Human Reproduction Vol.19, No.3 pp. 655±659, 2004
Advance Access publication 29 January, 2004
DOI: 10.1093/humrep/deh101
Polscope analysis of meiotic spindle changes in living
metaphase II human oocytes during the freezing and
thawing procedures
L.Rienzi1,3, F.Martinez1, F.Ubaldi1, M.G.Minasi1, M.Iacobelli1, J.Tesarik2 and E.Greco1
1
Centre for Reproductive Medicine, European Hospital, Via Portuense 700, 00149 Rome, Italy and 2MAR&Gen, Molecular Assisted
Reproduction & Genetics, Granada, Spain
3
To whom correspondence should be addressed. E-mail: [email protected]
BACKGROUND: The clinical ef®cacy of the current methods used for cryopreservation of metaphase II human
oocytes is low. Meiotic spindle disorders are thought to be largely responsible for this situation. METHODS:
Supernumerary fresh metaphase II human oocytes were cryopreserved in 1,2-propanediol with 0.1 M sucrose using
a slow freezing/rapid thawing programme. Meiotic spindles were analysed in these living metaphase II oocytes at
sequential steps of the freezing and thawing procedures with the use of a computer-assisted polarization microscopy
system (Polscope). RESULTS: The meiotic spindle was detected in all 56 oocytes (from 16 patients) before freezing
and remained visible in all these oocytes throughout the preparation for freezing up to the time that they were
loaded into cryopreservation straws. Immediately after thawing, the spindle was visible in 35.7% of oocytes, but it
disappeared in all of the thawed oocytes during the subsequent washing steps. However, the spindle reappeared in
all surviving thawed oocytes after washing (57.4%), by 3 h of incubation at 37°C in culture medium.
CONCLUSIONS: The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post-thaw reconstruction.
Key words: freezing/meiotic spindle/metaphase II/Polscope/thawing
Introduction
Oocyte cryopreservation has been used in human assisted
reproduction since the late 1980s (Chen, 1986; Al-Hasani et al.,
1987; Fabbri et al., 1998; Mandelbaum et al., 1988; Tucker
et al., 1998). However, this technique met with only limited
success when conventional IVF was used because the freezing
and thawing procedures caused the occurrence of a premature
cortical reaction (Schalkoff et al., 1989) leading to hardening
of the zona pellucida and reduced sperm penetration (Johnson
et al., 1988; Todorow et al., 1989). The introduction of ICSI
(Palermo et al, 1992) made it possible to circumvent the
negative consequences of zona pellucida hardening for
fertilization. However, cryopreservation has also been shown
to produce alterations in the meiotic spindle of metaphase II
mouse (Magistrini and Szollosi, 1980; Sathananthan et al.,
1988a; Van der Elst et al., 1992) and human (Sathananthan
et al., 1988b; Pickering et al., 1990; Gook et al., 1995) oocytes,
and this problem obviously cannot be overcome by the use of
micromanipulation-assisted fertilization.
Abnormalities of the metaphase II meiotic spindle can be
expected to lead to abnormal segregation of chromosomes after
fertilization of cryopreserved oocytes, resulting in aneuploid
embryos. However, a recent study failed to ®nd any signi®cant
differences in aneuploidy rate between embryos derived from
cryopreserved oocytes as compared with those developing
from fresh oocytes (Cobo et al., 2001). These surprising
®ndings could be explained either by the existence of a
mechanism preventing complete spindle disintegration during
the freezing and thawing procedures or by a rapid reconstruction of the cryo-damaged spindles.
Most studies dealing with the status of the meiotic spindle
carried out so far used electron microscopy or immunocytochemistry techniques (reviewed in Eichenlaub-Ritter et al.,
2002). However, this approach requires oocyte ®xation and
thus cannot be used to follow dynamic changes in the same
oocytes at different time points. In this study, we employed the
Polscope system, which permits non-invasive visualization of
spindles in living oocytes (Liu et al., 2000; Wang et al., 2001a;
Moon et al., 2003) to follow the behaviour of the metaphase II
meiotic spindle at different steps of the freezing and thawing
procedures.
