Human Reproduction Vol.21, No.7 pp. 1771–1776, 2006
doi:10.1093/humrep/del073
Advance Access publication March 20, 2006.
Sucrose concentration influences the rate of human oocytes
with normal spindle and chromosome configurations after
slow-cooling cryopreservation*
G.Coticchio1,8, L.De Santis2, G.Rossi3, A.Borini1, D.Albertini4, G.Scaravelli5, C.Alecci6,
V.Bianchi1, S.Nottola7 and S.Cecconi3
1
Tecnobios Procreazione, Bologna, 2Vita-Salute University, H.S.Raffaele, Milan, 3University of L’Aquila, L’Aquila, 4University of
Kansas Medical Center, Kansas City, KS, 5National Health Institute, Rome, 6UMR – Associazione HERA, Catania and 7‘La Sapienza’
University, Rome, Italy
8
To whom correspondence should be addressed at: Tecnobios Procreazione, Via Dante 15, 40125 Bologna, Italy.
E-mail: [email protected]
*On behalf of participants to the study on human oocyte cryopreservation supported by the Italian National Health Institute.
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.
Key words: oocyte cryopreservation/slow cooling/spindle apparatus/chromosomes/sucrose
Introduction
Oocyte cryopreservation has been a much anticipated advance
in human IVF and reproductive sciences that may extend
female reproductive potential, while avoiding ethical and legal
complications originating from the storage of embryos and fertilized oocytes. Because human oocytes are particularly sensitive to cryopreservation, irrespective of the employment of
either slow (controlled) cooling or vitrification as cryopreservation methods, this prospect has not been realized. During
slow cooling, the sources of cell damage are varied. Intracellular ice formation (IIF), cryoprotectant (CPA) toxicity, osmotic
stress, non-physiological supra-zero temperatures, pH instability and generation of reactive oxygen species (ROS) are factors
believed to affect oocyte viability (Fuller and Paynter, 2004).
Several oocyte organelles are suspected targets for cryodam-
age. Disruption or complete loss of the metaphase II (MII)
spindle is likely to compromise oocyte functionality. The
importance of this structure is well known, subserving not only
chromatid segregation at meiosis II but also the fertilization
process (Winston et al., 1995) and establishment of the cell
polarity (Albertini and Barrett, 2004). As a highly dynamic
structure originating from the reversible polymerization of
tubulin subunits, across mammalian species the oocyte MII
spindle appears to be prone to damage from non-physiological
depolymerization as a consequence to its extreme susceptibility to various extrinsic factors, especially temperature. In the
human, the MII spindle has been found to disassemble irreversibly in response to very short exposure to 0°C (Zenzes
et al., 2001) or even transient culture at a slightly lower, suboptimal temperature (Pickering et al., 1990). This has originated
© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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G.Coticchio et al.
the belief that damage to this structure is highly likely, if not
inevitable, as a result of the temperature changes involved in
cryostorage. In effect, Boiso et al. (2002) reported that chromosome and spindle organizations are extensively affected
by slow cooling. However, this matter remains highly controversial, because other authors have disputed such conclusions, suggesting that slow cooling does not compromise the
MII spindle and chromosome organizations (Gook et al.,
1993; Stachecki et al., 2004). Such a conflicting evidence
may have been generated in some cases by suboptimal quality
of the material examined, or inappropriate methodologies.
Clinical data are generally inadequate or too scarce to contribute to shed some light on the possible consequences of
cryopreservation on chromosome segregation at meiosis II.
Considering that chromosome anomalies are known to have
developmental consequences, an assessment of the comparative ability of embryos from fresh or frozen oocytes to
implant and give rise to viable, full-term pregnancies could
contribute to test this hypothesis, but unfortunately the studies conducted so far have not been designed with such an
intent.
Irrespective of the increasing number of reports of pregnancies from cryopreserved oocytes (Borini et al., 2004; Borini
et al., 2005; Chen et al., 2005; Levi Setti et al., 2006), because
the safety of oocyte cryopreservation remains an unresolved
concern, with this study we aimed at establishing the possible
impact on the MII spindle and chromosome configurations of
two alternative slow-cooling protocols differing in the concentration of the non-penetrating CPA sucrose in the freezing
solution. In a previous study (Fabbri et al., 2001), it was found
that increasing the sucrose concentration from 0.1 to 0.3 mol/l
dramatically improves the survival rate of human oocytes from
40 to about 80%. However, concerns have been raised that an
increase in CPA concentration, and hence in the extent of
osmotic stress to which the oocyte is exposed (Paynter et al.,
2005), may jeopardize viability, with possible, important consequences on the stability of the MII spindle.
