doi:10.1111/j.1447-0756.2010.01215.x
J. Obstet. Gynaecol. Res. Vol. 37, No. 1: 1–12, January 2011
Mouse ovarian follicle cryopreservation using vitrification
or slow programmed cooling: Assessment of in vitro
development, maturation, ultra-structure and meiotic
spindle organization
Nina Desai1, Faten AbdelHafez1,2, Mansour Y. Ali2, Ezzat H. Sayed2,
Ahmed M. Abu-Alhassan2, Tomasso Falcone1 and James Goldfarb1
1
Department of OB-GYN, Cleveland Clinic Foundation, Cleveland, Ohio, USA; and 2Department of OB-GYN, Women’s
Health Centre, Assiut University, Assiut, Egypt
Abstract
Aim: To compare different outcomes of vitrification and slow freezing of isolated pre-antral follicles and to
evaluate different cryo-devices for vitrification of isolated follicles.
Methods: Pre-antral follicles were isolated from mouse ovaries and cryopreserved using vitrification and slow
freezing. A preliminary experiment was carried out to select the optimal cryo-device for vitrification of isolated
follicles. A total of 414 follicles were randomly distributed among four groups: control (CT) fresh (n = 100),
nylon mesh (n = 96), electron microscopy grid (n = 102), and micro-capillary tips (n = 116). Subsequently, a total
of 979 follicles were randomly assigned to three different groups: CT fresh (n = 256), vitrification (n = 399) and
slow freezing (n = 324). CT and cryopreserved/thawed follicles were cultured in vitro and examined daily for
development. Final maturation was triggered with human chorionic gonadotrophin and rates of oocyte
maturation were calculated. The ultra-structure of cryopreserved/thawed follicles was studied using electron
microscopy. Meiotic spindle presence and organization in mature oocytes were examined using the Oosight
imaging system.
Results: Micro-capillary tips resulted in poor immediate post-warming survival but no differences were
observed in the subsequent in vitro development characteristics between different cryo-devices. Nylon mesh
proved to be the easiest carrier, particularly when large numbers of follicles were to be vitrified. Compared to
vitrification, slow freezing resulted in a significantly lower number of intact follicles at the end of the culture
period (P < 0.0001). However all other outcome measures were comparable between both techniques.
Conclusions: Isolated follicles were more vulnerable to cryodamage after slow freezing as compared to
vitrification.
Key words: in vitro maturation, meiotic spindle retardance, pre-antral follicle, ultra-structure, vitrification.
Received: June 16 2009.
Accepted: February 5 2010.
Reprint request to: Dr Nina Desai, The Cleveland Clinic Fertility Center, 26900 Cedar Road, Beachwood, OH 44122, USA.
Email: [email protected]
Financial support: None.
Capsule
In vitro cultured vitrified/warmed isolated pre-antral follicles have significantly higher survival rates but comparable maturation rates,
meiotic spindle assembly and chromosomal alignment compared to slowly frozen/thawed isolated pre-antral follicles.
© 2010 The Authors
Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology
1
N. Desai et al.
Introduction
Ovarian follicle/tissue cryopreservation has been proposed as an alternative fertility preservation option.1–6
Follicles can be cryopreserved in intact ovarian tissue
pieces3,7–9 or after isolation of individual follicles from
the fresh ovarian tissue using enzymatic or mechanical
techniques.10–16 In both situations, follicles would
require further maturation either in vivo or in vitro for
them to be useful in restoring fertility.
With ovarian tissue cryopreservation, follicle maturation upon thawing could be achieved in vivo with tissue
transplantation or alternatively in vitro by enzymatic
digestion of the cryopreserved/thawed ovarian tissue,
followed by in vitro maturation (IVM). As an alternative
strategy, cryopreservation of isolated ovarian follicles
has many potential advantages over ovarian tissue cryopreservation. First, the cryoprotectant agent (CPA) permeation through follicular suspensions is expected to
be more effective compared to ovarian tissue pieces.10 In
addition, ovarian tissue has more complex structure
with different cell types making the choice of a suitable
cryopreservation protocol even more challenging.
Moreover, the post-thaw in vitro assessment of the follicular pool will be easier with isolated follicles compared to the whole ovarian tissue pieces.17 More
importantly, cryopreserved/thawed follicles can be suspended in a plasma clot10,18 or collagen gel19 permitting
auto-transplantation via a minimally invasive approach.
Auto-transplantation of cryopreserved/thawed follicles could result in better survival compared to ovarian
cortical strips, as angiogenesis and revascularization
could be faster.20 Added to these benefits is the fact that
the basal lamina of isolated primordial follicles excludes
capillaries, white blood cells and nerve cells, serving as
a barrier to prevent cancer cell infiltration into the follicle.21 This could negate the risk of cancer recurrence
that has always been a concern when considering autotransplantation of cryopreserved/thawed ovarian
tissue in cured cancer patients.
In light of this concern, cryopreservation of isolated
ovarian follicles may provide an attractive fertility
preservation alternative. Isolated ovarian follicles can
be cryopreserved by vitrification12–15 as well as by
traditional programmed slow freezing methodology.10,11,16,19,22 Prevention of intra-cellular ice formation
is tantamount for the success with either methodology.
In addition, the cryopreservation technique must minimize disruption of the oocyte–granulosa cell (GC) connections within the follicular unit to allow further
maturation in vitro.
2
Slow freezing protocols require expensive instruments for controlled-rate freezing of cells/tissues. The
time required for cryopreservation and thawing with
slow cooling techniques is also substantially longer. In
contrast, vitrification is simpler, requires little specialized freezing equipment, and less procedural time.
