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com/Article/3237 on web 14 April 2008

Article
Blood clots in the cumulus–oocyte complex
predict poor oocyte quality and post-
fertilization development
Thomas Ebner PhD graduated with honours from the Paris Lodron University of Salzburg,
Austria, in 1992. He then worked on his thesis in cancer research at the Institute for
Pathology at the General Hospital, Salzburg until 1994. After a 2-year period as a researcher
in the Division of Animal Physiology, Salzburg University he began his IVF career in Linz.
His post-doctoral thesis on non-invasive selection at different stages of preimplantation
development qualified him as a university lecturer in Salzburg. He has published more than
60 papers and his research interests are non-invasive selection processes in IVF, apoptosis,
and culture media.

Dr Thomas Ebner
T Ebner1,3, M Moser1, O Shebl1, M Sommergruber1, C Yaman2, G Tews1,2
1
Landes-, Frauen- und Kinderklinik, IVF-Unit, Linz, Upper Austria, Austria; 2General Hospital, Department of Gynecology
and Obstetrics, Linz, Upper Austria, Austria
3
Correspondence: e-mail: thomas.ebner@gespag.at

Abstract
Assessment of oocyte maturity and quality (morphological appearance) at the time of retrieval is difficult as the egg is
obscured by a large cumulus mass that hinders adequate scoring. Since no data are available on the possible relationship
between the cumulus–oocyte complex (COC) and oocyte morphology, this prospective intracytoplasmic sperm injection
study was set up in 87 consecutive patients. COC were grouped according to expansion of both corona radiata and cumulus
matrix. Special emphasis was placed on recording morphological anomalies of COC (inclusion of blood clots and amorphous
clumps). For all mature ovae, quality was assessed and preimplantation development followed up to blastocyst stage if
fertilized. The risk of not harvesting an oocyte was higher in COC with blood clots compared with normal cumulus matrices
(P = 0.004). COC expansion did not allow for prediction of either nuclear status or quality of the egg. The presence of blood
clots within the cumulus matrix was associated with reduced oocyte quality (dense central granulation), fertilization rate and
blastocyst formation, compared with unaffected COC (P < 0.05). It may be postulated that COC showing blood inclusions
derive from poor quality follicles, which has a detrimental effect on oocyte quality and further cleavage to blastocyst stage.
Consequently, mechanical removal of blood clots cannot rescue the corresponding embryo.

Keywords: blastocyst formation, blood clots, central granulation, cumulus–oocyte complex, oocyte quality

Introduction
Oocyte maturity and quality at the time of retrieval are
difficult to assess as the egg is obscured by a large cumulus
mass that hinders adequate scoring. While the importance of
this is negligible in intracytoplasmic sperm injection (ICSI)
due to a preceding denudation process, it is of importance in
conventional IVF where harvested cumulus–oocyte complexes
(COC) are traditionally evaluated according to the appearance
and expansion of the corona radiata and the cumulus complex
(Veeck, 1986, 1990). Based on these criteria, oocytes within
cumulus matrix are roughly categorized as either mature
(metaphase II) or immature (prophase and metaphase I).
In detail, an expanded and luteinized cumulus complex
and a radiant corona radiata suggest completion of nuclear
maturation, while the absence of expanded cumulus or corona
cells is associated with total immaturity (prophase I). Thus, any
intermediate COC, in terms of expansion, would correlate to
metaphase I (Veeck, 1999).
However, since the early years of assisted reproduction treatment
it has been evident that assessment of maturation in stimulated
cycles is rather imprecise (Laufer et al., 1984; Hammitt et
al., 1992, 1993). The reported failure of adequate prognosis
has more recently been confirmed by Rattanachaiyanont et
al. (1999), who noted a discrepancy between the actual grade 801