Materials and methods
Source of oocytes
This study was approved by the Public Health Agency of the Italian
government, National Health Institute and the Institutional Review
Boards of the participating clinics. Oocytes were obtained from consenting couples undergoing assisted reproduction treatment from
January 2004 to April 2005. Only oocytes donated from women
younger than 36 years (mean ± SD: 32.6 ± 2.9) were used. Controlled
ovarian stimulation was induced with long protocols using GnRH
agonist and rFSH, according to the standard clinical protocols routinely employed by the participating clinics. HCG (10 000 IU) was
administered 36 h before oocyte collection. After retrieval, oocytes
were cultured in medium used for standard IVF for 3–4 h. Complete
removal of cumulus mass and corona cells was performed enzymatically using hyaluronidase (20–40 IU/ml) and mechanically by using
fine-bore glass pipettes. Only oocytes devoid of any sort of dysmorphisms, showing an extruded polar body I (PBI) thus presumably at
the MII stage, were assigned to the control or study groups. According
to their assignment, they were either fixed or frozen after a total
period of time of about 4 h following retrieval.
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Cryopreservation solutions
Oocytes were cryopreserved using a slow-cooling method. All cryopreservation solutions were prepared using Dulbecco’s phosphatebuffered solution (PBS) (Gibco, Life Technologies Ltd, Paisley, UK)
and a plasma protein supplement (PPS) (10 mg/ml), final concentration (Baxter AG, Vienna, Austria). The freezing solutions were 1.5
mol/l propane-1,2-diol (PrOH) + 20% PPS in PBS (equilibration solution) and 1.5 mol/l PrOH + either (a) 0.1 or (b) 0.3 mol/l sucrose + 20%
PPS in PBS (loading solutions), as described by Fabbri et al. (2001).
The thawing solutions were (i) 1.0 mol/l PrOH + sucrose + 20% PPS,
(ii) 0.5 mol/l PrOH + sucrose + 20% PPS and (iii) sucrose + 20% PPS.
Sucrose concentration in the thawing solutions was 0.2 and 0.3 mol/l,
depending of whether loading solutions (a) or (b) had been used for
freezing, respectively.
Freezing procedure
Oocytes were washed in PBS supplemented with 20% PPS. One or
two oocytes were subsequently placed in the equilibration solution
containing 1.5 mol/l PrOH + 20% PPS for 10 min. Afterwards,
oocytes were transferred to the solution containing 1.5 mol/l PrOH +
0.1 or 0.3 mol/l sucrose + 20% PPS, at room temperature (RT) for
5 min, then loaded into plastic straws (Paillettes Crystal 133 mm;
Cryo Bio System, France) and placed into an automated Kryo 10
series III biological freezer (Planer Kryo 10/1.7 GB). Temperature
was gradually lowered from 20 to 8°C at a rate of 2°C/min. Manual
seeding was induced during the 10-min holding ramp at –8°C. The
temperature was decreased then to –30°C at a rate of 0.3°C/min and
finally rapidly to –150°C at a rate of 50°C/min. The straws were
finally plunged into liquid nitrogen and stored for later use.
Thawing procedure
The straws were rapidly air-warmed for 30 s and then plunged into a
30°C water bath for 40 s. The CPA was removed at RT by step-wise
dilution. Oocytes were expelled in the first thawing solution, 1.0 mol/l
PrOH + sucrose + 20% PPS for 5 min, then equilibrated in 0.5 mol/l
PrOH + sucrose + 20% PPS for additional 5 min. Finally, they were
placed in sucrose + 20% PPS for 10 min before final dilution in PBS +
20% PPS for 20 min (10 min at RT and 10 min at 37°C). Before fixation, thawed oocytes were cultured for 3 h in 20 μl of drops of
glucose-free medium normally used for embryo culture under warm
mineral oil at 37°C in an atmosphere of 5% CO2 in air.
Immunofluorescence
Only morphologically normal oocytes were used for this study. After
freezing and thawing, oocytes showing swelling or shrinkage, vacuoles, membrane blebbing or other anomalies were not considered viable and suitable for microscopy analysis. Oocytes were fixed and
processed for microtubules and chromatin immunofluorescence as
previously described (Combelles et al., 2002).
