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com/Article/1889 on web 12 August 2005

Outlook
Criteria to assess human oocyte quality after
cryopreservation
After achieving his degree in Biological Sciences at the University of Palermo, Italy, in 1991
Giovanni Coticchio was awarded a PhD in cell and developmental biology, also at the
University of Palermo, Italy. In 1994–1995, he specialized in human IVF, achieving a Master
of Medical Sciences Degree in assisted reproduction technology at the Department of
Obstetrics and Gynaecology, University of Nottingham, UK. Whilst there between 1995 and
1998, he developed his research and clinical embryology interests as post-doctoral fellow.
In 1998 he joined Tecnobios Procreazione, Bologna, Italy, where he has been appointed
research supervisor. In 1999 he was visiting scientist at the Department of Obstetrics
and Gynaecology, University of Sydney, Australia. His major areas of interests are oocyte
cryopreservation and oocyte maturation.

Dr Giovanni Coticchio
G Coticchio1,3, MA Bonu1, V Bianchi1, C Flamigni2, A Borini1
1
Tecnobios Procreazione, Via Dante 15, 40125 Bologna, Italy; 2 University of Bologna, Bologna, Italy
3
Correspondence: coticchio@tecnobiosprocreazione.it

Abstract
Oocyte cryopreservation certainly represents one of the most attractive developments in the field of assisted reproduction,
with the aim of preserving female fertility and circumventing the ethical and legal drawbacks associated with embryo
freezing. Despite the achievement of the first pregnancy from frozen oocytes dating back as early as 1987, since then fewer
than 150 pregnancies have been reported. Over a long period of time, application of oocyte storage on a large scale has been
prevented by various factors, namely poor post-thaw survival. Fertilization rates remained low even after the introduction of
intracytoplasmic sperm injection. Modifications of slow-freezing protocols, mainly based on the increase of the concentration
of sucrose used as non-penetrating cryoprotectant (CPA) and the replacement of sodium with choline, appear to have decisively
improved survival rates to over 80%. Investigations at the cellular level on thawed oocytes are largely lacking. Fertilization
rates have also benefited from protocol modifications, reaching values indistinguishable from those normally obtained
with fresh material. Vitrification protocols have also been tested, giving rise to improvements whose reproducibility is still
uncertain. Data on the dynamics of fertilization and preimplantation development of embryos derived from frozen oocytes are
extremely scarce. At the moment, clinical efficiency of oocyte cryopreservation cannot be precisely assessed because of the
lack of controlled studies, although it appears to be considerably lower than that achieved with embryo freezing. In summary,
encouraging advances have been made in the field of oocyte cryopreservation, but presently no protocol can ensure standards
of success and safety comparable to those guaranteed by embryo storage.

