Objective
Synthetic glucocorticoid (sGC) administration to women threatening preterm delivery increases neonatal survival. Evidence shows that fetal exposure to glucocorticoid levels higher than appropriate for current maturation programs offspring development. We examined fetal sGC multigenerational effects on F1 and F2 female offspring hypothalamo-pituitary-adrenal axis (HPAA) function.
Study Design
At 0.7 gestation, pregnant F0 ewes received 4 dexamethasone injections (2 mg, approximately 60 μg/kg −1 per day −1 , 12 hours apart) or saline (control). F1 female offspring were bred to produce F2 female offspring. Postpubertal HPAA function was tested in F1 and F2 ewes.
Results
F1 and F2 ewe lambs showed reduced birthweight and morphometrics. Dexamethasone increased baseline but reduced stimulated HPAA activity in F1 and F2 female offspring.
Conclusion
This is the first demonstration that sGC doses in the clinical range have multigenerational effects on hypothalamo-pituitary-adrenal activity in a precocial species, indicating the need for the study of long-term effects of fetal sGC exposure.
The ground-breaking work of Liggins showed that glucocorticoids accelerate fetal lung development in sheep. A subsequent clinical trial demonstrated significant decreases in morbidity and mortality in premature babies whose mothers received synthetic glucocorticoids (sGCs) when threatening preterm delivery. It has now become routine clinical practice to treat women who threaten preterm delivery before 34 weeks of gestation with sGCs to improve outcomes in premature babies.
In the developing world, the incidence of preterm delivery is rising, with more than 10% of pregnancies resulting in preterm delivery, and these preterm deliveries account for 75% of all neonatal deaths. In one study in which the majority of patients received 4 or more weekly courses of glucocorticoids (GCs), delivery at less than 32 weeks occurred in only 24% of sGC-treated pregnancies and at less than 37 weeks in 63% of all treated pregnancies. Therefore, 76 % of all synthetic GC-exposed fetuses remained in utero for a significant period after fetal exposure and more than one third can be considered as delivering at term.
There is mounting evidence that in addition to the major benefits of accelerated lung maturation, fetal exposure to exogenous GC levels higher than appropriate for the current stage of fetal maturation produces intrauterine growth restriction in multiple species including sheep, nonhuman primates, and from data from retrospective analyses, in human pregnancy. Maternal administration of sGCs alters the trajectory of development of many fetal organ systems, and evidence is accumulating from animal studies that there are unwanted later-life developmental programming effects on offspring endocrine, renal, and metabolic function. These similarities across multiple species suggest common underlying mechanisms that result in adverse outcomes that need to be better understood.
Dexamethasone (DEX) treatment of F0 ewes in late gestation produces offspring (F1) who show fasting hyperglycemia and glucose intolerance and an increased glucose-stimulated insulin secretion in rats and sheep. Treatment of F0 pregnant ewes with sGCs early in the second half of gestation increased plasma adrenocorticotropic hormone (ACTH) and cortisol in their late-gestation fetuses. At 6 months, postnatal age F1 offspring from sCG-treated F0 mothers show no difference in hypothalamic-pituitary-adrenal-axis (HPAA) responsiveness to a combined challenge by corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP). However, at 1 year of age, the F1 sGC-exposed offspring showed elevated basal and stimulated cortisol levels with no change in ACTH response.
In 2 year old F1 offspring exposed to repeated maternal betamethasone injections, ACTH or cortisol levels were unaltered, but ACTH responses to CRH/AVP were increased. At 3 years of age, basal ACTH was elevated, and both basal and stimulated cortisol levels were suppressed, whereas basal and stimulated ACTH-cortisol ratios were suppressed following fetal exposure to repeated sGC injections to their mothers.
Female F1 rats exposed to DEX given to the F0 mother in her drinking water during days 15-21 of their own fetal life, although not treated with any agents during their own pregnancy, gave birth to male offspring who showed reduced birthweight, glucose intolerance, and elevated hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity. Multigenerational programming of metabolism was observed in offspring of male rats exposed prenatally to DEX mated with control females.