Fixation and extraction was carried out for 30–60 min at 37°C in a
microtubule-stabilizing buffer containing 100 mM PIPES, 5 mM
MgCl2, 2.5 mM EGTA, 2% formaldehyde, 0.1% Triton-X-100, 1 mM
taxol, 10 U/ml of aprotinin and 50% deuterium oxide (Cekleniak
et al., 2001). Washing and storage was conducted with a blocking
solution of PBS supplemented with 2% bovine serum albumin, 2%
powdered milk, 2% normal goat serum, 100 mM glycine and 0.01%
Triton-X-100 containing 0.2% sodium azide. After storage in blocking solution (for a few weeks maximum), oocytes were further processed for immunostaining, through serial incubations with primary
and secondary antibodies (1 h per antibody at 37°C with shaking) followed by washing (three washes of 15 min) in blocking solution after
each antibody incubation. Microtubule staining was performed by
Oocyte cryopreservation and meiotic spindle
using a mouse monoclonal anti-α/β tubulin antibody mixture (Sigma
Biosciences, Milan, Italy) diluted 1:150 in wash solution, followed by
treatment with a 1:50 dilution of an affinity-purified fluoresceinlabelled donkey anti-mouse IgG (Jackson Immuno Research Laboratories, West Growe, PA, USA). Chromatin staining was obtained by
using either propidium iodide or Hoechst 33258 (30 min), whereas
mounting was carried out with a mixture of 50% glycerol and 25 mg/ml
of the antifade agent sodium azide. Oocytes were analysed using
either a fluorescence microscope (AxioPlan 2, Zeiss; ×40 objective;
digital images collected with a Leica DFC350 FX camera interfaced
with IM500 Leica software) or a IX81 Olympus confocal microscope
(×60 objective; digital images collected with Fluoview FW500).
Statistical analysis
Statistical comparison between fresh and frozen groups was carried
out on pooled data using chi-square analysis. A P-value <0.05 was
considered significant.
Results
Three hundred and seventeen PBI oocytes were successfully
examined through confocal microscopy for the assessment of
the MII spindle and chromosome organizations. The survival
rates of the cryopreserved oocytes with either the 0.1 or the
0.3 mol/l sucrose protocol were 35 and 74%, respectively, an
outcome comparable to those published in previous articles
(Fabbri et al., 2001; Borini et al., 2004; Borini et al., 2005;
Chen et al., 2005; Levi Setti et al., 2006). The loss of viability
was observed in the large majority of cases (>80%) immediately after thawing or during rehydration, although it was
rarely observed during the 3 h of post-thaw culture.
Spindle organization was considered morphologically normal when a dense, bipolar (barrel-shaped or elliptical) array of
microtubles was observed. Chromosome distribution was
regarded regular when sister chromatids exhibited metaphase
alignment on the equatorial plane of a symmetric spindle. Basically, three different classes of spindle and chromosome configurations were observed using these criteria to define deviations
from the commonly observed MII phenotype (Battaglia et al.,
1996; Hodges et al., 2002; Combelles et al., 2003; Hunt et al.,
2003): (i) bipolar spindle, aligned chromosomes; (ii) disarranged spindle, non-aligned chromosomes and (iii) absent spindle, non-aligned or dispersed chromosomes (Figure 1).
Figure 1. Representative confocal pictures (×60 magnification) and
respective schematic descriptions of bipolar spindle and equatorial
chromosome alignment (left), disarranged spindle and irregular chromosome distribution (centre) and absent spindle and disorganized
chromosome distribution (right).
Of the 104 oocytes included in the unfrozen control, 76
(73.1%) displayed normal (bipolar) spindle and chromosome
configurations, 18 (17.3%) showed anomalies involving both
the spindle and the chromosomal arrays and 10 (9.6%)
appeared devoid of organized microtubules, although possessing non-aligned or dispersed chromosomes (Table I). In the
oocytes frozen with the 0.1 mol/l sucrose protocol (n = 61),
altered frequencies were found in the three classification categories. A bipolar spindle associated to an orderly array of chromosomes was observed in only 31 (50.8%) of oocytes.