Keywords: cryopreservation, fertility preservation, meiotic spindle, oocytes

of application does not correspond precisely with that of oocyte

Introduction
In human IVF, cryopreservation of cleavage-stage embryos is
fundamental for maximizing the cumulative clinical outcome per
started cycle (Veeck, 2003) and circumventing the pandemic of
multiple pregnancies (Gerris et al., 2003). However, it is hardly
arguable that the possibility of storing unfertilized oocytes would
represent a better option, solving the ethical, legal and – in some
cases – political complications associated with embryo freezing
(Pool and Leibo, 2004). Despite a number of fundamental
cryobiology and clinical studies pursued in the last several years,
the aim of establishing oocyte cryopreservation as a procedure
able to compete with embryo freezing in efficiency and safety
remains largely unaccomplished. Ovarian freezing has also been
proposed as a measure to preserve female fertility, but its field
storage, meeting more specifically the need of young women at
risk of premature ovarian failure (Hovatta, 2005). Attempts to
improve our understanding of oocyte cryobiology have been in
many cases empirical, while only limited experiences have been
gained by adopting models able to predict the intensity of various
forms of stress imposed on oocytes during freezing (Fuller and
Paynter, 2004; Paynter, 2005). Meanwhile, clinical experience
acquired in this field has been very limited, in the absence of
properly designed studies. While in the past the major obstacle
to the implementation of oocyte freezing was the inability to
achieve reproducible and high survival rates, recently developed
protocols can ensure consistent and high post-thaw recovery rates.
This has raised the hope that oocyte storage could at last become
a fully reliable practice, but it remains to be demonstrated that
high post-thaw survival coincides with unaltered developmental 421
 Outlook - Assessing human oocyte quality after cryopreservation - G Coticchio et al.
competence. However high the survival rates ensured by a novel
protocol, routine adoption of oocyte cryopreservation should
be preceded by both comprehensive cryodamage assessment
conducted at the cellular level, and controlled clinical trials. The
metaphase (MII) spindle, due to its sensitivity to low temperatures,
has always been suspected to undergo structural alterations
incompatible with normal chromosome segregation at meiosis
II. In this respect, evidence is highly controversial. Moreover,
the assumption that the quality of frozen–thawed oocytes can be
exhaustively ascertained by meiotic spindle analysis represents
an unacceptable oversimplification. In fact, the quality of
cryopreserved oocytes should be assessed through a whole
range of criteria. Further valuable information could be extracted
from careful monitoring of fertilization and cleavage events, in
order to pinpoint possible deviations from the normal pattern of
development. Efforts in this direction have been attempted, but
available evidence is still incomplete, scattered, and in some
cases unreproducible. Such a lack of information stems mainly
from the difficulty in obtaining donated good-quality oocytes for
research purposes. Alternative freezing protocols will have to be
compared under otherwise similar conditions of application, in
order to identify the procedure able to give the highest rate of
developmentally uncompromised oocytes after thawing. This
will involve taking account of a variety of parameters in addition
to the rate of survival.
Survival rates originating from
alternative cryopreservation
protocols
From the very first attempts aimed at storing mature oocytes, it
began to appear that the conventional slow-freezing protocols
originally developed for cleavage stage embryos were inadequate,
giving rise to survival rates not exceeding 40–50% (Chen, 1986;
Al-Hasani et al.,., 1987; Gook et al., 1994) (Table 1). However,
these initial experiences were conducted with a non-systematic
approach, in some cases by making use of scarce or poor-quality
material, such as oocytes left in culture for 24 h (Al-Hasani et al.,
1987). The poor outcome of the conventional protocol has been
confirmed by recent experience, based on the thawing of 737
oocytes, in which it was not possible to guarantee survival rates
in excess of 37% (Borini et al., 2004). In recent years, the study
with the largest number (1502) of oocytes frozen–thawed with
the protocol based on 1.5 mol/l propane-1,2-diol (PrOH) and 0.2
mol/l sucrose reported a 54.1% survival rate (Porcu et al., 2000).
Fabbri et al. (2001) published a study in which it was shown that
survival rates could be increased dramatically (above 80%) by
raising the concentration of sucrose to 0.3 mol/l (Table 1). Also,
this effect appeared unchanged by the presence or absence of a
few layers of cumulus cells left around the zona pellucida. It is
assumed that the beneficial effect of higher sucrose concentration
can be explained by a more pronounced degree of dehydration
(Paynter et al., 2005) that would reduce the risk of intracellular ice
formation, considered to be one of the major causes of cryoinjury.
The outcome of protocols based on high sucrose concentration
has been confirmed independently by other authors (Fosas et al.,
2003). Recently, we have tested the same freezing conditions,
observing survival rates (75–80%) comparable to those reported
by Fabbri et al. (Borini, unpublished).
In the attempt to improve survival rates, slow-freezing protocols
422 have been modified also on the basis of alternative rationales.
Stachecki et al. (1998) have tested the hypothesis that toxicity
derived from an increase in extracellular solutes, namely
Na+ ions, in the unfrozen fraction of the cryopreservation mix
during extracellular ice formation is an important source of
cryopreservation-induced damage. In order to reduce the
impact of this factor, by using mouse oocytes, they replaced
increasing percentages of Na+ with the less toxic choline,
obtaining proportionally improved survival rates (Stachecki et
al., 1998). Stachecki et al. (2004) also used similar choline-based
cryopreservation media for storing human mature oocytes via a
slow-freezing protocol involving a moderately elevated sucrose
concentration (0.2 mol/l). Under these conditions the survival rate
was very high (88.4%). This outcome has also been confirmed
by the study of Quintans et al. (2002). However, it should be
noted that in the work of Stachecki et al. (2004) the number of
frozen–thawed oocytes was small, without considering that the
incubation time in each of the two freezing solutions (PrOH 1.5
mol/l and PrOH 1.5 mol/l, 0.2 mol/l sucrose) varied between
10–20 and 5–10 min, respectively. Such variations are far from
being uninfluencial, giving rise to varying degrees of dehydration
(Paynter et al., 2005), a circumstance that imposes the need for
further tests conducted under stricter conditions.
Recently, increasingly more attention has been devoted to
vitrification protocols. While slow-freezing protocols aim
at preventing intracellular ice formation by dehydration,
vitrification attempts to interfere with ice crystal formation by
establishing a state of extreme viscosity at low temperatures, that
is achieved with very high cryoprotectant (CPA) concentrations
and cooling rates. Experiences based on vitrification protocols
are still very limited. The largest published study carried out
so far has involved the freezing and thawing of 474 oocytes, of
which 325 (68.6%) survived (Yoon et al., 2003). However, by
admission of the same authors, only 198 of the survived oocytes
were considered to have a morphologically normal cytoplasm
at the time of performing intracytoplasmic sperm injection
(ICSI), lowering the actual survival rate to 41.7% (198/474).
This evidence raises the point that even the most unsophisticated
criteria of oocyte viability, such as simple observation of the
cytoplasmic appearance, are not strictly codified, leaving room
for uncertainty. The same oocyte could be classified as normal
or non-viable, depending on whether its observation is carried
out immediately after thawing or following a short interval of
1–3 h, as reported also by Gook et al. (1994). Vitrification has also
been successfully tested on human pronuclear stage oocytes, with
high survival and developmental rates (Isachenko et al., 2003),
but it should be noticed that such a material cannot be adopted
as a model for mature oocytes. In fact, it is well established that
standard slow cooling, unable to preserve unfertilized oocytes,
can preserve efficiently pronuclear stage oocytes (Queenan et al.,
1997), suggesting that the two types of cells respond differently
to the same treatment conditions.
Towards more objective criteria of
oocyte viability
It has become progressively clear that survival after thawing,
intended as the absence of signs of degeneration or grossly
cytoplasmic abnormalities, such as extensive vacuolization, may
be not fully informative of the degree of oocyte viability. This
possibility has been described by Hunter et al. (1995), following
one of the first attempts of vitrification of human oocytes. Despite
 Outlook - Assessing human oocyte quality after cryopreservation - G Coticchio et al.