However, vitrification protocols do involve the use of
CPA at high concentrations and specialized cryodevices to achieve very high cooling rates.23 The high
concentrations of CPA used during vitrification may be
toxic to cells. Combining several CPA and/or exposing
cells to CPA at room temperature may help to decrease
the risk of toxicity.24 Although the vitrification technique appears to be simple, its outcomes are affected
by numerous factors such as technical skills, the type of
CPA and the cryo-device used for vitrification.25
Selection of the optimal cryopreservation technology
may affect the survival of intact follicles and their
ability to undergo IVM. To this end, we performed the
current study to evaluate the outcomes after vitrification or slow freezing of isolated mouse pre-antral follicles. The efficacy of different cryo-devices for isolated
follicle vitrification was tested. We assessed not only
the post-thaw survival of cryopreserved follicles but
also some functional parameters such as continued in
vitro growth (IVG), oocyte germinal vesicle breakdown
(GVBD), and maturation to the metaphase II (MII)
stage. Meiotic spindle organization in MII oocytes was
further scrutinized using the Oosight imaging system.
The ultra-structure of oocytes from pre-antral follicles
cryopreserved by both methodologies was also examined. To our knowledge this is the first comprehensive
study comparing the application of vitrification and
slow freezing techniques to the cryopreservation of isolated ovarian follicles. Understanding the limitations
and optimizing the current freezing methodology is a
necessary stepping stone to ultimately being able to
cryopreserve human ovarian follicles.
Methods
Animals and ovarian follicle isolation
The animal protocol was approved by the Cleveland
Clinic Foundation Institutional Animal Care and Use
Committee. Ovaries were harvested from B6D2F1 pups
(14–18 days old) and placed in Leibovitz media (L15)
supplemented with 10% synthetic serum substitute
(SSS). Ovarian tissue was enzymatically digested using
1 mg/mL collagenase Type I (132 U/mg) at 37°C. At
30-min intervals, ovaries were transferred to a new dish
containing L15/SSS and repeatedly pipetted using
© 2010 The Authors
Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology
Vitrification of isolated ovarian follicles
Eppendorf pipette tips of decreasing bore sizes to
facilitate the release of pre-antral follicles. Follicles
were collected, rinsed free of enzyme and examined
under a stereomicroscope. Intact pre-antral follicles
with two or more layers of GC surrounding the oocyte
were collected for the study. Primary follicles and follicles with antral cavities were excluded. Care was
taken to select follicles of a similar size range. Follicles
were first pooled before random distribution into treatment groups. Homogeneity of follicles was further confirmed by assessment at magnification ¥300 using an
inverted microscope with Hoffman Modulation optics
and the Microsuite Basic Imaging software. Follicles
selected for these experiments measured between 120
and 160 mm. Follicles exhibiting overt damage from the
enzymatic digestion and/or an incomplete collar of
GC surrounding the oocyte were excluded from the
study.
Follicle vitrification
Enzymatically isolated pre-antral follicles were vitrified
using an ethylene glycol (EG)-raffinose-based protocol.12 The basal media for the preparation of equilibration and vitrification solutions was L15 + 20%SSS.
Follicles were equilibrated for 5 min in 2M EG followed
by a 30–60-s incubation in vitrification solution containing 6M EG + 0.3M raffinose. All equilibration and vitrification steps were performed at room temperature. In
the first experiment, we compared the outcomes with
three different vitrification cryo-devices: nylon mesh
(4 mm pores, Cat# 146502), electron microscopy grid
(EM grid; Cat# G200T-Cu, Type: 200 mesh copper
square), and gel-loading micro-capillary tips (0.5–1 mL
in volume, 0.6 mm in diameter). The nylon mesh was
cut into 10 ¥ 10 mm sections that could be easily
handled and inserted into a cryovial.
Twenty pre-antral follicles in a fluid microdrop of
less than 3 mL were loaded on to either the nylon mesh
or EM grids and were immediately plunged into a
1.5-mL cryovial pre-filled with liquid nitrogen (LN2).
With micro-capillary tips, follicles were loaded with
the aid of a mouth pipette. After loading, the tip was
held for 2 min in the vapor phase, just above the surface
of the LN2, before immersion into a 3.5-mL cryovial
pre-filled with LN2.26 In the second series of experiments we elected to use a single carrier, the nylon
mesh, to compare follicle cryopreservation by slow programmed cooling versus vitrification.
Vitrified follicles were warmed by immersion in
basal medium containing 1M sucrose. Follicles were
left in the warming solution for 10 min at room tem-
perature and then washed at 37°C. After 5 min, follicles
were pipetted into fresh basal medium at 37°C for
another 5–10 min before placing in culture.
Slow freezing of isolated follicles
A dimethyl sulfoxide (DMSO)-based slow freezing
protocol was used.11 The basal medium for the preparation of freezing solutions was L15 + 20% SSS. The
freezing medium consisted of 1.5M DMSO. Twenty
follicles were pipetted and transferred into a cryovial
containing 150 mL freezing media at 4°C and equilibrated for 15 min. Vials were cooled in a Planer
Kryo-10 programmable freezer (UK) at a rate of
-2°C/minute until -7°C and manually seeded. Following seeding, samples were further cooled at -0.3°C/
min to -50°C and then at a rate of -50°C/min until
-110°C. Cryovials were plunged into LN2 to complete
the freezing process.
For thawing, cryovials were held at room temperature for 30 s before plunging into a water bath at 37°C,
with agitation, until ice melted. CPA was removed by
serial dilution in basal medium with decreasing concentrations of DMSO from 1.5 M to 1 M to 0.5 M to 0M
for 15 min intervals. The first two steps were carried
out at room temperature. The last step in basal media
was performed at 37°C.
