Increased myometrial contracture frequency at 96-140 days accelerates fetal cardiovascular maturation

¹ Laboratory for Pregnancy and Newborn Research, Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401; and ² Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Nagano 113-8655, Japan

	ABSTRACT

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Fetal cardiovascular responses to an altered intrauterine environment of increased myometrial contractures induced by oxytocin (OT) pulses to the ewe over the final 50 days of gestation were studied in chronically instrumented sheep. Ewes received saline (Cntl) or long-term OT treatment (LTOT, 600 µU · kg¹ · min¹ in 5-min pulses every 20 min) from 96 days gestational age. Fetal baroreflex responses to sodium nitroprusside (SNP) and phenylephrine (PE) were studied at 133 days gestation. OT increased contractures in LTOT ewes. Fetal blood pressure (FBP) was higher, and fetal heart rate (FHR) and slope of daily change in FBP and FHR were lower in LTOT fetuses. Fetal SNP-induced hypotension resulted in a narrow R-R interval variation range in LTOT fetuses; Cntl fetuses showed early breakdown in compensation. Baroreflex response slope during PE-induced fetal hypertension was lower in LTOT than in Cntl fetuses. Although the cortisol-to-ACTH ratio was lower in LTOT fetuses, fetal plasma ACTH and cortisol changes were similar in control and LTOT fetuses. We hypothesize that contracture-induced alterations in the intrauterine environment accelerate fetal cardiovascular development through mild hypoxemia, repetitive fetal pituitary-adrenal stimulation, and/or physical stimulation.

fetus; cardiovascular response; baroreflex; oxytocin; uterine contractures

	INTRODUCTION

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THERE IS CURRENTLY much interest in the effects of the quality of the intrauterine environment on the developing cardiovascular system (2, 3, 16). Recent epidemiological studies indicate that an adverse intrauterine environment affects fetal physiological development and may be the origins of later disease (2, 3). Restricted maternal nutrition was shown to have marked effects on fetal cardiovascular function (3). Studies of maternal nutrient restriction in rats and sheep demonstrated that differences in fetal growth are associated with changes in cardiovascular control and development of hypertension in the offspring (18, 26, 27). Prolonged hypoxia during fetal development produces long-term and short-term effects on fetal cardiovascular function in sheep (1, 21, 24, 25).

Myometrial contracture activity occurs throughout gestation and constitutes a major intrauterine influence acting on the developing fetus. Myometrial contractures are characterized by low-amplitude epochs of myometrial contractility that last at least 3 min. Contractures induce a fall in fetal arterial PO₂ (22, 23, 29, 31, 49), a decrease in uterine blood flow (28, 42), and a decrease in fetal activity and O₂ uptake (5, 28). Contractures also compress the fetus (31). Increased myometrial contractures induced at a critical period of maturation in the last third of gestation alter fetal pituitary-adrenal function (29, 32, 33, 38) and accelerate maturation of fetal electrocorticogram (ECoG) voltage amplitude (39).

In the present study, we sought to determine the fetal cardiovascular effects of increasing the frequency of contractures occurring throughout the majority of the final third of pregnancy. Contracture frequency was increased by pulsing the ewe with oxytocin (OT) for 5 min every 20 min. We previously demonstrated (14) that OT does not cross the ovine placenta. Thus direct effects of OT on the fetus are not responsible for any fetal changes (14). We also examined the ontogenic profile of fetal blood pressure (FBP), heart rate (FHR), and baroreflex responses evoked by sodium nitroprusside (SNP) and phenylephrine (PE).

	MATERIALS AND METHODS

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Care of Animals

Mature Rambouillet-Columbia cross-bred ewes (n = 11) bred on a single occasion (weighing 50-60 kg) and carrying a fetus of known gestational age were used. All procedures were approved by the Cornell University Animal Care and Use Committee. All facilities were approved by the American Association for the Accreditation of Laboratory Animal Care. For 7 days before surgery, ewes were housed in metabolic stalls with free access to food and water in a room with controlled light-dark cycles (lights on at 0700 and off at 2100).

