Molecular Human Reproduction, Vol. 5, No. 7, 651-655,
July 1999
© 1999 European Society of Human Reproduction and Embryology
Oestrogen receptor (ER)-
and ER-ß isoforms in normal endometrial and endometriosis-derived stromal cells
Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, CA 94143, USA
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Abstract |
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Several investigators have noted that hormone-dependent development of endometriosis implants lags behind that of simultaneously analysed eutopic endometrium. With the recent discovery of the oestrogen receptor-ß (ER-ß) isoform, the aim of this study was to investigate whether differences in the expression of ER-
and ER-ß might explain this observation. mRNA transcripts from endometrial stromal cells isolated from normal endometrium (NE) and from endometriomas (EI) were analysed using a semi-quantitative reverse transcription–polymerase chain reaction (RT–PCR) technique. RT–PCR and Southern blot analyses of the two ER isoforms indicated that NE and EI stromal cells predominantly express ER- mRNA, however the relative concentrations of ER isoform mRNA transcripts differed between the two cell types. Steady-state ER-:ER-ß mRNA ratios were 15.5 ± 2.8 and 5.2 ± 0.9 respectively for NE and EI cells (P = 0.02). NE and EI stromal cells expressed ER proteins with similar Kd (~0.9 nM) and densities (~24 500 binding sites/cell) respectively. Functional ER expression was indicated by an increase in progesterone receptor concentrations of ~60% (P = 0.03) after incubation with 10 nM oestradiol. We postulate that differential transcript processing, ligand specificity and biological actions of the ER- and -ß isoforms may influence differential growth responses in normal and ectopic endometrium.
cell cultures/endometriosis/oestrogen receptors and ß
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Introduction |
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Endometriosis is a common and often debilitating condition in women of reproductive age. Although multiple theories of the histogenesis of endometriosis exist, the implantation hypothesis of Sampson (1927) is the most widely accepted (Sampson, 1927
). Retrograde menstruation (Halme et al., 1984) and i.p. spillage of viable endometrial cells (Kruitwagen et al., 1991) occur frequently in cycling women. Syndromes of Müllerian tract outflow obstruction are associated with an increased prevalence of endometriosis (Cramer et al., 1996). The demographics of this disorder also support a close relationship between endometriosis and ovarian hormone production. Signs and symptoms do not appear before menarche and these typically regress spontaneously after menopause (Thomas, 1995). Conditions of oestrogen excess have been reported to exacerbate endometriosis, while medical interventions that inhibit oestrogen production or action, i.e. down-regulating doses of gonadotrophin-releasing hormone (GnRH) analogues (Schriock et al., 1985), progestins (Vercellini et al., 1997), androgens (Barbieri et al., 1982) and anti-oestrogens (Haber and Behelak, 1987), ameliorate its symptoms.
Despite their sensitivity to oestrogen, endometriosis lesions appear to have different hormonal responsiveness than that of eutopic endometrium. Simultaneous biopsies of ectopic and eutopic endometrium demonstrate different histochemical morphology and hormone receptor distribution (Lessey et al., 1989; Bergqvist and Ferno, 1993). Relative growth inhibition of the implants has been attributed to a suboptimal extrauterine environment and/or mitogenic interference by inflammatory cytokines (Zarmakoupis et al., 1995). It has been reported that the concentrations of oestrogen (ER) and progestin receptors (PR) in endometriosis lesion homogenates were lower than in extracts from normal eutopic endometrium (Jänne et al., 1981). However, a caveat of the observation is that peritoneal cells associated with endometriosis implants are ER- and PR-negative and would be expected to dilute receptor concentrations in these homogenates (Prentice et al., 1992).
The classical human oestrogen receptor (ER-), cloned in 1986 (Green et al. 1986), has been identified in both normal endometrium and endometriosis tissue by several techniques (Lyndrup et al., 1987; Lessey et al., 1989; Prentice et al., 1992; Bergqvist and Ferno, 1993; Jones et al., 1995). In 1996, a second oestrogen receptor, ER-ß was cloned from rat prostate (Kuiper et al., 1996) and human testis (Mosselman et al., 1996). Both ERs are members of the steroid receptor superfamily and consist of six regions, A–F, of which the DNA-binding domain (C) and ligand binding domain (E) are highly conserved with 96% and 58% amino acid sequence homology respectively. Both ERs act as transcription factors and are believed to play a key role in endometrial and endometriosis growth regulation. Recent studies indicate that the tissue distribution of ER- and ER-ß differs. It has previously been found that ER- and ER-ß mRNAs are present in human pre-menopausal and fetal uteri (Brandenberger et al., 1997). In cycling human endometrium, ER- is the predominant isoform (Enmark et al., 1997).
