L. J. Edwards

Impact of Maternal Undernutrition During the Periconceptional Period, Fetal Number, and Fetal Sex on the Development of the Hypothalamo-Pituitary Adrenal Axis in Sheep During Late Gestation

Abstract Evidence from epidemiologic, clinical, and experimental studies has shown that a suboptimal intrauterine environment during early pregnancy can alter fetal growth and gestation length and is associated with an increased prevalence of adult hypertension and cardiovascular disease. It has been postulated that maternal nutrient restriction may act to reprogram the development of the pituitary-adrenal axis, resulting in excess glucocorticoid exposure and adverse health outcomes in later life. It is unknown, however, whether maternal nutrient restriction during the periconceptional period alters the development of the fetal pituitary-adrenal axis or whether the effects of periconceptional undernutrition can be reversed by the provision of an adequate level of maternal nutrition throughout the remainder of pregnancy. We have investigated the effect of restricted periconceptional nutrition (70% of control feed allowance) from 60 days before until 7 days after mating and the effect of restricted gestational nutrition from Day 8 to 147 of gestation on the development of the fetal hypothalamo-pituitary adrenal (HPA) axis in the sheep. In these studies, we have also investigated the effects of fetal number and sex on the pituitary-adrenal responses to periconceptional and gestational undernutrition. In ewes maintained on a control diet throughout the periconceptional and gestational periods, fetal plasma ACTH concentrations were higher and the prepartum surge in cortisol occurred earlier in singletons compared with twins. Plasma ACTH concentrations were also significantly higher in male compared with female singletons, and in twin fetuses, the prepartum surge in cortisol concentrations occurred earlier in males than in females. Periconceptional undernutrition resulted in higher fetal plasma concentrations of ACTH between 110 and 145 days of gestation and a significantly greater cortisol response to a bolus dose of corticotropin-releasing hormone in twin, but not singleton, fetuses in late gestation. We have therefore demonstrated that fetal number and sex each has an impact on the timing of the prepartum activation of the HPA axis in the sheep. Restriction of the level of maternal nutrition before and in the first week of a twin pregnancy results in stimulation of the fetal pituitary-adrenal axis in late gestation, and this effect is not reversed by the provision of a maintenance control diet from the second week of pregnancy.

Restriction of placental and fetal growth in sheep alters fetal blood pressure responses to angiotensin II and captopril

1 We have measured arterial blood pressure between 115 and 145 days gestation in normally grown fetal sheep (control group; n= 16) and in fetal sheep in which growth was restricted by experimental restriction of placental growth and development (PR group; n= 13). There was no significant difference in the mean gestational arterial blood pressure between the PR (42.7 ± 2.6 mmHg) and control groups (37.7 ± 2.3 mmHg). Mean arterial blood pressure and arterial P  O 2 were significantly correlated in control animals (r= 0.53, P < 0.05, n= 16), but not in the PR group. 2 There were no changes in mean arterial blood pressure in either the PR or control groups in response to captopril (7.5 μg captopril min−1; PR group n= 7, control group n= 6) between 115 and 125 days gestation. After 135 days gestation, there was a significant decrease (P < 0.05) in the fetal arterial blood pressure in the PR group but not in the control group during the captopril infusion (15 μg captopril min−1; PR group n= 7, control group n= 6). 3 There was a significant effect (F= 14.75; P < 0.001) of increasing doses of angiotensin II on fetal diastolic blood pressure in the PR and control groups. The effects of angiotensin II were different (F= 8.67; P < 0.05) in the PR and control groups at both gestational age ranges. 4 These data indicate that arterial blood pressure may be maintained by different mechanisms in growth restricted fetuses and normally grown counterparts and suggests a role for the fetal renin‐angiotensin system in the maintenance of blood pressure in growth restricted fetuses.

