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a Howard Florey Institute of Experimental Physiology and Medicine and Department of Zoology, University of Melbourne, Parkville, Victoria 3010, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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conceptus, female reproductive tract, oxytocin, pregnancy, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is well established in many species that an increase in both OTR mRNA and receptor concentrations occurs in the myometrium at the end of pregnancy, with a marked up-regulation just before the onset of parturition (reviewed in [1–3]). However, there are clear species differences in the mechanisms controlling OTR expression. Administration of an estrogen receptor antagonist to rats during late pregnancy reduces OTR mRNA and OTR concentrations in the myometrium [4]; it is believed that estrogen activates a functional estrogen response element in the rat OTR promoter [5, 6]. Progesterone has a suppressing influence on OTRs in rats; ovariectomy or treatment with RU-486 results in a sharp increase in uterine OTRs and parturition [7, 8]. However, in many other species, steroid hormones are not the primary regulators of uterine OTRs [1, 2, 9]. The human and bovine promoter regions of the OTR gene lack classic steroid response elements [2], and there is no evidence to support a direct action of steroids in any cell culture system [10].
Uterine stretch is considered one of the factors that controls expression of OTRs in the myometrium. Early work in unilaterally pregnant rats reported elevated OTR concentrations in the gravid horn compared with the nongravid horn [11, 12]. More recently, using the same animal model, Ou et al. [8] demonstrated an up-regulation in OTR gene expression during labor in both gravid and artificially stretched nongravid horns. In contrast, OTR mRNA remained low in unstretched nongravid horns despite exposure to the same systemic endocrine environment as the gravid horn. Mechanical stretch also appears to be largely responsible for the up-regulation in OTR mRNA in the ovine myometrium of the gravid horn at the stage of gestation when the fetus enters its maximal growth phase, at around 130 days, and during labor [13]. These data imply that the effects of uterine stretch are not necessarily exerted only during labor. However, this hypothesis has not been examined in other animal models of parturition.
Female marsupials have two separate uteri, which open into the anterior vaginal expansion via separate cervices. Macropodid marsupials, such as the tammar wallaby (Macropus eugenii), are monovular species, and so only one uterus, the gravid uterus, will contain the single fetus in each pregnancy cycle, whereas the contralateral uterus is nongravid. The oxytocin-like neuropeptide mesotocin is one of the key hormones that regulates uterine contractile activity during parturition in marsupials [14]. As in eutherians, a large increase in uterine responsiveness to mesotocin at the end of pregnancy in the tammar is associated with a marked up-regulation in mesotocin receptor (MTR) mRNA and protein concentrations on Day 23 of the 26-day gestation, but in the myometrium of the gravid uterus only [15, 16]. In contrast, MTRs in the nongravid uterus are down-regulated compared with earlier pregnancy stages. It was suggested that the differential increase in myometrial MTRs in the gravid uterus is due to an endocrine stimulus that originates from the conceptus.
During early pregnancy in the tammar, both uteri are of similar size and weight, but from Day 20 of gestation, there is a large increase in intrauterine volume in the gravid uterus to accommodate the growing fetus. The volume of yolk sac fluid in the uterine cavity increases, and the gravid uterus becomes distended. Preliminary data demonstrated that MTR concentrations were up-regulated in a large, distended uterus despite the absence of a normal conceptus [14]. The aim of this study was to investigate whether or not distension of the gravid uterus is one of the factors involved in the regulation of MTRs in the myometrium of pregnant tammar wallabies. The unilateral gravid uterus in pregnant wallabies provides a unique system in which to examine fetal influences on uterine function. In this study, we removed the single conceptus from the gravid uterus to reduce uterine distension and compared myometrial MTRs with the nondistended, nongravid uterus in the same animal.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
Adult female tammars were transported from Kangaroo Island (South Australia) with approval from the Department for Environment and Heritage, South Australia (permit A23892). They were housed with males in open, grassed enclosures at the La Trobe University Wildlife Reserve with water and kangaroo pellets readily available. Pregnancies were synchronized by first removing the pouch young of tammars that were assumed to be carrying a blastocyst in embryonic diapause. Removal of the pouch young terminates embryonic diapause, and the blastocyst and corpus luteum resume development, with births expected 26–27 days later. The day of pouch young removal was designated Day 0 of gestation.
