Chorion releases a factor that inhibits oxytocin-stimulated myometrial contractility in the pregnant guinea pig

Jorge A. Carvajal, Irina A. Buhimschi, Loren P. Thompson, Kripamoy Aguan and Carl P. Weiner,1

Perinatal Research Laboratory, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
It was postulated that chorion releases a substance necessary for the maintenance of uterine quiescence during pregnancy. A decrease in the release of this substance at the end of the pregnancy would be necessary for normal myometrial activation. This hypothesis was tested by demonstrating the ability of chorion to inhibit oxytocin-stimulated myometrial contractility in vitro. Tissues were obtained from timed pregnant Duncan-Hartley guinea pigs either at pre-term or near-term gestation. Myometrial strips were placed in organ baths for isometric tension measurement and contractions stimulated by oxytocin (10–8 mol/l). Fetal membranes or conditioned medium from chorion were added directly to the organ bath. Near-term chorion and chorion conditioned-medium decreased oxytocin-stimulated contractile activity to 39% and 49% respectively. Neither pre-term nor near-term amnion reduced oxytocin-stimulated myometrial contractile activity. Relaxation induced by pre-term chorion was greater than near-term chorion (23% and 41% of the oxytocin-induced basal level respectively; P < 0.05). Further, chorion-induced relaxation was independent of the gestational age of the myometrium. Human chorion from a term, not-in-labour woman also inhibited oxytocin-stimulated guinea pig myometrial contractility. It was concluded that the chorion releases a substance or substances that reduce oxytocin-stimulated myometrial contractility and may be involved in the maintenance of uterine quiescence during pregnancy.

Key words: chorion/myometrial quiescence/paracrine regulation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The duration of gestation is tightly regulated in mammals to ensure the delivery of a newborn mature enough to survive the extra-uterine environment. A successful pregnancy requires near-complete relaxation of the uterus for more than 95% of gestation, overcoming the inherent tendency of the myometrium to contract in response to stretch from the growing fetus, until the appropriate onset of labour. This active and highly regulated process is called myometrial quiescence, and requires not only the near absence of myometrial contractions but also its refractoriness to contractile agents. Myometrial quiescence appears mediated by complex, but as yet poorly understood, molecular mechanisms despite much research in this area (Cunningham et al., 1993Go).

Myometrial quiescence precedes myometrial activation, the process of myometrial awakening that occurs late in pregnancy (Norwitz et al., 1999Go). During myometrial activation, the molecular mechanisms responsible for uterine quiescence must be nullified and the ability of the uterus to respond to uterotonins recovered (Cunningham et al., 1993Go).

The balance between substances that generate myometrial quiescence and activation no doubt impacts greatly on the timing of parturition. Numerous studies have confirmed in several animal models that a balance in the bioaction of progesterone (pro myometrial quiescence) and oestrogen (pro myometrial activation) are central to the initiation of parturition (Olson et al., 1995Go). However, such a relationship has been difficult to confirm in all mammals, including the human and guinea pig.

Several recent studies have focused on a paracrine rather than endocrine regulation of myometrial contractility. The amnion and chorion are of fetal origin, metabolically active, and located strategically adjacent to the decidua, a maternal tissue in direct contact with the myometrium. Thus, the amnion, chorion and decidua are potential sources of paracrine regulators of myometrial contractility (Bryant-Greenwood and Greenwood, 1998Go).

