from causing cell dehydration during freezing, possess the ability to stabilize the spindle structure (Joly et al., 1992Go). In our experience, immediately after thawing only a minority of oocytes display spindle birefringence, the intensity of which nevertheless appears in most cases weaker compared with the signal detected in the same oocytes before freezing. Subsequent culture for 3 h increases the overall incidence of oocytes with a visible spindle (67.5%), but the proportion of samples with pronounced spindles birefringence remains low (18.1%). Further culture for 2 h does not give rise to major changes in these percentages. High and weak signals imply differences in spindle organization, such as tubulin fibre density. Since different spindle configurations may reflect diverse functional capacity, this issue warrants further investigation in view of the fact that low spindle birefringence is represented in different proportions of oocytes before and after freezing. Preliminary observations indicate a correlation between birefringence detected with the Polscope and presence of an organized spindle analysed with confocal microscopy. Further studies with a significantly higher number of patients and oocytes are necessary to extend these findings and to assess more accurately not only the mere presence of the meiotic spindle, but also the fine details of its organization.

Rienzi et al. (2004)Go did not report differences in the intensity of spindle birefringence, although it is not clear whether they opted to not discriminate between different levels of intensity signal or in fact did not observe any obvious difference between oocytes showing spindle birefringence. The study by these authors suggests further reflections, compared with our results. With a standard protocol, Rienzi et al. (2004)Go reported a relatively low survival rate (50%) and the presence of the spindle in all surviving oocytes. By applying a modified protocol involving high sucrose concentration, our survival frequency was higher (83%), while the spindle was observed in 86% of oocytes. These data indicate that to assess the efficiency of a freezing method the relative importance of survival and cell integrity need to be observed carefully. Also, our data, as well as the work by Rienzi et al., describing a recovery of the spindle organization over a period of a few hours after thawing indicate some other important aspects. In particular, it appears obvious that for a clinical use, it would seem appropriate to culture frozen–thawed oocytes for 2–3 h before proceeding with microinjection. The necessity of such a measure is suggested by the observation that, irrespective of the aneuploidy risk, inability to visualize the spindle coincides with lower fertilization rates in fresh oocytes (Wang et al., 2001bGo). From our data it also appears that more extended incubation would not bring any obvious benefit. It should be considered, in fact, that prolonged culture in vitro exposes oocytes to a process of post-ovulatory ageing that may restrict their developmental ability (Fissore et al., 2002Go). Therefore, frozen–thawed oocytes should be allowed to recover their spindle organization, while making sure to coordinate the timing of insemination with the optimal ‘fertilization window’, which is believed to be only a few hours in duration. Also, while we have begun to shed some light on the kinetics of spindle reappearance after thawing, currently we have no information on whether and to what extent recovery after freezing procedure is required for other oocyte functions playing a role in the fertilization process. In view of these uncertainties, the optimal time for freezing with respect to HCG injection remains to be established.

Over 100 live births from frozen oocytes have been achieved so far. Our work indicates that the application of the Polscope can contribute to establish the clinical efficiency of this approach by making available an important criterion by which to assess a specific feature of oocyte viability before and after thawing.

Finally, future studies should investigate what other factors affect oocyte integrity following freezing and thawing. Assessment of oocyte viability should not be limited to the meiotic spindle, but rather broadened to other critical cellular attributes, such as subcortical actin and mitochondria. Studies are in progress to pursue this aim.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Dr Maria Luisa Vanelli, Department of Genetics University of Bologna for her assistance with statistical data analysis.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Baka SG, Toth TL, Veeck LL, Jones HW Jr, Muasher SJ and Lanzendorf SE (1995) Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation. Hum Reprod 10, 1816–1820.[Abstract]

Boiso I, Marti M, Santalo J, Ponsa M, Barri PN and Veiga A (2002) A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage. Hum Reprod 17, 1885–1891.[Abstract/Free Full Text]

Borini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M and Flamigni C (2004) Pregnancies and births after oocyte cryopreservation. Fertil Steril 82, 601–605.[CrossRef][ISI][Medline]

Bos-Mikich A and Whittingham DG (1995) Analysis of the chromosome complement of frozen–thawed mouse oocytes after parthenogenetic activation. Mol Reprod Dev 42, 254–260.[CrossRef][ISI][Medline]

Bos-Mikich A, Wood MJ, Candy CJ and Whittingham DG (1995) Cytogenetical analysis and developmental potential of vitrified mouse oocytes. Biol Reprod 53, 780–785.[Abstract]

Carroll J, Depypere H and Matthews CD (1990) Freeze–thaw-induced changes of the zona pellucida explains decreased rates of fertilization in frozen–thawed mouse oocytes. J Reprod Fertil 90, 547–553.[ISI][Medline]

Chen ZJ, Li M, Li Y, Zhao LX, Tang R, Sheng Y, Gao X, Chang CH and Feng HL (2004) Effects of sucrose concentration on the developmental potential of human frozen–thawed oocytes at different stages of maturity. Hum Reprod 19, 2345–2349.[Abstract/Free Full Text]

Cobo A, Rubio C, Gerli S, Ruiz A, Pellicer A and Remohi J (2001) Use of fluorescence in situ hybridization to assess the chromosomal status of embryos obtained from cryopreserved oocytes. Fertil Steril 75, 354–360.[CrossRef][ISI][Medline]

Coticchio G, Garetti S, Bonu MA and Borini A (2001) Cryopreservation of human oocytes. Hum Fertil (Camb) 4, 152–157.[Medline]

