PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Int J Obes (Lond). Author manuscript; available in PMC 2017 September 24.
Published in final edited form as:
PMCID: PMC5548625
NIHMSID: NIHMS861035

Prenatal Steroid Administration Leads to Adult Pericardial and Hepatic Steatosis in Male Baboons

Anderson H Kuo, MD,1 Junfei Li,2,4 Cun Li, MD,3,4 Hillary F Huber, PhD,3 Matthias Schwab, MD,5 Peter W Nathanielsz, MD, PhD, ScD, FRCOG,3,4 and Geoffrey D Clarke, PhD, FACR, FAAPM1,4

Abstract

Developmental programming studies indicate that glucocorticoids modify fetal development. We hypothesized that administration of the synthetic glucocorticoid (sGC) betamethasone to pregnant baboons at doses and stages of fetal life equivalent to human obstetric practice to decrease premature offspring morbidity and mortality, programs lipid metabolism. In 10-year-old male baboons (human equivalent 40) exposed in fetal life to betamethasone or saline, we quantified pericardial fat and hepatic lipid content with magnetic resonance imaging and spectroscopy. sGC offspring delivered at term as do most sGC exposed human neonates. Pericardial fat thickness (7.7 ± 3.6 mm vs. 3.1 ± 1.1 mm, M ± SD; p = 0.022; n=5) and hepatic fatty acids (13.3 ± 11.0 % vs. 2.5 ± 2.2 %; p = 0.046; n=5) increased following sGC without birth weight or current body morphometric differences. Our results indicate that antenatal sGC therapy caused abnormal fat deposition and adult body composition in mid-life primate offspring. The concern raised is that this degree of pericardial and hepatic lipid accumulation can lead to harmful local lipotoxicity. In summary, developmental programing by sGC produces a mid-life metabolically obese but normal weight phenotype. Prior studies show sexually dimorphic responses to some programming challenges thus female studies are necessary.

Keywords: Developmental programming, antenatal glucocorticoid, adiposity, baboons

Introduction

The developmental programming hypothesis states that challenges in critical fetal developmental time windows alter developing structure and function of fetal organs, predisposing to several chronic conditions including obesity and cardiovascular disease. Glucocorticoids play a central role in fetal cardiovascular development (1). Women at risk for premature delivery receive synthetic glucocorticoids (sGC) to accelerate fetal lung maturation, reducing perinatal mortality and morbidity in premature babies. Respiratory distress declines from about 70% to 12% (2). However, more women who receive a complete course of sGC deliver after 34 weeks than before by 4:1 (3). Human studies indicate adverse postnatal effects in offspring exposed to antenatal sGC delivering preterm, including cardiovascular (4) and attention problems (5). Given the well-established therapeutic benefits of sGC, information on long-term side effects need to be assessed in relation to cost-benefit. There is a need for more information. Current human studies are limited by focusing on sGC-exposed babies born prematurely, excluding the majority of exposed babies born at term. Multiple animal studies show that fetal exposure to glucocorticoid levels higher than appropriate for current fetal development can program obesity and cardiovascular function (6).

Little information is available on adverse effects of fetal primate exposure to sGC (7). A single course of maternal sGC alters fetal skeletal muscle metabolism (8). Early clinical studies showed that beneficial fetal effects of sGC only lasted a week (2), leading to some women threatening preterm delivery receiving multiple weekly sGC courses. We hypothesized that antenatal betamethasone administration to pregnant baboons in doses equivalent to those administered clinically results in increased adult offspring adiposity. Using magnetic resonance imaging and spectroscopy, we examined the extent of hepatic and pericardial lipid deposition in 10-year-old baboons (human equivalent 40 years) exposed as fetuses to the equivalent of three weekly sGC courses.

Materials and Methods

All procedures were approved by the Texas Biomedical Research Institute IACUC and conducted in AAALACi approved facilities.

Animal Model

Non-pregnant, female baboons (Papio species) of uniform phenotype were randomized into two groups (5 per group) and bred to the same male. Pregnant baboons were given intramuscular betamethasone phosphate (sGC; Celestan; Essex Pharma, Munich, Germany), two injections of 175 μg/kg spaced by 24h) or saline (CTL) at weekly intervals at 0.60, 0.64, and 0.68 gestation (pregnancy day 111/112, 118/119, and 125/126), equivalent to 24, 26, and 28 weeks of human pregnancy. This dose represents the human clinical dose, weight-adjusted for a 70kg woman. Offspring delivered spontaneously at term and were housed with their mothers until weaned and reared in juvenile group cages. Feed was Monkey Diet 5038 (Purina LabDiets, St Louis, MO) containing 13% calories from fat, 18% calories from protein, 69% calories from carbohydrates, mineral and vitamin additives, and metabolizable energy content of 3.22 kcal/g.