Translation of multigenerational findings of effects of sGC-induced developmental programming to possible effects of human fetal exposure requires studies in precocial species that more closely match the fetal maturational window of exposure and shorter duration, in both absolute terms and relative to the longer course of development, that occur during human clinical management of preterm labor. We hypothesized that exposure of F1 female fetal lambs to sGCs during their development at days 103-104 of gestation, equivalent of 27-28 weeks of human gestation, would have effects on both F1 and F2 female offspring basal HPAA and responses to challenges.
Materials and Methods
Care and use of animals
All procedures were approved by the University of Wyoming Animal Care and Use Committee. F0 nulliparous Rambouillet × Columbia cross-bred ewes (approximately 16 months of age) were used to produce the first filial generation (F1) ewe lambs studied. Founder generation (F0) ewes were bred to a single ram in 1 of 2 consecutive years. After natural mating, ewes were fed in accordance with National Research Council (NRC) requirements for maintenance. On day 103 and 104 of gestation, ewes averaged 73.6 ± 4.0 kg (mean ± SEM). DEX ewes (n = 10) received 4 injections of 2 mg of dexamethasone (intramuscularly; Vedco, St. Joseph, MO) 12 hours apart. Control (C) ewes (n = 12) received equal volumes of saline.
Ewes were allowed to lamb naturally and the flock that these ewes originated from has an approximate gestation length of 150 days. There were 8 C twin (2 sets were both females) and 4 C singles. In the DEX group, there were 7 twin (3 female twin sets) and 3 singles. After lambing, all F0 ewes were given free choice access to alfalfa hay.
Prior to 2 weeks of age, F1 female lambs were tail docked as per Federation of Animal Science Societies recommendations. F1 ewe lambs were given free access to a standard commercially available creep feed (Lamb Creep B30 w/Bovatec; Ranch-Way Feeds, Ft. Collins, CO) from birth to weaning at 4 months of age and placed in an outdoor housing facility with shelter and ad libitum water. These F1 ewe lambs were maintained in accordance with NRC requirements for replacement ewes. Diet consisted of alfalfa and corn to meet NRC requirements with ad libitum access to a trace mineral salt block. Diets were adjusted up for weight gain every month.
F1 ewe-lambs received maintenance requirements until either 16 or 28 months of age, depending on the year in which they were born. F1 ewes were then naturally mated to a single ram with the mating date noted using a marking ram harness. F1 ewes were allowed to lamb naturally. Second filial generation (F2) ewe lambs (n = 9 DEX and 8 C) were fed in a similar fashion as their F1 mothers, and postnatal body weights were recorded at the same times. There were 3 F2 C twin (all male female twin sets) and 5 F2 C singles. In the F2 DEX lambs, there were 5 twin (1 female twin sets and 3 male female twin sets) and 4 singles.
To determine the responsiveness of different components of the HPAA, a series of challenges were conducted in 6 randomly subsampled female F1 and F2 offspring, all from different mothers at 2.5 or 3.5 year of age (n = 3 per age and birth type in each treatment group) for F1 and approximately 6 months of age for F2 females (3 per birth type per treatment). F1 ewes were seasonally anestrous and F2 lambs had not obtained puberty when selected for challenges. A catheter (Abbocath, 16 gauge; Abbott Laboratories, North Chicago, IL) was placed aseptically into the jugular vein 48 hours prior to blood sampling. Catheters were sutured into the skin to secure them, and a 124.5 cm extension set (Seneca Medical, Tiffin, OH) was attached to the catheter to allow for infusion and sampling without disturbing the ewe. The neck and shoulder area of each animal were covered with netting material (Surgilast Tubular elastic dressing retainer; Derma Science Inc, Princeton NJ) to prevent the catheters from being damaged.
