Morphological and cytogenetic analysis of human giant oocytes and giant embryos*

Hanna Balakier1,4, Derek Bouman3, Agata Sojecki1, Clifford Librach1,2 and Jeremy A. Squire3

1 CReATe Program, Inc. and 2 Department of Obstetrics and Gynecology, Sunnybrook and Women's College Health Sciences Center, University of Toronto, Toronto and the 3 University Health Network and Department of Laboratory Medicine and Pathobiology, and Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, Canada


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Giant binuclear oocytes occur with considerable frequency in human ovaries, but their ultimate fate remains unknown. We report the morphology, cytogenetics and developmental potential of human giant oocytes from patients undergoing assisted reproductive technologies. METHODS AND RESULTS: A total of 44 giant oocytes was collected from patients aged 22–44 years old, with an overall frequency of 0.3% (44/14 272 oocytes). Giant oocytes were ~30% larger in diameter than normal oocytes (mean 200.4 versus 154.7 µm, P = 0.0001). Two different morphological patterns were observed among giant unfertilized and fertilized oocytes. All unfertilized oocytes appeared to be diploid and contained either one or two metaphase plates (46 or 2x23 chromosomes), and one or two polar bodies respectively. Consequently, fertilized giant oocytes exhibited either two or three pronuclei, or two or four polar bodies. Both types of giant zygotes were capable of normal cleavage and development to blastocyst stage. Four giant embryos were analysed by interphase fluorescence in-situ hybridization using probes for chromosomes 9, 22, X and Y, and all appeared chromosomally abnormal with numerical alterations indicative of ploidy change. CONCLUSIONS: Giant oocytes might be a possible source of human digynic triploidy. To avoid undesired miscarriages, giant embryos originated from either two- or three-pronuclear giant zygotes should be excluded from uterine transfers.

Key words: digynic triploidy/digyny/diploid oocytes/human triploidy/ploidy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Human triploidy, a pathological entity leading to multiple congenital malformations, is considered to be incompatible with life, and associated with spontaneous miscarriages, development of partial hydatidiform moles and blighted ova (Carr, 1971Go; Jacobs et al., 1978Go, 1982Go; Surti, 1987Go; Warburton et al., 1991Go; McFadden and Pantzar, 1996Go). This numerical abnormality of chromosomal segregation consists of an extra haploid set of chromosomes of either paternal (diandric) or maternal (digynic) origin in the genotype of the affected fetus. The proportion of diandric and dygynic cases is still unclear. However, as observed recently in abortion material, triploids of maternal origin, especially in the fetal period, are far more frequent than those of paternal origin (McFadden and Kalousek, 1991Go; McFadden et al., 1993Go; Dietzch et al., 1995; Miny et al., 1995Go; Baumer et al., 2000Go; McFadden and Langlois, 2000Go). The underlying mechanisms causing the development of these two classes of chromosomal disorder are dissimilar (Austin, 1960Go; Dyban and Baranov, 1987Go; Kaufman, 1991Go). Diandric triploids usually arise through dispermy of a normal ovum and rarely from penetration by a diploid sperm. By contrast, digynic triploids are derived as a consequence of monospermic fertilization of abnormal diploid ovum by a normal haploid sperm. Diploidization of the oocytes may occur due to failure in the extrusion of the first or the second polar body, which results in the retention of an extra maternal chromosomal complement within those cells. Interestingly, in the LT/Sv strain of mouse, when the primary metaphase I oocytes are ovulated, there is a high proportion of triploid offspring after fertilization (O'Neill and Kaufman, 1987). Another possible mechanism of increased oocyte ploidy is a formation of unusually large, diploid oocytes. These giant cells are believed to be produced by a lack of cytokinesis during mitotic divisions of oogonia or by cell fusion of two adjacent oogonia or oocytes at initial, or perhaps even more advance stages of, oogenesis (Austin, 1960Go). The development of fertilized giant, diploid oocytes has been described in some animals, but this phenomenon remains theoretical in humans (Funaki and Mikamo, 1980Go; O'Neil and Kaufman, 1987Go). Although, the error in the emission of the polar bodies during meiosis is proposed as the main causal mechanism of human digynic triploidy, the role of giant oocytes should not be neglected since multinucleate, especially binucleate, oocytes are present with considerable frequency in human ovaries (Kennedy and Donahue, 1969Go; Sherrer et al., 1977Go; Gougeon, 1981Go). The ultimate fate of giant oocytes remains unknown and in general they are assumed to be lost prematurely by atresia before reaching ovulation. For this reason, giant oocytes have not been considered a significant source of diploid oocytes and their involvement in the development of human digynic triploids generally has been overlooked (Gougeon, 1981Go), although some investigators acknowledge their role in this process (Kennedy and Donahue, 1969Go; Dyban and Baranow, 1987; Kaufman, 1991Go; Rosenbush, 2001).

