Placental glucose transport in growth-restricted pregnancies induced by overnourishing adolescent sheep

Abstract

Glucose clamp procedures were used to determine whether the slowing of fetal growth during the final third of gestation in overnourished adolescent ewes is due to a reduction in placental glucose transport capacity. Singleton pregnancies to a single sire were established by embryo transfer and thereafter adolescent dams were offered a high (n = 11) or moderate (n = 7) nutrient intake. Studies were conducted at 130 ± 0.5 days gestation. Uterine and umbilical blood flows were studied by the steady-state transplacental diffusion technique and glucose fluxes quantified by the Fick principle. To determine the relationship between the transplacental glucose gradient and umbilical (fetal) glucose uptake, studies were conducted with maternal arterial glucose clamped at 5 µmol ml⁻¹ and fetal glucose at spontaneously occurring and two additional higher levels. Maternal body weight gain during gestation averaged 282 and 57 g day⁻¹ for high- and moderate-intake dams, respectively. Total placentome weight (209 ± 23 vs. 386 ± 34 g) and fetal weight (3072 ± 266 vs. 4670 ± 196 g) were lower (P < 0.001) in high- than in moderate-intake groups. The growth-restricted pregnancies in the high-intake dams were associated with reduced uterine (P < 0.05) and umbilical (P < 0.02) blood flows and, in the non-perturbed state, the fetuses were relatively hypoxic (2.1 vs. 3.0 µmol ml⁻¹, P < 0.05) and hypoglycaemic (0.90 vs. 1.31 µmol ml⁻¹, P < 0.002). Linear regression analysis of umbilical glucose uptake at three steady-state uterine-umbilical arterial transplacental plasma glucose concentration gradients revealed that absolute placental glucose transport capacity was lower in high- than in moderate-intake dams (mean slope, 0.8 vs. 1.5 dl min⁻¹, P < 0.05; and mean intercept, 1.84 vs. 3.40 µmol ml⁻¹). However, glucose transfer capacity was not different between the two groups when expressed on a placental weight-specific basis. This confirms that the small size of the placenta per se is the major limitation to placental glucose transfer in the overnourished adolescent pregnant sheep.

Previous studies have shown that the hierarchy of nutrient partitioning during pregnancy can be dramatically altered in young growing females by manipulating maternal nutritional status. In our paradigm, we have demonstrated that overnourishing singleton-bearing adolescent sheep throughout pregnancy promotes rapid maternal growth at the expense of the gradually evolving nutrient requirements of the pregnant uterus. This results in a major reduction in placental mass and a significant decrease in lamb birth-weight at term relative to slow growing, moderate-intake, adolescent dams of equivalent gynaecological age (Wallace et al. 1996, 1997). In spite of the ready availability of nutrients in the maternal circulation (Wallace et al. 1999), the growth-restricted fetuses in the high-intake dams are relatively hypoxic, hypoglycaemic and hypoinsulinaemic during late gestation (Wallace et al. 2000, 2002). Moreover, fetal insulin-like growth factor 1 (IGF-1) concentrations are markedly lower while lactate and urea concentrations are elevated. This suggests a primary defect in uteroplacental uptake, metabolism or transport of essential nutrients resulting in a reduction in umbilical nutrient supply and hence a slowing of fetal growth during the final third of gestation. Indeed, we have recently examined spontaneous fetal and uteroplacental glucose uptakes in these growth-restricted pregnancies. In spite of major reductions in absolute uterine and umbilical blood flows and nutrient uptakes, fetal glucose and oxygen consumptions were normal when expressed on a fetal weight-specific basis (Wallace et al. 2002). Furthermore, we found no evidence that uteroplacental metabolism per unit placenta was altered at the expense of the fetus in that uteroplacental glucose and oxygen consumptions and uteroplacental lactate production were decreased in proportion to the observed reduction in placental mass (Wallace et al. 2002).

The present study used glucose clamp procedures to assess placental glucose transport over a range of maternal-fetal glucose concentration gradients to determine whether reduced placental glucose transfer capacity is responsible for the fetal hypoglycaemia and growth restriction observed previously in overnourished adolescent pregnancies.