Surgical Instrumentation

Maternal carotid arterial and jugular vein catheters were placed in all animals at 91 ± 1 (mean ± SE) days gestation (term = 148 days gestation). At 122 ± 1 days gestation, fetuses were instrumented with carotid arterial and jugular vein catheters as well as ECoG, electrocardiogram (ECG), and skeletal muscle electromyogram (EMG) leads for behavioral analyses. Myometrial EMG electrodes were also placed. All techniques have been described elsewhere in detail (10, 31, 40).

Before surgery, ewes received 1 g of ampicillin sodium (Polycillin-N, Bristol Laboratories, Syracuse, NY), ketamine (5-10 mg/kg), and 1 mg of glycopyrollate as premedication. Surgery was performed under halothane general anesthesia. For 4 days after surgery, 0.5 g of ampicillin sodium (AMP-Equine, SmithKline Beecham, West Chester, PA) prophylactic antibiotic treatment was administrated twice a day to the maternal jugular and fetal amniotic catheters. One gram of phenylbutazone (Equiphen, Pharma Cenricalas, Shirley, NY) was given to the ewe orally twice a day for pain relief.

Maternal and fetal arterial blood samples (0.5 ml) were taken every day after fetal surgery for blood gas analysis. Beginning at 128 days gestation, maternal and fetal blood samples (3 ml) were taken every 2 days to measure ACTH and cortisol. Heparin (10 U/ml of physiological saline) was continuously infused at a rate of 0.5 ml/h into each vascular catheter to ensure patency.

Vehicle or Long-Term Oxytocin Pulse Administration

Starting at 96 days gestation, ewes received either saline [control group (Cntl), n = 5] or 5-min OT [600 µU · kg¹ · min¹;long-term oxytocin pulsed group (LTOT), n = 6] pulses into the maternal jugular vein at 0.033 ml/min every 20 min. OT pulse administration was stopped a day before fetal surgery and recommenced 2 days after fetal surgery.

Data Acquisition

Beginning at least 3 days after surgery, myometrial EMG, maternal blood pressure (MBP), and FBP and FHR were recorded continuously using a data acquisition system as previously described (10, 40). FHR was calculated from the ECG signals. To study ontogenic development, FBP and FHR (beats/min) were averaged over 40 s and a daily average obtained from 126 to 140 days gestation. Data of adequate quality for analysis was obtained >96 ± 0.7% of the time in all animals.

Evaluation of Baroreflex Function

For the studies on baroreflex responses, FBP and ECG signals were recorded using an IBM-PC-based computer (80486 CPU at 33 MHz) with 16-bit analog-digital interface (CODAS system, DATAQ Instruments, Akron, OH) at a sampling speed of 250 Hz. Mean FBP and R-R intervals (ms) were calculated every 10 s.

Response to hypotension. At 133 days gestation, after 1-h basal recording, SNP (10 µg/ml) was infused intravenously to the fetus beginning at a rate of 0.1 ml/min. The rate of SNP infusion was doubled every 2 min until FBP had decreased by 30%, FHR fell below 100 beats/min, or arrhythmia appeared. Fetal arterial blood samples were taken at 5 min, at the end of the SNP infusion, and at +10 min for blood gas and hormone analysis.

Response to hypertension. After at least 1 h of recovery after the hypotension study, fetuses received PE (25 µg/ml) intravenously beginning at a rate of 0.1 ml/min. The rate was doubled every 2 min until FBP increased 30%, FHR fell below 100 beats/min, or arrhythmia appeared. Fetal arterial blood samples were taken at 5 min, at the end of the PE infusion, and at +10 min for blood gas and hormone analysis.

Blood Gas Measurements

Blood gases were measured using an ABL605 blood gas analyzer (Radiometer, Copenhagen, Denmark). Measurements were corrected to 39°C. O₂ saturation (%) and Hb (mg/dl) were measured with a hemoximeter (OSM2, Radiometer). O₂ content (ml/dl) was calculated from Hb, O₂ saturation and PO₂ values.

RIA for ACTH and Cortisol

Plasma ACTH and cortisol concentrations were measured using commercially available kits (ACTH ¹²⁵I RIA kits, INCstar, Stillwater, MN; double-antibody cortisol RIA kits, Diagnostic Products, Los Angeles, CA) as previously reported (43).

