Effect of moderate, 30 percent global maternal nutrient reduction on fetal and postnatal baboon phenotype

Abstract

Background

Most developmental programming studies on maternal nutrient reduction (MNR) are in altricial rodents whose maternal nutritional burden and offspring developmental trajectory differ from precocial nonhuman primates and humans.

Methods

Control (CTR) baboon mothers ate ad libitum; MNR mothers ate 70% global control diet in pregnancy and lactation.

Results

We present offspring morphometry, blood cortisol, and adrenocorticotropin (ACTH) during second half of gestation (G) and first three postnatal years. Moderate MNR produced intrauterine growth restriction (IUGR). IUGR males (n=43) and females (n=28) were smaller than CTR males (n=50) and females (n=47) in many measurements at many ages. In CTR, fetal ACTH increased 228% and cortisol 48% between 0.65G and 0.9G. IUGR ACTH was elevated at 0.65G and cortisol at 0.9G. 0.9G maternal gestational weight gain, fetal weight, and placenta weight were correlated.

Conclusions

Moderate IUGR decreased body weight and morphometric measurements at key timepoints and altered hypothalamo-pituitary-adrenal function.

Introduction

In human epidemiological and animal studies reduced maternal nutrition alters offspring organ structure and function, leading to Developmental Programming that predisposes offspring to chronic diseases, such as hypertension, obesity, and diabetes.^1,2 Developmental programming can be defined as responses to challenges during critical time windows that alter development with persistent effects on phenotype that may not emerge until later life. Life course altricial, polytocous rodent programming studies and data are extensive.^3–5 Fewer data are available in precocial, monotocous species. The two main precocial species studied are sheep^6–9 and guinea-pigs.^10–12 There is a need for data from nonhuman primates to translate to human development.

We have characterized the effects of moderate maternal nutrient reduction – and hence fetal nutrient reduction – on fetal growth and development of offspring from birth to three years of life. In this baboon (Papio hamadryas) model, control mothers are fed ad libitum while nutrient reduced mothers are fed 70% of the global diet eaten by controls throughout pregnancy and lactation. We have demonstrated that this moderate degree of maternal nutrient reduction leads to intrauterine growth restriction (IUGR) at term.¹³ We have also reported altered development of the placenta,^14–16 fetal kidney,^17–19 fetal brain,^20,21 fetal liver,^22,23 fetal skeletal muscle,²⁴ and fetal heart.²⁵ In postnatal life, these IUGR offspring show cognitive and behavioral changes,^26–28 accelerated aging of the heart,^29–31 and a pre-diabetic metabolic phenotype³² when compared to controls. We present here the weights and morphometrics of control and IUGR fetuses over the second half of gestation, at birth, and over the first three years of life. Because the fetal hypothalamo-pituitary-adrenal (HPA) axis plays a central role in balancing perinatal growth and differentiation in the second half of pregnancy,³³ we also measured plasma adrenocorticotropin (ACTH) in fetuses and cortisol levels in fetuses and juveniles.

Materials and methods

Humane care guidelines

All procedures were approved by the Texas Biomedical Research Institute Institutional Animal Care and Use Committee and conducted in AAALAC approved facilities.³⁴

Animal management and housing

Baboons were housed in group cages with normal physical and social interaction and allowing control and monitoring of food intake (Fig 1).³⁴ We recruited 156 non-pregnant female baboons and housed up to 16 per cage (Fig 1) with a male in custom-built group housing with full socialization, environmental enrichment, and physical activity at Southwest National Primate Research Center (SNPRC) under optimum conditions supervised by SNPRC veterinarians. Details of housing and breeding have been described previously.^34,35 After confirmation of pregnancy at 0.16 gestation, mothers were randomly assigned to the control group (CTR) or the maternal nutrient reduction group (MNR). Ad libitum food intake (Purina Monkey Diet 5038, Purina, St Louis, MO, USA) of CTR mothers was calculated weekly on a per kilogram basis. The MNR group received 70% of the average daily ad libitum amount eaten on a weight-adjusted basis by CTR animals of the same gestational age. Student's t-test showed that CTR and MNR mothers were similar in pre-pregnancy (30 days pre-conception) body weight (CTR 14.14 ± 0.41 kg; MNR 14.96 ± 0.54 kg; p = 0.24), age (CTR 11.18 ± 0.53 yrs; MNR 10.77 ± 0.78 yrs; p = 0.66), and food consumption (CTR 64.84 ± 3.80 kcal/kg/day; MNR 64.81 ± 3.45 kcal/kg/day; p = 0.10).

Figure 1.