© 2008 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
 Article - Blood clots in the COC predict gamete quality and subsequent development - T Ebner et al.
of the COC and the nuclear maturity of the corresponding
egg. Interestingly, some 60% of presumed immature oocytes
turned out to be at metaphase II, whereas approximately 8%
of expected metaphase II eggs did not show a first polar body
(Rattanachaiyanont et al., 1999).
In contrast to oocyte maturity, data on oocyte quality on the
basis of morphology and its possible dependence on COC
morphology are scarce. Though some studies postulated a
correlation between oocyte appearance and the morphology
of the corresponding cumulus matrix, their evidential value
is limited since fertilization rate (Ng et al., 1999), blastocyst
formation and quality (Lin et al., 2003) as well as pregnancy
rates (Ng et al., 1999) were used to retrospectively estimate
oocyte appearance. In this respect, it has to be kept in mind
that these publications exclusively dealt with IVF cases, which
usually do not require oocyte denudation.
In ICSI, however, a more explicit correlation between COC
morphology and gamete maturity and quality can be obtained
only a few hours after follicle aspiration. This is of special
interest in those COC that show certain types of anomalies,
such as blood clots (Motta et al., 1995) or amorphous clumps,
both of which could indicate suboptimal follicular maturation.
As far as is known, this is the first prospective ICSI study trying
to correlate COC morphology and the quality and maturational
status of the corresponding egg.
Materials and methods
In a 3-month study period, 87 consecutive ICSI patients were
recruited for this prospective analysis of COC morphology
and its possible prognostic value on oocyte maturity as well
as quality. The mean age of the female patients was 34.2 ± 5.2
years. Approximately half of them (n = 44) suffered from
primary infertility whereas the others had a history of at least
one biochemical or clinical pregnancy (no information on COC
morphology of previous cycles available). Though all couples
were confronted with male factor infertility, 16 couples (18%)
had a female indication as well (10 with tubal factors, four with
endometriosis, and two with polycystic ovaries). No correlation
between female indication and COC appearance could be
observed. Since this study required a slightly altered time
schedule compared with the routine procedure, Institutional
Review Board approval was sought and granted.
A total of 53 (60.9%) women were stimulated using an
antagonist protocol. Thus, human menopausal gonadotrophin
(Menogon; Ferring, Kiel, Germany) was started on day 2 of the
cycle. In addition, a gonadotrophin-releasing hormone (GnRH)
antagonist (Orgalutran; Organon, Vienna, Austria) was given
after 5–6 days of stimulation, depending on the presence of a
12–13 mm follicle in the ultrasound scan. The remaining 34
patients (39.1%) had their ovaries stimulated according to a
long protocol. Therefore, down-regulation of the pituitary was
achieved with the GnRH agonist buserelin (Suprecur; Aventis
Pharma, Vienna, Austria) and stimulation was initiated with
human menopausal gonadotrophin (Menogon).
In all patients, ovulation was induced with 5000 IU human
802 chorionic gonadotrophin (HCG, Pregnyl; Organon, Vienna,
Austria), providing that the leading follicle had reached an
appropriate diameter and serum oestradiol appeared adequate.
Follicle aspiration (aspiration vacuum of 200 mmHg) was
carried out transvaginally under ultrasound guidance 36 h
after HCG administration using a one-way follicle puncture
set (Reproline, Rheinbach, Germany) with an outer diameter
of 1.4 mm.
Immediately after oocyte retrieval, the COC collected were
transferred to Universal IVF Medium (MediCult, Copenhagen,
Denmark) and grouped according to their morphology. Instead
of evaluating the maturity of the whole complex, corona radiata
and cumulus were scored separately, essentially as reported by
Rattanachaiyanont et al. (1999); however, for ease of analysis it
was tightened when appropriate. It should be mentioned that the
optical set-up in this study did not allow for adequate counting
of cell layers (Ng et al., 1999), though no association between
this parameter and oocyte appearance has been suggested
(Rattanachaiyanont et al., 1999). According to the different
expansion patterns (dense or radiant) of both cell types (corona
and cumulus) four distinguishable grades were analysed: grade 1
(suspected mature), fluffy and radiant corona and cumulus with
visible oocyte; grade 2, dense corona (oocyte hardly visible)
but fluffy cumulus; grade 3, radiant corona (oocyte visible) but
rather dense cumulus; grade 4 (suspected immature), dense
corona and cumulus without visible oocyte (Figure 1).
In addition, special emphasis was placed on recording
morphological anomalies of COC since no ICSI data are
currently available dealing with certain COC dysmorphisms
and their potentially negative impact on the quality of the
corresponding gamete. In detail, it was planned to screen for
blood clots within the cumulus matrix (Daya et al., 1990) as
well as amorphous clumps, possibly being a sign of post-
maturity (Rattanachaiyanont et al., 1999).
Though the nature of amorphous clumps/pycnotic nuclei
is not fully understood, it can be assumed that these areas
originate from cumulus cells. However, due to their relatively
clear dissociation, it is easy to remove these anomalies using
syringe needles or hand-drawn glass pipettes for manipulation.
This is not the case for blood clots that sometimes adhere
to COC because the thin strands of blood are somewhat
interwoven with the cumulus matrix, making them more
difficult to dislodge.
Groups of COC of different grades were denuded (80 IU/
ml hyaluronidase, MediCult,) immediately after scoring
(within 30 min after collection). Attempts were made to
leave numerous cumulus cells attached during the denudation
process (Ebner et al., 2006a) in order to compensate for the
rather early manipulation, though no detrimental effect of
early denudation on outcome has been reported (Van de Velde
et al., 1998).
ICSI was performed within half an hour after denudation
according to routine technique (Ebner et al., 2001) using the 3
o’clock position for injection. Though oocyte quality (Ebner et
al., 2006b) was only checked in oocytes showing a first polar
body, attempts were made to fertilize metaphase I gametes
as well (but corresponding data were not incorporated into
the present analysis). To facilitate analysis of morphological
oocyte criteria on further outcome, these were classified as
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 Article - Blood clots in the COC predict gamete quality and subsequent development - T Ebner et al.