Evaluation of immediate
post-warming/thawing survival
Survival of cryopreserved/thawed follicles was
assessed microscopically based on morphology of the
follicle as viewed under the stereoscope and thereafter
at magnification ¥300 using an inverted microscope
with Hoffman modulation contrast. A follicle was considered to be intact if it possessed an oocyte surrounded by a complete tight collar of GC. Follicles with
partially or completely naked oocytes or large spaces
within the granulosa-oocyte complex were graded as
damaged. Any dark atretic-looking follicles were also
graded as damaged. Only undamaged, intact pre-antral
follicles were selected for further in vitro culture (IVC).
In vitro culture of follicles and oocyte maturation
Fresh and cryopreserved/thawed pre-antral follicles
were cultured in Minimum Essential Medium a
(a-MEM) supplemented with 1% Nu-serum, 100 mIU/
mL follicle-stimulating hormone (FSH), 10 mg/mL
insulin, 5.5 mg/mL transferrin and 0.67 mg/mL selenium (ITS). Pre-antral follicles were group-cultured on
© 2010 The Authors
Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology
3
N. Desai et al.
Transwell inserts (24 mm in diameter and 0.4 mm pore
size) in six-well plates. All cultures were performed at
37°C with 5.5% CO2 in air.
Serum concentration was reduced after overnight
culture to 0.1% Nu-Serum and medium was half
changed every other day. Follicles were cultured in
vitro for up to 10 days. The number of attached follicles
containing an oocyte was determined at the end of this
culture interval. Follicles were then placed in maturation medium to induce oocyte maturation. The maturation media consisted of a-MEM supplemented with
5% Nu-serum, 100 mIU/mL FSH, ITS (10 mg/mL
insulin, 5.5 mg/mL transferrin, 0.67 mg/mL selenium),
1 ng/mL epidermal growth factor (EGF) and
1.5 IU/mL human chorionic gonadotrophin. After
16–18 h, cultures were treated with 40 U/mL hyaluronidase to facilitate the removal of GC surrounding
the oocyte. Oocytes were examined under an inverted
microscope (¥300) and graded for nuclear maturity.
Meiotic spindle evaluation
Meiotic spindle organization was evaluated using both
polarized light retardance and immunochemical staining. For meiotic spindle imaging in living oocytes, we
applied computer-assisted polarized microscopy (Polscope) using the Oosight imaging system. MII oocytes
were placed in micro-droplets of pre-warmed medium
and covered with mineral oil on a glass-bottomed dish.
Care was taken to maintain temperature at 37°C
throughout the examination to prevent transient depolymerization of spindle microtubules through oocyte
cooling. Oocytes were imaged at magnification ¥400. In
cases where the spindle was not seen, the oocyte was
rotated two to three times using a holder and an intracytoplasmic sperm injection pipette, to make sure that
the spindle was not overlooked. Once the meiotic
spindle was visualized, an image was captured. The
spindle retardance was determined using the imaging
software. The retardance gave a quantitative measurement of the degree of order and density of the microtubules within the spindle.
For immunostaining of the meiotic spindle, MII
oocytes were first fixed and permeabilized at 37°C with
2% paraformaldehyde + 0.1% Triton X in phosphate
buffered saline (PBS) for 30 min.27 After rinsing in
PBS + 1% human serum albumin (HSA), oocytes were
incubated in 10% mouse serum + 1% HSA in PBS for
1 h. Oocytes were rinsed and then incubated in fluorescein isothiocyanate-conjugated anti-mouse alpha
and beta tubulin in a 1:1 ratio for 2 h. Following
another rinse, oocytes were incubated in 10 mg/ml pro-
4
pidium iodide in PBS for 45 min before being mounted
on glass slides. All steps were carried out at 37°C.
Meiotic spindles were visualized at magnification
¥1000 using an oil immersion lens and an Olympus BX
51 microscope fitted for reflected fluorescence. A
normal meiotic spindle system was defined as having
MII chromosomes aligned along the centre of a barrelshaped spindle. For evaluation of normality of the
spindle and chromatin, we used very strict criteria,
within the limits of our imaging capabilities. Variations
in spindle morphology such as displaced spindle
fibers, stray chromosomes, or chromosomal misalignments, all resulted in classification of the meiotic
spindle as abnormal.
Preparation of follicles for light and transmission
electron microscopy
All the transmission electron microscopy (TEM)
reagents were purchased from Electron Microscopy
Sciences. Cryopreserved pre-antral follicles were fixed
immediately upon thawing. Freshly isolated pre-antral
follicles were prepared in parallel as control (CT).
Fixation of follicles was performed using 2.5%
glutaraldehyde/4% paraformaldehyde in 0.2 M
cacodylate buffer overnight at 4°C. After being rinsed
in cacodylate buffer, follicles were post fixed in
osmium tetroxide. The samples were then dehydrated
in increasing concentrations of ethanol, embedded in
Epon and sectioned. Semi-thin sections were stained
with 1% toluidine blue and examined with the light
microscope (LM). Ultra-thin sections were stained with
uranyl acetate to be examined and photographed using
the TEM.
Statistical analysis
Each experiment was performed in two to four replicates. The data were pooled for subsequent analysis.
The initial outcome measures analyzed were the immediate post-thaw survival, morphology and ultrastructure. Further functional assessment included
sustained growth over the IVC period, antral cavity
formation and oocyte maturation as evidenced by
GVBD, progression to the MII stage and meiotic
spindle organization. Meiotic spindle analysis was conducted on all mature oocytes in a separate experiment
using either immunochemical staining or else the
Oosight imaging system. Pooled data was compared
and analyzed using c2 and anova tests as appropriate.
A P-value of <0.05 was considered significant. The
StatsDirect program was used for statistical analysis.