In subgroups of pregnant baboons, C-sections were performed^35,36 at the following percentages of gestation (term 180 - 185 days): 50 percent (n = 23; 13 male fetuses and 10 female fetuses), 65 percent (n = 22; 15 male fetuses and 7 female fetuses), 75 (n = 27; 16 male fetuses and 11 female fetuses), 90 percent (n = 44; 23 male fetuses and 21 female fetuses), and 95 percent (n = 12; 7 male and 5 female fetuses). Fetuses were euthanized while still under general anesthesia and a full morphometric examination (Table 1) was undertaken by the same team of investigators at all C-sections. Forty mothers delivered live newborns by spontaneous vaginal delivery. Mothers with suckling infants remained on their pregnancy diets and continued to be housed in the same group cages until offspring were self-weaned at approximately 9 months of age. After weaning, offspring were separated into male and female juvenile groups and ate normal monkey chow (Purina Monkey Diet 5038, Purina, St Louis, MO, USA).

Table 1.

Measurement	Method
Body length (cm)	largest protrusion of the coccyx to the heel of the right foot, added to crown-rump length
Chest circumference (cm)	around the chest at the level of the nipples
Crown-rump length (cm)	back of the head at the intersection of the parietal and lambdoidal sutures to the rump at the largest protrusion of the coccyx
Femur length (cm)	right side of the body from the most superior point on the head of the femur to the most inferior point on the distal condyles
Head circumference (cm)	over the most prominent part on the back of the head (occiput) and just above the eyebrows (supraorbital ridges), i.e., the largest circumference of the head
Hips circumference (cm)	around the hips at the level of the greatest protrusion of the innominate/pelvic bone (anterior superior iliac spine)
Abdominal circumference (cm)	around the waist at the level of the umbilicus

Morphometrics

Offspring of CTR and MNR mothers were weighed and body dimensions measured at C-section, birth, and at 0.5, 1, 2, 3, 6, 9, 12, 18, 24, 30, and 36 months of age. We measured total body weight with a digital scale. Body length, crown to rump length, head circumference, abdominal circumference, chest circumference, and hips circumference were assessed with a tape measure by trained and experienced technicians (Table 1). All measurements were taken three times and the means used as final values.

We tested inter-rater reliability of each of the six body dimension measurements (body length, crown to rump length, head circumference, abdominal circumference, chest circumference, hip circumference) in a set of juvenile baboons (n = 6 males, ages 1.1-1.3 years). Two technicians each measured all six baboons in all six dimensions three times. We analyzed the data using intraclass correlation coefficient (ICC).³⁷ ICC can be interpreted as follows: 0-0.2 indicates poor agreement; 0.3-0.4 indicates fair agreement; 0.5-0.6 indicates moderate agreement; 0.7-0.8 indicates strong agreement; and >0.8 indicates almost perfect agreement. While these values are arbitrary cutoffs, they have been used to interpret a wide variety of inter-rater reliability studies.^38–41 All six measurements produced ICC values ≥ 0.8, indicating strong reproducibility between raters.

Validation study to demonstrate that ACTH and cortisol do not rise within 10 minutes of ketamine tranquillization

To ensure that plasma ACTH and cortisol levels were not subject to elevation due to the necessity for ketamine immobilization of group-housed animals prior to venipuncture, we conducted a separate validation study on a subset of the same baboons at 3.5 ± 0.18 years of age on a tether system wearing a protective jacket and fitted with indwelling vascular catheters fitted at surgery under general anesthesia. This system allows baboons to be maintained with free movement in individual cages and their blood sampled without any exposure to restraint or pharmacological agents. Blood samples were taken at least a week after the surgery for placement of the vascular catheters. A baseline blood sample was taken in the fully conscious, unmedicated state immediately before tranquilization with ketamine (10mg/kg intramuscular, IM), and then two further samples 5 and 10 minutes later to observe any changes in cortisol and ACTH. Figure 2 shows that ketamine does not alter ACTH or cortisol in the first 10 minutes following the injection.

Figure 2.

Blood sampling

Blood samples were taken at birth and at 0.5, 1, 2, 3, 6, 9, 12, 18, 24, 30, and 36 months of age. Blood samples were taken in the group cage area within 5 minutes of isolation and tranquilization with 10mg/kg ketamine IM in the chute shown in Figure 1C. ACTH_1-39 was measured by a two-site ELISA and cortisol by chemiluminescent immunoassay on an Immulite® 1000 Immunoassay System (Siemens Healthcare Diagnostics). The intra-/inter-assay CVs for ACTH were 4.8 /7.2. The intra-/inter-assay CVs for cortisol were 5.6/8.4.

