Human Reproduction Vol.20, No.7 pp. 1915–1920, 2005
doi:10.1093/humrep/deh883
Advance Access publication April 28, 2005
Seminal haploid cell detection by flow cytometry
in non-obstructive azoospermia: a good predictive
parameter for testicular sperm extraction
I.Koscinski1,2,6, C.Wittemer2, J.M.Rigot3, M.De Almeida4, E.Hermant5 and A.Defossez5
1
Laboratoire de Biologie de la Reproduction, Hôpital Jeanne de Flandre, 59037 Lille cedex, 2Service de Biologie de la Reproduction,
Centre d’AMP, CMCO-SIHCUS, 19 rue Louis Pasteur, 67303 Schiltigheim cedex, 3Département d’Andrologie, Hôpital Calmette,
59037 Lille cedex, 4Département d’Histologie et de Biologie de la Reproduction, Faculté de Médecine Cochin Port Royal,
Université Descartes, Paris V, 75014 Paris and 5Département d’Histologie, Faculté de Médecine, Université Warembourg, Lille 2,
59045 Lille cedex, France
6
To whom correspondence should be addressed at: Service de Biologie de la Reproduction, Centre d’AMP, CMCO-SIHCUS,
19 rue Louis Pasteur, 67303 Schiltigheim cedex. E-mail: [email protected]
BACKGROUND: Testicular sperm extraction (TESE) associated with ICSI gives patients suffering from nonobstructive azoospermia (NOA) the possibility of becoming a father. The success rate of TESE based on sperm
recovery is ,50%, and the commonly used non-invasive parameters are not predictive enough. Only the invasive
testis biopsy has a good prognostic value. The aim of this study was to evaluate the prognostic value of the detection of seminal haploid cells by flow cytometry (FCM) in order to avoid unnecessary testicular biopsy. METHODS:
For 37 NOA patients undergoing testicular biopsy, we measured testis size, serum FSH and inhibin B levels and
carried out seminal cytology, seminal FCM analysis and histological examination. RESULTS: Sperm were found
in 18 biopsies. These results were correlated with cytology, FCM analysis and the histological examination. FCM
was more sensitive than cytology (100 versus 59%) but less specific (67 versus 83.5%) whereas the histological
observation of complete spermatogenesis appeared to be less sensitive (50%) but more specific (100%).
CONCLUSION: Detection of seminal haploid cells by FCM appears to be an interesting non-invasive technique
which can predict TESE results and improve the management of NOA patients.
Key words: azoospermia/flow cytometry/testicular biopsy
Introduction
In cases of non-obstructive azoospermia (NOA), testicular
sperm extraction (TESE) followed by ICSI gives azoospermic
patients the possibility of fathering a child (Devroey et al.,
1995). However, testicular biopsy is an invasive
procedure potentially leading to complications such as haematoma, inflammation, fibrosis, and even permanent devascularization and possible androgenous deficiency (Schlegel and Su,
1997; Manning et al., 1998; Schill et al., 2003). Moreover, it
is very difficult to predict the success of TESE. Several
studies have investigated the predictive value of various noninvasive parameters: testis size, FSH serum concentration
(Tournaye et al., 1997; Amer et al., 2001), inhibin B serum
concentration (Foresta et al., 1999), seminal anti-Müllerian
hormone (AMH) level as well as seminal inhibin B level
(Anderson et al., 1998; Fenichel et al., 1999; Anderson,
2001). Besides these non-invasive parameters, testicular histopathology is currently performed (Tournaye et al., 1996,
1997) and is regarded as the best predictor for
successful TESE (Su et al., 1999) especially in cases where
spermatids are observed (Ezeh et al., 1999). However, the
invasive character of this procedure can lead to the previously
described complications and hence it is no longer used by the
majority of fertility centres. TESE success seems to be correlated with the detection of round spermatids in seminal fluid
(Amer et al., 2001), but the identification of immature germinal cells in semen using classical cytological methods is
difficult. To facilitate cytological analysis, several staining
methods have been proposed, sometimes paired with a discontinuous density gradient separation step (Angelopoulos et al.,
1997; Gandini et al., 1999; Johanisson et al., 2000; Amer
et al., 2001). Immunocytochemistry techniques using monoclonal antibodies recognizing acrosomal antigens have also
been proposed (Kurth et al., 1991; Gallo et al., 1991;
Mendoza et al., 1996) but the sensitivity and the specificity of
this method are insufficient. Flow cytometry (FCM) can analyse the size and density of cells and has been used to identify
murine round spermatids in testis (Lassalle et al., 1999). This
method uses only physical cell parameters and thus hampers
the identification of human spermatids in semen (Ziyyat et al.,
q The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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1915
I.Koscinski et al.