METHODS

Animals and experimental design

All procedures were approved by the UK Home Office under the Animals (Scientific Procedures) Act 1986. Embryos from adult ewes (Border Leicester × Scottish Blackface), inseminated by a single sire (Dorset Horn), were recovered on day 4 after oestrus and transferred synchronously in singleton into the uteri of 21 recipient ewe lambs (Dorset Horn × Mule) exactly as described previously (Wallace et al. 1997). This technique removes the potentially confounding influence of partial embryo loss and variation in fetal number, and maximises the homogeneity of the resulting fetuses (Wallace et al. 1996). Embryo transfer was carried out during the breeding season and the animals were housed in individual pens under natural lighting conditions at the Rowett Research Institute (57 deg north, 2 deg west). At the time of embryo transfer, the recipient ewe lambs were peripubertal, approximately 7 months old, with a mean live weight of 45.6 ± 1.1 kg. After embryo transfer, the recipient ewes were individually offered either a high (n = 11) or moderate (n = 10) level of the same complete diet calculated to promote rapid or low maternal growth rates. The moderate dietary level was in fact a control group in that this intake level was predicted to optimise fetal growth in this genotype. The diet supplied 10.2 MJ of metabolisable energy and 137 g crude protein per kilogram and was offered in two equal feeds at 08.00 and 16.00 h daily. Animals offered moderate intakes were offered their entire ration immediately while those offered high intakes had the level of feed increased over a 2 week period until the level of daily feed refusal was approximately 15 % of the total offered (equivalent to ad libitum intakes). The level of feed offered was reviewed three times weekly and adjusted on an individual basis as appropriate on the basis of weight change data (recorded weekly) and the level of feed refused (recorded daily). After day 100 of gestation the feed intake of the moderate-intake group was adjusted weekly to meet the estimated increasing nutrient demands of the developing fetus during the final third of gestation by maintaining body condition score during this period. Ewes were body condition scored at fortnightly intervals throughout the study. Body condition score was assessed on a five point scale (1 = emaciated, 5 = obese), as described previously (Russell et al. 1969).

Surgery and animal care

Infusion and sampling catheters were surgically inserted at approximately 122 days of gestation. Water and food were withheld overnight prior to surgery. Anaesthesia was induced by intravenous (i.v.) administration of thiopentone (25 mg kg⁻¹, Intraval Sodium, Merial Animal Health Ltd, Essex, UK) and maintained by inhalation of halothane (Halothane-M&B, Rhone-Merieux Ltd, Essex, UK) in a mixture of oxygen and nitrous oxide. Just after anaesthesia induction, ewes received antibiotics (i.v.) in the form of ampicillin (fixed dose of 500 mg, Penbritin, Beecham Research, Hertfordshire, UK) and gentamicin (5 mg kg⁻¹, Pangram 5 %, Virbac Ltd, Cambridge, UK) and analgesics (i.v.) in the form of buprenorphine (0.006 mg kg⁻¹, Temgesic, Schering-Plough, Hertfordshire, UK) and carprofen (1.4 mg kg⁻¹, Rimadyl, Pfizer Ltd, Kent, UK). Using methods described previously (Hay et al. 1984), three polyvinyl catheters for infusion were placed, two into a fetal saphenous vein via a pedal vein and one into a maternal femoral vein. Catheters for sampling the uterine circulation were placed into a maternal femoral artery and the uterine vein draining the pregnant horn. Catheters for sampling the umbilical circulation were placed into the lower fetal aorta via a pedal artery and into the common umbilical vein. The catheters were tunnelled subcutaneously, exteriorised through a flank skin incision, and kept in a pouch stitched to the ewe's flank. Catheters were flushed daily with a heparin-saline solution (150 i.u. ml⁻¹, 0.9 % w/v sodium chloride solution). Ewes were housed individually in polypropylene floor level crates and allowed to recover from surgery for 6–9 days prior to study. The ewes had previously been acclimatised to these crates for short periods on several days prior to catheter insertion. During the recovery period, maternal and fetal arterial plasma glucose concentrations and blood oxygen contents were monitored daily. Ewes were transferred to a three times daily feeding regime and gradually re-alimented back to either a high or moderate dietary intake.

Study design

On day 5 post surgery, four high-intake dams were fed as normal at 07.00 h and then samples were withdrawn from the maternal arterial catheter into heparinised syringes at 30 min intervals between 10.00 and 15.00 h. Plasma glucose concentrations were determined in these samples as described below. The highest post-prandial maternal glucose concentration recorded was 4.16 µmol ml⁻¹.

The study protocol was designed to measure placental glucose transport at three different fetal glucose steady-state concentrations while maternal glucose concentration was maintained at a fixed level (4.94 ± 0.10 µmol ml⁻¹), thereby producing three different maternal-fetal arterial plasma glucose concentration gradients. Ewes were studied at 130 ± 0.5 days of gestation. To minimise stress, the ewes were fed as normal at 07.00 h on the day of study. The maternal plasma glucose concentration was established and maintained at the desired level by infusion of dextrose (50 % w/v) into the maternal femoral vein using a modified glucose clamp procedure described previously (Hay & Meznarich, 1986; Hay et al. 1988). The maternal glucose infusion rate was adjusted as required on the basis of maternal arterial glucose measurements made at 10–20 min intervals throughout the study period. After the maternal glucose infusion was started, tritiated water (³H₂O) was infused into the fetus to measure uterine and umbilical blood flows by application of the steady-state transplacental diffusion technique (Meschia et al. 1967). A solution of tritiated water (16.7 µCi ml⁻¹, Amersham Life Science Ltd, Buckinghamshire, UK) in saline was infused into the fetal vein catheter. A bolus of 3 ml (50 µCi) was administered over 1 min, after which the infusion rate was changed to 3 ml h⁻¹ (50 µCi h⁻¹). After both infusions had been running for at least 90 min to reach steady state, blood samples were withdrawn simultaneously into heparinised syringes from the maternal artery, uterine vein, fetal artery and umbilical vein catheters. Four sets of consecutive samples were withdrawn at 10–15 min intervals and analysed for whole blood oxygen content and plasma glucose, lactate and ³H₂O concentrations. In this initial experimental period, fetal glucose concentration was not perturbed (period 1 or level 1). This was followed by two further experimental periods, one during which fetal glucose concentrations were increased by ≈50 % relative to period 1 (level 2) and another during which fetal glucose concentrations were increased by ≈200 % relative to period 1 (level 3). Both these levels were established and maintained at the required steady state by infusion of dextrose (25 % w/v) into the fetal vein. Fetal arterial plasma glucose concentrations were measured every 10–15 min and the infusion rate adjusted accordingly. Levels 2 and 3 each required a mean of 145 and 95 min, respectively, to achieve the new steady state and a further 30–45 min to repeat the four sample sets. Fetuses were transfused isovolumetrically after each sampling period with freshly collected heparinised maternal whole blood to maintain blood volume and haemoglobin concentration. For maternal and fetal glucose concentrations steady state was defined during each sampling period as < 5 % variation of each sample value around the sampling period mean value, without a consistent trend to increase or decrease.