Fetectomy Surgery
Fetectomy surgeries were carried out at the stage of gestation just after the placenta forms, on Day 20. There were three surgical groups: 1) removal of the fetus, placenta, and all yolk sac fluid (complete fetectomy, n = 7); 2) removal of the fetus and placenta but not the yolk sac fluid (partial fetectomy, n = 5); and 3) sham-operated controls (n = 6). Anesthesia was induced with 4% (v/v) halothane (Zeneca Ltd., Allhank, South Melbourne, Australia) and was maintained between 2% and 3% during surgery, depending on the size of the animal. A midline incision was made through the pouch skin and underlying muscle layers to expose the reproductive tract. The gravid uterus was gently pulled out through the incision and oriented so the cervix could be held with forceps. A 5-mm incision was made in the uterine wall, close to the cervix in an area of minimal vascularization, and the fetus, yolk sac placenta, and uterine fluid were all removed (complete fetectomy). The yolk sac placenta in the tammar wallaby does not attach to or invade the maternal tissue, which facilitates its removal from the uterus. The yolk sac fluid was swabbed out of the uterus at the incision site with gentle pressure applied to the uterus itself. The uterine incision was then sutured with surgical cat gut (Ethicon; Johnson & Johnson, HSA, Mulgrave, Victoria, Australia), but all other incisions were closed with coated vicryl polyglactin sutures (910; Ethicon). The animals received a postoperative analgesic (Carprofen; Pfizer, Allhank, Australia; 4 mg/kg s.c.) and antibacterial injection (Baytril 50; Bayer, Allhank, Australia; 5 mg/kg s.c.), and were returned to open, grassed enclosures within 2 h of surgery. Thereafter, animals were checked daily for any signs of infection and were treated with a long-acting antibiotic (Benecillin; Troy Laboratories Pty. Ltd., Allhank, Australia; 1 ml i.v.).
In the second surgical group, the same procedure was followed as described above, except that the yolk sac fluid was left in the uterus (partial fetectomy). Initially, the six sham-operated controls were subjected to the same surgical procedures, and the uterus was pulled through the midline incision. However, in three of six animals, this caused developmental abnormalities in the fetus, including growth retardation, poor limb development, and no vascularization of the placenta. All these fetuses were dead in utero. The gravid uterus was, however, distended with yolk sac fluid. Tissue samples were also obtained from three wild shot, pregnant animals with similar fetal developmental abnormalities. The samples from these two groups were pooled (n = 6 in total) and designated "abnormal" fetuses. A further three animals were sham-operated subjected to anesthesia and an incision was made in the pouch skin.
Tissue Collection
Uterine tissues and blood samples (5 ml) were obtained from pregnant tammars 3 days after surgery on Day 23 of gestation in order to target the stage of gestation when the large increase in MTRs occurs in the gravid uterus only. The animals were killed by intracardiac injection of 3 ml pentobarbitone sodium (Lethabarb; Virbac Australia Pty. Ltd., Parkhurst, NSW, Australia; 325 mg/ml). The reproductive tracts were removed as quickly as possible after the animal was killed. The two uteri were then dissected from the extrauterine tissue, measured (length and width), and cut longitudinally to open the uterine cavity. A 5-mm strip from each uterus was placed in Zamboni fixative for histological analysis. The yolk sac placenta, or endometrium and cervix, or a combination of these were then separated from the myometrium, and all tissues were weighed. Tissues were snap-frozen and stored at -80°C until further processing. Blood samples were centrifuged at 1700 x g for 10 min at 4°C, and plasma was stored at -20°C. In this experiment, MTR mRNA and receptor concentrations were measured in the myometrium of the two uteri in each animal and compared between the three surgical groups.