1999). The haploid status of spermatids can be inferred from
observation of a single spot for each of the two probes used in
fluorescence in situ hybridization (FISH) experiments
(Mendoza et al., 1996). DNA content can also be stained with
a fluorescent intercalating agent and analysed by FCM. The
intensity of the fluorescent signal is related to DNA content
and accessibility which depends on chromatin condensation
(Evenson et al., 1980; Evenson and Melamed, 1983). For
each semen cell, FCM analyses the size, the granular cytoplasmic structure, and the intensity of the fluorescence signal,
which identifies any haploid cells in semen (Spano and
Evenson, 1993). FCM analysis has already been used on
human seminal cells to study the differentiation state of germ
cells and to evaluate the most current spermatogenesis
abnormalities (Hacker-Klom et al., 1999). FCM has not yet
been applied to the analysis of NOA semen samples. The aim
of the present work was to test the value of FCM identification
of haploid semen round cells as a predictive parameter
for TESE in cases of NOA. Our goal was to avoid useless
testicular biopsies.
Materials and methods
The local ethics committee gave its consent for the use of human
products according to the needs of the study.
Patients
Thirty-seven Caucasian infertile patients were diagnosed as NOA
based on the following factors: clinical examinations, echographical
determination of testis size, multiple semen analyses, seminal
concentration of citrate, acid phosphatases, fructose and a1 – 4glucosidase, serum FSH, inhibin B (Serotec; Oxford Bio Innovation,
UK) concentrations, as well as karyotype and research of microdeletions of the Y chromosome. Patients were informed about the nature
of this study and gave their consent.
Classical cytology
Each patient presented one ejaculate intended exclusively for cytological analysis. Semen was recovered by masturbation after 3 – 4
days of sexual abstinence and collected in a sterile container. After
liquefaction, 1 h at room temperature, seminal round cells were collected by centrifugation without culture medium (10 min, 500 g).
The pellet smears were stained according to Harris – Shorr technique
and examined for the presence of elongated or round spermatids
under a light microscope at £400 magnification.
Flow cytometric analysis
Patients produced a second ejaculate (several weeks after the
first) for FCM analysis. Seminal round cells were collected by
centrifugation (10 min, 300 g) of the whole ejaculate diluted five
times in culture medium (IVFe; Vitrolife AB, Sweden) and then in
1 £ phosphate-buffered saline (PBS) (Dulbecco’s; Gibco, Invitrogen Corp., UK) at a concentration of 0.1 – 2 £ 106 cells/ml.
The final pellet was resuspended and 0.5– 1 £ 106 round cells
in 30 ml of PBS were fixed and permeabilized using the
Cytofix-Cytoperm/Cytowash kite (BD Biosciences Pharmingen,
USA) according to the manufacturer’s instructions. Round cells
(0.5– 1 £ 106) were incubated for 5 min at 4 8C in the dark in 1 ml
of PBS 1 £ propidium iodide (PI) (1.5 mg/ml).
For haploid control cells, 0.5 – 1 £ 106 sperm were selected using
a two-layer density gradient (PureSperme; Nidacon international
1916
AB, Sweden). These sperm cells were provided by patients undergoing an IVF attempt for female infertility in our centre. For diploid
control cells, 0.5– 1 £ 106 lymphocytes from healthy donors
were selected using a density gradient (Lymphocyte Separation
Mediume; Eurobio, France). Control cells were fixed, permeabilized, and stained in the same conditions as the round cells. Flow
cytometric analysis was performed on a Coulter Epic XL (Beckman
Coulter Corp., USA). The cell size (Forward angle light scatter,
FSC) and the cell density (908 light scatter, SSC) were simultaneously measured. Instrument settings were adjusted using control
cells to observe every event (cells and debris) in the dot-blot
diagram. We used a resolution of 1024 channels. FSC detector and
SSC photodiode were set in linear mode. A total of 200 000–
500 000 events were acquired. FCM simultaneously measured the PI
fluorescence of stained control cells and patients’ seminal round
cells. Before staining, control cells and patients’ round cells were
analysed to assess the absence of spontaneous fluorescence. After
staining, the instrument settings were adjusted using the control
cells in order to obtain the signal peak of haploid and diploid
control cells on the same graph. The same settings were then used
for analysing the cell samples of the patients. Fragments of cells
contaminating the seminal cell preparations did not allow either
quantification or determination of the haploid cell proportion in the
semen.