Statistical analyses

Linear regression was used to provide correlation and hence potential evidence of causative effects during pregnancy of the variables maternal gestational weight gain, fetal weight, and placenta weight. All morphometric measurements and blood values were compared between CTR and IUGR at each time point using unpaired t-tests. One-way ANOVA with Tukey's multiple comparison tests were used to compare cortisol and ACTH levels of controls at different points during gestation. Data are presented as mean ± standard error of the mean (SEM).

Results

Relationships between maternal gestational weight gain, fetal weight, and placenta weight

Gestation duration was similar between groups (CTR 180.8 ± 1.58 days; MNR 183.1 ± 1.12 days, p = 0.27). CTR weight gain during pregnancy (1.96 ± 0.23 kg) was greater than MNR (0.12 ± 0.25 kg; p < 0.001). At 0.5 gestation (G), 0.65G, or 0.75G there were no significant correlations between fetal weight and maternal gestational weight gain, fetal weight and placenta weight, or gestational weight gain and placenta weight, whether the treatment groups were assessed separately or together. However, all three relationships were highly significant at 0.9G: R = 0.60, P < 0.0001; R = 0.71, P < 0.0001; R = 0.57, P = 0.0001, respectively (Fig 3).

Figure 3.

Fetal morphometrics

Mean body weights of IUGR fetuses were lower than CTR in both males (Table 2A) and females (Table 2B) at every stage of gestation. However, the difference in body weight between IUGR and CTR only approached or reached statistical significance in males at 75%, 90%, and 100% of gestation. Other morphometric measurements tended to be smaller in IUGR than CTR. In males, IUGR had smaller chest and head circumferences at 90%G (Table 2A); in females, IUGR body length was shorter at 65%G (Table 2B).

Table 2.

A
% Gest.	Group	Weight (g)	Length (cm)	Crown-rump (cm)	BMI (kg/m2)	Head circ. (cm)	Chest circ. (cm)	Abdomen circ. (cm)	Hips circ. (cm)
50	CTR	105.7 ± 5.25	17.3 ± 0.45		3.54 ± 0.13		9.14 ± 0.32	8.06 ± 0.25	7.38 ± 0.24
	IUGR	103.7 ± 4.69	17.5 ± 0.29		3.41 ± 0.22		9.00 ± 0.18	8.00 ± 0.26	7.00 ± 0.29
65	CTR	333.6 ± 29.3	27.5 ± 1.29	19.6 ± 1.21	4.39 ± 0.24	17.0 ± 0.54	12.4 ± 0.47	10.8 ± 0.45	11.5 ± 0.57
	IUGR	341.4 ± 13.20	28.1 ± 0.84	19.7 ± 0.49	4.35 ± 0.17	17.5 ± 0.18	12.5 ± 0.25	10.3 ± 0.29	11.2 ± 0.28
75	CTR	528.3 ± 13.0	31.6 ± 0.68	21.7 ± 0.44	5.32 ± 0.21	19.7 ± 0.31	14.2 ± 0.64	12.2 ± 0.49	13.4 ± 0.24
	IUGR	488.9 ± 13.7+	30.7 ± 0.83	22.7 ± 0.61	5.22 ± 0.20	19.2 ± 0.32	13.9 ± 0.32	11.5 ± 0.31	12.3 ± 0.87
90	CTR	828.0 ± 27.2	38.9 ± 0.67	26.6 ± 0.73	5.50 ± 0.19	22.5 ± 0.26	17.2 ± 0.27	14.7 ± 0.39	14.8 ± 0.71
	IUGR	745.2 ± 23.7*	37.7 ± 0.83	26.1 ± 0.72	5.28 ± 0.24	21.5 ± 0.30*	16.6 ± 0.27+	13.9 ± 0.30	14.7 ± 0.43
B
% Gest.	Group	Weight (g)	Length (cm)	Crown-rump (cm)	BMI (kg/m2)	Head circ. (cm)	Chest circ. (cm)	Abdomen circ. (cm)	Hips circ. (cm)
50	CTR	98.5 ± 2.98	17.9 ± 0.43		3.09 ± 0.13		8.70 ± 0.12	7.30 ± 0.25	32.0 ± 15.0
	IUGR	98.2 ± 3.34	17.5 ± 0.52		3.20 ± 0.12		9.35 ± 0.58	7.40 ± 0.37	6.67 ± 0.17
65	CTR	308.9 ± 19.1	26.5 ± 0.25	18.5 ± 0.20	4.39 ± 0.30	16.8 ± 0.46	11.5 ± 0.35	9.50 ± 0.2	11.25 ± 0.32
	IUGR	285.1 ± 11.2	25.0 ± 0.76	18.5 ± 0.76+	4.57 ± 0.14	17.1 ± 0.35	11.7 ± 0.44	8.83 ± 0.44	10.33 ± 0.44
75	CTR	445.4 ± 20.3	29.3 ± 0.30	19.9 ± 0.35	5.18 ± 0.21	18.9 ± 0.22	13.6 ± 0.18	11.4 ± 0.27	12.4 ± 0.20
	IUGR	464.6 ± 11.3	30.7 ± 2.42	21.2 ± 1.92	5.10 ± 0.68	19.7 ± 0.44	13.7 ± 0.60	10.88 ± 0.17	12.8 ± 0.17
90	CTR	756.9 ± 30.0	36.8 ± 0.78	25.4 ± 0.53	5.59 ± 0.16	21.6 ± 0.27	17.4 ± 0.41	14.8 ± 0.53	14.5 ± 0.53
	IUGR	704.3 ± 31.5	38.3 ± 2.22	25.7 ± 1.56	5.02 ± 0.42	21.4 ± 0.38	16.7 ± 0.34	13.7 ± 0.30	13.9 ± 0.52

