Elevation of insulin-like growth factor-binding protein-1 mRNA expression following hormone replacement therapy

Hsin-Shih Wang1, Tzu-Hao Wang and Yung-Kuei Soong

Department of Obstetrics & Gynecology, Chang-Gung Medical College, Chang-Gung Memorial Hospital, Lin-Kou Medical Center, Taipei, Taiwan, Republic of China


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The purpose of this study was to investigate the possible regulatory roles of insulin-like growth factors (IGF) and IGF binding protein-1 (IGFBP-1) on postmenopausal endometrium in women undergoing hormone replacement therapy (HRT). Endometrial tissues and blood samples were collected from 25 postmenopausal women with abnormal uterine bleeding before and after HRT. As a control, blood and endometrial samples were also obtained at mid-luteal phase (days 18–23 of the menstrual cycle) from 10 women with benign uterine leiomyoma during surgical intervention. Expression of mRNA for IGF-I, IGF-II, type 1-IGF receptor, IGFBP-1, oestrogen receptor (ER) and progesterone receptor (PR) in the endometrium was explored by a semiquantitative reverse transcription–polymerase chain reaction (RT-PCR). Serum concentrations of IGFBP-1, oestradiol, progesterone, follicle stimulating hormone (FSH) and sex hormone-binding globulin (SHBG) were also determined. In the endometrium obtained from postmenopausal women with abnormal uterine bleeding (before HRT), expression of IGFBP-1 was undetectable, whereas PR expression was abundant. HRT significantly up-regulated expression of IGFBP-1 but down-regulated PR. Moderate expression of IGF-I, IGF-II, type 1-IGF receptor and ER was identified in the postmenopausal endometrium before HRT. HRT significantly down-regulated IGF-I, up-regulated IGF-II, but had no effects on expression of type 1-IGF receptor and ER. In conclusion, up-regulation of IGFBP-1 collaboratively with down-regulation of IGF-I may account for the protective effect of progesterone in HRT on the endometrium.

Key words: endometrium/hormone replacement therapy/IGF/IGFBP-1/progesterone


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Insulin-like growth factor-binding protein-1 (IGFBP-1) is one of the specific binding proteins for insulin-like growth factors (IGF-I and IGF-II) (Wang et al., 1991Go; Wang and Chard, 1992Go) which exert the growth-promoting effects under the control of growth hormone (GH) through autocrine or paracrine mechanisms (Holly and Wass, 1989Go). Ontogenetically, synthesis of IGFBP-1 is regulated by GH (Norrelund et al., 1999Go), progesterone (Rutanen et al., 1997Go) and insulin (Suikkari et al., 1989Go). In the follicular phase of ovulatory cycles, endometrial growth of the uterus is promoted by ovarian oestradiol via local production of IGF-I, which in turn stimulates cell proliferation of the endometrium (Giudice et al., 1993Go). In the luteal phase, progesterone is prominent and stimulates the endometrium to secrete IGFBP-1, which promotes cell differentiation of the endometrium and modulates IGF-1 function (Rutanen and Pekonen, 1991Go). Although circulating IGFBP-1 concentrations decrease during the luteal phase (Wang et al., 1995Go), a striking increase in local production of IGFBP-1 by the endometrium in the presence of high progesterone concentrations has been observed (Rutanen and Pekonen, 1991Go).

IGFBP-1 mRNA is not detectable in the proliferative endometrium but is present in stromal cells of the secretory endometrium (Zhou et al., 1994Go; Rutanen et al., 1997Go). In addition, progesterone induces production of IGFBP-1 by proliferative phase endometrium and stimulates its secretion from secretory phase endometrium (Rutanen and Pekonen, 1991Go; Wang and Chard, 1999Go). Collectively, IGFBP-1 expression is believed to be only in secretory phase endometrium. On the other hand, IGF receptors are present in human endometrium throughout the menstrual cycle (Rutanen and Pekonen, 1991Go). It has been shown that IGFBP-1 inhibits receptor binding of IGF-I to endometrial membranes, suggesting a role as a paracrine inhibitor of IGF action (Rutanen and Pekonen, 1991Go). Recent studies have also demonstrated that the endometrium expresses IGF, and both the growth factors and their receptors in the endometrium are stimulated by oestrogen (Murphy and Ghahary, 1990Go).