The recovery of sporadic giant oocytes appears to be possible after hormonal stimulation for human assisted reproductive technologies. The occurrence of few individual cases of giant binucleated immature or mature oocytes with double sets of chromosomes has been described recently (Eichenlaub-Ritter et al., 1988Go; Mahadevan et al., 1988Go; Lim et al., 1995Go; Rosenbusch and Schneider, 1998Go; Veeck, 1999Go). Nevertheless, there is limited knowledge concerning the developmental capability, cytogenetics and chromosomal complements of human giant cells. In this study, we took an advantage of a unique opportunity to collect a number of giant oocytes and zygotes during human IVF procedures. The objective of this study was to examine the morphology and numerical chromosomal constitution of such abnormal cells during their maturation and early development.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
IVF procedures
Giant human oocytes were collected from infertility patients undergoing IVF (334 patients) and ICSI (769 patients). The mean age of the women was 34.3 years, range 22–44 years. The aetiology of infertility in the patients was male factor (36%), tubal anomaly (19%), endometriosis (16%), unexplained infertility (10%), polycystic ovarian syndrome (PCOS, 6%) and combination of these factors (13%). All patients were treated with the long protocol for ovarian stimulation. Briefly, the patients received luteal phase GnRH agonist (Lupron; Abbott Laboratories Ltd, Montreal, Quebec, Canada) followed by injections of hMG (Pergonal or Gonal-F; Serono Canada, Inc., Mississauga, Ontario, Canada; and Humagon or Puregon; Organon Canada Ltd, Scarborough, Ontario, Canada). The final stages of follicular maturation were induced by administration of hCG (Profasi HP; Serono), and oocyte retrieval was performed 36–38 h later by means of transvaginal ultrasound-guided aspiration. Oocytes were preincubated for 3–6 h before ICSI procedure or standard in-vitro insemination with 100 000 motile sperm. In the case of ICSI, oocytes were denuded of their surrounding cumulus cells using 80 IU/ml hyaluronidase (Type IV-S; Sigma, St Louis, MO, USA). Oocytes and embryos were cultured under oil (Sigma) in IVC-One medium (In Vitro Care, Inc., San Diego, CA, USA) supplemented with 10% synthetic serum supplement (SSS; Somagen, Edmonton, Alberta, Canada) in an atmosphere of 5% CO2, 5% O2 and 90% N2. Fertilization was confirmed 16–20 h post IVF and ICSI by the presence of two pronuclei (2PN) and two polar bodies (2PB). Embryo transfer was usually performed 72 h (day 3), and sometimes 120 h (day 5), after oocyte retrieval. Micronized vaginal progesterone (Medicine Shoppe, Toronto, Canada) or progesterone in oil (Cytex Pharmaceuticals Inc., Halifax, Canada) was used for luteal support.

Giant oocytes and embryos
Giant fertilized and unfertilized oocytes were identified either during the fertilization assessment of in-vitro inseminated oocytes or during a course of cumulus cell removal just before the ICSI procedure. The abnormally large oocytes were examined for the presence and number of the nuclei and polar bodies under an inverted microscope at x320 magnification (Leitz DMIL, relief contrast; Leica, Richmont Hill, Ontario, Canada). The diameters of giant oocytes and their normal siblings, serving as a control, were also measured. Additionally the volumes of cells were calculated according to the formula; V = (4/3)r3 (results in microns3, µm3). Patients consented to the chromosomal investigation of their giant cells. When the consents were not obtained, the abnormal oocytes of those patients were not submitted for further analysis.

