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a INRA, Unité Biologie du Développement, 78352 Jouy-en-Josas cedex, France
b UNCEIA, Services Techniques-13, 94703 Maisons-Alfort, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results show that the incidence of chromosomal abnormalities in 2-cell bovine IVF embryos varied from 28% to 44% according to the probes used for the analysis. Whereas the efficiency of FISH was high with somatic nuclei, it appeared to be highly variable with the 2-cell embryos. FISH efficiency depended firstly on the probe sequence (repetitive or unique sequence), secondly on the chromosomal target region (centromeric or telomeric regions), and thirdly on the embryo cell cycle phase.
With a unique sequence probe (19C10) specific to the subcentromeric regions, FISH efficiency was better on nuclei in the S-phase cycle than on those in the G-phase. In S-phase 2-cell embryos, the overall incidence of chromosomal abnormalities was more accurately assessed. It reached 13% and was represented by 1n/2n mixoploidies.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To date, most studies on the chromosomal constitution of embryos of domestic species have been conducted on blastocyst-stage embryos. The incidence of these aberrations has been estimated to be 40% and 20% in in vitro and in vivo bovine blastocysts, respectively [9]. Since the in vitro developmental potential of bovine IVF zygotes depends on the timing of first cleavage, and since delayed cleavage is associated with a poor blastocyst formation rate [10], it is important to evaluate the chromosomal constitution of embryos as soon as the first cleavage occurs. Such an evaluation will provide information about the incidence of chromosomal abnormalities that have occurred in the course of meiosis or at the first mitosis. This is well illustrated in humans by cytogenetic data derived from IVF programs [11, 12] indicating that the incidence of chromosomal aberration in zygotes fertilized in vitro is extremely high and that it declines in the course of development. In this species, a high incidence of polyploidy has been shown to reflect the spontaneous rate of nondisjunction in human gametes and also to be related to the IVF technique itself [13].
In this paper, we assess the effect of the in vitro maturation and fertilization process on the chromosome imbalance that occurs on embryos as early as completion of the first cell cycle. Since the probability of successful karyotyping of embryos is low, and since embryonic nuclei are difficult to arrest in metaphase and the chromosomes produced are short and difficult to band [14, 15], this study utilized the technique of FISH (fluorescent in situ hybridization), which enables the analysis of interphase nuclei. FISH with fluorescent target DNA probes [16] has been developed and is widely used in human clinical practice. It enables the detection of specific DNA sequences at the individual cell level [17, 18]. Because of the availability of a large number of probes specific to each chromosome in humans, the use of FISH is expanding rapidly in this species and has allowed characterization of numerous chromosome abnormalities in preimplantation embryos [19, 20]. This is not yet the case in the bovine species where most of the probes isolated are derived from a unique sequence.
In the present study, we have proceeded to FISH analysis of 2-cell-stage embryos using three different probes, the first from a repetitive sequence recently isolated in our laboratory and the other two derived from a unique sequence. The accuracy of these probes was first established on interphasic and metaphasic somatic cells. The incidence of chromosomal abnormalities was then assessed on 2-cell bovine IVF embryos, and the influence of the cell cycle phase on FISH efficiency was investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cumulus-oocyte complexes (COC) were aspirated from the ovaries of slaughtered cows and matured in M199 medium (Gibco-BRL, Merelbeke, Belgium) supplemented with 10% (v:v) fetal calf serum (FCS), 1% (w:v) FSH-LH, and 1 µg/ml estradiol-17ß for 24 h at 39°C in a humidified 5% CO2 atmosphere. Cumulus-expanded oocytes were then inseminated in vitro with frozen/thawed bovine sperm using the standard technique routinely used in the laboratory [21]. The same batch of frozen semen from a single bull was used throughout the experiment. Modified Tyrode's medium [22] was used for capacitation and fertilization. After thawing, motile spermatozoa were selected by the swim-up technique, concentrated by centrifugation, resuspended to a final concentration of 1 x 106 cells/ml in the fertilization medium containing 1% (w:v) of heparin, and incubated for 18 h at 39°C.
