Human Reproduction, Vol.27, No.1 pp. 106–113, 2012
Advanced Access publication on October 25, 2011 doi:10.1093/humrep/der344
ORIGINAL ARTICLE Embryology
Cytoskeletal analysis of human
blastocysts by confocal laser scanning
microscopy following vitrification
Katerina Chatzimeletiou 1,*, Ewan E Morrison 2, Yannis Panagiotidis 3,
Pierre Vanderzwalmen 4, Nikos Prapas 3, Yannis Prapas 3,
Basil C. Tarlatzis 1, and Alan H. Handyside 5
1
Section of Reproductive Medicine, First Department of Obstetrics & Gynaecology, Aristotle University Medical School, Papageorgiou
General Hospital, Thessaloniki 56403, Greece 2CRUK Clinical Centre at Leeds, St James’ University Hospital, Leeds LS9 7TF, UK 3Iakentro
Advanced Medical Centre, Thessaloniki 542 50, Greece 4IVF Centre Prof. Zech, Bregenz, Austria 5The London Bridge Fertility Gynaecology
and Genetics Centre, London SE1 9RY, UK
*Correspondence address. Tel: +30-231-332-3827; E-mail: [email protected]
Submitted on December 30, 2010; resubmitted on September 12, 2011; accepted on September 20, 2011
background: Vitrification of human blastocysts is being used increasingly to cryopreserve supernumerary embryos following IVF. In this
study, we investigate the effects of aseptic vitrification on the cytoskeleton and development of human blastocysts, by analysing survival rates
and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy.
methods: A total of 55 fresh blastocysts and 55 day 5 dimethylsulphoxide/ethylene glycol vitrified blastocysts, which were allowed to
remain in culture for 24 h post-warming, were rapidly fixed in ice cold methanol, and immunostained with an a-tubulin antibody to visualize
microtubules in combination with antibodies against acetylated tubulin (to visualize spindles, poles and mid bodies), gamma tubulin (to identify spindle poles) and 4(6-diamidino-2-phenylindole) to visualize DNA.
results: In total, 213 spindles were analysed in the control (fresh) group of which 183/213 (85.9%) were normal, 20/213 (9.4%) were
abnormally shaped, 9/213 (4.2%) were multipolar and 1/213 (0.5%) was monopolar. A total of 175 spindles were analysed in the vitrified
group, of which 120/175 (68.6%) were normal, 39/175 (22.3%) were abnormally shaped, 10/175 (5.7%) were multipolar and 6/175 (3.4%)
were monopolar. The incidence of multipolar spindles was similar in the two groups, but the level of abnormally shaped spindles, often
associated with chromosome lagging, or congression failure, was significantly higher in the vitrified group compared with the fresh group
(P , 0.05).
conclusions: The high survival rate following thawing and the large proportion of normal spindle/chromosome configurations
suggests that vitrification at the blastocyst stage on Day 5 does not adversely affect the development of human embryos and the ability
of spindles to form and continue normal cell divisions. However, there was a significantly higher incidence of abnormal spindles in the vitrified
group compared with the fresh group, notably of spindles with a focused and an unfocused pole as well as chromosome bridging and disorganized middle spindle fibres at telophase. Further investigation is warranted to elucidate the mitotic stages that are more vulnerable to
damage during vitrification, the fate of the abnormal spindles and any potential effects that may be reflected on the chromosomal constitution
of the developing blastocysts.
Key words: aseptic vitrification / chromosome bridging / confocal laser scanning microscopy / human blastocyst / spindle abnormalities
Introduction
Vitrification is an attractive ultrarapid cryopreservation technique
which has gained support as an alternative promising substitute for
slow freezing. It uses extremely high concentrations of cryoprotectants
and allows the solidification of a solution below the glass transition
temperature, without ice crystal formation (Liebermann, 2009; Vajta
et al., 2009). Vitrification has been successfully applied to both cleavage and blastocyst stage embryos and clinical trials have shown
high survival rates and promising implantation rates following transfer
of thawed embryos at all stages (Liebermann and Tucker, 2006;
Mukaida et al., 2006; Hong et al., 2009; Vanderzwalmen et al.,
2009, 2003; Desai et al., 2010; Wikland et al., 2010). The data on
the safety of vitrification in terms of obstetric and perinatal outcomes
& The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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107
Cytoskeletal analysis of human vitrified blastocysts
are also reassuring (Mukaida et al., 2008; Liebermann, 2009; Noyes
et al., 2009). However, blastocysts represent a unique challenge
because of the difficulty in accomplishing the required level of dehydration and high viscosity evenly in all blastomeres, due to their multicellular structure and presence of the water-filled blastocoele
(Vanderzwalmen et al., 2002).