Mesotocin Receptor Gene Expression
Real-time polymerase chain reaction (PCR) quantified MTR gene expression in the myometrium as described in Siebel et al. [16], using the same tammar-specific MTR primers and FAM-labeled probe (Keystone Division, Biosource International, Foster City, CA). For each sample, 600 ng of total RNA was reverse transcribed in a 30-µl reaction containing 1.25 U/µl MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA). The PCRs were carried out in triplicate using 96-well optical reaction plates (Applied Biosystems) in 25-µl volumes consisting of 1x TaqMan Universal PCR Master Mix (Applied Biosystems), 0.8 µM forward and reverse MTR primers, 0.4 µM MTR probe, and 2.5 µl of cDNA template. The amount of MTR gene expressed in each sample was calculated by dividing the log (MTR concentration) by the log (18S concentration) using the following formula: log (gene concentration) = (CT - b/a) where b = y-intercept of the standard curve and a = slope of the standard curve as described previously [16]. In this experiment the regression line for the MTR standard curve was y = -4.022x + 13.525, R2 = 0.991; and the 18S standard curve was y = -3.9136 + 15.082, R2 = 0.991.
Radioreceptor Assay
Radioreceptor assays were carried out as described by Parry et al. [15] using (125I)d(CH2)5 (Tyr(Me)2, Tyr4, Orn8, Tyr-NH29)-vasotocin (125I-OTA; Amrad Biotech, Boronia, Victoria, Australia) with a specific activity of 2200 Ci/mmol. The receptor assay mixture consisted of duplicate aliquots of 0.1 ml of diluted tissue suspension, 0.1 ml of (125I)-OTA (15 000–20 000 cpm/tube), and 0.1 ml of assay buffer (50 mM Tris-HCl, 5 mM MgCl2, and 0.2% BSA pH 7.6) containing a range of 0.01–1 pmol/tube unlabeled OTA (kindly provided by Dr. Maurice Manning, Medical College of Ohio, Toledo, OH). Protein concentrations in the resuspended membrane fractions were measured using a DC Protein Assay Kit (Bio-Rad, Regents Park, NSW, Australia) with BSA as the protein standard. Assay protein concentrations were in the range of 60–120 µg/ml, which was within the range at which specific binding is linearly correlated with protein concentration. Data were analyzed by nonlinear regression using the Ligand computer program [17] to obtain the binding affinity (Ka) and the receptor content (Ro) for radiolabeled ligand binding.
Progesterone Assay
To control for the potential effects of fetectomy on ovarian function, in particular luteolysis, plasma progesterone concentrations were measured by radioimmunoassay using a Spectria 125I-progesterone radioimmunoassay kit (Australian Laboratory Services Pty. Ltd., Rockdale, NSW, Australia). Progesterone was extracted from 750 µl of plasma with 8 ml of ethyl acetate and reconstituted in 250 µl of assay buffer. Duplicate 50-µl aliquots were all measured in the same assay. The recovery of 125I-progesterone from tammar plasma was 90.3%. The sensitivity of the assay was 0.15 ng/ml, and the intraassay coefficient of variation was 1.26%.
Statistical Analysis
Data for both MTR mRNA and receptor concentrations did not show homogeneity of variance and were analyzed using nonparametric statistical tests due to the relatively small sample sizes. To test for significant differences in MTRs between treatment groups, Mann-Whitney U-tests for independent samples were used, with a confidence level of 95% (SPSS 10.0; SPSS Inc., Chicago, IL). Wilcoxon signed rank tests for related samples were used to compare MTR mRNA and receptor concentrations between the two uteri (gravid versus nongravid). Differences in plasma progesterone between surgery groups were analyzed by univariate regression analysis (SPSS 10.0).
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RESULTS |
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Data from the real-time PCR experiments demonstrated that myometrial MTR mRNA concentrations were significantly (P = 0.032, Mann-Whitney U-test) lower in the gravid uterus of animals with complete fetectomy compared with concentrations in sham-operated controls (Fig. 3A). Whereas MTR gene expression in the gravid uterus of controls was significantly (P = 0.032, Wilcoxon signed rank test) greater than the nongravid uterus, there was no significant (P = 0.182) difference in MTR mRNA levels between the two uteri in animals with complete fetectomy (Fig. 3A). The pattern of MTR gene expression between these two treatment groups was matched by MTR concentrations that were determined by radioreceptor assay (Fig. 3B). Myometrial MTRs in the gravid uterus of animals with complete fetectomy were significantly (P = 0.007; Mann-Whitney U-test) lower compared with sham-operated controls and were not significantly (P = 0.064, Wilcoxon signed rank test) different from concentrations in the nongravid uterus (Fig. 3B).