After IVF, the presumptive zygotes were pooled and cumulus cells were removed mechanically by vortexing and pipetting. Denuded zygotes were cocultured under mineral oil in microdrops of B2 medium (CCD, Paris, France), previously seeded with Vero cells [23].
Embryos at the 2-cell stage were removed from the culture microdrops between 24 and 40 h postinsemination. They were fixed according to the Tarkowski technique [24] after a slight modification. In brief, 2-cell embryos were placed in a culture dish containing a hypotonic solution (0.017 M) of trisodium citrate for 1 h at room temperature and then transferred into a minimal volume of the same solution on a clean glass slide. Several drops of glacial fixative acetic acid/methanol (1:3, v:v) solution were poured onto the embryos while the cells were continuously observed under a stereomicroscope. The preparations were observed with a phase-contrast microscope to ensure that the nuclei were not lost and the cytoplasm was dissolved. The position of the nucleus on the slide was marked with a tungsten-carbide-tip pencil, and slides were then kept at 4°C until FISH analysis.
Characterization of the Cell Cycle Phase in 2-Cell Bovine IVF Embryos
Characterization of the cell cycle was achieved by immunocytochemical detection of 5'bromo-2'deoxyuridine (BrdU; Sigma, Saint Quentin Fallavier, France) incorporated in newly synthesized DNA strands. Therefore, embryos were incubated for 1 h at 39°C in PBS containing 10-3 M BrdU, either immediately after the first cleavage or at various time intervals (1, 3, 5, 6, or 7 h) after cleavage was observed. The occurrence of the first cleavage was checked by observation of the presumptive zygotes every 30 min from 20 h postinsemination), and the cleaved eggs were then isolated. For each time interval (1, 3, 5, 6, or 7 h postcleavage), a group of 10 2-cell-stage embryos was treated. The embryos were then washed in PBS for 15 min and fixed as previously described. The nuclei were denatured for 2 min at 70°C in a mixture (v:v) of 70% formamide (Coger, Noisy le Grand, France) and 30% double-strength saline-sodium citrate (SSC; single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate). In order to limit nonspecific labeling, the embryos were covered with 100 µl of 0.1% Triton X-100 (v:v) in PBS for 30 min at 39°C. Slides were then incubated with mouse IgG anti-BrdU antibody (Sigma), diluted at 1:500 (v:v) in PBS containing 2% (v:v) FCS, for 1 h at 39°C. After three intermediate rinses (5, 30, and 5 min) in PBS containing 2% FCS, they were incubated with goat immunoglobulin anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC) antibodies (Sigma) diluted at 1:400 (v:v) in PBS containing 2% (v:v) FCS for 1 h at 39°C. Finally, chromatin was stained with propidium iodide, mounted with antifade (PPD, p-phenylenediamine dihydrochloride; Sigma, France), and observed under a Leica (Milton Keynes, UK) fluorescence microscope equipped with a 490-nm excitation filter and a 550-nm emission filter. Fluorescence detection of the nucleus revealed BrdU incorporation and de novo DNA synthesis (S-phase).
According to the results, three groups of 2-cell embryos at different times after cleavage (< 1 h to 3 h to 7 h) were analyzed by FISH. They correspond to G1, S, and G2, respectively.
FISH
Three different probes (4.5-kilobase [kb] H1A, 37-kb D1S48, and 600-kb 19C10) were used for FISH experiments. Both D1S48 and 19C10 contain unique sequences specific to bovine chromosome 1. D1S48 is a cosmid clone [25] that hybridizes to the subtelomeric regions; 19C10 is a YAC clone [26] that hybridizes in the subcentromeric regions. H1A is a plasmid clone, generated from bovine genomic DNA (unpublished results); it hybridizes to the subcentromeric region of three chromosomes (ch 14, 20, and 25).
The efficiency and accuracy of each probe were first evaluated by analyzing FISH results on metaphase and interphase bovine fibroblast cells derived from a normal 58XX cell line. These cells were prepared using standard procedure [27].