It has been shown in animal models that inadequate intracellular
delivery of cryoprotectants and insufficient dehydration of blastocysts
can lead to a decreased survival rate and increased apoptosis (Kader
et al., 2010, 2009a,b; Morató et al., 2010). Bettencourt et al. (2009)
reported ultrastructural damage of mitochondria and cytoskeleton in
ovine vitrified blastocysts and recent studies in mice have shown an
alteration in methylation following vitrification (Wang et al., 2010).
Most studies in human vitrified blastocysts concentrate on assessing
the success of the procedure on survival rates post thaw and clinical
outcomes following transfer. No studies have looked into the dividing
cells and the effects vitrification may have on spindle structure,
chromosome alignment and ability of spindles to form and continue
normal cell division. Here, we have investigated the effects of vitrification on Day 5 on the cytoskeleton and development of human blastocysts, by analysing survival rates and spindle and chromosome
configurations by fluorescence and confocal laser scanning
microscopy.
Materials and Methods
Source of human preimplantation embryos
Surplus human blastocysts (n ¼ 110) were donated for research from consenting couples undergoing IVF treatment in the Assisted Conception Unit
of the Iakentro Advanced Medical Centre in Greece, where the embryos
were vitrified (n ¼ 55), fixed and labelled. The majority of the blastocysts
originated from oocyte donation cycles. This work was approved by the
ethical committee of the Aristotle University Medical School (Licence
no. A 10570).
Ovarian stimulation
A standard protocol was used to induce superovulation and control timing
of oocyte retrieval. Pituitary down-regulation was achieved by the administration of gonadotrophin-releasing hormone analogues (Suprefact;
Hoechst Marrion Roussel, Arvekap; Ipsen, Cetrotide; Serono or Orgalutran; MSD), for the first 7 – 14 days, followed by ovarian stimulation with
pure FSH (Metrodine; Serono, Middlesex, UK) or FSH and LH (Altermon;
IBSA) or recombinant FSH (Puregon; MSD or Gonal-F; Serono) for 12 – 14
days. The patients were monitored regularly by ultrasound, and by
measuring Estradiol levels, starting on Day 4 of gonadotrophin administration and when adequate follicular development had been demonstrated
(≥3 follicles of 17 mm in diameter), 10 000 IU of hCG (Ovitrelle; or
Pregnyl; MSD) was administered to trigger ovulation. Thirty-six hours
after the hCG administration, transvaginal ultrasound guided egg retrieval
was performed.
Oocyte retrieval
Oocytes were retrieved by flushing ovarian follicles with human tubal fluid
medium (Sage) incubated in 5% CO2 in air at 378C and subsequently fertilized by conventional IVF or ICSI and cultured until the day of transfer in
culture medium (SAGE or Vitrolife). Spare day 5 human blastocysts were
either vitrified or processed fresh for cytoskeletal analysis. The blastocysts
were assigned randomly in the fresh or vitrified group and they were of
similar quality.
Human blastocyst vitrification
Fifty-five human blastocysts were vitrified on Day 5 under aseptic conditions according to Vanderzwalmen et al. (2009). After gradual exposure
to dimethylsulphoxide/ethylene glycol (DMSO/EG) (5%/5%, 10%/10%
and 20%/20%), the blastocysts were loaded into the gutter of a plug (VitriSafe—MTG—Germany) before insertion into an outer protective straw
(CryoBioSystème, France) that was welded and plunged into liquid nitrogen (LN2). The vitrified blastocysts were warmed by immersing the tip of
the straw (Vitrisafe) in 1, 0.5 and 0.25 M sucrose solutions and were
allowed to recover in culture for 24 h before being treated for cytoskeletal
analysis.
