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. 2001 Jun 1;533(Pt 2):561–570. doi: 10.1111/j.1469-7793.2001.0561a.x

Maternal undernutrition increases arterial blood pressure in the sheep fetus during late gestation

L J Edwards 1, I C McMillen 1
PMCID: PMC2278632  PMID: 11389212

Abstract

  1. We have investigated the effect of a 50% reduction in maternal nutrient intake during the last 30 days of pregnancy on arterial blood pressure and on arterial blood pressure responses to angiotensin II (AII) and the angiotensin converting enzyme (ACE) inhibitor captopril in the sheep fetus at 115–125 and at 135–145 days gestation (term = 147 ± 3 days gestation).

  2. Fetal plasma glucose concentrations were lower in the undernourished (UN) group compared to the control animals. There was no difference, however, in fetal plasma cortisol or adrenocorticotrophic hormone (ACTH) concentrations between the UN and control groups between 115 and 145 days gestation.

  3. During the first 10 days of undernutrition, maternal plasma concentrations of cortisol were increased in the UN group compared to controls. At 115–125 days gestation, fetal arterial blood pressure was also higher in the UN group compared with controls and there was an inverse relationship (r =−0.62, P < 0.05) between mean arterial pressure and the fetal plasma concentrations of ACTH in the UN group. Fetal blood pressure responses to increasing doses of angiotensin II were also higher (P < 0.05) in UN compared to control animals at 115–125 days gestation.

  4. Between 135 and 145 days gestation, fetal arterial blood pressure was increased in UN fetal sheep and mean arterial blood pressure was correlated with fetal plasma concentrations of cortisol.

  5. Increased arterial blood pressure and responsiveness to AII measured in the fetuses of nutrient-restricted ewes may be related in part to fetal exposure to the actions of cortisol derived from transplacental transfer during the first 10 days after the start of the restricted feeding regime.

A range of epidemiological studies have shown that perturbed intrauterine growth is associated with an increased prevalence of hypertension, cardiovascular disease and non-insulin-dependent diabetes mellitus in adult life (Barker, 1999). It has been postulated that a reduced fetal nutrient supply perturbs fetal growth and concomitantly alters or programs the structure and function of the developing cardiovascular system predisposing the individual to adult hypertension and cardiovascular disease (Langley & Jackson, 1994). It has been proposed that one outcome of either a suboptimal placental or maternal nutrient supply is exposure of the fetus to excess glucocorticoids, which may then act to decrease fetal growth and to program permanent changes in the cardiovascular, neuroendocrine and metabolic systems (Barker et al. 1993).

In the pregnant rat, a global reduction in energy intake (Woodall et al. 1996) or a low protein diet (Langley & Jackson, 1994) results in reduced birth weight and persistent increased blood pressure in the offspring. Interestingly, blood pressure in the offspring of rats exposed to the low protein diet was not increased after maternal corticosterone biosynthesis was pharmacologically suppressed throughout pregnancy (Langley-Evans et al. 1996). These and other studies (Levitt et al. 1996) suggest that exposure to excess glucocorticoids in fetal life may program the development of the cardiovascular system to result in an increase in blood pressure in post-natal life. It has also been shown that the development of increased blood pressure in rat pups after exposure to a low protein diet in pregnancy was prevented if the pups were treated with an angiotensin converting enzyme (ACE) inhibitor during the early post-natal period (Langley-Evans & Jackson, 1995). Thus increases in blood pressure after fetal exposure either to excess glucocorticoids or to intrauterine nutrient restriction may be dependent on activation of the renin-angiotensin system (RAS).

Whilst it has been demonstrated that maternal nutrient restriction in the pregnant rat results in an increase in blood pressure during postnatal life, there are few data available on the impact of maternal nutrient restriction on blood pressure regulation before birth. It has been shown in the sheep that maternal undernutrition during the first 30 days of pregnancy results in lower blood pressure in the fetus during later gestation (Hawkins et al. 2000) and increased blood pressure in postnatal life (Hoet & Hanson, 1999). There are no studies, however, which have directly investigated the fetal blood pressure responses to maternal undernutrition during the period of rapid fetal growth which occurs during the last month of gestation in the sheep.

In the present study, therefore, we have investigated the effects of maternal undernutrition (50 % of feed intake) during the last 30 days of pregnancy on fetal arterial blood pressure and on the relationship between fetal blood pressure and circulating cortisol concentrations between 115 and 125 days, and between 135 and 145 days gestation, i.e. before and after the start of the prepartum increase in circulating cortisol concentrations (Challis & Brooks, 1989). We have also investigated the effects of maternal nutrient restriction on the fetal blood pressure responses to angiotensin II (AII) and the ACE inhibitor captopril, and determined whether these responses are related to the prevailing fetal plasma concentrations of cortisol.