For FISH, a total of 200 ng for H1A and D1S48, and 1 µg for 19C10, were labeled with biotin-14-dATP by nick translation using the Bionick labeling kit (Gibco-BRL). Labeled DNA samples were ethanol precipitated in the presence of 0.1 mg of sonicated herring sperm DNA (Sigma) as carrier and with calf thymus DNA as competitor (Sigma) at the same concentration. Precipitates were dissolved in 20 µl of hybridization mixture of 50% formamide (v:v) in saline-sodium phosphate and 10% (w:v) dextran sulfate (Pharmacia, Orsay, France). Probes were denatured for 10 min at 100°C and then prehybridized for 20 min at 37°C.
Before hybridization, slides were treated with RNase (0.01%, w:v in double-strength SSC) for 1 h at 37°C. They were then rinsed in double-strength SSC for 10 min, dehydrated in 50%, 75%, and 100% ethanol (10 min each), respectively, and air dried before denaturation. Slides were denatured for 2 min at 70°C (in 70% formamide, double-strength SSC), rinsed in glacial double-strength SSC for 2 min before dehydration in 50%, 75%, and 100% glacial ethanol (2 min each), and air dried. A total of 20 µl of denatured probe was deposited per slide, covered by a plastic coverslip, and incubated overnight at 37°C in a moist dark chamber (for H1A and D1S48) or for 3 days (for clone 19C10). After hybridization, the plastic coverslips were removed, and the slides were incubated twice in 50% (v:v) formamide, double-strength SSC at 43°C for 3 min; twice in double-strength SSC at 43°C for 3 min; and then twice in PBT (1 L PBS + 4 ml of 30% [v:v] BSA [Sigma]) + 1 ml of Tween 20 (Sigma) at room temperature for about 10 min. Slides were incubated with 4 µl/ml goat antibiotin (Biosys, Compiègne, France) for 45 min, rinsed twice for 10 min in PBT, and finally incubated with 60 µl/ml anti-goat-FITC (Tebu, Le Perray en Yvelines, France) for 45 min at 37°C. The slides were counterstained with propidium iodide, mounted with antifade, and observed with a fluorescence microscope as previously.
The preparations were examined, and the presence of green spots in the nuclei was noted.
Analytical Criteria
We will refer to FISH efficiency as the percentage of cells with detectable hybridization signals from the total number of nuclei observed. FISH accuracy is defined as the ratio of signals observed in diploid interphasic nuclei to that of the diploid metaphasic nuclei; hybridization signals from metaphases can be scored accurately as we can precisely check the labeled chromosome. According to the probe used and the number of signals detected, the nucleus was classified as shown in Table 1.
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For probe H1A, which hybridized to the subcentromeric regions of three chromosomes, the nucleus was considered diploid (2n), haploid (1n), hypoploid (n-1), or hyperploid (n+1) if it was possible to count 6 (Fig. 1A, 3) (Fig. 1B, 4 or 5) (Fig. 1C), or more than 6 signals, respectively; the embryo was considered diploid, haploid, hypoploid, or hyperploid if we could count 6, 3, 4 or 5, or more than 6 signals, respectively, on each of its two nuclei. When signals were intermingled, the nucleus was considered nonanalyzable.
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For probe D1S48 or 19C10, which hybridized to specific regions of chromosome 1, the nucleus was considered diploid, haploid, or polyploid if it was possible to count 2, 1, or more than 2 (Fig. 1D) signals, respectively, and the embryo itself was considered diploid, haploid, or polyploid if it was possible to count 2, 1 (Fig. 1E), or more than 2 signals, respectively, on each of its two nuclei. Regardless of the probe used, embryos showing a nonequivalent number of signals in the two nuclei were considered mixoploids (Fig. 1F).
All embryos that were diploid were classified as normal. In contrast, all embryos detected as haploid, mixoploid, hypoploid, or hyperploid were classified as abnormal. We made no distinction between haploidy and monosomy for the corresponding chromosomes. In the same way, polyploidy was not distinguished from multisomy.
Statistical Analysis
Significant differences between the mean value of each ploidy category detected by the three different probes were analyzed by the chi-square test. Differences were considered significant for P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ploidy distribution for metaphase and interphase somatic cells, using each of the three probes, is given in Table 2.