It has previously been postulated that the fetal membranes, and specifically the chorion, release a substance essential for the maintenance of uterine quiescence during pregnancy (Kim and Weiner, 1996Go; Tans et al., 1998Go). It has also been proposed that a reduction late in gestation in the release of the chorion-derived quiescent substance must occur for normal myometrial activation. In the current investigation, this hypothesis was tested by measuring and characterizing for the first time the effect of fetal membranes on oxytocin-stimulated myometrial contractility.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Animal model and tissue preparation
Timed pregnant, Duncan-Hartley guinea pigs (term = 65–68 days) were purchased from a commercial breeder (Harlan Sprague Dawley, Indianapolis, IN, USA). Myometrium was obtained from pre-term (43 ± 2 days gestation) and near-term (60 ± 2 days gestation), anaesthetized (xylazine 4 mg/kg, i.m.; ketamine hydrochloride 100–200 mg/kg, i.p.) guinea pigs that were respiring spontaneously. A hysterotomy was performed and the pups delivered. The fetal membranes (chorion and amnion) were carefully separated from the myometrium and from each other by gentle traction, immediately frozen in liquid nitrogen, and stored at –80°C until used. Full-thickness myometrial strips opposite the placental implantation site were excised including endometrium, but excluding fetal membranes. The myometrial strips were placed in Krebs buffer and stored on wet ice until used for isometric tension recording performed within 1h of removal from the animal. The protocol for these studies was approved by the Committee on Animal Care at the University of Maryland, Baltimore.

Isometric tension studies
Longitudinal strips (10 ± 2 mm lengthx2 mm width) of full- thickness myometrium were placed in organ chambers and attached to a force transducer (Grass Instruments, Quincy, MA, USA) for isometric tension recording. The chambers contained Krebs buffer composed of: 118 mmol/l NaCl, 4.7 mmol/l KCl, 1.18 mmol/l MgSO4, 1.18 mmol/l KH2PO4, 11.1 mmol/l D-glucose, 0.016 mmol/l EDTA, 2.2 mmol/l CaCl2, 15.8 mmol/l NaHCO3, pH 7.35–7.45; the chamber contents were maintained at 37°C and continuously bubbled with a 95% O2/5% CO2 mixture. The myometrial strips were equilibrated under 1 g of passive tension until a stable baseline was achieved (typically requiring 30 min). The bath buffer solution was changed every 5 min during equilibration. After the stabilization period, myometrial contractions were stimulated by 10–8 mol/l oxytocin (Sigma Chemical Co., St Louis, MO, USA). This concentration approximates the EC50 measured during previous concentration–response curves. The experiment was started after a pattern of regular contractions had been reproducibly achieved (typically 15–20min following the addition of oxytocin).

The myometrial contractions were recorded and analysed using a combination of PowerLab/800 hardware and Chart v3.4 software (AD Instruments, Mountain View, CA, USA). To quantify contractile activity, the integral area under the curve over 10 min intervals was measured and normalized for the cross-sectional area of the strip. The cross-sectional area was calculated as W/(LxD), where W = weight (g), L = length (cm) and D (density) = 1.05 g/cm3. The oxytocin-stimulated contractile level was calculated as the integral activity of stabilized contractions 10 min before the addition of fetal membranes to the tissue bath and designated as basal activity.

Experimental protocol
Fetal membranes
To determine the effect of the fetal membranes on oxytocin-stimulated myometrial contractility, either chorion or amnion was added directly to the organ bath containing the oxytocin-stimulated myometrial strips, and changes in contractile activity measured.

Previously stored frozen membranes (chorion or amnion) from a single near-term animal were weighed and thawed for 5 min in 37°C Krebs solution. Approximately 1000 mg was added directly to the 10 ml organ bath (100 mg membranes/ml buffer) after the establishment of regular contractions by oxytocin and the isometric tension had been recorded for an additional 30 min. The contractile activity after addition of the fetal membranes was measured at 10 min intervals, represented in the text and figures by its midpoint time (i.e. 5, 15 and 25 min). The contractility (area under the curve) of each interval was compared with the basal activity and expressed as a percentage. Thus, 100% denotes no change, while <100% denotes a decrease in contractile activity (myometrial relaxation). The pH of the muscle bath was measured at the end of several experiments and always found to be unchanged (7.45 ± 0.05).