Fetal ACTH and cortisol

Values for fetal ACTH and cortisol in CTR animals were distinct at different points in gestation (Fig 4). In CTR animals, ACTH was similar from 0.5G to 0.65G, increased from 0.65G to 0.9G, and was similar between 0.9 and 0.95G. Cortisol was also similar from 0.5G to 0.65G, and then increased at both 0.9G and 0.95G.

Figure 4.

Compared to CTR, IUGR showed higher ACTH at 0.65G, and higher cortisol at 0.9G (Fig 5).

Figure 5.

Maternal cortisol

There were no changes in maternal cortisol over the period studied and no differences between mothers of CTR and IUGR fetuses (Fig 6).

Figure 6.

Postnatal morphometrics

Mean body weight values were numerically lower for IUGR than CTR in both males (Table 3A) and females (Table 3B) at every age of measurement, with the difference approaching or reaching statistical significance at most ages. At birth and at 12 months of age, male IUGR weighed 13% less than did male CTR, with the level of difference fluctuating between those ages. Female IUGR weighed 12% less than female CTR at birth, and at 12 months of age, 15% less. By 18 months of age, the IUGR animals of both sexes had similar body weight. In both sexes, IUGR were significantly smaller than CTR in a variety of measurements at a variety of ages in the first year of life. IUGR males were smaller than CTR males at birth in abdominal size; at 0.5 months in chest size; at 2 months in body length, crown-rump length, and BMI; at 3 months in crown-rump-length; at 6 months in BMI and sizes of the head, chest, and hips; and at 30 months in body length and crown-rump length (Table 3A). IUGR females were smaller than CTR females at birth in abdominal and hip size; at 0.5 months in BMI; at 1 month in BMI and abdominal distance; at 2 months in head and chest size; at 3 months in BMI and sizes of the chest, abdomen, and hips; at 6 months in BMI, and sizes of the chest, abdomen, and hips; at 9 months in body length, crown-rump length, and sizes of the head, chest, and hips; at 12 months in body length and sizes of the chest and hips (Table 3B). After age 12 months, IUGR and CTR female morphometrics were similar (Table 3B). During the first year of life, growth appeared to be asymmetric in female offspring, as seen in the significantly different head to abdominal circumference ratios (Fig 7).

Table 3.

