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Regular Article |
a Department of Physiology, University of Adelaide, Adelaide 5005, South Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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adrenocorticotropic hormone, corticotropin-releasing hormone, cortisol, early development, pituitary hormones
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent findings from studies on the Dutch Winter Hunger Famine, a 5-mo period of malnutrition in 1944–1945, have provided evidence that the timing of nutrient restriction in pregnancy is important in determining subsequent pathologic outcomes [5–7]. Individuals exposed to famine in the first trimester only had an increased prevalence of coronary heart disease and a higher body mass index in adult life compared with individuals not exposed to the famine [5–7]. It therefore appears that nutrient restriction during early pregnancy, when the nutrient demands of the early conceptus are minimal, can have specific long-term consequences. There is also evidence from clinical studies that suboptimal growth in the first trimester is associated with an increased risk of low birth weight and premature delivery between 24 and 32 wk of gestation, suggesting a relationship between early development and gestation length [8]. Furthermore, in vitro culture of sheep embryos for 5 days [9] or progesterone treatment during the first 3 days of pregnancy [10] results in an increased fetal size and an increased length of gestation. Although there is evidence, therefore, that nutritional or hormonal manipulation of the intrauterine environment or of the embryo during early pregnancy results in altered fetal growth, gestation length, and adverse postnatal outcomes, the causal pathways by which these events occur are poorly defined. In the sheep, it is well established that the prepartum increase in fetal cortisol concentrations is essential for the normal timing of parturition [11] and for the maturation of a number of key fetal organ systems, such as the lungs, gut, and kidney, which is required for the fetus to make a successful transition to extrauterine life [12].
Embryo number and sex and the level of maternal nutrition during the periconceptional period are each important determinants of embryonic and fetal growth [13–15]. There have been no studies, however, that have determined the role of each of these variables in the prepartum activation of the fetal hypothalamo-pituitary adrenal (HPA) axis in late gestation. In the present study, we have investigated the hypotheses that fetal number, fetal sex, and maternal nutrient restriction during the periconceptional or later gestational periods may each alter the program of development of the HPA axis in the late-gestation sheep fetus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nutritional Management
Fifty-two Australian Border-Leicester-cross Merino ewes were used in this study. Sixty days before mating, ewes were randomly assigned to one of two feeding regimens, a control regimen (C, n = 23), during which ewes received 100% of nutritional requirements, or a restricted regimen (R, n = 29), during which ewes received 70% of the control allowance. The nutritional requirements for the control animals were calculated to provide sufficient energy for the maintenance of a nonpregnant ewe (7.8 MJ/day for a 60-kg ewe) [16]. All animals were housed in individual pens and had free access to water. The diet consisted of lucerne chaff and pellets containing straw, cereal, hay, clover, barley, oats, lupins, almond shells, oat husks, and limestone. Eighty percent of the total energy requirements were obtained from the lucerne chaff, and twenty percent of the energy requirements were obtained from the pellet mixture. The lucerne chaff provided 8.3 MJ/kg of metabolizable energy and 193 g/kg of crude protein and contained 85% dry matter. The pellets provided 8.0 MJ/kg of metabolizable energy and 110 g/kg of crude protein and contained 90% dry matter. All of the dietary components were reduced by an equal amount in the restricted diet.
After maintenance on either the control or restricted diet for a minimum period of 60 days, a ram was introduced, and 7 days after mating, ewes from each feeding regimen were randomly assigned to the control or restricted plane of nutrition for the remainder of pregnancy (term = 147 ± 3 days gestation). Therefore, animals were maintained either on a control or restricted diet during the periconceptional period (-60 to +7 days after mating) and then either on a control or restricted diet during the gestational period (+8 days until postmortem). Four treatment groups were therefore generated: control-control (C-C, n = 12); control-restricted (C-R, n = 11); restricted-restricted (R-R, n = 16), and restricted-control (R-C, n = 13).