a b

c d

Figure 1. Grading of cumulus–oocyte complexes according to the expansion of corona radiata and cumulus matrix (×40). (a) Grade
1 (suspected mature): fluffy and radiant corona and cumulus with visible oocyte; (b) Grade 2: dense corona (oocyte hardly visible)
but fluffy cumulus; (c) Grade 3: radiant corona (oocyte visible) and rather dense cumulus; (d) Grade 4 (suspected immature): dense
corona and cumulus without visible oocyte with blood clot (arrow).

extracytoplasmic or cytoplasmic anomalies (Mikkelsen and
Lindenberg, 2001).
On the morning of day 1 (17–20 h after injection) regular
fertilization was confirmed by the presence of two pronuclei
and polar bodies. On the following days (days 2 and 3), embryos
were scored in terms of number and size of blastomeres as well
as for the presence of fragments and multinucleation, signs of
compactation (day 4) and blastocyst development (day 5). The
routine media for culture were Blastassist System Medium 1
(MediCult) until day 3 and Blastassist System Medium 2 in case
of extended culture to day 5. During in-vitro culture, oocytes
and corresponding embryos were cultured in groups according
to their COC grade. The number of 8-cell embryos on day 3 was
the key determinant for selecting day 3 (n = 63) or day 5 (n =
24) transfer. On the day of transfer, embryo/blastocyst selection
was performed exclusively on the basis of the quality of the
conceptus and not on COC morphology; thus, only a limited
number of homogeneous transfers could be performed not
allowing for adequate analysis of pregnancy data. In detail, six
patients whose clinical parameters did not differ from those of
other patients had exclusive transfers of embryos deriving from
COC with blood clots; none of them became pregnant. It should
be noted that distribution of COC grades was comparable
between patients having day 3 and day 5 transfers.
Comparisons were performed using chi-squared tests and t-
tests. Statistical significance was defined as P < 0.05. COC
showing amorphous clumps did not show any deviation from
those with normal cumulus masses, so they were pooled in
order to increase sample number.
Results
In 87 patients, a total of 632 COC (7.3 ± 6.0 per patient) were
collected. In 25 (4.0%) of these, no oocyte or only an empty
zona pellucida was found within the cumulus mass. The latter
phenomenon was found to be higher in the presence of blood
clots (Table 1) due to difficulties during preparation. 803

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 Article - Blood clots in the COC predict gamete quality and subsequent development - T Ebner et al.

