Studies on human oocyte cryopreservation

Abstract
Background: This study examined the primary effect of selected cryoprotective agents (CPAs) on the meiotic
spindles of human oocytes during cooling.
Methods: Fresh metaphase II oocytes (n=26) donated from patients undergoing IVF treatment were analyzed
via Polscope. In experiment one, 16 oocytes with visible spindle at 37°C were cooled to 20°C and rewarmed
to 37°C to test the spindle response to cooling. They were then cooled to 20°C, 10°C, 0°C and rewarmed to
37°C after having been equilibrated with 1.5 M 1,2-propanediol (PROH), 1.5 M dimethyl sulfoxide (DMSO),
1.5 M ethylene glycol (EG) or 10 μM taxol at 37°C. In experiment two, 10 oocytes without visible spindles
at 37°C were cooled to 20°C and then equilibrated with PROH, EG and taxol at 20°C. Spindle images were
recorded at each temperature.
Results: Meiotic spindles remained visible or became more distinct during cooling to 20°C, 10°C and 0°C
when equilibrated with PROH, EG, DMSO and Taxol. Without these agents, meiotic spindles of the same
oocytes disappeared after cooling to 20°C.
Conclusion: The primary effect of PROH, EG and DMSO on the meiotic spindle is to stabilize and protect
it against low temperature disassembly. A higher equilibration temperature (≥33°C) for oocyte freezing is
recommended.
Keywords
cryoprotective agents; spindle; cryopreservation; human oocyte; cooling; Taxol
Introduction
Studies on human oocyte cryopreservation over the last
decade yielded only limited success, while human embryo
cryopreservation has been performed successfully around
the world for many years. The suboptimal results in oocyte
cryopreservation may be a result of damage to meiotic
spindle during freezing and thawing. The meiotic spindles
in mammalian oocytes, including the human, are
extremely sensitive to temperature change [1–4]. Exposure
of oocytes to room temperature (25°C) or lower for
as little as 1–5 min has been shown to cause depolymeriReceived
July 16, 2010.
Accepted August 12, 2010.
Published September 22, 2010.
© 2010 Yang et al.
This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/2.0/).
Florida Institute for Reproductive Medicine, 836 Prudential Drive, Suite 902, Jacksonville, Florida 32207, USA, E-mail: dunsong@aol.com, Fax:
+1 904 399 5645
Funding
This study was supported in part by Organon USA Inc., Roseland, New Jersey, USA. The sponsor had no involvement in study design, data collection,
analysis or interpretation of data, in the writing of the report, or in the decision to submit the paper for publication.
Journal of Experimental & Clinical Assisted Reproduction
zation of the meiotic spindle [4,5]. Cryoprotective agents
(CPAs) are essential components in freezing solutions, but
may also disrupt the integrity of meiotic spindle. Vincent
and colleagues [6,7] found that exposure of mouse oocytes
to 1.5M DMSO and PROH resulted in microfilament
depolymerization, and the absence of meiotic spindle in
oocytes after cryopreservation was attributed to the toxic
effect of cryoprotectants [8,9].
Due to concerns about toxicity of CPAs, oocytes are normally
cooled to room temperature (18°C to 24°C) or even
lower (4°C to 0°C) before equilibrating with CPAs [10–
14]. However, CPAs may have protective effects on the
meiotic spindle, and therefore should be applied prior to
cooling. Previous studies have shown the ability of both
DMSO [15,16] and glycerol [17–19] to enhance formation
of microtubules. Both DMSO and PROH have been reported
to have protective effects on the meiotic spindle of
mouse and human oocytes [20,21]. Several recent investigations
[22–28] incorporating traditional fixation and
immunostaining techniques have raised questions regarding
the protective effects of CPAs. Profound spindle alterations
and spindle disassembly in oocytes after cryopreservation
were displayed immediately upon thawing [23,
26,27], although partial spindle recovery may be achieved
after 1–3h of incubation [24]. In contrast, studies using a
computer-assisted polarization microscopy system (Polscope)
have provided compelling evidence for a protective
effect [29,30].
