A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage

Irene Boiso1,4, Mercè Martí2, Josep Santaló3, Montse Ponsá3, Pere N. Barri1 and Anna Veiga1

1 Servei de Medicina de la Reproducció, Dept Obtetrícia i Ginecologia, Institut Universitari Dexeus, 2 Servei de Microscòpia Electrònica and 3 Dept de Biologia Cel.lular, de Fisiologia i d'Immunologia, Universitat Autònoma de Barcelona, Barcelona, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Routine oocyte cryopreservation remains an elusive technique in the wide range of assisted reproductive technologies available. This study examines the effect of a cryopreservation protocol on the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle (GV) and metaphase II (MII) stage. METHODS: GV oocytes were randomly assigned to one of three groups: (i) control oocytes matured in vitro to MII stage (n = 156); (ii) oocytes cryopreserved at the GV stage and then matured in vitro (n = 90); (iii) oocytes cryopreserved at the MII stage (n = 147). Following cryopreservation and in-vitro maturation, immunostaining of tubulin and chromatin was performed, before visualization using confocal microscopy. RESULTS: A statistically significant increase was observed in the survival rate in group 2 (73.3%, 66/90) compared to group 3 (55.7%, 82/147) (P < 0.007). Exposure of oocytes to the cryoprotective solutions without freezing had no effect on the structure of their second meiotic spindle. However, statistically significant differences were observed on both spindle and chromosome configurations of oocytes from group 2 (5.2 and 5.2% respectively) and group 3 (16.2 and 18.8% respectively) compared with group 1 oocytes (71.6 and 82.0% respectively) (P < 0.001 in all cases). CONCLUSIONS: The protocol followed results in high rates of survival and potential for in-vitro maturation, but has a deleterious effect on the organization of the meiotic spindle of human oocytes cryopreserved at both the GV and MII stages.

Key words: chromosomal abnormalities/cryopreservation/human/oocyte/spindle


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Routine oocyte cryopreservation remains an elusive technique in the wide range of assisted reproductive technologies available. Successful cryopreservation of oocytes would avoid the ethical and legal problems associated with embryo freezing. It would be beneficial for women at risk of premature loss of ovarian function due to benign or malignant processes and would allow the establishment of oocyte banks for donation. The initial reports on pregnancies achieved using oocyte cryopreservation in conjunction with IVF (Chen, 1986Go; Al-Hasani et al., 1987Go; Van Uem et al., 1987Go) were encouraging. However, evaluation of the negative effects of cryopreservation on the integrity of several of the oocyte's unique features involved in normal fertilization and embryo development, such as premature cortical granule exocytosis leading to zona hardening, increased parthenogenic activation and damage to the cytoskeletal elements of the oocyte, in particular disruption of the meiotic spindle, led to a period of dormancy regarding research on the procedure. Ten years later, low fertilization rates (Pickering et al., 1991Go) and increased polyploidy (Al Hasani et al., 1987Go) associated with premature zona hardening have been overcome (Gook et al., 1995Go; Kazem et al., 1995Go) using ICSI (Palermo, 1992). Interest in human oocyte cryopreservation has reawakened and the procedure now is being applied clinically in some IVF programmes.

A growing number of pregnancies has been reported and ~30 healthy babies have been born around the world (Porcu et al., 1997aGo,bGo, 1999Go; Fabbri et al., 1998Go; Polak de Fried et al., 1998Go; Tucker et al., 1998aGo; Young et al., 1998Go) using ICSI combined with a slow freezing–rapid thawing cryopreservation protocol and 1,2-propanediol (1,2-PROH) as the cryoprotectant agent. However, despite considerable efforts (Gook et al., 1993Go, 1994Go; Tucker et al., 1998bGo) the efficiency of the technique is not adequate (Bernard and Fuller, 1996Go) as rates of survival and development in vitro and in vivo are still low (Mandelbaum et al., 1998Go; Ludwig et al., 1999Go).