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

We have investigated the effect of a 50% reduction in maternal nutrient intake during the last 30 days of pregnancy on arterial blood pressure and on arterial blood pressure responses to angiotensin II (AII) and the angiotensin converting enzyme (ACE) inhibitor captopril in the sheep fetus at 115–125 and at 135–145 days gestation (term = 147 ± 3 days gestation). Fetal plasma glucose concentrations were lower in the undernourished (UN) group compared to the control animals. There was no difference, however, in fetal plasma cortisol or adrenocorticotrophic hormone (ACTH) concentrations between the UN and control groups between 115 and 145 days gestation. During the first 10 days of undernutrition, maternal plasma concentrations of cortisol were increased in the UN group compared to controls. At 115–125 days gestation, fetal arterial blood pressure was also higher in the UN group compared with controls and there was an inverse relationship (r=−0.62, P < 0.05) between mean arterial pressure and the fetal plasma concentrations of ACTH in the UN group. Fetal blood pressure responses to increasing doses of angiotensin II were also higher (P < 0.05) in UN compared to control animals at 115–125 days gestation. Between 135 and 145 days gestation, fetal arterial blood pressure was increased in UN fetal sheep and mean arterial blood pressure was correlated with fetal plasma concentrations of cortisol. Increased arterial blood pressure and responsiveness to AII measured in the fetuses of nutrient‐restricted ewes may be related in part to fetal exposure to the actions of cortisol derived from transplacental transfer during the first 10 days after the start of the restricted feeding regime.

Prenatal Undernutrition, Glucocorticoids And The Programming Of Adult Hypertension

1. A range of epidemiological studies has shown that poor intra‐uterine growth is associated with an increased prevalence of cardiovascular disease, non‐insulin‐dependent diabetes mellitus and the Metabolic syndrome in adult life.

Effect of culturing mouse embryos under different oxygen concentrations on subsequent fetal and placental development.

The oxygen concentration used during embryo culture can influence embryo development and quality. Reducing the oxygen concentration in the atmosphere to 2% during post‐compaction culture of mouse embryos perturbs embryonic gene expression. This study examined the effect of culturing mouse embryos under different oxygen concentrations on subsequent fetal and placental development. Embryos were cultured from the zygote to morula stage under 7% oxygen, followed by 20, 7 or 2% oxygen to the blastocyst stage. Cultured and in vivo developed blastocysts were transferred into pseudopregnant recipients. Fetal and placental outcomes were analysed at day 18 of pregnancy. Implantation rate was not influenced by embryo culture conditions, but resorption rates were increased in embryos cultured under 2% oxygen, compared with 7% oxygen. Day 18 fetal weights were reduced following culture under 2%, compared with 7 or 20% oxygen, or in vivo development. Placental weight was not influenced by culture conditions. No differences in the proportion of junctional or labyrinthine exchange regions within the placenta or the morphometry of the labyrinthine region were detected. Surface density (surface area/gram labyrinth) of trophoblast available for exchange was reduced in placentas developed from embryos cultured under 2% oxygen, compared with 7% oxygen. Placental gene expression of Slc2a1, Slc2a3, Igf2, Igf2r and H19 was not influenced by oxygen conditions during embryo culture. Thus, exposure to 2% oxygen during post‐compaction pre‐implantation embryo development has adverse consequences for fetal development in the mouse. Oxygen is a significant component of the embryonic environment and reductions in oxygen availability can influence both embryonic gene expression and subsequent fetal development.

Circulating leptin concentrations are positively related to leptin messenger RNA expression in the adipose tissue of fetal sheep in the pregnant ewe fed at or below maintenance energy requirements during late gestation

