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BOR - Papers in Press, published online ahead of print May 2, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.061440
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BIOLOGY OF REPRODUCTION 77, 343–350 (2007)
DOI: 10.1095/biolreprod.107.061440
© 2007 by the Society for the Study of Reproduction, Inc.

Maternal and Fetal Growth, Body Composition, Endocrinology, and Metabolic Status in Undernourished Adolescent Sheep1

Justin Luther 3 4, Raymond Aitken 3, John Milne 3, Masatoshi Matsuzaki 5, Lawrence Reynolds 4, Dale Redmer 4, and Jacqueline Wallace 2 3

Rowett Research Institute,3 Bucksburn, Aberdeen AB21 9SB, United Kingdom Department of Animal and Range Sciences,4 North Dakota State University, Fargo, North Dakota 58105 Hirosaki University,5 Hirosaki 036-8561, Japan

ABSTRACT

The influence of relative maternal undernutrition on growth, endocrinology, and metabolic status in the adolescent ewe and her fetus were investigated at Days 90 and 130 of gestation. Singleton pregnancies to a single sire were established, and thereafter ewes were offered an optimal control (C; n = 14) or low (L [0.7 x C]; n = 21) dietary intake. Seven ewes receiving the L intake were switched to the C intake on Day 90 of gestation (L-C). At Day 90, live weight and adiposity score were reduced (P < 0.001) in L versus C dams. Plasma insulin and IGF1 concentrations were decreased (P < 0.02), whereas glucose concentrations were preserved in L relative to C intake dams. Fetal and placental mass was independent of maternal nutrition at this stage. By Day 130 of gestation, when compared to C and L-C dams, maternal adiposity was further depleted in L intake dams; concentrations of insulin, IGF1, and glucose were reduced; and nonesterified fatty acids increased. At Day 130, placental mass remained independent of maternal nutrition, but body weight was reduced (P < 0.01) in L compared with C fetuses (3555 g vs. 4273 g). Body weight was intermediate (3836 g) in L-C fetuses. Plasma glucose (P < 0.03), insulin (P < 0.07), and total liver glycogen content (P < 0.04) were attenuated in L fetuses. Fetal carcass analyses revealed absolute reductions (P < 0.05) in dry matter, crude protein, and fat, and a relative (g/kg) increase in carcass ash (P < 0.01) in L compared with C fetuses. Thus, limiting maternal intake during adolescent pregnancy gradually depleted maternal body reserves, impaired fetal nutrient supply, and slowed fetal soft tissue growth.

adolescent pregnancy,, endocrinology,, environment,, female reproductive tract,, fetus,, glucose,, placenta,, pregnancy,, sheep,, undernutrition

INTRODUCTION

Becoming pregnant during adolescent life (<19 yr of age) annually accounts for up to one fifth of all births worldwide [1] and is associated with a variety of negative outcomes for mother and child. The most serious and immediate of these include an increased risk of premature delivery, low birth weight, neonatal and infant mortality, and maternal death [2, 3]. There is evidence that both the growth and nutritional status of the mother may play an important role in adolescent pregnancy outcome. Data from the Camden Study in New Jersey reveal that up to 50% of adolescent humans continue to grow while pregnant and, in spite of larger pregnancy weight gains and increased fat stores, these girls deliver smaller babies compared with nongrowing adolescent mothers of equivalent age [4]. This alternation in the normal hierarchy of nutrient partitioning is attributed to a competition for nutrients between the maternal body and her gravid uterus and has been replicated in a highly controlled sheep paradigm. Using this paradigm, we have consistently shown that overnourishing the singleton-bearing adolescent throughout pregnancy to promote rapid maternal growth (primarily of adipose tissue) similarly results in the premature delivery of low-birth weight lambs [5–7] compared with control-fed adolescents of equivalent gynecological age. Additionally, we have been able to demonstrate that nutritionally mediated defects in early placental development (namely, reduced cellular proliferation, increased apoptosis, and impaired angiogenesis) compromise the placental growth trajectory and are the primary limitation to fetal growth in the rapidly growing adolescent [8–10]. Thus, by late gestation and in spite of the ready availability of nutrients in the maternal circulation, the small size of the placenta and the resultant attenuation of uteroplacental blood flows and nutrient uptakes limit fetal nutrient supply, resulting in asymmetric growth restriction and relative hypoxia and hypoglycemia [11, 12]. This robust alteration in nutrient partitioning during pregnancy in response to high dietary intakes is unique to the young adolescent in that it does not occur in overnourished primiparous adult sheep studied under identical experimental conditions [13]. At the other end of the nutritional spectrum, suboptimal dietary intakes are commonplace in sections of the general adolescent population, and many adolescent girls may be at risk of becoming pregnant with inadequate nutrient stores [14]. In particular, adolescent girls who have not achieved their predicted adult height at the time of conception and hence still have the potential to grow may be particularly vulnerable to poor pregnancy outcome if dietary intakes are inadequate [15] and, moreover, such a scenario may clearly benefit from targeted nutritional intervention. Indeed, insufficient pregnancy weight gains (a proxy indicator of maternal undernutrition) in human adolescent mothers have been associated with low birth weight in several studies [16–19].