METHODS

Animals and surgery

All procedures were approved by The University of Adelaide Animal Ethics Committee. Twenty-four pregnant Border-Leicester cross Merino ewes were used in this study.

Surgery was performed under aseptic conditions between 110 and 113 days gestation (term = 147 ± 3 days gestation) with general anaesthesia initially induced by an intravenous injection of sodium thiopentone (1.25 g; Pentothal, Rhone Merieux, Pinkenba, Queensland, Australia) and maintained with inhalational halothane (2.5-4 %; Fluothane, ICI, Melbourne, Victoria, Australia) in oxygen. In all ewes, vascular catheters were implanted in a fetal carotid artery and jugular vein, a maternal jugular vein and the amniotic cavity, as previously described (Edwards et al. 1999). All catheters were filled with heparinised saline and the fetal catheters exteriorised through an incision made in the ewes’ flank. All ewes and fetal sheep received a 2 ml intramuscular injection of antibiotics (procaine penicillin, 250 mg ml−1; dihydrostreptomycin, 250 mg ml−1; procaine hydrochloride, 20 mg ml−1; Penstrep Illium, Troy Laboratories, Smithfield, NSW, Australia) at the time of surgery. The ewes were housed in individual pens in animal holding rooms with a 12 h light-dark cycle and fed once daily at 11.00 h with water provided ad libitum. Animals were allowed to recover from surgery for at least 4 days prior to experimentation.

Nutritional regimes

All ewes were weighed once between 110 and 114 days gestation. From 115 days gestation ewes were fed either 20 g of lucerne per kg and 3 g of oats per kg (control group; n = 7), or 10 g of lucerne per kg and 1.5 g of oats per kg (undernutrition (UN) group; n = 17). In both groups, the feed allowance was increased by 15 % every 10 days until post mortem.

Blood sampling regime

Fetal arterial blood (0.5 ml) samples were collected every day for 4 days after surgery and then 3 times per week thereafter (UN n = 205 samples, control n = 102 samples) for the measurement of arterial PO2, PCO2, pH and oxygen saturation (ABL 520 analyser, Radiometer, Copenhagen, Denmark). Fetal arterial blood samples (3.5 ml) and maternal venous blood samples (5 ml) were also collected 3 times per week at 08.00-11.00 h, for the measurement of glucose, cortisol and adrenocorticotrophic hormone (ACTH) throughout late gestation. All blood samples were centrifuged at 1500 g for 10 min and plasma was separated into aliquots and stored at -20°C for subsequent assays.

Arterial blood pressure measurements

Fetal arterial blood pressure and intra-amniotic pressure were measured directly from the fetal carotid arterial catheter and amniotic catheter, which were connected to a MacLab data acquisition system via a MacLab 1050 displacement transducer and quad-bridge amplifier (ADInstruments, NSW, Australia). Arterial blood pressure, corrected for amniotic pressure, was measured using the MacLab Chart software on a Power Macintosh computer. Baseline arterial blood pressure was recorded continuously for periods up to 60 min before each experiment.

Angiotensin II

Bolus intravenous doses of AII (0.75, 1.5, 3.0, 5.0 and 10.0 mg, Peninsula Laboratories Inc., CA, USA) were administered in a random order with 20 min between each dose, at between 115 and 125 days gestation (UN group n = 15, control group n = 7) and similarly, at between 135 and 145 days gestation (UN group n = 6, control group n = 6). Angiotensin II experiments were performed once per animal in each gestational age range, and four animals in the UN group and six animals in the control group had experiments at both gestational age ranges. Fetal arterial blood pressure and intra-amniotic pressure were measured continuously throughout the AII dose-response experiments.

Captopril infusion

Captopril ([25]-1-[3-mercapto-2-methylpropionyl]-l-proline, Sigma Chemical Co., USA) was infused intravenously for 4 h (Graseby Medical syringe driver M5-10A, Selby Scientific & Medical, Australia) at between 115 and 125 days gestation (1.2 mg kg−1 captopril; UN group n = 11, control group n = 7) and at between 135 and 145 days gestation (1.2 mg kg−1 captopril; UN group n = 6, control group n = 6). Captopril infusions were carried out once in each animal in each gestational age range, and experiments were conducted on three UN animals and six control animals at both gestational age ranges. In control experiments, intravenous saline (3 ml (4 h)−1) was infused once per animal at between 123 and 133 days gestation (UN group n = 3, control group n = 4). Fetal arterial blood pressure and intra-amniotic pressure measurements were recorded continuously for 60 min before, during and for 240 min after the end of the infusion of either captopril or saline.