Statistical Analyses

Data are presented as means ± SEM. Analyses were carried out by SAS version 6 and JMP 3.1 software (SAS Institute, Cary, NC). Multivariate ANOVA was applied for data analysis, and differences were considered significant at P < 0.05.

	RESULTS

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OT pulses increased myometrial contracture frequency by slightly over 100% in LTOT ewes compared with Cntl ewes (Table 1). Fetal arterial blood gas values are summarized in Table 2. PO₂ was lower in LTOT at 126-135 days gestation, and O₂ content was higher in LTOT at 131-140 days gestation. Basal fetal plasma ACTH and cortisol values during the study period are presented in Fig 1. ACTH rose at the rate of 1.3 ± 0.5 pg · ml¹ · day¹ in Cntl fetuses and 1.1 ± 0.4 pg · ml¹ · day¹ in LTOT fetuses. These rates were not significantly different. There were no significant differences in plasma ACTH and cortisol values between Cntl and LTOT fetuses. However, cortisol-to-ACTH ratios (ng/pg) were significantly lower in LTOT fetuses compared with controls (0.06 ± 0.03 and 0.13 ± 0.02 at 128 days gestation; 0.08 ± 0.04 and 0.24 ± 0.09 at 130 days gestation; and 0.08 ± 0.04 and 0.21 ± 0.08 at 132 days gestation for LTOT and Cntl fetuses, respectively). Cortisol was higher at 140 days gestation in both Cntl and LTOT fetuses compared with values at 128 days gestation. No differences in fetal weight or organ weights were observed at necropsy at 141 days gestation between Cntl and LTOT (Table 3).

View this table: [in this window] [in a new window]   Table 1.   Effect of oxytocin pulses or vehicle control on number of contractures per hour

View this table: [in this window] [in a new window]   Table 2.   Fetal arterial blood gas values of control fetuses and fetuses of ewes pulsed with oxytocin

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Fetal plasma ACTH (left) and cortisol (right) concentrations in fetuses of control ewes (Cntl, open bars, n = 5) and ewes pulsed with oxytocin [long-term oxytocin (LTOT), filled bars, n = 6]. Values are means ± SE. a P < 0.05 compared with values in same group at 128-138 days gestational age.

View this table: [in this window] [in a new window]   Table 3.   Fetal body and organ weights

Ontogenic Changes in FBP and FHR

FBP was significantly higher in the fetuses of LTOT ewes compared with the fetuses of Cntl ewes at the start of the analysis period (126-128 days gestation). At this stage, OT had been pulsed for 30-32 days. FHR was significantly lower in the LTOT fetuses from 126-131 days gestation (Fig. 2). In addition, the slopes of daily FBP changes (Cntl 0.76 ± 0.21, LTOT 0.27 ± 0.06 mmHg/day gestation) and FHR (Cntl 1.91 ± 0.20, LTOT 0.70 ± 0.41 beats · min¹ · day gestation¹) were significantly lower in LTOT fetuses (Fig. 2).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Ontogenic changes in fetal blood pressure (FBP, top) and fetal heart rate (FHR, bottom) in Cntl (, n = 5) and LTOT (, n = 6) fetuses. Values are means ± SE. * P <0.05 compared with Cntl. bpm, Beats/min.