The present study aimed to model part of this problem by relatively undernourishing young adolescent sheep by maintaining maternal body weight at the level determined at conception and hence gradually depleting maternal nutrient reserves. Control-fed dams were nourished to maintain maternal adiposity throughout pregnancy. The sire, genotype, initial adiposity, and diet composition were identical to those used in previous adolescent studies to facilitate direct comparisons of key pregnancy outcome parameters. The impact of relatively underfeeding adolescent dams on maternal and fetal growth, body composition, and metabolic/endocrine status was determined at midgestation (Day 90) and late gestation (Day 130). A further group of undernourished adolescents was switched to control intakes from Days 90–130 of gestation to determine whether the predicted effects on fetal growth were reversible.

MATERIALS AND METHODS

Animals and Experimental Design

All procedures were licensed under the United Kingdom Animals (Scientific Procedures) Act of 1986 and approved by the Rowett Research Institute's Ethics Review Committee.

Embryos from superovulated adult ewes (Border Leicester x Scottish Blackface) inseminated by a single sire were recovered on Day 4 after estrus and were transferred synchronously in singleton into the uterus of recipient ewe lambs (Dorset Horn x Greyface). Donors were multiparous, between 3 and 4 yr of age, weighed 82.5 ± 2.03 kg, and had a body condition score of 2.3 ± 0.05 units at the time of embryo recovery. This protocol ensured that placental and/or fetal growth were not influenced by varying fetal number or partial embryo loss. In addition, the use of a single sire and a limited number of embryo donors maximized the homogeneity of the resulting fetuses. Embryo transfer was carried out during the midbreeding season, and animals were housed in individual pens under natural lighting conditions at the Rowett Research Institute (57°N, 2°W). At the time of embryo transfer, recipient ewe lambs were peripubertal (~8 mo of age) and had a mean live weight of 42.9 ± 0.34 kg, body condition score of 2.3 ± 0.02 units, and ovulation rate of 2.2 ± 0.21 corpora lutea. Based on these parameters, recipient ewe lambs were initially allocated to one of two nutritional treatments. Where possible, care was also taken to randomize for the maternity of the embryo. Recipients were individually offered an optimal control (C; n = 18) or low quantity (L; n = 28) of the same complete diet. The dietary level in the control group was calculated to maintain normal maternal adiposity throughout gestation and to provide 100% of the estimated metabolizable energy and protein requirement of the adolescent sheep carrying a singleton fetus according to stage of pregnancy [20]. Accordingly, previous studies have shown that this ration as fed optimizes placental and fetal growth in this genotype [7]. In practical terms, maximum placental and fetal growth is achieved in the C group by allowing a modest maternal weight gain (~50 g/day) during the first two thirds of gestation, followed by step-wise increases in maternal intake during the final third of gestation to meet the increasing nutrient demands of the developing fetus. In contrast, the dietary intake in the L group was calculated to maintain maternal live weight at embryo transfer, and hence gradually deplete maternal tissue reserves throughout gestation. In practical terms, we initially estimated the level of feed required to maintain maternal weight and prevent live weight gain from our previous experience using the control ration [7, 20]. At Day 90 of gestation, a representative group of seven L ewes was switched to the C intake (L-C) on the basis of maternal live weight, body condition score, ovulation rate at embryo transfer, and maternal live weight change from transfer to Day 90 of gestation. The complete diet supplied 10.2 MJ metabolizable energy and 137 g/kg crude protein and was offered in two equal feeds at 0800 h and 1600 h daily. The diet contained 30% (w/w) coarsely milled hay, 50% barley, 10% molasses, 9% soyabean meal, 0.3% salt, 0.5% dicalcium phosphate, and 0.2% of a vitamin-mineral supplement and had an average dry matter content of 86%. The level of feed offered was reviewed three times weekly and adjusted on the basis of body weight change data (determined weekly) on an individual basis as and when appropriate. Maternal body condition was subjectively assessed on a five-point scale (1 = emaciated, 5 = obese) every 2 wk [21] by the same experienced assessor. Pregnancy status was determined by transabdominal ultrasonography at approximately Day 45 of gestation (gestation length = 145 days) and revealed viable fetuses in 14 and 21 ewes in the C and L groups, respectively.