Hormone replacement therapy (HRT) relieves climacteric syndromes in perimenopausal women and benefits postmenopausal women through prevention of cardiovascular diseases and osteoporosis, having been accepted as a treatment to improve the health of menopausal women. Addition of progesterone into HRT regimens has been shown to protect the endometrium from hyperplasia and carcinoma (Wood, 1994Go; Writing Group, 1995). Immunoreactive IGFBP-1 has been detected in the endometrium after treatment with a combination of oestrogen and progesterone (Suvanto-Luukkonen et al., 1995Go). In addition, it has previously been reported that treatment with progesterone increases serum concentrations of IGFBP-1 (Wang and Soong, 1996Go). However, the mechanism of protective effects on the endometrium by progesterone and the role of IGFBP-1 in postmenopausal women undergoing HRT are not completely clear.

To investigate further the possible regulatory roles of IGF and IGFBP-1 on postmenopausal endometrium after HRT, expression of mRNA for IGF-I, IGF-II, type 1-IGF receptor, IGFBP-1, oestrogen receptor (ER) and progesterone receptor (PR) in the endometrium was explored by a semiquantitative reverse transcription–polymerase chain reaction (semiquantitative RT-PCR). In addition, serum concentrations of IGFBP-1, oestradiol, progesterone, follicle stimulating hormone (FSH) and sex hormone-binding globulin (SHBG) were also determined by radioimmunoassays and immunofluorometric assays.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects and protocols of HRT
Twenty-five postmenopausal women with abnormal uterine bleeding were recruited to this study. The age of patients ranged from 49 to 55 years. The body mass index [(body weight)/(body length)2; kg/m2] was 26.1 ± 2.8 (mean ± SD). The interval between the last uterine withdrawal bleeding and the present uterine bleeding or spotting was 19.4 ± 4.7 months (mean ± SD). On the first visit to the gynaecological outpatient clinic, the thickness of the uterine endometrium was measured by transvaginal ultrasonography. Blood samples and endometrial tissue were simultaneously collected. Women with dysplastic or malignant lesions of the endometrium were excluded from the present study. Following the first evaluation, these patients were treated with HRT using period-free conjugated oestrogens (Premarin®, 0.625 mg/day; Wyeth-Ayerst Canada Inc., Montreal, Canada) and medroxyprogesterone acetate (Provera®, 5 mg/day; Pharmacia and Upjohn NV/SA, Puurs, Belgium). As a control, blood and endometrial samples were obtained at luteal phase (between days 18 and 23 of the menstrual cycle) from 10 women with benign uterine leiomyoma during surgical intervention. Informed consent was obtained from each woman.

Collection of samples
Samples of blood were collected at the time of endometrial sampling (before HRT, and 6 and 12 months after HRT). Serum samples were stored at –20°C until assays for circulating oestradiol, progesterone, SHBG and FSH. Collection of endometrial samples was performed following cervical dilatation under general anaesthesia (i.v. injection of fentanyl and propofol). A part of endometrial tissue was sent to the pathology department for histological evaluation. The rest of the endometrial tissue was isolated from contaminated red blood cells (RBC) by density gradient centrifugation in 50% Percoll at 800 g for 15 min followed by immediate weighing and RNA extraction.

Assays for oestradiol, progesterone, FSH and SHBG
To verify the postmenopausal hormone status, serum concentrations of oestradiol and progesterone were determined by immunofluorometric assays (IFMA) (Pharmacia, Turku, Finland). The detection limit of the oestradiol assay was 13.6 pg/ml. The intra-assay coefficients of variation for oestradiol were 5.7% at 36 pg/ml and 3.1% at 91 pg/ml (n = 10). The inter-assay coefficients of variation for oestradiol were 8.4% at 36 pg/ml and 5.6% at 91 pg/ml (n = 6). The minimum detection limit of the assay for progesterone was 0.1 ng/ml. The intra-assay coefficients of variation for progesterone were 2.5% at 1.5 ng/ml and 6.8% at 11 ng/ml (n = 10). The inter-assay coefficients of variation for progesterone were 5.9% at 1.5 ng/ml and 9.3% at 11 ng/ml (n = 6).