Testicular biopsy
Biopsies were usually bilateral and consisted of a single large
biopsy taken from each testis. For each side, the biopsy was divided
into two parts for sperm extraction and for histological examination
but the part reserved for histological examination was five times
smaller than that used for sperm extraction.
Testicular sperm extraction (TESE)
TESE was performed by mincing and shredding the testicular tissue
in culture medium (IVFe; Vitrolife AB, Sweden). An aliquot was
examined under an inverted microscope using £400 magnification.
If no sperm cells were found, the preparation was centrifuged at
300 g for 10 min and the pellet re-examined for the presence of
sperm. TESE was considered positive when at least one living
sperm cell was observed (this was either spontaneously motile or
was shown to be viable by the hypo-osmotic swelling test).
Histological analysis
Histopathological examination was performed on the second part of
the biopsy after fixation in Bouin’s solution and embedding in paraffin. Sections 6 mm thick were stained (Trichrome of Masson) and
examined under a light microscope at £400 magnification. Four
types of pathology were quantified: SCO: Sertoli cells only, or total
absence of germ cells; MA: maturation arrest (often at the state
of primary spermatocyte); FCS: focal complete spermatogenesis
(a complete spermatogenesis is observed only in a small proportion
of the seminiferous tubules with a focused distribution); and DS:
diffused diminution of normal spermatogenesis (a complete spermatogenesis is observed in most of the seminiferous tubules, but with a
low density of germinal cells within each tubule).
Statistical analysis
Comparisons of means were performed using a Wilcoxon test (nonparametric analysis). Comparisons of frequencies were performed
using the x2-test or the Fisher exact test if necessary. Sensitivities
and specificities were expressed with their 95% confidence intervals.
Flow cytometry analysis in non-obstructive azoospermia
Results
Patients’ mean values
The mean age of patients was 32.9 ^ 4.6 (range 23 – 42)
years. The mean volume of both testes combined was
16.1 ^ 8.1 (range 2.7 – 33.0) ml and the mean volume of the
largest testis was 8.9 ^ 4.1 (range 1.5– 17.0) ml. The mean
FSH and inhibin B serum concentration was respectively
22.9 ^ 18.6 IU/l (range 1.9 –89.4; normal range 2.0 –10.0)
and 47.5 ^ 57.4 pg/ml (range 14 – 264; normal range
285 ^ 32). For all patients, the karyotype was normal and no
microdeletion of the Y chromosome was found.
TESE results and non-invasive classical parameters
Biopsies were taken from both testes in 36 patients, and from
only the right testis in one patient (who had no left testis).
Sperm were retrieved in 18 patients (49%); from only the
largest testis in three patients (with testis size of 4, 9 and
12 ml). TESE results according to the non-invasive parameters are presented in Table I. The age of patients in the
positive or negative sperm TESE groups was similar
(31.7 ^ 4.3 for positive TESE and 34.1 ^ 4.61 for negative
TESE, not significant). No significant differences were
observed with the other parameters: volume of both testes or
of the largest testis, FSH and inhibin B levels.
Histology and TESE results
Table II presents the results of the histological examination
according to TESE results. Usually, the histological pattern
was the same in both testes. When it was not the case, the
histology reported in Table II was taken from the side of the
Table I. Testicular sperm extraction (TESE) results according to noninvasive parameters
Age (years)
Volume of both testes (ml)
Volume of the largest
testis (ml)
Serum FSH (IU/l)
Serum inhibin B (pg/ml)
Positive seminal cytology
Positive seminal FCM
analysis
Positive TESE
(n ¼ 18)
Negative TESE
(n ¼ 19)
P
31.7 ^ 4.3
15.9 ^ 9.2
8.8 ^ 4.7
34.1 ^ 4.61
16.2 ^ 7.1
8.8 ^ 3.7
NS
NS
NS
24.7 ^ 22.86
43.4 ^ 72.5
10 (n ¼ 17)
18
21.2 ^ 13.5
51.1 ^ 41.7
3 (n ¼ 18)
6
NS
NS
0.01
0.0001
FCM ¼ flow cytometry; NS ¼ not significant.