Human embryo fixation, immunolabelling
and confocal imaging
Fifty-five blastocysts were used as controls (fresh) and were fixed and
labelled on Day 5, 6 or 7 and 55 vitrified blastocysts were fixed and
labelled 24 h post-warming (on Day 6).
Blastocyst immunostaining was performed according to Chatzimeletiou
et al. (2005a,b) using a primary rat monoclonal antibody specific for
a-tubulin (Serotec) to visualize microtubules in combination with mouse
monoclonal antibodies specific for gamma-tubulin (to identify spindle
poles) or acetylated tubulin (to visualize spindle poles and mid bodies)
(Sigma) and 4(6-diamidino-2-phenylindole) (DAPI) to visualize DNA. In
brief, all blastocysts were rapidly fixed in ice cold methanol, washed in
Ca++ /Mg++ free phosphate-buffered saline (PBS; Gibco BRL) containing
2% bovine serum albumin (BSA; Sigma) and transferred into 10 ml drops of
the primary antibodies (for antibody dilutions see Table I) under mineral
oil (Sigma) and incubated at 48C for 1 h. The blastocysts were then
washed in PBS/BSA and transferred into 10 ml drops of the secondary
antibodies [highly cross-adsorbed Alexa Fluor 488 or 594 conjugates
Table I Dilution of the primary and secondary antibodies used for microtubule immunostaining.
Primary antibodies
Dilution in 1 3 PBS/2%
BSA
Secondary antibodies
Dilution in 1 3 PBS/%
BSA
.............................................................................................................................................................................................
Rat anti-a-tubulin
1:800
Goat anti-rat Alexa 488 (FITC) or
1:500
Goat anti-rat Alexa 594 (tetramethylrhodamine isothiocyanate) 1:500
Mouse anti-g-tubulin
1:1000
Goat anti-mouse Alexa 594 (tetramethylrhodamine
isothiocyanate) or
1:500
Mouse anti-acetylated
tubulin
1:1000
Goat anti-mouse Alexa 488 (FITC)
1:500
108
Chatzimeletiou et al.
(Molecular Probes), containing 1 ng/ml DAPI; Sigma]. Following 1 h incubation in the secondary antibodies, the blastocysts were washed in PBS/
BSA and mounted on slides (BDH) in Vectashield antifade medium
(Vector laboratories, CA, USA) under a coverslip. The coverslips were
then sealed with nail varnish.
The blastocysts were examined using the Zeiss fluorescence microscope and/or the Leica TCS-SP laser scanning confocal microscope.
Standard fluorescence images were captured using the ISIS software
(Metasystems) and confocal image analysis was typically accomplished
by capturing a z-series stack of 1 mm thick sections encompassing
the entire blastocyst. The images were acquired sequentially to avoid
bleed-through artefacts using the 488 nm line of an argon laser to
image Alexa 488, the 568 nm laser line of a Kr laser to image Alexa
594 and an argon-UV laser to visualize DAPI staining of DNA. A
25x or a 100x UV-corrected oil immersion lens was used depending
on whether low or high magnification of the blastocyst to be captured
was desired.
Classification of spindle abnormalities
All interphase nuclei and metaphase/anaphase spindle and chromosome
configurations were carefully examined and counted in each blastocyst.
The criteria for classifying spindle abnormalities were as previously
described by Chatzimeletiou et al. (2005a). A spindle with astral shaped
or fusiform poles and with chromosomes aligned at the equator was classified as normal. A spindle with one or two poorly defined or apparently
absent poles, generally with misaligned chromosomes was classified as
having an abnormal shape. Spindles with more than two clearly defined
astral poles and the characteristic ‘Y’ or ‘X’ shaped arrangement of DAPIlabelled chromosomes were classified as multipolar. Multipolarity was also
confirmed in some cases by g-tubulin labelling of centrosomes at the
spindle poles. Finally, any DAPI-labelled presumptive chromosomes not
aligned with the other chromosomes on the spindle were classified as
lagging chromosomes (chromosome loss).