Table 1. Survival rates of human mature oocytes cryopreserved with different

protocols. Slow-cooling protocols differ in the use of choline as a substitute for
Na+ or the sucrose concentration in the loading solution.

Author Freezing method Oocytes

No. %
thawed survived

Al-Hasani et al., 1987 Slow cooling/0.1 sucrosea 159 33.9

Gook et al., 1994 Slow cooling/0.1 sucrose 134 41.0
Tucker et al., 1998 Slow cooling/0.1 sucrose 311 24.0
Borini et al., 2004 Slow cooling/0.1 sucrose 737 37.0
Yoon et al., 2000 Vitrification 90 63.3
Porcu et al., 2000 Slow cooling/0.1 sucrose 1502 54.1
Fabbri et al., 2001 Slow cooling/0.3 sucrose 224 82.0
Yang et al., 2002 Slow cooling/0.2 sucrose 158 70.9
Fosas et al., 2003 Slow cooling/0.3 sucrose 88 89.0
Boldt et al., 2003 Slow cooling/cholineb 90 74.4
Yoon et al., 2003 Vitrification 474 68.6
Stachecki et al., 2004 Slow cooling/cholineb 69 88.4
Chen et al., 2005 Slow cooling/0.3 sucrose 159 75.0
Borini, unpublished Slow cooling/0.3 sucrose 927 74.1
a
All sucrose concentrations are in units of mol/l.
b
Choline concentration was 137 μmol/l. It should be noted that this component was a replacement
for sodium.