The majority of the research concerning oocyte cryopreservation has centred on mature oocytes. The mature oocyte is arrested at the metaphase II (MII) stage, which means that the chromosomes are attached to the labile microtubules of the second meiotic spindle. Studies performed on animal models as well as in humans have shown that exposure to low temperatures (Pickering and Johnson, 1987Go; Van der Elst et al., 1988Go; Pickering et al., 1990Go; Almeida and Bolton, 1995Go) and to cryoprotectant agents (Johnson and Pickering, 1987Go; Van der Elst et al., 1988Go) result in depolymerization of the microtubules, sometimes with attendant dispersal of chromosomes. Once fertilized, sister chromatid non-disjunction may occur in the thawed oocyte giving rise to an aneuploid embryo.

One alternative approach to circumvent the problem of damaging the meiotic spindle would be freezing at the immature oocyte stage of development, when meiosis is arrested at the prophase I stage and the chromosomes are protected within the membrane of the germinal vesicle and when no microtubular structures have formed yet (Gallicano et al., 1994Go; Battaglia et al., 1996Go; Kim et al., 1998Go). Studies have demonstrated that immature oocytes can be matured and fertilized in vitro and can result in pregnancies (Cha et al., 1991Go; Trounson et al., 1994Go; Nagy et al., 1996Go; Russell et al., 1997Go). Immature human oocytes from stimulated (Mandelbaum et al., 1988Go; Toth et al., 1994aGo) and unstimulated (Toth et al., 1994bGo; Son et al., 1996Go) ovaries have been frozen, showing variable rates of survival and in-vitro maturation. To date, only one birth has been reported after the cryopreservation of immature oocytes collected in a stimulated cycle (Tucker et al., 1998aGo) using a slow freezing–rapid thawing protocol and 1,2-PROH as cryoprotectant.

The present study was undertaken to evaluate the effect of a cryopreservation protocol that has been clinically used, on the spindle and chromosome configurations of human oocytes cryopreserved at the GV and MII stages, as well as on their survival and potential for nuclear in-vitro maturation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Source of oocytes
Institutional approval by the Comité de Ética e Investigación Clínica del Institut Dexeus was obtained for the present study. Oocytes at the GV stage were obtained from consenting patients from our IVF–ICSI programme only when an adequate number of normal MII oocytes was retrieved. Ovarian hormonal stimulation, oocyte collection, culture and insemination have been described previously (Calderón et al., 1995Go).

Experimental design
After denudation, GV oocytes were randomly assigned to one of three experimental groups: (i) control, GV oocytes matured in vitro to MII (n = 156); (ii) oocytes cryopreserved at the GV stage and matured in vitro (n = 90); (iii) in-vitro matured oocytes cryopreserved at the MII stage (n = 147). Due to the reduced number available each day, oocytes were assigned to each experimental group in a linear way and not simultaneously, as would have been desirable. Additionally, a smaller group of oocytes was exposed to the cryoprotective solutions either at the GV (n = 10) or MII (n = 10) stage without freezing.

In-vitro culture
Only intact oocytes with a refringent cytoplasm and presenting no signs of atresia or degeneration were included. Oocytes were cultured in 4-well dishes (Nunc, Denmark) in maturation medium covered with oil (Ovoil-150; Vitrolife, Sweden). Maturation medium consisted of pooled inactivated follicular fluid from mature follicles, supplemented with 20% synthetic serum substitute (SSS; Irvine Scientific, USA). Serial observations under the stereomicroscope (Olympus SZH, Japan) were performed until extrusion of the first polar body was observed.

Freezing and thawing methods
Oocyte processing for cryopreservation (Lassalle et al., 1985Go) was done at room temperature. Oocytes were placed in Dulbecco's phosphate-buffered saline (PBS; Irvine Scientific) supplemented with 15% SSS. They were transferred immediately to a solution containing 1.5 mol/l 1,2-PROH (Sigma, USA) in Dulbecco's PBS–15% SSS and allowed to equilibrate for 15 min at room temperature. Finally, oocytes were transferred to a solution containing 0.2 mol/l sucrose (Sigma) and 1.5 mol/l 1,2-PROH in Dulbecco's PBS–15% SSS and loaded in 0.5 ml plastic straws (IMV, France). A maximum of three oocytes was loaded in each straw. The straws were placed in a programmable freezer (Kryo-10; Planer, UK) and cooled from 22°C to –7°C at a rate of –2°C/min. Straws were held at –7°C for 5 min and then seeded manually by touching them with pre-cooled forceps. After 5 additional min, straws were cooled to –30°C at a rate of –0.3°C/min and then to –150°C at a rate of –50°C/min, followed by plunging and storing in liquid nitrogen for variable periods of time (from 1 month to 3 years).