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For probe H1A, clear and distinct signals were obtained with the 54 analyzed metaphases, of which 92.5% were diploid (Fig. 1G), 1.8% haploid, and 5.5% hypoploid. All 123 interphasic nuclei of the same preparation exhibited the following distribution: 8 (6.5%) showed an overlapping of hybridization regions and could not be analyzed. For the others (n = 115), the number of signals was scored, showing that 86.0% were diploid, 9.5% were haploid, and 4.3% were hypoploid. It was concluded that the efficiency of probe H1A in both metaphase and interphase nuclei was 100%, since all the cells exhibited detectable hybridization signals; accuracy was 0.9, corresponding to the ratio of the rate of diploid interphasic nuclei to the rate of diploid metaphasic nuclei (86%/92.5%).
The same approach was used with the two other probes, each specific to chromosome 1. With probe D1S48, all 60 analyzed metaphases exhibited clear signals; 95.0% of them were diploid. Counts from 150 interphasic nuclei of the same preparation showed that 144 (96.0%) exhibited hybridization signals and 95.8% were diploid. The efficiency of probe D1S48 in metaphase and interphase nuclei was 100% and 96%, respectively, and accuracy was 1. With probe 19C10, 49 of 51 metaphases showed hybridization signals, and all of them were diploid (Fig. 1H). In interphasic nuclei, hybridization signals were detected in 97 of 105 nuclei, and 97.9% of them were diploid. Thus the efficiency in metaphase and interphase nuclei was 96.0% and 92.3%, respectively, and accuracy was 0.9.
We thus concluded that there was no significant difference in terms of efficiency between the three probes when metaphase plates were analyzed (
2 = 4.58; P > 0.05). For interphasic FISH, probe H1A was more efficient than D1S48 (P < 0.05), but no significant difference was observed between probes D1S48 and 19C10.
With regard to accuracy, no difference was observed in the diploidy rate in metaphase or interphase nuclei for each probe used. However, a higher proportion of abnormal interphase nuclei was detected with probe H1A than with D1S48 or 19C10.
2-Cell-Stage Embryo Analysis
Using probe H1A, hybridization signals were detected in all the analyzed embryos (n = 101). However, only 55.4% exhibited distinct signals that could be scored accurately. With D1S48 and 19C10, 34.6% and 20.9% of the embryos, respectively, exhibited hybridization signals, all of which were clearly detectable. A higher proportion of embryos could thus be analyzed with probe H1A than with D1S48 and 19C10 (P < 0.001), and probe D1S48 made it possible to analyze a higher proportion of embryos than probe 19C10 (P < 0.001). The chromosome constitution of embryos that could be analyzed after FISH is shown in Table 3. Among embryos analyzed with probe H1A, the overall rate of chromosomal abnormalities detected was 44.6%. These abnormalities included 26.7% haploidy, 5.3% hypoploidy, 7.1% hyperploidy, and 5.3% mixoploidy (1n/2n). When FISH was performed with probe D1S48, the total rate of abnormalities was 28.9%, and these abnormalities were represented by haploidy (14.4%), hyperploidy (4.6%), and mixoploidy (9.8%). Using probe 19C10, 36.2% of embryos showed chromosomal abnormalities, of which 30.1% were haploid and 6.0% were mixoploid. Comparison of the results obtained with each of the three probes showed that fewer diploid embryos could be detected by probe H1A than by D1S48 (P < 0.05), but nonsignificant differences in diploid rates could be determined between results obtained by the analysis with probes 19C10 and D1S48. For each of the three probes used, the most frequently detected abnormality was haploidy. This represented 60.0% (15 of 25) versus 50.0% (22 of 44) versus 83.3% (35 of 42) of all the aberrations observed after analysis of embryos with probes H1A, D1S48, and 19C10, respectively. Thus the haploidies were less well detected with probe D1S48 than with 19C10 (P < 0.01) or H1A (P < 0.05).
