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Regular article |
a MRC Group in Fetal and Neonatal Health and Development,
b Departments of Physiology and Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada M5S 1A8
c Mt. Sinai Hospital, Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada M5G 1X5
d University of Western Ontario and Lawson Research Institute, London, Ontario, Canada N6A 5B8
e MRC Group in Development and Fetal Health, Department of Obstetrics and Gynecology, University of British Columbia, British Columbia, Canada V6T 1Z3
f
g Faculty of Pharmaceutical Sciences, University of British Columbia, British Columbia, Canada V6T 1Z3
ABSTRACT
Term and preterm labor are associated with increased fetal hypothalamic-pituitary-adrenal (HPA) activation and synthesis of prostaglandins (PGs) generated through the increased expression of prostaglandin H synthase-II (PGHS-II) in the placenta. Inhibition of PGHS-II has been advocated as a means of producing uterine tocolysis, but the effects of such treatment on fetal endocrine functions have not been thoroughly examined. Because PGE2 is known to activate the fetal HPA axis, we hypothesized that administration of meloxicam, a PGHS-II inhibitor, to sheep in induced labor would suppress fetal HPA function. Chronically catheterized pregnant ewes were treated with RU486, a progesterone receptor antagonist, to produce active labor, and then treated with either high-maintenance-dose meloxicam, graded-maintenance-dose meloxicam, or a saline infusion. Maternal uterine contraction frequency increased 24 h after the RU486 injection and the animals were in active labor by 48 ± 4 h. RU486 injection led to increased concentrations of PGE2, ACTH, and cortisol in the fetal circulation, and increased concentrations of 13,14 dihydro 15-ketoprostaglandin F2
(PGFM) in the maternal circulation. Uterine activity was inhibited within 12 h of beginning meloxicam infusion at both infusion regimes. During meloxicam infusion there were significant decreases in fetal plasma PGE2, ACTH, and cortisol concentrations, and PGFM concentrations in maternal plasma. In control animals, frequency of uterine contractions, maternal plasma PGFM, fetal plasma PGE2, ACTH, and cortisol concentrations increased after RU486 administration, and continued to rise during saline infusion until delivery occurred. We conclude that RU486-provoked labor in sheep is associated with activation of fetal HPA function, and that this is attenuated during meloxicam treatment to a level considered compatible with pregnancy maintenance.
ACTH, cortisol, parturition
INTRODUCTION
Preterm labor occurs in 5%–10% of all pregnancies and accounts for 70% of neonatal morbidities and mortality not attributable to congenital anomalies [1]. Long-term morbidities, including neurological handicap and learning difficulties, impose a substantial cost on the health care and education systems as well as placing emotional hardship and stress on families [2]. Currently, there is no treatment for preterm labor that is safe and effective for more than 48 h [3]. A greater understanding of the mechanisms of term and the pathogenesis of preterm labor should lead to the development of improved prevention and treatment strategies.
The onset and progression of labor across a variety of species is associated with increased intrauterine prostaglandin (PG) production; in particular PGE2 and PGF2 [4, 5]. These PGs mediate cervical dilation and uterine contractility and may play a role in fetal adaptation to labor [4]. PGs are formed from arachadonic acid released from membrane stores by the action of PGH synthase (PGHS), which catalyzes a cyclo-oxygenation-peroxidation reaction to form the PG intermediate, PGH2 [6, 7]. PGH2 is then converted to a variety of primary PGs through the action of different PG isomerases [7, 8]. There are two PGHS isoforms. Type I is a constitutively expressed enzyme with a wide tissue distribution that is responsible for cellular housekeeping functions [8]. PGHS type II is a highly inducible enzyme that is responsible for mediating acute-phase reactions, including inflammation [7]. Although both PGHS-I and PGHS-II have been identified within the human and ovine intrauterine tissues, it is the expression and activity of PGHS-II that increases toward the end of gestation and has been directly correlated with the onset of labor [9–12]. It has been suggested that inhibition of PG production may provide an effective method for the treatment of preterm labor. Nonsteroidal anti-inflammatory drugs are effective inhibitors of PGHS activity [13, 14]. Indomethacin has been identified as a nonspecific inhibitor of both PGHS-I and PGHS-II and tocolysis has been achieved when the drug is administered in active labor [15, 16]. Although this drug has demonstrated favorable tocolytic effects, there is a high incidence of undesirable fetal side effects, including premature closure of the ductus arteriosus and intraventricular hemorrhage [17–19]. It has been suggested that the main cause of these fetal side effects is the inhibition of the PGHS-I enzyme [20, 21]. Recently, specific PGHS-II inhibitors have become available; however, their safety and efficacy as tocolytic agents have yet to be demonstrated.