To determine whether the effect of chorion on oxytocin- stimulated myometrial contractions was gestational age-dependent, the membranes of pre-term and near-term animals were thawed as previously described and added to different myometrial strips from one animal in a single experiment.

To test whether the response of the myometrium to the factor(s) released by chorion changes with the gestational age of the myometrium, near-term membranes from the same animal were added to myometrium from pre-term and near-term animals.

To establish whether the effect of chorion is dose-dependent, the chorion-induced relaxation of the oxytocin-stimulated myometrium was compared in the presence of either ~50 mg tissue/ml or ~100 mg tissue/ml bath in separate experiments using chorion from the same animal.

Chorion-conditioned medium
To determine whether chorion-induced relaxation of oxytocin-stimulated myometrium was the product of a substance released into the medium, chorion-conditioned medium was prepared as follows and its effect on myometrial contractility evaluated.

A sample of chorion (1500 mg) from an animal was thawed for 5 min in 37°C Krebs buffer. The membranes were then incubated an additional 30 min in a glass container with 5 ml of 37°C Krebs buffer. The membranes were discarded and the 5 ml freshly prepared supernatant added directly to the 10 ml organ bath (final ~100 mg chorion/ml buffer). The effect of chorion-conditioned medium on oxytocin-stimulated myometrial contractions was measured as described for chorion membranes.

Spontaneous contractility
The myometrial strips of pregnant guinea pig generally do not exhibit a regular pattern of spontaneous contractility. However, 3–5% of myometrial strips presented regular spontaneous activity. To determine the effect of the fetal membranes on spontaneous myometrial contractility, either chorion or amnion was added directly to the organ bath containing the spontaneously contracting myometrial strips and changes in contractile activity measured.

Human chorion
To determine whether the chorion of humans had a similar impact on oxytocin-stimulated contractions of guinea pig myometrium, chorion from a single, near-term Caesarean delivery without labour was obtained and frozen in liquid nitrogen until studied. The protocol for these studies was approved by the Institutional Review Board at the University of Maryland, Baltimore.

The previously stored frozen human chorion was weighed and thawed for 5min in 37°C Krebs solution. Approximately 1000mg was directly added to the 10 ml organ bath (100 mg membranes/ml buffer) after the establishment of regular contractions by oxytocin, and the isometric tension recorded an additional 30 min; the same protocol was used as for examination of the effects of guinea pig chorion.

Statistical analysis
All data were presented as average responses of five to eight animals from which the fetal membranes were obtained. All data sets (mean ± SEM) were subjected to a test of normalcy (Shapiro–Wilk test), and parametric or non-parametric tests performed when appropriate. Statistical comparisons between two groups were conducted using Student's t-test. Among multiple groups, a one-way analysis of variance (ANOVA) followed by post-hoc Student–Newman–Keuls (parametric) or a Kruskal–Wallis one-way ANOVA on ranks followed by Dunn's multiple comparison tests (non-parametric) was used. A two-tailed P < 0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Guinea pig myometrium as a model for isometric tension studies
The spontaneous contractile activity of isolated guinea pig myometrial strips was characterized by irregular contractions of long duration (~3 min for each contraction), high amplitude and variable frequency (from zero to four in 10min). The addition of oxytocin initially produced a tetanic contraction lasting 3–5 min, followed by regular contractions of short duration (30–60 s), low amplitude compared with the spontaneous contractions, and a frequency of 10–15 in 10 min (Figure 1Go). This regular pattern of activity was maintained with little variation for about 30 min. Thereafter, contractile activity slowly declined a non-significant degree reaching ~80% of the oxytocin-stimulated basal activity 60 min after initiating the experiment. In preliminary studies, it was determined that 10–8 mol/l oxytocin was the optimal concentration to produce a regular pattern of contractions.



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Figure 1. Representative tracing of the effect of chorion on oxytocin-stimulated contractility of the myometrium from the pregnant guinea pig.