The ewe and fetus were humanely killed by intravenous administration of an overdose of sodium pentobarbitone (200 mg ml⁻¹ Euthesate, Willows Francis Veterinary, Crawley, UK) on 133 ± 0.5 days of gestation. The fetus and placental cotyledons were weighed and catheter location verified. Fetal brain and liver were dissected and weighed, as was the maternal perirenal fat.

Chemical analyses

The plasma ³H₂O concentrations were measured in triplicate using 200 µl plasma, 500 µl distilled water and 15 ml Ultima Gold scintillation fluid (Packard Bioscience B.V., The Netherlands) and counted on a Packard scintillation counter (Tri-carb 1900TR with internal quench correction). Plasma ³H₂O concentrations were converted to whole blood concentrations according to Van Veen et al. (1984). Plasma glucose and lactate concentrations were measured in duplicate with a Yellow Springs Instruments (YSI, Yellow Springs, OH, USA) dual biochemistry analyser (model 2700). The YSI instrument was calibrated with known standards after every fourth determination. Whole blood oxygen content was measured in 0.2 ml blood sampled into heparinised syringes using a Radiometer OSM-3 hemoximeter (Radiometer, Copenhagen, Denmark).

Calculations and data analysis

Blood flow and net substrate uptake rates were calculated as described previously (Meschia et al. 1980) and according to the following equations.

Umbilical blood flow = net transplacental diffusion rate of ³H₂O/umbilical arteriovenous blood concentration difference of ³H₂O, where net transplacental diffusion rate of ³H₂O is calculated according to the Fick principle as fetal ³H₂O infusion rate minus the rates of accumulation and metabolism in the fetus.

Uterine blood flow = net transplacental diffusion rate of ³H₂O/uterine venoarterial blood concentration difference of ³H₂O.

Net uterine uptake of substrate = uterine blood flow × uterine arteriovenous blood substrate concentration difference.

Net umbilical uptake of substrate = umbilical blood flow × umbilical venoarterial blood substrate concentration difference.

Net uteroplacental consumption of substrate = uterine uptake - umbilical uptake.

Net uteroplacental production of substrate = −(uterine uptake) + umbilical uptake.

The significance of differences between the high and moderate nutritional treatment groups was determined by Student's unpaired t test while differences within animals were assessed by Student's paired t test. Regression analysis was by the least squares method and correlation analysis was by Pearson's Product Moment Test, where appropriate. Significance was accepted when P < 0.05.

RESULTS

Full catheter patency was maintained in 18 of 21 pregnancies (11 high- and 7 moderate-intake dams) and only data pertaining to these animals are reported hereafter.

Maternal dietary intakes and maternal weight and condition score changes in relation to pregnancy outcome

Mean weekly maternal feed intakes from embryo transfer to day 119 of gestation are shown in Fig. 1. Maternal dietary intakes were elevated in high- compared with moderate-intake dams throughout this period (P < 0.001). On the day prior to study (≈day 129 of gestation), the dry matter feed intake was 1545 ± 61 and 1229 ± 60 g in high- and moderate-intake groups, respectively (P < 0.01).

Figure 1.

Weekly dry matter intakes from embryo transfer on day 4 of the cycle until day 119 of gestation in singleton-bearing adolescent dams offered a high (□) or moderate (▪) nutrient intake. In this and subsequent figures, values are given as means ± s.e.m.; where error bars are not shown, they are smaller than the symbol size.

The dietary-induced changes in maternal weight and body condition score and morphometric data relating to pregnancy outcome in high- and moderate-intake adolescent dams are detailed in Table 1. In the high dietary intake group, live weight and body condition score were significantly elevated by the end of the first third of pregnancy and remained higher throughout the study (P < 0.001). Mean daily live weight gain during the first two-thirds of gestation was 282 ± 15 and 57 ± 5 g day⁻¹ in the high- and moderate-intake groups, respectively. Similarly, at autopsy the absolute and relative perirenal fat mass of the dams was markedly elevated (P < 0.001 and P < 0.01, respectively) in the high-intake group.