Statistical analysis
Statistical analysis of the results was carried out with the SPSS 9 (SPSS Inc.,
Headquarters, Chicago, IL, USA) statistical package for Windows. Fisher’s
exact test was used to determine whether the incidence of spindle
abnormalities differs in the fresh and vitrified blastocysts.
Results
Spindle abnormalities in fresh blastocysts
Fifty-five fresh human blastocysts were processed for cytoskeletal
analysis on Day 5 (n ¼ 26), Day 6 (n ¼ 22) and Day 7 (n ¼ 7).
All 55 fresh blastocysts had cells with mitotic spindles and were
analysed. The range of mitotic spindles was 1 – 11 per fresh blastocyst. The mean cell number of the blastocysts analysed on Days 5,
6 and 7 was 80.4 + 5.4, 172.8 + 15.3 and 281.7 + 29.1, respectively. Cytoskeletal analysis revealed that overall 37/55 (67.3%)
fresh blastocysts had only normal spindles, while 18/55 (32.7%)
had multipolar or abnormally shaped spindles in addition to
normal spindles. In total, 213 spindles were analysed, of which
183/213(85.9%) were normal, 20/213 (9.4%) were abnormally
shaped, 9/213 (4.2%) were multipolar and 1/213 (0.5%) was
monopolar (Table II).
Table II Spindle abnormalities in vitrified and fresh human blastocysts.
No. of blastocysts
analysed
No. of
blastocysts with
abnormal
spindles (%)
Total no. of
spindles analysed
Normal
spindles (%)
........................
0
Abnormal spindles (%)
.......................................................
≥1
Abnormal
Multi-
Mono-polar
shape
polar
...........................
.............................................................................................................................................................................................
Vitrified
Day 6
Grade A 4
Grade B 39
Grade C 4
Vitrified Day 6 total
Fresh Day
5
Fresh Day 5 total
Fresh Day
6
22
Grade A —
Grade B 7
Grade C —
Fresh Day 7 total
Fresh total
26
Grade A 4
Grade B 15
Grade C 3
Fresh Day 6 total
Fresh Day
7
47
Grade A 3
Grade B 22
Grade C 1
7
55
1
13
0
3
26
4
15
153
7
14 (29.8) 33 (70.2) 175
10
109
1
4
34
1
120 (68.6)
39 (22.3)
10 (5.7)
6 (3.4)
1
5
1
11
46
3
10
41
2
19 (73.1)
7 (26.9)
60
53 (88.3)
2 (3.3)
5 (8.3)
0
2
10
2
2
5
1
33
64
9
30
53
7
2
9
2
1
1
0
0
1
0
8 (36.4) 106
90 (84.9)
13 (12.3)
—
40
—
—
5
—
—
3
—
—
4
—
—
47
—
3
4
47
40 (85.1)
37 (67.3) 18 (32.7) 213
183 (85.9)
5 (10.6)
20 (9.4)
Blastocyst classification—Grade A: Top quality with well defined ICM and TE, Grade B: Medium quality, Grade C: poor quality.
1
3
1
0
3
3
2
17
0
14 (63.6)
0
2
0
1
7
2
2 (1.9)
—
2
—
0
0
0
1 (0.9)
—
0
—
2 (4.3)
0
9 (4.2)
1 (0.5)
109
Cytoskeletal analysis of human vitrified blastocysts
Table III Distribution of spindle abnormalities in the ICM and TE of vitrified and fresh human blastocysts.
No. of blastocysts
analysed
Total no. of
spindles analysed
Spindles
......................................................................................
Normal
Abnormal
shape
Multi-polar
Mono-polar
................... ................. ................. .................
ICM
TE
ICM
TE
ICM
TE
ICM
TE
.....................................
.............................................................................................................................................................................................