Fetal and maternal outcome

In the UN group, eight fetal sheep died or delivered before 144 days (non-survivors: 3 before 125 days gestation, 4 between 130 and 140 days and 1 between 141 and 144 days). In the control group, one fetus died before 125 days gestation. Within 48 h of fetal death, ewes were killed with an overdose of sodium pentobarbitone (Virbac, Peakhurst, NSW, Australia). All other ewes (UN survivors n = 9, control n = 6) were killed with an overdose of sodium pentobarbitone at either 144 or 145 days gestation and fetal sheep were delivered by hysterotomy, weighed and killed by decapitation.

Plasma glucose analysis

Fetal arterial plasma glucose concentrations were determined by enzymatic analysis using hexokinase and glucose-6-phosphate dehydrogenase, measuring the formation of NADH photometrically at 340 nm (COBAS MIRA automated analysis system, Roche Diagnostic, Basle, Switzerland). The intra- and interassay coefficients of variation were both < 5 %.

Cortisol

Cortisol was extracted from fetal and maternal plasma using dichloromethane as previously described (Bocking et al. 1986). The efficiency of recovery of 125I-labelled cortisol from fetal plasma using this extraction procedure was always greater than 90 %. Cortisol concentrations were then measured (UN group: n = 61 fetal samples and 71 maternal samples; control group: n = 37 fetal samples and 27 maternal samples) by radioimmunoassay using an Orion Diagnostica kit (Orion Diagnostica, Turku, Finland) (Phillips et al. 1996). The sensitivity of the assay was 0.078 nmol l−1, and the cross-reactivity of the rabbit anti-cortisol was < 1 % with pregnenolone, aldosterone, progesterone and oestradiol. The interassay coefficient of variation was 11.2 % and the intraassay coefficient of variation was < 10 %.

ACTH

Immunoreactive ACTH concentrations in fetal sheep plasma (UN group, n = 117 samples; control group, n = 69) were measured by radioimmunoassay using an ICN Biomedicals kit (ICN Biomedicals, Seven Hills, NSW, Australia) (Phillips et al. 1996). The sensitivity of the assay was 0.9 pg ml−1, and the rabbit anti-human ACTH 1-39 had a cross-reactivity of < 0.1 % with β-endorphin, α-melanocyte-stimulating hormone, α-lipotrophin, and β-lipotrophin. The interassay coefficient of variation was 11.2 % and the intra-assay coefficient of variation was < 10 %.

Statistical analysis

Data are shown as the mean ± standard error of the mean (s.e.m.). Fetal body weights and placental weights, measured at 144- 145 days gestation, were compared between UN and control groups using Student's unpaired t test.

Values for arterial PO2, pH and PCO2 and for plasma glucose and cortisol concentrations between 115 and 125 days were compared between fetal sheep in the UN group which did not survive past 144 days gestation (n = 8) with respective values from fetal sheep in the UN group which did survive past 144 days gestation (n = 9) using Student's unpaired t test. There was no significant difference between mean arterial blood gas, glucose and cortisol values in the survivor and non-survivor groups and the data from the UN group were therefore combined for subsequent analyses.

Arterial PO2, PCO2, pH and plasma glucose concentrations in the UN (n = 17) and control (n = 7) groups were compared using a multifactorial ANOVA with repeated measures using the Statistical Package for Social Sciences (SPSSX; SPSS Inc., Chicago, IL, USA) on a Vax mainframe computer. Specified factors for the ANOVA included nutritional group (UN vs. control), gestational age (115-120, 121-125, 126-130, 131-135, 136-140 and 141-145 days gestation) and animal.

Maternal and fetal plasma cortisol concentrations and fetal plasma ACTH concentrations in the UN and control groups were also compared using a multifactorial ANOVA with repeated measures. Specified factors for the ANOVA included nutritional group (UN vs. control), gestational age (115-125, 126-134 and 135-145 days gestation) and animal.

Mean arterial blood pressure (diastolic blood pressure + 0.4 (systolic - diastolic blood pressure)) was calculated between 115 and 125 days, and between 135 and 145 days gestation, as the mean of six values over either the 2 min period preceding administration of AII or the 60 min period preceding captopril administration. If blood pressure was measured on more than one occasion in any sheep within an age range, the mean of the available values was used.