Sample Collection and Analyses

Weekly blood samples were collected from Day 7 of gestation onward by jugular venepuncture ~3 h after the morning feed. A further blood sample was collected 1 h prior to necropsy on either Day 90 or 130 of gestation. Killing was achieved by intravenous administration of an overdose of sodium pentobarbitone (20 ml Euthesate; 200 mg pentobarbitone/ml; Willows Francis Veterinary, Crawley, UK) and exsanguination (by severing the main vessels of the neck). The gravid uterus was removed, weighed, and opened, and a fetal blood sample was collected by cardiac puncture immediately before intracardiac administration of sodium pentobarbitone (3 ml Euthesate) to kill the fetus. After clamping the cord, the fetus was removed, dried, and weighed. All major fetal organs were dissected and weighed. Selected organs, including the liver, were sampled and snap frozen in liquid nitrogen-chilled isopentone and stored at –80°C until analysis. Intact placentomes were dissected, and their number and weight were recorded.

The maternal perirenal fat was dissected and weighed. The omental and mesenteric fat was stripped from the gastrointestinal tract and, together with the perirenal fat weight, provided an index of the maternal internal fat depot. The maternal body was divided into carcass and noncarcass components, and the chemical composition of the carcass was determined after removing the pelt, head, and feet. The composition of the fetal carcass was determined after removing the head only. The carcasses were individually homogenized in a Wolfking (Slagelse, Denmark) mincer [22]. Three samples of each homogenate were then freeze dried, ground in a laboratory mincer, and subsampled for analyses. The residual moisture content of the carcass remaining after freeze drying was obtained by drying to constant weight at 100°C. The fat content was determined by the chloroform-methanol technique [23], and the nitrogen by the Kjeldahl method [24]. The ash content of the samples was determined by ashing at 600°C to constant weight.

To determine fetal liver glycogen stores, glycogen was hydrolysed to release glucose, and then glucose was measured using glucose oxidase with glucose as the reference standard [25].

Maternal and fetal plasma glucose concentrations at necropsy were determined in duplicate using a Yellow Springs Instruments dual biochemistry analyzer (model 2700; Yellow Springs, OH) as previously described [26]. Maternal and fetal insulin, insulin-like growth factor 1 (IGF1), and cortisol concentrations were measured in duplicate by radioimmunoassay as described previously [27–29]. The sensitivities of the assays were 4 µU insulin/ml, 6 pmol IGF1/ml, and 2 ng cortisol/ml. The concentration of each hormone was measured within a single assay, and the intraassay coefficients of variations were <10%. Maternal weekly blood samples were analyzed for glucose as above and for nonesterified fatty acids (NEFA) as previously described [30].

Data Analysis

Data are presented as means ± standard error of the mean (SEM). Differences between treatments for data collected weekly or at Day 90 or 130 of gestation were analyzed by a one-way ANOVA (General Linear Models, Minitab 13; Minitab Inc., State College, PA), and then the least significant difference was determined between individual treatment means. In specified instances, a similar approach was used to analyze weekly data collected throughout gestation after calculating a mean individual value spanning the first (from Day 4 to Day 49), second (from Day 50 to Day 90), and final third (Day 91 to Day 130) of gestation. Data collected weekly are presented with all animals in each necropsy group up to Day 90 of gestation, as there were no significant differences in any of the parameters measured between pregnancies terminated at Day 90 versus Day 130 within groups up to this point. Maternal endocrinology data collected at Day 90 or 130 of gestation from the C versus L groups were analyzed by two-way ANOVA to determine the main effects of nutrition and stage of gestation and the interaction between these factors.