Table 1.

Maternal weight and body condition score and morphometric data relating to pregnancy outcome in adolescent dams offered a high or moderate nutrient intake from day 4 of gestation

	Maternal nutrient intake
	Moderate (n = 7)	High (n = 11)	Significance
Live weight (kg) at:
Embryo transfer	47.8 ± 1.6	44.2 ± 1.3	n.s.
Day 100 of gestation	53.7 ± 1.5	73.0 ± 1.7	P < 0.001
Autopsy on ˜day 133	60.3 ± 1.7	75.8 ± 1.7	P < 0.001
Body condition score at:
Embryo transfer	2.40 ± 0.05	2.30 ± 0.04	n.s.
Day 100 of gestation	2.20 ± 0.05	3.10 ± 0.05	P < 0.001
Autopsy on ˜day 133	2.10 ± 0.05	3.30 ± 0.06	P < 0.001
At autopsy
Maternal perirenal fat mass (g)	524 ± 95	1420 ± 115	P < 0.001
Perirenal fat per maternal autopsy weight (g g−1)	0.012 ± 0.002	0.019 ± 0.001	P < 0.01
Gestational age (days)	133 ± 1	133 ± 1	—
Total placentome weight (g)	386 ± 34	209 ± 23	P < 0.001
Average placentome weight (g)	4.47 ± 0.55	2.91 ± 0.24	P < 0.02
Placentome number	88 ± 7	75 ± 5	n.s.
Fetal weight (g)	4670 ± 196	3072 ± 266	P < 0.001
Fetal:placental weight ratio	12.5 ± 0.9	15.3 ± 0.9	P < 0.05
Gravid uterus weight (kg)	7.4 ± 0.4	5.0 ± 0.4	P < 0.001

Values are means ± S.E.M. n.s., not significant.

Both total and average placentome weights were reduced in high- compared with moderate-intake groups (P < 0.001 and P < 0.02, respectively) and this reduction in placental mass (46 %) resulted in a significant decrease (P < 0.001) in fetal weight of 34 % when compared with the moderate-intake group. At this stage of gestation the placenta was relatively more perturbed than the fetus, resulting in a significantly higher fetal:placental weight ratio (P < 0.05) in the overnourished group. Irrespective of nutritional treatment group, placental weight was strongly correlated with fetal weight (r = 0.891, n = 18, P < 0.001). Fetal liver weights but not brain weights were significantly smaller in high- vs. moderate-intake group fetuses (91 ± 11.1 vs. 143 ± 11.6 g, P < 0.001, and 37.9 ± 1.4 vs. 41.6 ± 1.9 g, n.s., respectively). Consequently, the brain:liver weight ratio was elevated in high- compared with moderate-intake group fetuses (0.5 ± 0.05 vs. 0.3 ± 0.03, P < 0.01). The relationships between placental weight and the weights of the fetal body, liver and brain are depicted in Fig. 2.

Figure 2.

Relationships between total placentome mass and fetal brain (A), liver (B) and body (C) weight in fetuses from adolescent dams offered a high (□) or moderate (▪) nutrient intake throughout pregnancy.

Uterine and umbilical blood flows

Absolute uterine and umbilical blood flow rates in the growth-restricted pregnancies of high-intake dams were reduced by 29 % (P < 0.05) and 33 % (P < 0.02), respectively, compared with the moderate-intake group (Table 2). However, when uterine blood flow was normalised for the weight of the conceptus (uterus + placenta + fetus) there was no difference between treatment groups. Similarly, umbilical blood flow was proportional to both placental and fetal weight. Furthermore, the uterine to umbilical blood flow ratio was not perturbed in high- vs. moderate-intake groups (Table 2). Irrespective of maternal nutritional treatment, uterine and umbilical blood flows were positively correlated with both placental (r = 0.845 and 0.813, respectively, n = 18, P < 0.001) and fetal (0.809 and 0.821, respectively, n = 18, P < 0.001) weight.

Table 2.

Uterine and umbilical blood flows in adolescent dams offered a high or moderate nutrient intake from day 4 of gestation

	Maternal nutrient intake
	Moderate (n = 7)	High (n = 11)	Significance
Uterine blood flow (ml min−1)	1656 ± 189	1174 ± 114	P < 0.05
Uterine blood flow per kilogram conceptus (ml min−1 kg1)	262 ± 30	301 ± 13	n.s
Umbilical blood flow (ml min−1)	869 ± 84	585 ± 63	P < 0.02
Umbilical blood flow per kilogram fetus (ml min−1 kg1)	192 ± 13	181 ± 12	n.s
Uterine:umbilical blood flow ratio	1.85 ± 0.20	2.28 ± 0.20	n.s.

