Hum. Reprod. Advance Access published online on July 29, 2008
Human Reproduction, doi:10.1093/humrep/den292
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Homocysteine metabolism in the pre-ovulatory follicle during ovarian stimulation
1 Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Centre, Rotterdam, The Netherlands 2 Department of Epidemiology, Erasmus MC, University Medical Centre, Rotterdam, The Netherlands 3 Department of Pediatric Cardiology, Erasmus MC, University Medical Centre, Rotterdam, The Netherlands 4 Department of Clinical Genetics, Erasmus MC, University Medical Centre, Rotterdam, The Netherlands 5 Department of Clinical Chemistry, Erasmus MC, University Medical Centre, Rotterdam, The Netherlands 6 Department of Urology, Erasmus MC, University Medical Centre, Rotterdam, The Netherlands 7 Department of Reproductive Medicine and Gynaecology, University Medical Centre, Utrecht, The Netherlands
8 Correspondence address. Department of Reproductive Medicine and Gynaecology, University Medical Centre, PO Box 85500, 3508 GA Utrecht, The Netherlands. Tel: +31-30-2507524; E-mail: n.s.macklon{at}umcutrecht.nl
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Abstract |
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BACKGROUND: Ovarian stimulation gives rise to supraphysiological estradiol levels, which may affect oocyte quality. This study aims to investigate whether ovarian stimulation deranges the homocysteine pathway thereby affecting the pre-ovulatory follicle.
METHODS: Blood samples were collected on cycle day 2 and the day of hCG administration in 181 women undergoing ovarian stimulation for IVF. In each subject, the diameter of the two leading follicles was measured and the corresponding follicular fluids were collected. In blood and follicular fluid samples, total homocysteine (tHcy), folate, cobalamin and pyridoxal’5-phosphate (PLP) were determined. According to the blood folate levels, women were classified as either folic acid supplemented (n = 113) or non-supplemented (n = 32).
RESULTS: Ovarian stimulation resulted in a significant decrease in blood tHcy and cobalamin levels (both P 
0.001). The blood concentrations of tHcy, folate, cobalamin and PLP were significantly correlated with the corresponding follicular fluid concentrations (all P 0.001). Follicular fluid tHcy concentrations were inversely correlated with follicular diameter (P 0.05). In folic acid supplemented women, follicular fluid folate was inversely correlated with follicular diameter (P 0.05).
CONCLUSIONS: Ovarian stimulation deranges blood and follicular fluid biomarkers of the homocysteine pathway. High ovarian follicular fluid tHcy and folate levels may have detrimental effects on follicular development.
Key words: folic acid/cobalamin/pyridoxine/assisted reproduction/follicular fluid
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Introduction |
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Involuntary childlessness imposes a heavy burden on most subfertile couples (Verhaak et al., 2002
; Lechner et al., 2007). Many couples therefore seek help by means of artificial reproduction techniques such as in vitro fertilization (IVF) to achieve pregnancy. Although these techniques have improved the treatment of subfertile couples, the chance of achieving a clinical pregnancy remains around 25% per started cycle (Andersen et al., 2007). In order to obtain multiple embryos from which to select for transfer to the uterus, exogenous gonadotrophins are administered to stimulate multifollicular development in the ovaries. This results in supraphysiological serum levels of gonadotrophins and estradiol which themselves may have a detrimental effect on oocyte and embryo quality (Macklon et al., 2006). Although the mechanisms of this effect remain to be elucidated, derangement of intrafollicular homocysteine pathway may be involved. Mild to moderate hyperhomocysteinemia is associated with detrimental effects on reproductive outcome, ranging from congenital malformations and miscarriages to pregnancy induced hypertension and low birthweight (Steegers-Theunissen et al., 1992; Wouters et al., 1993; Hague, 2003). The main causes of hyperhomocysteinemia include a dysbalance between the intake of folate, cobalamin, pyridoxine and methionine, metabolic derangements and related genetic variations (Refsum et al., 2004).