Pregnancy and fetal number were confirmed by ultrasound at 60 days of gestation. The nutritional intake for animals on the restricted diet was maintained at 70% of control energy requirements, and both nutritional regimens were adjusted for gestational age and fetal number, as outlined by the Ministry of Agriculture, Fisheries and Food [16].
Animals and Surgery
Pregnant ewes were transported into the Animal House between 90 and 100 days of gestation. Surgery was performed under aseptic conditions between 105 and 110 days of gestation with general anesthesia initially induced by an i.v. injection of sodium thiopentone (1.25 g, Pentothal; Rhone Merieux, Pinkenba, Qld, Australia) and maintained with inhalational halothane (2.5%–4%, Fluothane; ICI, Melbourne, Vic, Australia) in oxygen. In all ewes, vascular catheters were implanted in a fetal carotid artery and jugular vein, a maternal jugular vein, and the amniotic cavity, as previously described [17]. Vascular catheters were inserted into only 1 fetus in twin pregnancies. All catheters were filled with heparinized saline, and the fetal catheters exteriorized through an incision made in the ewes' flank. All ewes and fetal sheep received a 2-ml i.m. injection of antibiotics (procaine penicillin 250 mg/ml, dihydrostreptomycin 250 mg/ml, and procaine hydrochloride 20 mg/ml; Penstrep Illium; Troy Laboratories, Smithfield, NSW, Australia) at the time of surgery. The ewes were housed in individual pens in animal holding rooms with a 12L:12D cycle and fed once daily at 1100 h with water provided ad libitum. Animals were allowed to recover from surgery for at least 4 days before experimentation.
Blood Sample Collection
Fetal arterial blood samples (0.5 ml) were collected every day for 4 days after surgery and then 3 times per week thereafter for the measurement of arterial PO2, PCO2, pH, oxygen saturation, and hemoglobin (ABL 520 blood gas analyzer; Radiometer, Copenhagen, Denmark). The fetal arterial blood gas variables measured across late gestation in fetuses in all of the nutritional groups were in the normal range previously reported for healthy fetuses in late gestation [17, 18]. Fetal arterial blood samples (3.5 ml) were collected in chilled tubes 3 times per week between 0800 and 1100 h for the measurement of plasma glucose, ACTH, and cortisol concentrations throughout late gestation. Similarly, maternal venous blood samples (5 ml) were collected in chilled tubes 3 times per week between 0800 and 1100 h for the measurement of plasma glucose and cortisol concentrations. All blood samples were centrifuged at 1500 x g for 10 min, and plasma was separated into aliquots and stored at -20°C for subsequent hormone and metabolite assays.
Corticotropin-Releasing Hormone
Corticotropin-releasing hormone (CRH) (a 1-µg bolus in 1 ml saline; Peninsula Laboratories Inc., San Carlos, CA) was injected i.v. in 13 fetal sheep between 139 and 144 days of gestation (all twin fetuses; C-C plus C-R, n = 8; R-R plus R-C, n = 5). Fetal arterial blood samples (2.5 ml) were collected at 30 and 5 min before and 10, 20, 40, 60, 120, and 240 min after the injection of CRH. Blood samples were centrifuged at 1500 x g for 10 min, and plasma was separated into aliquots and stored at -20°C for ACTH and cortisol RIAs.
Postmortem
Ewes were killed with an overdose of sodium pentobarbitone (Virbac, Peakhurst, NSW, Australia) between 140 and 147 days of gestation, and the fetuses was delivered by hysterotomy, weighed, and killed by decapitation. Fetal adrenal glands were then collected and weighed; in the case of twins, adrenal glands were collected and weighed from both fetuses.
Plasma Glucose Determination
Plasma concentrations of glucose were determined by enzymatic analysis using hexokinase and glucose-6-phosphate dehydrogenase to measure the formation of NADH photometrically at 340 nm (COBAS MIRA automated analysis system; Roche Diagnostica, Basel, Switzerland). The sensitivity of the assay was 0.5 mmol/L, and the intraassay and interassay coefficients of variation were both less than 5%.