© 2010 The Authors
Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology
Vitrification of isolated ovarian follicles
Table 1 Post-warming survival and development of isolated pre-antral follicles
vitrified using different cryo-devices
Total follicles cryopreserved
Post-warming survival rate (%)†
Number of follicles cultured
Intact follicles at end of culture (%)§
Antral cavity formation (%)¶
GVBD (%)¶
MII maturation (%)¶
CT
Nylon
mesh
EM grid
Tips
NA
NA
100
73%
56%
79%
52%
96
95%
88
55%
52%
90%
58%
102
91%
86
62%
45%
79%
51%
116
72%‡
77
61%
66%
91%
60%
†Post-warming survival was defined as follicles that were intact on warming. Only intact
pre-antral follicles were plated for further culture. Survival rate is expressed as a percentage
of the number of follicles initially cryopreserved. ‡Survival rate was significantly lower with
the microcapillary tips (P < 0.001). §The rate is expressed as a percentage of the number of
cultured follicles. ¶The rates of antral cavity formation, GVBD and MII maturation are
expressed as a percentage of the number of intact follicles at the end of the culture interval.
CT, non-frozen control group; EM grid, electron microscopy grid; GVBD, germinal vesicle
breakdown; Tips, micro-capillary tips; NA, not applicable.
Results
Vitrification and cryo-device
A total of 414 pre-antral follicles were utilized in this
experiment. The CT group consisted of fresh nonvitrified follicles (n = 100). The remaining follicles were
vitrified using one of three cryo-devices: nylon mesh
(n = 96), EM grid (n = 102) and micro-capillary tips
(n = 116).
The nylon mesh proved to be the easiest of the tested
cryo-devices for loading as many as 20 follicles at a
time. The mesh could be easily cut into a variety of
sizes, which facilitated its handling and manipulation.
In this experiment, we cut the nylon mesh into squares
(10 ¥ 10 mm). At the time of loading, the edge of the
nylon mesh was held above the dish surface with a
hemostat and the follicles were easily pipetted on to the
surface in a miniscule volume of fluid. The EM grid was
loaded similarly but proved more tedious to handle
due to its extremely small size (3 mm diameter disc).
The micro-capillary tip also proved to be more difficult
to control. It was technically challenging to quickly load
20 follicles into the tip. Moreover inadvertent damage
to the tip was possible during immersion into the vial
containing LN2.
On warming, the micro-capillary tip resulted in the
lowest percentage of intact follicles compared to the
nylon mesh and EM grid (P < 0.0001 and P = 0.001,
respectively). We plated and monitored the subsequent
growth of all intact vitrified/warmed follicles. Nonvitrified follicles were cultured as a CT group. Plated
follicles from all three vitrification cryo-devices developed similarly. There was no statistically significant dif-
ference in the subsequent development and maturation
of all surviving pre-antral follicles among the groups
(Table 1). Follicle survival at the end of the IVC period
was 55–62%. Antral cavity formation ranged from 45%
to 66%. No differences were observed between the treatment groups. GVBD occurred in 79% of oocytes from
CT follicles and in 79–91% of vitrified/warmed follicles.
The rate of maturation to the MII stage was also not
significantly different from that observed in the CT
group (Table 1). Based on these data and the ease of
handling experienced with the nylon mesh, the cryodevice was selected for all subsequent experiments.
Vitrification and slow freezing
A total of 979 pre-antral follicles were utilized in this
experiment. The CT group consisted of fresh follicles
(n = 256). The remaining follicles were either vitrified
using nylon mesh (n = 399), or frozen using slow programmed cooling (n = 324).
Survival, in vitro development and maturation of isolated
pre-antral follicles
Immediate post-thaw survival (Fig. 1) did not differ
with vitrification versus slow cooling (Table 2).
However with prolonged IVC, the CT and vitrification
treatment groups had a significantly higher proportion
of follicles that survived to the end of the culture interval as compared to the slow freeze group (71%, 65%
and 48%, respectively, P < 0.0001). Other outcome measures such as antral cavity formation, GVBD and MII
formation were similar with both cryopreservation
techniques and in the CT group (Table 2).
© 2010 The Authors
Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology
5
N. Desai et al.
Figure 1 Isolated pre-antral follicles photographed immediately after warming/thawing (¥150). (a) Vitrified/warmed follicles. (b) Frozen/thawed follicles.
Table 2 Summary of results for vitrified and slowly frozen follicles
Total follicles cryopreserved
Post-warming/thawing survival rate (%)†
Number of follicles cultured
Intact follicles at end of culture (%)‡
Antral cavity formation (%)¶
GVBD (%)¶
MII maturation (%)¶
Control
Vitrification
Slow
Freeze
NA
NA
256
71%
57%
77%
40%
399
95%
366
65%
64%
75%
41%
324
92%
280
48%§
61%
78%
40%
†Post-warming/thawing survival was defined as follicles that were intact on thaw. Only
intact pre-antral follicles were plated for further culture. Survival rate is expressed as a
percentage of the number of follicles initially cryopreserved. ‡The rate is expressed as a
percentage of the number of cultured follicles. §Significantly lower than control and vitrified
treatment groups (P < 0.0001). ¶The rates of antral cavity formation, GVBD and MII maturation are expressed as a percentage of the number of intact follicles at the end of the culture
interval. NA, not applicable.
Meiotic spindle assessment
In a separate experiment, meiotic spindles were analyzed using either immunochemical techniques or
polarized light imaging. All mature oocytes were prepared for microscopy (n = 194). Using immunochemical staining techniques we could not distinguish any
difference in morphology of meiotic spindles after
slow freezing as compared to vitrification. The majority
of oocytes derived from IVC/IVM follicles in all three
groups displayed barrel-shaped meiotic spindles with
centrally arranged chromosomes typical of normal
spindles (Fig. 2a). Figure 2b depicts an example of an
oocyte with spindle abnormalities and disorganized
chromosomal alignment. The percentage of normal
6
spindles observed was similar in all three groups
(Table 3).
A total of 110 MII oocytes were evaluated using
polarized light microscopy and the Oosight imaging
software (Fig. 3). This system allowed us to not only
monitor the visual presence of the spindle but also to
make morphometric measurements of the spindle
assembly. Retardance values of meiotic spindles in CT
and oocytes derived from the two cryopreservation
techniques were similar (Table 3).