Abstract We have investigated the effects of maternal undernutrition during late gestation on maternal and fetal plasma concentrations of leptin and on leptin gene expression in fetal perirenal adipose tissue. Pregnant ewes were randomly assigned at 115 days of gestation (term = 147 ± 3 days [mean ± SEM]) to either a control group (n = 13) or an undernourished group (n = 16) that received ∼50% of the control diet until 144–147 days of gestation. Maternal plasma glucose, but not leptin, concentrations were lower in the undernourished ewes. A significant correlation was found, however, between mean maternal plasma leptin (y) and glucose (x) concentrations (y = 2.9x − 2.4; r = 0.51, P < 0.02) when the control and undernourished groups were combined. Fetal plasma glucose and insulin, but not fetal leptin, concentrations were lower in the undernourished ewes, and no correlation was found between mean fetal leptin concentrations and either mean fetal glucose or insulin concentrations. A positive relationship, however, was found between mean fetal (y) and maternal (x) plasma leptin concentrations (y = 0.18x + 0.45; r = 0.66, P < 0.003). No significant difference was found in the relative abundance of leptin mRNA in fetal perirenal fat between the undernourished (0.60 ± 0.09, n = 10) and control (0.70 ± 0.08, n = 10) groups. Fetal plasma concentrations of leptin (y) and leptin mRNA levels (x) in perirenal adipose tissue were significantly correlated (y = 1.5x ± 0.3; r = 0.69, P < 0.05). In summary, the capacity of leptin to act as a signal of moderate maternal undernutrition may be limited before birth in the sheep.

Restriction of placental growth in sheep impairs insulin secretion but not sensitivity before birth

Restricted growth before birth is associated with impaired insulin secretion but with initially enhanced insulin sensitivity in early postnatal life, which then progresses to insulin resistance and impaired glucose homeostasis by adulthood. This suggests that prenatal restraint impairs insulin secretion, but increases insulin sensitivity, before birth. Poor placental growth and function are major causes of restricted fetal growth in humans. We have therefore investigated the effects of restricted placental growth and function on plasma glucose, α‐amino nitrogen and insulin concentrations and glucose‐ and arginine‐stimulated insulin secretion in the fetal sheep at 120 and 140 days gestational age, and on insulin sensitivity, measured by hyperinsulinaemic euglycaemic clamp, at 130 days gestational age. Placental restriction decreased fetal blood pH and oxygen content, and weight in late gestation by ∼20%. Reduced fetal and placental weights and indices of poor placental function, in particular fetal hypoxia and hypoglycaemia, were associated with impaired glucose‐ and arginine‐stimulated insulin secretion, but not with changes in insulin sensitivity in the fetal sheep. We conclude that the impaired insulin secretion capacity reported in children and adults after intrauterine growth restriction, and in the neonatal and young adult sheep which is small at birth, is present in utero and persists. Whether this reflects the actions of the adverse intrauterine environment or changes to intrinsic capacity is unclear, but in utero interventions may be necessary to improve postnatal insulin secretion in the infant who is growth‐restricted before birth.

Nutritional Manipulation of Fetal Adipose Tissue Deposition and Uncoupling Protein 1 Messenger RNA Abundance in the Sheep: Differential Effects of Timing and Duration

Abstract A range of epidemiological and experimental studies have indicated that suboptimal nutrition at different stages of gestation is associated with an increased prevalence of adult hypertension, cardiovascular disease, and obesity. The timing of prenatal nutrient restriction is important in determining postnatal outcomes—including obesity. The present study, aimed to determine the extent to which fetal adiposity and expression of the key thermogenic protein, uncoupling protein (UCP)1, are altered by restriction of maternal nutrient intake imposed during four different periods, starting from before conception. Maternal nutrient intake was restricted from 60 days before until 8 days after mating (periconceptional nutrient restriction; R-C), from 60 days before mating and throughout gestation (R-R), from 8 days gestation until term (C-R), or from 115 days gestation until term. Fetal perirenal adipose tissue (PAT) was sampled near to term at ∼143 days. UCP1 mRNA, but not protein, abundance in PAT was increased in fetuses in the R-R group (C‐C 63 ± 18; R-C 83 ± 43; C-R 103 ± 38; R-R 167 ± 50 arbitrary units (P < 0.05)). In contrast, the abundance of UCP1 mRNA, but not protein, in fetal PAT was decreased when maternal nutrition was restricted from 115 days gestation. The major effect of maternal nutrient restriction on adipose tissue deposition occurred in the C-R group, in which the proportion of fetal fat was doubled, whereas maternal nutrient restriction from 115 days gestation reduced fetal fat deposition. In conclusion, there are differential effects of maternal and therefore fetal nutrient restriction on UCP1 mRNA expression and fetal fat mass and these effects are dependent on the timing and duration of nutrient restriction.