 
Effect of chorion and amnion on myometrial contractile activity
The addition of chorion, but not amnion, to the organ bath produced a time-dependent decrease in the oxytocin-stimulated contractions of myometrium obtained from near-term pregnant guinea pigs (Figure 1Go). As shown in Figure 2Go, chorion significantly (P < 0.05 versus temporal control) reduced the contractile activity to 58 and 39% of the basal activity at 15 and 25 min respectively. There was no significant change in myometrial contractions without the addition of chorion (temporal control) (Figure 2Go). The contractility returned to a near-basal level (oxytocin-induced basal level) within 30 min after removing the membranes from the bath and replacing the existing solution with fresh buffer.



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Figure 2. Effect of near-term chorion membranes and chorion-conditioned medium on oxytocin-stimulated myometrial contractility. Values are mean ± SEM of eight experiments (chorion from eight different guinea pigs). *P < 0.05 compared with temporal control.

 
In contrast to chorion, the addition of amnion to the organ bath, either from pre-term or near-term animals, had no significant effect on the oxytocin-stimulated myometrial contractions (Figure 3Go).



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Figure 3. Effect of amniotic membranes from pre-term and near-term animals on oxytocin-stimulated contractility of near-term myometrium. Values are mean ± SEM of five experiments (amnion from five near-term and five pre-term guinea pigs).

 
As was demonstrated for oxytocin-stimulated myometrial contractility, the chorion—but not the amnion—induced a decrease in spontaneous myometrial contractility in four strips evaluated. After addition of chorion membranes (obtained from two different animals), the contractility decreased to 78, 68 and 56% of basal at 5, 15 and 25 min respectively, while after amnion addition, contractility was 95, 89 and 86% respectively.

The inhibitory effect of chorion on oxytocin-stimulated myometrial contraction was gestational age-dependent (Figure 4Go). Pre-term chorion produced a significantly greater (P < 0.05) reduction in contractile activity (41 and 23% of the basal activity at 15 and 25 min respectively) compared with that measured after adding near-term chorion (60 and 41% from baseline).



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Figure 4. Effect of chorion membranes from near-term and pre-term animals on oxytocin-stimulated contractility of near-term myometrium. Values are mean ± SEM of five experiments (chorion from five near-term and five pre-term guinea pigs). *P < 0.05 compared with near-term membranes.

 
Chorion induced-relaxation was independent of the gestational age of the myometrium. There were no significant differences in the ability of near-term chorion to inhibit oxytocin-stimulated contraction of myometrium obtained from pre-term and near-term animals (Figure 5Go).



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Figure 5. Effect of near-term chorion on oxytocin-stimulated contractile activity of myometrium from near-term and pre-term animals. Values are mean ± SEM of eight experiments (myometrium from eight near-term and eight pre-term guinea pigs; chorion from eight guinea pigs).

 
Relaxation induced by chorion was dose-dependent (Figure 6Go). Oxytocin-stimulated contractility was reduced to 75 and 61% of baseline at 15 and 25 min respectively after adding ~50 mg of chorion membranes per ml. The reduction in activity was significantly greater in the presence of ~100 mg membranes/ml, declining to 58 and 39% of baseline respectively at 15 and 25 min (P < 0.05).



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Figure 6. Effect of variable amounts of chorion on oxytocin-stimulated myometrial contractility. Values are mean ± SEM of eight experiments (chorion from eight different guinea pigs used in both amounts). *P < 0.05 compared with 50 mg chorion/ml.

 
Effect of chorion-conditioned medium on myometrial contractile activity
Chorion-conditioned medium had the same effect as the whole chorion on oxytocin-stimulated myometrial contractions (see Figure 2Go). The addition of chorion-conditioned medium reduced contractility to 63 and 49% of the basal activity at 15 and 25 min (P < 0.05 versus temporal control). Similar to chorion-induced responses, the effect of chorion-conditioned medium was reversed by washing the organ bath and replacing it with fresh buffer.