Adrenocorticotropic Hormone
Immunoreactive ACTH concentrations in fetal sheep plasma were measured with an RIA (ICN Biomedicals, Seven Hills, NSW, Australia) previously validated for fetal sheep plasma [19]. The sensitivity of the assay was 0.9 pg/ml, and the interassay and intraassay coefficients of variation were 10.3% and <10%, respectively.
Cortisol
Cortisol was extracted from fetal plasma using dichloromethane as previously described [20]. The efficiency of recovery of 125I-cortisol from fetal plasma using this extraction procedure was always greater than 90%. Maternal and fetal cortisol concentrations were then measured using an Orion Diagnostica Radioimmunoassay kit (Orion Diagnostica, Turku, Finland) previously validated for fetal sheep plasma [21]. The sensitivity of the assay was 0.078 pmol per tube, and the interassay and intraassay coefficients of variation were 20.6% and <10%, respectively.
Statistical Analysis
Data are shown as the mean ± SEM. The effect of periconceptional nutrition on conception rates was compared using a chi-square test. The effects of periconceptional and gestational nutrition on fetal survival rates were also compared using a chi-square test. Hormonal data were log transformed where required, to normalize data variance for parametric analysis. When a significant interaction between major factors was identified by ANOVA, the data were split on the basis of the interacting factor and reanalyzed using ANOVA. The Duncan New Multiple Range Test was used after ANOVA to identify significant differences between mean values, and a probability level of 5% (P < 0.05) was taken as significant. The weights of nonpregnant ewes assigned to the restricted and control periconceptional nutrition groups were compared using an unpaired Student t-test. The effects of periconceptional and gestational nutrition on fetal weight and fetal adrenal weight, expressed as a percentage of body weight, were compared separately in singleton and twin fetuses using a multifactorial ANOVA. Specified factors for the ANOVA included periconceptional nutrition (C or R) and gestational nutrition (C or R). The effects of periconceptional and gestational nutrition on maternal and fetal plasma concentrations of glucose were compared using a multifactorial ANOVA with repeated measures using the Statistical Package for Social Sciences (SPSSX; SPSS Science, Chicago, IL) on a Vax mainframe computer (Adelaide University). Specified factors for the ANOVA included group (C-C, C-R, R-R, R-C), fetal number (singleton or twin), and gestational age (115–118, 119–122, 123–126, 127–130, 131–134, 135–138, 139–142, and 143–146 days of gestation). The effects of periconceptional and gestational nutrition on maternal and fetal plasma glucose concentrations were then compared separately in singleton- and twin-bearing ewes and fetuses using a multifactorial ANOVA with repeated measures. Specified factors for the ANOVA included periconceptional nutrition (C or R), gestational nutrition (C or R), and gestational age.
Plasma concentrations of ACTH and cortisol in twin and singleton fetuses in the C-C group were also compared using a multifactorial ANOVA (including fetal number and gestational age) and repeated measures. The effect of fetal number on the timing of the prepartum surge in fetal plasma cortisol concentrations was also compared in the C-C animals. The age of onset of the prepartum surge in cortisol was defined as the first day on which plasma cortisol concentrations were greater than 10 nmol/L. The age at the time of the prepartum surge in cortisol was then compared in singleton and twin fetuses using an unpaired Student t-test.
The effects of periconceptional and gestational nutrition on fetal plasma ACTH, fetal plasma cortisol, and maternal plasma cortisol concentrations were compared separately in singleton- and twin-bearing ewes using a multifactorial ANOVA with repeated measures. Specified factors included nutritional group, gestational age, and sex of the fetus (male or female).