Dal Prato L, Borini A, Trevisi MR, Bonu MA, Sereni E and Flamigni C (2001) Effect of reduced dose of triptorelin at the start of ovarian stimulation on the outcome of IVF: a randomized study. Hum Reprod 16, 1409–1414.[Abstract/Free Full Text]

Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S and Flamigni C (2001) Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 16, 411–416.[Abstract/Free Full Text]

Fissore RA, Kurokawa M, Knott J, Zhang M and Smyth J (2002) Mechanisms underlying oocyte activation and postovulatory ageing. Reproduction 124, 745–754.[Abstract/Free Full Text]

Fuku E, Xia L and Downey BR (1995) Ultrastructural changes in bovine oocytes cryopreserved by vitrification. Cryobiology 32, 139–156.[CrossRef][ISI][Medline]

Gook DA, Osborn SM and Johnston WI (1993) Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 8, 1101–1109.[Abstract]

Hunter JE, Fuller BJ, Bernard A, Jackson A and Shaw RW (1995) Vitrification of human oocytes following minimal exposure to cryoprotectants; initial studies on fertilization and embryonic development. Hum Reprod 10, 1184–1188.[Abstract]

Joly C, Bchini O, Boulekbache H, Testart J and Maro B (1992) Effects of 1,2-propanediol on the cytoskeletal organization of the mouse oocyte. Hum Reprod 7, 374–378.[Abstract]

Jones A, Van Blerkom J, Davis P and Toledo AA (2004) Cryopreservation of metaphase II human oocytes effects mitochondrial membrane potential: implications for developmental competence. Hum Reprod 19, 1861–1866.[Abstract/Free Full Text]

Keefe D, Liu L, Wang W and Silva C (2003) Imaging meiotic spindles by polarization light microscopy: principles and applications to IVF. Reprod Biomed Online 7, 24–29.[Medline]

Litkouhi B, Winlow W and Gosden RG (1999) Impact of cryoprotective agent exposure on intracellular calcium in mouse oocytes at metaphase II. Cryo Letters 20, 353–362.

Moon JH, Hyun CS, Lee SW, Son WY, Yoon SH and Lim JH (2003) Visualization of the metaphase II meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI. Hum Reprod 18, 817–820.[Abstract/Free Full Text]

Oldenbourg R (1999) Polarized light microscopy of spindles. Methods Cell Biol 61, 175–208.[ISI][Medline]

Paynter SJ, O'Neil L, Fuller BJ and Shaw RW (2001) Membrane permeability of human oocytes in the presence of the cryoprotectant propane-1,2-diol. Fertil Steril 75, 532–538.[CrossRef][ISI][Medline]

Pickering SJ and Johnson MH (1987) The influence of cooling on the organization of the meiotic spindle of the mouse oocyte. Hum Reprod 2, 207–216.[Abstract]

Pickering SJ, Braude PR, Johnson MH, Cant A and Currie J (1990) Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 54, 102–108.[ISI][Medline]

Porcu E (2001) Oocyte freezing. Semin Reprod Med 19, 221–230.[CrossRef][ISI][Medline]

Porcu E, Fabbri R, Damiano G, Giunchi S, Fratto R, Ciotti PM, Venturoli S and Flamigni C (2000) Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol 169, 33–37.[CrossRef][ISI][Medline]

Quintans CJ, Donaldson MJ, Bertolino MV and Pasqualini RS (2002) Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium. Hum Reprod 17, 3149–3152.[Abstract/Free Full Text]

Rienzi L, Ubaldi F, Martinez F, Iacobelli M, Minasi MG, Ferrero S, Tesarik J and Greco E (2003) Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI. Hum Reprod 18, 1289–1293.[Abstract/Free Full Text]

Rienzi L, Martinez F, Ubaldi F, Minasi MG, Iacobelli M, Tesarik J and Greco E (2004) Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures. Hum Reprod 19, 655–659.[Abstract/Free Full Text]

Sato H, Ellis GW and Inoue S (1975) Microtubular origin of mitotic spindle form birefringence. Demonstration of the applicability of Wiener's equation. J Cell Biol 67, 501–517.[Abstract]

Stachecki JJ, Cohen J and Willadsen SM (1998) Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium. Cryobiology 37, 346–354.[CrossRef][ISI][Medline]

Stachecki JJ, Munne S and Cohen J (2004) Spindle organization after cryopreservation of mouse, human, and bovine oocytes. Reprod Biomed Online 8, 664–672.[ISI][Medline]

Van Blerkom J and Davis PW (1994) Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microsc Res Tech 27, 165–193.[ISI][Medline]

Wang WH, Meng L, Hackett RJ and Keefe DL (2001a) Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination. Hum Reprod 16, 1464–1468.[Abstract/Free Full Text]

Wang WH, Meng L, Hackett RJ, Odenbourg R and Keefe DL (2001b) The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil Steril 75, 348–353.[CrossRef][ISI][Medline]

Wang WH, Meng L, Hackett RJ, Odenbourg R and Keefe DL (2001c) Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy. Hum Reprod 16, 2374–2378.[Abstract/Free Full Text]

Zenzes MT, Bielecki R, Casper RF and Leibo SP (2001) Effects of chilling to 0 degrees C on the morphology of meiotic spindles in human metaphase II oocytes. Fertil Steril 75, 769–777.[CrossRef][ISI][Medline]

Submitted on October 19, 2004; resubmitted on December 12, 2004; accepted on December 12, 2004.