The risk of harvesting no oocyte was significantly higher if blood
clots were present within the COC (P = 0.004). The oocytes
found were at metaphase II in 78.5% of the cases (n = 496), at
metaphase I in 9.3% (n = 59) and completely immature in 8.2%
(n = 52). With respect to anomalies in the cellular matrix, blood
clots were present in 97 COC (15.3%) and amorphous clumps
in 83 (13.1%).
COC morphology and oocyte quality
Table 2 indicates that oocyte morphology was not predicted
by the radiation and expansion of the corona/cumulus matrix.
This is supported by the fact that comparable rates of normal
gametes were found in the four groups analysed, which allowed
for pooling these data.

Interestingly, neither presence of blood clots (Table 1) nor
amorphous clumps were related to morphology of the COC on
the basis of cumulus and/or corona expansion, i.e. grades 1–4
had 16%, 10%, 9%, and 15% of blood clots, respectively.
The presence of blood clots or amorphous clumps was not
related to demographic and stimulation data except the
expected correlation (P = 0.0029) between the possibility
of having at least one COC with blood clots and oestradiol
concentration on the day of ovulation induction (1660 ± 1254
pg/ml oestradiol in blood clot positive cycles versus 900 ± 772
pg/ml in negative ones).
COC morphology and oocyte maturity
Of all normal COC expected to contain metaphase II gametes
(group 1) almost 90% were actually mature; but approximately
3% showed a germinal vesicle (Table 1). In the other extreme
case (group 4), an immature egg was predicted; however, only
half of the gametes showed a germinal vesicle but 25% had
already completed nuclear maturation, as indicated in Table 1.
Intermediate degrees of expansion did not differ significantly,
but COC with a fluffy corona radiata (group 3) tended to allow
for a better prediction of MII oocytes than COC with a fluffy
cumulus complex (group 2).
Focusing on the presence of anomalies and their possible
association with oocyte quality, it was found that in the group
of COC with amorphous clumps (n = 83), a similar rate of
normal oocytes was found as compared with COC without
dysmorphisms. Blood clot positive COC, on the other hand,
yielded a significantly lower number of normal oocytes as
indicated in Table 2 (P = 0.043). In detail, in these COC a
significantly higher percentage of oocytes showing dense
central granulation was found (24.3%) as compared with COC
without any anomaly (8.7%) (P < 0.001).
COC morphology and fertilization
outcome
All groups analysed showed similar fertilization rates. In
detail, 74.0%, 64.0%, 87.5% and 75.0% of the MII oocytes in
groups 1–4, respectively, showed two pronuclei. The same was
observed for MI oocytes (25.0%, 40.0%, 25.0% and 50.0%).
Table 3 indicates that MII oocytes from COC presenting blood
clots had a significant lower fertilizability compared with
oocytes from normal COC (P < 0.05).
No correlation could be observed between the four different
grades of COC and further cleavage behaviour up to day 4.
Blood clots in the COC, however, led to significantly lower
blastulation rates as compared with all other COC (Table 3).

Table 1. Relationship between cumulus–oocyte complex morphology and maturity

of the corresponding gamete evaluated within half an hour after collection.

COC n Metaphase II Metaphase I Prophase I No oocyte

morphology or empty
group zona pellucida

Group 1 421 375 (89.1) 27 (6.4) 10 (2.4) 9 (2.1)

Group 2 47 24 (51.1) 10 (21.3) 10 (21.3) 3 (6.4)
Group 3 21 15 (71.4) 4 (19.0) 1 (4.8) 1 (4.8)
Group 4 46 12 (26.1) 9 (19.6) 22 (47.8) 3 (6.5)
Groups 1–4 515 426a (82.7) 50 (9.7) 43 (8.4) 16b (3.1)
(without blood)
Groups 1–4 97 70a (72.2) 9 (9.3) 9 (9.3) 9b (9.3)
(with blood)

Values in parentheses are percentages; values with the same superscript letter are significantly different.
a
P < 0.05; bP < 0.01.

804

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Table 2. Correlation between cumulus–oocyte complex morphology and quality of the

corresponding metaphase II gamete evaluated immediately after denudation.