Chromosomal preparations of unfertilized giant oocytes were performed usually 5 h and in some cases 24 h after ovum retrieval, according to a published method (Kamiguchi et al., 1993Go). The zona pellucida was removed by acid Tyrode solution (pH 2; Sigma) and the oocytes were treated with hypotonic sodium citrate (1%), followed by gradual fixation and staining with Giemsa.

Two-colour interphase fluorescence in-situ hybridization (FISH) analysis was applied to investigate the ploidy of embryos derived from giant zygotes. The fixation and FISH techniques used were as described previously (Liu et al., 1998Go). Two commercial probe sets were used to evaluate the genetic constitution of giant embryos. The first probe set (Ventana Medical Systems, Tucson, AZ, USA) was a cocktail that hybridizes with the ABL, gene on chromosome 9 at band 9q34 (green fluorescent) and also hybridizes with the BCR gene on chromosome 22 at band 22q11.2 (red fluorescence). The second probe set used to evaluate the sex chromosome constitution of the embryos was a commercially available cocktail probe (Vysis, Inc., Downers Grove, IL, USA) that hybridizes with the centromeric region of chromosome Y (green fluorescence) and with the centromeric region of chromosome X (red fluorescence). The slides were denatured at 70°C in 70% formamide/2xSSC for 5–15 s and hybridized overnight. Then they were washed in 0.4xstandard saline citrate (SSC)/0.3% NP-40 at 70°C for 2 min followed by 2xSSC/0.1% NP-40 at room temperature for 1 min and subsequently counterstained with 4,6-diamino-2-phenylindole (1.5 mg/ml). The probe sets were applied sequentially. Slides were evaluated on an epifluorescence microscope (Zeiss; Don Mills, Ontario, Canada) with software and hardware provided by Applied Imagining (Santa Clara, CA, USA). Hybridization sensitivities with all four probes demonstrated an excellent signal:noise ratio, low background and an efficiency of >98% using normal control lymphocyte preparations.

Statistical analysis
Statistical analysis was performed using two-sample t-test to compare the diameters of giant and control, normal oocytes. Two-way ANOVA was also applied to evaluate the significant differences in the estradiol (E2) levels and the number of retrieved eggs between the patients that possessed and those that did not have giant oocytes. P < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A total of 44 giant oocytes from 43 patients (one patient had two oocytes) were detected among 14 272 oocytes that were retrieved from 1103 patients (Figure 1Go). This frequency measured ~0.3% of the occurrence of abnormally large cells in the study. Twenty-four of the giant oocytes were derived from women aged 22–35 years, 15 from 36–39 years and five from women >=40 years. They were found in the patients of all aetiological groups of infertility problems: male factor (n = 19), tubal abnormality (n = 7), PCOS (n = 5), unexplained (n = 4), endometriosis (n = 3) and combination of these factors (n = 5). Table IGo contains the information regarding the number of retrieved oocytes and the level of E2 at the day of hCG for the group of 43 patients that had, and those who did not have, giant oocytes (n = 1060). The overall mean number of oocytes/patient (19.4 versus 12.6, P = 0.007) and the mean E2 value (2803 versus 1786 pg/ml, P = 0.002) were significantly higher in the patients with giant oocytes than in the patients without those cells. Additionally, in general the patients aged <40 years compared with those >=40 years had higher levels of E2 (P = 0.007) as well as increased number of oocytes (P = 0.03).