For thawing, straws were removed from liquid nitrogen and air-warmed, followed by immersion in a water bath at 30°C until ice crystals melted. The cryoprotectant was removed by stepwise dilutions in 1.0, 0.5 and 0.0 mol/l 1,2-PROH in the presence of 0.2 mol/l sucrose. After washing in Dulbecco's PBS supplemented with 20% SSS, surviving oocytes (with an intact zona pellucida and plasma membrane and with a refringent cytoplasm without evidence of lysis) were either transferred to maturation (GV oocytes) or to culture medium (IVF-50; Vitrolife) (MII oocytes). Oocytes were cultured at 37°C, 5% CO2, until polar body extrusion was observed in the case of GV oocytes and for 4 h in the case of MII oocytes, before fixing. A smaller group of oocytes was exposed sequentially to the freezing and thawing solutions without freezing. Surviving oocytes were cultured as explained before.

Immunostaining
Oocytes from group 1, as well as surviving oocytes from groups 2 and 3, were immunostained for tubulin and chromatin detection (Pickering et al., 1988Go) (modified). A control oocyte was included in each experimental session that involved only cryopreserved oocytes. Fixation and all subsequent incubations were carried out at 37°C. Oocytes were fixed in formaldehyde–PBS 2% and permeabilized using Triton X-100, 0.02% in Dulbecco's PBS and then incubated in anti-{alpha}-tubulin monoclonal antibody (Sigma) (1:250) for 1 h, followed by incubation in anti-mouse IgG antibody–biotin (Boehringer Mannheim Biochemica, Germany) (1:5000) for 1 h and extravidin–fluoroscein isothiocyanate (FITC) conjugate (Sigma) (1:50) for 30 min. Chromosomes were counterstained by incubating the oocytes in propidium iodide (Sigma) (5 mg/ml) for 15 min. Between incubations, oocytes were washed three times in pre-warmed Dulbecco's PBS supplemented with 5% human serum albumin (Grifols, Spain) for 5 min. Oocytes (a maximum of two) were mounted on poly-L-lysine-treated coverslips that had a self-adhesive reinforcement ring (commonly used to reinforce holes on paper sheets) attached to it, covered with antifade mounting medium (glycerol, n-propyl-gallate and sodium azide in PBS) to avoid photobleaching, and then with a slide. The preparation was sealed with clear nail varnish and kept frozen and protected from light until observation. The localization of tubulin and chromatin revealed by FITC and propidium fluorescence was made on a laser-scanning confocal microscope (Leica TCS-4D) provided with an argon–krypton laser. The images were recorded on a host computer.

Statistical analysis
Statistical comparisons between goups 1, 2 and 3 were carried out using the {chi}2-test for qualitative variables and the ANOVA test for quantitative variables. In both cases, differences were considered significant when P <= 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Mean age of the patients was 33.4 ± 3.8 years (range 27–44) in group 1, 33.7 ± 3.4 years (range 28–41) in group 2 and 34.0 ± 4.1 years (range 26–46) in group 3. No statistically significant differences were observed when the mean age of the patients was compared between groups 1, 2 and 3.

Results of the survival and maturation rates in the different experimental groups are summarized in Table IGo. A total of 90 GV oocytes (group 2) were cryopreserved, with 66 intact upon thawing resulting in a survival rate of 73.3%. Forty-eight surviving GV oocytes matured in vitro to the MII stage representing a maturation rate of 72.7%. No significant differences were observed in maturation rates between control and cryopreserved GV oocytes. A total of 147 oocytes were cryopreserved at MII stage (group 3) and 82 survived the cryopreservation procedure resulting in a survival rate of 55.7%. A statistically significant increase was observed in the survival rate in group 2 compared to group 3 (P < 0.007).