A
Age (months)	Group	Weight (kg)	Length (cm)	Crown-rump (cm)	BMI (kg/m2)	Head circ. (cm)	Chest cire, (cm)	Abdomen circ. (cm)	Hips circ. (cm)
0	CTR	0.93 ± 0.04	38.5 ± 0.73	25.9 ± 0.78	6.10 ± 0.12	22.5 ± 0.51	19.8 ± 0.36	16.3 ± 0.38	16.2 ± 0.59
	IUGR	0.82 ± 0.03*	38.0 ± 0.82	24.9 ± 1.18	5.69 ± 0.21	22.5 ± 0.50	20.1 ± 0.39	17.8 ± 0.81±	16.0 ± 0.46
0.5	CTR	1.07 ± 0.04	39.2 ± 1.29	27.6 ± 0.36	7.22 ± 0.63	23.7 ± 0.35	21.1 ± 0.37	18.6 ± 0.72	17.4 ± 0.90
	IUGR	0.89 ± 0.05*	39.0 ± 1.20	26.1 ± 1.14	5.92 ± 0.41	22.8 ± 0.42	19.5 ± 0.67*	18.5 ± 1.17	15.9 ± 1.01
1	CTR	1.13 ± 0.05	41.4 ± 0.87	28.2 ± 0.65	6.67 ± 0.33	24.4 ± 0.49	21.4 ± 0.35	19.3 ± 0.85	17.8 ± 0.68
	IUGR	1.10 ± 0.05	41.1 ± 0.84	27.9 ± 1.44	6.57 ± 0.34	24.1 ± 0.54	21.1 ± 0.47	18.3 ± 0.68	17.7 ± 0.81
2	CTR	1.36 ± 0.07	47.5 ± 1.02	32.6 ± 0.86	6.04 ± 0.26	25.4 ± 0.31	22.9 ± 0.5	18.2 ± 0.63	18.7 ± 0.86
	IUGR	1.28 ± 0.07	43.2 ± 0.71**	29.9 ± 1.10+	6.9 ± 0.33+	24.7 ± 0.85	22.6 ± 0.60	20.4 ± 3.13	17.5 ± 0.68
3	CTR	1.59 ± 0.08	49.8 ± 1.05	34.1 ± 0.96	6.42 ± 0.31	25.7 ± 0.33	24.0 ± 0.49	18.6 ± 0.69	18.9 ± 0.42
	IUGR	1.41 ± 0.08	49.1 ± 1.39	31.1 ± 1.13+	5.89 ± 0.30	25.4 ± 0.40	22.8 ± 0.50	18.4 ± 0.81	17.7 ± 0.66
6	CTR	2.34 ± 0.05	56.7 ± 0.83	38.0 ± 0.55	7.29 ± 0.20	27.7 ± 0.21	27.0 ± 0.31	22.8 ± 0.84	22.0 ± 0.67
	IUGR	1.89 ± 0.13**	55.0 ± 1.47	36.0 ± 1.36	6.20 ± 0.23**	26.7 ± 0.34*	24.8 ± 0.79*	21.1 ± 0.66	20.2 ± 0.53+
9	CTR	3.08 ± 0.05	62.8 ± 1.54	41.1 ± 0.82	7.93 ± 0.40	27.9 ± 0.94	30.0 ± 0.26	25.6 ± 0.64	24.1 ± 0.65
	IUGR	2.85 ± 0.11+	62.0 ± 1.53	40.9 ± 1.08	7.45 ± 0.28	28.3 ± 0.30	29.2 ± 0.70	25.3 ± 0.63	22.7 ± 0.87
12	CTR	4.24 ± 0.16	69.1 ± 0.85	43.7 ± 0.97	8.94 ± 0.45	29.5 ± 0.43	33.3 ± 0.68	29.5 ± 1.23	26.8 ± 0.98
	IUGR	3.73 ± 0.16*	66.9 ± 1.61	44.3 ± 1.04	8.31 ± 0.19	29.0 ± 0.23	33.5 ± 1.20	29.3 ± 1.47	25.8 ± 1.30
18	CTR	5.52 ± 0.24	75.4 ± 0.83	50.2 ± 1.35	9.67 ± 0.34	30.7 ± 0.51	37.0 ± 0.56	32.8 ± 0.89	30.5 ± 0.62
	IUGR	5.11 ± 0.25	73.7 ± 0.52	49.4 ± 0.98	9.41 ± 0.54	30.1 ± 0.15	36.4 ± 0.67	32.3 ± 0.78	29.1 ± 0.67
24	CTR	6.92 ± 0.27	82.0 ± 1.30	52.2 ± 0.76	10.28 ± 0.27	31.3 ± 0.56	39.2 ± 0.81	34.8 ± 0.72	33.6 ± 0.98
	IUGR	6.52 ± 0.17	79.7 ± 1.22	51.9 ± 1.37	10.13 ± 0.28	30.7 ± 0.61	39.5 ± 0.88	34.3 ± 0.96	32.2 ± 0.76
30	CTR	8.28 ± 0.37	87.6 ± 1.66	54.8 ± 1.02	10.81 ± 0.45	32.6 ± 0.38	43.2 ± 0.86	36.3 ± 1.06	35.2 ± 0.84
	IUGR	7.58 ± 0.23	83.5 ± 0.97+	52.3 ± 0.87+	10.89 ± 0.37	31.4 ± 0.84	42.2 ± 0.91	35.0 ± 1.07	34.0 ± 0.89
36	CTR	9.53 ± 0.47	92.4 ± 1.88	58.8 ± 1.82	11.12 ± 0.26	33.2 ± 0.66	45.4 ± 1.2	36.2 ± 1.19	36.3 ± 1.32
	IUGR	8.98 ± 0.35	90.6 ± 1.72	55.6 ± 1.59	10.96 ± 0.43	33.6 ± 0.6	44.3 ± 0.43	35.6 ± 0.69	38.1 ± 1.43
B