In the UN group, mean arterial blood pressure measurements between 115 and 125 days in fetal sheep which did not survive past 144 days gestation (n = 8) were compared with those made in fetal sheep which did survive past 144 days (n = 7) using Student's unpaired t test. There was no significant difference between mean arterial blood pressure values in the survivor and non-survivor groups and these data from the UN group were therefore combined for subsequent analyses.

Mean arterial blood pressure measurements in the UN (n = 15) and control (n = 7) groups before 125 days gestation were compared using Student's unpaired t test. Mean arterial blood pressure for each of the five UN and the seven control fetal sheep which had blood pressure measurements available both before 125 and after 135 days gestation were compared using a multifactorial ANOVA with repeated measures. Specified factors for the ANOVA included nutritional group (UN vs. control), gestational age (< 125 days vs. > 135 days) and animal. Linear regression analyses were used to determine the relationships between mean arterial blood pressure and plasma glucose, ACTH or cortisol concentrations (measured on or within 2 days of the fetal blood pressure measurement) at 115-125 or at 135-145 days gestation.

The fetal diastolic and systolic blood pressure responses to increasing doses of AII were calculated as the difference between basal blood pressure and the maximal blood pressure response to any given dose of AII. In the UN group, the diastolic and systolic blood pressure responses to AII between 115 and 125 days in fetal sheep which did not survive past 144 days gestation (n = 8) were compared with those made in the fetal sheep which did survive past 144 days (n = 7) using a multifactorial ANOVA with repeated measures. Specified factors for the ANOVA included nutritional group (UN vs. control), AII dose (0.75-10 mg) and animal. There was no significant difference between the systolic or diastolic blood pressure responses to increasing doses of AII in the survivor and non-survivor groups and these data from the UN group were therefore combined for subsequent analyses.

The diastolic and systolic blood pressure responses to AII in the combined UN group and in the control group were compared using a multifactorial ANOVA with repeated measures. The specified factors for the analysis included nutritional group (UN vs. control), AII dose (0.75-10 mg), gestational age range (< 125 days vs. > 135 days) and animal. The change from baseline in fetal arterial blood pressure in response to either captopril or saline infusion was analysed similarly using a multifactorial ANOVA with group (UN vs. control), time (in relation to captopril administration) and animal as the specified factors. Linear regression analyses were used to determine the relationship between the arterial blood pressure responses to a submaximal dose of AII (3 μg) or to captopril and the fetal plasma glucose or cortisol concentrations measured on or within 2 days of the relevant experimental protocol. Logarithmic transformation of hormonal or blood pressure data was used where required to reduce heterogeneity of variance.

When a significant interaction between major factors was identified by ANOVA, the data were split on the basis of the interacting factor and reanalysed. Duncan's new multiple range test was used post ANOVA to identify significant differences between mean values and a probability level of 5 % (P < 0.05) was taken as significant.

RESULTS

Fetal outcome

There was no difference in fetal body weights (UN, 4.56 ± 0.2 kg, n = 9; control, 4.85 ± 0.2 kg, n = 6) or in placental weights (UN, 432.1 ± 29.4 kg, n = 9; control, 497.7 ± 69.8 kg, n = 6) between the UN and control groups at 144-145 days gestation.

Arterial blood gas status

There was no significant difference in the arterial PO2 and PCO2 measured between 115 and 125 days in fetal sheep in the UN group which did not survive past 144 days gestation (n = 8) compared with the fetal sheep in the UN group which did survive past 144 days (n = 9) (Table 1). There was a small but significant difference, however, in arterial pH measured between 115 and 125 days in fetal sheep in the UN group which did not survive past 144 days gestation (7.37 ± 0.01) compared with those which survived past 144 days (7.40 ± 0.01) (Table 1).

Table 1.

Fetal arterial blood gas status, plasma glucose and cortisol concentrations and mean arterial blood pressure in fetal sheep in the UN group which survived past 144 days and in fetal sheep which did not survive past 144 days

Survived past 144 days (n = 9) Did not survive past 144 days (n = 8)
Arterial PO2 (mmHg) 24.2 ± 1.0 21.7 ± 0.7
Arterial PCO2 (mmHg) 46.0 ± 1.3 46.7 ± 2.4
pH 7.40 ± 0.01 7.37 ± 0.01*
[Glucose] (mmol l−1) 1.3 ± 0.1 1.2 ± 0.1
[Fetal cortisol] (nmol l−1) 3.2 ± 0.6 2.5 ± 0.3
Mean arterial blood pressure (mmHg) 45.1 ± 3.0 45.0 ± 3.3
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Values are means ±s.e.m.