Effect of human chorion on myometrial contractile activity
The result of one experiment in duplicate testing the effect of human chorion on oxytocin-stimulated contraction of guinea pig myometrium is shown in Figure 7Go. After addition of the chorion membranes to the organ bath, the contractility decreased to 69, 47 and 39% of basal activity during the 10, 20 and 30 min periods following membrane addition. The effect was reversed by removing the membranes and replacing the solution with fresh buffer. Thus, human chorion had virtually an identical effect on oxytocin-stimulated contraction of guinea pig myometrium as did the guinea pig chorion.



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Figure 7. Representative tracing of the effect of human chorion on oxytocin-stimulated contractility of the myometrium obtained from a near-term pregnant guinea pig.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
It has been demonstrated for the first time that the chorion, but not the amnion, decreases oxytocin-stimulated myometrial contractions. The effect was dependent on the gestational age of the chorion (declining as pregnancy progressed), independent of the gestational age of the myometrium, and reproduced by chorion-conditioned medium.

Chorion from pre-term animals produced greater inhibition of oxytocin-stimulated myometrial contractions than chorion from near-term animals. The pre-term membranes were obtained from pregnant guinea pigs at 0.7 gestation (corresponding to 28 weeks of human pregnancy), a period under normal conditions of near-absent myometrial contractile activity. The near-term membranes approximated 0.9 gestation—a period equivalent to 36 weeks human pregnancy. By this time, the process of myometrial activation has begun and the uterus is more sensitive to contractile agonists (Norwitz et al., 1999Go). Since relaxation of oxytocin-stimulated myometrium by chorion is similar regardless of the gestational age of the myometrium, the gestational age differences of chorion-induced relaxation are due to differences in the capability of the chorion to produce relaxation rather than to differences in the ability of myometrium to relax.

Based on these data, it is suggested that the chorion produces one or more factors which inhibit oxytocin-stimulated myometrial contraction, and that the chorion-derived substance(s) is produced in greater amounts during the period of uterine quiescence. These observations support the hypothesis that chorion produces a substance that may have a role in the maintenance of uterine quiescence during pregnancy and that the generation of this substance(s) decreases near-term to allow myometrial activation.

Additionally, it has been demonstrated that the effect of chorion-conditioned medium is similar to the effect of whole chorion on oxytocin-stimulated myometrial contraction, suggesting that the inhibitory substance is released by the chorion and therefore capable of acting in a paracrine fashion.

Previous studies have also reported that the human chorion releases a substance that inhibits spontaneous and prostaglandin-stimulated but not oxytocin-stimulated myometrial contractility of the rat uterus, suggesting a role for the fetal membranes in the maintenance of uterine quiescence (Collins et al., 1993Go, 1995Go, 1996Go; Emery et al., 1998Go). In isolated strips of non-pregnant rat uterus, human chorion membranes were not able to inhibit oxytocin-induced contractions, but were shown to inhibit spontaneous contractions (Collins et al., 1993Go) and agonist-induced contractions to prostaglandin F2{alpha} (Collins et al., 1995Go) and the L-type calcium channel opener BAY K 8644 (Collins et al., 1996Go). From these studies it was proposed that a Ca2+-channel blocker was released from chorion that inhibits the activation of L-type Ca2+ channels of myometrium (Emery et al., 1998Go).

In the present study, we report a new finding that guinea pig chorion releases a factor that inhibits oxytocin-induced myometrial contractility in the pregnant guinea pig myometrium. It was found that spontaneous contractions were also inhibited by the chorion, but not by the amnion. The factor characterized appears different from the one described by Collins' group since, first, our model uses myometrium obtained from pregnant animals, and second, it was possible to inhibit contractions stimulated by oxytocin. Further, the human chorion, which did not relax rat myometrium (Collins et al., 1995Go), inhibited oxytocin-stimulated contraction of myometrium obtained from pregnant guinea pigs. While the two experimental designs of our group and Collins' group are similar, it is presumed that the apparently divergent findings are most likely due to differences in the model (pregnant versus non-pregnant myometrium) and species (guinea pig versus rat).