Age (months)	Group	Weight (kg)	Length (cm)	Crown-rump (cm)	BMI (kg/m2)	Head circ. (cm)	Chest circ. (cm)	Abdomen circ. (cm)	Hips circ. (cm)
0	CTR	0.82 ± 0.04	36.7 ± 1.05	24.4 ± 0.65	6.33 ± 0.52	22.0 ± 0.41	19.5 ± 0.45	17.3 ± 0.57	15.8 ± 0.62
	IUGR	0.73 ± 0.04	35.6 ± 1.07	24.6 ± 0.78	5.86 ± 0.48	21.7 ± 0.44	18.8 ± 0.68	15.0 ± 0.76*	14.3 ± 0.37+
0.5	CTR	0.97 ± 0.03	37.4 ± 1.13	25.1 ± 1.17	7.18 ± 0.61	22.9 ± 0.30	20.0 ± 0.58	17.7 ± 0.63	16.1 ± 0.70
	IUGR	0.81 ± 0.06*	37.5 ± 1.52	24.9 ± 1.06	5.74 ± 0.21+	22.5 ± 0.57	19.3 ± 0.49	16.5 ± 0.58	15.2 ± 0.67
1	CTR	1.05 ± 0.04	40.9 ± 0.91	28.0 ± 0.82	6.40 ± 0.32	23.6 ± 0.29	21.0 ± 0.51	18.1 ± 0.55	17.4 ± 0.72
	IUGR	0.89 ± 0.06*	40.7 ± 0.91	26.7 ± 0.93	5.35 ± 0.22*	23.6 ± 0.28	20.2 ± 0.99	17.4 ± 0.79	16.0 ± 0.86
2	CTR	1.29 ± 0.06	43.1 ± 1.23	29.6 ± 1.24	7.21 ± 0.66	24.7 ± 0.21	22.5 ± 0.51	18.8 ± 0.63	17.8 ± 0.61
	IUGR	1.07 ± 0.07*	41.8 ± 0.57	28.8 ± 1.08	6.07 ± 0.29	23.8 ± 0.40*	21.2 ± 0.56+	18.3 ± 0.71	16.4 ± 0.77
3	CTR	1.52 ± 0.06	47.5 ± 0.95	32.4 ± 1.52	6.80 ± 0.31	25.2 ± 0.27	23.6 ± 0.53	19.3 ± 0.57	18.8 ± 0.48
	IUGR	1.18 ± 0.09**	45.4 ± 1.45	30.5 ± 1.12	5.83 ± 0.51+	24.8 ± 0.24	21.2 ± 0.60**	16.8 ± 0.88*	16.4 ± 0.67**
6	CTR	2.24 ± 0.07	55.5 ± 1.23	37.2 ± 1.01	7.36 ± 0.31	26.0 ± 0.59	27.0 ± 0.53	23.0 ± 0.66	21.5 ± 0.68
	IUGR	1.62 ± 0.11**	53.5 ± 1.24	35.0 ± 1.30	5.57 ± 0.17**	25.7 ± 0.28	23.3 ± 0.86**	18.8 ± 0.71**	17.3 ± 0.71**
9	CTR	2.87 ± 0.10	62.4 ± 1.00	42.4 ± 0.71	7.41 ± 0.28	27.7 ± 0.21	29.4 ± 0.39	24.2 ± 0.43	23.1 ± 0.47
	IUGR	2.25 ± 0.11**	57.8 ± 1.47*	37.8 ± 0.96**	6.80 ± 0.40	26.9 ± 0.24*	26.7 ± 0.56**	22.6 ± 0.98	20.8 ± 0.73*
12	CTR	3.65 ± 0.13	65.3 ± 1.29	42.9 ± 1.36	8.52 ± 0.40	27.9 ± 0.3	31.6 ± 0.57	28.0 ± 0.89	25.4 ± 0.73
	IUGR	3.14 ± 0.14*	62.0 ± 1.28+	40.8 ± 0.92	8.24 ± 0.44	28.0 ± 0.38	29.6 ± 0.55*	27.5 ± 1.23	23.2 ± 0.72*
18	CTR	4.90 ± 0.20	72.6 ± 1.47	46.3 ± 1.41	9.15 ± 0.38	28.8 ± 0.42	35.5 ± 0.90	30.9 ± 0.93	28.0 ± 0.87
	IUGR	4.80 ± 0.24	71.7 ± 1.59	46.4 ± 1.38	8.97 ± 0.35	29.5 ± 0.29	34.7 ± 0.54	31.5 ± 0.65	27.5 ± 0.89
24	CTR	6.01 ± 0.20	78.6 ± 1.17	48.9 ± 1.17	9.75 ± 0.26	30.2 ± 0.38	37.9 ± 0.61	32.0 ± 0.66	30.2 ± 0.59
	IUGR	5.74 ± 0.33	77.3 ± 1.98	49.5 ± 1.24	9.61 ± 0.41	30.3 ± 0.38	37.8 ± 1.25	32.9 ± 1.06	30.6 ± 1.11
30	CTR	7.60 ± 0.20	84.5 ± 1.12	52.6 ± 1.01	10.55 ± 0.34	31.8 ± 0.44	42.0 ± 0.63	35.5 ± 0.72	34.0 ± 0.74
	IUGR	7.20 ± 0.40	82.4 ± 1.67	52.2 ± 1.12	10.54 ± 0.24	31.3 ± 0.31	40.6 ± 1.00	33.8 ± 0.87	31.5 ± 0.66*
36	CTR	8.74 ± 0.18	88.9 ± 1.18	54.8 ± 0.91	10.99 ± 0.29	32.2 ± 0.51	43.1 ± 0.61	35.6 ± 0.94	35.3 ± 0.84
	IUGR	8.55 ± 0.49	89.8 ± 1.83	55.0 ± 2.04	10.6 ± 0.56	32.5 ± 0.55	43.3 ± 1.44	33.7 ± 1.42	36.3 ± 1.68