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In contrast, partial fetectomy (in which yolk sac fluid was retained without the conceptus) did not significantly affect MTR expression compared with sham-operated controls. There was no significant (P > 0.05) difference in either myometrial MTR mRNA levels (Fig. 4A) or MTR concentrations (Fig. 4B) between the gravid uterus of partially fetectomized animals and sham-operated controls. Unlike the animals with complete fetectomy, MTR mRNA and receptor concentrations in those with partial fetectomy remained significantly (P = 0.03, Wilcoxon signed rank test) higher in the gravid uterus compared with the nongravid uterus (Fig. 4). However, a comparison between animals with partial and complete fetectomy demonstrated no significant difference in either MTR mRNA (0.61 ± 0.24 vs. 0.41 ± 0.14 MTR:18S ratio) or receptor concentrations (449.53 ± 87.32 vs. 360.51 ± 54.45 fmol/mg protein).
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Fetectomy had no marked effect on the general morphology of the endometrium (Fig. 5, A–D). In the gravid uterus of control animals, endometrial glands were dense in number and regularly dispersed throughout the stromal tissue (Fig. 5A). The glands consisted of tall columnar cells with round basal nuclei. Numerous small capillaries were present in the endometrial epithelium, with larger blood vessels in the stromal tissue. The cuboidal cells of the epithelium lining the uterine lumen were loosely arranged and flattened. The nongravid uterus (Fig. 5B) had fewer endometrial glands in the subepithelial stroma. The glands and columnar cells were also smaller. Unlike the gravid uterus, the nongravid uterus had fewer capillaries in the endometrial epithelium or the subepithelial stroma, and the cuboidal cells lining the uterine lumen were tightly packed and cobbled in appearance. In both fetectomized groups of animals, neither the density nor the size of the endometrial glands were affected by removing the conceptus (Fig. 5, C and D). Moreover, the epithelium lining the uterine lumen had loosely arranged and flattened cuboidal cells, which was similar to controls, indicating that tissue damage to the endometrium after fetectomy was minimal in both surgical groups. However, vascularization in the epithelial layer of the endometrium was not as extensive in animals with complete fetectomy (Fig. 5D).
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The potential negative effects of fetectomy on ovarian function, in particular luteolysis, were assessed by measuring peripheral progesterone concentrations. Plasma progesterone concentrations in sham-operated controls were 0.81 ± 0.18 ng/ml and did not differ significantly (P = 0.128; ANOVA) from animals with either partial (0.61 ± 0.26 ng/ml) or complete (0.41 ± 0.20 ng/ml) fetectomy (Fig. 6), indicating that ovarian function was not affected. However, the sample sizes were relatively small and there was a trend toward lower progesterone concentrations in the experimental animals. Histological examination of the corpora lutea revealed no signs of early luteolysis in fetectomized animals (Fig. 5, E and F). The luteal cells remained large and cytoplasmic, with regular shaped nuclei. Thus, the effects of complete fetectomy on myometrial MTRs cannot be attributed to an indirect action on the corpus luteum.