*

significant difference (P < 0.05).

Arterial PO2 was higher (F = 7.97, P < 0.001) at 115- 125 days and at 141-145 days gestation compared to 136-140 days gestation in both the UN and control groups (Fig. 1). Arterial PCO2 was also higher (F = 13.0, P < 0.001) at 115-135 days and at 141-145 days gestation compared to between 136 and 140 days gestation (Fig. 1). There was no effect, however, of nutritional regime or gestational age on fetal arterial pH.

Figure 1. Mean arterial PO2, PCO2, pH and glucose concentrations in control and UN fetal sheep.

Figure 1

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There was no significant difference in PO2, PCO2 and pH between control (□) and UN (▪) fetal sheep between 115 and 145 days gestation. There was a significant effect of gestational age on arterial PO2 and PCO2 levels (a and b denote differences in mean values; P < 0.05) at different gestational age ranges in each of the control and UN groups. Plasma glucose concentrations were significantly decreased (F = 4.74, P < 0.05) in the UN fetal sheep (▪) compared to the control group (□) between 115 and 145 days gestation. * Significant difference (P < 0.05) from control glucose concentrations.

Maternal and fetal plasma glucose

Maternal plasma concentrations of glucose were not different in the UN and control groups throughout late gestation. Maternal plasma glucose concentrations ranged between 2.5 ± 0.1 mmol l−1 (115-120 days) and 2.4 ± 0.2 mmol l−1 (141-145 days) in the UN group and between 2.8 ± 0.2 mmol l−1 (115-120 days) and 2.5 ± 0.2 mmol l−1 (141-145 days) in the control group. In the UN group, there was no significant difference in the fetal plasma glucose concentrations measured between 115 and 125 days in fetuses which did not survive past 144 days gestation compared with those which did survive (Table 1). Fetal plasma glucose concentrations were lower (F = 4.74, P < 0.05), however, in the UN group compared to the control animals between 115 and 145 days gestation. There was also a significant effect of gestational age (F = 4.51, P < 0.01) on fetal plasma glucose concentrations in both the UN and control groups. Fetal plasma glucose concentrations were significantly lower after 131 days compared to between 126 and 130 days gestation (Fig. 1).

Maternal and fetal plasma cortisol

In the UN group, there was no significant difference between maternal plasma concentrations of cortisol measured between 115 and 125 days in ewes carrying fetuses which did not survive (39.8 ± 6.8 nmol l−1) and those which did survive (51.7±6.5 nmol l−1) past 144 days gestation. There was a significant interaction (F = 5.59, P < 0.05), however, between the effects of nutritional regime and gestational age on maternal cortisol concentrations. Maternal plasma cortisol concentrations were significantly higher in the UN (45.1 ± 9.2 nmol l−1) compared to the control group (26.6 ± 6.6 nmol l−1) at between 115 and 125 days gestation. There were no differences between the UN and control groups, however, in maternal cortisol concentrations after 125 days gestation (126-134 days gestation: UN, 27.1 ± 5.8 nmol l−1; control, 35.2 ± 10.2 nmol l−1: 135-145 days gestation: UN, 28.4 ± 8.7 nmol l−1; control, 29.0 ± 6.0 nmol l−1).

Fetal plasma concentrations of cortisol were not different between 115 and 125 days gestation in those fetuses which did not survive past 144 days compared with those that did (Table 1). There was no significant difference between the UN and control groups in the fetal plasma cortisol concentrations between 115 and 145 days gestation. Fetal plasma concentrations of cortisol were 2.7 ± 0.4 nmol l−1 (UN group) and 1.8 ± 0.2 nmol l−1 (control group) at 115-125 days gestation and increased (F = 24.52, P < 0.001) to 24.0 ± 6.8 nmol l−1 (UN group) and 17.9 ± 4.6 nmol l−1) (control group) after 135 days gestation.

Fetal plasma ACTH

There was no significant difference between fetal plasma ACTH concentrations in the UN and control groups at 115-125 days gestation (UN, 54.6 ± 5.9 pg ml−1, n = 13; control, 53.5 ± 5.6 pg ml−1, n = 7), 126-134 days gestation (UN, 66.2 ± 5.82 pg ml−1, n = 13; control, 63.5 ± 6.7 pg ml−1, n = 7) or at 135-145 days gestation (UN, 105.8 ± 13.2 pg ml−1, n = 10; control, 75.2 ± 8.2 pg ml−1, n = 6; P = 0.07). Fetal plasma ACTH concentrations were higher (F = 26.68, P < 0.001) at 135-145 days gestation than at any earlier stage in gestation in both the UN and control groups.