To assess the relative hormonal responsiveness and growth characteristics of endometrial and endometriosis cells an in-vitro model of proliferating, primary stromal cells from these two tissue sources was developed (Ryan et al., 1994). The aim of the current study was to investigate whether differences in the expression of ER- and ER-ß genes might explain the observation that endometriosis lesions experience relative growth inhibition compared to eutopic tissue.
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Materials and methods |
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Subjects
All the subjects enrolled in this study were healthy women reporting molimina and symptoms of ovulatory menstrual cycles, and who had not used hormones or gonadotrophin-releasing hormone (GnRH) agonists for at least 3 months prior to surgery. Subjects undergoing elective laparoscopic surgery for tubal sterilization or evaluation of pelvic pain without evidence of endometriosis or other pelvic pathology were the source of normal endometrial biopsies (n = 11). Subjects with an echogenic adnexal mass on ultrasonography and evidence of an endometrioma at surgery were the source of endometriosis implants (n = 9). Informed consent and biopsy specimens were obtained in compliance with the University of California, San Francisco, Committee on Human Research guidelines. Laparoscopy was performed and all tissue samples were collected under anaesthesia during the mid to late proliferative phase of the menstrual cycle (days 7–12). The phase of the ovulatory cycle was assessed by preoperative history and ultrasonography and confirmed histomorphometrically in the endometrial biopsies according to previously published criteria (Noyes et al., 1950
). The diagnosis of endometriosis also was confirmed histologically.
Isolation of stromal cells from normal endometrium (NE) and endometriosis implants (EI)
Endometrial stromal cell cultures were prepared from normal eutopic endometrial (NE) tissues and endometriosis implant (EI) tissues as described previously (Ryan et al., 1994). Briefly, NE and EI cultures were prepared from Pipelle biopsies of normal fundal endometrium and ovarian endometrioma cyst linings respectively, collected under sterile conditions and carefully oriented with respect to the luminal surface. The tissues were carefully dissected free from underlying parenchyma, minced and digested with collagenase (2 mg/ml) at 37°C for 60–90 min. Stromal cells were separated from epithelial glands and debris by serial filtration using narrow gauge sieves with apertures of 105–38 µm. The stromal cells were plated and allowed to proliferate in minimal essential medium (MEM)- supplemented with 10% fetal calf serum (FCS), nucleosides and amino acids. All experiments were performed between the second and third cell culture passages to eliminate immunocyte contamination and preserve endogenous ER and PR expression. Previous immunocytochemistry studies from our laboratory indicate that >95% of the NE and EI stromal cells are vimentin positive and that these are CD3, CD11b, CD45 and cytokeratin negative (Ryan et al., 1994). The latter findings exclude contamination of the stromal cell cultures by immune cells or endometrial or ovarian surface epithelial cells respectively.
RNA extraction and analysis
Total RNA from cell cultures was extracted using the guanidine isothiocyanate method (Trizol®; Gibco BRL, Grand Island, NY, USA) (Chomczynski and Sacchi 1987), treated with amplification-grade DNase I (Gibco BRL), followed by proteinase K digestion, phenol extraction and ethanol precipitation. Integrity of the isolated RNA was assessed by electrophoresis in ethidium bromide-stained 1% agarose–tetrabromoethane (TBE) gels.
RT–PCR and Southern blotting
Reverse transcription–polymerase chain reaction (RT–PCR) and Southern blotting were performed as previously described (Brandenberger et al., 1997). Total RNA (1 µg) was reverse transcribed at 42°C for 1 h in a 10 µl reaction containing 20 mM Tris–HCl, 50 mM KCl, 2.5 mM MgCl2, 5 mM dithiothreitol (DTT), 0.5 mM dNTPs, 50 ng random hexamers and 200 IU of Superscript II reverse transcriptase. PCR reactions contained 0.4 µM ER- or -ß specific primers, each spanning intron 1 of the poorly conserved A/B region of the two respective ERs, [nucleotides 546–817 of the ER-, (Green et al., 1986) and nucleotides 33–291, (Mosselman et al., 1996), of ER-ß]. Platinum Taq DNA polymerase (Gibco BRL) was used to perform automatic hot start PCR following an initial denaturation step at 94°C for 2 min. PCR was performed for 30–40 cycles. Amplification was shown to be in the linear range using 30 cycles for the low concentrations of cDNA detected in endometrial samples (Brandenberger et al., 1997), so all phosphoimaging calculations were performed under these conditions (see below).