Gamete parameter Group 1 Group2 Group 3 Group 4 Groups 1–4

with blood

Total 375 24 15 12 70
Normal 243 (64.8) 11 (45.8) 10 (66.6) 9 (75.0) 36 (51.4)c
Cytoplasmic anomalies
sER 9 (2.4) 0 (0.0) 1 (6.7) 0 (0.0) 1 (1.4)
Vacuoles 22 (5.9) 2 (8.3) 3 (20) 0 (0.0) 4 (5.7)
Central granulation 31 (8.3) 5 (20.8) 0 (0.0) 1 (8.3) 17 (24.3)d
Incorporationsa 43 (11.5) 4 (16.7) 1 (6.7) 0 (0.0) 3 (4.3)
Extracytoplasmic anomalies
Ovoid shape 8 (2.1) 1 (4.2) 0 (0.0) 1 (8.3) 2 (2.9)
Discolouration 11 (2.9) 0 (0.0) 0 (0.0) 1 (8.3) 4 (5.7)
PVS anomaliesb 8 (2.1) 1 (4.2) 0 (0.0) 0 (0.0) 3 (4.3)

Values in parentheses are percentages; PVS = perivitelline space; sER = aggregation of smooth endoplasmic reticulum.
a
Incorporations include refractile bodies; bPVS anomalies include giant first polar bodies; cP < 0.05 compared with pooled
groups 1–4 without blood clots; dP < 0.001 compared with pooled groups 1–4 without blood clots.

Table 3. Preimplantation development of cumulus–oocyte complexes (COC)

with or without blood clots.

Parameter COC with COC without P-value

blood clots blood clots

Total 97 535 –
MII oocytes 70 (72.2) 426 (79.6) NS
2 pronuclei 45 (64.3) 323 (75.8) 0.041
Cleavage 45 (64.3) 313 (73.5) NS
Good-quality day 2 embryos 19/45 (42.2) 153/313 (48.9) NS
Good-quality day 3 embryos 21/45 (46.7) 154/314 (49.0) NS
No. of blastocyst cultures 30 178 –
Compacting day 4 10/30 (33.3) 66/178 (37.1) NS
Blastocyst formation day 5 7/30 (23.3) 78/178 (43.8) 0.035

Values in parentheses are percentages; MII = metaphase II; NS = not statistically significant.

Discussion
Within the cumulus oophorus, which protrudes into the
follicular cavity, the oocyte is located surrounded by the corona
radiata, the innermost layers of cumulus cells. Maturation and
growth of the egg is dependent on the nurturing capacity of
the follicle, especially of the cumulus granulosa cells. In this
respect, bidirectional communication between gamete and
somatic cells occurs via paracrine and gap-junctional signalling
(for reviews, see Canipari, 2000; Sutton et al., 2003).
The latter mode is facilitated since highly specialized cells
of the corona radiata have cytoplasmic processes penetrating
the zona pellucida and reaching the oolemma. At the points of
contact, a network of gap junctions unites the corona cells with
the ovum as a structural and functional ‘syncytium’, which is
essential for gamete growth (Eppig, 1977). The rate of growth
is likely to be directly related to the number of coupled somatic
cumulus cells (Brower and Schultz, 1982) and follicle size
(Han et al., 2006).
Since the vast majority of trans-zonal processes withdraw
successively around the time of resumption of meiosis (induced
by the preovulatory surge of gonadotrophins), the light-
microscopical appearance (Goud et al., 1998; Sato et al., 2007)
changes from a rather compact corona radiata to a less dense
one. In parallel, considerable production of hyaluronic acid
causes further dispersion of cumulus cells resulting in COC 805

