doi:10.1093/humrep/deh736
Human Reproduction Vol.20, No.4 pp. 1078–1083, 2005
Advance Access publication March 10, 2005
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 2University of Bologna, 40125 Bologna, Italy
3
To whom correspondence should be addressed. E-mail: [email protected]
Key words: human oocyte/meiotic spindle/oocyte cryopreservation/Polscope/thawing
Introduction
In human IVF, oocyte cryopreservation so far has been
applied with limited success. Various studies (Porcu et al.,
2000; Borini et al., 2004; Chen et al., 2004) 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.,
2001) or the replacement of sodium with choline in the freezing mixtures (Stachecki et al., 1998; Quintans et al., 2002).
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., 1995), jeopardizing the establishment of a viable
pregnancy.
Oocyte cryopreservation has been associated with hardening (Carroll et al., 1990) or fracturing (Fuku et al., 1995) 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., 1999). 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.,
2004). Alterations in mitochondrial function in cryopreserved
human oocytes have also been documented (Jones et al.,
2004). Because of its sensitivity to low temperatures
(Pickering et al., 1990; Zenzes et al., 2001), 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., 2002), other studies have suggested that
1078 q The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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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 6 1.5 mol/l
SD 5 2.58 6 0.1 nm), assessed through analysis, were included in this study. Oocytes were cryopreserved with a
1.5mol/l 1,2-propanediol 10.3 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 6 0.3 nm) or high (2.50 6 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.
Spindle visualization after oocyte cryopreservation
Materials and methods
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., 2001). 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 ml 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 8C
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 200£ 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), 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 þ30% PPS in PBS (equilibration solution) and (ii) 1.5 mol/l
PROH þ0.3 mol/l sucrose þ 30% PPS in PBS (loading solution), as
described by Fabbri et al. (2001).
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 8C).
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 8C then slowly reduced to 2 7 8C
at a rate of 2 2 8C/min. Ice seeding was induced manually at 2 7 8C;
after a hold ramp at 27 8C for 10 min, the straws were cooled
slowly to 230 8C at a rate of 20.3 8C/min and then rapidly to
2150 8C at a rate of 2 50 8C/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 8C 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 þ0.3 mol/l sucrose þ30% PPS) (5 min), then equilibrated in 0.5 mol/l PROH þ0.3 mol/l sucrose þ30% PPS for
another 5 min. Finally they were placed in a 0.3 mol/l sucrose
þ30% PPS for 10 min before final dilution in PBS solution þ30%
PPS for 20 min (10 min at RT and 10 min at 37 8C). The oocytes
were placed in 20 ml drops of cleavage medium (Cook IVF) under
warm mineral oil (Cook IVF) at 37 8C 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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this structure can survive freezing– thawing without consequences (Gook et al., 1993; Stachecki et al., 2004) in the
absence of chromosome dispersal in the oocyte or an increase
in aneuploidies in the resulting embryo (Cobo et al., 2001).
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 Polscopew, a recently introduced microscopy apparatus that, using polarized light, allows the observation of
highly ordered subcellular structures such as the spindle
microtubules (Oldenbourg, 1999; Wang et al., 2001a). Compared with immunostaining or other microscopy methods, the
Polscope is totally non-invasive, and therefore oocyte viability can be preserved (Keefe et al., 2003). 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., 2001b), as
well as embryo quality (Moon et al., 2003), are to some
extent dependent on spindle presence and localization.
V.Bianchi et al.
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 8C for 1 h.
Treatment with FITC-conjugated secondary antibody diluted 1:50
was performed in the dark at 37 8C 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 x2-test for qualitative
variables. Differences were considered significant when a P-value
was , 0.05.
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).
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).
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
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 8C for 30 min
with a buffer containing 3.7% formaldehyde. Afterwards, they were
1080
Figure 2. Diagram showing different rates of birefringence signal
intensity at different time points after thawing.
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Results
Spindle visualization after oocyte cryopreservation
Table I. Birefringence at different times of observation
Time (h)
Signal
Absent
0
3
5
a
63
27b
20b
Weak
a
19
41b
43b
High
1a
15b
20b
a,b
Within each category of signal intensity (absent, weak, high) values with
different superscripts are statistically different (P , 0.001).
Discussion
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., 2001).
Fabbri et al. (2001) 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.
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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.
On the other hand, treatment with elevated cryoprotectant
concentrations coincides with fluctuations in cell volume
(Paynter et al., 2001) 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 8C causes spindle
disassembly and a series of cytoskeletal changes derived by
the increase in monomeric tubulin (Pickering and Johnson,
1987). To a large extent, however, in most of these oocytes
spindle organization can be re-established following re-incubation at 37 8C. 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., 1990). More recently, testing human oocytes with the Polscope, Wang et al. (2001c)
found that a slight temperature reduction to 33 8C 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 8C,
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., 2002). 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., 1993; Stachecki et al., 2004). 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., 1995). In
contrast, Boiso et al. (2002) 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, 1995) or vitrification (BosMikich et al., 1995) 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
V.Bianchi et al.
1082
Rienzi et al. (2004) 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) 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., 2001b). 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., 2002). 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
We thank Dr Maria Luisa Vanelli, Department of Genetics
University of Bologna for her assistance with statistical data
analysis.
Downloaded from http://humrep.oxfordjournals.org/ at Pennsylvania State University on March 3, 2014
Davis, 1994). 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., 2001).
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, 2001), 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., 2003). Some authors have
reported lower incidence of spindle visualization in fresh
oocytes (Wang et al., 2001b), but this may reflect technical
limitations of the early Polscope prototype, as well as patientspecific 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), 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., 1992). 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.
Spindle visualization after oocyte cryopreservation
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Submitted on October 19, 2004; resubmitted on December 12, 2004;
accepted on December 12, 2004
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