Internal standards of ER- and ER-ß cDNAs were added in 10-fold dilutions of 102 to 106 copies to determine the relative quantification of the amplification signal. The PCR products were subjected to electrophoresis in 2% agarose–TBE gels and transferred to nylon membranes for 4 h. The blots were hybridized overnight in solutions containing 0.5 M Na2HPO4, 7% sodium dodecyl sulphate (SDS), 1 mM EDTA, 100 µg/ml salmon sperm DNA and [32P]-labelled ER- and -ß riboprobes as previously described (Brandenberger et al., 1997). After stringency washes the blots were exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, NY, USA) and then phosphoimaged (see below). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used as internal controls for equal cDNA amounts and PCR was carried out under identical conditions to the above. Negative controls lacking cDNA were included in all reactions to ensure that there was no genomic DNA contamination.
Quantification of Southern blots
After 30 cycles of PCR and Southern blotting, the signals of ER- and ER-ß cDNA were measured using a Molecular Dynamics PhosphoImager and Image Quant software (Molecular Dynamics, Sunnyvale, CA, USA). These signals were normalized to the corresponding intensity of the GAPDH cDNA signal for each sample. The normalized amounts of ER- and ER-ß mRNA were compared with the intensities obtained with 104 copies of the respective ER plasmid DNA and expressed as a ratio of the latter value (see Table I). Efforts to assure similar amplification efficiencies for ER- and ER-ß cDNA included the selection of similar amplicon sizes and use of similar PCR conditions. The analysis was limited to the relative expression of ER-:ER-ß transcripts to avoid problems of absolute quantification. For statistical comparisons, only Southern blots after 30 cycles of PCR were used, as this was shown to be in the linear range of the assay (Brandenberger et al., 1997).
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Equilibrium saturation radioligand binding and Scatchard analysis
Oestrogen and progestin receptors were measured using whole cell radioligand binding assays in stromal cells plated at a density of 40 000 cells/cm2. When the cells reached 90% confluence they were quiesced overnight in Phenol Red-free MEM-
supplemented with charcoal-stripped 10% FCS and insulin (200 ng/ml). The cells were incubated with 0.15–10 nM [2,4, 6,7–3H]-17ß-oestradiol (88 Ci/mmol; New England Nuclear, Boston, MA, USA) or 0.50–50 nM [17-methyl-3H]-promegestone (R5020, 82 Ci/mmol, NEN, Boston, MA, USA) for 90 min at room temperature. Free label was removed by two washes with ice-cold phosphate-buffered saline (PBS), and bound, intracellular [3H]-steroid was extracted with cold ethanol and quantified in a ß-scintillation counter at 35% efficiency. Binding data were analysed by the method of Scatchard using an iterative, non-linear curve fitting program which analyses bound ligand as a function of free ligand (Kilpatrick et al., 1992). Steroid receptor concentrations were normalized to total cellular DNA content using a sensitive fluorimetric assay (Setaro and Morley, 1976), assuming 6.5 pg DNA/human diploid cell. Kd values were calculated as nM and the receptor concentrations as numbers of receptors per cell.
Biostatistical analysis
RT–PCR and ligand binding experiments presented were repeated in a minimum of three independent experiments. Because of the limited numbers of cells isolated per specimen, each analysis represents a single biopsy. Data are presented as the mean ± SE of n independent cell preparations. Given the relatively small sample sizes, and difficulty verifying Gaussian distributions of all the study parameters, conservative non-parametric statistics were used. Comparisons between NE and EI stromal cells are based on Mann–Whitney tests. Wilcoxon signed-rank tests were used for paired determinations of PR before and after incubation with 10 nM oestradiol. Sample size calculations assumed parametric distributions. For all analyses, a significant difference was accepted when two-tailed tests yielded a probability of P < 0.05.
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Results |
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Estimation of ER-
and ER-ß mRNA quantities in NE and EI cellsTo examine the relative expression of ER-
and ER-ß mRNA in NE and EI stromal cells an established, semi-quantitative RT–PCR assay was used. After 30 cycles of amplification using oligonucleotide primers specific for the two human ER isoforms, the cDNA products were blotted onto nylon membranes and probed with specific [32P]-labelled riboprobes. In parallel reactions, 10-fold dilutions of specific ER- and ER-ß plasmid vectors were amplified and Southern blotted to allow quantification of the signals (Figure 1). As an internal control for cDNA quantity and integrity, amplification of the constitutive GAPDH cDNA also was performed. The results, shown in Table I
, indicate that ER- is the predominant transcript in both cell types. NE cells contained higher steady-state concentrations of ER- mRNA than EI cells (Z = 2.24, P = 0.04) whereas ER-ß mRNA concentrations did not differ between NE and EI cells (Z = 1.05, P = 0.27). Thus, the ratio of ER-:ER-ß in stromal cells from the two different sources were 15.5 ± 2.8 and 5.2 ± 0.9 respectively, for NE and EI cells (Mann–Whitney test, Z = 2.24, P = 0.02).