The effects of periconceptional and gestational nutrition on the plasma ACTH and cortisol responses to CRH in twin fetuses were also compared using a multifactorial ANOVA with repeated measures. Specified factors for the ANOVA included periconceptional nutrition (C or R), gestational nutrition (C or R), and time.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The weights of the nonpregnant ewes assigned to the control (55.7 ± 0.9 kg, n = 23) or restricted (56.6 ± 0.8 kg, n = 29) periconceptional nutrition groups were not different before the start of the feeding regimen. Ewes in the restricted periconceptional nutrition group lost significantly more weight (-2.2 ± 0.4 kg, n = 29) than those in the control group (-0.5 ± 0.5 kg, n = 23) during the prepregnancy period. Restricted periconceptional nutrition did not significantly affect the conception rates, which were 86% in the restricted periconceptional group and 67% in the control periconceptional group.
Effect of Periconceptional and Gestational Undernutrition on Maternal and Fetal Glucose Concentrations
Plasma glucose concentrations were significantly lower (F = 13.4, P < 0.005) in ewes carrying twins compared with ewes carrying singletons between 115 and 147 days of gestation in all nutritional groups. There was no effect of restricted periconceptional nutrition on maternal plasma concentrations of glucose measured between 115 and 146 days of gestation. Restricted gestational nutrition resulted in lower plasma glucose concentrations in ewes carrying either singleton (F = 8.4, P < 0.01) or twin (F = 9.9, P < 0.01) fetuses between 115 and 146 days of gestation (Table 1).
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Plasma glucose concentrations were also significantly lower (F = 35.2, P < 0.001) in twin compared with singleton fetuses between 115 and 147 days of gestation in all nutritional groups. There was no effect of restricted periconceptional nutrition on plasma glucose concentrations measured in either single or twin fetuses throughout late gestation. Restricted gestational nutrition, however, resulted in lower fetal glucose concentrations in both singleton (F = 7.6, P < 0.05) and twin (F = 24.9, P < 0.0001) fetuses between 115 and 147 days of gestation (Table 1).
Effect of Fetal Number on the Prepartum Changes in Plasma ACTH and Cortisol in the C-C Group
In ewes on a maintenance control diet before and throughout the whole of pregnancy (the C-C group), plasma ACTH concentrations were higher (F = 17.7, P < 0.005) in singleton fetuses (4 male, 1 female) between 115 and 146 days of gestation than those in twin fetal sheep (4 male, 2 female) (Fig. 1A). In the C-C group, plasma cortisol concentrations were also higher (F = 5.4, P < 0.05) in singleton compared with twin fetuses between 127 and 146 days of gestation (Fig. 1B). In this group, the onset of the prepartum surge in cortisol also occurred significantly earlier in singletons (128 1.7 days of gestation, n = 5) compared with twins (135 1.2 days of gestation, n = 6).
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Effect of Fetal Sex on Prepartum Changes in Fetal Plasma ACTH and Cortisol Concentrations
There was no interaction between the effects of sex and either periconceptional or gestational nutrition on plasma ACTH or cortisol concentrations in singleton or twin fetal sheep. In singletons, however, there was a significant interaction between the effects of sex and gestational age on plasma ACTH concentrations, and this effect was present in all nutritional groups. There was no difference in plasma ACTH concentrations between male and female singleton fetuses before 130 days of gestation (males, 45.0 ± 3.7 pg/ml; females, 60.2 ± 19.7 pg/ml). After 130 days of gestation, however, plasma ACTH concentrations were significantly higher in male (75.1 ± 8.8 pg/ml) compared with female (45.9 ± 7.7 pg/ml) singleton fetuses. There was no effect, however, of sex on plasma cortisol concentrations in singleton fetal sheep.
In contrast to the pattern noted in singleton fetuses, there was no effect of sex on plasma ACTH concentrations in twin fetuses either before or after 130 days. There was, however, a significant interaction between the effects of sex and gestational age on plasma cortisol concentrations in twins. The prepartum surge in cortisol concentrations occurred earlier in male twin fetuses (131–134 days: 15.0 ± 4.2 nmol/L; 135–138 days: 33.0 ± 12.1 nmol/L) than in female twin fetuses (131–134 days: 8.1 ± 2.7 nmol/L; 135–138 days: 17.6 ± 6.8 nmol/L).