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Figure 1. (A) A giant binucleated oocyte at the germinal vesicle stage (left) containing two nuclei in contrast to and a normal, sibling oocyte with one nucleus (right). (B) A giant metaphase II (MII) oocyte with one polar body (left) and a normal MI oocyte without the polar body (right). (C) A giant, MII oocyte with two polar bodies (left) and a normal MII oocyte with one polar body (right). (D) A giant `normal type' zygote showing two pronuclei and two polar bodies. (E) A giant `polyploidy type' zygote with three pronuclei and two polar bodies (left) and a normal zygote with two pronuclei and two polar bodies (right). Note several small vacuoles in a giant zygote. (F) A giant `polyploidy type' zygote containing three pronuclei (one significantly smaller) and four polar bodies.

 

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Table I. Age, number of oocytes and estradiol (E2) value for patients with and without giant oocytes
 
The giant oocytes were ~30% or 1.3-fold larger than normal ones (P = 0.0001) and sometimes showed distinct oval shape (Figure 1Go). The mean diameters were respectively 147.73 µm (SD = 8.09), when measured without the zona pellucida (i.e. just vitellus alone) and 200.4 µm (SD = 11.6) when the zona pellucida was included. This contrasted with similar measurements for their normal counterparts (113.79 and 154.66 µm; SD = 5.45 and 6.71). In consequence, the volume of giant oocytes was estimated to be ~1.6-fold larger than normal and their mean values varied between 19 223 721 and 42 018 181 µm3 (without and with zona pellucida respectively). The mean volume of normal oocytes was calculated to be between 13 463 367 and 26 664 263 µm3 respectively.

Table IIGo presents the stage of chromosomal constitution of giant oocytes at retrieval, and after fertilization/in-vitro culture. Among 29 unfertilized giant oocytes, 12 were immature and had either two germinal vesicles (GV; n = 7; Figure 1AGo) or were at metaphase of the first meiotic division (MI; n = 5) and did not display any nuclei and polar bodies. The remaining 17 giant cells were classified as mature oocytes that were arrested at the second meiotic division (MII). Seven of them possessed one polar body, which is considered to be normal, and the others had two polar bodies (Figure 1B, CGo). The polar body size was similar to those observed in the control mature oocytes. Chromosomal preparations from four MI oocytes indicated the presence of two separate metaphase plates in each oocyte. In two cases, individual plates contained haploid number of 23 chromosomes, and in two others the chromosomes were compact and because of overlaps accurate numerical counts were not possible. Analysis of four MII mature oocytes that contained only one polar body revealed the presence of a single diploid metaphase plate comprising 46 chromosomes (Figure 2AGo). Among seven, MII oocytes displaying two polar bodies, four oocytes showed double sets of 23 chromosomes (Figure 2BGo), one oocyte had two compact groups of chromatin, and two oocytes contained a common group with 46 chromosomes.


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Table II. The morphology and ploidy of giant oocytes and zygotes
 


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Figure 2. The chromosomal plates from two different types of giant metaphase II oocytes. (A) One diploid meiotic plate containing 46 chromosomes. (B) Two chromosomal plates with 23 chromosomes on each plate.

 
In accordance with the two types of MII oocytes, two morphologically distinct types of giant zygotes were also observed (Table IIGo). In the first group, apart from their large size, the eight giant zygotes looked normal and possessed two pronuclei and two polar bodies, (Figure 1D, EGo). In the second group, seven giant zygotes were polyploid and contained either three (n = 6) or four (n = 1) pronuclei and four or two polar bodies respectively (Figure 1E, FGo). During 72 h of in-vitro culture, embryos derived from both types of giant zygotes underwent normal cleavage divisions, reaching the stage of 6–12 cells (n = 15). In general they were of good quality (equal cells, low fragmentation), although ~40% of them contained micronuclei in some blastomeres (Figure 3AGo). Three out of six embryos cultured for 120–144 h formed high grade blastocysts (Figure 3B–EGo), one embryo was arrested at morula stage (20–22 cells) and two stopped development at 10–12 cells. The other embryos (9/15; Table IIGo) that were cultured for only 3 days were used for FISH analysis or were frozen according to the patient's request.



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Figure 3. (A) One giant 2-cell stage embryo exhibiting multiple subnuclei in both blastomeres (left) and two normal mononucleated embryos (right). (BE) Four pictures showing a giant embryo (left) obtained after IVF procedure (many sperm on zona pellucida), and a normal embryo after ICSI (right) during subsequent development from day 3 to day 6.