Table II. Testicular sperm extraction (TESE) results according to
histopathological analysis
Positive TESE
Sertoli cell-only
Maturation arrest
Focal complete
spermatogenesis
Diffused diminution
of normal
spermatogenesis
Total
Total
4
5
2
13
6
0
17
11
2
7
0
7
19
Seminal cytology and TESE results
Cytology was carried out on 35 patients and detected spermatids in 13 cases (37.1%). The results according to the TESE
outcome are presented in Table III. There was a statistically
significant correlation between the detection of spermatids
with classical cytology and the result of TESE (P ¼ 0.01,
Table I). This technique had a low sensitivity: 58% (95% CI
33– 82), but a high specificity: 83% (95% CI 59– 96).
Seminal FCM analysis and TESE results
FCM was carried out on 37 patients and detected haploid
cells in 24 patients. In 13 cases only were diploid cells
detected. Positive FCM analysis positively correlated with
successful TESE (P ¼ 0.0001). Table III also presents the
FCM results according to the TESE: FCM had a high sensitivity: 100% (95% CI 81 –100), and a lower specificity: 68%
(95% CI 43– 87).
The cytometry graph shown in Figure 1 displays the level
of IP fluorescence according to the size of the analysed cells.
Several fluorescence levels could be determined: the lowest
fluorescence level corresponded to the cell fragments and
debris, a higher fluorescence level to the haploid control cells
(sperm); the diploid control cells showed the highest fluorescence level on this graph. The spermatids, which share the
ploidia of sperm, with a lower level of condensation in the
chromatin, should be detected between haploid and diploid
control cells. Spermatocytes I, having a quadriploidia, were
distinguished at an upper level in some samples (not shown).
These graphs were obtained for each patient. When an FCM
result was positive, several aspects of the graphs could be
observed: either there was an important and/or very individualized cell contingent with the same fluorescent signal as
Table III. Seminal spermatid detection by classical cytology or flow
cytometry (FCM) and testicular sperm extraction (TESE) results
Negative TESE
18
positive TESE. When the TESE was negative, the histology
was homogeneous in both testes. According to the previously
described histological classification, 17 SCO, 11 MA, two
FCS and seven DS were observed. In the 17 patients with
SCO, sperm were found in four patients (23.5%). In the 11
cases of MA, sperm were retrieved from five biopsies
(45.4%). The histological evidence of a complete spermatogenesis even reduced (DS) or very focalized (FCS) was confirmed by the presence of sperm in all the nine biopsies
(100%). There was a significant difference in the results of
TESE between patients with complete spermatogenesis (FCS
and DS) and patients with an absence of MA (100 and
32.14% of positive TESE respectively, P ¼ 0.0012).
37
Cytology
Positive
Negative
Total
FCM
Positive
Negative
Total
Positive TESE
Negative TESE
Total
10
7
17
3
15
18
13
22
35
18
0
18
6
13
19
24
13
37
1917
I.Koscinski et al.
Table V. Testicular sperm extraction (TESE) results according to flow
cytometry (FCM) result and histopathology
Sertoli cell-only
Maturation arrest
Focal complete
spermatogenesis
Diffused diminution of
normal spermatogenesis
Total
Positive TESE
Negative TESE
Positive
FCM
Negative
FCM
Positive
FCM
Negative
FCM
4
5
2
0
0
0
3
3
0
10
3
0
7
0
0
0
18
0
6
13
Discussion
Figure 1. IP fluorescence levels according to the cell size. Haploid
c ¼ control haploid cells (sperm cells); diploid c ¼ control diploid
cells (lymphocytes); patient round c ¼ patient seminal round cells;
debris ¼ cell debris.
sperm cells (10 cases), or the fluorescent signal of round
cells was intermediate between sperm and lymphocytes
(eight cases). Sometimes a small number of intact cells was
analysed in comparison with the amount of debris and the
presence of the haploid peak was not so evident (six cases).