Effect of Periconceptional or Gestational Undernutrition on Maternal Plasma Cortisol
There was no effect of either restricted periconceptional or gestational nutrition on maternal plasma cortisol concentrations during late gestation in ewes bearing either singleton or twin pregnancies (Table 2). There was also no effect of gestational age on maternal cortisol concentrations.
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Effect of Periconceptional or Gestational Undernutrition on Plasma ACTH and Cortisol Concentrations in Singleton Fetuses
In singleton fetuses in all nutritional groups, plasma ACTH concentrations were higher (F = 6.77, P < 0.001) between 135 and 146 days compared with between 119 and 122 days of gestation. There was no effect of either periconceptional or gestational undernutrition on plasma ACTH concentrations in singleton fetuses (Fig. 2A). In singleton fetuses in all nutritional groups, fetal plasma concentrations of cortisol were higher (F = 45.7, P < 0.001) between 127 and 146 days than between 115 and 122 days of gestation. There was no effect of either restricted periconceptional or gestational nutrition or any interaction between these nutritional treatments on fetal plasma cortisol concentrations in singleton fetuses (Fig. 3A).
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Effect of Periconceptional or Gestational Undernutrition on Plasma ACTH and Cortisol in Twin Fetuses
In twin fetuses, plasma ACTH concentrations were higher (F = 3.34, P < 0.005) between 139 and 142 days compared with between 123 and 126 days of gestation in all nutritional groups. In twin fetuses, restricted periconceptional nutrition resulted in higher (F = 10.95, P < 0.005) plasma ACTH concentrations between 115 and 146 days of gestation when compared with the control periconceptional nutrition group (Fig. 2B). There was no effect, however, of restricted gestational nutrition or any interaction between the effects of restricted periconceptional and gestational nutrition on plasma ACTH concentrations in twin fetuses during late gestation.
In twin fetuses in all nutrition groups, plasma cortisol concentrations were higher (F = 48.73, P < 0.001) between 131 and 146 days compared with between 115 and 130 days of gestation (Fig. 3B). There was no effect of restricted periconceptional or gestational nutrition or any interaction between the effects of periconceptional and gestational nutrition on plasma cortisol concentrations in twin fetuses between 115 and 146 days of gestation. There was also no effect of either periconceptional or gestational nutrition or an interaction (F = 4.0, P = 0.07) between these factors when cortisol concentrations were compared after the onset of the prepartum increase in cortisol.
Effect of Periconceptional or Gestational Undernutrition on ACTH and Cortisol Responses to CRH Stimulation in Twin Fetuses
In twin fetuses, fetal plasma concentrations of ACTH increased (F = 15.7, P < 0.0001) between 45 and 240 min after CRH administration in all nutritional groups. The fetal ACTH response (expressed as a change from baseline concentrations) was higher (F = 12.0, P < 0.0001) between 60 and 240 min compared with between 10 and 45 min after CRH administration in all nutritional treatment groups (Fig. 4). There was no separate or interacting effect, however, of restricted periconceptional or gestational nutrition on the fetal ACTH response to CRH.
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There was an increase in plasma cortisol concentrations in response to CRH between 10 and 240 min after CRH administration in all nutritional treatment groups. There was also a greater increase (F = 4.18, P < 0.005) in the fetal cortisol response to CRH in the restricted periconceptional nutrition group when compared with the control periconceptional nutrition group (Fig. 4).
Fetal Outcomes
Restricted periconceptional or gestational nutrition did not affect fetal survival to postmortem between 140 and 147 days (number of fetal sheep to survive to postmortem: C-C, n = 9/12; C-R, n = 6/11; R-R, n = 9/16; R-C, n = 5/13). There was no significant effect of either restricted periconceptional or gestational nutrition on fetal weight in singleton fetal sheep. In twin fetal sheep, there was an interaction between the effects of periconceptional and gestational nutrition on fetal weight. Twin fetuses in the C-R group (2.8 ± 1.8 kg) were significantly smaller (P < 0.05) compared with those in the C-C group (4.5 ± 2.7 kg), whereas there was no difference in the fetal weights between twins in the R-C (4.3 ± 2.3 kg) and R-R (3.9 ± 2.1 kg) groups.