RESULTS

Maternal Dietary Intake, Live Weight, External Adiposity Score, and Carcass Composition

Mean weekly dry matter intake (DMI) was determined for the first (Days 4–49), second (Days 50–90), and third (Days 91–130) periods of gestation for C, L, and L-C dietary intake groups (Fig. 1). DMI was reduced in the L versus the C group during all three periods of gestation (P < 0.001). During period three, DMI in the C group was increased to maintain maternal body condition and meet the increasing nutrient demands of the developing conceptus. Thus, during the third period of gestation, DMI in the L group was approximately 60% of the C group. Averaged throughout pregnancy, overall DMI in the L group was approximately 73% of the C group. The design of the study ensured that DMI in the L-C switchover group was equivalent to the L group during the first and second periods and equivalent to the C group during the third period. Consequently, DMI of the L-C group was lower (P < 0.001) than the C group during the first and second periods and greater (P < 0.001) than the L group during the third period.


Figure 01
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  		FIG. 1. Average weekly DMIs during the first (Days 4–49), second (Days 50–90) and third (Days 91–130) periods of gestation in C (black bars), L (white bars), and L-C (hatched bars) intake adolescent dams (values are means ± SEM, n = 7 per group; different letters for each group within each period represent statistical significance at P < 0.001).

Weekly changes in maternal live weight are presented in Figure 2. Maternal live weight remained equivalent (P > 0.05) among all groups during the first third of gestation (Days 4–49). Thereafter, live weight in the C group diverged and was greater (P < 0.05) than the L group for the remainder of the study. After switching to the C intake at Day 90 of gestation, maternal live weight in the L-C group diverged from that in the L-only group, reached statistical significance by Day 105 (P < 0.02), and remained significantly greater until Day 130 of gestation. The increase in live weight in the L-C group reflected an increase in DMI and paralleled both DMI and live weight in the C group.


Figure 02
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  		FIG. 2. Weekly maternal live weight changes throughout gestation in C (white triangle), L (black square), and L-C (white square) intake adolescent dams (values are means ± SEM, n = 7 per group).

Biweekly changes in maternal adiposity, as indicated by external body condition score, are presented in Figure 3. Maternal adiposity was maintained from embryo transfer (Day 4) to necropsy (Days 90–130) in the C group. At Day 35 of gestation, adiposity was lower (P < 0.05) in the L versus the C group, and it continued to decrease as gestation advanced. Adiposity in the L-C group was equivalent to that in the L group up to the switch in DMI at Day 90 of gestation. Thereafter, adiposity in the L-C group was maintained and became significantly greater (P < 0.05) from Days 119 to 130 of gestation compared with the L group. Despite the maintenance of maternal adiposity in the L-C group, it remained considerably lower (P < 0.001) than the C group for the duration of the study.


Figure 03
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  		FIG. 3. Biweekly maternal body condition score changes throughout gestation in C (white triangle), L (black square), and L-C (white square) intake adolescent dams (values are means ± SEM, n = 7 per group).

At Day 90 of gestation, maternal carcass weight was higher in C versus L dams (23.4 ± 0.48 kg and 20.5 ± 0.71 kg, respectively; P < 0.005), but the absolute weight of the internal fat depots (omental, mesenteric, and perirenal fat) were not significantly different (C: 2.4 ± 0.22 kg vs. L: 2.2 ± 0.18 kg). By Day 130 of gestation, the difference in maternal carcass weights between the C and L groups was even more pronounced (Table 1), whereas carcass mass of the L-C group was intermediate and different from both the C (P < 0.05) and the L (P < 0.001) groups. As presented in Table 1, chemical analyses of the maternal carcass revealed absolute carcass dry matter (DM) content followed a similar pattern to carcass weight, but were not different between dietary groups when expressed as a percentage of carcass weight. Maternal nutrient restriction throughout pregnancy (L group) decreased absolute fat (P < 0.02), crude protein (CP; P < 0.001), and ash (P < 0.04) contents of the carcass compared with the C group. Similarly, the absolute weight of the internal fat depots was reduced (P < 0.05) in L compared with C groups. Absolute ash (P < 0.04) and CP (P < 0.001) contents were higher in L-C than in L groups, whereas weight of the internal fat depot was intermediate and statistically similar to the C and L groups. When expressed as a percentage relative to maternal carcass weight, none of the chemical parameters analyzed were significantly affected by maternal diet, and thus further investigation into maternal carcass composition at Day 90 of gestation was deemed unnecessary.