Responses to Hypotension Induced by SNP

No differences were observed between the two groups of fetuses with respect to the fall in FBP at the end of SNP infusion. FBP dropped from 47.3 ± 1.0. to 29.6 ± 0.5 mmHg in Cntl fetuses and from 47.5 ± 1.3 to 30.9 ± 5.8 mmHg in LTOT fetuses. This represents a fall of approximately one-third. However, the total SNP dosage (adjusted by fetal weight) required to produce this fall in FBP was significantly higher in LTOT fetuses (34.7 ± 4.6 µg/kg) compared with Cntl fetuses (21.1 ± 3.7 µg/kg). Initially, the fetus attempts to compensate for the FBP fall by increasing FHR. Cntl fetuses showed an early breakdown of this compensatory tachycardia with the appearance of a marked bradycardia. In contrast, LTOT fetuses showed a much narrower range in R-R interval (Figs. 3 and 4). The slope of the baroreflex response before the onset of bradycardia was significantly lower in LTOT fetuses (3.1 ± 0.7 ms/mmHg) compared with Cntl fetuses (9.3 ± 2.5 ms/mmHg). PO₂ and O₂ saturation values, on the other hand, were significantly lower at the end of SNP infusion in Cntl fetuses. Fetal blood gas values did not deviate from normal values during hypotension in LTOT fetuses (Table 4). ACTH and cortisol responses to hypotension in fetuses of LTOT ewes were attenuated compared with fetuses of Cntl ewes. The difference was significant at +10 min for cortisol (P < 0.05), whereas for ACTH the P value was 0.07 (Fig. 5A). However, no differences were found in cortisol-to-ACTH ratios (ng/pg) between Cntl and LTOT fetuses (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   FBP and FHR (R-R interval) during hypotension induced by sodium nitroprusside (SNP) infusion directly to Cntl (, n = 5) and LTOT (, n = 6) fetuses. Values are means ± SE. * P <0.05 compared with LTOT.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   A: baroreflex response during hypotension induced by SNP infusion to Cntl (n = 5) and LTOT (n = 6) fetuses. Data are plotted every 1 min. Solid line shows mean value, and enclosed areas represent SE range. B: baroreflex response during hypertension induced by phenylephrine (PE) infusion to Cntl (n = 5) and LTOT (n = 6) fetuses. Data are plotted every 1 min. Solid line shows mean values, and enclosed areas represent SE range.

View this table: [in this window] [in a new window]   Table 4.   Arterial blood gas values of Cntl and LTOT fetuses during sodium nitroprusside-induced hypotension

View larger version (26K): [in this window] [in a new window]   Fig. 5.   ACTH and cortisol response during hypotension induced by SNP infusion (A) and hypertension induced by PE infusion (B) in Cntl (, n = 5) and LTOT (, n = 6) fetuses. Values are means ± SE. a P < 0.05 compared with baseline; b P < 0.05, Cntl vs. LTOT. End, end of infusion; +10 min, 10 min after infusion.

Responses to Hypertension Induced by PE

PE infusion increased FBP in Cntl fetuses from 48.0 ± 1.7 to 60.1 ± 3.3 mmHg and in LTOT fetuses from 47.4 ± 0.9 to 60.1 ± 1.5 mmHg. This represents an approximately one-third increase and was not different between groups. The total PE dosage (adjusted by fetal weight) required to produce the rise in FBP was significantly higher in LTOT (34.0 ± 3.4 µg/kg) compared with Cntl (22.8 ± 2.5 µg/kg). Although there was no difference in the final FBP rise between Cntl and LTOT, the final R-R interval was significantly higher in Cntl fetuses (631 ± 72 ms) than in LTOT fetuses (501 ± 51 ms) (Fig. 6). The slope of the baroreflex response (ms/mmHg) was significantly lower in LTOT (11.2 ± 2.9) compared with Cntl (17.2 ± 1.5). Blood gas values were similar in Cntl and LTOT fetuses (Table 5). No significant ACTH or cortisol response occurred during PE-induced hypertension (Fig 5B).

View larger version (29K): [in this window] [in a new window]   Fig. 6.   FBP and FHR (R-R interval) during hypotension induced by PE infusion directly to Cntl (, n = 5) and LTOT (, n = 6) fetuses. Values are means ± SE. * P <0.05 compared with LTOT.

View this table: [in this window] [in a new window]   Table 5.   Arterial blood gas values of Cntl and LTOT fetuses during phenylephrine-induced hypertension

	DISCUSSION

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Baseline Fetal Arterial Blood Gas Values and Plasma ACTH and Cortisol

At 126-135 days gestation, fetal PO₂ was lower and O₂ content higher in LTOT fetuses than Cntl fetuses. The effect of the increased contracture activity in the LTOT fetuses was to lower fetal arterial PO₂ and increase O₂ content. This finding shows that the LTOT fetuses attempted to adapt to the mild stress of acute episodes of hypoxemia. We previously reported (41) that exposure to repeated contractures results in altered oxygenation by shifting the O₂ dissociation curve to the left. Fetal PO₂ was consistently lower in the LTOT fetuses, and fetal hemoglobin concentrations were consistently higher in the LTOT fetuses, although this was not significant. These findings strongly suggest that the LTOT fetuses were attempting to compensate for decreased O₂ delivery to the tissues. Further studies will be necessary to evaluate the role of the different factors associated with the increased contracture activity in stimulating fetal O₂ delivery functions. It is important to note that a contracture frequency of 3.2/h, the average in the LTOT group, is within the upper range of normally observed contractures.