Figure 7.

Postnatal cortisol

There were no differences between males and females so cortisol data were pooled. Cortisol was not different between CTR and IUGR at postnatal months 1, 2, 3, 6, 12, 18, 24, 30, or 36 (Fig 8A). In both IUGR and CTR, cortisol values were higher after weaning (Fig 8B).

Figure 8.

Discussion

Around 4-8% of human infants in the developed world are small for gestational age.^42,43 Among the unwanted consequences are predispositions to chronic conditions that decrease lifespan and impair the quality of life, such as hypertension, stroke, and adult onset diabetes.^43–46 There are many causes of IUGR, including maternal nutritional deficiency, placental insufficiency, maternal disease (e.g., hypertension and renal disease), and tobacco, alcohol, and recreational drug use.⁴³ The concept of developmental programming came to the fore in the 1990s.^1,2 Data from the Dutch Hunger Winter (November 1944 to May 1945) show that birth weight declined by about 10% when diet was restricted to a very low level.^47,48 Children born to the food deprived mothers of the Dutch Hunger Winter are predisposed to several life course chronic diseases, such as type 2 diabetes,^49,50 obesity,^51,52 and cardiovascular disease.^53,54 The reduction in birth weight observed is similar to the level of IUGR observed in the baboon fetuses in this study, just over 10%. A common limitation of human epidemiological studies is that the background health, nutrition, and disease burden of the mothers cannot be controlled. In addition, the burden of investigation that is tolerated by parents and children is limited in both extent and frequency. Maternal, fetal, and postnatal offspring tissues and blood samples are not available as easily as in animal studies.

To overcome these limitations and investigate the challenges, mechanisms, and outcomes of exposure to reduced nutrient availability during development, animal models have been extensively studied, including mice,^55–57 rats,^4,58–61 guinea pigs,⁶² and sheep.^63,64 However, the need for studies in a nonhuman primate to assist extrapolation from sheep and rodents to humans led us to develop the baboon model described here. To our knowledge, although effects of maternal obesity have been extensively investigated in a Japanese macaque model,^65–68 maternal nutrient reduction leading to IUGR has received less attention in NHP.

The morphometric data presented in this paper demonstrate that in our baboon model of IUGR, males and females are smaller than CTR at a variety of fetal and postnatal ages, in several dimensions. The difference in postnatal body weight is particularly pronounced. From birth through one year of age, male and female IUGR were lighter than CTR. By 18 months of age, the IUGR animals of both sexes had caught up, and body weight was similar between IUGR and CTR at older ages. Since offspring were self-weaned at around nine months of age and had free access to food thereafter, it seems likely their growth accelerated due to improved resource access after weaning. Catch-up growth after IUGR is a strong risk factor for later life type 2 diabetes, hypertension, obesity, osteoporosis, and cardiovascular disease (reviewed in ^44–46).