Vitrified Day 6
Grade A
Grade B
Grade C
Vitrified Day 6 total
Fresh Day 5
Grade A
Grade B
Grade C
Fresh Day 5 total
Fresh Day 6
Grade A
Grade B
Grade C
Fresh Day 6 total
Fresh Day 7
Fresh Day 7 total
Fresh total
Grade A
Grade B
Grade C
4
39
4
15
153
7
1
17
0
9
92
1
1
9
0
3
25
1
0
0
0
1
7
2
47
175
18
102
10
3
22
1
11
46
3
2
6
0
8
35
2
0
1
0
26
60
8
45
4
15
3
33
64
9
6
11
0
24
42
7
22
106
17
—
7
—
—
47
—
—
7
—
7
47
55
213
0
1
0
0
2
3
29
0
10
1
5
0
1
0
0
0
0
1
3
1
0
0
0
0
0
0
1
1
0
5
0
0
1
2
0
1
7
2
0
0
0
1
1
0
0
1
0
0
0
0
73
3
10
0
2
1
0
33
—
1
—
4
—
0
—
2
—
0
—
0
7
33
1
4
0
2
0
0
32
151
5
15
0
9
1
0
Grade A: Top quality with well defined ICM and TE, Grade B: Medium quality, Grade C: poor quality.
Post-warming survival and spindle
abnormalities in vitrified blastocysts
Fifty-one out of 55 (92.7%) human blastocysts that were vitrified on
Day 5 survived post-warming and 47/51 vitrified blastocysts had
cells with mitotic spindles and were analysed. The range of mitotic
spindles was 1–10 per human vitrified blastocyst. The mean cell
number of the blastocysts analysed 24 h post thaw, on Day 6, was
163 + 9.3. Cytoskeletal analysis showed that 14/47 (29.8%) vitrified
blastocysts had only normal spindles, 5/47 (10.6%) had only abnormal
spindles and 28/47 (59.6%) had a mixture of normal and abnormal
spindles, including abnormally shaped, monopolar and multipolar spindles, disorganized prometaphases, chromosome lagging, congression
failure and chromosome bridging (Tables II and III, Figs. 1 and 2). A
total of 175 spindles were analysed in total in the vitrified group, of
which 120/175 (68.6%) were normal (Fig. 1A), 39/175 (22.3%)
were abnormally shaped (Fig. 1F –H and Fig. 2I), 10/175 (5.7%)
were multipolar and 6/175 (3.4%) were monopolar (Fig. 1C).
Abnormalities in fresh versus vitrified
blastocysts: comparison of results
The embryos were randomly assigned to the fresh or the vitrified
groups and they were of similar quality (Table II). Overall, the vitrified
blastocysts showed excellent survival post-warming, but the cytoskeletal analysis detected specific spindle abnormalities that were
not observed as frequently in fresh blastocysts. The incidence of multipolar spindles was similar in the two groups, but the level of abnormally shaped spindles, often associated with chromosome lagging or
congression failure, was significantly higher in the vitrified group compared with the fresh group (P , 0.05). The most frequently observed
type of spindle abnormality in the vitrified group was that of a spindle
with one well-focused pole and another more rounded (unfocused)
pole (Fig. 1G, and Fig. 2I). Some spindles also appeared more
elongated (Fig. 1H) and occasionally had lagging chromosomes. Disorganized middle spindle fibres at telophase were also observed
(Fig. 1B). It is of particular interest to note that these abnormalities
were present even in good quality blastocysts, such as the blastocyst
in Fig. 2 (Grade A), which survived in an excellent way after
warming but (i) had the most frequently observed type of spindle
abnormality with one well-focused pole and an unfocused pole and
(ii) a spindle with chromosome bridging.
Distribution of acetylated and g-tubulin
Acetylated tubulin antibodies strongly labelled spindle midbodies
during telophase in both fresh and vitrified/thawed blastocysts. In
some cases disorganized middle spindle fibres at telophase were
evident, which would inevitably result in binucleation. Acetylated
tubulin also strongly labelled the spindle poles during both metaphase
and anaphase and the tails of any extra sperm bound on the zona of
IVF embryos (Fig. 1G and H). g-tubulin was detectable at the spindle
poles and confirmed bipolarity, multipolarity or the absence of poles in
certain cases.
Discussion
This study provides the first cytoskeletal analysis of human vitrified
blastocysts and compares the type and incidence of spindle abnormalities in those, to that observed in fresh blastocysts. The high survival
rate following warming (92.7%) suggests that vitrification at the blastocyst stage does not adversely affect the development of human
110
Chatzimeletiou et al.