There was no effect of periconceptional nutrition alone or any interaction between the effects of periconceptional and gestational nutrition on the relative adrenal weight in either singleton or twin fetal sheep. There was no effect of restricted gestational nutrition on relative adrenal gland weight in singleton fetuses (C-R and R-R, 0.0109% ± 0.001%; C-C and R-C, 0.0117% ± 0.001%). The relative adrenal gland weight was significantly greater, however, in twin fetal sheep in the restricted gestational nutrition groups (C-R and R-R, 0.0167% ± 0.005%) when compared with twins maintained on the control level of maternal nutrition during the gestational period (C-C and R-C, 0.0099% ± 0.002%).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the current study, the well-characterized prepartum rise in plasma ACTH and cortisol concentrations [22, 23] was present in both twin and singleton fetuses in all nutritional groups. Interestingly, plasma ACTH concentrations in the C-C group were significantly higher in singleton compared with twin fetal sheep between 115 and 146 days of gestation. Furthermore, the timing of the prepartum surge in fetal cortisol occurred earlier in singletons, and plasma cortisol concentrations were also higher in singleton compared with twin fetuses between 127 and 146 days of gestation. It is unlikely that sex contributed to this effect of fetal number on plasma ACTH and cortisol concentrations as 80% and 67%, respectively, of the control fetuses in the singleton and twin groups were males. The finding that the cortisol rise occurs earlier in singletons compared with twins is somewhat surprising because in humans, twin fetuses deliver earlier than singletons [24]. Interpretation of data from human pregnancies may be confounded, however, by the greater probability of an association between fetal hypoxemia and hypoglycemia and twin pregnancies. Several studies in the sheep have demonstrated that the fetal HPA response to chronic or intermittent hypoxemia varies according to the duration, intensity, and frequency of the hypoxemic episodes [17, 25, 26]. Our data in normoxemic fetal sheep highlight that there is a delay in the timing of the normal prepartum activation of the HPA axis in twin compared with singleton fetal sheep.
In the current study, we also found an effect of sex on the fetal HPA axis in singleton and twin fetuses that was independent of prior nutritional history. In singletons, plasma ACTH, but not cortisol, concentrations were higher in male than female fetuses after 130 days of gestation. As fetal sex did not influence plasma cortisol concentrations in singleton fetuses, it may be the case that after 130 days of gestation, the fetal ACTH concentration may exceed the level required to activate adrenal growth and steroidogenesis in both male and female singleton fetuses. In the twin fetuses, plasma cortisol, but not ACTH, concentrations increased earlier in males than in females. These results are consistent with previous reports in the sheep that the rise in fetal cortisol concentrations occurs earlier in male compared with female twin fetuses and that there is a greater cortisol response to a bolus dose of ACTH in the twin in which the earlier cortisol rise occurs compared with the second twin [27, 28]. It may be that factors associated with male sex, including fetal growth rate or gonadal factors, result in earlier activation of the fetal HPA axis.
There was a differential effect of periconceptional undernutrition on the HPA axis of singleton and twin fetal sheep. Restricted nutrition during the periconceptional period specifically increased plasma ACTH concentrations in twins. The timing of the prepartum increase in plasma ACTH concentrations in twin fetuses was not altered, however, by restricted periconceptional nutrition. This suggests that periconceptional undernutrition may result in an increase in the corticotropic synthetic and secretory capacity of the fetal pituitary, rather than an alteration of the hypothalamic mechanisms that initiate the prepartum activation of the fetal HPA axis. This is the first report, to the best of our knowledge, of an effect of nutrient restriction during the periconceptional period on the subsequent development of the fetal HPA axis, and the underlying mechanisms clearly require further investigation. It has previously been shown that maternal undernutrition during the preimplantation period in the guinea pig and rat alters the allocation of cells between the trophectoderm and the inner cell mass of the embryo [29, 30]. Such alterations of the development of the trophectoderm may result in a change in the secretion of placental hormones such as prostaglandin E2, which have been implicated in the control of ACTH secretion in the late-gestation fetus [31].