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  		TABLE 1. Maternal carcass composition and internal fat depots at Day 130 of gestation in control (C), low (L) and low-control (L-C) dietary intake groups (values are means ± SEM, n = 7 per group).

Pregnancy Outcome at Necropsy

At necropsy on Day 90 of gestation, mean fetal (C: 657 ± 30.0 g vs. L: 597 ± 42.7 g) and placental (C: 665 ± 75.6 g vs. L: 607 ± 28.0 g) weights were unaffected by maternal intake. Individual fetal organ weights were similarly unperturbed (data not shown). In contrast, by Day 130 of gestation, maternal nutrient restriction (L group) resulted in a significant reduction (P < 0.01) in fetal weight (–17%) compared with the C group (Table 2). This reduction in body weight in L fetuses was independent of placental mass and was associated with a statistically (P < 0.05 or less) smaller mean biparietal head diameter and crown-rump length, as well as lower absolute liver, kidney, lung, and spleen weights compared with normally growing C fetuses. In contrast, growth of the brain was preserved in L fetuses, resulting in both a higher body weight-specific brain weight and a higher brain:liver weight ratio compared with the control group. At Day 130, fetal body weight in the L-C dietary switchover group was intermediate between the C and L groups but was only statistically different (P < 0.05) from the C group. With respect to the other parameters measured, mean head diameter and crown-rump length for the L-C fetuses were most similar to the L fetuses, whereas liver and lung weights and brain:liver weight ratio were most similar to the C fetuses.


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  		TABLE 2. Placental and fetal parameters on Day 130 of gestation in control (C), low (L), and low-control (L-C) dietary intake groups (values are means ± SEM, n = 7 per group).

Fetal carcass composition at Day 130 of gestation is presented in Table 3. Maternal nutrient restriction during the initial 90 days of gestation only (L-C group) or all of gestation (L group) reduced absolute fetal perirenal fat mass relative to the C group (P < 0.04 and P < 0.002, respectively). Fetal carcass weight followed a similar pattern to fetal weight and was decreased in L (P < 0.005) and L-C (P < 0.05) groups compared with the C group. Absolute fetal carcass CP content was attenuated (P < 0.03) as a result of maternal nutrient restriction to Day 130 of gestation (L group) compared with the C group. In addition, when compared to the C (P < 0.005) and L-C (P < 0.05) groups, the L group had lower absolute carcass fat content. When these parameters were expressed relative to fetal carcass weight, fetal perirenal fat was reduced (P < 0.04) and fetal ash content was increased (P < 0.01) in the L versus the C group (Table 3). In contrast, relative fetal carcass fat and carcass CP were not significantly altered by maternal diet.


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  		TABLE 3. Fetal carcass composition on Day 130 of gestation in control (C), low (L), and low-control (L-C) dietary intake groups (values are means ± SEM, n = 7 per group).