Postnatal body size and growth in the IUGR offspring were asymmetric. Asymmetrically small offspring are those in which weight is reduced proportionally more than length (i.e., reduced weight but normal length for gestational age).⁴³ IUGR offspring showed reduced weight at most ages of investigation, but not reduced body length compared to CTR. Additionally, there was a difference between IUGR and CTR in head/abdominal ratio at birth, with male IUGR showing reduced ratio compared to CTR, and female IUGR showing increased ratio compared to CTR. In females, this difference persisted until 6 months of age. The symmetry or asymmetry of individual IUGR offspring is a reflection of timing of the challenge to growth and development.⁴³ Symmetric IUGR tends to occur when the challenge comes early in gestation because the fetus responds with decreased cell number and cell size, resulting in overall small body size. In asymmetric IUGR, challenge to growth late in gestation leads to normal cell number but decreased cell size.⁴³ Risk of morbidity and mortality is higher in symmetric IUGR due to the increased incidence of congenital abnormalities,⁴³ but both types are subject to increased risk compared to offspring of normal size.^43–46 In this study, maternal nutrient reduction began at 0.16 gestation. Mothers had free access to food and were of normal weight and good health prior to pregnancy. As a result they were able to sustain normal growth and development in the early stages of pregnancy, but as their nutrient status declined, so did nutritional support for their fetuses.⁴³ Figure 6 shows the stress of this level of maternal nutrient reduction did not lead to increased maternal cortisol levels during pregnancy; therefore, maternal cortisol was unlikely to be the cause of the rise in fetal cortisol in the IUGR fetuses.

Cortisol and ACTH are controlled by the hypothalamo-pituitary-adrenal (HPA) axis, which regulates the stress response. The HPA axis is extremely sensitive to environmental perturbations.⁷¹ When the brain perceives stressors, the hypothalamus releases corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), prompting release of ACTH, which stimulates adrenal glucocorticoid secretion. Fetal adrenal function is relatively unresponsive to ACTH until late gestation when it increases dramatically.^72,73 This is shown clearly in this study by comparing the changes in ACTH and cortisol between 0.65-0.9G and 0.9-0.95G. In CTR fetal baboons, ACTH increased 228% between 0.65G and 0.9G while cortisol only increased by 48% (Fig 4). Between 0.9G and 0.95G mean ACTH remained statistically similar, while mean cortisol rose significantly by 46% (Fig 4). ACTH was higher in IUGR than CTR at 0.65G and fetal cortisol was higher in IUGR than CTR at 0.9G (Fig 5). These data suggest that the stress of maternal, and consequently fetal, nutrient reduction prematurely activates the HPA axis in the IUGR fetuses. The normal increase in cortisol in the late stages of gestation plays a central role in preparing the fetus for birth and adaptations needed for an independent extra-uterine existence.⁶⁹ Cortisol also increased substantially after weaning. This may arise from the stress associated with weaning, a well-established stressful process for primate mothers and infants.⁷⁰ These features of normal baboon hormonal development are important to consider when investigating effects of developmental programming, as they set the normative profile.

Cortisol levels in IUGR and CTR offspring were similar by one month of postnatal Iife, indicating removal of some of the potential factors contributing to stress in the IUGR fetuses, even though mothers were still receiving reduced nutrition. Our previous report from immunohistochemical studies in this baboon model of IUGR showed upregulation of the HPA axis at the hypothalamic level.⁷⁴ The altered function observed in the IUGR baboons implies overexposure during critical developmental windows to glucocorticoid levels inappropriate for the current stage of development.

These changes have implications for development of multiple organ systems. As mentioned above, in late gestation, cortisol plays a vital role in preparing the fetus for extrauterine life.⁶⁹ with maturational effects on major organs, including the thyroid, lungs, intestines, liver, kidney, and brain that favor cellular differentiation over proliferation.⁶⁹ We have reported impaired fetal neurodevelopment,^13,20,74 altered behavior,^26–28 increased insulin resistance,³² and accelerated aging of heart function in IUGR offspring.^29–31 These various studies show that challenges to the normal developmental trajectory of the fetus have both immediate and long-term consequences in the same critical organs whose development is shaped by perinatal cortisol.

In summary, the IUGR baboon cohort described in this study was exposed to a moderate level of maternal nutrient reduction (30%) during fetal and neonatal life until weaned. Even at this moderate level, numerous changes to pre- and post-natal offspring phenotype were observed. Our findings will focus attention on critical periods of development and potential involvement of the HPA axis. IUGR is a persistent problem for humans, and studies with NHP are necessary to determine the origins and outcomes of IUGR.