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FISH According to the Cell Cycle Phase in 2-Cell-Stage Bovine IVF Embryos
Since FISH with probes 19C10 and D1S48 did not reveal any hybridization signal in a high percentage of embryos (79% and 65%, respectively), we assessed FISH efficiency according to the cell cycle phase. This was done because it had been clearly shown in humans that the detection of hybridization signals could be highly dependent on the cell cycle phase. Thus we first characterized the timing of the cell cycle phase during the 2-cell-stage using BrdU incorporation and FITC labeling (see Materials and Methods). When embryos were observed at the occurrence of first mitosis (t = 0, n = 10), no FITC labeling could be detected. In contrast, all nuclei from embryos treated during the first 5 h after cleavage were FITC positive (Fig. 2A). At more advanced timings (6 or 7 h), labeling was only slightly detectable (Fig. 2B). Therefore, we concluded that in our IVF conditions, the S-phase had already started 1 h after the first cleavage and that it lasted for about 5–6 h. During the first hour postcleaving, the embryos were considered to be in the G1-phase. Six hours after cleavage, the G2-phase had already begun in most embryos. FISH efficiency according to the cell cycle was then assessed at less than 1 h (G1-S), 3 h (S), and 7 h (G2) after the onset of cleavage with probes D1S48 (n = 73) and 19C10 (n = 86) (Table 4).
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When FISH was performed with probe D1S48, we did not detect any difference in hybridization efficiency according to the cell cycle phase. By contrast, using probe 19C10, a significantly higher proportion of nuclei showed evidence of hybridization when in the S- or G1-S-phase than when in the G2-phase (P < 0.01).
With probe D1S48, there was apparently no difference in the incidence of chromosomal abnormalities between embryos according to the cell cycle phase of the embryo (Table 5). In contrast, the incidence of chromosomal abnormalities detected by 19C10 (Table 6) in the G2-phase tended to be more important than when observed in S-phase embryos.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We cannot exclude the possibility that the high proportion of chromosomal abnormalities detected in our study could be partly related to the bull used for IVF as reported by Kawarsky et al. [32]. Although we used a single batch of frozen semen for the entire experiment, this batch of semen is known to yield a high IVF rate (95%), in vitro development rate to the blastocyst stage (36%), and a 48% calving rate after transfer of the blastocysts (data not shown). Moreover, the detection rate for chromosomal abnormalities in embryos varied from 20% to 44% according to the probe used. More abnormalities were detected by probe H1A than by 19C10 or D1S48. This was also the case when probes were first tested in somatic cells, with results showing that the incidence of abnormal cells in interphase nuclei detected by probe H1A was higher than that with either probe D1S48 or 19C10 despite the use of the same cell line. This difference, however, is not observed in metaphase analysis. This suggests that the number of abnormal cells detected by probe H1A in interphase nuclei was overestimated and could be due to the overlapping of two adjoining signals that may have been counted as a single signal. Caution is thus warranted in interpretation of the results on 2-cell embryos, particularly since a high proportion of embryos could not be analyzed by probe H1A because of the intermingling of hybridization signals. The hybridization specificity of probe H1A, quite intense, and typical of repetitive sequence probes that are often required as the signals can be easily detected, has not been beneficial in our study. Because this probe hybridizes several chromosomes (n = 6 for 3 chromosome pairs), overlapping signals often occurred, preventing an accurate counting of the hybridization signals. This is in agreement with the results of Viuff et al. [30]. After hybridization with repetitive sequence probes, these authors detected diffuse signals that made it difficult to distinguish between two distinct signals in some nuclei. This drawback was not observed with the other two probes (D1S48 and 19C10). As they were derived from a unique sequence, the hybridization signals were very weak. This advantage is, however, counterbalanced by the fact that the efficiency of signal detection is considerably reduced. Consequently, the hybridization signals could not be detected in a high proportion of embryos. The lack of hybridization signals is usually attributed to a loss of target DNA, poor probe penetration, or incomplete or inefficient hybridization [33]. However, we can partly discard the hypothesis of an artifact of hybridization, since some embryos on the same slide were hybridized whereas others were not, even though the conditions of hybridization were the same. According to Eastmond et al. [33], cellular phenomena can influence the frequency of nuclei that contain 0 or 1 hybridization region. One such phenomenon, as reported in a recent study by Matsuta et al. [34], is related to the chromosomal locations within the nucleus depending on the cell cycle phase. After FISH with a centromeric repetitive probe specific to chromosome 17, and with use of a confocal microscope, these authors demonstrated that the centromeres of both chromosomes 17 were located at the periphery of the nuclei of both G1- and S-phases, and moved toward the center of the nuclei in G2-phase cells. Therefore, the intensity of binding sites in the optical planes was different; this was quite visible during the S-phase, less important in the G1-phase, and hardly distinguishable, or even absent, in the G2-phase.
