John S. Milne

Nutritionally Mediated Placental Growth Restriction in the Growing Adolescent: Consequences for the Fetus

Abstract Human adolescent pregnancy is characterized by poor pregnancy outcome; the risks of spontaneous miscarriage, prematurity, and low birth weight are particularly acute in girls who are still growing at the time of conception. Studies using a highly controlled sheep paradigm demonstrate that, in growing adolescents who are overnourished throughout pregnancy, growth of the placenta is impaired, resulting in a decrease in lamb birth weight relative to control-fed adolescents of equivalent age. Rapid maternal growth is also associated with increased spontaneous abortion rates in late gestation and a reduction in gestation length. Nutritionally sensitive hormones of the maternal somatotrophic axis may orchestrate nutrient partitioning in this paradigm and the particular role of growth hormone is discussed. At midgestation, the placentae of rapidly growing dams exhibit less proliferation in the fetal trophectoderm and reduced placental mRNA expression of a range of angiogenic factors. These changes occur before differences in placental size are apparent but may impact on subsequent vascularity. By late pregnancy, placental mass in the rapidly growing versus the control dams is reduced by approximately 45%; the fetuses display asymmetric growth restriction and are hypoxic and hypoglycemic. These growth-restricted pregnancies are associated with major reductions in absolute uterine and umbilical blood flows, leading to attenuated fetal oxygen, glucose, and amino acid uptakes. Placental glucose transport capacity is markedly reduced in the rapidly growing dams but is normal when expressed on a weight-specific placental basis. Thus, it is the small size of the placenta per se rather than alterations in its nutrient metabolism or transfer capacity that is the major limitation to fetal growth in the growing adolescent sheep. Information obtained from this highly controlled paradigm is clearly relevant to the clinical management of human adolescent pregnancies. In addition, the paradigm provides a robust model of placental growth restriction that replicates many of the key features of human intrauterine growth restriction per se.

Influence of Maternal Nutrition on Messenger RNA Expression of Placental Angiogenic Factors and Their Receptors at Midgestation in Adolescent Sheep

Abstract Previous studies have shown that placental growth and pregnancy outcome are severely compromised in adolescent ewes overnourished to promote rapid maternal growth. Using this paradigm, the aim of the present study was to investigate expression of the major angiogenic factors and their receptors in the placenta at the onset of the most rapid phase of fetal growth. Singleton pregnancies to a single sire were established by embryo transfer, and thereafter, adolescent dams were offered a high or moderate nutrient intake predicted to induce compromised or normal fetoplacental size at term, respectively. Ovine-specific oligonucleotide probe and primer sets for several angiogenic factors and their receptors were developed for quantitative real-time reverse transcription-polymerase chain reaction determination of placentome mRNA expression at Day 81 of gestation. Total placentome weight and fetal weight were equivalent in high- compared with moderate-intake groups at this stage of gestation. Placentome expression of the angiogenic factors, vascular endothelial growth factor, angiopoietins 1 and 2, and nitric oxide synthase 3, were reduced in overfed ewes. Similarly, level of expression of vascular endothelial growth factor/vascular permeability factor receptor (FLT1) was less in overfed ewes. Thus, in the adolescent, maternal overnutrition has a negative impact on midgestation placental angiogenic factor/ receptor expression. This may impact placental vascularity and explain why uteroplacental mass, blood flow, and nutrient uptake are compromised in late pregnancy, resulting in low-birth-weight offspring.

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

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.

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

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 × 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.

The effect of overnourishing singleton-bearing adult ewes on nutrient partitioning to the gravid uterus.