Values are means ± S.E.M. Individual average uterine and umbilical blood flows were calculated from the flows measured on three occasions during the maternal glucose clamp.

Oxygen data

Maternal arterial and venous blood oxygen contents were similar in both nutritional treatment groups (Table 3). In contrast both fetal arterial and umbilical vein oxygen contents were significantly lower (P < 0.05) in the growth-restricted pregnancies of the high-intake dams. Absolute uterine and umbilical oxygen uptake values were lower (P < 0.05) in high- than in moderate-intake groups but were in proportion to the weight of the conceptus and fetus, respectively.

Table 3.

Maternal and fetal blood oxygen contents and uterine and umbilical oxygen uptakes in adolescent dams offered a high or moderate nutrient intake from day 4 of gestation

	Maternal nutrient intake
	Moderate (n = 7)	High (n = 11)	Significance
Maternal arterial blood oxygen content (mM)	4.8 ± 0.4	5.3 ± 0.2	n.s.
Uterine venous blood oxygen content (mM)	4.0 ± 0.7	4.0 ± 0.2	n.s.
Fetal arterial blood oxygen content (mM)	3.0 ± 0.3	2.1 ± 0.2	P < 0.05
Fetal venous blood oxygen content (mM)	4.9 ± 0.2	4.1 ± 0.2	P < 0.05
Uterine oxygen uptake (mmol min−1)	2460 ± 377	1462 ± 141	P < 0.05
Uterine oxygen uptake per kilogram conceptus (mmol min−1 kg−1)	385 ± 33	374 ± 19	n.s.
Umbilical oxygen uptake (μmmol min−1)	1637 ± 239	1030 ± 101	P < 0.05
Umbilical oxygen uptake per kilogram fetus (mmol min−1 kg1)	357 ± 41	350 ± 17	n.s.

Values are means ± S.E.M.

Glucose concentrations and uptakes

During period 1, the maternal plasma glucose concentration was similar in the high and moderate dietary intake groups due to the maternal glucose clamp (Table 4). In contrast the spontaneous fetal arterial plasma glucose concentrations (level 1) were lower (P < 0.002) in the growth-restricted fetuses of the high-intake group. In this non-perturbed state, within the high-intake (but not the moderate-intake) group, fetal arterial glucose concentrations were positively correlated with fetal and placental mass (r = 0.842 and 0.923, respectively, P < 0.001). Both absolute uterine (P < 0.06) and umbilical glucose (P < 0.01) uptakes were lower in high- than in moderate-intake groups, by an average of 40 %. However, these reductions in glucose uptake were proportional to the weight of the conceptus and fetus, respectively (Table 4). Uterine glucose extraction was not different between groups but fetal glucose extraction was significantly increased (P < 0.02) in the high- compared with the moderate-intake group. Uteroplacental glucose consumption was on average 57 % lower (P = 0.09) in high- compared with moderate-intake groups but was largely in proportion to the difference in placental weights between groups.

Table 4.

Maternal and fetal plasma glucose concentrations and extractions, and uterine, umbilical and uteroplacental glucose uptakes in adolescent dams offered a high or moderate nutrient intake from day 4 of gestation

	Maternal nutrient intake
	Moderate (n = 7)	High (n = 11)	Significance
Maternal arterial plasma glucose (μmol ml−1)	5.10 ± 0.20	4.88 ± 0.09	n.s.
Fetal arterial plasma glucose (μmol ml−1)	1.32 ± 0.05	0.90 ± 0.10	P < 0.002
Arterial plasma glucose difference (M-F) (μmol ml−1)	3.76 ± 0.19	4.00 ± 0.12	n.s.
Uterine glucose uptake (μmol min−1)	346 ± 60	206 ± 32	P < 0.06
Uterine glucose uptake per kilogram conceptus (mmol min−1 kg−1)	55.8 ± 6.6	50.7 ± 6.0	n.s.
Umbilical glucose uptake (μmol min−1)	206 ± 25	120 ± 13	P < 0.01
Umbilical glucose uptake per kilogram fetus (mmol min−1 kg1)	45.2 ± 4.3	40.3 ± 2.2	n.s.
Uteroplacental glucose consumption (mmol min−1)	199 ± 58	86 ± 23	P = 0.09
Uteroplacental glucose consumption per kilogram placenta (mmol min−1 kg−1)	474 ± 97	401 ± 91	n.s.
Uterine glucose extraction (%)	5.3 ± 0.7	4.6 ± 0.5	n.s.
Fetal glucose extraction (%)	17.3 ± 0.8	24.1 ± 2.3	P < 0.02

M, maternal; F, fetal. Values are means ± S.E.M.