Morphometric Measurements, Magnetic Resonance Imaging, and Spectroscopy

Morphometric measurements and MRI were performed on two groups of baboons: sGC (5 males, age = 10.5 ± 0.7 yr; mean ± SD) and age-matched control baboons (CTL, 5 males, age = 10.4 ± 0.4 yr. Morphometric evaluation included measurement of body length, crown-rump length, chest circumference, waist circumference, and hip circumference. Body mass index was calculated using the standard formula

BMI=Weight(TotalBodyLength)2

weight (kilograms), and body length (meters). MRI was performed using a 3.0 Tesla MR scanner (TIM Trio, Siemens Healthcare, Malvern, PA). T1 weighted inversion recovery short axis cardiac images were obtained centered at the midventricular level (TE/TR/TI 1.75/934/480 ms). Spatial localized magnetic resonance 1H spectroscopy (MRS) was performed with regions of interest defined in the right hepatic lobe, avoiding visible portal bundles. All studies were conducted between 9 and 11 am after overnight fast. Anesthesia was induced with ketamine hydrochloride (12 mg/kg, i.m.) and maintained with isoflurane (0.8–1.0%).

Image Processing and Analysis

The CMR42 image analysis package (Circle Cardiovascular, Calgary, AB) was used for assessment of pericardial fat. Using short axis images, thickness of pericardial fat was measured at the cardiac mid-ventricular level anteriorly. The MRS data were fitted using jMRUI software with Lorentzian shape and the Advanced Magnetic Resonance (AMARES) algorithm to measure concentration of intrahepatic triglycerides.

Data were analyzed using GraphPad Prism 7. Grubbs’ test (extreme Studentized deviate) was used to determine outliers. Normal distribution was assessed by the d’Agostino-Pearson test. Student’s t test and Pearson’s correlation were performed. Data are presented as mean ± SD, N = 5 per group; significance at p < 0.05.

Results

Birth weight (CTL 1.0 ± 0.07 kg, sGC 1.0 ± 0.05 kg, M ± SD) and body weight at MRI (34 ± 3.1 kg, 33 ± 4.0 kg) were similar between groups. Body mass index (22 ± 2.2, 22 ± 2.2), whole body length (124 ± 5.3 cm, 122 ± 4.3 cm), crown-rump length (74 ± 4.9 cm, 73 ± 3.6 cm), chest circumference (68 ± 6.8 cm, 67 ± 2.8 cm), waist circumference (62 ± 9.4 cm, 62 ± 8.5 cm), and hip circumference (54 ± 3.7 cm, 51 ± 3.6 cm) were similar.

Mid-ventricular pericardial fat thickness was 148% higher in sGC (7.7 ± 3.6 mm, M ± SD) vs. CTL (3.1 ± 1.1 mm, p = 0.022), Figure 1. Hepatic parenchymal lipid content increased 432% in sGC (13.3 ± 11.0 %) vs. CTL (2.5 ± 2.2 %, p = 0.046), Figure 2. Anterior pericardial fat and intrahepatic lipid content correlated positively (r = 0.66, p < 0.05).

Figure 1
Prenatal betamethasone administration results in increased pericardial fat deposition
Figure 2
Prenatal betamethasone administration results in increased hepatic steatosis

Discussion

We demonstrated increased pericardial and hepatic fat deposition in baboons exposed to prenatal sGC without difference in birth weight, current body weight, or other body measurements, indicating programming of adiposity. Our results represent a major concern and indicate long-term alteration in lipid metabolism without discernible change in external phenotype following fetal sGC exposure at a clinical equivalent weight-adjusted dose. Though not NIH recommended, multiple courses are often given to women threatening preterm delivery not delivering within a week or as rescue therapy in late pregnancy.

Our finding of sGC induced pericardial and hepatic steatosis confirmed rodent studies showing metabolic dysregulation. Administration of dexamethasone in late gestation causes glucose intolerance (9) and alters GC receptor expression in hepatic and visceral fat (10) in adult rat offspring. Increased intra-abdominal fat deposition has been documented in prenatal dexamethasone exposed rats with concurrent serum leptin increase (11). Our findings suggest similar abnormal adult primate fat deposition.