Oocyte spindles were identified during room-temperature
equilibration with PROH and sucrose as cryoprotectants
and immediately after thawing; spindles disappeared with
subsequent removal of cryoprotectants and reappeared
after 1–3h of culture [29]. Ice formation and excessive
dehydration during oocyte freezing and thawing may also
damage spindles and oocyte chromatin [31,32]. It remains
unclear if disappearance of spindles after thawing results
from a toxic effect of cryoprotectants and/or the physical
stress of the freeze-thaw sequence. Understanding the primary
effects of CPAs on the meiotic spindles is therefore
essential in developing improved cryopreservation protocols.
The present research using a Polscope system and a cooling
stage attached to the same microscope was designed
to assess the impact of commonly used penetrating CPAs,
(i.e., PROH, EG and DMSO) on meiotic spindles of
human oocytes by cooling (without freezing) and rewarming
the same oocytes with and without one of the
cryoprotectants. Taxol, a known spindle stabilizing agent
was tested as a reference reagent. Based on these findings,
a higher equilibration temperature (≥33°C) for oocyte
freezing is recommended.
Materials and methods
Oocyte procurement and reagents
Twelve patients (four of whom were oocyte donors) provided
a total of 50 oocytes for this study. Eight of them
who had 20 to 40 oocytes (mean 30.6 oocytes) available
for IVF donated 3 to 5 oocytes each, two oocyte donors
underwent split cycles without a second recipient donated
half of the retrieved oocytes, and two patients who chose
to inseminate limited number of oocytes for transfer donated
half of their oocytes. Mean (±SD) age of patients was
30.58 ± 9.44 years. After screening oocytes for visible
spindles using Polscope, all oocytes without visible spindle
(n=10) and 16 of the 40 oocytes with spindle were
randomly selected for study. The remaining oocytes were
discarded because only a maximum of 3 oocytes from the
same patient could be analyzed within 40–44h after hCG
injection. The study was approved by the Institutional
Review Board (IRB) of Baptist Medical Center, Jacksonville,
Florida and written informed consent was obtained
from all study subjects.
Controlled ovarian hyperstimulation was preceded by
pituitary downregulation with GnRH agonist (Lupron;
TAP, Deerfield, IL), followed by recombinant FSH (Follistim;
Organon, Roseland, NJ) and hMG (Pergonal;
Serono). 10,000IU hCG was used for triggering. Transvaginal
oocyte aspiration was performed under i.v. sedation
35h after hCG injection. Following retrieval, oocytes
were stripped of cumulus cells after being cultured in IVCONE
medium (InVitroCare, Frederick, MD) supplemented
with serum substitute (SSS; Irvine Scientific, Santa
Ana, CA) for 2–4h.
Unless stated otherwise, all reagents were purchased from
Sigma Chemical Company (St. Louis, MO, USA). All
oocyte treatment solutions were prepared using Dulbecco’s
phosphate-buffered solution (PBS) (Sage Biopharma,
Bedminster, NJ) and contained a final concentration
of 20% SSS (Irvine Scientific). Treatment solutions
used were: (1)1.5M 1,2-propanediol (PROH); (2)
1.5M dimethyl sulfoxide (DMSO); (3) 1.5M ethylene glycol
(EG) and (4) 10μM taxol.
Spindle examination
For spindle imaging, each oocyte was placed in10μl of
treatment solution covered with mineral oil (Medicult,
Denmark) in a glass-bottomed culture dish (Willco Wells,
Amsterdam, The Netherlands). An inverted microscope
equipped with a Peltier system (PE100, Linkam Scientific
Instruments Ltd, UK) was used for oocyte assessment and
spindle examination. The Peltier system includes a biological
warming and cooling microscope stage attached to
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Journal of Experimental & Clinical Assisted Reproduction
a temperature controller with a range of −5°C to 99°C
(PE-94, Linkam Scientific Instruments Ltd). Dishes were
placed on the stage during equilibration and examination.
During examination, oocytes were manipulated by holding
pipette (Sunlight Medical, Jacksonville, FL) and/or
partial zona dissection pipette (Sunlight Medical). The
meiotic spindle visualization was performed at 200X magnification
with LC Polscope optics and controller (SpindleView;
CRI, Woburn, MA, USA) fitted to a computerized
image analysis system (SpindleView software; CRI).