Figure 1 Confocal sections of human vitrified blastocysts showing (A) normal astral bipolar spindle, (B) disorganized middle spindle fibres at telophase, (C) monopolar spindle, (D) normal anaphase spindle, (E) normal telophase, (F) abnormally shaped spindle with misaligned lagging chromosomes (arrow), (G) abnormally shaped spindle with a well focused and an unfocused pole (short arrow) and monopolar spindle (long arrow). Note
the strong staining of midbodies with acetylated tubulin (arrow heads), (H) abnormally shaped elongated spindle. Note the strong staining of sperm
tails (arrow) and midbodies with acetylated tubulin (arrow heads). DAPI (blue), a-tubulin (red) (images A, B, E, F, G and H) or green (images C, D),
acetylated tubulin (green) (images G, H).
embryos. This is in agreement with clinical trials that have shown high
survival rates and promising implantation rates following transfer of
thawed blastocysts (Liebermann and Tucker, 2006; Mukaida et al.,
2006; Hong et al., 2009; Vanderzwalmen et al., 2009, 2002;
Wikland et al., 2010).
Detailed cell count revealed no statistically significant difference in
the total number of nuclei between the vitrified blastocysts, 24 h post-
warming on Day 6 and fresh blastocysts on Day 6 (163 + 9.3 versus
172.8 + 15.3). The majority of mitotic spindles examined by confocal
laser scanning microscopy in both groups had normal astral or fusiform
shaped poles and were bipolar (68.6% vitrified versus 85.9% fresh).
The level of multipolar spindles (tripolar and tetrapolar spindles with
the characteristic ‘Y’ and cruciform ‘X’ shaped organization) was
similar in vitrified and fresh blastocysts (5.7 versus 4.2%). However,
Cytoskeletal analysis of human vitrified blastocysts
111
Figure 2 (A– F) Confocal sections of a human vitrified blastocyst showing staining with a-tubulin (red) and DAPI (blue). Note the chromosome
bridging shown in high magnification in (G) and under DAPI only filter in (H) (arrow). Also note in high magnification in (I) a spindle with one
focused (long arrow) and an unfocused pole (short arrow). The blastocyst is adapted from the Journal Zygote with permission.
there was a significantly higher incidence of abnormally shaped spindles (lacking well-defined poles, and/or one or more chromosomes
separate from the spindle resulting presumably either from congression failure or anaphase lag) in the vitrified group (P , 0.05), with
more prominent the type of spindles with a well focused and an unfocused pole (Figs. 1G and 2I). For the first time, the present study also
provides unique observations of chromosome bridging (Fig. 2G and H)
which is associated with chromosome breakage and micronucleation
and disorganized middle spindle fibres at telophase (Fig. 1B) which
can lead to binucleation, extending our previous work into the mechanisms that lead to postzygotic chromosomal abnormalities in early
human development (Chatzimeletiou et al., 2005a,b, 2008).
It is unclear whether the increase in spindle abnormalities following
vitrification is due to toxicity or mechanical stress following exposure
to the high concentrations of cryoprotectants or due to the submersion into liquid nitrogen. However, our recent pilot data (Chatzimeletiou et al., 2010) suggested that it is the exposure to the
cryoprotectants, which causes the cells to shrink, that gives rise to
the abnormally shaped spindles observed. This was concluded by
the fact that when human blastocysts were analysed cytoskeletally,
4 h following exposure to the cryoprotectants, but without being submerged into LN2, they had the same type of abnormally shaped spindles as those that were vitrified and submerged in LN2
(Chatzimeletiou et al., 2010 and unpublished data). It is possible
that if cells are caught at a vulnerable stage of mitosis during vitrification, spindle damage will be inevitable. Whether it is the metaphase/
anaphase stage that is more vulnerable to damage than the point of
centrosome duplication and movement to opposite poles, or any
other mitotic stage warrants further investigation.