Compared to the fresh control, such a difference was found to
be statistically significant (P < 0.05). In the same treatment
group, 26 oocytes (42.6%; P < 0.05) were classified as having
non-bipolar or grossly abnormal spindles with disarranged
chromosomes, and four (6.6%) exhibited only disarranged or
dispersed chromosomes without microtubule staining. In the
treatment group corresponding to the 0.3 mol/l sucrose protocol, oocytes (n = 152) were classified according to the categories (i), (ii) and (iii) with frequencies comparable to those of
the control group. In particular, regular spindle and chromosome arrangement was ascertained in 106 (69.7%; P = NS)
oocytes, whereas the disruption or total loss of spindle microtubules was detected in 35 (23.0%) and 11 (7.3%) oocytes,
respectively.
Discussion
Initial attempts at adopting oocyte cryopreservation for storing
the reproductive potential of IVF patients date back as early as
1986 (Chen, 1986; Al-Hasani et al., 1987), but current cryopreservation techniques remain unable to generate clinical
results consistently comparable to those achievable with
embryo freezing. Protocols used lately have improved survival
frequencies (Fabbri et al., 2001; Boldt et al., 2003), thereby
overcoming a major hurdle that limited widespread utilization
of oocyte storage. Despite promising advances, it remains
unclear why cryopreserved oocytes can survive, fertilize,
cleave with high rates and yet implant with rates that appear
compromised (Borini et al., 2005; Levi Setti et al., 2006). It
has been suggested repeatedly that the loss of functionality of
frozen oocytes may be secondary to the meiotic spindle sensitivity to the processes of freezing and thawing, a circumstance
that could cause increased aneuploidy rates in the embryos
thereby generated. The data illustrated in this study indicate
that, in the oocytes that are found functional after thawing with
a protocol including a high concentration of the non-penetrating
CPA in the freezing solution, the meiotic spindle organization
and the alignment of associated chromosomes are unaltered.
However, our methodology did not allow us to establish
whether these structures are affected in the cryopreserved
oocytes that do not survive. Our observations also suggest that,
to a certain extent, a specific protocol characteristic, i.e. low
sucrose concentration, can compromise the chromosome segregation apparatus.
It is not surprising that cytoskeletal damage has been
assumed to be an obvious consequence of cryopreservation,
considering the detrimental effects on the MII spindle of transient exposure to lower, suboptimal temperatures (Pickering
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G.Coticchio et al.
Table I. Frequencies of meiotic spindle configuration and chromosome organization in fresh oocytes or in the surviving morphologically normal fraction after
cryopreservation with alternative protocols
Treatment (number of oocytes)
Bipolar spindle and
aligned chromosomes (%)
Disarranged spindle,
non-aligned chromosomes (%)
Absent spindle, non-aligned
or dispersed chromosomes (%)
Fresh (104)
Frozen, 0.1 mol/l sucrose (61)
Frozen, 0.3 mol/l sucrose (152)
76 (73.1)a
31 (50.8)b
106 (69.7)a
18 (17.3)a
26 (42.6)b
35 (23.0)a
10 (9.6)
4 (6.6)
11 (7.3)
a versus b: P < 0.05 within each column.
et al., 1990; Wang et al., 2001). Specific studies on cryopreserved
oocytes, on the contrary, have not been always consistent with
this evidence. Using the classical PrOH-based slow-cooling
method (Lassalle et al., 1985), Gook et al. (1993, 1994) were
the first to challenge the prevailing dogma, reporting that
freezing with the conventional slow-cooling protocol does not
reduce, in a statistically significant fashion, the rate of oocytes
with a normal spindle, nor does it cause aneuploidies after fertilization. Cobo et al. (2001) have further strengthened the
hypothesis that chromosome segregation is unaffected in frozen oocytes. Using probes for chromosomes 13, 18, 21, X and
Y, these authors conducted a PGD assessment, ascertaining
that embryos developed from oocytes frozen with a 1.5 mol/l
PrOH and 0.2 mol/l sucrose protocol had a rate of aneuploidy
similar to the one of embryos from fresh oocytes (28 and 26%,
respectively). However, it should be noticed that only 21
embryos from frozen oocytes were examined in this study.