By using the two different techniques for meiotic
spindle analysis, we had both morphological and morphometric data (retardance values) to support the ‘normalcy’ of the spindles. The fresh in vitro matured
follicles served as a CT. These data suggest that the
© 2010 The Authors
Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology
Vitrification of isolated ovarian follicles
(a)
(b)
Figure 2 (a) Immunocytochemical
staining of the meiotic spindle
showing a normal barrel-shaped
spindle with the chromosomes
well aligned along the center. (b)
Example of an MII oocyte with
an abnormal spindle and disrupted chromosomal alignment.
Magnification: ¥630; scale bar:
20 mm.
Table 3 Evaluation of meiotic spindle system in MII oocytes derived from cryopreserved/thawed in vitro matured preantral follicles using immunocytochemistry and polarized light imaging system
Technique
Immunochemical
Number of
Normal spindles
MII oocytes
& chromosomes
Number of
MII oocytes
Control
28
43
2.54 ⫾ 0.52
Vitrification
31
44
2.55 ⫾ 0.65
Slow freeze
25
23
2.32 ⫾ 0.64
P-value
–
–
0.22
19/28
(67.9%)
18/31
(58.1%)
16/25
(64.0%)
0.73
Polarized light imaging
Spindle retardance
(Mean ⫾ SD nm)
No spindle
found
6/43
(7%)
3/44
(7%)
3/23
(13%)
0.5
SD, standard deviation.
cryopreservation technique did not affect the meiotic
spindle assembly.
Light microscopic (LM) and TEM evaluation
The LM examination of the semi-thin sections of CT
and cryopreserved/thawed follicles showed intact follicles with a centrally located oocyte and intact basement membrane surrounded by a flattened thecal cell
layer(s) (Fig. 4).
TEM examination of vitrified and slowly frozen follicles was similar to that of their fresh CT counterparts.
Cytoplasmic organelles in the oocyte and GC were
well preserved. Also, finger-like processes (microvilli)
were seen projecting into the zona pellucida. In cases
where the ultra-thin sections were taken through the
nucleus, the germinal vesicle and its nucleolus
appeared normal (Fig. 5).
Discussion
We have shown that enzymatically isolated ovarian
pre-antral follicles can be quite successfully vitrified
using at least two of the tested cryo-devices, the nylon
mesh and the EM grid. Pre-antral follicle cryopreservation by vitrification as compared to slow programmed
cooling may better preserve the integrity of the follicular unit allowing continued in vitro development and
preventing premature ovulation of the enclosed
oocyte.
The vitrification process can be affected by the type of
the cryo-device. Cryo-devices that permit the loading of
a very minimal amount of fluid expedite the rate of
temperature drop, which is particularly important
during vitrification. Examples of such carriers include
the cryoloop,28 cryotop,7 open pulled straw29and EM
grid.30 To date, conventional straws,12,14 EM grids13 and
solid surface vitrification (SSV)15 have been attempted
towards vitrification of isolated ovarian follicles, with
variable results. Although the cryotop was successfully
applied for ovarian tissue vitrification,7,9 it has not been
used for isolated ovarian follicles. In the current study,
the type of cryo-device did have an impact on immediate post-warming survival. However, continued development in vitro, antral cavity development and oocyte
© 2010 The Authors
Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology
7
N. Desai et al.
(a)
(a)
(b)
(b)
(c)
(c)
Figure 3 The meiotic spindle birefringence shown by
Oosight spindle view (¥400). (a) Control group (b) vitrification group, and (c) slow freezing group.
maturation were comparable between different
cryo-devices.
Another consideration in evaluating the different
cryo-devices is whether they are ‘closed’ or ‘open’
systems. Open systems allow direct contact between the
sample and the LN2. To date, only two closed-system
vitrification vessels are commercially available: cryotips31,32 and high-security vitrification (HSV) straws.33
Neither permits easy loading of large numbers of cells
8
Figure 4 Thin sections of embedded pre-antral follicles
stained with Toluidine blue. (a) Control fresh (b)
vitrified/warmed, and (c) slowly frozen/thawed. O,
Oocyte; GC, granulosa cells; TC, theca cells.
© 2010 The Authors
Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology
Vitrification of isolated ovarian follicles
(a) 8,000x
(c) 3,000x
Figure 5 Electron micrographs of
(a,b) control (c,d) vitrified/
warmed and (e,f) frozen/thawed
pre-antral
follicles.
(a,c,e)
Showing
the
relationship
between oocyte (O) and surrounding granulosa cells (GC).
Note
finger-like
process
(microvilli, arrow) projecting
into the zona pellucida (ZP).
When the follicle was sectioned
at the level of the germinal
vesicle, the oocyte nucleus (N)
and nucleolus (NU) were also
seen. Note the areas of contact
between the oocyte and GC (*).
(b,d,f) Showing the relationship
between adjacent GC, GC
nucleus (GCN) and GC cytoplasm. Note the abundant mitochondria (M) populating the
cytoplasm. The contacts between
adjacent GC were also preserved
(arrow head).
(e) 3,000x
(unpublished data), which is an important practical
consideration for follicle preservation. Dela Pena et al.
(2002)12 used a 0.25-mL sealed plastic straw for vitrification. All other reports on vitrification of isolated
ovarian follicles have involved open carrier systems.
The potential for cross contamination during storage
and theoretical safety risks have been debated.34,35 New
Food and Drug Administration and European Tissue
Directive regulations may require the exclusive use of
closed systems for any type of cryopreservation as a
(b) 17,000x
(d) 17,000x
(f) 17,000x
safety precaution. Modification and/or development
of ‘closed’ cryo-devices specifically for isolated follicles
may ultimately be necessary for long-term storage.
