Human Reproduction, Vol.26, No.3 pp. 535–544, 2011
Advanced Access publication on January 12, 2011 doi:10.1093/humrep/deq376
ORIGINAL ARTICLE Embryology
Efficiency of polarized microscopy as a
predictive tool for human oocyte
quality
B. Heindryckx *, S. De Gheselle, S. Lierman, J. Gerris, and P. De Sutter
Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium
*Correspondence address. Tel: +32-9-332-4748; Fax: +32-9-332-4972; E-mail: [email protected]
Submitted on September 1, 2010; resubmitted on October 29, 2010; accepted on December 3, 2010
background: Conflicting results have been reported regarding the use of polarized microscopy as a predictive tool for human oocyte
quality.
methods: Oocytes from 121 ICSI cycles were analysed with polarized microscopy. Both qualitative (spindle presence) and quantitative
(retardance) data were correlated to the key assisted reproduction technology outcome parameters. Second, polarized microscopy was
applied on in vitro matured (IVM) oocytes from germinal vesicle oocytes that matured after 24 or 48 h and from metaphase I oocytes
matured after 3 or 24 h. These data were correlated with confocal analysis of spindle-chromosome complex.
results: Spindles were detected in 82% of in vivo matured oocytes and in 64% adjacent to the first polar body (PB). Fertilization rate
was higher in oocytes with a visible spindle (P ¼ 0.0002). In patients aged over 35 years, the percentage of a visible spindle and mean
spindle retardance was lower than in younger patients (P , 0.03). A higher number of spindles were located adjacent to the first PB in IVM
matured oocytes (94%) versus in vivo matured oocytes (P , 0.0001). Confocal imaging revealed that spindle absent IVM metaphase II (MII)
oocytes had a higher degree of aberrant spindle and chromosomal configurations versus IVM MII oocytes with a visible spindle (P ¼ 0.002).
conclusions: Oocytes with absent spindles were associated with lower fertilization rates and advanced female age. Other important
outcome parameters (embryo quality, pregnancy rates) were not correlated to spindle nor zona inner layer analysis. Interestingly, confocal
imaging showed that polarized microscopy might be used as a qualitative predictive tool of human oocyte quality but no correlation could
be demonstrated with quantitative polarized microscopy.
Key words: in vitro maturation / oocyte quality / polarized microscopy / predictive tool / spindle
Introduction
The number of collected oocytes that are intrinsically capable of producing an embryo with full developmental potential is limited. It is well
known that over 80% of the detected aneuploidies in embryos originate from meiotic errors in the oocyte (Hassold and Hunt, 2001).
Current oocyte morphology scoring systems, such as the size of the
polar body (PB), cytoplasm granularity, extra- and intra-cellular
oocyte dysmorphisms, zona pellucida thickness and expanded perivitelline space, lack a strong predictive potential of implantation
(Balaban and Urman, 2006; Yakin et al., 2007). Therefore, noninvasive assessment of oocyte quality could help to increasing
success rates after assisted reproduction technology (ART). The introduction of a novel polarized microscopy system coupled with imageprocessing software has allowed the visualization of the meiotic
spindle and the different layers of the zona pellucida in human
oocytes on the basis of birefringence in a non-destructive way
(Keefe et al., 2003; Montag and van der Ven, 2008). Birefringence
refers to an inherent optical property of highly ordered molecules
such as microtubules in spindles or the oriented filamentous glycoproteins that form the tri-laminar zona pellucida. Once birefringent
structures are illuminated using polarized microscopy, they can be
visualized as bright structures, while the light beams are differentially
slowed down which can be measured as retardance (nm) in a quantitative manner by a computerized image-analysis system (Keefe et al.,
2003).
Conflicting results have been reported regarding the predictive
value of spindle or zona analysis of human oocytes by polarized
microscopy in ICSI cycles. Some studies showed higher fertilization
(Wang et al., 2001; Rienzi et al., 2003; Cohen et al., 2004; Shen
et al., 2006; Rama Raju et al., 2007; Madaschi et al., 2008; Woodward
et al., 2008), higher embryonic quality (Moon et al., 2003; Shen et al.,
2006; Rama Raju et al., 2007), better blastocyst-formation rates
(Wang et al., 2001; Rama Raju et al., 2007) and higher pregnancy
& The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email:
[email protected]
536
rates (Shen et al., 2006; Rama Raju et al., 2007; Madaschi et al., 2008)
for oocytes with a visible spindle or a given retardance of the spindle
or inner zona layer, whereas others did not show a difference in all or
some of these outcome parameters (Moon et al., 2003; Cohen et al.,
2004; Chamayou et al., 2006; Woodward et al., 2008).