Maternal and Fetal Endocrine and Metabolic Status

Maternal endocrine and metabolic status at necropsy on Day 90 or 130 of gestation in C and L group dams is detailed in Table 4. Nutrient-restricted dams (L group) were in a relatively catabolic state, as indicated by higher (P < 0.001) NEFA concentrations compared with the C group at both gestational timepoints. NEFA concentrations remained elevated as gestation advanced in the L group, but they declined between Days 90 and 130 of gestation in the C group (Nutrition x Day; P < 0.001). At Day 130 of gestation, NEFA concentrations in the L-C group (0.09 ± 0.011 mmol/ml) were lower than in the L (P < 0.001) and C (P < 0.04) groups. Analyses of weekly maternal blood samples (Fig. 4) further revealed that NEFA concentrations were similar between groups throughout the first period of gestation (Weeks 1–7) and increased in the nutrient-restricted group (L: P < 0.001; L-C: P< 0.04) versus the control group during the second period of gestation (Weeks 8–13). In contrast, maternal glucose concentrations did not diverge between groups until the final period of gestation (Weeks 14–19; Fig. 5). At necropsy, maternal glucose concentrations were lower (P < 0.001) in L versus C dams, but this difference was most pronounced at Day 130 of gestation, which resulted in a Nutrition x Day interaction (P < 0.06; Table 4). Maternal glucose concentrations in the L-C dams (3.43 ± 0.205 µmol/ml) at Day 130 of gestation were greater (P < 0.01) than those in the L group and were similar to those in the C group. Maternal insulin concentrations were attenuated (P < 0.001) in the L versus C dams at both stages of gestation. At Day 130 of gestation, maternal insulin (16.7 ± 1.40 IU/ml) in the L-C group was greater (P < 0.008) than it was in the L group and was similar to the C group. Maternal IGF1 concentrations were higher (P < 0.001) in the C group compared with the L group. As gestation advanced, IGF1 concentrations increased in the C group but declined in the L group (Nutrition x Day; P < 0.04). At Day 130 of gestation, maternal IGF1 concentrations in the L-C group (49.2 ± 4.25 pmol/ml) were similar to those in the C group and greater (P < 0.008) than those in the L group. At Day 130 of gestation, maternal cortisol concentrations were similar among C (33.7 ± 6.19 ng/ml), L (31.1 ± 2.87 ng/ml), and L-C (29.5 ± 5.47 ng/ml) dietary groups.


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  		TABLE 4. Maternal metabolic and endocrine status at Day 90 or 130 of gestation in control (C) and low (L) dietary intake groups (values are means ± SEM, n = 7 per group).


Figure 04
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  		FIG. 4. Weekly maternal plasma NEFA concentrations throughout gestation in C (white triangle), L (black square), and L-C (white square) intake adolescent dams (values are means ± SEM, n = 7 per group).


Figure 05
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  		FIG. 5. Average maternal plasma glucose concentrations during the first (Weeks 1–7), second (Weeks 8–13), and third (Weeks 14–19) periods of gestation in C (black bars), L (white bars), and L-C (hatched bars) intake adolescent dams (values are means ± SEM, n = 7 per group; different letters for each group within each period represent statistical significance at P < 0.01).

At necropsy on Day 90 of gestation, fetal glucose (C: 0.49 ± 0.045 µmol/ml; L: 0.47 ± 0.067 µmol/ml), insulin (C: 10.6 ± 0.54 IU/ml; L: 10.6 ± 0.48 IU/ml), IGF1 (C: 21.9 ± 0.89 pmol/ml; L: 23.8 ± 2.66 pmol/ml), and liver glycogen (C: 12.6 ± 0.87 mg/g; L: 14.9 ± 0.98 mg/g) concentrations were not influenced by maternal diet. Fetal metabolic and endocrine status at necropsy on Day 130 of gestation is detailed in Table 5. Fetal glucose was lower (P < 0.03) in L versus C group fetuses at this stage. Fetal glucose concentrations in the L-C switchover group were intermediate, but were statistically similar to the other groups. The highly significant decrease in maternal glucose concentrations in the L group during late gestation resulted in a lower transplacental glucose concentration gradient (i.e., maternal minus fetal glucose concentration) compared with C (P < 0.08) and L-C (P < 0.03) groups at this stage. Fetal insulin concentrations followed a similar pattern; L fetuses had lower concentrations relative to C (P < 0.07) and L-C (P < 0.06) groups. In contrast, fetal IGF1 concentrations were similar in C and L groups but were markedly higher (P < 0.01) in the dietary switchover group. Although fetal liver glycogen concentrations were not affected by maternal diet, total fetal liver glycogen stores were greater in C (P < 0.04) and L-C (P < 0.02) groups relative to the undernourished L group. Fetal cortisol was independent of maternal nutrition at the Day 130 timepoint.


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  		TABLE 5. Fetal metabolic/endocrine status at Day 130 of gestation in control (C), low (L), and low-control (L-C) dietary intake groups (values are means ± SEM, n = 7 per group).