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Isolated NE and EI stromal cells express functional oestrogen and progestin receptor proteins
To verify that the ER mRNA was effectively translated into biologically active receptor proteins, functional receptor assays were performed (Table II
). Equilibrium saturation radioligand binding assays, used to characterize ER affinity and concentration, revealed that these were very similar in purified NE and EI stromal cells. The Kd values for [3H]-oestradiol were 1.0 ± 0.4 and 0.8 ± 0.2 nM and the receptor concentrations were 24 538 ± 10 169 and 24 417 ± 7174 receptors/cell respectively (n = 5). The ER affinities and concentrations did not differ statistically (Mann–Whitney tests, Z = 0.25 and Z = 0.10, P = 0.81 and P = 0.92 respectively). Under the same conditions, PRs were analysed. NE cells had a Kd for [3H]-R5020 of 0.8 ± 0.3 nM and 99 516 ± 27 568 PR/cell. EI cells had a Kd for [3H]-R5020 of 0.5 ± 0.2 nM and 111 778 ± 49 446 PR/cell (n = 4). The PR affinities and concentrations did not differ statistically between NE and EI cells (Mann–Whitney tests, Z = 0.71 and Z = 0.00, P = 0.33 and P = 1.00 respectively).
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Oestradiol up-regulates progestin receptorexpression in NE and EI cells
Up-regulation of PR in these cells was used as an indicator of physiological ER function (Table II
). Dose–response experiments revealed an EC50 of 1 nM oestradiol, corresponding well to the experimentally determined Kd for the stromal cell ER. Incubation of NE and EI stromal cells with 10 nM oestradiol for 24 h increased PR concentrations to 165 096 ± 38 568 and 148 584 ± 64 428 receptors/cell respectively, without appreciable changes in affinity (Kd = 0.5 ± 0.2 and 0.4 ± 0.2 nM respectively). The oestradiol-induced increase (~60%) in PR concentration was statistically significant (Wilcoxon test, Z = 2.20, P = 0.03). |
Discussion |
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This study confirmed previous findings that ERs are expressed in NE and EI stromal cells and has shown that these receptors are biologically functional, promoting the up-regulation of cellular PR. Moreover, we have established the relative expression of mRNA transcripts encoding ER-
and ß-receptor isoforms in NE and EI cells. Our findings help to explain some of the discrepancies reported in previous studies of ER expression in endometriosis. As [3H]-oestradiol binds to both human ER isoforms with similar affinity (Kuiper et al., 1997), this approach cannot be used to distinguish between the - and ß-receptors. The semi-quantitative RT–PCR data demonstrate that despite similar total ER protein concentrations, the distribution of ER- and ER-ß mRNA transcripts differs between NE and EI stromal cells. The latter cells have a lower concentration of ER- mRNA and a decreased ER-:ER-ß transcript ratio.
At the time of completion of these studies, the co-expression of ER- wild-type and ER- splicing variant mRNAs in endometria from normal fertile women, those with endometriosis and those with unexplained infertility was reported (Rey et al., 1998). Splicing variants involving exons 2, 3, 4 and 7 of ER- were identified. Based on the design of our ER- primers, it is possible that the apparent decrease in ER- mRNA in EI cells might be due to an increase in exon 2 deleted transcripts. However, this and other ER- splice variants will need to be investigated by rigorous, quantitative methods to test this hypothesis. Similar to our own findings in endometrial tissues (A.W.Brandenberger, D.I.Lebovic and R.N.Taylor, unpublished observations), the expression of ER-ß transcripts in normal, endometriosis and infertile endometria was observed, albeit at lower concentrations than ER- mRNA (Rey et al., 1998).
Heterogeneity of ER in the human uterus was demonstrated >20 years ago (Smith et al., 1979). Now, with the availability of cloned ER- and ER-ß molecules, the distinctive functions of the two ER isoforms can be investigated. Gene transfection experiments have shown that cells expressing ER-ß isoforms have a lower transcriptional response to oestradiol at nuclear activator protan-1 (AP-1) sites than when the same cells are transfected with similar concentrations of an ER- expression vector (Paech et al., 1997). It also has been proposed that heterodimers of ER- and ER-ß can associate with oestrogen responsive elements in vitro, potentially modulating transcriptional activation (Cowley et al., 1997). Thus, altered ER-:ER-ß ratios in target tissues could influence endogenous gene expression. We postulate that a decreased ER-:ER-ß ratio might favour local expression of proinflammatory cytokines, which are noted to be increased in endometriosis (Taylor et al., 1997).