The mean maternal and fetal arterial glucose concentrations maintained during the three different fetal glucose steady-state levels are presented in Table 5 together with the umbilical glucose flux rates. The individual arterial glucose levels for each sampling period represent the mean of four consecutive samples. For the maternal samples the overall mean variance in glucose concentration between these four samples was 1.8 and 1.6 % for the moderate- and high-intake dams, respectively. Similarly, for the fetal samples the mean variance was 2.0 and 2.8 % for fetuses in the moderate- and high-intake groups, respectively. The maternal glucose clamp was maintained during all three study periods and maternal arterial glucose concentrations were similar in both groups throughout. As expected, fetal arterial glucose concentrations increased significantly in both groups during the two different levels of fetal glucose infusion. This rise in fetal arterial glucose concentration was associated with a significant decrease in absolute umbilical glucose uptake and in placental- and fetal weight-specific umbilical glucose uptakes (Table 5).

Table 5.

Placental glucose transport in adolescent dams offered a high or moderate nutrient intake throughout gestation

	Level 1		Level 2		Level 3
	Moderate	High	Moderate	High	Moderate	High
Arterial plasma glucose (μmol ml−1)
Maternal	5.05 ± 0.22	4.90 ± 0.09	5.02 ± 0.16	4.84 ± 0.10	5.17 ± 0.14	4.92 ± 0.07
Fetal	1.29 ± 0.06	0.91 ± 0.10	1.87 ± 0.07 ‡	1.41 ± 0.12 †	2.48 ± 0.06 ‡	1.97 ± 0.14 ‡
Arterial plasma glucose difference (M–F)
(μmol ml−1)	3.77 ± 0.19	4.00 ± 0.12	3.15 ± 0.13 *	3.43 ± 0.16 *	2.68 ± 0.15 †	2.95 ± 0.15 ‡
Umbilical glucose uptake
(μmol min−1)	206 ± 25	120 ± 13	105 ± 10 †	78 ± 10 *	54 ± 12 †	30 ± 7 ‡
(μmol min−1 (kg fetus)−1)	45.2 ± 4.3	40.3 ± 2.2	23.6 ± 2.2 †	26.8 ± 2.7 †	9.8 ± 3.0 ‡	11.4 ± 2.8 ‡
(μmol min−1 (kg placenta)1)	542 ± 58	585 ± 35	283 ± 34 †	396 ± 43 †	154 ± 38 †	182 ± 47 ‡
Uteroplacental glucose uptake
(mmol min−1 (kg fetus)−1)	43.1 ± 12.8	28.7 ± 6.7	50 ± 17	42.5 ± 6.1	52.2 ± 13.5	41.6 ± 5.8

High dietary intake, n = 11. Moderate dietary intake, n = 7.Values are means ± S.E.M.P values were determined by Student's paired t test comparing level 1 with either level 2 or level 3.

^*P < 0.05

^†P < 0.01

^‡P < 0.001.

The relationship between the transplacental plasma arterial glucose concentration difference and umbilical glucose uptake in high- and moderate-intake pregnancies is shown in Fig. 3. For the moderate-intake group:

Figure 3.

Relationships between umbilical glucose uptake and the transplacental plasma arterial glucose concentration difference in adolescent dams offered a high (□) or moderate (▪) nutrient intake throughout pregnancy. See text for linear regression equations.

The slope and y-intercept of this equation are similar to the mean slope (1.5 ± 0.28 dl min⁻¹) and y-intercept (3.40 ± 0.78 µmol ml⁻¹) calculated by linear regression analysis of the data for each animal separately. For the high-intake group:

which is also similar to the mean slope (0.8 ± 0.10 dl min⁻¹) and y-intercept (1.84 ± 0.25 µmol ml⁻¹) of the individual animal data. The mean slope values were significantly (P < 0.05) different between treatment groups and there was also a clear trend for a difference in the y-intercept (P < 0.08). Therefore in absolute terms placental glucose transport capacity was reduced in the high- compared with the moderate-intake group. Thus, as illustrated in Fig. 3, if the mean maternal-fetal arterial plasma glucose difference in the moderate group (3.20 µmol ml⁻¹) is used to construct the dashed line which intersects with the high-intake group regression line, the growth-restricted fetuses of the high-intake group had a predicted umbilical uptake of 55.6 µmol min⁻¹, which was equivalent to 53 % of the moderate group value (105.6 µmol min⁻¹).

The data presented in Table 5 were further analysed by plotting the transplacental plasma glucose concentration difference against (a) umbilical glucose uptake per kilogram fetus (Fig. 4), and (b) umbilical uptake per kilogram placenta (Fig. 5). In Fig. 4, the slopes and intercepts of the regression lines were not significantly different, indicating that the growth-restricted fetus largely achieves normal weight-specific glucose consumption. Thus at the mean transplacental glucose gradient of 3.20 µmol ml⁻¹ (indicated by the dashed line), the predicted umbilical uptakes per kilogram fetus would have been ≈19.2 vs. 25.6 µmol min⁻¹ kg⁻¹ for the high- and moderate-intake groups, respectively, and were not significantly different from each other. Similarly, as shown in Fig. 5, there was no difference in the linear regression analysis between groups indicating that the growth-restricted placenta of the high-intake dams has a normal weight-specific glucose transfer capacity.

Figure 4.