Fetal arterial blood pressure

There was no significant difference in the mean arterial blood pressure measured in the UN group between 115 and 125 days in fetal sheep which did not survive past 144 days gestation compared with those which did (Table 1).

At 115-125 days gestation, mean arterial blood pressure was significantly higher (P < 0.05) in fetal sheep in the UN group (45 ± 2.2 mmHg, n = 15) when compared with control animals (38 ± 0.7 mmHg, n = 7). In this gestational age range, there was a significant inverse correlation between mean arterial blood pressure (BP) and plasma glucose concentrations in the UN group (mean arterial BP = 67-17 (plasma glucose), r = 0.4, P < 0.05, n = 13) which was not present in the control animals. There was also a significant inverse correlation between mean arterial blood pressure and plasma ACTH concentrations in the UN group at 115-125 days gestation (mean arterial BP = 75-0.5 (plasma ACTH), r = -0.62, P < 0.05, n = 10) which was also not present in the control animals (n = 6) (Fig. 2). There was no relationship, however, between mean arterial blood pressure and fetal plasma cortisol concentrations in this gestational age range.

Figure 2. The relationship between fetal plasma ACTH concentrations and mean arterial blood pressure in control and UN fetal sheep between 115 and 125 days gestation.

Figure 2

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There was a significant correlation between fetal plasma ACTH concentrations and blood pressure in the UN fetal sheep (▴, n = 10) (mean arterial BP = 75 - 0.5 (plasma ACTH), r = -0.62, P < 0.05) between 115 and 125 days gestation but not in the control fetal sheep (○, n = 6).

Mean arterial blood pressure was compared in those fetuses in the UN and control groups which had measurements at both 115-125 and 135-145 days gestation. Mean arterial blood pressure was significantly higher in the UN fetuses compared with the control group at both 115-125 days gestation (UN, 46.1 ± 3.1 mmHg, n = 5; control, 38.3 ± 1.3 mmHg, n = 7) and 135-145 days gestation (UN, 49.5 ± 3.5 mmHg, n = 5; control 44.8 ± 1.8 mmHg, n = 7) (Fig. 3).

Figure 3. Mean arterial blood pressure in control and UN fetal sheep.

Figure 3

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Mean arterial blood pressure was significantly greater in UN fetal sheep (▪, n = 5) compared to control fetal sheep (□, n = 7) at both 115-125 and 135-145 days gestation. * Significant difference (P < 0.05) from control blood pressure values.

There was no significant effect of gestational age on the mean arterial blood pressure in either UN or control groups. After 135 days gestation, there was no correlation between mean arterial blood pressure and ACTH or glucose in either UN or control groups. There was, however, a significant positive correlation between mean arterial basal BP and plasma cortisol concentrations in the combined UN and control groups between 135 and 145 days gestation (r = 0.58, P < 0.05; UN n = 7, control n = 6).

Fetal arterial BP responses to angiotensin II

There was no significant difference in the systolic or diastolic blood pressure responses to increasing doses of AII in the UN group at 115-125 days in fetal sheep which did not survive compared with those which did survive past 144 days and the blood pressure responses were therefore combined for subsequent analyses.

There was a significant effect of increasing doses of AII on the fetal diastolic (F = 36.4, P < 0.001) and systolic blood pressure (F = 68.1, P < 0.001) in the UN and control groups. There was also a significant interaction (F = 5.4, P < 0.05) between the effects of gestational age, AII and the different nutritional regimes on the fetal diastolic and systolic blood pressure responses. At 115-125 days gestation, the diastolic and systolic blood pressure responses to increasing doses of AII were significantly higher (P < 0.05) in the UN group when compared with the control fetuses (Fig. 4). Between 135 and 145 days gestation, however, there was no significant difference between the UN and control groups in either the diastolic or the systolic blood pressure responses to AII (Fig. 4). There was also no correlation between the fetal blood pressure response to a submaximal dose of AII (1.5 μg) and the prevailing plasma glucose, ACTH or cortisol concentrations in the UN or control fetal sheep at either gestational age range.

Figure 4. The effect of angiotensin II on systolic and diastolic blood pressure in control and UN fetal sheep.