Besides implicating metabolic derangement, increased hepatic and pericardial fat deposition raise cardiovascular concerns. The Framingham Heart Study reported association between pericardial adiposity and coronary artery calcification, thought to reflect local toxic pericardial fat effects on adjacent vasculature (12). Likewise, the Multi-Ethnic Study of Atherosclerosis (MESA) concluded that pericardial fat predicts coronary heart disease incidence independently of conventional risk factors (13). Hepatic steatosis is the first sign of nonalcoholic fatty liver disease affecting up to one third of Americans (14). Excess hepatic lipid deposition leads to oxidative stress, mitochondrial dysfunction, and hepatic lipid peroxidation, potentiating nonalcoholic steatohepatitis, presaging end-stage liver disease.

Our finding of unchanged birth weight is noteworthy because it suggests the lack of change in birth weight alone should not be considered evidence for lack of adverse programming. Studies on asthmatic pregnant mothers (15) and fetuses suspected of congenital adrenal hyperplasia (16) revealed no decrease in birth weight with antenatal glucocorticoid treatment, interpreted as an indicator of treatment safety. Further, our findings of unchanged birth weight or current weight indicate a potentially large at-risk population. Our results agree with the Auckland Steroid Trial, which reported insulin resistance but not birth weight and current weight difference in adult offspring who received antenatal betamethasone treatment (17). The concurrent increase in hepatic and pericardial fat deposition and unchanged body weight are consistent with alteration in body composition. We postulate prenatal programming may contribute to the curious “metabolically obese but normal weight” phenotype (18). Our study highlights the need to consider prenatal steroid exposure as part of offspring medical history even if the child is delivered at term, as programming is likely to predispose to lipid metabolic dysfunction and affect life course health. Our study also indicates the need for evaluating pathophysiological levels of maternal GC resulting from maternal stress in pregnancy on offspring obesity phenotype.

In summary, we report a metabolically vulnerable phenotype in adult male baboons exposed to human clinical equivalent antenatal sGC treatment, evidenced by increased hepatic and pericardial fat deposition without changes in body weight. The presence of pericardial and hepatic lipid abnormalities is of concern as they can exert local cytotoxic effects. While the number of subjects is only 5, careful standardization of pre-pregnant maternal phenotype and weight adjustment of the sGC doses allowed demonstration of a highly statistically significant (p =0.022) 150% increase in pericardial fat thickness and 432% increase in liver lipid (p=0.046) in baboons exposed as fetuses to equivalent of three weekly human clinical courses of sGC therapy. Importantly we adjusted the sGC dose for maternal weight a standardization not performed in clinical obstetric practice. Our findings agree with the MACS study that multiple courses of sGC are not recommended (19). Sexually dimorphic responses to antenatal sGC treatment have been documented in rats (20), and studies in females are needed to determine whether sexual dimorphism exists in primates.

Acknowledgments

Funding

This work was supported by the National Institutes of Health (5P01HD021350 to P.W.N., 5R24OD011183 to P.W.N., 5K25DK089012 to G.D.C., 1R25EB016631 to A.H.K., and OD P51 OD011133 from the Office of Research Infrastructure Programs/Office of the Director); EU FP 7/HEALTH/GA No.: 279281: BrainAge- Impact of Prenatal Stress on BRAINAGEing.

The authors thank Dr. Robert Lanford and the Southwest National Primate Center Staff for their ongoing technical support of our baboon research program described in this article. The authors also acknowledge the technical support of Steven Rios, Sam Vega, McKenna Considine, and Susan Jenkins, as well as the administrative support of Karen Moore.

Footnotes

Competing interests

The authors have no potential conflict of interest, financial or otherwise, to disclose.