Materials and methods
Oocyte source and preparation
This study was approved by the Ethical Committee of the European
Hospital.
Human Reproduction vol. 19 no. 3 ã European Society of Human Reproduction and Embryology 2004; all rights reserved
655
L.Rienzi et al.
Metaphase II oocytes were obtained from consenting patients from
our IVF-ICSI programme only when an adequate number of oocytes
was retrieved.
Ovarian stimulation was conducted in all patients as described
previously (Ubaldi et al., 1999) using a combination of GnRH agonist
(subcutaneous buserelin acetate 0.2 mg twice daily; Suprefact;
Hoechst, Marion Roussel, Milan, Italy) started in late luteal phase of
the previous cycle, recombinant FSH (Gonal-F, 75 IU, Serono, Rome
Italy or Puregon 100 IU, Organon, Oss, The Netherlands) and HCG
(Profasi; Serono).
Oocyte decumulation has been described extensively elsewhere
(Rienzi et al., 1998). Brie¯y, the cumulus and corona radiata were
removed immediately after ovum pick-up by a brief exposure to
HEPES-buffered medium (Gamete, Vitrolife, Gothenburg, Sweden)
containing 20 IU/ml hyaluronidase fraction VIII (Hyase-10X;
Vitrolife) and by gentle aspiration in and out of a plastic pipette
(Flexipet, 130 mm i.d., COOK, Australia). The denuded oocytes were
then evaluated to assess their nuclear maturation stage. Only oocytes
that had released the ®rst polar body (metaphase II) were included in
this study. Immediately after decumulation, the supernumerary
metaphase II oocytes obtained were used for meiotic spindle
observation and the freezing procedure.
Freezing and thawing procedures
The freezing solution consisted of phosphate-buffer saline (PBS) with
25 mg/ml human serum albumin (HSA; Cryo-PBS, Vitrolife), freezing
solution 1 containing 1.5 M 1,2-propanediol (PrOH) in Cryo-PBS
(FS1, Vitrolife) and freezing solution 2 containing 1.5 M PrOH + 0.1 M
sucrose in Cryo-PBS (FS2, Vitrolife). A maximum of three oocytes
were frozen per straw. Equilibration with the freezing solutions was
carried out at room temperature. Oocytes were ®rst incubated for 2 min
in Cryo-PBS, then for 10 min in freezing solution 1 and ®nally in
freezing solution 2, and immediately loaded into straws (paillette
cristal, Cryo Bio System, IMV Technologies, France). The straws
were placed into a freezing chamber (CL-863, Cryologic, Australia) at
room temperature. The temperature was decreased to ±7°C at a rate of
2°C/min. Seeding was done manually by touching the straw close to
the cotton plug with a nitrogen-cooled forceps. After holding for
10 min at ±7°C, the temperature was lowered slowly at a rate of 0.3°C/
min to ±30°C, followed by rapid cooling (±10°C/min) to ±110°C. The
straws were then plunged into liquid nitrogen (LN2), and stored
submerged in LN2 until thawing.
For thawing, the straws were removed from LN2 and rapidly
warmed to room temperature. The retrieved oocytes were then
transferred to a plastic 4-well dish where they were washed free from
the cryoprotectant at room temperature by stepwise dilutions in 1.0,
0.5 and 0.0 M PROH in the presence of 0.1 M sucrose and Cryo-PBS
(Thaw Kit 1, Vitrolife). The exposure times for thawing solutions were
5 min (solution 1), 10 min (solution 2), 10 min (solution 3) and 10 min
(PBS). All surviving oocytes (with an intact membrane and clear
cytoplasm) were cultured further in IVF-medium (Vitrolife) at 37°C,
5% CO2 for 3 h.