There was no effect of restricted periconceptional nutrition on plasma cortisol concentrations measured during late gestation in either singleton or twin fetuses. It has been shown that expression of adrenal steroidogenic enzymes, including the rate-limiting cytochrome P450 enzyme 17alpha-hydroxylase (CYP17) is relatively low and that the fetal sheep adrenal gland is therefore relatively unresponsive to ACTH stimulation before 130 days of gestation [11, 32–34]. It is surprising, however, that fetal cortisol concentrations were not higher after 130 days of gestation in the twin fetuses in the periconceptional undernutrition group. One possibility is that the increased plasma immunoreactive ACTH concentrations measured during late gestation in twin fetuses after exposure to periconceptional undernutrition reflects an increase in higher molecular weight forms of ACTH rather than bioactive ACTH1–39.
In the present study, the ACTH secretory response of the fetal pituitary to CRH was not altered by restricted periconceptional nutrition in twins. The cortisol response to CRH was greater, however, in those twin fetuses exposed to restricted periconceptional nutrition, independent of any subsequent exposure to gestational undernutrition. Although it is possible that CRH may be acting directly at the fetal adrenal gland to enhance adrenal cortisol output, a more likely explanation is that periconceptional undernutrition results in an increased responsiveness of the fetal adrenal gland to ACTH stimulation. An increase in the potential exposure of the fetus to excess glucocorticoid concentrations may represent one mechanism whereby maternal undernutrition before or in early pregnancy results in adverse cardiovascular outcomes in later life [5–7]. Maternal protein restriction during early, mid, or late pregnancy in the rat results in high blood pressure in the offspring [35, 36], and this effect is prevented by the inhibition of maternal corticosterone biosynthesis during pregnancy [37]. Furthermore, administration of synthetic glucocorticoids to pregnant ewes in early pregnancy results in the birth of lambs that develop hypertension in adult life [38]. Interestingly, growth restriction in utero is also associated with an enhanced activity of the HPA axis in later life. A study of approximately 200 men in the U.K. found that there was an association between low birth weight and increased activity of the HPA axis at 66–70 yr of age and that there was also an association between features of the metabolic syndrome (raised blood pressure, glucose intolerance, and increased plasma triglyceride concentrations) and increased HPA activity in this cohort [39]. Thus, the imposition of maternal nutrient restriction during either the periconceptional period or during late gestation may each result in exposure of the fetus to excess glucocorticoids, which act to program adverse cardiovascular and metabolic outcomes.
In the present study, we found that restricted periconceptional nutrition had no effect on fetal weight in singleton or twin fetuses between 140 and 147 days of gestation. Fetal weight was decreased, however, in those twin fetuses exposed to restricted gestational nutrition after maintenance on a control diet during the periconceptional period, compared with fetuses that were maintained on a control level of nutrition for the entire periconceptional and gestational periods. Although the decreased fetal weight in the twin but not singleton pregnancies in the C-R group may reflect the lower plasma glucose concentrations measured in the twin fetuses in late gestation, fetal weight in twins with low glucose concentrations in the R-R group was not decreased. This suggests therefore that in twin fetuses, the impact of restricted gestational nutrition on fetal growth in late gestation may be dependent on the prior level of periconceptional nutrition. Maternal undernutrition before and during the first 30 days of gestation has previously been reported to decrease the subsequent rate of fetal growth in late gestation [40]; however, the present study suggests that undernutrition before and during the first week of pregnancy may be sufficient to determine the fetal growth trajectory. Interestingly, studies in multifetal human pregnancies have also found that after embryo reduction in the first trimester, the birth weights of the remaining twin pregnancies were significantly reduced compared with the birth weights from nonreduced twin pregnancies [13]. This suggests that the fetal growth trajectory in multifetal pregnancies may be set early in gestation.