In sheep, sustained activation of the fetal hypothalamic-pituitary-adrenal (HPA) axis and the subsequent rise in fetal circulating cortisol concentrations provides the trigger for intrauterine PG production [4]. Several studies have shown that intrafetal cortisol administration increases fetal plasma PGE2 concentration and intrauterine PGHS-II expression [9, 22]. Fetal plasma cortisol and PGE2 concentrations both increase from approximately 110–115 days of gestation (term 
150 days), suggesting that fetal HPA function and placental PGE2 production may be linked in a positive feedback manner [23, 24]. Previously, it has been shown that intrafetal PGE2 infusion leads to increased fetal HPA function, suggesting that intrauterine PG production may be responsible for stimulating or maintaining the sustained increase in fetal HPA activity observed toward the end of gestation and through the progression of labor [25, 26]. Inhibition of intrauterine PG production would therefore inhibit uterine contractions but should also attenuate fetal HPA activation, which is the proposed trigger of intrauterine PG production. Therefore, we examined effects of specific PGHS-II inhibition in sheep during induced labor and hypothesized that this should produce uterine tocolysis during preterm labor and also lead to a decrease in fetal HPA activity.
MATERIALS AND METHODS
Animal Preparation
Pregnant ewes of mixed breed and known gestational age were used. Catheters were implanted into the maternal femoral artery and vein, and fetal carotid artery and fetal jugular vein under general anesthesia at Day 120 of gestation as previously described [27]. Electromyogram (EMG) electrodes were implanted into the myometrium to monitor myometrial activity and a catheter was placed in the amniotic cavity to measure intrauterine pressure. A 5-day period of postoperative recovery was allowed prior to initiating the experimental protocol.
Experimental Protocol
All animals (n = 12) received a maternal s.c. injection of RU486 (10 mg/kg), a progesterone receptor antagonist on Day 127 of gestation. RU486 has been shown to induce labor in sheep within 44 ± 4.1 h [28]. Once the animals were determined to be in active labor, which was defined as contractions of frequency greater than 20 per 1 h, duration less than 1 min, and amplitude greater than 5 mm Hg, they were divided into 3 groups of 4 animals each: 1) controls, infused with maternal i.v. saline (n = 4); 2) high-maintenance dose (HMD) meloxicam maternal i.v. infusion 2 µg/kg per minute for 2 h, then 4 µg/kg per minute for 2 h, then increased by 4 µg/kg per minute until the uterine activity pattern returned to a baseline contracture pattern of fewer than two contractions per hour. Meloxicam was continued at this dose until termination of the protocol (n = 4); 3) graded-maintenance-dose (GMD) meloxicam infusion given as in group 2 until contractions returned to baseline of fewer than two contractions per hour. Then the meloxicam was decreased by 4 µg/kg per minute until a maintenance dose of 2 µg/kg per minute was reached for the remainder of the protocol (n = 4). A second injection of RU486 was given within 12 h of starting the saline or meloxicam infusion. Following 48 h of infusion, the animals were killed with an overdose of sodium pentobarbital (Euthanyl, MTC Pharmaceuticals, Cambridge, ON, Canada).
Starting 48 h prior to RU486 administration and continuing throughout the infusion period, maternal (4–5 ml) and fetal (2–3 ml) arterial blood samples were collected at 8-h intervals. Blood to be used for the determination of ACTH and cortisol levels was collected in syringes that had been rinsed with heparinized saline; blood which was to be subsequently used for the determination of PGE2 and 13,14 dihydro 15-ketoprostaglandin F2 (PGFM) were collected in nonheparinized syringes and then transferred to vials containing indomethacin (150 µl indomethacin/EDTA per 1.5 ml blood). Plasma was separated from red blood cells by centrifugation at 1500 x g for 10 min at 4°C and stored at -20°C in three separate aliquots.