Overnourishing the singleton-bearing adolescent sheep throughout pregnancy promotes maternal tissue synthesis at the expense of the nutrient requirements of the gravid uterus. Consequently, the growth of the placenta is impaired and results in the premature delivery of low-birth-weight lambs relative to moderately fed adolescents of equivalent age. To establish if this phenomenon is unique to the growing animal, singleton pregnancies to a single sire were established by embryo transfer into primiparous adult ewes who had attained the normal mature body size for their genotype. Thereafter ewes were offered a maintenance or a high level of a complete diet throughout gestation. High maternal intakes resulted in elevated maternal insulin, no significant change in growth hormone or glucose, and attenuated progesterone and NEFA concentrations. Live weight gain during the first 93 d of gestation was 48 and 244 g/d, and adiposity score at term was 2.4 and 3.7 in the maintenance and high groups, respectively (P<0.001). In spite of achieving levels of adiposity similar to overnourished adolescents, placental (477 (sem 30) v. 518 (sem 41) g) and fetal (5190 (sem 320) v. 5420 (sem 250) g) weights were equivalent in maintenance and high groups. Gestation length was shorter (P<0.01) and colostrum yield at parturition lower (P<0.05) in high v. maintenance dams. Thus, adult sheep appear to be relatively insensitive to the oversupply of nutrients during pregnancy and have the ability to meet the nutrient requirements for normal conceptus growth in spite of their increased adiposity.

Fetoplacental growth and vascular development in overnourished adolescent sheep at day 50, 90 and 130 of gestation

To establish the basis for altered placental development and function previously observed at late gestation, fetoplacental growth and placental vascular development were measured at three stages of gestation in a nutritional paradigm of compromised pregnancy. Singleton pregnancies to a single sire were established and thereafter adolescent ewes were offered an optimal control (C) or a high (H) dietary intake. At day 50, the H group had elevated maternal insulin and amniotic glucose, whereas mass of the fetus and placenta were unaltered. At day 90, the H group exhibited elevated maternal insulin, IGF1 and glucose; fetal weight and glucose concentrations in H were increased relative to C, but placental weight was independent of nutrition. By day 130, total placentome weight in the H group was reduced by 46% and was associated with lower fetal glucose and a 20% reduction in fetal weight. As pregnancy progressed from day 50 to 130, the parameters of vascular development in the maternal and fetal components of the placenta increased. In the fetal cotyledon, high dietary intakes were associated with impaired vascular development at day 50 and an increase in capillary number at day 90. At day 130, all vascular indices were independent of nutrition. Thus, high dietary intakes to promote rapid maternal growth influence capillary development in the fetal portion of the placenta during early to mid-pregnancy and may underlie the subsequent reduction in placental mass and hence fetal nutrient supply observed during the final third of gestation.

Maternal Growth Hormone Treatment from Day 35 to 80 of Gestation Alters Nutrient Partitioning in Favor of Uteroplacental Growth in the Overnourished Adolescent Sheep

Abstract Overnourishing the pregnant adolescent ewe promotes maternal tissue synthesis at the expense of placental growth and leads to a major reduction in lamb birth weight at term. Growth hormone (GH) secretion is attenuated in these overnourished dams and the maternal somatotrophic axis may play a key role in coordinating nutrient usage in the pregnant adolescent. Thus we investigated whether increasing maternal GH during the period of rapid placental proliferation alters nutrient partitioning between the maternal, placental, and fetal tissues as assessed at Day 81 of gestation. Adolescent recipient ewes were implanted with singleton embryos, derived from superovulated dams and a single sire on Day 4 postestrus. Thereafter, the ewes were offered either a high (H) or moderate intake (M) of the same complete diet. From Day 35 to 80 of gestation, ewes were either injected twice daily (s.c. at 0800 and 1800 h) with recombinant bovine GH (bGH, 0.14 mg/kg live weight/day) or remained untreated (n = 8 ewes per group). Maternal concentrations of GH, insulin, insulin-like growth factor (IGF-1), glucose, and non-esterified fatty acids (NEFAs) were higher, and leptin secretion lower, in bGH-treated dams from both nutritional groups. Maternal body weight gain was higher in H versus M groups and was independent of bGH treatment. Treatment with bGH reduced relative perirenal and carcass fat deposition and increased carcass protein content in both H and M dams. Uteroplacental mass (uterus + placentomes + fetal membranes) averaged 1099, 1069, 1112, and 1754 g in M, H, M+GH, and H+GH groups. This significant increase in uteroplacental development in the H+GH group was associated with higher fetal kidney and liver weights and elevated fetal insulin, glucose, and lactate concentrations. Treatment with bGH also induced polyhydramnios in the H group. The transplacental glucose gradient was increased twofold in the H+GH group but placental GLUT- 1 and GLUT-3 expression was unaffected. In conclusion, administration of GH during the period of rapid placental proliferation alters endocrine status and thus nutrient partitioning in the overnourished adolescent dam in favor of uteroplacental and fetal growth. It remains to be established whether these effects are due wholly to alterations in maternal metabolism or if they also reflect an effect of bGH and/or the IGF system at the level of the uteroplacenta.