Summary
In summary, we have found that fetal number and sex each has an effect on the timing of the prepartum activation of the fetal HPA axis. Plasma ACTH concentrations are higher in singleton compared with twin fetuses, and the onset of the prepartum surge in plasma cortisol concentrations occurs earlier in singletons. The prepartum activation of the fetal HPA axis also occurs earlier in male than in female fetuses. In contrast, the impact of periconceptional undernutrition in twin fetuses appears to reflect an increase in fetal ACTH throughout the whole of late gestation. The differential effect of periconceptional undernutrition in twin and singleton fetuses indicates that the development of the HPA axis may differ in twins and singletons from as early as the first week of pregnancy. The present study also clearly demonstrates that periconceptional undernutrition before and during the preimplantation period results in altered development of the HPA axis and is relevant in the context of the epidemiologic data that provide evidence that events in early pregnancy result in adverse health outcomes in later life.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The authors are grateful to the Government Health Employees Health Research Fund for their financial support of this work. 
2 Correspondence. FAX: 61 8 8303 3356; caroline.mcmillen{at}adelaide.edu.au
Accepted: December 19, 2001.
Received: November 6, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 S E Chadio, B Kotsampasi, G Papadomichelakis, S Deligeorgis, D Kalogiannis, I Menegatos, and G Zervas Impact of maternal undernutrition on the hypothalamic-pituitary-adrenal axis responsiveness in sheep at different ages postnatal J. Endocrinol., March 1, 2007; 192(3): 495 - 503. [Abstract] [Full Text] [PDF]
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 F. H. Bloomfield, M. H. Oliver, and J. E. Harding Effects of twinning, birth size, and postnatal growth on glucose tolerance and hypothalamic-pituitary-adrenal function in postpubertal sheep Am J Physiol Endocrinol Metab, January 1, 2007; 292(1): E231 - E237. [Abstract] [Full Text] [PDF]
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 H. Dickinson, D. W. Walker, E. M. Wintour, and K. Moritz Maternal dexamethasone treatment at midgestation reduces nephron number and alters renal gene expression in the fetal spiny mouse Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R453 - R461. [Abstract] [Full Text] [PDF]
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S. Gentili, J. S. Schwartz, M. J. Waters, and I. C. McMillen Prolactin and the expression of suppressor of cytokine signaling-3 in the sheep adrenal gland before birth Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2006; 291(5): R1399 - R1405. [Abstract] [Full Text] [PDF]
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D S Gardner, B W M Van Bon, J Dandrea, P J Goddard, S F May, V Wilson, T Stephenson, and M E Symonds Effect of periconceptional undernutrition and gender on hypothalamic-pituitary-adrenal axis function in young adult sheep. J. Endocrinol., August 1, 2006; 190(2): 203 - 212. [Abstract] [Full Text] [PDF]
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L. C. Carey, Y. Su, N. K. Valego, and J. C. Rose Infusion of ACTH stimulates expression of adrenal ACTH receptor and steroidogenic acute regulatory protein mRNA in fetal sheep Am J Physiol Endocrinol Metab, August 1, 2006; 291(2): E214 - E220. [Abstract] [Full Text] [PDF]
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S. Gentili, M. J. Waters, and I. C. McMillen Differential regulation of suppressor of cytokine signaling-3 in the liver and adipose tissue of the sheep fetus in late gestation Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2006; 290(4): R1044 - R1051. [Abstract] [Full Text] [PDF]
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A I Turner, B J Hosking, R A Parr, and A J Tilbrook A sex difference in the cortisol response to tail docking and ACTH develops between 1 and 8 weeks of age in lambs. J. Endocrinol., March 1, 2006; 188(3): 443 - 449. [Abstract] [Full Text] [PDF] < |