To search for additional differences between our model and the rat, we sought to study the effect of another contractile agonist. However, as previously reported by others, the guinea pig myometrium failed to generate regular contractions in response to other uterotonins such as prostaglandins, endothelin and platelet activating factor (Montrucchio et al., 1986Go; Coleman and Parkington, 1988Go). In our own experiments, only at a very high concentration (10–4 mol/l) did these agents produce a single, tetanic contraction lasting 3–5 min, followed by the absence of contractile activity.

The guinea pig has several strengths in the search for an understanding of human pregnancy compared with rats. First, it has haemomonochorial placentation, which is the most similar to human placenta among all non-primate mammals (Pijnenborg et al., 1981Go). Second, the sex hormone profiles of its oestrous cycle and pregnancy are similar to those of humans (Challis et al., 1971Go; Martensson, 1984Go). Third, the pattern of in-vitro contractility of myometrium from pregnant guinea pig is more similar to human than to rat myometrium. Finally, the relatively long and stable gestational period (65 ± 2 days from copulation) compared with 22 days for the rat, permits easy comparisons to be made between different gestational ages. Additionally, we believe that our model has an added advantage of using myometrium from pregnant animals, which may be central to the differences because of the important modifications occurring during pregnancy.

However, it is recognized that the human and guinea pig chorion are embryologically and structurally different (Ramsey, 1975Go). Thus, it is reasonable to ask if the present findings in the guinea pig chorion are applicable to humans. Our preliminary data indicate that human chorion obtained from a woman at term, prior to labour is—like the guinea pig chorion—able to relax oxytocin-stimulated contractions of the pregnant, guinea pig myometrium (Figure 7Go). As a consequence, the guinea pig myometrium can be used as a biological model to explore physiology of human pregnancy. Our preliminary data also suggest that a chorion-derived substance may modulate myometrial contractility during human pregnancy.

The chemical identity of the substance(s) released by the guinea pig chorion and perhaps the human chorion that inhibits oxytocin-stimulated contractions has (have) not yet been elucidated. However, several substances produced by the fetal membranes, decidua and myometrium itself have been studied and proposed as possible regulators of uterine quiescence based on their known ability to relax smooth muscle (Downing and Hollingsworth, 1992Go; Ferguson et al., 1992Go; Itoh et al., 1993Go; Bansal et al., 1997Go; Acevedo and Ahmed, 1998Go; Brodt-Eppley and Myatt, 1999Go). Also, a line of evidence has suggested that the inhibitory role of the fetal membranes, particularly the chorion, on uterine contractile activity is based in the capacity of degrading locally produced uterotonins, thus preventing their action on the myometrium (Germain et al., 1994Go). Our data refute this possibility as an explanation for the chorion-induced relaxation of oxytocin-stimulated contractions, since after washing the strips and replacing the bath with fresh buffer (not containing oxytocin) the contractility returned to a near-oxytocin-induced basal level.

In summary, the chorion may release one or multiple factors that mediate uterine quiescence during pregnancy in a paracrine fashion. While we document the ability of the chorion to inhibit oxytocin-stimulated myometrial contractility in vitro, the exact nature of the relaxing factor (or factors) released by chorion during gestation remains to be elucidated. The identification and characterization of the mechanism of action of this substance(s) will be important for understanding the process of parturition and for the development of pharmacological tools useful in the management of pre-term labour in humans.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported by grants from the National Institutes of Health: Fogarty International Center 5 F05 TW05442-02 (J.A.C.); HL49999 (L.P.T.) and HL49041, HD24492, HL51735 (C.P.W.).


    Notes
 
1 To whom correspondence should be addressed. E-mail: cweiner{at}umm.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on September 14, 2000; accepted on January 4, 2001.





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