Serum concentration of FSH was measured by radioimmunoassays using a commercial kit (Nichols Institute Diagnostics, San Juan Capistrano, CA, USA). The lowest measurable concentration for FSH was 0.5 mIU/ml. The intra-assay coefficients of variation for FSH were 4.5% at 2.5 mIU/ml and 8.7% at 6.9 mIU/ml (n = 12). The inter-assay coefficients of variation for FSH were 8.7% at 2.5 mIU/ml and 13.3% at 6.9 mIU/ml (n = 22).

Concentrations of SHBG were determined by an immunoradiometric assay (IRMA) using a commercially available kit (Diagnostic Systems Laboratories, Inc., Webster, TX, USA). The minimum detection limit for SHBG was 3 nmol/l. The intra-assay coefficients of variation for SHBG were 4.2% at 60 nmol/l and 8.8% at 130 nmol/l (n = 8). The inter-assay coefficients of variation for SHBG were 9.4% at 60 nmol/l and 13.3% at 130 nmol/l (n = 12).

Extraction of RNA
Total RNA in the endometrium was isolated using a guanidium thiocyanate-phenol-chloroform procedure (Chomczynski and Sacchi, 1987Go). Briefly, homogenized endometrial tissue was lysed in 0.5 ml denaturing solution (4 mol/l guanidium thiocyanate, 25 mmol/l sodium citrate, 0.5% sarcosyl, 0.1 mol/l 2-mercaptoethanol) followed by the addition of 0.05 ml of 2 mol/l sodium acetate, 0.5 ml of phenol and 0.1 ml of chloroform/isoamyl alcohol (49:1). After vigorous shaking, the tubes were incubated on ice for 15 min and centrifuged at 10 000 g at 4°C for 20 min. The aqueous layer was collected followed by an addition of equal volume of cold isopropanol. RNA was pelleted, re-suspended in 0.3 ml of denaturing solution and re-precipitated with isopropanol. The RNA pellet was then washed twice with 75% ethanol and dissolved in diethyl pyrocarbonate (DEPC)-treated water. The amount of RNA was quantified spectrophotometrically and stored at –70°C until analysis.

Semiquantitative RT-PCR with multiple primers
In semiquantitative RT-PCR, two sets of primers were used simultaneously in each tube (one for target mRNA and the other for ß-actin mRNA used as an internal control) (Table IGo). Aliquots of 2 µg of total RNA from each case were used to run reverse transcription using a Gene Amp RNA PCR kit (Perkin-Elmer Cetus, AT, USA). In each tube, total RNA was reversely transcribed with murine leukaemia virus (MuLV) reverse transcriptase (50 IU) as well as the downstream primers for both ß-actin mRNA and one of target mRNA in a final volume of 40 µl. Using a DNA thermal cycler (Perkin-Elmer Cetus), reagents were incubated at 42°C for 15 min, heated to 99°C for 1 min to denature the MuLV reverse transcriptase, rapidly cooled to 4°C and stored at 4°C.


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Table I. Primers for human mRNA used in reverse transcription–polymerase chain reaction (RT-PCR)
 
The cDNA pools from the reverse transcription reaction were subjected to 25 cycles of PCR containing second primers for both ß-actin mRNA and one of target mRNA. Each cycle consisted of 94°C for 1 min (denature); 60°C (annealing temperature for IGF-I, IGF-II, and type 1-IGF receptor) or 55°C (annealing temperature for IGFBP-1, ER and PR) for 1 min; and then extension for 2 min at 72°C. After 25 cycles, the mixtures were treated at 72°C for 7 min, then cooled rapidly and stored at 4°C.