Ewes and lambs were maintained in neighboring individual pens with free access to water. Bovine CRH, human AVP, and human ACTH were all purchased from Sigma Chemicals (Sigma-Aldrich, St. Louis, MO; catalog nos. C2671, V9879, and 02275, respectively). Two days after catheter placement, an ACTH challenge was conducted using similar procedures to Turner et al with slight modifications as previously published.
Jugular blood samples were obtained into chilled tubes (heparin plus NaF; 2.5 mg/mL; Sigma, St. Louis, MO) at –30, –15, and 0 minutes relative to a 0.2 μg/kg intravenous bolus injection of ACTH in saline (2.0-3.0 mL) over 2 seconds. Blood samples were collected at 2, 5, 10, 15, 20, 30, 45, 60, 80, 100, 120, and 150 minutes after ACTH administration. All blood samples were immediately placed on ice, centrifuged at 2500 × g and plasma collected and stored at −80°C.
On the following day, a CRH/AVP challenge was conducted as previously reported by Bloomfield et al with the following modifications we previously reported. Jugular blood samples were collected at −30, −15, and 0 minutes relative to injection of 0.5 μg/kg of CRH and 0.1 μg/kg of AVP in saline as an intravenous bolus (2.0-3.0 mL) over 2 seconds. Jugular blood samples were obtained at 2, 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, and 240 minutes after the CRH/AVP infusion. Blood samples were processed as for the ACTH challenge. Following the ACTH challenge, blood from the catheter in 1 F2 C lamb was not accessible.
Hormone quantification
Concentrations of ACTH were determined as previously described by Gilbert et al using an Immulite 1000 immunoassay analyzer (Siemens Medical Solutions, Malvern, PA) in a single assay with interassay coefficients of variation (CVs) of less than 5%. Concentrations of cortisol were determined as previously described by Dong et al using Coat-A Count Cortisol RIA (Siemens Medical Solutions Diagnostics, Los Angeles, CA) with an intraassay and interassay CV less than 7%.
Statistical analysis
Data are presented as means ± least square SEM, and differences were considered significant at P ≤ .05, with a tendency at P ≤ .10. GraphPad Prism (GraphPad Software Inc, La Jolla, CA) was used to calculate the area under the curve (AUC) for plasma cortisol and ACTH responses following CRH/AVP infusion and plasma cortisol response following the ACTH challenge. Baseline concentrations of ACTH and cortisol before infusion were obtained by averaging the 3 samples before both challenges.
Plasma cortisol and ACTH response following CRH/AVP administration and plasma cortisol response following the ACTH challenge were analyzed as repeated measures using the MIXED procedure of SAS (SAS Institute, Cary, NC) with treatment and time and their interaction in the model statement. Areas under the curves for ACTH and cortisol during CRH/AVP and cortisol release during the ACTH challenge along with baseline concentrations of cortisol and ACTH were analyzed using the GLM procedure of SAS with treatment in the model. A cortisol to ACTH ratio was calculated for baseline and for AUC during the CRH/AVP challenge and the ratio was analyzed using the GLM procedure of SAS with treatment in the model.
F1 ewe maternal age was initially included in all F1 ewe models but was found to be nonsignificant ( P > .52), and birth type was also included in both the F1 and F2 animals but was found to be nonsignificant ( P > .20) and both were therefore removed from the final models.
Results
In the F1 generation, the gestation length was shorter for DEX ewes compared with C ewes ( P < .05; 152.3 ± 0.5 vs 155.4 ± 0.5) and tended to be shorted in the F2 generation ( P < .06; 147.6 ± 0.7 vs 149.6 ± 0.6). Birthweight was also reduced in F1 lambs ( P = .04) from DEX-treated mothers compared with C-treated mothers (5.03 ± 0.18 vs 5.59 ± 0.17), and this birthweight reduction was maintained into the F2 generation ( P = .03), with lambs born to ewes whose mothers were treated with DEX having smaller lambs compared with ewes whose grandmothers were C (5.72 ± 0.26 vs 6.62 ± 0.29). Body weights of F1 DEX ewes at catheterization were less ( P < .05) than F1 C ewes (F1 65.1 ± 2.5 vs 72.2 ± 2.5 kg, respectively).