The oocyte spindle is crucial for ensuring proper homologous
chromosome and sister chromatid segregation during oocyte meiotic
transitions. Spindle disturbances can result from intrinsic factors, as
reported by Battaglia et al. (1996), who showed significantly more
abnormal spindles in oocytes from women aged 40 –45 years (79%
abnormal) compared with women aged 20–25 years (17% abnormal).
Inadequate culture conditions may represent an extrinsic source of
spindle damage as spindle structures are highly temperature-sensitive,
and small drops in temperature can cause microtubule depolymerization
(Pickering et al., 1990). In this way, non-invasive spindle imaging using
polarized microscopy may also be used as a quality control tool in an
IVF setting, as IVF requires strict temperature and pH control (Montag
and van der Ven, 2008). Detailed assessment of the metaphase II
(MII) spindle quality in oocytes is classically performed by immunofluorescence microscopy, making the microtubules and chromosome alignment visible. However, this method causes oocyte lysis during
fixation, rendering it unusable as a predictive tool for oocyte quality in
an IVF setting (Coticchio et al., 2004; Borini et al., 2005). Obviously,
high-performance confocal microscopy allows for a more accurate
determination of spindle normalcy and is able to detect even minor chromosomal disorders, in contrast to non-invasive polarized microscopy;
the correlation between results obtained using these two techniques
has not been properly investigated yet.
A possible explanation for contradictory data about the presence of
the spindle as well as its predictive potential for oocyte quality is its
dynamic nature during oocyte maturation, which may account for
the reported absence of spindles in in vivo matured oocytes (Montag
et al., 2006). Another subject of controversy is whether the deviation
of the spindle relative to the first PB is correlated to several embryological outcomes and what causes this deviation (Rienzi et al., 2005).
Detailed comparisons of a large number of in vivo and IVM oocytes
within the same study will help us to clarify these matters. It is generally accepted that the developmental potential of IVM human oocytes
from stimulated cycles is inferior to their in vivo matured counterparts
(Heindryckx et al., 2009). The most abundant source of human
oocytes for research includes the supernumerary germinal vesicle
(GV) or metaphase I (MI) oocytes following infertility treatment. It
would be of interest to investigate whether both qualitative (spindle
presence) and/or quantitative (spindle and inner layer zona retardance) polarized microscopy is able to distinguish between the
quality of both GV and MI oocytes. A few studies have used polarized
microscopy imaging on IVM oocytes but none of them have specifically
compared GV with MI oocytes and their in vivo matured counterparts
within the same study.
The first objective of the present study was to assess the predictive
capacity of spindle presence in in vivo matured oocytes in patients
undergoing an ICSI cycle: spindle presence was correlated with
maternal age, fertilization, embryo quality and pregnancy outcome.
Additionally, quantitative polarized microscopy of the spindle and
zona inner layer was correlated with the above outcome parameters.
In a second step, both qualitative (spindle presence) and quantitative
(retardance) polarized microscopy analysis was applied to IVM
Heindryckx et al.
oocytes of GV or MI origin, to better understand the dynamic behaviour of spindles during oocyte maturation.
Materials and Methods
Source of oocytes
Approval was obtained from the Ethical Committee of the Ghent University Hospital to use polarized microscopy on in vivo matured and donated
IVM oocytes (of GV or MI origin) after obtaining written informed consent.
Oocyte handling and spindle visualization in
in vivo matured oocytes
A total of 121 ICSI cycles with a mean (+SD) female age of 34.2 + 4.9
years were included. Two to three hours after oocyte collection,
cumulus cells were removed using 80 IU/ml hyaluronidase (Irvine Scientific, Santa Ana, CA, USA) and mechanical pipetting, and oocytes were
assessed for maturation either at (i) GV stage, with an intact GV; (ii) MI
stage, showing absence of a GV or first PB and (iii) MII stage, with the presence of the first PB. Oocytes were maintained in Cook Fertilization
Medium (Cook Ireland Ltd., Ireland) at 378C in 6% CO2 and air atmosphere until further processing. For polarized microscopy (Oosight
Imaging System, CRI Inc., Woburn, MA, USA), only oocytes in MII stage
at denudation were incorporated in the data analysis. Oocytes were
placed individually into 5-ml drops of Gamete buffer (Cook) in glassbottom dishes (Willco Wells, Amsterdam, The Netherlands) 3 – 4 h
after denudation on a heated microscope stage (Tokaihit, Shizuoka-ken,
Japan). In order to position the oocyte spindle perpendicular to the light
path (Shen et al., 2006), a partial zona dissection needle was used to
rotate the oocyte. Several rotation attempts were carried out and pictures
were taken at ×400 magnification and subsequently saved for further
analysis. Spindle position relative to the first PB (defined as being at 6
o’clock) was assessed using an analogue clock face as an approximation.