Intrauterine pressure and myometrial activity were continuously monitored starting on Day 125 of gestation. Uterine EMG activity was processed by a Grass wide-band AC preamplifier, Model 7P511J. The EMG signal was recorded using a Grass 78 D electroencephalogram and polygraph data recording system (Grass Instruments, Quincy, MA).
PGE2; PGFM, ACTH, and Cortisol Radioimmunoassay
Extraction of plasma samples and radioimmunoassay (RIA) for fetal plasma PGE2, cortisol, and maternal plasma PGFM were conducted as previously described [29–31]. The intra-assay coefficients of variation were 8%, 1%, and 4% respectively; the assay sensitivities were 6.5 pg/ml, 2 pg/ml, and 19 pg/ml, respectively. The RIA for fetal plasma ACTH was performed using a commercially available 125I RIA kit (DiaDsorin Inc., Stillwater, MN), which was validated for use with ovine plasma [31]. The intra-assay coefficient of variation was 1% and the sensitivity was 5.9 pg/ml.
Statistical Analysis
Data are presented as the mean ± SEM. In the control group of animals, delivery occurred within 18 h of saline infusion. Given that animals that did not receive meloxicam delivered prematurely after RU486 alone, as expected, the effects of meloxicam on the plasma profiles of PGE2, cortisol, and uterine contractions were assessed within group using one-way ANOVA for repeated measures followed by posthoc analysis by Bonferroni correction with significance set at P < 0.05. Fetal plasma ACTH and maternal PGFM levels were evaluated within group using one-way ANOVA for repeated measures followed by posthoc analysis by the Fisher test of least significant difference with significance set at P < 0.05.
RESULTS
Uterine Contractility
Labor-type uterine contractions were induced after RU486 administration and these contractions were increased significantly to a mean frequency of 22.6 ± 2.2 contractions per hour (range 20 to 26) at 48 ± 4 h after the RU486 injection (0 h in Table 1) in all three treatment groups. Both HMD and GMD meloxicam administration, beginning at 0 h (Table 1) significantly decreased uterine contraction frequency; following 18 h of meloxicam treatment, uterine contraction frequency had returned to basal level and was maintained at that level until termination of the protocol. Control animals delivered within 18 ± 4 h of maternal saline infusion starting at time 0 h (Table 1).
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Maternal and Fetal Plasma PG Levels
Maternal plasma concentrations of PGFM in the control group increased after RU486 treatment from 150 ± 17 pg/ml to 1188 ± 350 pg/ml within 18 h prior to delivery (Fig. 1). Animals in the HMD and GMD meloxicam treatment groups also exhibited a significant increase in PGFM plasma concentration 48 h after the RU486 injection (Fig. 1) and a significant decrease in PGFM concentration following 48 h of meloxicam infusion (Table 1).
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Mean fetal plasma PGE2 concentration was significantly increased in all three treatment groups 48 h after the animals received RU486 (Fig. 2). The saline-treatment animals had a peak PGE2 plasma concentration of 3740 ± 1100 pg/ml within 18 h prior to delivery. Fetuses in the HMD and GMD treatment groups showed a significant decrease in PGE2 concentrations, which returned to basal levels (Table 1) following 48 h of meloxicam infusion (Fig. 2).
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Fetal Plasma ACTH and Cortisol Levels
Mean fetal plasma ACTH concentrations in all three groups of animals increased significantly 48 h after RU486 injection (Fig. 3). Plasma ACTH concentration in the control group increased from basal (34.0 ± 3.3 pg/ml) to 110 ± 31 pg/ml 48 h after RU486 injection. Fetuses in the HMD and GMD meloxicam treatment groups showed a significant increase in plasma ACTH concentration to similar values 48 h after RU486 injection (Fig. 3) and a significant decrease in ACTH concentration during meloxicam infusion and prior to the termination of the protocol (Table 1).