Reassuring evidence has been recently described also by
Stachecki et al. (2004), whose data suggest that the proportion
of oocytes with barrel-shaped spindles and chromosomes regularly aligned on the spindle equatorial plane is unchanged after
cryopreservation (76.7 and 76.7% versus 69.7 and 71.2%, in
fresh and frozen oocytes, respectively). It is interesting to note
that these authors used a slow-cooling protocol involving the
replacement of sodium with the less toxic cation choline, to
reduce the so-called solution effect occurring during freezing.
Also, it is not clear whether the authors’ choice of exposing
oocytes to freezing solutions for varying times (10–20 min or
5–10 min, depending of the freezing solution) may have influenced the outcome of this study. In effect, we have recently
shown that exposure times to CPAs may significantly influence the rate of dehydration (Paynter et al., 2005), with possible implications for the risk of IIF. The issue of the quality of
oocytes assigned to experiments on cryopreservation is of critical importance. As an example, it has become progressively
clearer that inappropriate in vitro conditions have major implications for many oocyte attributes, including the structure and
possibly the function of the meiotic spindle (Cekleniak et al.,
2001; Sanfins et al., 2003). Therefore, we reckon that oocytes
matured in vitro cannot be considered a suitable model for
establishing the response of mature oocytes to cryopreservation. Furthermore, polarized light microscopy provides evidence that human oocytes with an extruded PBI may not have
reached full meiotic maturation, with their microtubular apparatus still not organized in a properly formed MII spindle (De
Santis et al., 2005). Moreover, the same microscopy technique
has ascertained that spindle presence is less frequent in oocytes
1774
showing the PBI observed at <38 h following HCG injection
(Cohen et al., 2004). Importantly, in our study, we observed
morphologically normal, supernumerary oocytes that would
otherwise have been used for treatment and that were fixed at
appropriate times from oocyte recovery and thawing.
Our data indicate that normal MII spindle configuration and
chromosome alignment are not disturbed by the processes of
freezing and thawing during the application of a modified version of the slow-cooling method including 0.3 mol/l sucrose in
the freezing solution. In control oocytes, we ascertained tubulin staining in about 90% of oocytes, although only in a smaller
fraction (73.1% of the total) we observed an orderly array of
microtubules and a regular chromosome distribution. These
figures are comparable to the previously cited findings of
Stachecki et al. (2004). It seems established, then, that about
30% of fresh oocytes are found with disorganized microtubules
or, in extreme case (5–10%), without a visible spindle. The
fraction of oocytes with abnormal spindles varies depending on
age (Battaglia et al., 1996), a circumstance that suggested us to
limit our observations to oocytes donated by patients younger
than 36 years. In our experience, cryopreservation with a slowcooling method in which sucrose concentration in the loading
solution was 0.3 mol/l did not essentially affect the regularity
of spindle and chromosome arrangement in the oocytes that
maintained a normal morphology after freezing and thawing.
This finding confirms and completes previous observations
that had documented via polarized light analysis the presence
of the MII spindle in oocytes stored with the same protocol
(Bianchi et al., 2005). It is difficult to speculate on the reason
why transient exposure to just a few degrees below optimal
temperature can disrupt irreversibly the spindle, although, during cryopreservation, exposure to much lower temperatures
does not cause the same effect. It is possible that permeating
CPAs, irrespective of their action of dehydration and mitigation of the solution effect, can influence microtubular polymerization (Johnson and Pickering, 1987). By contrast, the
application of the protocol involving a lower sucrose concentration (0.1 mol/l) was associated to a limited but statistically
significant reduction in the rate (50.8%) of oocytes with normal spindle and chromosome configurations. Sucrose is a nonpenetrating CPA whose concentration in the freezing solution
influences the magnitude of the osmotic gradient that draws
water from the oocyte. The effect of non-penetrating CPA concentration (0.1 versus 0.3 mol/l) on the rate of dehydration in
the pre-cooling phase of exposure to the freezing solution has
been described in detail (Paynter et al., 2001; Paynter et al.,
2005). Because during slow cooling the amount of residual
Oocyte cryopreservation and meiotic spindle
water in the intracellular environment is associated to the risk
of mechanical damage caused by IIF, it is tempting to speculate that the 0.1 mol/l sucrose protocol is inadequate to ensure
sufficient rates of dehydration. Consequently, the MII spindle
may be more likely to be disrupted by ice crystals. Conversely,
with the 0.3 mol/l sucrose protocol, it may be possible to reach
the levels of dehydration that prevent the formation of significant intracellular ice, leaving the spindle relatively undisturbed.