When spindles were detectable, retardance was measured with the automatic tool provided by the software (Oosight Imaging System). In other
cases, manual drawing around the spindle was carried out according to
the manufacturers’ instructions. The same procedure was used to
measure the birefringence quantitatively from the inner layers of the
zona pellucida, with the exception that when the automatic tool was
not accurately enclosing the full inner layer of the zona pellucida, four
regions of the zona pellucida were measured followed by a mean retardance. In some of the analysed oocytes, we verified whether the angle
of the spindle relative to the light path had an influence on quantitative
measurements of birefringence, and whether the automatic tool measurement differed from manual measuring. To assess the predictive capacity of
polarized microscopy, the following parameters were analysed: (i) spindle
visibility; (ii) spindle location relative to the first PB; (iii) correlation
between the presence or absence of the spindle and fertilization rate,
embryo quality, freezing utilization rate (number of embryos that could
be frozen) and pregnancy rate; (iv) comparison of mean spindle and
zona retardances in oocytes with or without a detectable spindle; (v) comparison of mean spindle and zona retardances in oocytes from women of
different age groups (,30, 30 –35, .35 years) and (vi) spindle visibility
and mean spindle and zona retardances in conception versus nonconception cycles.
Fertilization, embryo quality and embryo
transfer
ICSI was performed with the first PB located at 6 o’clock, irrespective of
the spindle position. Injected oocytes were cultured in Cook Cleavage
537
Predictive capacity of polarized microscopy
medium under 6% CO2, at 378C in air. Fertilization was assessed between
16 and 18 h post-ICSI and oocytes showing two distinct pronuclei (PN)
with extrusion of the second PB were considered to be fertilized normally.
Embryo quality was evaluated on Day 2 after ICSI and the following parameters were recorded: number of blastomeres, percentage of fragmentation, variation in blastomere symmetry and the presence of
multinucleation. Accordingly, the embryos were subdivided into three
main categories: (i) top-quality embryos, (ii) good-quality embryos and
(iii) poor-quality embryos (Laverge et al., 2001). Embryo transfer was
carried out on Day 2 or 3, and pregnancy rates were defined as the
number of clinical pregnancies (gestational sac with foetal heartbeat at
6 – 7 weeks) per fresh cycle.
IVM and spindle imaging
Stimulation was performed with the short GnRH agonist protocol for 7
days (Decapeptylw, Ipsen, Paris, France) and hMG (Menopurw, Ferring,
Saint-Prex, Switzerland) or recombinant FSH (Puregonw, Organon, Oss,
The Netherlands or Gonal-Fw, Merck-Serono, Geneva, Switzerland)
until hCG administration (Pregnylw, Organon, Oss, The Netherlands)
when at least half of the follicles were 18– 20 mm in diameter.
Cumulus – oocyte complexes were collected by ultrasound-guided transvaginal aspiration 36 h post-hCG, donated immature oocytes (GV and MI)
originated from these stimulated ICSI cycles and were cultured in vitro.
GV oocytes were matured in TCM 199 supplemented with 10 ng/ml
epidermal growth factor, 1 mg/ml oestradiol, 75 mIU/ml FSH (Puregon,
Organon, The Netherlands), 0.5 IU/ml hCG (Pregnyl, Organon), 1 mM
L-glutamine, 0.3 mM sodium pyruvate, 0.8% human serum albumin
(Belgian Red Cross, Brussels, Belgium) and antibiotics. Maturation was
evaluated after 24 and 48 h of in vitro culture. MI oocytes were collected
from patients who had at least six MII oocytes available for ICSI treatment
and cultured in vitro in Cook Fertilization Medium for 3 or 24 h. Spindle
detection and quantitative polarized microscopy analysis of IVM MII-stage
oocytes was performed as for in vivo matured oocytes. In order to assess
spindle dynamics during meiotic progression, a distinction was made
between GV oocytes that either matured after 24 h (GV-24 h) or 48 h
(GV-48 h) and MI oocytes that progressed to MII stage after 3 h of in
vitro culture (MI-3 h) or 24 h (MI-24 h).