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Mean fetal plasma cortisol concentrations in the three groups were significantly increased 48 h after the RU486 injection (Fig. 4). The plasma cortisol concentration in the control group increased from basal (3.1 ± 0.4 ng/ml) to 31.5 ± 7.0 ng/ml within the 18 h prior to delivery. Fetuses in both the HMD and GMD meloxicam treatment groups showed a significant increase in plasma cortisol concentration 48 h after RU486 injection, followed by a significant decrease following 24 h of meloxicam infusion (Fig. 4). This significant decrease in plasma cortisol was maintained for the remainder of the 48-h infusion period in the HMD group; however, the fetal plasma cortisol concentration in the GMD group was no longer significantly decreased by the termination of protocol (Table 1).
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DISCUSSION
In this study we have found that specific PGHS-II inhibition with meloxicam effectively decreased uterine contractility and attenuated fetal HPA activation induced by administration of the progesterone receptor antagonist, RU486. Meloxicam infusion significantly decreased fetal plasma cortisol and ACTH concentrations with time courses that were similar and related temporally with the attenuation of fetal plasma PGE2 concentration. These data are consistent with changes in intrauterine PG synthesis playing an important role in the maintenance of fetal HPA activation as well as uterine contractility.
We recognize that labor induction by RU486 may not exactly mimic the endocrine events of spontaneous term or preterm labor. RU486 is a progesterone receptor antagonist that has been shown to induce ovine parturition in a highly reproducible manner, apparently indistinguishable from spontaneous term labor [28]. RU486 also has been shown to have antiglucocorticoid effects in addition to its antiprogesterone effects [32]. However, it has been demonstrated in vivo that these antiglucocorticoid effects are obtained by administration of RU486 at doses far greater than are needed to obtain the antiprogesterone effects and the onset of labor [32]. Therefore, it is probable that the amount of RU486 used in this study induced primarily antiprogesterone effects without altering glucocorticoid metabolism or potential glucocorticoid effects on intrauterine PG production and HPA axis activity.
A current hypothesis of ovine parturition suggests that the surge in fetal adrenal cortisol production at the end of gestation, which results from sustained activation of the fetal HPA axis, leads to increased placental trophoblast PGHS-II expression and PGE2 production [9, 22]. PGE2 in turn stimulates placental P450C17 hydroxylase activity, thereby contributing to placental estradiol production. Estradiol up-regulates maternal intrauterine tissue PGHS-II expression and PGF2 production and triggers the expression of a specific cassette of contraction-associated proteins (CAPs) within the myometrium [9, 22, 33, 34]. Consequently, labor is initiated. In addition to mediating placental steroidogenesis, PGE2 may play a role in regulating the sustained activation of the fetal HPA axis observed at the end of gestation and through the progression of labor [25, 26]. We have found that infusion of a specific PGHS-II inhibitor leads to a decrease in circulating PGE2 concentration as well as decreases in fetal plasma cortisol and ACTH concentrations. The decrease in HPA activity was related in time with the decline in PGE2 production. These data suggest, therefore, that placental PGE2 may be responsible for maintaining fetal HPA axis activation. In support of this hypothesis, studies have demonstrated similar attenuation of fetal HPA activity in sheep with maternal administration of another PGHS inhibitor, nimesulide, during spontaneous term labor [33, 34]. However, the effect of nimesulide that was reported is based on basal HPA activity, whereas in the present study, we examined effects of PGHS-II inhibition in the presence of stimulated HPA activity, as one may predict at a time of preterm labor [4, 9]. It is also possible that the increase in uterine activity after RU486 administration provides a direct stimulus to fetal HPA function that was reversed by meloxicam after uterine inhibition, rather than HPA activity being attributable to changing output of placental PGE2. Other studies, however, referred to later, support the latter relationship.