Following amplification, 15 µl of the reaction products mixed with 3 µl of loading dye [0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol and 30% (v/v) glycerol in water] was applied to a 2% agarose (Promega Corporation, Madison, WI, USA) gel containing a minimal amount of ethidium bromide. Molecular weight standards included 1 µg of `100 bp DNA Ladder®' (Fermentas Ltd, Vilnius, Lithuania). At the end of electrophoresis, the intensity of RT-PCR products was visualized on a UV box and analysed by using an image analyser (Gel Doc 1000, Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analysis
Changes in serum hormonal concentrations and intensity of PCR products were statistically analysed using Student's t-test (two-tailed) for paired and unpaired data. In all cases, P values <0.05 were considered to indicate significance.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A significant decrease in the thickness of the uterine endometrium measured by transvaginal ultrasonography was identified after HRT (4.1 ± 0.5 mm; mean ± SD) as compared to that before HRT (8.5 ± 2.2 mm; mean ± SD) [P < 0.00001, t = 9.75, degrees of freedom (d.f.) = 48]. Before HRT, three out of 25 postmenopausal women with abnormal uterine bleeding exhibited histologically simple hyperplasia of the endometrium. After 6 and 12 months of HRT, none of the women studied developed endometrial hyperplasia. Abnormal uterine bleeding of these patients subsided after HRT.

Elevated serum oestradiol concentrations were found 6 and 12 months after HRT in comparison with those before HRT (P < 0.0005). Similarly, circulating concentrations of SHBG 6 and 12 months after HRT were higher than those before HRT (P < 0.02 and P < 0.01 respectively). In addition, substantial elevation in serum concentrations of progesterone was found 6 and 12 months after HRT (6.7 ± 2.3 and 7.3 ± 2.8 ng/ml; mean ± SD). By contrast, decreased serum FSH was detected 6 and 12 months after HRT (P < 0.01 and P < 0.005 respectively) (Table IIGo).


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Table II. Serum hormone concentrations in controls and alteration before and after hormonal replacement therapy (HRT). Values are means ± SD
 
In semiquantitative RT-PCR, a set of primers for ß-actin mRNA was used simultaneously in each reaction tube as an internal control in RT-PCR to verify that the amounts of target mRNA (e.g. IGFBP-1, IGF-I, IGF-II, IGFR-1, ER and PR) analysed were on the basis of the same amount of total RNA. In order to determine if the number of cycles of PCR following reverse transcription (RT) was appropriate for the present study, serial dilutions (1:50, 1:100, 1:1000, and 1:10 000; in distilled water) of total RNA from pooled endometrial cells were subjected to different numbers of PCR cycles (20, 25, 30 and 35 cycles) using ß-actin primer sets. The linear correlation between intensity and RNA dilution in logarithm scales was only observed in PCR products after 25 cycles (data not shown). No such correlation was detected in PCR products after 20, 30 and 35 cycles. Thus, 25-cycle PCR was used as a standard procedure for the entire study.

After amplification with semiquantitative RT-PCR, a band at 838 bp on the agarose gel represented the expression of ß-actin mRNA (Figures 1 and 2GoGo). In the endometrium obtained from postmenopausal patients with abnormal uterine bleeding before HRT, expression of IGFBP-1 mRNA (bands at 417 bp) was not detected using semiquantitative RT-PCR, whereas substantial expression of IGFBP-1 mRNA was detected after HRT (Figures 1, 2 and 3GoGoGo). In contrast, expression of progesterone receptor (PR) mRNA (bands at 630 bp) was abundant before HRT while it became undetectable after HRT (Figures 1, 2 and 3GoGoGo). In addition, elevated expression of IGF-II mRNA (bands at 538 bp) and decreased expression of IGF-I mRNA (bands at 514 bp) by semiquantitative RT-PCR were observed after HRT (P < 0.0005 and P < 0.00001 respectively) (Figure 3Go). In the endometrium from both controls and postmenopausal women undergoing HRT for 12 months, findings after semiquantitative RT-PCR were similar to those 6 months after HRT (Figure 2Go).