Confocal imaging of IVM oocytes
Immediately after polarized microscopy analysis, IVM MII oocytes were
simultaneously fixed and extracted in a microtubule-stabilizing buffer, as
described elsewhere (Mattson and Albertini, 1990). To visualize microtubules, oocytes were incubated overnight at 48C in the presence of a 1:1
mixture of mouse monoclonal anti-a, b-tubulin, followed by Alexa Fluor
conjugated goat-anti-mouse immunoglobulin (Molecular Probes, Eugene,
OR, USA) for 2 h at 378C. Chromatin was stained with ethidium
homodimer-2 (Molecular Probes) for 1 h at 378C. Labelled oocytes
were mounted on microscope slides in 90% glycerol-phosphate-buffered
saline solution containing 0.2% 1, 4-diazabicyclo [2.2.2] octane as an antifading reagent. Preparations were observed using a laser scanning confocal
microscope (Biorad Radiance 2000 mounted on a Nikon inverted microscope, Tokyo, Japan) equipped with an Argon-ion/Helium Neon (488/
543) laser and selective filter sets for Alexa Fluor 488 and ethidium
homodimer-2. Images were obtained using a 60× plan oil immersion
objective. A three-dimensional image of the microtubular structure and
chromosomes was generated from the collected data by using ImageJ software. Z-axis stacks and three-dimensional reconstructions were obtained
by using 0.5-to 0.75-mm steps. This allowed a detailed analysis of spindle
shape and chromosome alignment according to three categories: (i)
bipolar spindle formation with chromosomes aligned on the equatorial
metaphase plate, (ii) bipolar spindle with non-aligned or dispersed
chromosomes and (iii) disorganized spindles with dispersed or chaotic
chromosome configuration. Data from confocal analysis were correlated
to spindle visibility, and to spindle and inner zona layer retardances.
Statistical analysis
All data were analysed by contingency table analysis followed by chi-square
for independence. The level of significance was set at P ≤ 0.05. Mean
retardance values were analysed using one-way analysis of variance followed by Tukey post test when the level of significance reached P ≤ 0.05.
Results
Assessment of spindle presence and
correlation with ICSI outcome
Spindles were detected in 82% of the injected in vivo matured MII
oocytes (Table I). When giant oocytes were present, two spindles
were always observed located adjacent to their respective two PBs
(Fig. 1). After ICSI, the two PN fertilization rate was significantly
higher in the oocytes with a visible spindle (78.2%) compared with
oocytes without detectable spindle (65.2%; P ¼ 0.0002). Of the
former, 64% of the in vivo matured oocytes showed the spindle
located directly adjacent to the first PB (between 5.30 and 6.30
o’clock if the first PB is considered at 6 o’clock position). Embryo
developmental rates, based on Day-2 scoring and embryo utilization
rates, were comparable between oocytes with or without a spindle
(Table I). When comparing conception and non-conception cycles,
82.4% of the transferred oocytes possessed a visible spindle, which
was similar to the non-conception cycles (81.8%) (Table II). Regarding
age, oocytes from patients under 30 years (85.3%) and from those
aged 30 –35 years (81.6%) contained a significantly higher rate of
visible spindles compared with older women (.35 years, 74.1%;
P , 0.03).
Table I Presence of a spindle in in vivo matured
oocytes from 121 ICSI cycles, and correlation with key
parameters
Parameter
Spindle
present n
(%)
Spindle
absent n
(%)
Significance
P
........................................................................................
Injected MII
oocytes (n ¼ 1089)
891 (81.8)
198 (18.2)
NA
Fertilization rate
697 (78.2)
129 (65.2)
0.0002
Top
214 (30.7)
35 (27.1)
NS
Good
213 (30.6)
39 (30.2)
NS
Poor
269 (38.6)
55 (42.7)
NS
Number of
embryos used for
transfer
173 (24.8)
38 (29.5)
NS
Number of
embryos frozen
244 (35)
37 (28.7)
NS
Embryo quality
MII, metaphase II.
538
Heindryckx et al.
Quantitative polarized microscopy in ICSI
cycles
zona retardances were comparable between the three different age
categories.
Measurement of the spindle and inner layer of the zona pellucida was
taken both manually and automatically (Fig. 2) and the results did not
differ (data not shown). It must be taken into account that minimal
rotations of the oocyte to place the spindle as close as possible to perpendicular to the light path results in different spindle retardances
(Fig. 3). To avoid a long-time exposure to suboptimal environmental
conditions outside the incubator, only one picture per oocyte was
captured for further quantitative analysis. Quantitative analysis of
spindle and zona retardances from transferred oocytes (n ¼ 211) in
conception versus non-conception cycles showed no differences
(Table II). Maternal age and the number of transferred embryos
were comparable in both groups. No correlation could be demonstrated between mean spindle and zona inner layer retardances and
embryo quality on Day 2. With respect to the different age categories,
the mean spindle retardance of patients over 35 years (1.51 + 0.4)
was lower than patients ,30 years (1.62 + 0.4; P ¼ 0.006), while
Spindle presence, quantitative polarized
microscopy versus confocal imaging in IVM
oocytes
Figure 1 Giant human oocyte showing two birefringent spindles
(arrow) adjacent to their two respective PBs.