Figure 4

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Between 115 and 125 days gestation, the systolic (A) and diastolic (B) blood pressure responses (mean ±s.e.m.) to AII were significantly greater (systolic: F = 7.27; diastolic: F = 4.32, P < 0.05) in the UN (▪, n = 15) compared to the control group (□, n = 7). In both the UN and control groups, the systolic and diastolic blood pressure responses to 3-10 mg AII were significantly greater than to 0.75 mg AII. Between 135 and 145 days gestation, there was no difference between the UN (▪, n = 6) and control groups (□, n = 6) in the systolic (C) and diastolic (D) blood pressure responses to AII. In both the UN and control groups, the systolic and diastolic blood pressure responses to 3-10 mg AII were significantly greater than to 0.75 mg AII.

Captopril infusion

There was no significant effect of saline infusion on the fetal diastolic and systolic blood pressure in the UN and control groups.

There was a similar and significant decrease (P < 0.05) in fetal diastolic and systolic blood pressure during infusion of captopril in the UN and control groups between 115 and 125 days gestation and between 135 and 145 days gestation (Fig. 5). There was no significant correlation between the maximal fetal arterial blood pressure response to captopril and the prevailing fetal plasma concentrations of glucose, ACTH or cortisol at either gestational age range.

Figure 5. The effect of captopril on arterial blood pressure in control and UN fetal sheep.

Figure 5

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Between 115 and 125 days gestation, there was no significant difference in the systolic (A) and diastolic (B) blood pressure responses (mean ±s.e.m.) to a 4 h captopril infusion in control (○, n = 11) and UN (▴, n = 7) fetal sheep. In both the control and UN groups, systolic blood pressure was significantly lower at +150, +240 and +300 min, relative to the start of the captopril infusion compared with preinfusion values. Diastolic blood pressure was significantly lower between +90 and +300 min relative to the start of the captopril infusion compared with preinfusion values. Between 135 and 145 days gestation, there was no significant difference in the systolic (C) and diastolic (D) blood pressure responses to a 4 h captopril infusion in control (○, n = 6) and UN (▴, n = 6) fetal sheep. Systolic and diastolic blood pressures were significantly lower between +30 and +240 min relative to the start of the captopril infusion compared with preinfusion values in the control and UN groups.

DISCUSSION

In this study, a 50 % reduction in maternal nutrient intake during the last month of gestation resulted in an increase in mean arterial blood pressure in the sheep fetus both between 115 and 125 days and between 135 and 145 days gestation. Maternal cortisol concentrations were increased in the first 10 days after the start of the restricted feeding regime, and during this period fetal arterial blood pressure was inversely correlated with fetal plasma glucose and ACTH concentrations. Fetal blood pressure responses to increasing doses of angiotensin II were also higher in fetuses of undernourished ewes compared to fetuses in control ewes between 115 and 125 days, but not between 135 and 145 days gestation. Between 135 and 145 days gestation, there was a direct relationship between mean arterial blood pressure and the fetal plasma concentrations of cortisol when the undernourished and control groups were combined.

Fetal outcome

Chronic maternal hypoglycaemia, induced by insulin infusion, has been found to decrease placental GLUT-1 protein expression, which would result in a decreased glucose transfer to the fetus (Das et al. 1998). In the present study it is possible, therefore, that chronic maternal nutrient restriction also resulted in a reduced transfer of glucose to the fetus resulting in decreased fetal plasma concentrations of glucose throughout late gestation. In the undernourished group, 8 of the 17 fetuses died or delivered before post mortem at 144-145 days gestation and there was no difference in the placental or body weight of the fetuses in the undernourished group which survived until 144-145 days when compared with control animals. Maternal nutrient restriction may have had a greater impact on the growth and development and hence survival of those fetuses which died before post mortem, compared with the fetuses which survived. Previous studies in the sheep have reported variable effects of maternal undernutrition on fetal body weight depending on the timing and degree of the nutritional insult (Mellor & Murray, 1982; Hawkins et al. 1999). Whilst fetal plasma glucose concentrations were significantly decreased in the undernourished group, there was no difference in either the fetal plasma ACTH or cortisol concentrations between the undernourished and control groups throughout late gestation. It has been demonstrated that acute insulin-induced hypoglycaemia stimulates the fetal hypothalamo-pituitary-adrenal (HPA) axis (Ozolins et al. 1992), but it is possible that the degree of chronic fetal hypoglycaemia in this study was not sufficient to stimulate the fetal HPA axis.