Experimental designs
Details of the oocyte spindle status, experimental assignment
and treatment subgroups are summarized in Figure
1. Oocytes were first placed in a drop of PBS in a glassbottom
dish at 37°C on the microscope stage. Spindle
images were recorded and oocytes were placed in two
study groups as a function of spindle visualization as follows:
If a clear oocyte spindle was visible, oocytes were
entered into Experiment 1; if no spindle was noted, then
oocytes were used for Experiment 2. As stated previously,
only 16 of 40 MII oocytes with a visible spindle were
actually used in Experiment 1.
Experiment 1
Oocytes in Experiment 1 were cooled to 20°C at −5°C per
minute, held for 5min and then rewarmed to 37°C at 5°C
per minute, to measure spindle response to temperature
change. Oocytes showing a positive response to temperature
change (i.e., spindle loss at 20°C and recovery after
rewarming to 37°C) were continued in Experiment 1.
Spindle images were recorded after reaching the desired
temperature and at 5min. These images were used as controls
for comparison with treatment by the different chemical
agents.
Oocytes were then exposed to one of four treatment solutions
at 37°C x 10min. The stage temperature was then
dropped to and held for 10min at 20°C, 10°C, 0°C and then
Figure 1. Oocyte allocation for Experiments 1 and 2 with summary of response to selected cryoprotectants.
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rewarmed to 37°C. Spindle images were taken 5 and
10min after reaching the desired temperatures. The experiment
was repeated on at least three oocytes for each agent.
Experiment 2
Oocytes without visible spindle at 37°C in PBS were
cooled to 20°C and then exposed to PROH, EG and taxol
at 20°C x 10min. Polscope images were taken before
treatments, at 5 and 10 min after equilibration in each
treatment solution at 20°C. Cryoprotectants were removed
by washing oocytes in PBS three times. The oocytes were
then maintained in PBS at 20°C for 10 min before rewarming
to 37°C. Next, oocytes were cultured 18–20h in our
embryo culture medium (IVC-ONE supplemented with
5% SSS) at 37°C. Polscope images were recorded 5 and
10min after removal of cryoprotectants at 20°C, after 30
minutes in culture at 37°C, and after overnight culture
before discarding. The experiment was repeated on at least
three oocytes for each agent. DMSO was omitted from this
experiment since it induced an extra spindle structure in 2
of 4 treated oocytes. It was our concern that DMSO may
not be a suitable human oocyte freezing reagent given this
finding.
Results
Oocyte assignment to each treatment group, responses to
temperature modifications and various cryoprotectants are
illustrated in Figure 1. All oocytes in both experiments
showed positive responses to temperature change and
treatment reagents.
Experiment 1
Figure 2 shows spindle dynamics after cooling and treatment
with selected cryoprotectants. All oocytes showed
dynamic changes in response to cooling from 37°C to
20°C and then rewarming to 37°C in PBS. Spindles which
were visible in all oocytes at 37°C (Fig. 2, row 1) became
faint (Fig. 2, A2), or disappeared entirely (B2, C2 and D2),
at 20°C. Spindles recovered 5min (Fig. 2, row 3) after
being warmed to 37°C.
The spindle dynamics in response to temperature change
without any CPA served as a control for each oocyte
group. The oocytes were then equilibrated with one of the
four treatment solutions (PROH, EG, DMSO and taxol) at
37°C. Within 5min after oocyte equilibration in each
reagent, the spindle images became larger (A4, C4) and/
or more intense (B4, D4) than in PBS. Two of the four
oocytes treated with DMSO developed an extra spindle
structure (C4). Oocyte spindles in all four treatment solutions
were clearly visible after the temperature was dropped
to 20°C (row 5), 10°C (row 6) and even 0°C (row 7).
Multiple irregular spindles were observed in the taxol
group at temperatures down to 0°C (D6, D7), and this formation
continued following oocyte rewarming to 37°C
(D8). Spindle images of oocytes in EG became weaker at
10°C (B6) and 0°C (B7) compared to 20°C (B5) and 37°C
(B8), while in PROH and DMSO spindles showed no
obvious changes at 20°C (A5, C5), 10°C (A6, C6), 0°C
(A7, C7) or after warming to 37°C (A8, C8) (including
extra spindle structure in the DMSO treated oocyte).