For the IVM MII oocytes, spindles were present, absent or in telophase I (Table III). In GV oocytes that matured after 48 h and MI
oocytes that developed to MII after 24 h, more detectable spindles
were observed compared with the other groups (P , 0.0001). Alternatively, a higher proportion of MI oocytes that matured after 3 h contained spindles in telophase I. Of the total of 178 IVM MII oocytes, 166
oocytes showed the spindle directly adjacent to the first PB (93%),
which is higher than in vivo matured oocytes (P , 0.0001). A quantitative polarized microscopy analysis revealed higher spindle and zona
retardances in GV-24 h and GV-48 h compared with MI-24 h group
of oocytes (Table III). The highest spindle retardance was observed
in the MI-3 h group of IVM oocytes. This can be explained by the
very high spindle retardance of telophase oocytes (2.96 + 0.77),
which were a major part of these MI-3 h oocytes (29%). All spindle
and zona retardances of IVM MII oocytes were significantly higher
than in vivo matured oocytes, except for the MI-24 h IVM oocytes.
Detailed assessment of spindle configuration and chromosomal alignment in the different groups of IVM oocytes is presented in Table IV.
Owing to the smaller sample size and the many categories of spindle
configuration, we can only observe trends in the different groups of
IVM oocytes. GV oocytes matured for 48 h showing a visible spindle
tended to have more oocytes in the normal spindle and chromosome
configuration (category A, Fig. 4A). It was confirmed by confocal
imaging that a major group of MI oocytes that matured after 3 h contained telophase I spindles (Fig. 4D). Overall, IVM oocytes without a
visible spindle had significantly fewer spindle/chromosome configurations of category A compared with IVM MII oocytes with a detectable spindle (P ¼ 0.002). Only in oocytes without a detectable
spindle, did we observe abnormal spindle formations and chaotic
chromosome alignment of category C (Fig. 4C) in contrast to IVM
MII oocytes possessing a visible spindle. There was no correlation
between quantitative measurements of spindles and zona pellucida
inner layers and the respective categories of spindle/chromosome
configurations.
Table II Comparison of ICSI cycles: conception versus non-conception cycles.
Conception cycles
Non-conception cycle
Significance
.............................................................................................................................................................................................
No. of patients
42
79
Maternal age
32.5 + 4.7
31.9 + 4.5
NS
Mean no. of oocytes per patient
10.8 + 5.1
10.3 + 4.9
NS
No. fertilized (%)
299 (77.9)
526 (74.6)
NS
No. of oocytes transferred
74
137
NS
No. of oocytes with visible spindle (%)
61 (82.4)
112 (81.8)
NS
Mean number of embryos/patient
1.8 + 0.8
1.7 + 0.9
NS
Retardance spindle (mean + SD)
1.48 + 0.4
1.51 + 0.4
NS
Retardance zona (mean + SD)
1.85 + 0.5
1.91 + 0.6
NS
Predictive capacity of polarized microscopy
539
Discussion
The non-invasive selection of good-quality gametes and embryos has
gained increasing interest during recent years in order to increase ART
efficiency and reducing the need for multiple embryo transfers. For
non-invasive oocyte selection, polarized microscopy was demonstrated to be highly predictive of human oocyte quality but conflicting
results have been reported (Petersen et al., 2009). In the present
study, the predictive potential of polarized microscopy was verified
in in vivo matured oocytes; we also investigated whether the analysis
of IVM oocytes would clarify some of the existing conflicts in results
using polarized microscopy, and we compared polarized microscopy
with confocal imaging to asses its predictive capacity.
Figure 2 Mean retardance of spindle and zona pellucida inner layer
within the same oocyte using automatic (A) and manual (B) measurement. Spindle and inner layer retardance were 2.73 and 3.31 (A) and
2.80 and 3.38 (B), respectively.
Figure 3 Three captured images from the same oocyte but after
different small rotations of the oocyte relative to the light path:
respective spindle retardances are measured using the automatic
tool function.
540
Heindryckx et al.
Table III Spindle imaging and quantitative polarized microscopy in IVM MII oocytes.
IVM
oocyte
n
Spindle present n
(%)
Spindle
absent
n (%)
Telophase
n (%)
Mean retardance spindle
(nm)
Mean retardance zona inner
layer (nm)
.............................................................................................................................................................................................
57
36 (63)b
GV-48 h
47
a
MI-3 h
28
6 (21)c
46
a
GV-24 h
MI-24 h
16 (28)
42 (89)
3 (6)
14 (50)
43 (93)
3 (7)
5 (9)a
1.98 + 0.56a
2.57 + 0.41a
a
b
2 (4)
1.87 + 0.43
2.73 + 0.62b
8 (29)b
2.47 + 0.91a,b,c
2.46 + 0.46
0
a
a,b,c
1.64 + 0.44
2.18 + 0.47a,b
Data with the same superscripts are significantly different from each other (P , 0.05).
GV, germinal vesicle; MI, metaphase I.