 
Four out of six giant embryos were successfully analysed using interphase FISH probes for chromosomes 9, 22, X and Y, and additional signals indicative of an abnormal chromosome constitution were apparent (Table IIIGo, Figure 4Go). The embryos were in general mosaic for the chromosomes tested and in some nuclei the presence of trisomy for all four chromosomes was apparent and suggested triploid cells may be present. In other nuclei, a mixed chromosome constitution was detected, indicative of double trisomies or a more complex constitution. However, the vast majority of the nuclei were uniformly XXY or XXX (Table IIIGo), indicating that 3n was probably the most prevalent ploidy in these cells.


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Table III. Interphase fluorescence in-situ hybridization (FISH) analysis of nuclei derived from human giant zygotes
 


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Figure 4. Fluorescent in-situ hybridization (FISH) analysis of nuclei from human embryo derived from `normal' type of giant zygote (2PN, 2PB). Sequential application of probes to the nuclei from embryo no. 1 (Table IIIGo). Only four representative nuclei of this embryo are shown. (A) Green signals indicate chromosome 9, and red signals indicate chromosome 22. (B) The probe from preparation shown in (A) was removed and FISH was performed with the second probe cocktail. Red signals indicate chromosome X, and green signals indicate chromosome Y. Note that the majority of nuclei are triploid for all chromosomes investigated. Some nuclei are mosaics.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The occurrence of giant oocytes is considered a relatively rare event that is attributed to some invertebrates (sea-urchin, anura) and several mammalian species, including humans (Austin and Braden, 1954Go; Austin, 1961Go; Uzzell et al., 1975Go; Berger and Uzzell, 1977Go; Funaki and Mikamo, 1980Go; Rosenbusch and Scheider, 1998). In animals, giant oocytes may appear as binuclear or mononuclear cells, and the embryos resulting from their fertilization are triploid. The incidence of post-ovulatory giant oocytes in hamster, rabbit, rats and mice has been found to vary from ~0.1 to ~0.5% (Austin and Braden, 1954Go; Austin, 1961Go). The observed frequency in the present study of 0.3% of human giant oocytes retrieved during IVF procedures in women of all ages (22–44 years old) is therefore in a similar range to that observed in the above mammals. This is also consistent with the fact that such abnormally large oocytes, especially binuclear, persist in human ovaries throughout reproductive life in most adult women (18–52 years old) and their frequency oscillates between 0.2 and 1.03% (Kennedy and Donahue, 1969Go; Sherrer et al., 1977Go; Gougeon, 1981Go). Although our data is limited to 44 giant oocytes, it seems that ovarian development of those cells and their recurrence during IVF procedures may be linked with augmented response to gonadotrophin therapy. As we recorded, the patients with giant oocytes exhibited significantly higher E2 levels (P = 0.002) and had the increased number of retrieved oocytes (P = 0.007), when compared with the patients that did not have those abnormal cells. It is also worth mentioning that hormonal stimulation has previously been suggested to increase the occurrence of human multinucleated ova (Kennedy and Donahue, 1969Go) and to increase the incidence of triploidy in human spontaneous abortuses (Boue et al., 1973Go) as well as in rabbit and mouse embryos (Fujimoto et al., 1974Go; Takagi and Sasaki, 1976Go).