the fact that in such a study survival rate was relatively high
(65%), fertilized oocytes failed to cleave, indicating major, and
yet cryptic, damage to the cell cycle machinery. Ideally, criteria to
assess post-thaw viability should be conducted with conservative
techniques, in order to compare different protocols, as well as
selecting and using individual oocytes. This objective appears
difficult because non-invasive assessment of oocyte quality is
per se an unresolved matter, irrespective of the possible damage
induced by freezing. On the other hand, non-conservative
techniques, such as fluorescence microscopy and biochemical
analysis, while unable to assist in the selection of the most
viable oocytes, offer the possibility of assessing objectively the
relative state of post-thaw viability, providing clues for setting the
appropriate standards of quality.
Conservative assessment of post-
thaw oocyte viability
PolScope analysis of the meiotic spindle
It is well established that the oocyte MII spindle is extremely
sensitive to lower temperatures, undergoing depolymerization
just a few degrees below the optimal thermal conditions. While
in the mouse this change is fully reversible once oocytes are
returned to 37°C (Pickering and Johnson, 1987), in the human
even a short incubation at 25–27°C causes irreversible loss of the
spindle microtubules (Pickering et al., 1990; Wang et al., 2001b).
On the other hand, the physical–chemical conditions taking place
during freezing may influence the spindle response to temperature,
preventing permanent depolymerization. For example, exposure
to PrOH prevents spindle loss that normally occurs after
maintenance at room temperature for 10–15 min (Rienzi et al.,
2004), suggesting that CPAs may have biological effects on
the spindle apart from interfering with ice formation, reducing
the accumulation of extracellular solutes, and dehydrating the
intracellular environment.
In the last few years, visualization of the oocyte meiotic spindle has
been carried out with the polarized-based microscopy apparatus
referred to as the PolScope. This type of analysis does not provide
detailed information on the spindle organization, nevertheless
it offers the unique possibility of detecting non-invasively and
dynamically the presence of this cytoskeletal structure (Keefe et
al., 2003), so important not only for chromosome segregation,
but also for fertilization and early developmental stages. From
studies conducted on fresh oocytes, the general consensus has
emerged that meiotic spindle presence is associated with higher
fertilization and cleavage rates (Wang et al., 2001a), confirming
the usefulness of this type of oocyte assessment. Using the
PolScope, with a standard slow-freezing protocol, Rienzi et al.
(2004) reported that immediately after thawing and CPA removal
the spindle is not detectable, while it reappears after culture under
standard conditions. This suggests that thawed oocytes should be
incubated for a few hours before being injected. Likewise, using
a slow-freezing protocol involving high sucrose concentration,
we have observed that the percentage of oocytes showing spindle
birefringence increases from 24% following CPA removal to 76%
within the following three hours of incubation at 37°C (Bianchi
et al., 2005). However, under these conditions spindle retardance,
that is positively associated to the degree of microtubule
organization, is not fully restored compared with the values
observed before freezing. Although the functional significance of
spindle retardance has not been clearly elucidated, it is warranted
that quantitative PolScope analysis of the meiotic spindle (Sun et 423
 Outlook - Assessing human oocyte quality after cryopreservation - G Coticchio et al.
al., 2004) appears to be an important area of investigation for the
assessment of oocyte quality after thawing.
Non-conservative assessment of
post-thaw oocyte viability
Fluorescence microscopy analysis of the
meiotic spindle
Although the PolScope is a valid tool to monitor over time the
MII spindle during and after the freezing–thawing process, it
does not allow accurate analysis of the microtubular structure
and chromosome alignment, criteria upon which a more accurate
estimate of the risk of abnormal chromosome segregation
may be made. Evidence based on fluorescence microscopy
is controversial. It has been found that, after freezing with a
conventional slow-freezing protocol, the proportion of MII oocytes
with a morphologically normal spindle is reduced (61% and 81%
of normal oocytes in test and control groups, respectively) (Gook
et al., 1993a), although this difference does not reach statistical
significance, presumably because of the size of samples. In
another study, Boiso et al. (2002) have come to a different
conclusion, finding that oocytes frozen after maturation in vitro
with a 0.2 mol/l sucrose slow-freezing protocol display very low
frequencies of normal chromosome (18.8%) and microtubular
(16.2%) configurations. In agreement with this, Baka et al. (1995)
have reported that in vitro maturation prior to freezing decreases
the rate of occurrence of oocytes with a normal spindle. However,
it should be noted that the design of studies aiming at assessing
spindle organization should not involve experiments of in vitro