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Table I. Survival and maturation rates in the three experimental groups
 
Results of the spindle and chromosome configurations were obtained in 67 oocytes from group 1, 38 from group 2 and 37 from group 3. The spindle structure was located in all cases at the periphery of the oocyte, and oriented perpendicular to the plasma membrane. Spindle configuration was regarded as morphologically normal when a barrel-shaped structure with slightly pointed poles formed by organized microtubules traversing from one pole to another was observed. Chromosomal configuration was regarded as normal when chromosomes were arranged on a compact metaphase plate at the equator of the structure (Figure 1Go). Slightly abnormal spindle structures included rounded poles and reduction in the longitudinal dimension of the spindle. Spindle structure was regarded as abnormal when there was partial or total disorganization or complete lack of microtubules. The chromosomal organization was regarded as slightly abnormal when a single chromosome or chromatid was displaced from the plane of the metaphase plate and abnormal when it showed chromosome dispersal or chromosomes with an aberrant, less condensed appearance. Details of the abnormal patterns found are given in Figure 1Go.



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Figure 1. Oocytes stained immunocytochemically with an anti-{alpha}-tubulin monoclonal antibody and fluoroscein isothiocyanate to visualize the spindle (green) and counterstained with propidium iodide to visualize the chromosomes (orange/red). (a) Normal spindle configuration with chromosomes arranged on a compact plate at the equator of the structure. (b) Slightly abnormal configuration with a single chromosome displaced from the plane of the metaphase plate (arrow). (c) Slightly abnormal spindle structure with rounded poles. (d) Abnormal spindle structure associated with an otherwise organized metaphase plate; arrows indicate tubulin fibres that come out from the spindle. (e) Abnormal chromosomal organization associated with an otherwise well-structured spindle. (fh) Abnormal spindle structures associated with disorganized chromosomes. (i and j) Chromosomes with an aberrant, less condensed appearance. (k) Slightly abnormal spindle structure showing reduction in the longitudinal dimension of the spindle and compact metaphase plate. (l) Absent spindle and dispersed chromosomes.

 
Results of the spindle analysis are shown in Table IIGo and results of the chromosomal organization analysis are shown in Table IIIGo. Statistically significant differences were observed on both spindle and chromosome normal configurations of oocytes from group 2 (5.2 and 5.2% respectively) and group 3 (16.2 and 18.8% respectively) compared with group 1 oocytes (71.6 and 82.0% respectively) (P < 0.001 in all the cases). No statistically significant differences in either spindle or chromosome configurations were observed when groups 2 and 3 were compared. Also, statistically significant differences were observed when the incidence of absent spindles was compared in groups 2 (76.3%) and 3 (72.9%) with respect to the controls (17.9%) (P < 0.001).


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Table II. Spindle analysis in the three experimental groups
 

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Table III. Analysis of the chromosomal organization in the three experimental groups
 
Ten oocytes at the GV stage were exposed to the cryoprotective solutions and all of them survived. Eight surviving GV oocytes matured to the MII stage. Ten oocytes at the MII stage were exposed to the cryoprotective solutions and all of them survived. Surviving oocytes showed no abnormalities of the spindle or chromosomal configuration at either stage.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study analyses the effect of cryopreservation on the spindle and chromosome configurations of human oocytes frozen at the GV and MII stages, as well as on their survival and potential for nuclear in-vitro maturation. Our results show that immature oocytes collected from stimulated ovaries are more resistant to the cyopreservation process than mature oocytes, and that the process of cryopreservation does not impair their potential to mature to the MII stage as they show similar maturation rates as controls. However, our results also demonstrate that cryopreserving immature or fully mature oocytes using a slow freezing–rapid thawing cryopreservation protocol and 1,2-PROH has a deleterious effect on the spindle and chromosome configurations of the subsequently thawed oocytes.