Previously, it has been shown that intrafetal PGE2 infusion stimulated fetal HPA activity [25]. The site or sites of PGE2 action within the fetal HPA axis are not well established but could include one or all of the hippocampus, hypothalamus, pituitary, and adrenal cortex. One study compared the effect of PGE2 administration to intact ovine fetuses with that in fetuses in which the pituitary had been surgically disconnected from hypothalamic control [35]. Intrafetal PGE2 infusion caused a significant increase in plasma ACTH and cortisol levels in the intact fetuses at several times in gestation but did not change cortisol or ACTH levels in the hypothalamic-disconnected fetuses. The authors concluded that PGE2 must influence the fetal HPA axis predominantly at or above the level of the pituitary [35]. Previously, it has also been shown that PGE2 can increase P450C17 hydroxylase mRNA and protein expression in cultured bovine adrenal cells and thereby lead to increased cortisol production [36]. These data suggest that in addition to its effects on the pituitary, PGE2 could affect fetal adrenal cortisol production. It should be noted that in the present study, in the animals treated with graded-dose meloxicam, a gradual increase in fetal plasma cortisol and ACTH was observed as the dose of meloxicam was lowered (data not shown). We suggest that this may reveal incomplete inhibition of PGHS-II activity due to a subtherapeutic maintenance dose of meloxicam. In a previous study, a similar increase in fetal plasma cortisol but not fetal ACTH following nimesulide administration was observed [34]. Nimesulide inhibits both PGHS isoforms with selectivity for the type II enzyme [37, 38]. Thus, the dose of nimesulide used may not have been great enough to provide effective and sustained PGHS-II inhibition, similar to the effects we observed with the lower dose of meloxicam.
We also demonstrated that specific PGHS-II inhibition decreased intrauterine PGF2 production, which was reflected in maternal plasma PGFM concentrations. Previous studies have shown a direct correlation between increased PGF2 production and the spontaneous onset of labor contractions at term [22, 39, 40]. During preterm labor induced by infusing cortisol to the fetal sheep, inhibition of PGF2 output but not PGE2, led to an attenuation of uterine contractility [22]. We speculate that PGF2 is a key uterotonin and perhaps a more potent stimulant of uterine contractility in sheep than PGE2. It has been found that inhibition of intrauterine PG production by nimesulide, another PGHS inhibitor, decreased uterine CAP gene expression, suggesting that PGs may also contribute to uterine contractility through the up-regulation of CAPs [34]. Thus, the inhibition of PGHS-II activity may attenuate uterine activity through decreases in both uterotonin production and uterine CAP gene expression.
In summary, we have shown that inhibition of PGHS-II by meloxicam decreased the uterine contractility and fetal HPA axis activity produced by administration of RU486. Because PGE2 may act to maintain fetal HPA axis function through a positive feedback mechanism, inhibition of PGE2 production would not only attenuate HPA axis function but also abolish the stimulus for further intrauterine PG production. PGF2 has been shown to be a potent uterotonic agent as well as a contributor to the stimulus of myometrial CAP expression. Therefore, inhibition of uterine PGF2 production would limit uterine activity through two separate mechanisms. Thus, meloxicam may prove to be an effective tocolytic agent because of its ability to inhibit the mechanisms of parturition on several levels: the HPA trigger, the production of uterotonins, and the expression of CAPs. However, the safety of this potential tocolytic agent to mother and fetus remains to be evaluated.
ACKNOWLEDGMENTS
We thank S. Diamant for her support of this work.
FOOTNOTES
1 We gratefully acknowledge the support of the Medical Research Council of Canada (Operating Grant MT-14395; Group Grant in Development and Fetal Health; Group Grant in Fetal and Neonatal Health and Development). 
2 Correspondence: Kevin McKeown, Department of Physiology, Medical Sciences Building, University of Toronto, 1 King's College Circle, Toronto, ON, Canada M5S 1A8. FAX: 416 978 4940; kevin.mckeown{at}utoronto.ca
Accepted: July 31, 2000.
Received: April 11, 2000.
REFERENCES
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D. M. Olson The Promise of Prostaglandins: Have They Fulfilled Their Potential as Therapeutic Targets for the Delay of Preterm Birth? Reproductive Sciences, October 1, 2005; 12(7): 466 - 478. [Abstract] [PDF]
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