Spindle visualization by Polscope
Meiotic spindles were imaged at sequential steps during the freezing±
thawing procedure. Brie¯y, the oocytes were placed in a 5 ml drop of
the corresponding medium covered with mineral oil (ovoil, Vitrolife,
Gothemburg, Sweeden) in a glass-bottomed culture dish (Willco
Wells, Amsterdam, The Netherlands). The meiotic spindle visualization was performed at 203 magni®cation with LC Polscope optics and
controller (SpindleView, CRI, Woburn, MA), combined with a
computerized image analysis system (SpindleView software, CRI).
For this purpose, each oocyte was rotated with the use of the injection
656
Figure 1. Sequential Polscope images of a metaphase II human
oocyte at different steps of the freezing procedure. The meiotic
spindle (arrow) is visible during incubation in PBS (A), in freezing
solution 1 (B) and in freezing solution 2 (C). Scale bar = 50 mm.
pipette until both the meiotic spindle and the polar body were clearly
in focus in the oocyte equatorial plane.
The oocytes were maintained at 37°C on a heated stage (Linkam
Scienti®c instruments Ltd, UK) during the spindle assessment in
HEPES-buffered culture medium (Gamete, Vitrolife) performed
before starting the freezing procedure and at the end of the thawing
procedure. All meiotic spindle observations at different steps of the
freezing±thawing procedure were performed at room temperature.
Spindle visualization by immunocytochemistry
All chemicals used were purchased from Sigma (St Louis, MO).
Oocytes were treated with 0.5% pronase E from Streptomyces griseus
type XIV and ®xed with 4% formaldehyde in PBS. The ®xed oocytes
were then rinsed in PBS and permeabilized using Triton X-100, 0.02%
in PBS. After washing in PBS, the oocytes were incubated in blocking
solution containing 5% HSA. Oocytes were double-stained for
microtubules and chromosomes by incubation in ¯uorescein isothiocyanate (FITC)-conjugated monoclonal anti-a-tubulin diluted 1:100
in PBS followed by incubation in 10 mg/ml of propidium iodide in
PBS. Oocytes were then mounted on poly-L-lysine-treated slides that
had a peripheral epoxy ring, covered and sealed with clear nail
varnish, and examined in a Nikon Eclipse E600 ¯uorescence
microscope.
Results
The meiotic spindle was visualized in all of the 56 metaphase II
oocytes used in this study obtained from 16 different patients.
As the oocytes became progressively dehydrated during
equilibration with the freezing solutions, the spindles not
only remained detectable in all oocytes in spite of the
temperature decrease but even became brighter, with the
maximum brightness observed in freezing solution 2 (Figure 1).
Thereafter, the oocytes were loaded into cryopreservation
straws and the behaviour of their spindles could not be
evaluated any more.
Immediately after the removal of the straws from liquid
nitrogen and expulsion of the oocytes from the straws, a
meiotic spindle was detected in 20 out of the 56 oocytes
examined (35.7%). However, 10 (50%) of the oocytes did not
survive throughout the subsequent steps of the thawing
procedure, during which cryoprotectant was progressively
washed out, and these oocytes were discarded from further
analysis. When the oocytes that survived the entire thawing
procedure were taken into account, the overall percentage of
oocytes in which the meiotic spindle was detected was 31.3%
in thawing solution 1, 25.0% in thawing solution 2, 0% in
Meiotic spindle and oocyte cryopreservation
Table I. Spindle visualization at different stages of the thawing procedure in in vivo matured metaphase II oocytes that remained alive throughout the
procedure
Meiotic spindle
Solution 1
Solution 2
Solution 3
PBS
Medium (37°C)
Detected (%)
Not detected (%)
10 (31.3)
22 (68.7)
8 (25.0)
24 (75.0)
0 (0)
32 (100)
0 (0)
32 (100)
32 (100)
0 (0)
Figure 4. Polscope image of a fresh metaphase II human oocyte
incubated for 10 min in thawing solution 3. A bright meiotic spindle
(arrow) is clearly visible. Scale bar = 50 mm.
Figure 2. Sequential Polscope images of a metaphase II human
oocyte at different steps of the thawing procedure. The meiotic
spindle (arrow) is visible in thawing solution 1 (A) and in thawing
solution 2 (B), but it disappears during incubation in thawing
solution 3 (C) and PBS (D). The meiotic spindle is visible again
after 3 h of incubation at 37°C in culture medium (E). Scale
bar = 50 mm.