Relationships between umbilical glucose uptake per kilogram fetus and the transplacental plasma arterial glucose concentration difference in adolescent dams offered a high (□) or moderate (▪) nutrient intake throughout pregnancy. For high-intake pregnancies, y = −0.68 + 0.272x. For moderate-intake pregnancies, y = −0.78 + 0.327x.

Figure 5.

Relationships between umbilical glucose uptake per kilogram placenta and the transplacental arterial glucose concentration difference in adolescent dams offered a high (□) or moderate (▪) nutrient intake throughout pregnancy.

Uteroplacental glucose consumption did not vary with increasing fetal arterial glucose concentrations and when expressed relative to fetal weight was not significantly different between high- and moderate-intake groups during any of the three clamp periods studied (Table 5).

DISCUSSION

Overnourishing the singleton-bearing adolescent ewe throughout pregnancy results in significant placental and fetal growth restriction (Wallace et al. 1996, 1997). In the present study, blood flows and nutrient fluxes were measured at a fixed maternal glucose concentration. The growth-restricted pregnancies in the high-intake dams were associated with major reductions in absolute (a) uterine and umbilical blood flows, (b) uterine and umbilical glucose and oxygen uptakes, and (c) uteroplacental glucose consumption. This agrees with our previous observations at spontaneous maternal glucose concentrations (Wallace et al. 2002). In both the original and present study, the blood flow and nutrient uptake parameters were largely not different when normalised for the weight of the conceptus, placenta or fetus as appropriate. The present study further demonstrates that, in spite of normal weight-specific nutrient uptakes, the fetuses themselves remain relatively hypoglycaemic in the presence of a physiologically normal and fixed maternal glucose concentration. This suggests that a reduction in the ability of the placenta to supply glucose may be a primary effect of overnourishing the pregnant adolescent.

Indeed, the major aim of the present study was to accurately determine placental glucose transport capacity in our unique paradigm. The transport of glucose by the placenta to the fetus is determined by the arterial glucose concentration difference between the maternal (uterine artery) and fetal (umbilical artery) plasma, according to saturation-limited kinetics (Hay et al. 1990; Hay, 1995). Under spontaneously occurring variations in maternal and fetal glucose concentrations, the relationship between the transplacental glucose gradient and fetal glucose uptake is difficult to quantify because transport is mediated by saturable membrane-localised glucose transporters on both the maternal-facing microvillus and fetal-facing basal trophoblast membranes (GLUT1 and GLUT3; Ehrhardt & Bell, 1997). Furthermore changes in glucose concentration have previously been shown to alter the rate of placental glucose metabolism (Hay et al. 1990). The relationship can be studied if maternal plasma glucose is maintained at a physiological level and the transplacental arterial glucose concentration gradient is manipulated by varying fetal plasma glucose over a relatively narrow range. Under such conditions, the dependence of placental transport rate on the transplacental glucose concentration difference is virtually linear (Hay et al. 1990), thus making relatively few measurements an acceptable basis for comparing glucose transport between growth-restricted and normal pregnancies. The comparison shows that at a given transplacental glucose concentration difference, the absolute transport of glucose from the placenta to the fetus in the overnourished dams with growth-restricted pregnancies was approximately half of that measured in the moderate-intake (control) group (Fig. 3). However, the difference in placental mass at autopsy between these groups was 46 % and when umbilical uptake was expressed as per kilogram placenta, weight-specific placental transport capacity was shown to be similar in both groups (Fig. 5). The decreased transport capacity of the growth-restricted placentas in the high-intake group, therefore, is the result of a smaller placenta, and not the capacity of this smaller placenta to transfer glucose.

As indicated above, glucose transport across the uteroplacenta is mediated by the steady-state expression and activity of GLUT1 and GLUT3. While the relative concentrations of these glucose transporters have not been quantified in the placentae in the present study, the normal placental weight-specific transport capacity measured in vivo suggests that the concentration of these transporters per unit membrane is unlikely to differ between maternal nutritional groups in the baseline state. Furthermore, since transport capacity did not diverge between groups at any of the levels of fetal glycaemia, it is unlikely that there was an acute change in either or both transporters of sufficient magnitude to affect transport.

The results of the present study contrast with the only other experimental paradigm of placental growth restriction for which strictly comparable glucose transport capacity data are available, namely the hyperthermic model (Thureen et al. 1992). In this model, placental and fetal growth restriction are produced by exposure of the pregnant ewe to naturally hyperthermic conditions of ≈40 °C for 12 h day⁻¹ between days 39 and 125 of gestation. Placental glucose transfer in this model, measured as umbilical glucose uptake per kilogram placenta, is significantly reduced at the mean maternal-fetal arterial plasma glucose difference of the control group. Thus, transfer capacity is decreased even more than can be accounted for simply because the placenta is smaller. The mechanisms responsible for such transport deficiencies in the hyperthermic model are not known. Such dissimilar adaptations of placental glucose transport function indicate that the physiological processes underlying altered placental development and growth restriction in the two models are fundamentally different. This is intriguing in view of the fact that the adolescent and heat stress models exhibit many similarities with respect to maternal endocrinology, placental and fetal phenotype, and uteroplacental metabolism (Bell et al. 1987, 1989; Early et al. 1991; Vatnick et al. 1991; Thureen et al. 1992; McCrabb & Bortolussi 1996; Wallace, 2000).