The EG-raffinose vitrification protocol selected for
use in this study resulted in excellent post-warming
survival. About 95% of follicles were morphologically
intact immediately post warming and 65% survived to
the end of the IVC interval period, with 41% of oocytes
maturing to the MII stage. Our results are comparable
to those reported by others.12,14 In vitro survival of
© 2010 The Authors
Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology
9
N. Desai et al.
plated follicles was 71% and 50% of IVM oocytes that
reached the MII stage.12,14 Lower in vitro development
rates were obtained by Choi et al. (2007) using EM grids
(40%).13 No information was presented on the final
oocyte maturation. More recently, Lin et al. (2008)15 successfully vitrified isolated pre-antral follicles on a metal
surface (SSV). Post-warming survival was 91–96%.
After IVC and IVM, a 66% MII rate was reported.
It should however be noted that all of the abovementioned studies used the mechanical isolation
method for recovering individual pre-antral follicles
from the ovary. In the current work we used an enzymatic technique for follicle isolation. The enzymatic
technique is less labor-intensive, allowing the harvest
of a large number of follicles. However, enzymatically
isolated follicles may be more vulnerable during freezing. The oocyte–GC connections within the follicular
unit may be weakened by the enzymatic treatment.
Nagano et al. (2007)14 reported lower post-warming
survival rates (49–57%) with this method of follicle
isolation.
Enzymatically isolated ovarian follicles cryopreserved by slow cooling also have less developmental
capacity.22 Despite high post-thaw survival, only 58%
remained intact during culture with only 7.7% resuming
meiosis and only 3% developing to the MII stage.22 This
has not been the case with slow freezing of mechanically
isolated follicles. Xu et al. (2009)16 achieved quite excellent results with frozen/thawed secondary follicles cultured in alginate beads. After 12 days of culture, 74% of
follicles remained intact and the maturation rate was
64%. Cortvindt et al. (1996)11 also found that mechanically isolated follicles responded well to slow freezing
and at least 80% were able to grow as intact units to the
end of the culture interval, with 45% of IVM oocytes
extruding a polar body.
Follicle segregation by enzymatic isolation may
however prove necessary when dealing with human
and large mammalian ovaries that have a dense ovarian
cortex.36 Establishing cryopreservation techniques that
give good outcomes with enzymatically isolated follicles would therefore be an essential stepping stone in
human fertility preservation. The current investigation
certainly indicates that enzymatically isolated follicles
can be successfully vitrified and in vitro matured. The
rapid cooling rates associated with vitrification may be
essential in preventing any further weakening or disruption of the oocyte–GC complex.
We applied TEM to further evaluate the efficacy of the
two cryopreservation techniques at the ultra-structural
level.37 To our knowledge, this is the first report on
10
ultra-structure of vitrified isolated pre-antral follicles.
Ultra-structural differences specific to the freezing technique were not in evidence in the present study. TEM of
isolated pre-antral follicles after slow cooling11 also
indicated little ultra-structural changes when compared
to fresh controls. Other researchers comparing ultrastructure of vitrified and slowly frozen ovarian tissue
from mouse38 and human39 reported similar findings.
To complete this comparison on pre-antral follicle
cryopreservation by vitrification versus programmed
slow freezing, we examined the meiotic spindle morphology in the resultant IVM oocytes. The formation of
a normal meiotic spindle with well-aligned chromosomes is vital for the normal functioning of the oocyte
and the subsequent embryo.40,41 Immunochemical
staining of fixed oocytes allows morphological assessment of meiotic spindle assemblies and evaluation of
normalcy.27 Several studies have suggested that spindle
assembly and chromosome alignment is better after
vitrification as compared to slow freezing of human42
as well as mouse oocytes.43,44 This difference was not
observed by Cobo et al. (2008)32 or by our group in the
present work.
The availability of computer-assisted polarized light
microscopy has opened up a new avenue for studying
meiotic spindles. Real-time non-invasive spindle
imaging technology allows evaluation of spindle position and organization in living oocytes.45 Quantitative
measurement of spindle birefringence or retardance
may be valuable in comparing different cryopreservation protocols and their ability to preserve the order
and density of microtubules within the spindle. With
mature human oocytes, investigators using polarized
light microscopy were able to demonstrate faster
recovery of the meiotic spindle after vitrification as
compared to traditional slow freezing.46,47 To our
knowledge, the current study is the first to analyze
microtubule birefringence in isolated pre-antral follicles after vitrification and IVC/IVM. The molecular
integrity and density of spindles in oocytes from cryopreserved pre-antral follicles appeared to be unaffected by the cryopreservation technique.
One of the limitations of the present work may have
been the optimization of IVC conditions and the timing
for IVM. The number of days selected for IVC before
final maturation has varied amongst investigators
ranging from 8 to 14 days.48–50 Increasing the culture
period to 16 days may lead to better results.8 We are
currently exploring this possibility. Further grouping
of follicles into small and large categories during
culture could also be beneficial in improving the
© 2010 The Authors
Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology
Vitrification of isolated ovarian follicles
maturation rates. In the present work, the size of follicles ranged from 120 to 160 mm in all groups.
The sum of the data presented indicate that although
follicle structure may not be optimally preserved by
slow cryopreservation, resulting in lower yields of
mature oocytes after IVM, oocyte quality based on
meiotic spindle morphology is not different to that of
vitrification. The current data add to the limited pool of
information documenting outcomes with isolated preantral follicle cryopreservation. Further work in this
area and functional analysis of the developmental
capacity of embryos arising from both cryopreservation
methodologies are needed to establish the superiority
of one or the other technique. Efforts to maximize the
developmental competence of oocytes also need to
focus on the culture aspects of the maturation process
and conditions for in vitro fertilization.