Serial Measurement of Uterine Blood Flow From Mid to Late Gestation in Growth Restricted Pregnancies Induced by Overnourishing Adolescent Sheep Dams

Uterine blood flow (UtBF) is a major regulator of transplacental fetal nutrient supply. The aim was to serially measure uterine blood flow from mid to late pregnancy in a paradigm of relatively late onset placental and fetal growth restriction. Singleton bearing adolescent dams was fed high (H) or control (C) nutrient intakes to induce putatively compromised or normal pregnancies, respectively. A perivascular flow probe was attached to the uterine artery of the gravid horn on Day 83 of gestation and UtBF was then recorded continuously for 2h, three times weekly until approximately Day 135, when pregnancies were either terminated or ewes allowed to deliver at term (approximately Day 145). Pregnancy outcome was determined at term in contemporaneous ewes without UtBF assessment. Placental and fetal weights were lower (P<0.001) in H compared with C intake groups and were independent of flow probe surgery and monitoring. Uterine blood flow was lower in H compared with C groups at the first assessment (Day 88, P<0.001) and was positively correlated with adjusted fetal weight at term, irrespective of treatment group (P<0.01). UtBF increased throughout the second half of gestation in both groups. Linear regression analysis of UtBF against day of gestation revealed that the slope was equivalent (5.5 vs. 5.3ml/min per day) and the mean intercept lower (212 vs. 370ml/min, P<0.001) in H compared with C groups, respectively. This study demonstrates the feasibility of serially measuring UtBF within the same individual sheep for a protracted period during the second half of gestation. UtBF was already lower at mid gestation in putatively growth restricted compared with control pregnancies, ahead of any reduction in placental and fetal weight, but increased similarly during the second half of gestation in both groups. These data are commensurate with the reported decrease in placental angiogenic growth factor expression at mid gestation, and, indicate that attenuated UtBF is an early defect in this adolescent paradigm.

Effect of Weight and Adiposity at Conception and Wide Variations in Gestational Dietary Intake on Pregnancy Outcome and Early Postnatal Performance in Young Adolescent Sheep

Abstract Nutritional backgrounds prior to pregnancy may interact with subsequent gestational intake to influence pregnancy outcome, particularly in young, growing adolescents. To investigate this interaction, singleton pregnancies were established in two groups of adolescent sheep of identical age but different initial weight and adiposity score, classified as good (G) and poor (P) body mass index (BMI). Thereafter, ewes were offered either an optimal control (C) intake to maintain adiposity throughout pregnancy, undernourished (UN) to maintain weight at conception but deplete maternal body reserves, or overnourished (ON) to promote rapid maternal growth and adiposity, resulting in a 2 × 3 factorial design. Gestation length was independent of BMI and reduced in ON dams. Average placental and lamb birth weights were influenced by initial BMI (G > P) and gestational intake (C > UN > ON), with the highest incidence of growth restriction in ON groups. Metabolic challenges at two thirds of gestation revealed enhanced insulin insensitivity in ON dams (higher glucose postinsulin challenge and higher insulin postglucose challenge), but nevertheless fetal growth was constrained. Initial colostrum yield, total IgG, and nutrient supply were reduced in ON groups, but these low-birth-weight lambs exhibited rapid catch-up growth to weaning. Thus, both maternal BMI at conception and gestational intake have a profound influence on pregnancy outcome in young, putatively growing adolescent sheep and may have implications for the nutritional management of pregnant adolescent humans.

Bone mass and metabolism in women aged 45–55

OBJECTIVE Changes in calcium homeostasis and bone mass around the climacteric are poorly understood. We examined relations between endocrine factors and indices of bone mass and metabolism in healthy women approaching the menopause.