1. family and parenting
2. motherhood
3. pregnancy

BIOLOGY OF REPRODUCTION 66, 1562–1569 (2002)
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 Gestation1
L.J. Edwards and I.C. McMillen2
Department of Physiology, University of Adelaide, Adelaide 5005, South Australia
ABSTRACT
INTRODUCTION
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.
A worldwide series of epidemiologic studies has demonstrated that there are significant associations between low
birth weight and a range of poor adult health outcomes
including increased blood pressure, obesity, insulin resistance, and coronary heart disease [1–3]. These associations
have lead to the articulation of the ‘‘fetal origins of adult
disease hypothesis,’’ which states that fetal adaptations to
a period of intrauterine deprivation result in a permanent
reprogramming of the development of key tissue and organ
systems and pathophysiologic outcomes in later life [3, 4].
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.
adrenocorticotropic hormone, corticotropin-releasing hormone,
cortisol, early development, pituitary hormones
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;
e-mail: caroline.mcmillen@adelaide.edu.au
1
Received: 6 November 2001.
First decision: 26 November 2001.
Accepted: 19 December 2001.
Q 2002 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
1562
UNDERNUTRITION AND THE FETAL HPA AXIS
MATERIALS AND METHODS
All procedures were approved by The University of Adelaide Standing
Committee on Animal Ethics and Experimentation.
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 5 23), during which
ewes received 100% of nutritional requirements, or a restricted regimen
(R, n 5 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 5
147 6 3 days gestation). Therefore, animals were maintained either on a
control or restricted diet during the periconceptional period (260 to 17
days after mating) and then either on a control or restricted diet during
the gestational period (18 days until postmortem). Four treatment groups
were therefore generated: control-control (C-C, n 5 12); control-restricted
(C-R, n 5 11); restricted-restricted (R-R, n 5 16), and restricted-control
(R-C, n 5 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
1563
concentrations. All blood samples were centrifuged at 1500 3 g for 10
min, and plasma was separated into aliquots and stored at 2208C for
subsequent hormone and metabolite assays.
Corticotropin-Releasing Hormone
Corticotropin-releasing hormone (CRH) (a 1-mg 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 5 8; R-R plus R-C, n 5 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
3 g for 10 min, and plasma was separated into aliquots and stored at
2208C 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 6 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
1564
EDWARDS AND MCMILLEN
TABLE 1. Effect of gestational nutrition and fetal number on maternal and fetal plasma glucose concentrations in late gestation.
Fetal
number
Level of gestational
nutrition
Maternal glucose
concentration
(mmol/L)
Singletons
Fetal glucose
concentration
(mmol/L)
Singletons
Control (n 5 10)
Restricted (n 5 12)
Control (n 5 12)
Restricted (n 5 12)
Control (n 5 12)
Restricted (n 5 15)
Control (n 5 13)
Restricted (n 5 11)
Twins
Twins
Gestational age (days)
115–125
2.90
2.39
2.39
2.00
1.67
1.38
1.33
1.08
6
6
6
6
6
6
6
6
0.12
0.09a
0.09b
0.13a, b
0.06
0.05a
0.06b
0.07a, b
126–135
2.67
2.33
2.39
1.91
1.62
1.37
1.32
1.00
6
6
6
6
6
6
6
6
0.20
0.15a
0.11b
0.15a, b
0.12
0.08a
0.05b
0.06a, b
136–146
2.85
2.36
2.39
2.10
1.72
1.48
1.28
1.03
6
6
6
6
6
6
6
6
0.28
0.15a
0.10b
0.18a, b
0.13
0.07a
0.04b
0.06a, b
a
Restricted gestational nutrition (C-R, R-R) significantly decreased plasma concentrations of maternal glucose in ewes carrying singleton and twin fetuses
and fetal glucose in singleton and twin fetuses, compared with control gestational nutrition between 115 and 146 days gestation (P , 0.05).
b Glucose concentrations were significantly lower in ewes carrying twin pregnancies compared with ewes carrying singleton pregnancies and in twin
fetuses compared with singleton fetuses (P , 0.05).
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.
RESULTS
Ewe Weights
The weights of the nonpregnant ewes assigned to the
control (55.7 6 0.9 kg, n 5 23) or restricted (56.6 6 0.8
kg, n 5 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 (22.2 6 0.4 kg, n 5 29) than those in the
control group (20.5 6 0.5 kg, n 5 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 5 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 5 8.4, P , 0.01) or twin (F 5 9.9, P
, 0.01) fetuses between 115 and 146 days of gestation
(Table 1).
Plasma glucose concentrations were also significantly
lower (F 5 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
5 7.6, P , 0.05) and twin (F 5 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 5 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 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 5) compared
with twins (135 1.2 days of gestation, n 5 6).
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 6 3.7 pg/
ml; females, 60.2 6 19.7 pg/ml). After 130 days of gestation, however, plasma ACTH concentrations were significantly higher in male (75.1 6 8.8 pg/ml) compared with
female (45.9 6 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,
1565
UNDERNUTRITION AND THE FETAL HPA AXIS
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 6
4.2 nmol/L; 135–138 days: 33.0 6 12.1 nmol/L) than in
female twin fetuses (131–134 days: 8.1 6 2.7 nmol/L; 135–
138 days: 17.6 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.
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 5 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 5 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).