Fetal arterial blood pressure responses before 125 days gestation

The mean arterial blood pressure in fetuses of undernourished ewes was higher than that of fetuses in well nourished ewes between 115 and 125 days gestation. There was no evidence that the mean arterial blood pressure was higher in this group as a consequence of either fetal hypoxaemia or acidosis. There was a relationship between fetal blood pressure and plasma ACTH concentrations at 115-125 days gestation, such that fetal blood pressure was high when fetal ACTH was low. One possible explanation of these data is that low fetal ACTH concentrations reflect the negative feedback actions of an increased transplacental transfer of free cortisol which would not be detected in an assay which measures total cortisol concentrations. Fetal blood pressure responses to increasing doses of AII were also greater in the fetuses of undernourished ewes at 115-125 days gestation. Whilst a number of previous studies have reported that intrafetal administration of AII results in an increase in fetal arterial blood pressure (Ismay et al. 1979; Iwamoto & Rudolph, 1981; Tangalakis et al. 1992; Rosenfeld et al. 1995; Stevenson & Lumbers, 1995), the site of action of AII in the fetal circulation is unclear. One study using tissue autoradiography reported that AT1 receptors, which mediate arteriolar vasoconstriction, were only present in the sheep in external umbilical and primary placental arteries during late gestation and were not present in other circulatory regions until 2-4 weeks after birth (Cox & Rosenfeld, 1999). The AT1 receptor mRNA is also expressed throughout the fetal heart (Segar et al. 1995). The increased blood pressure responses to AII in the fetuses of undernourished ewes between 115 and 125 days gestation may therefore be a result of an increase in the expression or activity of AT1 receptors within the fetal cardiovascular system.

Interestingly intrafetal infusion of cortisol also results in an increased expression of AT1 receptor mRNA within the fetal heart (Segar et al. 1995) and there is a greater hypotensive effect after blockade of AT1 receptors in fetal sheep which have been infused with cortisol (Forhead et al. 2000).

Whilst there was an increased fetal blood pressure response to AII in fetal sheep during the first 10 days after the start of the restricted feeding regime, there was no difference between the undernourished and control groups in the effects of infusion of an ACE inhibitor on fetal blood pressure. Consistent with previous studies (Robillard et al. 1983), there was a small decrease in blood pressure in response to captopril in fetuses of both undernourished and well nourished ewes. It does not appear likely, therefore, that the increased fetal arterial blood pressure in the undernourished group results from changes in ACE activity or from an increase in circulating AII concentrations.

Fetal arterial blood pressure responses after 135 days gestation

Fetal arterial blood pressure was also higher in the fetuses of undernourished ewes when compared with control ewes after 135 days gestation and interestingly, there was a direct relationship between fetal arterial blood pressure and plasma cortisol concentrations when animals from the undernourished and control groups were combined. At this stage of gestation, however, there was no difference between the undernourished and control groups in the fetal arterial blood pressure responses to either increasing doses of AII or captopril infusion. The increase in fetal blood pressure in the undernourished group after 135 days gestation is therefore unlikely to be the result of activation of the renin-angiotensin system.

Fetal nutrition and adult hypertension

We have previously reported that the regulation of fetal arterial blood pressure was also altered after restriction of placental growth and development in the sheep (Edwards et al. 1999). In placentally restricted fetuses, infusion of captopril results in a significantly greater fall in fetal arterial blood pressure after, but not before, 135 days gestation, which suggests that blood pressure in these growth restricted fetuses is maintained by activation of the renin-angiotensin system in late gestation (Edwards et al. 1999). Thus, in contrast to the fetus in the undernourished ewe, changes in the regulation of the fetal cardiovascular system in the placentally restricted fetus occur only after the prepartum activation of the fetal pituitary-adrenal axis. Interestingly, it has also been shown in the sheep that maternal undernutrition during the first 70 days of pregnancy results in lower blood pressure in the fetus during later gestation (Hawkins et al. 2000). Differences between the effects of maternal undernutrition on the fetal cardiovascular system during the first half of gestation compared with later gestation may be explained by the impact of undernutrition on the umbilical-placental vasculature at these different stages of gestation. Thus the timing, type and duration of fetal nutrient restriction are each important in determining the nature of the fetal neuroendocrine and cardiovascular adaptive responses and their interactions. Clearly, further clarification of the molecular and cellular mechanisms which underpin these adaptive responses and interactions is required in order to elucidate the physiological mechanisms whereby poor fetal nutrient supply predisposes the individual to adult hypertension and cardiovascular disease.

Acknowledgments

We are grateful to the National Heart Foundation and the National Health and Medical Research Council of Australia for financial support of this work. We thank Anne Jurisevic for her expert technical assistance with fetal sheep surgery.

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