Baseline plasma ACTH was similar in F1 DEX and C ewes ( P = 0.26; 30.1 ± 9.1 vs 36.8 ± 9.1 pg/mL, respectively), but cortisol was greater ( P < .05) in F1 DEX ewes compared with C F1 ewes (1.8 ± 0.1 vs 0.8 ± 0.1 mg/dL, respectively). In the F1 ewes, there was a treatment * time interaction ( P = .04) for the cortisol response ( Figure 1 ) during the ACTH challenge. F1 ewes from mothers treated with dexamethasone had reduced ( P < .05) cortisol release at 2, 20, and 30 minutes after intravenous ACTH compared with ewes from C mothers. However, there was no difference ( P = .37, Figure 1 ) in the area under the curve for cortisol.
There was a significant ( P ≤ .04) treatment * time interaction for plasma ACTH and cortisol during the CRH/AVP challenge ( Figure 2 ), with F1 DEX offspring showing reduced ( P < .01) plasma ACTH and cortisol at 5-15 and 10-30 minutes after infusion, respectively. The decreased plasma ACTH and cortisol response to the CRH/AVP challenge was confirmed by reduced ( P = .04) area under the curve for both ACTH and cortisol. The cortisol to ACTH ratio was not different between DEX and C F1ewes during baseline sampling prior to CRH/AVP challenge ( P = .06; 0.035 ± 0.007 vs 0.056 ± 0.007, respectively). During the CRH/AVP challenge, the AUC cortisol to ACTH ratio was similar between DEX and C F1 ewes ( P = .09; 0.164 ± 0.014 vs 0.186 ± 0.014, respectively).
Body weights of F2 DEX lambs at catheterization were less ( P < .05) than F2 C lambs (44.8 ± 2.4 vs. 51.2 ± 2.4 kg, respectively). Baseline ACTH was higher ( P < .05) in F2 DEX lambs than F2 C lambs (56.2 ± 6.4 vs 35.4 ± 6.4 pg/mL, respectively), and cortisol was also higher ( P < .05) in F2 DEX lambs compared with F2 C lambs(1.8 ± 0.1 vs 0.9 ± 0.1 mg/dL, respectively). In the F2 lambs at 6 months of age during the ACTH challenge, there was a significant treatment * time interaction ( P < .001, Figure 3 ), with F2 DEX lambs having increased ( P < .05) plasma cortisol before the ACTH administration and reduced ( P < .05) plasma cortisol at 10, 15, and 20 minute after ACTH administration compared with F2 C lambs. There was no difference ( P = .22, Figure 3 ) in the area under the curve for plasma cortisol during the ACTH challenge.
During the CRH/AVP challenge, F2 offspring from DEX treated grandmothers had a significant treatment * time interaction for ACTH response, with DEX-treated F2 lambs having reduced ( P < .05, Figure 4 , A) plasma ACTH at 5-30 minutes after the infusion compared with C F2 lambs. Plasma cortisol was reduced ( P < .01, Figure 4 , B) in F2 DEX lambs compared with F2 C lambs during the CRH/AVP challenge. There was significantly reduced ( P < .05) plasma ACTH and cortisol area under the curve ( Figure 4 ) in the F2 DEX lambs compared with the F2 C lambs. The cortisol to ACTH ratio during baseline sampling prior to the CRH/AVP challenge was not different between the DEX and C F2 lambs ( P = .48; 0.024 ± 0.004 vs 0.023 ± 0.004, respectively). During the CRH/AVP challenge, the AUC cortisol to ACTH ratio was similar between DEX and C F2 lambs ( P = .16; 0.009 ± 0.001 vs 0.010 ± 0.001, respectively).