Exogeneous estrogens have been shown to affect several endocrine and metabolic pathways in women, including homocysteine metabolism. For instance, artificial estrogen treatment of post-menopausal women significantly lowers the total homocysteine (tHcy) levels (Madsen et al., 2002). Further evidence for sex steroid modulation of tHcy levels comes from studies showing levels to be significantly lower in the luteal phase than in the follicular phase of the normovulatory cycle (Tallova et al., 1999). Moreover, blood tHcy levels decrease during pregnancy from preconception onwards (Cikot et al., 2001; Murphy et al., 2002; Velzing-Aarts et al., 2005).
Whether estrogens affect the homocysteine pathway directly and/or via intermediates and cofactors is not clear. In some studies, a significant decrease of pyridoxine (Smolders et al., 2005) or cobalamin (Cagnacci et al., 2006) was observed in post-menopausal women taking estradiol supplements, but this could not be confirmed by others (Berger et al., 2000; Smolders et al., 2005).
Given the previously described detrimental effects of hyperhomocysteinemia on embryo quality (Ebisch et al., 2006), suppression of tHcy concentrations by increasing estradiol levels might be expected to have a beneficial effect. However, recent data has indicated that supraphysiological estradiol levels are correlated to higher levels of embryo aneuploidy (Baart et al., 2007) suggesting a negative effect on oocyte quality. At present, little is known about the influence of ovarian stimulation treatment on the homocysteine pathway in the direct environment of the maturing follicle and oocyte in human, or the relationship between follicular tHcy concentrations and follicular growth. Moreover, the extent to which folate supplementation modifies these relationships is not known. Therefore, the principal aims of the present study were to investigate the influence of ovarian stimulation with exogenous gonadotrophins on the levels of biomarkers of the homocysteine pathway in blood and the follicular fluid of the maturing oocyte, and to study the correlations between the levels of these biomarkers and the follicular diameter. Finally, the impact of periconception folate supplementation on these correlations was determined.
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Materials and Methods |
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Population
The FOod, Lifestyle and Fertility Outcome-study (FOLFO-study) is a periconception cohort study focused on the influence of the preconception health of the couple on fertility parameters and pregnancy outcome. Between September 2004 and October 2006, subfertile couples undergoing an IVF with or without intracytoplasmic sperm injection (ICSI) procedure at the Erasmus MC, University Medical Centre, Rotterdam, The Netherlands, were invited to participate. Exclusion criteria were oocyte donation cycles, the diagnosis of endometriosis or hydrosalpinx and age above 43 years. Seventy-four percent of eligible couples consented to participate in the study. Data from these women have not been previously published. All obtained materials and questionnaires were processed anonymously. The study protocol was approved by the Dutch Central Committee for Human Research (CCMO) and the Medical Ethical and Institutional Review Board of the Erasmus MC, University Medical Centre in the Netherlands.
In vitro fertilization procedure
All women started daily subcutaneous injections of 150 IU recombinant FSH, on the second day of a spontaneous cycle (Puregon®, NV Organon, Oss, The Netherlands or Gonal-F®, Serono Benelux BV, The Hague, The Netherlands). Daily subcutaneous administration of 0.25 mg GnRH antagonist (Orgalutran®, NV Organon, or Cetrotide®, Serono Benelux BV) was started when at least one follicle measured 
14 mm. To induce final oocyte maturation, a single dose of 5000 or 10 000 IU human chorionic gonadotrophin s.c. (hCG, Pregnyl®, NV Organon) was administered as soon as the largest follicle reached at least 18 mm in diameter and at least 1 additional follicle of >15 mm was present. Oocyte retrieval was carried out 35 h after hCG injection by transvaginal ultrasound-guided puncture of the follicles. Immediately prior to this procedure, the diameter of the leading follicle in both the right and left ovary was measured in three-dimensions. Fluid from each of these two follicles was aspirated and stored separately before continuing with the oocyte retrieval procedure. After oocyte retrieval, the follicular fluid samples were centrifuged for 10 min at 1700g to separate red blood cells (RBCs), leucocytes and granulosa cells. The samples were frozen without preservatives and stored at –20°C until assayed. Venous blood samples were drawn from each woman on cycle day 2 and the day of hCG administration. At both time-points, tHcy, folate, cobalamin, pyridoxine, total protein and estradiol were determined. In addition, on cycle day 2, FSH was determined. Blood was collected in dry vacutainer tubes, ethylenediamine tetra-acetate (EDTA) containing vacutainer tubes and lithium heparin containing vacutainers.