References

1. Nathanielsz P, Berghorn K, Derks J, Giussani D, Docherty C, Unno N, et al. Life before birth: effects of cortisol on future cardiovascular and metabolic function. Acta Paediatrica. 2003;92:766–772. [PubMed]
2. Liggins GC, Howie RN. A controlled trial of antepartum glucocorticoid treatment for prevention of the respiratory distress syndrome in premature infants. Pediatrics. 1972;50:515–525. [PubMed]
3. Mahony R, McKeating A, Murphy T, McAuliffe F, O’Herlihy C, Foley M. Appropriate antenatal corticosteroid use in women at risk for preterm birth before 34 weeks of gestation. BJOG. 2010;117:963–967. [PubMed]
4. Karemaker R, Karemaker JM, Kavelaars A, Tersteeg-Kamperman M, Baerts W, Veen S, et al. Effects of neonatal dexamethasone treatment on the cardiovascular stress response of children at school age. Pediatrics. 2008;122:978–987. [PubMed]
5. Crowther CA, Doyle LW, Haslam RR, Hiller JE, Harding JE, Robinson JS. Outcomes at 2 years of age after repeat doses of antenatal corticosteroids. N Engl J Med. 2007;357:1179–1189. [PubMed]
6. Fletcher AJ, McGarrigle HH, Edwards CMB, Fowden AL, Giussani DA. Effects of low dose dexamethasone treatment on basal cardiovascular and endocrine function in fetal sheep during late gestation. J Physiol (Lond) 2002;545:649–660. [PubMed]
7. Uno H, Lohmiller L, Thieme C, Kemnitz JW, Engle MJ, Roecker EB, et al. Brain damage induced by prenatal exposure to dexamethasone in fetal rhesus macaques. I. Hippocampus. Dev Brain Res. 1990;53:157–167. [PubMed]
8. Blanco CL, Moreira AG, McGill-Vargas LL, Anzueto DG, Nathanielsz P, Musi N. Antenatal corticosteroids alter insulin signaling pathways in fetal baboon skeletal muscle. J Endocrinol. 2014;221:253–260. [PMC free article] [PubMed]
9. Nyirenda MJ, Lindsay RS, Kenyon CJ, Burchell A, Seckl JR. Glucocorticoid exposure in late gestation permanently programs rat hepatic phosphoenolpyruvate carboxykinase and glucocorticoid receptor expression and causes glucose intolerance in adult offspring. J Clin Invest. 1998;101:2174–2181. [PMC free article] [PubMed]
10. Cleasby ME, Livingstone DE, Nyirenda MJ, Seckl JR, Walker BR. Is programming of glucocorticoid receptor expression by prenatal dexamethasone in the rat secondary to metabolic derangement in adulthood? Eur J Endocrinol. 2003;148:129–138. [PubMed]
11. Dahlgren J, Nilsson C, Jennische E, Ho HP, Eriksson E, Niklasson A, et al. Prenatal cytokine exposure results in obesity and gender-specific programming. Am J Physiol Endocrinol Metab. 2001;281:E326–34. [PubMed]
12. Rosito GA, Massaro JM, Hoffmann U, Ruberg FL, Mahabadi AA, Vasan RS, et al. Pericardial fat, visceral abdominal fat, cardiovascular disease risk factors, and vascular calcification in a community-based sample: the Framingham Heart Study. Circulation. 2008;117:605–613. [PubMed]
13. Ding J, Hsu FC, Harris TB, Liu Y, Kritchevsky SB, Szklo M, et al. The association of pericardial fat with incident coronary heart disease: the Multi-Ethnic Study of Atherosclerosis (MESA) Am J Clin Nutr. 2009;90:499–504. [PubMed]
14. Browning JD, Szczepaniak LS, Dobbins R, Horton JD, Cohen JC, Grundy SM, et al. Prevalence of hepatic steatosis in an urban population in the United States: impact of ethnicity. Hepatology. 2004;40:1387–1395. [PubMed]
15. Murphy VE, Zakar T, Smith R, Giles WB, Gibson PG, Clifton VL. Reduced 11beta-hydroxysteroid dehydrogenase type 2 activity is associated withdecreased birth weight centile in pregnancies complicated by asthma. J Clin Endocrinol Metab. 1:1660–1668. [PubMed]
16. Mercado AB, Wilson RC, Cheng KC, Wei JQ, New MI. Prenatal treatment and diagnosis of congenital adrenal hyperplasia owing to steroid 21-hydroxylase deficiency. J Clin Endocrinol Metab. 1995;80:2014–2020. [PubMed]
17. Dalziel SR, Walker NK, Parag V, Mantell C, Rea HH, Rodgers A, et al. Cardiovascular risk factors after antenatal exposure to betamethasone: 30-year follow-up of a randomised controlled trial. Lancet. 2005;365:1856–1862. [PubMed]
18. Karelis AD, St-Pierre DH, Conus F, Rabasa-Lhoret R, Poehlman ET. Metabolic and body composition factors in subgroups of obesity: what do we know? J Clin Endocrinol Metab. 2004;89:2569–2575. [PubMed]
19. Murphy KE, Hannah ME, Willan AR, Hewson SA, Ohlsson A, Kelly EN, et al. Multiple courses of antenatal corticosteroids for preterm birth (MACS): a randomised controlled trial. Lancet. 2009;372:2143–2151. [PubMed]
20. O’Regan D, Kenyon CJ, Seckl JR, Holmes MC. Glucocorticoid exposure in late gestation in the rat permanently programs gender-specific differences in adult cardiovascular and metabolic physiology. Am J Physiol Endocrinol Metab. 2004;287:E863–70. [PubMed]