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In a second study, MTR mRNA and receptor concentrations were examined in the uteri of animals with an "abnormal" fetus. Although these animals appeared to be pregnant with a large, distended, gravid uterus, the fetuses of these animals had developmental abnormalities, including lack of growth, no limb development, and no vascularization of the placenta. All these fetuses were dead in utero. Dimensions of the distended uterus in these animals were not significantly different from those of the gravid uterus in sham-operated controls (Fig. 1). Both MTR mRNA and MTR concentrations in the distended uterus of animals with developmentally abnormal fetuses were significantly (P < 0.03, Wilcoxon signed rank test) higher compared with those of the nondistended uterus and did not differ from MTR expression in the gravid uterus of sham-operated controls (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Complete removal of the conceptus and all yolk sac fluid from the gravid uterus on Day 20 of gestation reduced uterine distension and resulted in lower MTR mRNA and receptor concentrations on Day 23 compared with the marked increase observed in sham-operated controls. In fact, MTR expression in the gravid uterus of these fetectomized animals did not differ from that of the nongravid uterus. However, although data from this experimental group demonstrate that removal of the conceptus affects MTR expression within 3 days, it was not possible to isolate mechanical versus endocrine inputs from the fetus because both potential regulatory factors were removed. Previous studies in rats showed that mechanical uterine stretch is one of the factors involved in regulating myometrial OTRs at term [8, 11, 12], but the influence of stretch is observed only during labor. This is not the case in sheep because an increase in myometrial OTR mRNA is observed in the distended gravid horn on Day 130 of gestation, when there is maximal fetal growth, as well as during labor [13]. The first part of the current study demonstrates that uterine occupancy in the tammar wallaby is essential for the up-regulation of myometrial MTRs in the gravid uterus. As in sheep, the influence of the tammar wallaby conceptus on MTRs is not restricted to labor, but it occurs 4–5 days before the day of expected birth, in the presence of high circulating progesterone and low estradiol concentrations.
The novel aspect of this study was the ability to distinguish between mechanical and endocrine inputs by examining MTRs in animals with "abnormal" fetuses and using the partial fetectomy experimental model. In both cases, the gravid uterus was distended due to the presence of yolk sac fluid, but the conceptus was either removed or had developmental abnormalities such as growth retardation, no limb development, and no vascularization of the placenta. The important finding was that MTR mRNA and MTR concentrations in the distended uterus of animals with abnormal dead fetuses did not differ significantly from those of the gravid uterus of controls and were higher than MTRs in the contralateral, nondistended uterus. These data imply that endocrine signals from the conceptus are not essential for the increase in MTRs on Day 23 of gestation. In partially fetectomized animals with some degree of uterine distension, MTR expression did not differ from that of sham-operated controls, but MTRs were not significantly higher than they were in animals with complete fetectomy. Thus, we have not clearly isolated the mechanical inputs from the endocrine inputs and can only suggest that uterine distension is one of the factors involved in MTR up-regulation.
To date, no other fetectomy experiments have examined the influence of the conceptus versus uterine distension on myometrial OTRs or other contractile-associated proteins. A recent fetectomy study in rats demonstrated that uterine occupancy is essential for the full induction of myometrial 11ß-hydroxysteroid dehydrogenase type 1 expression, but the placenta, rather than uterine stretch, provides the key stimulus for this [19]. In the tammar wallaby, it would appear that a stimulus from the conceptus between Days 20 and 23 of gestation is not essential for MTR expression in the myometrium, and that uterine stretch is probably one of the key factors involved. However, this does not rule out the possibility that other endocrine factors contribute in some way to this system. Twenty days before fetectomy surgeries, the uterus held an embryo and the yolk sac placenta had formed, so presumably, some degree of fetal-maternal recognition, involving local endocrine factors, will have occurred. This could have had an early priming effect on myometrial MTRs so that the response to uterine stretch still occurs, even without a conceptus for 3 days. This would also explain why MTRs are still up-regulated in the distended uterus of animals with an abnormal fetus and avascular placenta, assuming that at some point there was an early priming stimulus. However, without any uterine distension (as in animals with complete fetectomy), the early priming stimulus is not sufficient per se to cause the marked up-regulation in MTRs on Day 23 of gestation.
It is likely that a pregnancy-specific endocrine factor is important for OTR/MTR up-regulation prior to the stretch stimulus, but the nature and temporal expression of this factor may not be the same. In rats there is strong evidence that ovarian estrogen is this priming factor [4–6], but in sheep, the ability of uterine distension to cause an increase in OTRs appears to be independent of estrogen [13]. This is also the case in the tammar wallaby. Increases in both peripheral and utero-ovarian estradiol-17ß concentrations are detected immediately before birth but are highest 1–2 days postpartum to coincide with estrus [20]. The increase in both MTR mRNA and receptor concentrations in the gravid uterus is independent of high circulating estrogen [16]. There also appears to be no correlation between myometrial MTR and steroid receptor concentrations in the tammar. Renfree and Blanden [21] showed that at the time of the unilateral increase in MTRs in the gravid uterus, progesterone and estrogen receptor concentrations are both extremely low. This strengthens our hypothesis that steroids or their receptors are not mediating the differential up-regulation in myometrial MTRs in the pregnant tammar wallaby. In this experiment, we did not examine the potential influence of other ovarian factors versus uterine stretch on myometrial MTRs. Preliminary data indicate that removal of the corpus luteum on Day 17 of gestation in the tammar wallaby does not affect uterine MTR concentrations (personal observations). Ovarian factors are unlikely to be the main regulatory element in this species because despite normal ovarian function in animals with complete fetectomy, MTRs were not up-regulated on Day 23 of gestation.