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Figure 1. Agarose gel (2%) electrophoresis of polymerase chain reaction (PCR) products of insulin-like growth factor binding protein-1 (IGFBP-1), IGF-I, IGF-II, type 1-IGF receptor, oestrogen receptor and progesterone receptor in the endometrium from a postmenopausal woman with abnormal bleeding before hormone replacement therapy (HRT). A band corresponding to the PCR products of ß-actin (838 bp) is shown in each lane. A 630 bp band corresponding to the PCR products of progesterone receptor is shown in lane 6. However, no band corresponding to the PCR products of IGFBP-1 (417 bp) was detected in lane 1. In addition to the internal control (ß-actin) in the upper band, PCR products shown are: IGFBP-1 (lane 1); IGF-I (514 bp, lane 2); IGF-II (538 bp, lane 3); type 1-IGF receptor (540 bp, lane 4); oestrogen receptor (532 bp, lane 5); and progesterone receptor (lane 6). Molecular markers (100-bp ladder) are shown in lane M.

 


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Figure 2. Agarose gel (2%) electrophoresis of PCR products of IGFBP-1, IGF-I, IGF-II, type 1-IGF receptor, oestrogen receptor and progesterone receptor in the endometrium from a postmenopausal woman undergoing hormone replacement therapy (HRT) for 6 months. A band corresponding to the PCR products of ß-actin (838 bp) is shown in each lane. A 417 bp band corresponding to the PCR products of IGFBP-1 is shown in lane 1. By contrast, no band corresponding to the PCR products of progesterone receptor (630 bp) was detected in lane 6. In addition to the internal control (ß-actin) in the upper band, PCR products shown are: IGFBP-1 (lane 1); IGF-I (514 bp, lane 2); IGF-II (538 bp, lane 3); type 1-IGF receptor (540 bp, lane 4); oestrogen receptor (532 bp, lane 5); and progesterone receptor (lane 6). Molecular markers (100-bp ladder) are shown in lane M. Figures 1 and 2GoGo show data from the same patient before and after HRT. Similar trends in changes of gene expression were observed in all patients included in this study.

 


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Figure 3. The changes in densitometric intensity of PCR products for IGFBP-1 (IBP-1), IGF-I, IGF-II, type 1-IGF receptor (IGFR-1), oestrogen receptor (ER) and progesterone receptor (PR) from the paired endometrium before HRT and 6 months after HRT. Before HRT, expression of IGFBP-1 mRNA was hardly detected. On the contrary, expression of PR mRNA was almost undetectable after HRT. The data were normalized by intensity of ß-actin, which was set as 100 arbitrary unit. Data shown are the means ± SD (n = 25). *P < 0.00001, t = 11.6, degree of freedom (d.f.) = 48. **P < 0.0005, t = 5.42, d.f. = 48.

 
The expression of mRNA for type 1-IGF receptor (IGFR-1) and oestrogen receptor (ER) was unchanged before and after HRT (Figure 3Go).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
IGFBP-1 is present in abundance in decidualized stromal cells of the endometrium in women with a progestogen (levonorgestrel)-releasing intrauterine contraceptive device (IUCD) (Pekonen et al., 1992Go; Suhonen et al., 1996Go). Additionally, the decidual reaction and epithelial atrophy induced by intrauterine progestogen (levonorgestrel) are associated with expression of IGFBP-1 in decidualized stromal cells (Suhonen et al., 1996Go; Rutanen et al., 1997Go). In contrast, a study using immunohistochemical techniques has shown that administration of micronized progesterone (100 mg/day orally, or 100–200 mg/day vaginally) fails to neutralize the proliferative effect induced by oestrogen (Suvanto-Luukkonen et al., 1995Go). In endometrial samples from women treated with micronized progesterone, no histological signs of progestogen effect are detected by microscopic examination; in addition, immunohistochemical staining of IGFBP-1 is completely negative in endometrial stromal cells (Suvanto-Luukkonen et al., 1995Go). In the present study, IGFBP-1 was significantly up-regulated by oral administration of medroxyprogesterone acetate (Provera®, 5 mg per day) (Figure 2Go). By contrast, no expression of IGFBP-1 was detected in the endometrium in the absence of progesterone (before HRT) (Figure 1Go). These observations indicate that progestogen (levonorgestrel)-releasing IUCD and oral administration of medroxyprogesterone acetate (Provera®, 5 mg per day) may stimulate the local production of IGFBP-1 in the endometrium, whereas administration of micronized progesterone (100 mg/day orally, or 100–200 mg/day vaginally) fails to induce such an effect. This lack of an effect by microionized progesterone may be due to its low absorption rate and low bioavailability.