FCM and seminal cytology
The correlation between FCM and classical cytology results
(Table IV) was statistically significant (P ¼ 0.0034): when
cytology analysis was positive, the FCM was always positive
(13 cases) and when FCM was negative, the cytology was
always negative (13 cases). The combination of negative
cytology and negative FCM predicted a negative TESE in all
nine cases (100%). The combination of positive cytology and
positive FCM predicted a successful TESE in seven out of
nine cases (78%).
FCM and histopathology
The comparison of FCM and histopathology (Table V)
showed that the presence of complete spermatogenesis (FCS
or DS) in histopathology was always associated with a positive FCM and a positive TESE (nine cases). The six positive
FCM followed by a negative TESE corresponded with SCO
in three cases and MA in three cases. Moreover, whatever
the result of histopathological analysis, a negative FCM
result always predicted a negative TESE (13 cases), leading
to a negative predictive value of 100% for FCM.
Table IV. Comparison of flow cytometry (FCM) and cytology for the
detection of seminal spermatids
Positive cytology
Negative cytology
Total
Positive FCM
Negative FCM
Total
13 (59)
9 (41)
22
0
13 (100)
13
13
22
35
Values in parentheses are percentages.
Sensitivity: 59% (95% confidence interval 36–79).
Specificity: 100% (95% confidence interval 75–100).
1918
For patients presenting an NOA, ICSI using TESE has
become a standard therapy (Devroey et al., 1995). But TESE
is an invasive procedure with potential complications,
especially when TESE is linked to multiple extensive testicular biopsies as is often necessary for these patients (Schlegel
and Su, 1997; Manning et al., 1998).
In order to avoid useless TESE, different non-invasive
parameters have been proposed to predict the outcome of
TESE. In our study, we confirmed that the combined volume
of both testes or of the largest testis did not predict TESE
outcome (Devroey et al., 1995; Amer et al., 2001; Seo and
Ko, 2001). Similarly, FSH or inhibin B serum concentrations
did not predict TESE results (Kim et al., 1997; Mulhall et al.,
1997; Amer et al., 2001; Seo and Ko, 2001; Vernaeve et al.,
2002). The seminal inhibin B level (not measured in this
study) has been described to be a more reliable predictor
(Anderson et al., 1998; Frydelund-Larsen et al., 2002; Bailly
et al., 2003) but the wide range of concentrations complicates
the interpretation. Moreover, the regulation of the seminal
concentration of inhibin B secreted by Sertoli cells seems to
be complex with a probable contribution of accessory sex
glands (Garem et al., 2002).
As reported in previous literature, the histology was correlated with the TESE outcome. The best prognosis was
observed with complete spermatogenesis (focal spermatogenesis or diffused diminution in the degree of normal spermatogenesis). Maturation arrest had a better prognostic value
(positive TESE in 57% of cases) than germinal cell aplasia
(positive TESE in only 24% of cases) which agrees with
other studies (Tournaye et al., 1997; Silber et al., 1997; Su
et al., 1999; Sousa et al., 2000; Amer et al., 2001; Seo and
Ko, 2001). For some authors, the observation of testicular
round spermatids (Silber et al., 1997) or late spermatid forms
(Mulhall et al., 1997) was considered as the best predictive
element of a positive TESE.
It has been suggested (Ezeh et al., 1998) that the presence
of spermatids in the seminal fluid may reflect their presence
in the testis and could be a non-invasive predictive factor
before TESE. Therefore, there may be a threshold level of
spermatogenesis below which no spermatid could be detected
in semen (Ezeh et al., 1998) in analogy with the histopathological threshold of six mature spermatids per seminiferous
tubule that is correlated with the presence of sperm in the
seminal fluid (Silber et al., 1997).
Flow cytometry analysis in non-obstructive azoospermia
In this study, a significant correlation was found between
the detection of seminal spermatids by Harris– Shorr staining
and TESE results, as in the studies using May – Grünwald –
Giemsa (MGG) staining (Amer et al., 2001). Immunostaining
with acrosome-specific monoclonal antibodies reaches a sensitivity of 75%, and a specificity of 69% (Ezeh et al., 1998)
which is similar to the use of the anti-proacrosin antibody
(4D4). The detection of this antigen from the spermatocyte I
stage onwards leads to false positive results and the loss of
this antigen by the cell seems to be usual (Mendoza et al.,
1996).