Cryopreservation of GV oocytes collected from stimulated ovaries has been carried out before with variable success. It was reported that there were no differences in the survival rate of frozen–thawed GV and MII oocytes (37% and 36% respectively), while the rate of maturation for surviving GV oocytes was 20% (Mandelbaum et al., 1988Go).

More recently, it was found that the survival rate for GV cryopreserved oocytes was 58.5% (Toth et al., 1994aGo), a value which is slightly lower than the one obtained in this study, and that there were no significant differences in the maturation rate of cryopreserved GV oocytes compared with controls, in agreement with our results. Contrary to our results, another study (Goud et al., 2000Go) obtained a lower survival rate of 48.4% for GV frozen oocytes while a higher survival rate (84.4%) for frozen in-vitro matured MII oocytes was obtained. No differences in the respective maturation rates were found by these authors when GV frozen oocytes were compared with controls, as in the present study.

Reports on the survival of cryopreserved MII oocytes using a variety of methods are much more abundant in the literature, with values ranging from 34 to 95% (Trounson, 1986Go; Al-Hasani et al., 1987Go; Gook et al., 1993Go, 1994Go, 1995Go; Kazem et al., 1995Go; Porcu et al., 1997aGo, 1999Go; Tucker et al., 1998bGo; Yang et al., 1999Go; Goud et al., 2000Go). Fluctuating oocyte quality has been indicted as an explanation for variations in survival rate (Gook et al., 1994Go, 1995Go; Toth et al., 1994bGo). However, in the majority of reports, survival rates are ~50–60%, a value not different from the one obtained in this study. A recent publication (Fabbri et al., 2001Go) reports higher survival rates when MII oocytes are frozen using an increased concentration of sucrose.

Injury to the second meiotic spindle can lead to non-disjunction of chromatids and result in aneuploid embryos. Studies performed on animal models as well as in humans have shown that the spindle is sensitive to low temperatures (Pickering and Johnson, 1987Go; Van der Elst et al., 1988Go; Pickering et al., 1990Go; Almeida and Bolton, 1995Go) and to cryoprotectant agent exposure (Johnson and Pickering, 1987Go; Van der Elst et al., 1988Go), sometimes resulting in irreversible damage. The consequences of such damage could not be obvious until fertilization and second meiosis reinitiation (Pickering et al., 1990Go; Almeida and Bolton, 1995Go). The absence of spindle at the GV stage would therefore be advantageous to circumvent this problem. When this study was undertaken, it was expected that oocytes cryopreserved at the GV stage would show more stability regarding cryoinjury to the meiotic spindle than those cryopreserved at the MII stage.

On the contrary, our results reveal that only 5.2% of the analysed oocytes cryopreserved at the GV stage show a well-structured spindle with the chromosomes aligned on the metaphase plate in contrast with 71.6% in the control group. While the number of abnormal detectable spindles is relatively low (18.4%), absence of detectable microtubules is significantly higher (76.3%) in this group of oocytes with respect to the controls. Oocytes cryopreserved at the GV stage that displayed abnormal configurations had either disorganized chromosomes or chromosomes clustered in a discrete group with an aberrant, less condensed appearance (a morphological feature not found in abnormal cryopreserved MII oocytes) as previously described (Park et al., 1997Go).

Van der Elst et al. did not find abnormalities in the morphology of the second spindle after ultra-rapid freezing of GV mouse oocytes (Van der Elst et al., 1992Go). Baka et al. found that cryopreservation of human immature oocytes from stimulated ovaries did not increase the rate of abnormalities in the resulting meiotic spindle (Baka et al., 1995Go). By contrast, it was shown that cryopreserved immature human oocytes from unstimulated ovaries displayed an increased incidence of chromosomal and spindle abnormalities after in-vitro maturation, in agreement with our results (Park et al., 1997Go). These authors evaluated the incidence of chromosomal abnormalities by fluorescence in-situ hybridization using a total DNA probe and found a high rate of aneuploidy and polyploidy in cryopreserved GV oocytes. This finding is coincident with the high rate of spindle and metaphase plate abnormalities observed in cryopreserved immature oocytes and indicates that meiotic progression does not occur normally in oocytes cryopreserved at the GV stage. During the freeze–thawing process, irreversible changes in nuclear and cytoplasmic organization may take place, in spite of an apparent normal progression to the MII stage evidenced by polar body extrusion. This is in agreement with the report (Van Blerkom and Davis, 1994Go) describing nucleolar fragmentation and premature chromatin condensation in cryopreserved GV oocytes, which despite this, were capable of reaching the MII stage.