Irrespective of IIF during cooling, the MII spindle may be
affected during other phases of the cryopreservation process.
When oocytes are stored with the 0.1 mol/l sucrose protocol,
immediately after removal from liquid nitrogen and rewarming
at room temperature, the spindle, which is initially found in a
reduced but significant proportion of oocytes, undergoes depolymerization during rehydration and CPA removal, for reappearing after the completion of the thawing process and
incubation for 3 h (Rienzi et al., 2004; Bianchi et al., 2005). It
is plausible to suppose, therefore, that the irreversible loss of
spindle organization may occur also during this final phase of
rehydration after recovery from low temperature storage,
although it is not obvious how inappropriate thawing and CPA
extraction conditions can influence spindle dynamics.
It is interesting to note that the different ability of the two protocols tested in our study to preserve the microtubular apparatus
and associated chromosomal array is consistent with clinical
evidence obtained through the application of the same procedures. In particular, fertilization rates in microinjected oocytes
stored with the 0.1 mol/l sucrose protocol rarely exceed 45–50%
(Tucker et al., 1998; Borini et al., 2004), whereas in oocytes cryopreserved with the alternative protocol these rates (65–75%)
are indistinguishable from normal values (Borini et al., 2005;
Chen et al., 2005; Levi Setti et al., 2006). The lower incidence of
normal spindle organization after freezing with the 0.1 mol/l
sucrose protocol may play a causal role in the establishment of
such a difference in fertilization rate, in the light of the fact that
the meiotic spindle, in addition to co-ordinating chromosome
segregation, is also critical for the regulation of meiosis. In fact,
in mouse oocytes, alterations (Brunet et al., 2003) or complete
loss (Winston et al., 1995) of the spindle have been shown to
prevent chromosome segregation and/or exit from meiotic
M-phase. The frequency of frozen oocytes with normal meiotic
spindle could also have an influence on the chances that clinical
pregnancies can evolve to full term. Some oocytes with abnormal spindles could undergo fertilization and generate aneuploid
pregnancies. Considering our data, it is interesting to observe
that cryopreservation with either the 0.1 or the 0.3 mol/l sucrose
protocols has given rise to different abortion rates (20.0 and
14.2%, respectively) (Borini et al., 2004; Borini et al., 2005), but
presently it is not possible to draw definite conclusions because
of the limited numerical significance of the clinical experiences
and the lack of information on the causes of the developmental
failures described in these studies.
We reckon that our data represent a significant contribution
to the assessment of oocyte quality following cryopreservation,
especially with reference to a protocol (0.3 mol/l sucrose) that
has already proven to generate high survival rates and that is
being adopted for clinical purposes, especially in Italy where
embryo freezing is prohibited. However, assessment of the
chromosome segregation apparatus cannot be considered
exhaustive to the aim of evaluating oocyte viability. This opinion is confirmed by the observation that oocytes cryopreserved
with the 0.3 mol/l sucrose protocol, although possessing unaffected spindle and chromosome organizations, develop into
embryos that implant with frequencies that have not exceeded
5.7% in at least two independent studies (Borini et al., 2005;
Levi Setti et al., 2006). Clearly, other factors other than damage to the cytoskeletal apparatus may be responsible for the
poor developmental performance of these oocytes. We are currently involved in investigations using other criteria to evaluate
oocyte post-thaw quality. In effect, ultrastructural analysis of
oocytes stored with the 0.3 mol/l sucrose protocol has revealed
abnormalities of the cytoplasm that may be interpreted as a
possible cause of compromised viability (S. Nottola, unpublished data).
In conclusion, in this article, we provided evidence that during slow cooling spindle and chromosome configurations are
safely preserved provided that a higher sucrose concentration
in the freezing solution is used. Our data also suggest that each
individual protocol should be tested, considering that limited
changes in the technical procedure may have significant consequences on the process of freezing and ultimately on oocyte
quality. However, analysis on spindle and chromosome
arrangement appears of relative value, being unable to provide
full explanation to the clinical data that have been recently
described. This calls for broader spectrum investigations on the
quality of frozen oocytes.
Acknowledgements
This work was partly funded by the Italian National Institute of Health
and MIUR. We are very grateful to Dr Ilaria Cino for her technical
support.
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Submitted on December 7, 2005; resubmitted on February 11, 2006; accepted
on February 15, 2006