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growth as nuclear maturation proceeds (Eppig, 1981; Chen et
al., 1990; Han et al., 2006).
While it is suspected that in natural cycles the pattern of changes
in COC closely parallels the corresponding meiotic maturation
of the egg (Ng et al., 1999), stimulated cycles show varying
degrees of asynchrony between COC appearance and actual
oocyte maturity (Laufer et al., 1984; Hammitt et al., 1992,
1993; Rattanachaiyanont et al., 1999).
Since morphological changes and proliferation of cells of the
cumulus oophorus were seen to differ between patients and
between sibling oocytes of the same cohort (Gregory et al.,
1994) a variety of scoring systems for COC have been introduced
(Veeck, 1986; Wolf, 1988; Daya et al., 1990; Ng et al., 1999;
Rattanachaiyanont et al., 1999; Lin et al., 2003; Balaban and
Urman, 2006) in order to predict the maturational stage of the
corresponding oocyte for proper timing of insemination. All of
them dealt with the individual expansion of the cumulus mass
and/or corona radiata, but only few took into consideration the
possible negative effect of morphological COC deviations, e.g.
presence of blood clots or amorphous clumps (Hyttel et al.,
1986; Daya et al., 1990; Rattanachaiyanont et al., 1999).
The present modified approach to grade COC according to the
expansion of the corona and cumulus turned out to be insufficient
in terms of predicting nuclear status of the corresponding
gamete. This is in line with literature (Laufer et al., 1984;
Hammitt et al., 1992, 1993); however, the present data provide
the first evidence that adequate expansion of the corona radiata,
i.e. oocyte visible within the COC (groups 1 and 3), is superior
in predicting nuclear maturation of the egg compared with the
appearance of the cumulus (groups 2 and 4). This supports a
suspected correlation between corona appearance and cleavage
behaviour (Daya et al., 1990).
Early denudation allowed for a precise assessment of oocyte
appearance immediately after retrieval (36 h after HCG).
It should be noted that COC expansion did not allow for
prediction of oocyte quality based on morphology, indicating
that maturational processes of both gamete and somatic cells
are not automatically synchronized. It has been suggested
that this asynchrony could be due to a different sensitivity
of cumulus cells and oocytes to gonadotrophin-induced
maturation or to other intrafollicular factors (Laufer et al.,
1984; Bar-Ami et al., 1989).
Interestingly, those oocytes derived from COC showing distinct
blood clots (but not with amorphous clumps) had a significantly
higher rate of changes in cytoplasmic texture (Kahraman et al.,
2000). This might be the reason for the present reduction in
rates of fertilization and blastulation as compared with ovae
from COC without blood clots. These observed declines are
perfectly consistent with the hypothesis of Van Blerkom and
Henry (1992) who suggested that anomalies developing early
in maturation (e.g. extensive central granulation) may be
associated with failed fertilization and aneuploidy (not analysed
in this study).
Any causal connection between blood clots and oocyte
quality, however, would indicate that blood found within the
cumulus mass is not necessarily an artefact caused by the
806 ovarian puncture (Daya et al., 1990). This is further supported
by the unpublished observation that a minor proportion of
COC showing blood clots had actually been collected from
follicular fluid contaminated by blood and the fact that red
blood cells have also been reported in COC harvested from
oviducts of unstimulated women (Motta et al., 1995). As the
mentioned paper (Daya et al., 1990) was published more than
25 years ago, it could indeed be that follicle puncture sets used
in those days were more invasive than nowadays. At least,
there is evidence that technical details of aspiration procedure
can change COC morphology (Bols et al., 1996, 1997). As a
matter of fact, the observed (Daya et al., 1990) proportion of
COC with blood inclusions (21%) was much higher than in
the present study (15%).
Apart from a suspected blocking function of blood clots
preventing direct contact between spermatozoa and the zona
pellucida (not relevant in ICSI cycles), Daya et al. (1990)
blamed shifts in pH and temperature for the observed reduction
in fertilization and cleavage. In addition, some negative impact
could also be attributed to the production of reactive oxygen
species induced by exogenous factors, e.g. blood components
(Attaran et al., 2000; Guérin et al., 2001). Based on the present
data, the presence of blood clots were not associated with
atretic or luteinized follicles (no mucified COC observed) since
demographic (low LH on the day of ovulation induction) and
stimulation data were similar in all patients.
To conclude, it may be postulated that COC showing blood
inclusions derive from poor quality follicles that have a
detrimental effect on oocyte quality and further cleavage to
blastocyst stage. It is likely that these gametes are already harmed
during folliculogenesis and, thus, their developmental capacity
may not be retained by mechanical removal of blood clots.
Based on preliminary implantation data, with no pregnancies
after exclusive transfer of embryos/blastocysts deriving from
COC showing blood clots, it may further be postulated that
the implantation potential of such concepti could be impaired.
Consequently, it can be suggested that affected COC should be
cultured separately and corresponding embryos should only be
transferred if no others of comparable quality are available.
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Declaration: The authors report no financial or commercial
conflicts of interest.
Received 11 October 2007; referred 16 November 2007; accepted 18
January 2008.
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