In our study, the influence of the embryo cell cycle phase on the hybridization rate was tested with both probes D1S48 and 19C10. The interpretation of results was difficult. We found no influence of the cell cycle phase on the embryo hybridization rate with probe D1S48. In contrast, with 19C10, the hybridization rate was significantly higher in embryos at the S-phase than in those at the G2-phase. Thus the variation in the ability of the embryos to be hybridized according to the cell cycle phase should not be discarded. However, this hypothesis has to be taken with caution: it seems to depend on the probe target region, as the results obtained with the two probes were not consistent. Although the probes hybridized to the same chromosome (chromosome 1), they differed in their target region on the same chromosome: it must be kept in mind that D1S48 is a subtelomeric probe while 19C10 is a subcentromeric probe. In human T-lymphocytes, centromeric regions of a dozen examined chromosomes have been localized at the nuclear periphery in the G1-phase [35], whereas telomeric domains are consistently localized within the inner 50% of the nuclear volume. However, it is not yet known whether telomeres remain within the central part of the nuclei throughout the various cell cycle phases. If this is the case, it is evident that we cannot accurately detect hybridization signals with probe D1S48; therefore the risk exists of overestimating the incidence of chromosomal abnormalities. However, we did observe that when using probe 19C10 in embryos at different stages of their cycle phase, the incidence of chromosomal abnormalities detected in S-phase embryos (13.3%) was similar to that reported by Iwasaki et al. [28]. These abnormalities were represented by 1n/2n mixoploid embryos. The fact that only one signal has been detected in one of the two nuclei does not allow us to distinguish between an aneusomy specific for chromosome 1 and a complete haploidy. Even if chromosome 1 is known to be involved in the 1/29 Robertsonian translocation [36, 37] that leads to the trisomy and monosomy for chromosome number 1, we believe that the rate of 13% of chromosomal abnormalities detected in this study rather corresponds to the haploid set of chromosomes. In fact, when we karyotyped a small sample of 2-cell embryos derived from the same IVF batch, we found that 20% (6 of 30) of embryos had haploid nuclei (data not shown). A similar rate was also reported for 2- to 8-cell embryos by Kawarsky [32].
From this study, we can conclude that the performance of the FISH technique for embryo chromosome analysis depends on the type of probe used. The three different probes used in this study made it possible to conclude the following. 1) Repetitive sequence probes like H1A exhibit such bright and large signals that they are easily detected. By contrast, the risk of signal overlapping increases with the number of hybridization regions. 2) Unique sequence probes like D1S48 and 19C10 have the advantage of exhibiting weak signals that reduce the risk of an overlapping, but the intensity of hybridization detection remains weak or even absent in some cell cycle phases such as the G2-phase, with subcentromeric probes resulting in a lower proportion of embryos that can be analyzed.
It would therefore be most appropriate to use repetitive sequence probes that hybridize only to single chromosome. In this case, signal hybridizations would be intense enough to be easily detected in every phase of the cell cycle, and the risk of overlapping would be less important, as the number of spots would be low. Instead, the use of a unique sequence probe, specific to the peri-centromeric regions, could be an efficient tool for detecting chromosomal abnormalities in embryos, but at their S-cycle phase.
Our work demonstrates the feasibility of FISH for analyzing interphasic nuclei of 2-cell-stage IVF embryos. Using a centromeric probe at the S-phase of the embryo offers the most accurate estimation of chromosomal abnormalities. For our in vitro-produced embryos, this was assessed to be 13%.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This research was partly funded by a grant from the European contract CT950190. 
2 Correspondence. FAX: 33 1 34 65 26 77; wafa{at}biotec.jouy.inra.fr
Accepted: October 22, 1999.
Received: August 4, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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