Follicle cryopreservation may serve as a ‘bridge’ technology until such time that we are more successful with
ovarian transplantation or more capable of in vitro
maturing primordial follicles from the human ovary.
Indeed much more work is needed in this area
and proof of concept through ovarian follicle transplantation in animal models is necessary. Autotransplantation of cryopreserved/thawed follicles
embedded in collagen matrices or plasma clots may
prove to be more effective than transplantation of
cryopreserved/thawed ovarian cortical strips, permitting faster neo-vascularization and slowing down
atresia. Progress in this area is necessary if follicle cryopreservation is ever to be considered a clinically viable
treatment option. Given the current limitation of
ovarian tissue cryopreservation and transplantation, vitrification of isolated ovarian follicles could be an invaluable alternative approach for fertility preservation for
patients at risk of premature ovarian failure.
Acknowledgments
The authors would like to thank Dr Judith A Drazba
PhD and Mrs Mei Yin at the Imaging Core facility of
Cleveland Clinic Foundation for their expert technical
assistance with the transmission electron microscopy.
References
1. Oktay K, Buyuk E. The potential of ovarian tissue transplant
to preserve fertility. Expert Opin Biol Ther 2002; 2: 361–370.
2. Bedaiwy MA, Falcone T. Ovarian tissue banking for cancer
patients: Reduction of post-transplantation ischaemic injury:
Intact ovary freezing and transplantation. Hum Reprod 2004;
19: 1242–1244.
3. Gook DA, Edgar DH, Stern C. Cryopreservation of human
ovarian tissue. Eur J Obstet Gynecol Reprod Biol 2004; 113
(Suppl 1): S41–S44.
4. Donnez J, Dolmans MM, Martinez-Madrid B et al. The role of
cryopreservation for women prior to treatment of malignancy. Curr Opin Obstet Gynecol 2005; 17: 333–338.
5. Falcone T, Bedaiwy MA. Fertility preservation and pregnancy
outcome after malignancy. Curr Opin Obstet Gynecol 2005; 17:
21–26.
6. Meirow D, Levron J, Eldar-Geva T et al. Monitoring the
ovaries after autotransplantation of cryopreserved ovarian
tissue: Endocrine studies, in vitro fertilization cycles, and live
birth. Fertil Steril 2007; 87: e7–e15.
7. Hasegawa A, Hamada Y, Mehandjiev T et al. In vitro growth
and maturation as well as fertilization of mouse preantral
oocytes from vitrified ovaries. Fertil Steril 2004; 81 (Suppl 1):
824–830.
8. Segino M, Ikeda M, Hirahara F et al. In vitro follicular development of cryopreserved mouse ovarian tissue. Reproduction
2005; 130: 187–192.
9. Hasegawa A, Mochida N, Ogasawara T et al. Pup birth from
mouse oocytes in preantral follicles derived from vitrified
and warmed ovaries followed by in vitro growth, in vitro
maturation, and in vitro fertilization. Fertil Steril 2006; 86
(Suppl 4): 1182–1192.
10. Carroll J, Gosden RG. Transplantation of frozen-thawed
mouse primordial follicles. Hum Reprod 1993; 8: 1163–1167.
11. Cortvrindt R, Smitz J, Van Steirteghem AC. A morphological
and functional study of the effect of slow freezing followed by
complete in-vitro maturation of primary mouse ovarian follicles. Hum Reprod 1996; 11: 2648–2655.
12. Dela Pena EC, Takahashi Y, Katagiri S et al. Birth of pups after
transfer of mouse embryos derived from vitrified preantral
follicles. Reproduction 2002; 123: 593–600.
13. Choi WJ, Yeo HJ, Shin JK et al. Effect of vitrification method
on survivability, follicular growth and ovulation of preantral
follicles in mice. J Obstet Gynaecol Res 2007; 33: 128–133.
14. Nagano M, Atabay EP, Atabay EC et al. Effects of isolation
method and pre-treatment with ethylene glycol or raffinose
before vitrification on in vitro viability of mouse preantral
follicles. Biomed Res 2007; 28: 153–160.
15. Lin TC, Yen JM, Kuo TC et al. Comparison of the developmental potential of 2-week-old preantral follicles derived from
vitrified ovarian tissue slices, vitrified whole ovaries and
vitrified/transplanted newborn mouse ovaries using the
metal surface method. BMC Biotechnol 2008; 8: 38.
16. Xu M, Banc A, Woodruff TK et al. Secondary follicle
growth and oocyte maturation by culture in alginate hydrogel
following cryopreservation of the ovary or individual follicles.
Biotechnol Bioeng 2009; 103: 378–386.
17. Shaw JM, Cox SL, Trounson AO et al. Evaluation of the longterm function of cryopreserved ovarian grafts in the mouse,
implications for human applications. Mol Cell Endocrinol 2000;
161: 103–110.
18. Dolmans MM, Martinez-Madrid B, Gadisseux E et al.
Short-term transplantation of isolated human ovarian follicles
and cortical tissue into nude mice. Reproduction 2007; 134:
253–262.
19. Carroll J, Whittingham DG, Wood MJ et al. Extra-ovarian production of mature viable mouse oocytes from frozen primary
follicles. J Reprod Fertil 1990; 90: 321–327.
© 2010 The Authors
Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology
11
N. Desai et al.
20. Laschke MW, Menger MD, Vollmar B. Ovariectomy improves
neovascularization and microcirculation of freely transplanted ovarian follicles. J Endocrinol 2002; 172: 535–544.
21. Rodgers RJ, Irving-Rodgers HF, Russell DL. Extracellular
matrix of the developing ovarian follicle. Reproduction 2003;
126: 415–424.
22. Carroll J, Whittingham DG, Wood MJ. Growth in vitro and
acquisition of meiotic competence after the cryopreservation
of isolated mouse primary ovarian follicles. Reprod Fertil Dev
1991; 3: 593–599.