DISCUSSION

This study demonstrates that limiting maternal intake in young, singleton-bearing adolescent sheep results in a gradual depletion of maternal nutrient reserves and a slowing of fetal growth by late gestation. In direct contrast to the overnourished paradigm described in the Introduction, this alteration in the fetal growth trajectory was independent of changes in placental mass per se. Furthermore, the progressive reduction in maternal anabolic hormone concentrations and circulating glucose levels, as well as the correspondingly low fetal insulin and glucose concentrations, suggests that the slowing of fetal growth observed in the underfed dams is largely due to reduced nutrient availability in the maternal circulation. Support for this concept comes from the observation that when undernourished dams were offered a dietary level equivalent to the control group from Days 90–130 of gestation, mean fetal weight was intermediate between the control (–9%) and low-intake (+8%) group values. Thus, while underfeeding the adolescent dam during the first two thirds of gestation had clearly had an impact on the growth of these fetuses, similarities with respect to fetal insulin and glucose and the higher fetal IGF1 concentrations in the L-C versus C group imply that compensatory fetal growth following maternal refeeding may have completely restored fetal body mass in the switchover group had the pregnancies been allowed to progress to term.

Although the adolescent dams were already relatively catabolic by Day 90 of gestation (as indicated by low anabolic hormone concentrations, elevated NEFA levels, and reduced external adiposity scores), maternal and fetal glucose concentrations and fetal mass were not significantly perturbed at this stage of pregnancy. This is, perhaps, unsurprising, as the fetus has only achieved 12% of its birth weight at this time and absolute glucose requirements of the conceptus as a whole are low, and are only one sixth of those required for normal growth during late gestation [31]. This is the first study in young adolescent animals, but our observations at Day 90 contrast with an earlier study in adult sheep [32]. In the latter study, ewes were nutrient restricted from Days 28 to 78 of gestation and were immediately necropsied. Fetal weight was significantly reduced (twins and singles) and was associated with significant reductions in maternal (–12%) and fetal (–23%) glucose concentrations. These contrasting effects on fetal nutrient supply and growth at midpregnancy can largely be explained by the differences in the degree of nutrient restriction applied. In the adult study, the ewes lost 7.5% of their initial body weight by Day 78 of gestation and were 15% lighter than the corresponding control group [32]. In contrast, in the adolescent study, maternal weight at conception was maintained, and the restricted ewes were only 8% lighter than the control group at Day 90 of gestation. In designing this study we were cognizant of the fact that although nutrient intakes, and hence pregnancy weight gains are likely to be inadequate during human adolescent pregnancy, these young mothers are unlikely to actively lose weight while pregnant, and hence prepregnancy weights will largely be maintained [16–19].

By late gestation (Day 130), the adolescent dams who were continuously undernourished by maintaining their weight at conception were severely catabolic, and circulating maternal glucose concentrations were significantly reduced (–17% relative to controls). Glucose is the primary fetal fuel, and transport to the fetus is dependent on the maternal-fetal transplacental glucose concentration gradient and the number and activity of the placental glucose transporters [33–35]. While the latter were not measured in the current study, the major decrease in maternal glucose concentrations did decrease the transplacental glucose gradient, and hence glucose supply, to the fetus at a time when absolute requirements for body growth are at their highest. In adult sheep there are few comparable studies in which nutrient restriction was applied over a similarly prolonged period of pregnancy. Nevertheless, collectively, the available data for nutrient restriction from midpregnancy to late pregnancy, together with this adolescent study, suggest that in the absence of placental growth restriction, maternal glucose levels must fall below a threshold of ~3.0 µmol/ml before growth of the fetus is significantly perturbed [36, 37].

Analyses of maternal body composition at Day 130 of gestation revealed proportionate decreases in internal fat depots and carcass fat, protein, and ash content in the low-intake, and hence growth-constrained, adolescent dams, and are consistent with the differences in maternal endocrine and metabolic indicators reported. As pregnancy progressed, maternal adipose tissue was gradually depleted and mobilized to supply NEFA as an alternative energy source for maternal metabolism [38]. Carcass fat content in the dietary switchover dams (L-C) was intermediate, yet statistically similar to, the moderate intake control and nutrient-restricted dams during late gestation. A larger-scale study with more animals per treatment group may have revealed whether or not the intermediate carcass fat content of these dams was significantly different from the undernourished or control dams, respectively. Reduced glucose utilization by maternal skeletal muscle in response to attenuated insulin secretion prevented maternal protein accretion and led to a reduced total carcass CP content [39]. The decreased carcass ash content represents slower skeletal growth in the undernourished adolescent dam and is commensurate with the reduction in maternal plasma IGF1 concentrations [40]. The body composition data in the current study contrast markedly with our overnourished model, in which high nutrient intakes promote rapid maternal growth and a disproportionate or relative increase in both internal and carcass fat content [41]. The latter paradigm is characterized by high maternal insulin, IGF-1, and glucose concentrations while NEFA remains low [42, 43] and, as such, is in direct contrast to the current study.