There is evidence that expression of IGF-I is prominent in proliferative phase endometrium (Zhou et al., 1994Go), and that the cyclic changes of IGF-I mRNA in the proliferative phase of the endometrium are coincident with serum oestradiol concentrations (Giudice et al., 1993Go). In addition, IGF binds to both cell membrane receptors and soluble IGFBP-1 with similar affinity in the endometrium (Giudice et al., 1993Go). In postmenopausal women after HRT, expression of IGFBP-1 was substantially increased and that of IGF-I was decreased in the endometrium, whereas there was no change in type 1-IGF receptor (Figure 3Go). In postmenopausal women enrolled in the present study, symptoms of abnormal uterine bleeding subsided and thickness of the endometrium was significantly suppressed after HRT. Thus, it is plausible that the protective effects of progesterone from possible endometrial overgrowth or hyperplasia induced by unopposed oestrogens might, in addition to direct inhibition of expression of IGF-I mRNA, be indirectly mediated through neutralizing the actions of IGF-I by increasing the expression of IGFBP-1 without affecting the type 1-IGF receptor (IGFR-1) in the endometrium. Furthermore, the data presented here suggest that detection of IGFBP-1 mRNA in the endometrium by semiquantitative RT-PCR may be a useful assessment of the progestogen effects on the endometrium during HRT.

In human endometrium, IGF-I expression is substantially higher during the proliferative than the secretory phase, whereas the converse is true for IGF-II (Zhou et al., 1994Go; Gao et al., 1995Go). In the present study, expression of IGF-I was decreased while that of IGF-II was enhanced after HRT (Figure 3Go). This further confirms previous studies that gene expression of IGF-I is prominent in the presence of oestrogen (proliferative endometrium) and abundant IGF-II gene expression is found in the presence of both oestrogen and progesterone (secretory endometrium) (Giudice et al., 1993Go). Furthermore, the decrease in expression of IGF-I and the preferential expression of IGF-II and IGFBP-1 mRNA in secretory endometrium suggests that the differentiation of the endometrium might also be mediated by IGF-II under the regulation of progesterone.

Serum concentrations of progesterone and oestradiol were substantially increased in women after HRT (Table IIGo). The principal metabolite of medroxyprogesterone acetate (MPA, Provera®) is a 3-enol form of MPA glucuronide. Thus, the progesterone antibodies in the immunofluorometric assay (IFMA) used to determine serum progesterone concentrations in the present study might have partial cross-reaction with the 3-enol form of MPA glucuronide. Using RT-PCR, expression of IGFBP-1 mRNA was detected only in the endometrium from postmenopausal women after HRT, but not before HRT (Figures 1 and 2GoGo). None of the women investigated complained of abnormal uterine bleeding after HRT. Histologically, no simple hyperplasia of the endometrium was observed in the studies women after HRT. These clinical observations suggest that medroxyprogesterone acetate (Provera®, 5 mg per day) may elicit a protective effect on the endometrium even in the milieu of relatively high concentrations of oestrogen in postmenopausal women undergoing HRT (Table IIGo). Thus, it is possible that medroxyprogesterone acetate (Provera®, 5 mg per day) exerts anti-oestrogenic effects through regulation of the endometrial IGF/IGFBP system.


    Acknowledgments
 
This work was supported by a grant from Chang-Gung Memorial Hospital, Taipei, Taiwan, Republic of China (CMRP-0611).


    Notes
 
1 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on April 19, 1999; accepted on August 17, 1999.