FCM can analyse several thousand events quickly with a
good reproducibility. The ploı̈dia analysis requires cell fixation and permeabilization. After a stay of several days in
the genital tractus, however, round cells were weakened by
the fixation/permeabilization procedure. This probably
explains the large amount of cellular fragments and debris in
the samples. Therefore the proportion of haploid cells among
all seminal round cells could not be quantified.
In the present study, the sensitivity of FCM was 100% and
the specificity 68%. This mild specificity was due to six
patients having a negative TESE despite a positive FCM.
These six false positive FCM presented a germ cell aplasia in
three cases and a maturation arrest in three cases. The apparent absence of spermatogenesis could reflect a partial or
incomplete defect, which is why one single large biopsy may
not be representative of the functionality of the whole of the
testis (Amer et al., 1999). Patients with positive FCM and
negative TESE could present a very focal spermatogenesis
which could be identified only by multifocal biopsy. This is
why more and more teams practice a multifocal needle
aspiration. Using such a technique, it seems possible to exhibit a very great heterogeneity in histological patterns
observed in the aspiration products at different sites of the
testes and to establish a histological mapping of the testes
(Meng et al., 2000). So, it was possible to find sperm cells in
one or several sites (Turek et al., 1999) which is in accordance with our observation that sperm could be retrieved in
24% of patients presenting an SCO.
Moreover, repeated or multiple needle or open testicular
biopsies required for such mapping, diagnosis and TESE,
could subject the patients to potential risks of vascular injuries and further to androgenic defect or testis atrophy
(Harrington et al., 1996; Friedler et al., 1997; Schlegel and
Su, 1997). The defenders of the needle aspiration therefore
considered that the amount of valuable tissue removed from
the failing testes was minimized, and they observed a high
clinical retrieval rate of sperm obtained by post-needle
aspiration TESE (Turek et al., 1999).
The comparison of classical cytology with the FCM
showed a better sensitivity for FCM (100 versus 59% for
cytology) and a better specificity for classical cytology (83
versus 68% for FCM). Classical cytology is a cheap and
simple technique which does not require any special high
cost equipment: in this study, this technique revealed the
presence of spermatids in the majority of positive FCM
cases. But a negative classical cytology agreed with the
results of the FCM in only 50% of the cases.
Figure 2. Proposition of non-obstructive azoospermia (NOA) for
the management of NOA patients.
These observations have led us to propose a strategy for
the management of NOA patients. The detection of round
spermatids in semen must be initially conducted by classical
cytology. If this detection is positive, then the TESE is
proposed without doing any FCM assay. In case of a negative
result, the FCM assay should be carried out: if FCM is also
negative, the indication for TESE would be reconsidered and
perhaps abandoned. If FCM is positive for the presence of
haploid round cells, we would propose to carry out a TESE,
accepting a risk of 22% of having an unsuccessful TESE.
This proposition for the management of NOA patients is
summarized in Figure 2.
It would also be interesting to reconsider the method of
TESE in patients having a negative classical cytology and a
positive FCM. Indeed, these patients, presenting an SCO
histology, may have some very localized spots of complete
spermatogenesis justifying a multifocal TESE or a fine
needle aspiration mapping before TESE which could be more
efficient than a direct single large biopsy in each testis.
In conclusion, we have evaluated the detection of seminal
spermatids as a prognostic factor of TESE in cases of NOA.
The detection of seminal haploid round cells using FCM
ploı̈dia analysis offers a good predictive parameter for successful TESE compared with testicular size, serum FSH and
inhibin B concentrations as well as histopathological findings. FCM appeared to be more sensitive than classical
cytology with Harris –Shorr staining but less specific. By
combining these two techniques, cytology and FCM analysis,
an accurate management of non-obstructive azoospermic
patients could be conducted. This strategy would allow us
to prevent some unnecessary biopsies with their possible
deleterious effects for the patient.
Acknowledgements
We cordially thank C.Grussmacher for cytometry technical
assistance, P.Devos for helpful statistical suggestions, R.Dolan and
Gita for language editing.
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Submitted on September 8, 2004; resubmitted on February 21, 2005;
accepted on February 25, 2005