Further analysis is also needed to establish if the morphological
change in the structure of the spindle, that is so frequently observed
in the vitrified group, has a functional effect on the way the spindle
progresses into mitosis. If this abnormality in morphology is superficial
and these abnormally shaped spindles complete mitosis normally it is
likely that the derivative cells will be normal. However, if this abnormality in the shape of the spindle is conjugated with a function abnormality, it is possible that the spindle will either fail to progress further or
if it progresses through mitosis the derivative cells may have an abnormal chromosomal constitution. This is certainly the case for the spindles that are associated with chromosome lagging, in which the
derivative cells will be affected by chromosome loss, and consequently
will become monosomic.
Although cell cycle checkpoints are thought not to operate during
cleavage before global activation of the embryonic genome, a functional spindle assembly checkpoint at the blastocyst stage, should minimize any deleterious effects by arresting mitosis until the defect is
112
corrected or by eliminating mitotically arrested cells by apoptosis
(Rieder and Palazzo, 1992; Musacchio and Hardwick, 2002). In vertebrate somatic cells and sea urchin zygotes, mitosis is prolonged
2–3-fold when monopolar spindles assemble, but not when multipolar spindle assemble, suggesting that the checkpoint control for
metaphase-anaphase transition does not monitor excess spindle
poles or bipolar spindle symmetry. (Sluder and Begg, 1983; Wang
et al., 1983; Sluder et al., 1997). Therefore tripolar and tetrapolar spindles are likely to progress, giving rise to cells with chaotic chromosomal constitutions, as the segregating chromosomes would be pulled in
three or four directions respectively.
The effects spindle abnormalities may have on the development of
human blastocysts will depend on the cell lineage they occur, the proportion of cells affected and the potential for further division. The total
number of spindles analysed per blastocyst ranged from 1 to 10 and
although several of them were in the inner cell mass (ICM) the
majority of the spindles were in the trophectoderm (TE) (Table III).
It is therefore possible that even if these abnormal spindles are functional and produce chromosomally abnormal cells, if they are located
in the TE, there will be no detrimental effect, as TE cells only give rise
to the placenta. Even in the case of abnormal spindles progressing into
mitosis in the ICM, there may be no detrimental effect if the abnormal
cells produced give rise to the extrafetal membranes. The only case in
which a detrimental effect may occur is if the abnormal cells give rise
to the fetus. However, low level mosaicim localized in specific tissues
of the fetus may remain phenotypically invisible in adulthood.
In conclusion, the high survival rate following warming (92.7%) and
the large proportion of normal spindle/chromosome configurations
observed in the human blastocysts that were vitrified in the present
study suggest that vitrification at the blastocyst stage does not
adversely affect the development of human embryos and the ability
of spindles to form and continue normal cell divisions. However,
detailed examination by confocal laser scanning microscopy revealed
a significantly higher incidence of spindle abnormalities in the vitrified
group compared with the fresh group, notably of spindles with a
focused and an unfocused pole and uniquely identified various other
abnormalities, reflecting mechanisms that can lead to chromosomal
mosaicism in early human development. Further investigation is warranted to elucidate the mitotic stages that are more vulnerable to
damage during vitrification, the fate of the abnormal spindles and
the potential effects these may have on the chromosomal constitution
of the developing blastocysts. It is suggested that vitrification is always
applied with caution to avoid any potential risks arising from abnormal
divisions of affected spindles, which if in the ICM, may lead to mosaicism and affect embryo development.
Authors’ roles
K.C., P.V. and A.H.H. designed the study. K.C., Y.P. and P.V. performed blastocyst vitrification/warming, K.C. performed antibody labelling of both fresh and vitrified blastocysts, K.C. and E.E.M. performed
analysis by confocal and fluorescence microscopy, image capturing and
statistical interpretation of the results. All authors (K.C., E.E.M., Y.P.,
P.V., N.P., Y.P., B.C.T. and A.H.H.) contributed to manuscript drafting
and critical discussion and approved of the final version of the
manuscript.
Chatzimeletiou et al.
Conflict of interest
B.C.T. has received Merck Serono Unrestricted Research grants, travel
grants and Honorarium, Merck sharp and Dohme Unrestricted
research grants, travel grants and Honorarium, BSA & Ferring Travel
grants and Honorona.
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