The modest fetal growth restriction observed in low-intake adolescents at Day 130 in the present study (–17% relative to controls) is less than that previously reported for overnourished adolescents at the equivalent stage of pregnancy in three individual studies (–28% to –37% relative to controls [11, 44, 45]). Late-gestation fetuses from both the undernourished and overnourished dams exhibit relative preservation of brain growth, and hence an elevated brain:liver weight ratio as shown above and by Wallace et al. [11]. In both sheep and humans, this asymmetric growth restriction is indicative of a limited nutrient supply in utero [13, 46]. However, comparison of body composition in fetuses from these contrasting models indicates that they exhibit a subtle difference in phenotype. Fetuses from undernourished adolescent dams had lower absolute carcass fat and protein contents and reduced perirenal adipose tissue mass indicative of a "thin" phenotype. These fetuses also had higher weight-specific ash levels (an index of bone mass), and hence preserved skeletal growth relative to the reduction in fat and muscle mass. In contrast, growth-restricted fetuses from overnourished dams have a higher fetal weight-specific perirenal fat mass and carcass fat content than contemporaneous controls, indicative of a "fat" phenotype [45]. These differences in body composition may have implications beyond the fetal period with regard to both neonatal survival [47] and longer-term growth and metabolism [48].

The pregnancies in this initial nutrient restriction study were not allowed to progress to term. However, in marked contrast to the overnourished paradigm [7], we have recently shown that modest fetal growth restriction in undernourished adolescents is not associated with either spontaneous miscarriage or premature delivery. Gestation length was equivalent in control and undernourished dams (147.3 and 147.5 days) and was, on average, 4 days longer than in overnourished dams (143.0 days; P < 0.001; n = 18 to 22 pregnancies per group; Wallace et al, unpublished data). These results are perhaps surprising in view of reports of extreme premature delivery in adult ewes that were nutrient restricted during the periconceptual period [49, 50]. Premature deliveries in these latter pregnancies have been attributed to accelerated maturation of the fetal hypothalamic-pituitary-adrenal axis in late pregnancy [51]; however, neither fetal nor maternal cortisol concentrations were altered by maternal nutrition in the present study.

The results of this nutrient restriction study, together with our earlier work in overnourished adolescents [7, 10], demonstrate that when gynecological age and maternal weight and adiposity at conception are controlled, dietary intake at both extremes of the nutritional scale is a powerful regulator of fetal growth in young adolescent sheep. This study further suggests that targeted nutritional supplementation may benefit fetal growth in undernourished adolescents. Although a strong relationship between inadequate gestational weight gains and reduced infant birth weight has already been established [16–19], there is also good evidence that competition for nutrients between the maternal and fetal compartments will impair nutrient supply in girls who are still growing during pregnancy [4]. Thus, formulating correct dietary advice for these young adolescent girls is likely to be complex, particularly if the mother has not completed her own growth. Our results suggest that biomarkers of growth and nutritional status at the time of conception and at midpregnancy, as well as the use of ultrasound to detect whether placental growth has been perturbed, may prove beneficial in the optimal management of adolescent pregnancies.

ACKNOWLEDGMENTS

We thank Terry Atkinson, Christine Horrocks, and Maureen Annand for technical assistance and Graham Horgan (Bioss) for statistical advice.

FOOTNOTES

1Supported by the Scottish Executive Environment and Rural Affairs Department and the National Institutes of Health. Back

Correspondence: 2FAX: 44 1224 716686; e-mail: Jacqueline.Wallace{at}rowett.ac.uk

Received: 11 March 2007.

First decision: 7 April 2007.

Accepted: 27 April 2007.

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J. Luther, J. Milne, R. Aitken, M. Matsuzaki, L. Reynolds, D. Redmer, and J. Wallace
Placental Growth, Angiogenic Gene Expression, and Vascular Development in Undernourished Adolescent Sheep
Biol Reprod, August 1, 2007; 77(2): 351 - 357.
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