Early Embryonic Environment, The Fetal Pituitary‐Adrenal Axis and the Timing of Parturition

It is well established in the sheep, that the normal timing of parturition is dependent on a prepartum activation of the fetal pituitary‐adrenal axis. We have recently demonstrated for the first time that embryo number, embryo sex, and alterations in the environment of the early embryo, including exposure to maternal undernutrition during the periconceptional period, alter the timing and level of activation of the pituitary‐adrenal axis in the sheep fetus during late gestation. There is a delay in activation of the fetal HPA axis in twin fetuses and we speculate that the diminished adrenocortical responsiveness in the twin fetus may be an adaptive response, which counters the impact of the potential enhanced intrauterine stress experienced by a twin fetus, thereby reducing the possibility of preterm delivery. We have also reported that a moderate restriction of maternal nutrition to during the periconceptional period (from 60 days before and for one week after conception) resulted in an earlier activation of the pituitary‐adrenal axis of twin, but not singleton, fetuses during late gestation. A series of studies using assisted reproductive technologies have also found that perturbation of the early embryonic environment results in a dysregulation of placental and fetal growth and development and in the timing of normal parturition. In summary, after several decades of work focussed on events in late gestation associated with the prepartum activation and stress responsiveness of the fetal HPA axis, our recent studies indicate that the environment of the early embryo may have a significant role to play in determining the timing and level of the prepartum activation of this axis and potentially on the functional capacity of the axis to respond to acute or chronic stress in later life.

47. Recombinant human FSH induced ovarian stimulation impairs in vitro embryo development in the mouse

Gonadotrophins are routinely used in animals and humans to induce multiple ovulations and thus increase the number of oocytes available for techniques such as in vitro fertilisation (IVF). Studies in the mouse, using equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG), have reported a reduction in embryo quality compared to naturally conceived embryos. The impact of recombinant human follicle stimulating hormone (rhFSH), which is routinely used during human infertility treatment, on subsequent embryo development and quality is largely unknown due to its limited use in animal models. The aim of this study therefore was to develop a novel model of rhFSH induced ovarian stimulation in the mouse and investigate the impact of rhFSH on embryo development. One-cell embryos were collected from adult female C57Bl/6 × CBA F1 mice treated with rhFSH (0, 2.5, 5.0, 10.0 or 20.0 IU) or 5 IU eCG. All groups received 5 IU hCG 48 h after the start of gonadotrophin treatment. One-cell embryos were also recovered from non-treated control mice. Embryos were cultured in vitro for 88 h under 5% O2, 6% CO2, 89% N2 and the stage of development was morphologically assessed. Differences between groups were determined by one-way ANOVA and Bonferroni’s test for multiple comparisons. We found an increased proportion (P<0.05) of abnormal one-cell embryos recovered from mice treated with 10 IU (13.1 ± 3.6%) and 20 IU (11.5 ± 3.6%) rhFSH and eCG (19.7 ± 2.0%) compared to control embryos (0.7 ± 0.5%). Furthermore, blastocyst development was reduced in the 10 IU (72.3 ± 5.1%) and 20 IU (77.3 ± 5.6%) rhFSH groups compared to the control group (96.7 ± 1.0%). In conclusion, ovarian stimulation with rhFSH and eCG impairs the in vitro development of preimplantation mouse embryos. These results have potential implications for clinical ovarian stimulation during infertility treatment and subsequent embryo quality. L Edwards is supported by an NHMRC Peter Doherty Fellowship.