maturation, because it is known that when oocytes are matured
in the absence of cumulus cells, under certain culture conditions
spindle organization may be profoundly affected (Cekleniak et al.,
2001). Therefore, after cryopreservation at the germinal vesicle
stage and maturation in vitro, abnormal spindle configuration may
reflect the absence or loss of cumulus cell viability, rather than
damage to the oocyte itself. Cytogenetic investigation suggests
that slow freezing does not affect the rate of aneuploidy in human
oocytes (Van Blerkom and Davis, 1994a). Besides, by performing
a fluorescence in-situ hybridization analysis and employing
specific probes for chromosomes 13, 18, 21, X and Y, Cobo et
al. (2001) have found comparable aneuploidy frequencies in
embryos obtained from fresh and frozen oocytes (28% and 26%,
respectively). These data should be interpreted with caution,
considering that only 21 embryos from frozen–thawed oocytes
were subjected to chromosome analysis. Although these studies
suggest similar rates, using the five probes commonly used for
IVF embryos, the lack of repolymerization of a spindle tubule is
likely to be random, and therefore full chromosomal analysis is
required to answer this question. One of the most encouraging
sets of data on chromosome and spindle configuration concerns
human oocytes cryopreserved with a slow-freezing method
involving choline as a substitute for sodium in association with
a 0.2 mol/l concentration of sucrose (Stachecki et al., 2004).
Under such conditions, spindles were not detectable immediately
after thawing and rehydration, in agreement with the evidence
generated via the PolScope. However, after 1 h of incubation,
oocytes with a visible spindle and a normal barrel-shaped
microtubular organization had a frequency of 95.5% and 69.7%
respectively, frequencies statistically comparable to those of non-
424 frozen controls (96.7% and 76.7%, respectively). Similarly, the
percentages of fresh and stored oocytes with regular chromosome
alignment were not statistically different (76.7% and 71.2%,
respectively). The overall outcome of spindle analysis, including
PolScope and immunofluorescence data, indicates that spindle
and chromosome constitutions are not significantly affected by
cryopreservation. However, these data do not appear conclusive
and call for further and larger studies.
Mitochondrial function
Considering the essential role that mitochondria play in normal
cell function and specifically in fertilization and early embryo
development, it is surprising that these organelles have been almost
completely neglected in studies of oocyte cryopreservation. Based
on the hypothesis that mitochondrial hyperpolarization reflects the
Ca2+-sequestering ability and ATP-production potential, activities
that are crucial for normal fertilization and early development,
Jones et al. (2004) have analysed the polarization state of mature
oocytes cryopreserved with a slow-cooling protocol involving
1.5 mol/l and 0.2 mol/l sucrose. This analysis has shown that
hyperpolarization of pericortical mitochondria is lost during the
actual cooling procedure, but not during previous CPA exposure.
This result indicates that during cryopreservation, mitochondria
may represent one of the oocyte soft spots, However, the functional
implications of these findings are still unclear, considering that
loss of mitochondrial hyperpolarization does not coincide with
a decrease in the oocyte ATP content. Clearly, evidence on
mitochondrial function in frozen oocytes is largely insufficient
and needs a more systematic approach.
Calcium stores
Regulation of intracellular free Ca2+ is a signal essential for
promoting events of the fertilization process, such as cortical
granule release, reinitiation and completion of meiosis, and
pronuclear formation. The importance of Ca2+ oscillations may
not be restricted to the fertilization process, since modulation of
this signal has been shown to influence pre- and post-implantation
development (Ozil and Huneau, 2001). Apart from causing
loss of mitochondrial hyperpolarization, cryopreservation with
the 1.5 mol/l and 0.2 mol/l sucrose protocol compromises the
human oocyte Ca2+-releasing activity in response to ionophore
(Jones et al., 2004), suggesting that the Ca2+-sequestering
activity of mitochondria may be affected. However, intracellular
Ca2+ regulation is principally based on the release and uptake
from the smooth endoplasmic reticulum (SER). The function
of this organelle depends critically on the redistribution of its
compartments in the subcortical region during final maturation, and
the functional and topographical association with mitochondria.
Surprisingly, no data are available on the localization of SER
elements in frozen oocytes, either in human or animal material.
Cortical granules
It has been postulated that volume changes and other forms of stress
occurring during the cryopreservation process lead to illegitimate
release of cortical granules and consequent modifications of the
zona pellucida that prevent sperm penetration. This is apparently
in contrast with the fact that some pregnancies from frozen
oocytes were achieved before the advent of the ICSI era. Direct
observation of cortical granules is not necessarily consistent