The tammar wallaby is a good experimental model in which to examine the effects of fetectomy on uterine function because there is a single, small (300–400 mg at term) fetus in the gravid uterus. Moreover, the chorioepithelial-like placenta does not invade the maternal tissue and can be pulled away from the uterus, leaving the uterine epithelium and endometrium intact. For these reasons, removal of the conceptus is a relatively simple procedure and results in minimal trauma to the maternal uterine tissue. Removal of the conceptus did not result in luteolysis because circulating progesterone concentrations were not significantly affected between the three surgical groups. There was also no histological evidence of early luteolysis in the ovary ipsilateral to the uterus from which the conceptus was removed. The density and size of the uterine endometrial glands were not affected by removing the conceptus in animals with either partial or complete fetectomy. Moreover, the epithelium lining the uterine lumen was similar in appearance to that of controls, indicating that tissue damage after fetectomy was minimal in both surgical groups. However, there were fewer capillaries in the endometrial epithelium of animals with complete fetectomy, which suggests that removal of the placenta and yolk sac fluid had some effect on vascularization.
An interesting observation was that anesthesia and laparotomy on Day 20 of gestation resulted in abnormal development of fetuses in 50% of the control animals and is in agreement with earlier studies [22]. This is the stage at which the yolk sac placenta undergoes a large increase in vascularization, but in animals with abnormal fetuses, the yolk sac placenta remained completely avascular. It would appear that lack of placental vascularization resulted in abnormal fetal growth and development. Despite this abnormality, MTRs were still up-regulated in the distended uterus.
The MTR is not the only contractile-associated protein differentially expressed and up-regulated in the gravid uterus of the tammar wallaby. Prostaglandin H synthase-2 mRNA levels are higher in the endometrium of the gravid uterus only, in late gestation (Sebastian and Parry, personnel communication). A recent study demonstrated that nitric oxide (NO) induced a marked relaxation via a guanylyl-cyclase mediated pathway and completely inhibited mesotocin-induced myometrial contractions in the gravid uterus of pregnant tammars [23]. In contrast, the nongravid myometrium was relatively insensitive to NO. The influence of uterine stretch on either PGHS-2 or NO expression has not been investigated.
In summary, we have demonstrated that uterine distension contributes to the mechanisms involved in the up-regulation of myometrial MTRs in the gravid uterus of tammar wallabies. Removal of the conceptus and yolk sac fluid on Day 20 of gestation resulted in significantly lower MTR mRNA and receptor concentrations compared with sham-operated controls. Moreover, MTRs were up-regulated in the distended uterus of animals with an abnormally developed fetus and avascular placenta. It would appear that endocrine signals from the conceptus are not essential for the maintenance of MTR expression at this stage of gestation. These data in the tammar wallaby also strengthen the hypothesis that differential regulation of contractile-associated proteins in the gravid uterus is due to the unilateral influence of the conceptus, including uterine distension.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Laura J. Parry, Howard Florey Institute, University of Melbourne, Parkville, Victoria 3010, Australia. FAX: 61 3 9348 1707; l.parry{at}hfi.unimelb.edu.au
Received: 1 March 2002.
First decision: 26 March 2002.
Accepted: 6 May 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  A. L Siebel, R. A D Bathgate, and L. J Parry Differential expression of mesotocin receptors in the uterus and ovary of the pregnant tammar wallaby Reproduction, May 1, 2005; 129(5): 639 - 649. [Abstract] [Full Text] [PDF]
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