Table IV Correlation between spindle visualization and
confocal microscopy classified according to the
categories: (A) bipolar spindle, normal chromosome
alignment; (B) bipolar spindle, chromosomal nonalignment, (C) disorganized spindle, chromosomal
non-alignment or dispersed chromosomes and (D)
telophase I spindle.
a
Spindle and chromosome
configuration
(A)
(B)
(C)
(D)
........................................................................................
GV-24 h (n ¼ 25)
2
2
5
3
0
+
7
5
0
3
2
0
2
0
0
+
14
9
0
1
2
0
3
0
0
+
8
13
0
0
2
0
5
4
0
+
0
0
0
8
15
7a
0
27
0a
12
GV-48 h (n ¼ 26)
MI-24 h (n ¼ 24)
MI-3 h (n ¼ 17)
Total IVM MII oocytes (n ¼ 92)
2 (n ¼ 24)
+ (n ¼ 68)
2a
a
29
Data with equal superscripts are significantly different from each other (P , 0.05).
a
+ denotes presence of a spindle, 2 denotes absence of a spindle.
The percentage of visible spindles in in vivo human matured oocytes
is reported to range from 63% (Madaschi et al., 2008) to 91% (Rienzi
et al., 2003). It was suggested that these differences might be
explained by variation in the patient population and ovarian stimulation
protocols, or an inefficient culture environment, such as incorrect
temperature or pH control during spindle visualization (Wang and
Keefe, 2002a; Cooke et al., 2003). Still, nowadays IVF laboratories
are fully equipped to ensure stable environmental conditions. Most
plausibly, this high range of spindle visibility is a result of the way
that spindles are visualized. Rienzi et al. (2004) reported that rotation
of the oocytes improves the detection of spindles, even those which
are not very bright. We used micromanipulation tools to rotate the
oocyte, that resulted in 82% of the MII oocytes showing a visible
spindle.
In most reports, MII oocytes with a visible spindle during ICSI
showed significantly higher fertilization rates as demonstrated by a
recent meta-analysis (Petersen et al., 2009) and confirmed in this
study. Contradictory observations have been reported regarding the
spindle presence in oocytes and subsequent embryo quality.
Oocytes with a visible spindle produced significantly more goodquality embryos on Day 3 or Day 5 (Wang et al., 2001; Moon
et al., 2003; Shen et al., 2006; Rama Raju et al., 2007) than oocytes
where the spindle was not visible, while others did not find this correlation (Cohen et al., 2004; Woodward et al., 2008). These conflicting results may be influenced by the wide variety in scoring systems for
embryo quality between IVF laboratories (Woodward et al., 2008) or
the difference in embryo scoring day that was used. As we evaluated
embryo quality only at Day 2, it is difficult to compare our data with
the above studies.
A correlation between pregnancy outcome and the presence of
spindles in oocytes has only been addressed in two studies, and
these gave contradictory results. In one study, no relationship was
found (Chamayou et al., 2006) while the other showed significantly
higher pregnancy and implantation rates when only embryos originating from oocytes with a detectable spindle were transferred (Madaschi
et al., 2008). Still the number of patients analysed in the latter study
was very low and meta-analysis confirmed the absence of a correlation
(Petersen et al., 2009). Furthermore, when the visibility of spindles of
transferred oocytes was compared between conception versus nonconception cycles in the present study, no correlation was found.
In order to explain the biological meaning of spindle absence in MII
oocytes, one has to take into account the dynamic nature of the
spindle during final meiotic maturation. Given that during the MI and
MII transition, the spindle completely disappears after PB extrusion
for a certain time interval (Montag et al., 2006), one could state
that the lower fertilization and embryo developmental rates observed
from oocytes without a detectable spindle are related to cytoplasmic
immaturity, indicative for inferior oocyte quality. Time-lapse studies
showed that when MI oocytes extrude the PB, the spindle forms a
connective strand between the extruded PB and the oocyte cytoplasm
for 75 –90 min (Montag et al., 2006): this is what we observed as telophase I by polarized microscopy in IVM oocytes containing very
Predictive capacity of polarized microscopy
541
Figure 4 Immunostaining for chromosomes (red) and microtubules (green) in IVM MII oocytes viewed using confocal microscopy: (A) bipolar
spindle with chromosomal alignment, (B) bipolar spindle with chromosomal non-alignment, (C) disorganised spindle showing chaotic chromosomal
dispersion, (D) telophase I spindle.
bright spindle structures. This is followed by a time of spindle depolymerization (40–60 min) after which the MII spindle reassembles
underneath the first PB, 115 –150 min after extrusion of the first PB.