Although ER mRNA patterns seem different in the two types of stromal cells, some reservations apply to these findings. The abundance of ER-ß mRNA in both NE and EI cells was low. With a small sample size and variation among specimens, it is possible that significant differences in ER-ß transcript concentration were obscured by a type II error. Based on the variability observed in the current study, sample size calculations were performed assuming an unpaired t-test model with - and ß- parameters of 0.05 and 0.20 respectively. The analyses indicate that four and 30 specimens each of NE and EI cells would be needed to detect a 50% difference in ER- and ER-ß mRNA concentrations respectively.
In summary, this study indicates that isolated endometrial stromal cells, like their tissue of origin, express lower concentrations of ER-ß mRNA relative to ER- mRNA. This finding also held true for cells derived from endometriotic implants. In cultured EI stromal cells there was a significant decrease in the ER-:ER-ß transcript ratio compared to NE cells. Whether this could reflect differential effects on post-transcriptional processing of the mRNA of the two ER (Rey et al., 1998) must await future studies. Differences in the ER-:ER-ß ratio between the two stromal cell types could have important functional implications, since these ERs have different ligand binding characteristics (Kuiper et al., 1997; Tong and Perkins, 1997). ER- has a five-fold higher affinity for 17ß-oestradiol, whereas the phytoestrogen genistein binds seven times better to ER-ß. Evolving details about the ligand specificity of the ER- and ER-ß isoforms may make it possible to therapeutically manipulate endometriosis implant growth by exploiting isoform selective signalling pathways.
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Acknowledgments |
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The authors appreciate the assistance and suggestions of Victor Chao and Dale Leitman, M.D., Ph.D. (Reproductive Endocrinology Center, UCSF). These studies were supported by National Institutes of Health grant R01-HD33238 to R.N.T, and by a stipend to A.W.B. from the Department of Obstetrics and Gynecology, University of Bern, Switzerland.
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Notes |
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1 To whom correspondence should be addressed
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References |
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Submitted on November 10, 1998; accepted on April 8, 1999.
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 H. Brady, S. Desai, L. M. Gayo-Fung, S. Khammungkhune, J. A. McKie, E. O'Leary, L. Pascasio, M. K. Sutherland, D. W. Anderson, S. S. Bhagwat, et al. Effects of SP500263, a Novel, Potent Antiestrogen, on Breast Cancer Cells and in Xenograft Models Cancer Res., March 1, 2002; 62(5): 1439 - 1442. [Abstract] [Full Text] [PDF]
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H. O. D. Critchley, R. M. Brenner, T. A. Henderson, K. Williams, N. R. Nayak, O. D. Slayden, M. R. Millar, and P. T. K. Saunders Estrogen Receptor {beta}, But Not Estrogen Receptor {{alpha}}, Is Present in the Vascular Endothelium of the Human and Nonhuman Primate Endometrium J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1370 - 1378. [Abstract] [Full Text]
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G. LECCE, G. MEDURI, M. ANCELIN, C. BERGERON, and M. PERROT-APPLANAT Presence of Estrogen Receptor {beta} in the Human Endometrium through the Cycle: Expression in Glandular, Stromal, and Vascular Cells J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1379 - 1386. [Abstract] [Full Text]
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J. Kitawaki, H. Obayashi, H. Ishihara, H. Koshiba, I. Kusuki, N. Kado, K. Tsukamoto, G. Hasegawa, N. Nakamura, and H. Honjo Oestrogen receptor-alpha gene polymorphism is associated with endometriosis, adenomyosis and leiomyomata Hum. Reprod., January 1, 2001; 16(1): 51 - 55. [Abstract] [Full Text] [PDF]
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 M. D. Mueller, J.-L. Vigne, A. Minchenko, D. I. Lebovic, D. C. Leitman, and R. N. Taylor Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta PNAS, September 19, 2000; (2000) 200377097. [Abstract] [Full Text]
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M. D. Mueller, J.-L. Vigne, A. Minchenko, D. I. Lebovic, D. C. Leitman, and R. N. Taylor Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta PNAS, September 26, 2000; 97(20): 10972 - 10977. [Abstract] [Full Text] [PDF]
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