23. Tanasawa I. Things we do not know about cryopreservation of
biological organs. Ann N Y Acad Sci 1998; 858: 227–234.
24. Vajta G, Nagy ZP. Are programmable freezers still needed in
the embryo laboratory? Review on vitrification. Reprod Biomed
Online 2006; 12: 779–796.
25. Liebermann J, Dietl J, Vanderzwalmen P et al. Recent
developments in human oocyte, embryo and blastocyst vitrification: Where are we now? Reprod Biomed Online 2003; 7:
623–633.
26. Cremades N, Sousa M, Silva J et al. Experimental vitrification
of human compacted morulae and early blastocysts using fine
diameter plastic micropipettes. Hum Reprod 2004; 19: 300–305.
27. Stachecki JJ, Munne S, Cohen J. Spindle organization after
cryopreservation of mouse, human, and bovine oocytes.
Reprod Biomed Online 2004; 8: 664–672.
28. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse
and human blastocysts using a novel cryoloop container-less
technique. Fertil Steril 1999; 72: 1073–1078.
29. Vajta G, Holm P, Kuwayama M et al. Open Pulled Straw (OPS)
vitrification: A new way to reduce cryoinjuries of bovine ova
and embryos. Mol Reprod Dev 1998; 51: 53–58.
30. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling.
Biol Reprod 1996; 54: 1059–1069.
31. Kuwayama M, Vajta G, Ieda S et al. Comparison of open and
closed methods for vitrification of human embryos and the
elimination of potential contamination. Reprod Biomed Online
2005; 11: 608–614.
32. Cobo A, Perez S, De Los Santos MJ et al. Effect of different
cryopreservation protocols on the metaphase II spindle in
human oocytes. Reprod Biomed Online 2008; 17: 350–359.
33. Camus A, Clairaz P, Ersham A et al. The comparison of the
process of five different vitrification devices. Gynecol Obstet
Fertil 2006; 34: 737–745.
34. Bielanski A, Nadin-Davis S, Sapp T et al. Viral contamination
of embryos cryopreserved in liquid nitrogen. Cryobiology
2000; 40: 110–116.
35. Bielanski A, Bergeron H, Lau PC et al. Microbial contamination of embryos and semen during long term banking in
liquid nitrogen. Cryobiology 2003; 46: 146–152.
36. Demeestere I, Delbaere A, Gervy C et al. Effect of preantral
follicle isolation technique on in-vitro follicular growth,
oocyte maturation and embryo development in mice. Hum
Reprod 2002; 17: 2152–2159.
12
37. Camboni A, Martinez-Madrid B, Dolmans MM et al. Preservation of fertility in young cancer patients: Contribution of
transmission electron microscopy. Reprod Biomed Online 2008;
17: 136–150.
38. Wang Y, Xiao Z, Li L et al. Novel needle immersed vitrification: A practical and convenient method with potential
advantages in mouse and human ovarian tissue cryopreservation. Hum Reprod 2008; 23: 2256–2265.
39. Keros V, Xella S, Hultenby K et al. Vitrification versus
controlled-rate freezing in cryopreservation of human ovarian
tissue. Hum Reprod 2009; 24: 1670–1683.
40. Eichenlaub-Ritter U, Chandley AC, Gosden RG. Alterations
to the microtubular cytoskeleton and increased disorder of
chromosome alignment in spontaneously ovulated mouse
oocytes aged in vivo: An immunofluorescence study. Chromosoma 1986; 94: 337–345.
41. Volarcik K, Sheean L, Goldfarb J et al. The meiotic competence
of in-vitro matured human oocytes is influenced by donor
age: Evidence that folliculogenesis is compromised in the
reproductively aged ovary. Hum Reprod 1998; 13: 154–160.
42. Cao YX, Xing Q, Li L et al. Comparison of survival and embryonic development in human oocytes cryopreserved by
slow-freezing and vitrification. Fertil Steril 2009; 92: 1306–
1311.
43. Huang JY, Chen HY, Tan SL et al. Effect of cholinesupplemented sodium-depleted slow freezing versus
vitrification on mouse oocyte meiotic spindles and chromosome abnormalities. Fertil Steril 2007; 88: 1093–1100.
44. Ko CS, Ding DC, Chu TW et al. Changes to the meiotic spindle
and zona pellucida of mature mouse oocytes following different cryopreservation methods. Anim Reprod Sci 2008; 105:
272–282.
45. Wang WH, Meng L, Hackett RJ et al. Limited recovery of
meiotic spindles in living human oocytes after coolingrewarming observed using polarized light microscopy. Hum
Reprod 2001; 16: 2374–2378.
46. Larman MG, Minasi MG, Rienzi L et al. Maintenance of the
meiotic spindle during vitrification in human and mouse
oocytes. Reprod Biomed Online 2007; 15: 692–700.
47. Ciotti PM, Porcu E, Notarangelo L et al. Meiotic spindle recovery is faster in vitrification of human oocytes compared to
slow freezing. Fertil Steril 2009; 91: 2399–2407.
48. Mousset-Simeon N, Jouannet P, Le Cointre L et al. Comparison of three in vitro culture systems for maturation of
early preantral mouse ovarian follicles. Zygote 2005; 13: 167–
175.
49. Cortvrindt R, Smitz J, Van Steirteghem AC. In-vitro maturation, fertilization and embryo development of immature
oocytes from early preantral follicles from prepubertal mice
in a simplified culture system. Hum Reprod 1996; 11: 2656–
2666.
50. Kreeger PK, Deck JW, Woodruff TK et al. The in vitro
regulation of ovarian follicle development using alginateextracellular matrix gels. Biomaterials 2006; 27: 714–723.
© 2010 The Authors
Journal of Obstetrics and Gynaecology Research © 2010 Japan Society of Obstetrics and Gynecology