After clotting, the blood collected in dry vacutainer tubes was centrifuged at 2000g and the sera were stored at 4°C before being assayed. The venous blood samples collected in the EDTA containing vacutainer tubes were kept on ice for a maximum of 1 h after which the plasma was separated after centrifugation and stored at 4°C before being assayed. Blood samples drawn into lithium heparin containing vacutainers were stored at 4°C before being assayed.
tHcy in EDTA plasma and follicular fluid and pyridoxine as pyridoxal’5-phosphate (PLP) in whole blood and follicular fluid were determined using high-performance liquid chromatography with reversed phase separation and fluorescence detection (Schrijver et al., 1981; Pfeiffer et al., 1999). For the determination of folate and cobalamin, an immunoelectrochemoluminescence immunoassay was used (Roche Modular E170, Roche Diagnostics GmbH, Mannheim, Germany). For the determination of RBC folate, 100 µl blood out of one EDTA tube was hemolyzed with 2 ml freshly prepared ascorbic acid (0.05 g ascorbic acid in 25 ml aqua dest) directly after blood sampling. Subsequently, the hematocrit of the EDTA blood was determined on a Sysmex XE-2100 (Groffin Meyvis, Etten-Leur, The Netherlands). The hemolysate was centrifuged for 10 min at 2000g shortly before the folate measurement. The folate concentration in the hemolysate was calculated in RBC folate using the following formula: (nmol hemolysate folate x 21) – (nmol/l serum folate x (1 – hematocrit))/hematocrit = nmol/l RBC folate. Total protein concentrations were determined photometrically on a Hitachi 917 (Roche Diagnostics GmbH). Levels of FSH were measured by luminescence-based immunometric assay (Immulite 2000, Diagnostic Products Corporation, Los Angeles, USA). Estradiol was measured using coated tube radioimmunoassay obtained from the same supplier.
Inter-assay coefficients of variation for tHcy were 4.8% at 14.6 µmol/l and 3.3% at 34.2 µmol/l, for folate 4.5% at 13 nmol/l and 5.7% at 23 nmol/l, for cobalamin 3.6% at 258 pmol/l and 2.2% at 832 pmol/l, for PLP 1.8% at 40 nmol/l and 1.3% at 115 nmol/l, for total protein 1.5% at 55 g/l and 1.3% at 84 g/l, for FSH <5.8% and for estradiol <8.8%. The detection limit for tHcy was 4 µmol/l, for folate 1.36 nmol/l, for cobalamin 22 pmol/l, for pyridoxine 5 nmol/l, for total protein 0.1 g/l, for FSH 0.1 U/l and for estradiol 10 pmol/l.
Questionnaires
After oocyte retrieval, women filled out a general questionnaire from which the following data were extracted: medical history, body length and weight, ethnicity and lifestyle factors, such as smoking and the use of vitamin supplements. Ethnicity was classified according to the definitions of Statistics Netherlands (http://www.cbs.nl/en-GB/default.htm, 2007).
Women were considered to be taking folic acid supplements when folate levels in serum were >22.4 nmol/l on both cycle day 2 and the day of hCG administration (Brouwer et al., 1999).