A single contracture produced in response to a single pulse of OT administered to the pregnant ewe in late gestation stimulates the release of fetal ACTH (29, 49). This contracture-induced ACTH secretion can be abolished by preventing the contracture-induced fall in PO₂ (49). In a previous study (38), we demonstrated that 6 days of OT pulses (5 min of OT every 30 min) beginning at 127 days gestation results in increased basal fetal plasma cortisol concentration but no change in basal fetal plasma ACTH. This observation suggests that at this stage of fetal development, increased contracture frequency leads to increased sensitivity of the adrenal gland to ACTH. In contrast, we previously demonstrated (32, 33) that a longer period of fetal exposure to contractures induced by OT pulses begun at an earlier stage of maturation, ~91 days gestation, decreases the sensitivity of the fetal adrenal to ACTH both under basal conditions and after exposure to hypoxemia and hypotension at ~130 days gestation. The differences between the two studies could be caused by the timing of the extra stimulation of the fetus by the contractures, the extended duration of this extra stimulation, or a combination of these two features in the intrauterine environment. Opposing effects on the development of the pituitary-adrenal axis have been shown in the rat according to the critical period of development at which the rat is exposed to various stimuli that act on the hypothalamo-pituitary-adrenal system (30). It will be of interest to determine whether the contracture-induced changes we have observed in pituitary-adrenal development are continued into later life.

Ontogenic Changes in FBP and FHR

Fetal cardiovascular maturation during the final month of pregnancy is characterized by a rise in FBP and a fall in FHR (7, 8, 15, 48). We observed the same pattern of maturation of FBP and FHR in Cntl fetuses (43). When related to body weight, fetal cardiac output is higher in the fetus than in the adult (6, 35) and increases in proportion to fetal weight (35). The gradual rise in FBP in late gestation has been attributed to both a rise in peripheral vascular resistance and an increase in fetal cardiac output (17, 43). Several mechanisms have been suggested to account for the decrease in FHR: a baroreflex-mediated response to the rise in FBP (20) and/or altered sympathetic and parasympathetic autonomic nervous system tone. An increased parasympathetic influence has been considered more important than sympathetic activity (44, 46).

Alteration in FBP and FHR in LTOT fetuses suggests an acceleration of fetal cardiovascular maturation. However, final FBP and FHR at the end of gestation were similar in the two groups. This finding indicates that maturation of the Cntl fetuses eventually catches up with respect to absolute values of FBP and FHR. However, the fact that Cntl and LTOT fetuses are functioning at the same level at the end of the study period does not mean that their regulating mechanisms are the same.

Effect of Long-Term Exposure to Increased Contractures on Fetal Baroreflex Responses

The ACTH-cortisol response during SNP infusion showed a less "stressed" response in LTOT. However, the ratio of cortisol to ACTH was similar in both Cntl and LTOT fetuses.

Baroreflex Study

Normal development of fetal cardiovascular reflexes is vital to an independent extrauterine existence. The fetal cardiovascular response to acute hypoxia has been the most extensively studied perturbation during fetal life. The cardiovascular response to fetal hypoxemia is composed of an initial neural reflex response followed by endocrine changes (13, 16). Several slightly different methods have been used to study the developing fetal baroreflex response. The sensitivity of the baroreflex observed depends on the method used for evaluation (9). We used SNP to induce hypotension and PE to induce hypertension. SNP acts as a vasodilator, acting on both arterial and venous vessels. As a result, both peripheral vascular resistance and venous return are reduced (4). PE acts as an -agonist, increasing the force of myocardial contraction and vascular smooth muscle contraction, thereby increasing peripheral resistance and producing a reflex vagal bradycardia (19).

The fetal lamb heart operates near the upper limit of the Frank-Starling curve (12, 45). Changes in cardiac output are induced almost solely by alteration of heart rate (37, 48), because the fetal heart possesses a very limited ability to change stroke volume (34, 36). However, as shown by Downing et al. (11), the newborn lamb has the ability to alter the inotropic state of the heart. Thus the near-term fetus may develop the ability to increase the inotropic action of the heart when FBP deviates from the physiological range.