In order to avoid misinterpretation of spindle detection as a result
of this kinetic behaviour during final meiotic progression, one should
first assess the nuclear maturity of oocytes and wait several hours
before spindle visualization. Consequently, oocytes with no detectable
spindle owing to inherent inferior quality can be distinguished from
oocytes with missing spindles because they are undergoing meiotic
progression. Alternatively, spindle visualizations can be repeated to
allow the spindle to form during final maturation, as reported by
Montag et al. (2006). In our study, maturity of the oocytes was
assessed 3 –4 h before spindle evaluation and only oocytes with a
visible first PB at denudation were included in the in vivo matured
oocytes. Consequently, oocytes with no visible spindle might represent inferior quality oocytes that fail to complete final meiotic progression. Data on the time-intervals between nuclear maturity
assessment and eventual polarized microscopy imaging of spindle
are mostly lacking but are important to clarify the observed differences
or the biological relevance of oocytes with no visible spindle.
The presence of a visible spindle was highly variable in our IVM MII
oocytes, depending on the GV or MI origin and the time needed for
IVM to MII stage. MI oocytes that matured after 24 h and GV
oocytes that matured after 48 h of in vitro culture contained significantly more visible spindles than MI oocytes matured after 3 h and
GV oocytes matured after 24 h. These findings reflect the kinetics
and dynamic behaviour of spindles during final meiotic progression,
as shown by Montag et al. (2006). Braga et al. (2008) reported detectable spindles in 73.8% of oocytes after 24 h of IVM, which is comparable to our overall rate of spindle detection in IVM oocytes (79.8%).
Still, it has to be taken into account that our immature IVM oocytes
originated from stimulated ICSI cycles, while in the Braga et al.
(2008) study, immature oocytes originated from controlled ovarian
stimulation (COS) cycles where the immature oocytes were recovered 3 –6 h after hCG administration. In addition, other supplements
were used for IVM in the Braga et al. (2008) study compared with our
IVM system. Of interest, IVM MI oocytes derived from non-stimulated
cycles all showed telophase I spindle within 1 h after first PB extrusion
while the spindle was located adjacent to the first PB after 1 h (Hyun
et al., 2007), which is similar to our findings for IVM MI oocytes
derived from stimulated cycles. The overall quality of IVM oocytes
from stimulated cycles is likely different from IVM oocytes originating
from non-stimulated or COS cycles as recent studies have shown that
IVM oocytes from stimulated cycles have lower embryonic
542
developmental potential and might even lack implantation potential
(Reichman et al., 2010), in agreement with our experience. In contrast,
although IVM oocytes derived from non-stimulated cycles show
mostly delayed embryonic development, they do possess reasonably
high implantation potential (Bos-Mikich et al., 2010); therefore, comparisons between these two origins of IVM oocytes might be biased.
Conflicting results have also been reported regarding the utility of
spindle location relative to the first PB. Lower fertilization rates
were reported when a spindle deviation of more than 908 relative
to the first PB was present (Rienzi et al., 2003). In contrast,
Woodward et al. (2008) found no difference in fertilization or
embryo quality when spindles were displaced from the first PB.
Rienzi et al. (2004) suggested that PB displacement accounts for
spindle relocations related to manipulations during oocyte handling,
mainly during denudation. In contrast, a study in mouse postulated
that spindle displacement involves a time-dependent process related
to a deficiency in GV oocyte maturation (Moon et al., 2003). Given
that the majority of spindles were located adjacent to the first PB in
the IVM oocytes (93%) that were denuded before PB extrusion, in
contrast to in vivo matured oocytes (64%), our data support the
theory of Rienzi et al. (2004).
Retardance values from the spindle are thought to reflect the
density of microtubules and therefore a higher degree of order of
the spindle may reflect normal spindles and thus better-quality
oocytes. Shen et al. (2006) found that high retardance spindle values
are positively correlated with a good pronuclear score as well as
with transfer cycles giving rise to a pregnancy. Two studies demonstrated a trend for an inverse relationship between spindle retardance
and advanced maternal age (De Santis et al., 2005; Shen et al., 2006),
which was confirmed in our study. A positive correlation was reported
between spindle retardance and embryonic developmental potential
at Day 3 (Trimarchi et al., 2004) and blastocyst formation rate
(Rama Raju et al., 2007). Moreover, high zona inner layer retardances
have been positively correlated with embryonic development and
even implantation rates (Montag et al., 2008). In the latter prospective
study, oocytes were classified as having a high or lower zona birefringence using the OCTAX polarized microscopy system coupled with
software that allows a fast subjective zona judgement (Octax Microscience GmbH, Altdorf, Germany). These data were confirmed in a
recent study showing a positive correlation of blastulation with pregnancy rates in oocytes where the system’s automatic detection of
the inner zona layer did not succeed (Ebner et al., 2010). An
updated version of the OCTAX system was used in the latter study
(OCTAX Polar Aide coupled with software), which generated a
single score value, mainly based on the intensity of birefringence and
the local width of the zona inner layer, resulting in a more objective
judgement. Our data did not show any positive correlation between
quantitative polarized microscopy and embryo developmental and
pregnancy rates. The polarized microscopy system that we used
measures only zona inner layer retardances, and does not take into
account other parameters, such as the width of the zona layer, and
it remains to be elucidated whether this is responsible for the lack
of predictive potential of quantitative polarized microscopy in our
study. It would therefore be of interest to test both polarized
systems in the same setting. Almost all spindle and zona retardances
of IVM MII oocytes were significantly higher than in vivo matured
oocytes in our study, although it is well known that the quality of
Heindryckx et al.