Statistical methods
Follicular fluid total protein concentrations vary with follicular maturity (Spitzer et al., 1996). Concentrations of tHcy and B-vitamins in follicular fluid are therefore also expressed in concentration per gram of protein. The results were expressed in median (range), because of the skewed distributions of tHcy, B-vitamins, FSH and estradiol. Consequently, these variables were log transformed before statistical testing. Because the distribution was normal after log transformation, paired t-tests were performed to determine differences between biomarker concentrations on cycle day 2 and the day of hCG administration and to determine differences between biomarker concentrations in blood and follicular fluid. Pearson’s correlation coefficients were calculated to determine correlations between the deltas of the estradiol, tHcy and B-vitamin concentrations.
In most subjects, but not all, two monofollicular fluid samples were obtained. In order to avoid a possible statistical bias arising from subjects producing just one fluid sample, a mixed model analysis was performed to determine correlations between the biomarker concentrations in blood and follicular fluid, and the diameter of the follicles. An independent t-test was performed with one follicle per women to determine differences between biomarkers in follicular fluid of supplemented and non-supplemented women. A P-value of 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 14.0 for Windows software (SPSS Inc., Chicago, IL, USA).
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Results |
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The flow chart of the study is presented in Fig. 1, and the baseline characteristics of the participating women are given in Table I. Of the 216 included couples, 10 dropped out before commencing the study. Five women conceived spontaneously before the start of IVF treatment, and five were diagnosed with a visible endometrioma or hydrosalpinx at the start of the IVF treatment. Four couples were excluded from the analysis due to incorrect use of the IVF-medication, and one couple withdrew. In the remaining 201 couples, treatments were cancelled before oocyte retrieval due to a poor response (n = 16), the threat of ovarian hyperstimulation syndrome (OHSS) (n = 3) and the occurrence of ovulation before oocyte retrieval (n = 1). Data on BMI, ethnicity, folic acid supplementation and smoking were missing in respectively 15, 24, 17 and 14 women. The mean number of follicles >10 mm measure on the day of hCG administration was 8 (SD 5) and the mean number of retrieved oocytes was 7 (SD 5). In four cases, blood samples were not taken at the correct day. Consequently, 177 cycle day 2 blood samples and 177 samples taken on the day of hCG administration were available for analysis.
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Of the 299 collected monofollicular fluid samples, 20 samples were excluded from the analysis due to contamination with the culture medium, which contains very high concentrations of cobalamin (>14 000 pmol/l) and folate (>950 nmol/l). The mean diameter of the follicles from which fluid was collected was 19.2 mm (SD 2.8 mm). This mean diameter was similar in the subgroups of folic acid supplemented and non-supplemented. Moreover, no significant differences were observed between young and old participants (19.0 versus 19.4 in the subgroups of
36 and >36 years, respectively).
Blood and follicular fluid concentrations of estradiol, total protein and biomarkers of the homocysteine pathway are presented in Table II. One hundred and thirteen women (67%) were classified as folic acid supplement users, with median folate concentrations of 36.6 nmol/l (range 22.9–908.0) on cycle day 2 and 38.9 nmol/l (range 22.8–174.3) on the day of hCG administration. Thirty-two women (19%) were classified as non-supplemented, with median folate concentration of 14.8 nmol/l (range 6.4–20.7) on cycle day 2, and 15.3 nmol/l (range 6.6–22.3) on the day of hCG administration. The median age of non-supplemented and supplemented women was not significantly different. In folic acid non-supplemented women, subfertility was more often caused by both a female and a male factor compared with the supplemented women (28% versus 3%) (P 0.01). Other causes for subfertility were not significantly different between these two subgroups. In the remaining 24 women, serum concentrations reflected sporadic folate intake, and the associated data were considered non-informative in this case.