Our findings also revealed the existence of two different morphological types of human giant unfertilized and fertilized oocytes. All giant, immature GV oocytes were binucleated and MI/MII oocytes appeared to be diploid. About half of MII oocytes contained a single, diploid group of chromosomes while another half displayed two separate haploid sets of chromosomes, resulting in a diploid state as well. Consequently, giant fertilized oocytes, obtained after standard in-vitro insemination, showed normal or typical polyploid number of pronuclei. In the first case, giant zygotes resembled normal ones with two pronuclei and two polar bodies. In contrast, polyploid types of zygotes contained three or four pronuclei and four or two polar bodies. Thus, this indicates that giant zygotes showing normal nuclear morphology derive from fertilization of giant MII oocytes with a single set of chromosomes, whereas atypical polyploidy zygotes are formed due to monospermic or dispermic fertilization of giant oocytes possessing either one (3PN, 2PB, dispermic) or two (3 or 4PN, 4PB) groups of meiotic chromosomes. Interestingly, cytogenetic studies on hamster have shown that although both mononuclear and binuclear giant immature eggs can be found, the binucleate state is rarely maintained to MII (Funaki and Mikamo, 1980Go; Funaki, 1981Go). For this reason, the majority of hamster giant MII oocytes contain a diploid set of chromosomes arranged in a single spindle and accordingly most giant zygotes exhibit a normal number of pronuclei comprising one female and one male pronucleus. This behaviour contrasts with that of human giant oocytes in which both chromosomal anomalies occurred with similar frequency while binuclear germinal vesicle oocytes were exclusively observed. Interestingly, mouse giant oocytes produced by cell fusion (Karnikova et al., 2000Go) revealed distribution patterns of metaphase plates and polar bodies (one or two plates and, respectively, one or two polar bodies) similar to our observations in human giant oocytes.

In this study, human giant oocytes demonstrated full viability during the preimplantation period. Fertilization was compromised (15 giant oocytes from IVF procedure) and thereafter giant zygotes cleaved normally and some were capable of reaching blastocyst stage (Figure 3Go). On the other hand, giant embryos showed numerical chromosomal abnormalities and FISH analysis suggested the presence of triploid or tetraploid nuclei as well as different types of mosaics. It should be noted that, in a previous report on humans, four abnormally large embryos were identified as triploid or triploid mosaic by means of FISH involving chromosomes 18, X and Y, and it was concluded that these embryos most likely originated from diploid oocytes (Munné et al., 1994Go). Indeed, based on our observations, it is evident that those atypical embryos could develop from giant zygotes containing 2PN, 2PB. The preimplantation development of giant oocytes into triploid 4-cell or blastocyst stage embryos has also been described in the past in hamster and rat (Funaki, 1981Go). Taking into consideration the obvious chromosomal abnormalities present in giant oocytes and embryos, we conclude that all giant cells obtained during IVF procedures should be excluded from uterine transfers.

Although there is no a single proven human digyny originated in vivo from giant oocytes, the contribution of diploid oocytes to human triploidy cannot be completely excluded. First, giant oocytes exist in human ovaries and show full developmental capability when retrieved and cultured in vitro. Second, in contrast to early studies that were based on the analysis of cytogenetic heteromorphisms (Kajii and Nikawa, 1977; Jacobs et al., 1982Go; Procter et al., 1984Go; Uchida and Freeman, 1985Go), several recent reports on the aetiology of human aborted material have revealed that digyny rather than diandry is the most common cause of human triploidy (Dietzsch et al., 1995Go; Miny et al., 1995Go; Baumer et al., 2000Go; McFadden and Langlois, 2000Go). However, in the light of the complex allelic segregation patterns possible for giant oocytes, it is apparent that to distinguish between triploids caused by meiotic failure and those derived from giant diploid gametes would require comprehensive analysis with various genetic markers. Otherwise triploids could be mistakenly interpreted as suppression of the first or second polar bodies because a similar distribution of maternal alleles could take place. The investigation of the involvement of giant diploid oocytes in the development of human triploids remains an intriguing challenge for future genetic studies.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The material of this study was collected at the START IVF clinic and we would like to thank the staff for their kind assistance. We also would like to thank Jana Karaskova for her technical help.


    Notes
 
* The results of this study were presented at the 57th Annual Meeting of the ASRM, Orlando, Florida, USA, 2001. Back

4 To whom correspondence should be addressed at: CReATe (Canadian Reproductive Assisted Technology) Program, Inc., 790 Bay Street, suite 1020, Toronto, Ontario M5G 1N8, Canada. E-mail: hbalakier{at}sympatico.ca Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on February 8, 2002; accepted on May 10, 2002.