Experiment 2
Spindle dynamics of human oocytes equilibrated with
PROH, EG and taxol at 20°C (and their response to cooling
and removal of the protective compounds) were studied
in Experiment 2. None of the oocytes showed a spindle
structure after being cooled to 20°C before equilibration
with one of the three treatment solutions. Spindles became
clearly visible 5min after being placed in each treatment
solution (row 2). The spindles disappeared within 5min
following removal of PROH (P3) and EG (E3), but not in
the oocyte treated with taxol (T3). Spindle formation continued
even after taxol was washed out and cultured in
PBS at 37°C for 30 minutes (T3). Spindles in oocytes
treated with EG (E4) and taxol (T4) appeared normal after
overnight culture. No spindle could be identified in two of
the four oocytes treated with PROH after overnight culture
(P4). Weak spindle images were seen in the other two
oocytes treated with PROH.
Discussion
Cryoprotective agents have been reported to have beneficial
effects on the meiotic spindle of mouse and human
oocytes [20,21]. However, some recent studies [22–28]
using traditional fixation and immunostaining technique,
provide limited support on the protective effects of CPAs
on meiotic spindle. On the other hand, cryoprotective
agents used in freezing solutions have been regarded as
toxic and may disrupt the integrity of meiotic spindle by
other investigators [6–9]. Such contradictory findings
have complicated the understanding of the effect of CPAs
on meiotic spindle. More recent research using a computer-assisted
polarization microscopy system (Polscope)
[29,30] has showed visible spindles in oocytes during
equilibration with PROH and sucrose at room temperature
and immediately after thawing, but the spindles disappeared
during the subsequent removal of cryoprotectants.
Ice formation and excessive dehydration during freezing
and thawing may also cause damage to spindles and chromosomes
in oocytes [31,32]. It remains unclear if spindle
disappearance after thawing results from toxic effects of
cryoprotectants and/or the physiologic stress associat
with the freeze/thaw sequence. To eliminate the possibility
of freezing and dehydration damage to the spindle, the
present study analyzed the dynamic change of spindle
during cooling up to 0°C with and without the presence of
PROH, EG, DMSO or taxol. We found meiotic spindles
of human oocyte were visible or became more intense
Figure 2. Spindle images of human oocytes following cooling and treatment with cryoprotective agents (Experiment 1). Images in column A,
B, C, and D (representing four treatment groups, PROH, EG, DMSO and taxol respectively) were taken after oocytes were maintained in PBS
at 37°C (row 1), after temperature dropped to 20°C (row 2), then rewarmed to 37°C (row 3), after having been equilibrated with the cryoprotective
agents at 37°C (row 4), after temperature dropped to 20°C (row 5), 10°C (row 6), 0°C (row 7) and then rewarmed to 37°C (row 8). Arrow outlines
show spindles while solid arrows in column C indicate newly formed extra spindle structures [original magnification = 200X].
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Journal of Experimental & Clinical Assisted Reproduction
when cooled to 20°C, 10°C and 0°C after equilibrating
with each of the tested CPAs at 37°C (Fig. 2). Without
CPAs, spindles of control oocytes disappeared after cooling
to 20°C. We have demonstrated the ability of PROH,
EG and DMSO to stabilize and protect the meiotic spindle
against cold induced depolymerization. Interestingly, in
all oocytes that did not initially show spindles at 37°C,
spindles did become detectable after exposure to PROH,
and EG (Fig. 3). It is possible that dynamics of the meiotic
spindles in such oocytes (even at 37°C) favored depolymerization.
The exposure of the oocytes to PROH and EG
may reverse the depolymerization and make spindles
detectable, and supports the notion of a stabilizing effect
of PROH and EG on meiotic spindle.