with this hypothesis either. Application of the conventional
 Outlook - Assessing human oocyte quality after cryopreservation - G Coticchio et al.
slow-freezing protocol (with 0.1 mol/l sucrose) does not induce
exocytosis of cortical granules, as revealed by fluorescence
microscopy (Gook et al., 1993b). This has been confirmed by
Jones et al. (2004), who used confocal microscopy and observed
unaltered density of cortical granules in oocytes frozen with a
1.5 mol/l, 0.2 mol/l sucrose protocol. Therefore reduced fertilization
rates reported in association with some cryopreservation
protocols may depend on other more fundamental reasons than
zona pellucida hardening.
Ultrastructural analysis
Electron microscopy observation is one of most valuable tools for
a global assessment of cell organization. Such a powerful method
has not been applied to the field of oocyte cryopreservation
as it should have been. Apart from observations conducted on
diverse animal species, only two studies have concerned the
ultrastructural analysis of frozen–thawed human oocytes. In a
study dating back to almost two decades ago (Sathananthan et
al., 1987), oocytes stored with different methods showed signs of
cryoinjury consisting of cracks in the zona pellucida, disruption
of the plasma membrane and extensive disorganization of the
ooplasm. However, sperm interaction and penetration into the
oocyte were not compromised. Apart from a study of Van Blerkom
and Davis performed on immature oocytes (Van Blerkom and
Davis, 1994b), no other analysis has been performed on stored
human oocytes in the last decade. This is rather disappointing,
in view of the fact that recently developed protocols can ensure
survival rates that are much higher than those applied in previous
years.
Developmental ability of stored
oocytes
Fertilization
From the evidence presented in the previous sections it is clear
that survival may coincide with various forms of cryodamage
that cannot be assessed via routine microscopy observation
and yet may compromise developmental ability. In response
to the suspected cortical granule release and zona hardening in
frozen–thawed oocytes, ICSI has been proposed as the method
able to improve fertilization rates that appear compromised after
storage with conventional slow freezing (Gook et al., 1995).
Such a prediction has not been confirmed by various studies in
which, even after ICSI, fertilization rates of oocytes frozen with
the conventional slow-freezing protocol have been reported to
be below 50–55% (Borini et al., 2004). Therefore, in this case
reduced fertilization ability reflects the perturbation of more
fundamental aspects of the cell machinery rather than simple
dysfunction of the zona. After storage with a 1.5 mol/l and 0.2
mol/l sucrose mixture, inadequate fertilization rates may depend
on the above-mentioned impairment of mitochondrial function.
The application of vitrification methods (Yoon et al., 2003) or
improved slow-freezing protocols (Fabbri et al., 2001) that can
ensure high survival rates (67% and over 80%, respectively)
coincides also with the achievement of fertilization rates (65–
75%) virtually equivalent to those observed in fresh oocytes.
This is a sign that under such conditions, at least as far as the
fertilization process is concerned, the overall oocyte viability is
more efficiently preserved. However, little if anything is known
about whether the pace and choreography of the events directly
involved in the fertilization programme occur unperturbed in
these conditions, compared with unfrozen oocytes.
Cleavage and post-implantation
development
Despite cellular and cytoplasmic characteristics being of limited
value for the prediction of the developmental ability, nevertheless
they provide a useful tool to assess at least the overall embryo
viability. It has already been mentioned that inadequate
vitrification protocols can generate relatively high survival and
fertilization and yet profoundly compromise further development
to such an extent that even the first cleavage is inhibited (Hunter et
al., 1995). After storage at the mature unfertilized stage, nothing
is known about the timing of the first cleavage, a parameter that
has been suggested by several authors to be a significant predictor
of implantation ability. Apparently high cleavage rates (86.3%,
91.2%, and 91.0%) have been achieved with slow-freezing
protocols using 1.5 mol/l PrOH and increasing doses (0.1, 0.2
and 0.3 mol/l, respectively) of sucrose (Porcu et al., 2000; Borini
et al., 2004; Chen et al., 2005), or choline (Quintans et al., 2002)
as a replacement for Na+ (84.6%). Certain vitrification conditions
can also ensure high cleavage rates (Yoon et al., 2003). However,
it should be noticed that in the absence of control groups of fresh
oocytes it is difficult to calculate whether these percentages
are either within the normal ranges or suggestive of reduced
developmental ability.
Virtually no published information is available on the
characteristics of embryos derived from frozen oocytes. Even the
largest studies do not describe embryo population distribution
in terms of blastomere number, degree of fragmentation,
multinucleation or other cytoplasmic anomalies. An exception to
this is the data of Boldt et al. (2003) who described a mean cell