The sensitivity of the MII spindle to the different steps involved in cryopreservation is well studied. In the present study, only 16.2% of the MII cryopreserved oocytes had a normally structured spindle. In those MII frozen oocytes that displayed a normal spindle, tubulin fluorescence was less intense than that observed in controls, indicating a lower number of polymerized microtubules (Pickering et al., 1988Go). One oocyte cryopreserved at MII showed an extensive mesh of tubulin in the cortical region. Similar findings had been described in oocytes aged in vitro (George et al., 1996Go) or treated with the drug taxol (Pickering et al., 1988Go). In those oocytes where polymerized microtubules were not detected, chromosomes were found forming a disorganized group, but in no case were chromosomes isolated in the cytoplasm observed. In contrast with the present results, another study (Gook et al., 1993Go) reported a 60% rate of normal spindles in oocytes cryopreserved at the MII stage.

Exposure of oocytes to the cryoprotective solutions either at the GV or MII stage at room temperature showed no effect on the spindle and chromosome configurations. A protective effect of PROH at room temperature on the spindle has been suggested (Gook et al., 1993Go). No detrimental effect of 1,2-PROH was found on the chromosomal and spindle normality of GV oocytes exposed (Park et al., 1997Go). In the present study, these findings are confirmed. Therefore the high rate of spindle and chromosome abnormalities observed may be attributed to the cryopreservation itself.

In the literature, the majority of the births after oocyte cryopreservation have been obtained with oocytes frozen at the MII stage. This is an indication that after thawing, a number of oocytes have normal functional spindles that give rise to euploid embryos. A study performed in live oocytes showed that 44.2% of oocytes in which the birefringent spindle was absent at the time of ICSI were, apparently, normally fertilized (Wang et al., 2001Go). It is possible that in some of the oocytes cryopreserved at MII the spindle repolymerizes when returned to culture conditions. In that sense it must be noted that in the present study, surviving oocytes were submitted to a 4 h culture before fixing. In spite of an apparent normal fertilization, unbalanced disjunction could occur resulting in an aneuploid embryo. Aneuploidy could be one of the reasons for the poor performance in vitro and in vivo of embryos derived from frozen oocytes (Ludwig et al., 1999Go). Apart from the reassurances given in a few reports (Gook et al., 1994Go; Cobo et al., 2001Go) (four and 12 embryos analysed respectively) large scale studies stating the chromosomal normality of embryos derived from frozen oocytes are lacking. Preimplantation genetic diagnosis should be part of clinical oocyte freezing programmes.

A deleterious effect on the spindle and chromosome configurations of oocytes cryopreserved using a slow freezing–rapid thawing and 1,2-PROH is described in the present study. New methods of oocyte cryopreservation must be developed. Areas for future research include use of low-sodium cryopreservation media (Stachecki et al., 1998Go; Goud et al., 2000Go) and vitrification (Martino et al., 1996Go; Hong et al., 1999Go; Kuleshova et al., 1999Go; Chung et al., 2000Go). An option for young cancer patients, although it would not contribute to a greater flexibility in the IVF laboratory, may be the cryopreservation of ovarian tissue (Newton et al., 1996Go).


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank Dr Joan Carles Suris for his assistance in data analysis.


    Notes
 
4 To whom correspondence should be addressed at: Servei de Medicina de la Reproducció, Institut Universitari Dexeus, P. Bonanova 89–91, Barcelona 08017, Spain. E-mail: ireboi{at}iudexeus.uab.es Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on January 9, 2002; accepted on March 18, 2002.