Rienzi et al [29] reported that spindles not only remained
detectable in all oocytes during equilibration with a freezing
solution containing PROH and sucrose at room temperature,
but became even more intense. These investigators
could not identify the protective component in their
cryopreservation media, however. Because sucrose is a
non-permeable agent, it is possible that PROH is the protective
element. The beneficial effects of PROH and
DMSO on oocyte cooling and cryopreservation have been
reported previously in oocytes. Specifically, Van de Elst
et al [20] and Gook et al [21] found a significantly greater
portion of oocytes with normal spindles in the presence of
DMSO and PROH than without these reagents, suggesting
a protective effect of PROH and DMSO.
Rienzi et al [29] reported that the spindle of the surviving
oocytes disappeared almost immediately after PROH
removal. Spindles reappeared in all oocytes within 3h of
culture. Analyzing mouse thawed oocytes after vitrificaFigure
3. Spindle images of human oocytes equilibrated with cryoprotective agents at 20°C (Experiment 2). Polscope images of oocytes in
PROH, EG and taxol groups (Column P = PROH, E = EG and T= taxol) were recorded at 37°C in PBS (row 1), after equilibration with
cryoprotective agents at 20°C (row 2), after cryoprotective agents were removed (row 3), and after being cultured overnight at 37°C (row 4).
Spindles are designated by white arrows [original magnification = 200X].
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Journal of Experimental & Clinical Assisted Reproduction
tion also showed disappearance of spindles after cryoprotectant
(EG) was removed [33]; recovery of spindles was
documented in 75% of surviving oocytes. In this study,
disappearance of spindles was also recorded after PROH
and EG were removed even when oocytes were not frozen
or dehydrated (Fig. 3), ruling out the possible spindle
damage caused by ice formation and dehydration [32]. In
contrast to taxol, which showed continued growth of the
spindle after the chemical was removed (Experiment 2),
the disappearance of spindle immediately after removal of
PROH and EG is reassuring in terms of continued normal
oocyte development.
The disappearance of spindle after thaw is of clinical concern.
Embryos and oocytes are normally thawed at room
temperature and cultured through a sequence of media to
remove CPAs before they are cultured in an incubator at
37°C or transferred. The meiotic spindle is a dynamic
structure of cytoskeletal filaments, undergoing rapid
structural reorganization including filament disassembly
at one site and reassembly at another [34]. It is conceivable
that without the stabilizing effect of CPAs during and after
thawing and when the reassembling activities are not
recovered, an accelerated disassembly of spindle microtubules
may occur. Although the spindle can be recovered
after 1–3h of culture at 37°C in some thawed oocytes, loss
of spindle bipolarity and chromosome alignment has been
reported [24]. Loss of spindle bipolarity and chromosome
alignment may contribute to the poor pregnancy and high
miscarriage rates (21–38%) as reported by others [11,35–
37]. Prevention of oocyte spindle disassembly during thaw
and removal of CPAs could therefore represent an important
clinical advance.
It is common practice to cool oocytes to room temperature
(18–24°C) [10,11,35] or even lower (0–4°C) [12–14]
before equilibrating with CPAs because of concerns over
CAP toxicity. This may subject the spindle to a cycle of
disassembly and recovery thus altering spindle and chromosome
configuration, and ultimately resulting in poor
pregnancy outcome.
Reports on CPA effects on the microfilament system are
varied, and may be influenced by reagent concentration
[38,39], temperature [40], cell type and species [6,41]. At
low concentration (1M), PROH was associated with meiotic
spindle disruption in mouse oocytes, although it had
a stabilizing effect at 1.5 and 2.0 M [38]. Chen et al [42]
found that the spindles of mouse oocytes disorganized or
disappeared when exposed to 5.5M ethylene glycol. Vincent
et al [7] demonstrated a depolymerizing effect of
PROH on actin filaments of early stage rabbit embryos,
and even suggested that the actin depolymerization might
be one of the reasons for the efficiency of PROH. Exposure
of oocytes to 1.5M DMSO caused depolymerization
of microtubules in mouse oocytes [6], but showed stabilizing
effects on human oocyte spindles during cooling
[41]. It is possible that these opposing effects of CPAs on
the microfilament system may be influenced by the immunocytochemical
technique, i.e., washing, fixation and
labeling [7,20] and possible variations among individual
technicians. The status of the dynamic structure of the
microtubules may be changed during the process, and then
captured at the moment of fixation. The advantage of Polscope
imaging is the ability to observe the complete range
of spindle dynamics in the course of in vitro treatment in
a noninvasive fashion, eliminating any question of artifact
introduced by the immunocytochemical technique. Using
the Polscope, we have demonstrated that the primary
effect of PROH and EG on meiotic spindle is to stabilize
and protect it from depolymerization. We consider the
depolymerizing effect reported by Vincent et al [6,43]
after thawing and removal of CPA as a “withdrawal
effect” of the reagent.