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Samples taken following ovarian stimulation (on the day of hCG administration) revealed tHcy concentrations to be significantly lower in follicular fluid than in blood. Women taking folic acid supplementation had significantly lower follicular fluid levels of tHcy, median 6.4 µmol/l (range 3.5–73.6 µmol/l) than non-supplemented women, median 7.1 µmol/l (range 4.0–47.0) (P
0.005). Folate concentrations were also observed to be consistently higher in blood than in follicular fluid. Folate concentrations in follicular fluid were significantly higher in supplemented women, median 35.8 nmol/l (range 13.0–234.2), compared with non-supplemented women, median 15.1 nmol/l (range 6.3–39.8) (P 0.001). Cobalamin and pyridoxine concentrations were also significantly lower in follicular fluid than in blood (P 0.001). All biomarker concentrations in blood were significantly correlated with follicular fluid levels (all P 0.001) (Table II). During ovarian stimulation, serum estradiol concentrations significantly increased and blood tHcy, cobalamin and total protein levels all decreased significantly (Table II). However, no significant correlations were found between the increase of the estradiol concentrations and the change in those biomarker concentrations in the blood. Likewise, the increase of the estradiol concentrations were not correlated with the biomarker concentrations in follicular fluid.
In the group as a whole, a significant inverse correlation between follicular fluid tHcy concentration and the follicular diameter was demonstrated (Table III). A 2-fold increase of the tHcy concentration was associated with a 0.06 mm decrease of the follicular diameter (P 0.05). In non-supplemented women, this inverse correlation was much stronger (1.64 mm; P 0.01). In supplemented women, a 2-fold increase of the follicular folate concentration was associated with a 0.74 mm decrease of the follicular diameter (P 0.05).
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Discussion |
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This study demonstrates that ovarian stimulation in women undergoing IVF treatment is associated with a decrease of tHcy and cobalamin levels both in blood and in follicular fluid. Furthermore, inverse correlations are shown between follicular fluid tHcy and folate concentrations, and follicular diameter.
The observed decline in blood levels of tHcy and cobalamin levels during ovarian stimulation treatment may be due to estradiol mediated induction of general enzyme activity through which the metabolization and clearance of both biomarkers might be increased. Two smaller studies reported no significant effect of ovarian stimulation on biomarkers of the homocysteine pathway (Bettahar-Lebugle et al., 2002; Roopnarinesingh et al., 2006). However, the inverse effect of rising estradiol levels on tHcy concentration is consistent with the results of studies comparing pre- and post-menopausal women (Hak et al., 2000; Morris et al., 2000) and trials in which the administration of estrogen was compared with placebo in post-menopausal women (van Baal et al., 1999; Madsen et al., 2002; Smolders et al., 2005). Similarly, a decrease in the cobalamin levels in estrogen supplemented post-menopausal women has been recently demonstrated (Cagnacci et al., 2006).
In the present study, the concentration of tHcy in follicular fluid was inversely correlated with the follicular diameter. Since tHcy levels were adjusted for maturation by calculating the protein ratio (Spitzer et al., 1996), our data suggest that exposure of the follicles to high blood concentrations of tHcy may contribute to restricting follicular growth. The strong correlations between tHcy in blood and follicular fluid and the stronger correlation between tHcy and follicular diameter in non-supplemented women are consistent with this observation. Previously, our group demonstrated a positive correlation between tHcy in follicular fluid and follicular diameter (Boxmeer et al., 2007). The levels of tHcy in follicular fluid in that study and the present study were similar. The discrepancy in results may reflect the small number of samples in our previous study.
On the basis of the present findings, it is not possible to determine whether high tHcy levels in follicular fluid are the cause or result of limited follicular growth. However, the present findings are consistent with our previously reported observation that a high tHcy in follicular fluid reduces embryo quality (Ebisch et al., 2006) and other reported associations between mild to moderate hyperhomocysteinemia and detrimental effects on reproductive outcome (Steegers-Theunissen et al., 1992; Wouters et al., 1993; Hague, 2003).
The toxic effects of tHcy may be due to the production of reactive oxygen species (ROS). A certain level of ROS in follicular fluid is necessary for oocyte maturation (Riley and Behrman, 1991) and ovulation (Miyazaki et al., 1991). Moreover, in a previous study, patients who became pregnant after IVF had significantly higher levels of ROS in their follicular fluid compared with non-pregnant women (Attaran et al., 2000; Bedaiwy et al., 2002). An excess of ROS results in a state of oxidative stress. High levels of ROS in culture medium of embryos are associated with low cleavage rates and high embryonic fragmentation rates (Bedaiwy et al., 2006). The results of our study seem to suggest that high levels of tHcy have a detrimental effect on follicular maturation as reflected by the follicle diameter. This may be a consequence of oxidative stress. The decline of blood tHcy concentrations during ovarian stimulation may suggest a beneficial effect of this hormonal treatment on follicle maturation. However, it is plausible that there is an optimal level of tHcy.