In both the present overnourished adolescent pregnant ewe model and the hyperthermic model, however, there is a similar fetal response to the decreased ability of the placenta to supply glucose. In both cases, fetal glucose utilisation remains at normal levels per fetal weight, resulting in increased fetal glucose extraction and a decrease in fetal glucose concentration. This widens the transplacental glucose concentration difference, thereby helping to maintain the flux of maternal glucose into the umbilical circulation. Such similar adaptations indicate that the physiological processes underlying fetal growth restriction in the two models are fundamentally the same. The nature of the adaptive mechanisms that maintain fetal weight-specific glucose utilisation are not known, but should involve increased glucose uptake and/or metabolic capacity or increased insulin sensitivity, or both.

Placental metabolism and glucose transfer capacity have also been studied in the pre-mating carunclectomy model of reduced placental mass. In the carunclectomised ewes, the fetal to maternal clearance of 3-O-methyl glucose, a non-metabolisable glucose analogue, was increased when expressed as per unit placental mass implying an enhanced efficiency of placental glucose transfer (Owens et al. 1987b). In addition, relative uterine extraction of glucose across the uterine circulation was increased (Owens et al. 1989) and in the most severely growth-restricted pregnancies, uteroplacental glucose consumption per kilogram placenta was reduced while lactate production per kilogram placenta was increased resulting in a preferential distribution of glucose to the fetus (Owens et al. 1987a). These adaptations, which try to preserve glucose supply to the fetus, result in the maintenance of normal fetal growth in a proportion of animals studied (Robinson et al. 1979). This adaptation may partially reflect the different placental phenotype in the carunclectomy model, which is characterised by marked placental hypertrophy and overgrowth of the remaining fetal cotyledons (Robinson et al. 1979; Chidzanja et al. 1992). In contrast, in the overnourished adolescent ewe, placental growth restriction is characterised by a major reduction in individual placentome size and cell number (Wallace et al. 2000) and placental mass accounts for a high proportion (70 %) of the variability in fetal weight (Wallace et al. 2001). In the present study, we have not attempted to relate individual placental glucose transport capacity to placentome phenotype as the placentae of both high- and moderate-intake groups rarely comprise a single phenotype.

In this study the different transplacental glucose gradients did not impact significantly on uteroplacental metabolism in either group (Table 5). This was somewhat unexpected as a previous series of glucose and insulin clamps in normally growing fetuses had shown that uteroplacental glucose consumption increased in response to an increase in fetal glucose concentration, that is, as the maternal to fetal glucose gradient was reduced towards zero (Hay et al. 1990). There are several possible reasons, yet to be studied or determined, for this discrepancy between studies. The adolescent pregnancies in general might have a relatively restricted capacity for glucose uptake by the placenta from the fetus. This could be due to decreased numbers of glucose transporters on the fetal-facing, basal surface of the placental cells that ordinarily would act to transport glucose directly and relatively unidirectionally into placental cellular metabolism. Alternatively placental glucose metabolic capacity is in some way limited. It is also possible that the range of fetal glucose concentrations studied in the present investigation was not wide enough to detect the relationship described in the earlier study.

The results of this study clearly demonstrate that it is the small size of the placenta per se rather than alterations in placental nutrient uptake, metabolism or transfer that limits fetal growth in the overnourished adolescent ewe. The mechanisms regulating ovine placental growth remain obscure but in the adolescent ewe model they are likely to involve nutritionally mediated alterations in endocrine hormones or growth factors of maternal, placental or fetal origin. In overnourished compared with moderate-intake adolescent dams, peripheral concentrations of insulin, IGF-1, leptin and thyroid hormones are elevated while progesterone, growth hormone and placental lactogen concentrations are low (see Wallace et al. 2001 for review). These endocrine perturbations, which are evident from early pregnancy onwards, may in turn impact on placental cell proliferation and/or apoptosis. They may also play a role in orchestrating both the initial development of the placental vascular bed and its structural remodelling during late pregnancy, hence laying the haemodynamic foundations for the changes in uterine and umbilical blood flow known to occur at this time (Molina et al. 1990). Indeed, uteroplacental blood flows are critical regulators of nutrient partitioning during the last third of pregnancy (Carter & Myatt, 1995) and in this and other models of placental growth restriction uteroplacental blood flows are attenuated and positively associated with placental mass (Owens et al. 1986; Bell et al. 1987; Thureen et al. 1992). This study does not establish when during pregnancy uteroplacental blood flows diverge, or whether they drive or merely reflect the observed placental growth restriction. Nevertheless, recent analysis of placental data from adolescent dams throughout gestation suggests that the relative restriction in placental mass in high- compared with moderate-intake dams increases as pregnancy progresses (Wallace, 2000) and this may well be blood flow regulated.