Besides stabilizing the microfilament system, DMSO was
associated with the formation of an extra spindle, suggesting
a direct or indirect ability to promote polymerization.
DMSO has been reported to form multiple cytoplasmic
microtubular asters in mouse oocytes [44] as well as
promoting rapid formation of microtubules [45] which
may favor development of abnormal spindle structures.
However, many oocyte vitrification protocols include
DMSO as CPA [46–48] with no increased risk of abnormality
[46]. This may be helped by the fact that vitrification
involves a very short time culture of oocytes with
CPAs at low temperature before they are vitrified and that
the DMSO is removed right after thawing. A long period
of contact with DMSO at physical temperature should be
avoided.
The protective effect of CPAs is poorly understood. It has
been postulated that CPAs may act by reducing the concentration
of intracellular electrolytes, by stabilizing
plasma membrane by electrostatic interactions, and by
reducing the rates of ice nucleation and crystal growth by
increasing the viscosity of extra- and intracellular solutions
[49]. Surprisingly little is known about their mode
of action at the molecular level. Doebbler and Rinfret
[50] observed some correlation between hydrogen bonding
capacities of some cryoprotective agents (i.e., glycerol,
EG) and their protective capacities, suggesting that
perhaps the ability to bind or to substitute for water might
be an important factor. Hydrogen bonding sites may serve
not only to bind water but also in forming and stabilizing
an extended region of oriented water around each molecule.
The H-bound water oriented around protective solute
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Journal of Experimental & Clinical Assisted Reproduction
molecules may be less capable of participating in ice crystal
formation. This mechanism may also be responsible
for stabilizing the meiotic spindle against cooling. It is our
hypothesis that the H-bonding sites of the CPAs may form
multiple H-bonds between microtubule and CPA molecules
to increase the stability of the microtubule against
cold induced depolymerization.
Taxol, an antitumor drug [51], was tested as a reference
reagent because it is known to enhance microtubule
assembly through a combination of elongation of existing
microtubules and spontaneous nucleation of new microtubules
in vitro [52,53]. Microtubules polymerized in the
presence of taxol are resistant to depolymerization by cold
(4°C) [54]. In our study, Polscope imaging demonstrated
rapid growth of the spindle structure with multiple poles
extending radially, even at 0°C (Fig. 1) and for at least
30min after reagent removal (Fig. 3). Although taxol has
been shown to improve preimplantation development of
cryopreserved mouse [55], porcine [56], bovine [57] and
human [58] oocytes, we believe it should not be used clinically
because of abnormal microtubule formation. Further
studies may be needed to confirm the finding.
This study examined spindle responses to cooling up to
0°C in presence of selected CPAs. It may not represent
spindle response to freezing at −196°C when a few CPAs
are often used in clinical oocyte cryopreservation. The
reason for the weak or no spindle formation among
oocytes treated with PROH (compared to EG and taxol
treated oocytes) after overnight culture (Experiment 2) is
unknown. More research is needed to identify the cause(s)
of such spindle loss.
The present study demonstrates the ability of CPAs to stabilize
and protect the meiotic spindle of human oocytes
against cooling. It is recommended that oocytes should
always be equilibrated with freezing solutions at physiological
temperature (37°C), or at least 33°C [4] before
cooling. Our preliminary experiments included equilibrating
human unfertilized oocytes with PROH at physiological
temperature (37°C), and showed improved oocyte
survival [59]. The fertilization, survival, embryo development,
and pregnancy rates were comparable to fresh
oocytes and cryopreserved embryos [60]. The benefits of
higher pre-equilibration temperature on oocyte cryopreservation
outcome have also been reported by Keskintepe
et al using vitrification [48]. We believe oocytes should
never be cooled without prior protection.
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