In the present study, a detrimental effect of both low and high folate levels on follicular growth is suggested. Folate supplementation appeared to protect follicular growth against the observed detrimental effects of high tHcy levels. Folic acid supplementation lowers tHcy levels by remethylation of tHcy into methionine. Surprisingly, we also observed that rising folate levels in follicular fluids of folic acid supplemented women, median 35.8 nmol/l, are associated with a significant smaller follicular diameter. There is an ongoing discussion about possible adverse effects of excessive folate levels with respect to increased twinning rates (Boxmeer et al., 2006; Haggarty et al., 2006; Muggli and Halliday, 2007). Our results are consistent with the previously proposed concept of an ‘optimal’ level of supplementation above which reproductive outcomes may deteriorate. Such a concept would also reconcile two apparently contradictory observations that supraphysiological estradiol concentrations are associated with suppression of tHcy concentrations in the follicle, yet may be detrimental to oocyte and embryo quality. It would appear that excessive suppression of tHcy levels by high estrogens may indeed be harmful to gamete and embryo quality.
Some limitations of this study have to be addressed. Our own study lacks a control group, because follicular fluid is not available from spontaneous cycles. There is little data about the spontaneous cycle, and only a decline of tHcy has been reported before (Tallova et al., 1999). However, the extent of tHcy decrease as reported by Tallova et al. cannot be compared with the present results because of the different timing of sampling. The women recruited for the present study constitute a heterogeneous group in terms of race, age and indication for treatment. The effect of ovarian stimulation treatment on tHcy, folate, cobalamin and pyridoxine and the associations between the same biomarkers and follicular diameter may be more prominent in certain subgroups. However, the aim of our study was to investigate this in a representative population of women undergoing IVF or ICSI. The strength of our study is the standardized design, the large sample size and the biomarkers determined in blood and monofollicular fluids. Moreover, all participants were treated according to the same ovarian stimulation protocol. Future studies should address possible associations between nutritional biomarker concentrations in the follicle and serum, and clinical outcomes of IVF. Moreover, maternal gene polymorphisms involved in the homocysteine pathway have also been associated with an increased risk of offspring with Down's syndrome possibly due to chromosomal damage (Scala et al., 2006; Coppede et al., 2007; Zintzaras, 2007). The findings of the present study indicate that further studies are required to address whether polymorphisms may also be associated with follicular growth.
In conclusion, ovarian stimulation resulted in a significant decline in tHcy and cobalamin blood concentrations but did not affect folate or pyridoxine levels. Follicular fluid tHcy concentrations were inversely correlated with follicular diameter, and this association was much stronger in women who did not use a folic acid supplement. In folic acid supplemented women, follicular folate concentrations were inversely correlated with follicular diameter. This suggests that both high tHcy and high folate concentrations may be detrimental for follicular growth. This supports the need for finding the optimal folic acid dose not only to prevent neural tube defects but also to improve the quality of oogenesis.
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Funding |
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This study was supported by funding from the Divisions of Reproductive Medicine and of Obstetrics and Perinatal Medicine within the Department of Obstetrics and Gynaecology, Erasmus MC, Rotterdam, The Netherlands.
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Acknowledgements |
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The authors gratefully acknowledge the contribution of the IVF team of the division of Reproductive Medicine, and the laboratory technicians and research assistants of the Laboratory of Clinical Chemistry and the laboratory of Endocrinology of the Erasmus MC, University Medical Center in Rotterdam, The Netherlands for the laboratory determinations.
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Submitted on March 8, 2008; resubmitted on June 20, 2008; accepted on July 3, 2008.
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