IVM oocytes, especially from stimulated cycles, is inferior when compared with their in vivo matured counterparts (Heindryckx et al.,
2009). Moreover, we have shown that a minor deviation of the
spindle angle relative to the light path can have an influence on the
absolute values of spindle retardance, which needs to be considered
when performing retardance measurements. Regarding zona analysis,
we have to accentuate that it is difficult to compare our findings with
reports that used another system of polarized microscopy. Still, in our
setting, quantitative polarized microscopy lacked a predictive capacity
of oocyte quality.
To further elucidate the predictive capacity of polarized microscopy
analysis of oocyte quality, we correlated these findings with confocal
microscopy. Wang and Keefe (2002b) were the first to investigate
this in IVM human oocytes of both GV and MI origin from stimulated
cycles, as in our study: of the 27 oocytes that matured to MII stage
after 24 h, 52% showed a visible spindle. No distinction was made
as to whether the oocytes originated from MI or GV stage. Of the
oocytes with a visible spindle, 71% showed normal chromosome alignment while IVM oocytes without a detectable spindle all showed
abnormal chromosome alignment after fixation. Recently, Coticchio
et al. (2010) verified the correlation of polarized microscopy and confocal staining in both fresh and frozen/thawed oocytes: the oocytes
were in vivo matured, fresh oocytes (n ¼ 22) that were donated for
research, and MI oocytes that matured after 3 h of in vitro culture
(n ¼ 16) donated after hyperstimulation. No correlation could be
found between retardance measurements of the spindles and the concomitant normalcy of spindle configuration and chromosome alignment after confocal analysis (Coticchio et al., 2010). These authors
concluded that the polarized microscopy might still be rather inefficient for non-invasive oocyte selection in an IVF setting. However, a
comparison of qualitative polarized microscopy and confocal staining
was made only on six oocytes. In our study, a larger number of IVM
MII oocytes without a detectable spindle possessed significantly
fewer normal bipolar spindle configurations and normal chromosome
alignment than IVM MII oocytes with a visible spindle. Interestingly,
highly abnormal disorganized spindle-chromosome complexes were
only observed in oocytes with no spindle, comparable to Wang and
Keefe (2002b). However, no correlation could be demonstrated
between either spindle or zona layer retardances and their respective
normalcy of spindle configuration and chromosome alignment after
confocal staining, similar to Coticchio et al. (2010).
Finally, we want to bring attention to one published case report
(Heindryckx et al., 2008). This patient had a history of low normal fertilization (32%) with a high rate of abnormally fertilized zygotes (50%)
showing ≥3PN in three ICSI cycle attempts. In the fourth cycle, polarized microscopy was applied and revealed only one oocyte with a
visible spindle of the 14 MII oocytes retrieved. Although fertilization
could be achieved by the application of assisted oocyte activation
(AOA) on half of the oocytes, only one oocyte with a detectable
spindle gave rise to normal two PN formation without AOA in the
remaining oocytes, while the rest showed abnormal fertilization.
In the present study, in vivo matured oocytes with no spindles were
associated with lower fertilization rates and advanced female age.
Other important outcome parameters (embryo quality, pregnancy
rates) were not correlated to spindle or zona inner layer analysis.
Polarized microscopy of IVM MII oocytes revealed important kinetics
in the process and visualisation of spindles. Interestingly, spindle
Predictive capacity of polarized microscopy
kinetics do not account for the absence of spindle detection in in vivo
matured oocytes. Polarized microscopy also revealed that spindle
deviation relative to the first PB is likely caused by PB displacement
during oocyte denudation. More importantly, confocal imaging in
IVM oocytes showed that qualitative polarized microscopy might be
used as a qualitative predictive tool for assessment of human oocyte
quality, in contrast to quantitative polarized microscopy.
Authors’ roles
B.H. was involved in study design, execution, analysing data, manuscript writing; S.D.G. framed study design, carried out polarized
microscopy, analysed the data; S.L. did confocal staining and analysed
data; J.G. drafted the manuscript and took part in the discussion.
P.D.S. framed the study design and drafed the manuscript.
Funding
P.D.S. is holder of a fundamental clinical research mandate by the
Flemish foundation of Scientific Research (FWO-Vlaanderen).
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