Figure 3. Sequential Polscope images of a metaphase II human
oocyte in which the meiotic spindle was undetectable immediately
after thawing in thawing solution 1 (A) and re-appeared after
cryoprotectant removal during incubation at 37°C in culture medium
(B). Immunocytochemical visualization of residual tubulin
immunoreactivity associated with chromosomes of a frozen±thawed
oocyte lacking the meiotic spindle (C).
thawing solution 3, 0% in PBS and 100% after an additional 3 h
of incubation at 37°C in culture medium.
The timing of the changes in meiotic spindle morphology
was not uniform in individual oocytes (Table I). In most
oocytes in which the spindle was visualized in thawing solution
1 immediately after expulsion from cryopreservation straws,
the spindle disappeared during incubation in thawing solution 3
(Figure 2). However, the disappearance of the spindle as early
as during incubation in thawing solution 2 was also observed
(Table I). At the end of incubation in thawing solution 3, there
was not a single oocyte in which the spindle could be detected.
However, the spindle reappeared again by 3 h of incubation at
37°C in culture medium in all of the oocytes that survived the
entire procedure and in which it was observed immediately
after the expulsion from the cryopreservation straws (Table I).
Meiotic spindle re-appearance after thawing solution removal
was also observed in all oocytes in which the spindle was
undetectable in thawing solution 1 immediately after thawing
(Figure 3, Table I). The position of the reconstructed spindle
with regard to the ®rst polar body was the same as that of the
original, pre-freeze spindle position in all oocytes (Figure 2).
To assess the possible effects of the thawing media on
meiotic spindle visualization, fresh oocytes were incubated for
10 min in thawing solutions 1, 2 and 3 (three oocytes in each).
The spindle remained clearly visible in all of these oocytes at
the end of incubation (Figure 4), and its brightness was even
greater than before incubation in all cases. Thus, the disappearance of the spindles during thawing of frozen oocytes was
not caused by optical properties of the thawing solutions.
In all oocytes (n = 12) that were examined by immunocytochemistry with anti-tubulin antibody after previous Polscope
imaging, the ®ndings obtained by both methods were consistent. However, immunocytochemistry revealed residual tubulin
immunoreactivity in association with chromosomes in most
oocytes that lacked the spindle (Figure 3).
Discussion
In this study, the meiotic spindle was detected in 100% of
in vivo matured metaphase II oocytes before freezing. This
®nding is in agreement with our previously published series of
observations (Rienzi et al., 2003) but in disagreement with
several other studies using the Polscope, in which the
percentage of oocytes with a detectable spindle varied between
657
L.Rienzi et al.
60 and 80% (Wang et al., 2001b,c, 2002; Moon et al., 2003).
This difference may be due to the fact that we rotated each
oocyte with an injection pipette controlled by a micromanipulator during observation until the spindle became visible, which
was not done in the other studies. This manipulation made it
possible to detect even spindles with relatively low brightness.
It cannot be excluded that spindles that can be visualized
relatively easily are functionally superior to poorly detectable
spindles whose visualization requires extensive oocyte rotation
with the injection pipette. This would explain the ®ndings of
poor developmental quality of oocytes with non-visualized
spindles by the Polscope after ICSI (Wang et al., 2002; Moon
et al., 2003).
Interestingly, in all oocytes that showed the meiotic spindle
before freezing, the spindle remained detectable throughout the
freezing procedure up to the step at which the oocytes were
loaded into the cryopreservation straws, a period during which
the temperature decreased from 37°C to room temperature.
This observation contrasts with the ®nding that even transient
cooling to the room temperature for only 10 min causes
irreversible disruption of the meiotic spindle in the human
oocyte (Pickering et al., 1990; Wang et al., 2001a; Zenzes et al.,
2001). The present observation, showing that the spindle can
persist during prolonged incubation of metaphase II human
oocytes exposed to laboratory temperature while in the
cryoprotecting medium, suggests that some component of
this medium also protects the spindle against the temperatureinduced disintegration.