Effect of Periconceptional or Gestational Undernutrition
on Plasma ACTH and Cortisol in Twin Fetuses
In twin fetuses, plasma ACTH concentrations were higher (F 5 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 5 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 5 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 restrict-
FIG. 1. A) Plasma concentrations of ACTH were significantly higher in
singleton fetuses in the C-C group (n 5 6) between 115 and 146 days of
gestation compared with twin fetuses in the C-C group (n 5 6). B) Plasma
concentrations of cortisol were significantly higher in singleton fetuses in
the C-C group (n 5 6) between 127 and 146 days gestation compared
with twin fetuses in the C-C group (n 5 6). If there were fewer than 3
animals per group, no SEM bars are given. * Significant effect of fetal
number (P , 0.05).
ed 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 5 4.0, P 5 0.07) between these factors
when cortisol concentrations were compared after the onset
of the prepartum increase in cortisol.
TABLE 2. Effect of periconceptional nutriton and fetal number on maternal plasma cortisol concentrations in late gestation.a
Maternal cortisol
concentration
(nmol/L)
Maternal cortisol
concentration
(nmol/L)
a
b
Gestational age (days)b
Fetal
number
Level of periconceptional
nutrition
115–125
126–135
136–146
Singletons
Control (n 5 6)
Restricted (n 5 11)
18.9 6 3.7
16.7 6 3.2
11.2 6 3.7
23.7 6 8.1
11.4
11.2 6 2.6
Twins
Control (n 5 11)
Restricted (n 5 11)
18.8 6 3.4
18.9 6 4.4
20.0 6 5.0
14.8 6 2.9
13.8 6 3.5
14.9 6 3.3
There was no significant effect on periconceptional nutrition or fetal number on maternal cortisol concentrations in late gestation.
If less than 3 ewes per group, there are no SEM bars.
1566
EDWARDS AND MCMILLEN
FIG. 2. A) There was no significant effect of nutritional group on plasma
ACTH concentrations in singleton fetuses (C-C, n 5 6; C-R, n 5 4; R-R,
n 5 10; R-C, n 5 6) between 115 and 146 days of gestation; however,
plasma ACTH concentrations increased significantly (P , 0.001) between
135 and 146 days compared with between 119 and 122 days of gestation
across all nutritional groups. If there were fewer than three animals per
group, no SEM bars are given. B) In twin fetuses, restricted periconceptional nutrition (R-R, n 5 5; R-C, n 5 7) significantly increased plasma
ACTH concentrations between 115 and 146 days of gestation compared
with control periconceptional nutrition (C-C, n 5 6; C-R, n 5 6) (*, P ,
0.05). In twin fetuses, plasma ACTH concentrations were increased significantly (P , 0.005) between 139 and 142 days compared with between
123 and 126 days of gestation across all nutritional groups.
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 5 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 5 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.
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
FIG. 3. There was no significant effect of nutritional group on plasma
cortisol concentrations in singleton (A) (C-C, n 5 6; C-R, n 5 4; R-R, n
5 10; R-C, n 5 6) or twin (B) (C-C, n 5 6; C-R, n 5 6; R-R, n 5 5; RC, n 5 7) fetuses between 115 and 146 days of gestation. Fetal plasma
concentrations of cortisol were increased (P , 0.001) between 127 and
146 days compared with between 115 and 122 days of gestation in singleton fetuses across all nutritional groups. If there were fewer than three
animals per group, no SEM bars are given. In twin fetuses, plasma cortisol
concentrations were increased (P , 0.001) between 131 and 146 days
compared with between 115 and 130 days of gestation across all nutrition
groups.
was also a greater increase (F 5 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 5 9/12; C-R, n 5 6/11; R-R, n 5 9/16; R-C, n 5
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
UNDERNUTRITION AND THE FETAL HPA AXIS
1567
group (2.8 6 1.8 kg) were significantly smaller (P , 0.05)
compared with those in the C-C group (4.5 6 2.7 kg),
whereas there was no difference in the fetal weights between twins in the R-C (4.3 6 2.3 kg) and R-R (3.9 6 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% 6
0.001%; C-C and R-C, 0.0117% 6 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% 6 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% 6
0.002%).
DISCUSSION
In this study, we have demonstrated for the first time
that there are important and separate effects of fetal number,
fetal sex, and the level of periconceptional nutrition on the
development of the fetal HPA axis during late gestation.
These results are relevant in the context of the series of
epidemiologic and clinical studies that show that either restriction of maternal nutrient intake or a low rate of fetal
growth during early pregnancy is associated with changes
in gestation length and birth weight and specific adverse
health outcomes in later life [5–9].
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 steroidogen-
FIG. 4. A) There was no significant difference in the change from baseline in fetal ACTH responses to a 1-mg bolus dose of CRH injected at
time point 0 between the restricted periconceptional nutrition group (RR and R-C, n 5 5) and the control periconceptional nutrition group (C-C
and C-R, n 5 8) in twin fetuses between 139 and 144 days of gestation.
There was however a significant effect of time in both the restricted and
control periconceptional nutrition groups (#, P , 0.05). B) Restricted periconceptional nutrition (R-R and R-C, n 5 5) resulted in a significantly
greater change from baseline in fetal cortisol responses to a 1-mg bolus
dose of CRH compared with control periconceptional nutrition (C-C and
C-R, n 5 8) in twin fetuses (*, P , 0.05).
esis 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, how-
1568
EDWARDS AND MCMILLEN
ever, 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 E 2, 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 periconcep-
tional 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.
ACKNOWLEDGMENTS
We are indebted to Dr. Simon Walker and staff at the South Australian
Research and Development Institute for their invaluable assistance with
the nutritional regimens and sheep mating programs. We would like to
thank Dr. Julie Owens and Professor Jeffrey Robinson for their helpful
advice during the early phases of this work.
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