During hypotension induced by SNP infusion to the fetus, the initial baroreflex action is to increase FHR to oppose the falling FBP that results from vasodilatation. This increase in FHR is the result of increased sympathetic activity (47). The fetal tachycardia eventually breaks down as compensation fails, and fetal bradycardia ensues. Differences in sympathetic activity between Cntl and LTOT fetuses may explain the difference in baroreflex slope before breakdown between the two groups. Tachycardia was reversed as SNP dosage increased. Although this phenomena is similar in both Cntl and LTOT, the breakdown point and magnitude of the slope were quite different in Cntl and LTOT fetuses. Breakdown is likely to be caused by a decreased venous return and increased vagal activity. Walker et al. (47) reported different patterns of baroreflex response to hypotension in fetal and newborn lambs. In contrast to the newborn lamb, which develops tachycardia at a 30% fall in blood pressure, the fetus develops tachycardia at a 15% fall in blood pressure and FHR reverses at a 30% fall in blood pressure. These authors concluded that fetal reversal of tachycardia in severe hypotension was caused by counteraction of sympathetic acceleration by increased vagal activity. The difference in response to SNP infusion in LTOT fetuses compared with Cntl fetuses reveals significantly altered sympathetic and parasympathetic control of cardiac function in LTOT fetuses. Because cardiac output is defined by stroke volume and heart rate, the effect of altered inotropic function, even if it is limited, must be taken into consideration (11). Our results suggest that the myocardium of LTOT fetuses may have a more mature inotropic function than that of Cntl fetuses.

Arterial blood gas values at the end of SNP infusion in Cntl fetuses show an obvious failure in circulatory compensation mechanisms compared with LTOT fetuses. The improved ability of LTOT fetuses to compensate for hypotension is shown by the baroreflex response, the total dosage of SNP used, and the arterial blood gas values, indicating that cardiovascular function is more mature in LTOT fetuses than in Cntl fetuses.

During PE-induced hypertension, PE acts mainly as an -agonist and a fall in heart rate is induced by the vagal reflex (19). Therefore, the main reason for the difference in the baroreflex slope between LTOT and Cntl fetuses is likely the alteration of vagal activity in LTOT fetuses (9, 15). However, cardiac contractility must also be considered. The baroreflex slope is known to change with gestational age or postnatal age (9, 15). The fact that the baroreflex slope is lower in LTOT fetuses again indicates that the cardiovascular baroreflex response is more mature than that of Cntl fetuses. The change in baroceptor slope may be caused by effects on either or both the neural and the endocrine component of the reflex. The higher total PE dose required by LTOT fetuses also indicates contracture-induced accelerated maturation of the fetal cardiovascular function in relation to the challenges we studied.

In conclusion, increased uterine contracture frequency over the period of 45 days of late gestation alters ontogenic changes in FBP and FHR as well as baroreflex response. We speculate that long-term contracture-induced changes in the intrauterine environment accelerate fetal cardiovascular functional development through the mild repetitive epochs of hypoxemia that we have shown to be critical in the contracture-related release of ACTH (49) and activation of the fetal pituitary adrenal system and/or the physical stimulation of the contracture. Programming of cardiovascular function in adult life by the environment experienced during fetal life results from several nutritional and endocrine factors operating during development. In keeping with data we previously presented on the direct and indirect effects of contractures on the maturation of various physiological functions, we propose that contractures are a significant factor that influences normal and abnormal cardiovascular maturation.

	ACKNOWLEDGEMENTS

This study was supported by National Institute of Child Health and Human Development Grant HD-28014. The authors thank Dr. Xiu-Ying Ding for surgical assistance and Ms. Karen Moore for help in preparing this manuscript.

	FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. W. Nathanielsz, Laboratory for Pregnancy and Newborn Research, Dept. of Biomedical Sciences, College of Veterinary Medicine, Cornell Univ., Ithaca, New York 14853-6401 (E-mail: pwn1{at}cornell.edu).

Received 28 January 1999; accepted in final form 13 July 1999.

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