In fact, simple incubation of metaphase II human oocytes in
cryoprotective solutions without freezing has been shown
previously to have no effect on the structure of the meiotic
spindle (Boiso et al., 2002). In the absence of any direct
deleterious effect of the cryoprotective solutions on the spindle,
the progressive replacement of water molecules by those of
cryoprotectant, leading to an increase in the concentration of
tubulin monomer by reducing the volume of the aqueous
solvent, might shift the equilibrium of the temperaturedependent tubulin polymerization/depolymerization reaction
in favour of polymerization, thus slowing down the physical,
temperature-sensitive tubulin depolymerization process.
Accordingly, cryoprotective media may in fact also act as
cooling-protective media for metaphase II human oocytes.
Little is known about what happens with the spindles after
oocyte loading into the cryopreservation straws, during the rest
of the freezing procedure. However, the observation of the
absence of spindles in most of the surviving oocytes immediately after thawing suggests that additional destabilization of
the meiotic spindle occurs during the ®nal phase of oocyte
cooling (after transfer to straws) and subsequent freezing.
This study has shown that, unlike oocyte freezing, the
thawing procedure causes severe damage to the oocyte meiotic
spindle. In fact, all oocytes lose their spindles during washing
from the cryopreservation medium. The loss of the spindles
during the thawing procedure was con®rmed by immunocytochemistry with anti-tubulin antibody. In fact, immunocytochemistry and Polscope ®ndings were consistent in all oocytes
that were examined by both methods. These data are in
agreement with those of Wang and Keefe (2002) who found a
658
good correlation between Polscope and immunocytochemistry
®ndings in human in vitro matured oocytes. The disintegration
of the meioitic spindle during the thawing procedure may be
explained by the fact that, contrary to the freezing sequence,
depolymerized tubulin in the oocyte cytoplasm is progressively
diluted by water entering the oocyte during this step, thus
shifting the equilibrium of the tubulin polymerization/depolymerization reaction in favour of depolymerization. The known
rapidity of microtubule turnover in metaphase-arrested oocytes
(Gorbsky et al., 1990) explains the rapidity with which the
spindle disappears during this period. Consequently, all
spindles eventually present in frozen and thawed metaphase
II oocytes were products of de novo assembly in the post-thaw
period.
These observations have several biological and clinical
implications. If all oocytes lose their spindles during the
thawing procedures followed by de novo spindle reconstruction, a question arises of whether the thawing methods could be
modi®ed so as to protect the original spindle and prevent its
disappearance. The use of microtubule-stabilizing agents, such
as taxol (Mailhes et al., 1999), might be one possibility, but
toxicity by this kind of drug remains a problem. Alternatively,
if we accept the destruction of the oocyte's original spindle as
an unavoidable fact, future research might focus on improving
the conditions in which the new spindle is formed in the postthaw period. The Polscope system will be useful in both of
these research directions.
From the clinical point of view, the fact that the spindle
function in cryopreserved metaphase II oocytes is entirely
dependent on spindle reconstruction in the post-thaw period
raises some concern about the functional capacity of these
newly formed spindles. Thus, further research is needed to
evaluate the potential risks of the application of oocyte
cryopreservation in assisted reproduction treatment. Since
spindle dysfunction may lead to abnormal segregation of
chromosomes between the oocyte and the second polar body
after oocyte activation, the use of the Polscope system to
control the presence and location of the meiotic spindle in
cryopreserved oocytes will enable the elimination of oocytes
with spindle anomalies that could be at the origin of
aneuploidy. In cases in which oocyte cryopreservation is
suggested as an alternative to embryo cryopreservation, when
the latter is prohibited by law or rejected by the infertile couple
for ethical or religious reasons, these uncertainties should be
seriously taken into account and explained to the patients.
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Submitted on May 30, 2003; resubmitted on October 21, 2003; accepted on
October 31, 2003
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