proportion of 5.6% and a mean embryo score of 2.7 (scale 3–1,
best–worst), but these figures were generated by observing only
30 embryos. No data are known on the ability of embryos derived
from frozen–thawed oocytes to reach the blastocyst stage in vitro,
apart from a study of Gook et al. (1995), conducted at a time
when blastocyst culture could not benefit from the sequential
media culture systems. In an unpublished observation we have
noticed that using a 1.5 mol/l PrOH, 0.3 mol/l sucrose slow-
freezing protocol, while fertilization rates are indistinguishable
between fresh and frozen groups, embryos developing from stored
oocytes tend to have lower cleavage rates, fewer cells and higher
degree of fragmentation at 44–46 h post-insemination (Figure 1).
Certainly, thorough investigation of this matter is warranted.
Implantation
Implantation, pregnancy and live birth rates are critical to assess
the relative success of different treatments. When calculated
on the number of embryos transferred, even the application of
slow-freezing protocols that do not guarantee high survival rates
may be compatible with implantation rates as high as 16.4%
(Borini et al., 2004). While in terms of clinical efficiency this
information needs to be carefully interpreted, considering the
high oocyte loss and the consequent number of treatments in
which embryo transfer is not achieved, on the other hand this
is indicative of the fact that despite low survival rates, those
oocytes that endure the double insult of freezing and subsequent 425
 Outlook - Assessing human oocyte quality after cryopreservation - G Coticchio et al.
thawing may develop with a potential not too dissimilar from
that of their fresh counterparts. The implantation rate derived
from vitrified oocytes does not appear particularly impressive,
amounting to only 6.4% (Yoon et al., 2003). While pregnancy
rates are not particularly informative in cases in which a relatively
high number of embryos are transferred, up to 4.5 ± 1.9 in the
work of Yoon et al. (2003), perhaps a more reliable parameter
for measuring the efficiency of a method is the percentage of
implantations as a fraction of the number of oocytes thawed.
Under this criterion, both slow-freezing protocols based on
1.5 mol/l PrOH and 0.1 or 0.2 mol/l sucrose, as well as
vitrification have low efficiency rates (2.3%, 1.1% and 1.7%,
respectively) (Borini et al., 2004; Porcu et al., 2000; Yoon et
al., 2003). To better appreciate these figures, in embryo freezing
programmes the rate of implantation calculated on the basis of
the number of oocytes used to generate supernumerary embryos
is in general at least 5% (Edgar et al., 2000). It is interesting
to note that after oocyte storage with a 1.5 mol/l PrOH, 0.3
mol/l sucrose, recently Chen et al. (2005) have achieved a 5%
implantation per thawed oocyte. This figure has to be confirmed
by a larger study, as it was obtained by the thawing of only 159
frozen oocytes.
Abortions are not systematically reported and for that reason
we are not aware of their incidence in relation to each protocol.
This is a crucial point, on the basis that chromosome or
other cytoplasmic anomalies may be compatible with the
establishment of a pregnancy and yet cause fetal loss at later
stages.
Conclusions
In recent years, modified versions of the conventional slow-
freezing protocol have overcome one of the major restrictions
to the application of oocyte cryopreservation, i.e. poor post-
thaw survival. It is not clear whether this goal has also been
reproducibly achieved with vitrification protocols. Information
generated on possible cryodamage to the meiotic spindle does
not support the view that this cytoskeletal element is necessarily
affected by storage conditions. Studies on other cellular
components that are fundamental for fertilization and early
cleavage are scant. In particular, electron microscopy studies,
that could importantly contribute to an objective comparison of
oocyte viability after storage with different protocols, have been
disappointingly episodic. Finally, implantation rates calculated
426 on the basis of thawed oocytes are generally low with almost
Figure 1. Comparison of the rates of fertilization
(2PN = 2 pronuclei), cleavage, embryos with three or
more blastomeres and high-quality embryos (Grade
A) between fresh (n = 210, open bars) and frozen
(n = 250, solid bars) oocytes. In each category,
frequencies with different letters are significantly
different (P < 0.05).
all protocols. A recent report describing a much higher value of
this parameter will require validation through larger numbers
of patients treated. Overall, the need for studies on oocyte
viability should be met through a wide range of methodological
approaches, even though the achievement of higher survival
rates has increased the number of attempts for the adoption of
oocyte cryopreservation in the clinical field.
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Paper based on contribution presented at the Tecnobios
Procreazione meeting ‘Assisted Conception and Reproductive
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November, 2004.
Received 27 May 2005; refereed 10 June 2005; accepted 5 July 2005.
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