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The Liggins Institute (C.W.H.R., M.H.O., E.B.T., A.L.J., J.E.H., F.H.B.), University of Auckland, Private Bag 92019, Auckland, New Zealand; and Department of Basic Animal and Veterinary Science (S.M.H.), University of Copenhagen, DK-1870 Frederiksberg C, Denmark
Address all correspondence and requests for reprints to: Dr. Frank Bloomfield, Liggins Institute, University of Auckland, Private Bag 92019, Auckland, New Zealand. E-mail: f.bloomfield{at}auckland.ac.nz.
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Introduction |
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We have previously shown in singleton fetal sheep that periconceptional undernutrition (PCUN) increases pituitary ACTH production in response to decreased negative feedback by cortisol (5) and leads to a precocious rise in plasma ACTH and cortisol levels and preterm delivery (6). Others have shown that PCUN also results in increased basal activity of the HPA axis in postnatal life (7).
Twinning is also a periconceptional event. However, in contrast to the effects of PCUN, in sheep, there is evidence of reduced responsiveness of the HPA axis at the adrenal level in twins in late gestation (8, 9), and gestation may be prolonged (Oliver, M. H., unpublished data) (10), although there are no data regarding the effect of twinning either on activity of the hypothalamus and pituitary gland or on negative feedback from the hippocampus, hypothalamus, or pituitary gland. Furthermore, the prepartum cortisol surge appears to happen asynchronously in twin pairs (11) and is related to changes in adrenal sensitivity to ACTH (12). Thus, in twins, one fetus may be born without experiencing a substantial cortisol surge. The factors determining which fetus generates the prepartum cortisol surge in twins are not known. In contrast to reduced adrenal sensitivity in twin fetal sheep before birth, we have shown that after birth, there is increased responsiveness of the central HPA axis in twins compared with singletons (13). This responsiveness is strongly associated with the within-twin coefficient for birth weight, rather than the between-twin coefficient, suggesting an effect of factors related to the growth of individual fetuses rather than to their shared maternal environment.
Thus, twinning and PCUN both appear to increase activity of the HPA axis after birth in sheep, but before birth they have opposite effects, with twinning decreasing but PCUN increasing HPA axis activity. The similarity of effects after birth may be related to the decreased fetal growth trajectory seen both in twins (14) and after PCUN (15), because there is an association between reduced birth weights, increased HPA axis responsiveness (16, 17), and risk of metabolic diseases in later life in human singletons (18). However, the literature on humans is inconsistent regarding the relationship between birth weight and disease risk in twins (19), with one explanation being that biology of growth is substantially different in twins from that of singletons (20).
Understanding how these two periconceptional events affect function of the higher aspects of the HPA axis might help explain how twinning and PCUN both result in increased postnatal HPA axis activity but opposite effects on adrenal function in fetal life. We therefore investigated the higher centers of the HPA axis during late gestation in singleton and twin fetuses of ewes that had either been well fed throughout pregnancy or been exposed to PCUN. We studied HPA axis responses to an acute maternal nutritional deprivation and to central stimulation of the fetal HPA axis with CRH and arginine vasopressin (AVP) and also the negative feedback response to decreased cortisol production by the adrenal gland.
In addition, because exposure of the fetus to inappropriate levels of maternal glucocorticoid has been proposed as a common mechanism underlying the relationship between undernutrition, reduced size at birth, and increased risk of disease (21), and we have previously demonstrated that PCUN profoundly down-regulates the maternal HPA axis (5, 22), we also studied the effect of twinning and PCUN on basal maternal HPA axis activity and on the maternal HPA axis response to the physiological stimulus of fasting.
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Materials and Methods |
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). Nutritional management in the PCUN group consisted of a 2-d fast followed by individually adjusted intake of concentrate feeds to achieve and then maintain a maternal body weight reduction of 10–15%. Initially, feed intake in the PCUN group was at 1–2% of body weight/d but by mating was 80% of intake in control ewes. After the period of undernutrition, all ewes were fed maintenance feeds. A fortnight before mating with Dorset rams, the estrous cycle of all ewes was synchronized (23). Fetal number was established by ultrasound scanning at 55 d. Maternal blood samples were taken by jugular puncture at regular intervals from –71 d gestation until transport to the laboratory at 104 d, and ewes were weighed at least twice weekly.
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Ewes were fasted from d 121–124, during which time maternal and fetal blood samples were taken daily. On d 124, ewes were refed and also given an iv glucose infusion of 25 g over 8 h, aiming to restore maternal, and hence fetal, blood glucose concentrations as rapidly as possible to prefasting levels. Additional blood samples were collected 8 h after the start of the glucose infusion and at 24 and 48 h of refeeding.
On d 127, a pituitary stimulation challenge was performed using 2.0 µg bovine CRH (Sigma Chemical Co., St. Louis, MO) in 0.2 ml sterile saline and 0.4 µg AVP (Sigma) in 0.2 ml sterile saline. After baseline fetal blood samples at –15 and 0 min, the combined solution was administered to the fetus by iv injection, and arterial samples were taken at 15, 30, 45, 60, 120, and 240 min.
On d 128, a metyrapone challenge was performed on the fetuses. Metyrapone (Novartis Pharma, Basel, Switzerland) was dissolved in sterile normal saline at a concentration of 20 mg/ml, and passed through a 0.2-µm filter. After baseline blood samples at –30 and 0 min, 3 ml solution (60 mg metyrapone) was administered to the fetus by iv injection, and arterial blood samples were taken at 30, 60, and 120 min.
Challenges were performed simultaneously in twin fetuses. Blood samples were drawn into chilled tubes containing lithium heparin, stored on ice during the experiments, and then centrifuged at 2500 x g for 10 min at 4 C and plasma stored at –80 C.
The sheep were euthanized with an overdose of pentobarbitone on d 132 (term = 147 d) and a postmortem performed.
Hormone and metabolite assays
Glucose concentrations were measured on a Hitachi 902 autoanalyzer (Hitachi High Technologies Corp., Tokyo, Japan) by enzymatic colorimetric assay (Roche, Mannheim, Germany). ACTH was measured with a commercial RIA (Diasorin, Stillwater, MN) with intra- and interassay coefficients of variation of 16.1 and 15.2%, respectively.
Steroids were measured using mass spectrometry. The internal standards were cortisol-d2 for cortisol; corticosterone-d8 for cortisone, dehydroepiandrosterone (DHEA), and progesterone; and 11-deoxycortisol-d2 for 11-deoxycortisol. A 100-µl volume of internal standard (20 ng/ml in water) was added to 200 µl plasma. Steroids were extracted using 1 ml ethyl acetate. After removal of the organic supernatant, samples were dried, resuspended in 100 µl mobile phase (80% methanol and 20% water), and transferred to HPLC injector vials. A 25-µl volume was injected onto an HPLC mass spectrometer system consisting of a Surveyor MS pump and autosampler followed by an Ion Max APCI source on a Finnigan TSQ Quantum Ultra AM triple-quadrapole mass spectrometer all controlled by Finnigan Xcaliber software (Thermo Electron Corp., San Jose, CA). The mobile phase was isocratic, flowing at 600 µl/min through a Luna 3µC18(2) 100A 250 x 4.6 column at 40 C (Phenomenex, Auckland, New Zealand). Retention times were as follows: cortisol, 6.1 min; cortisone, 5.7 min; DHEA, 9.3 min; progesterone, 11.4 min; and 11-deoxycortisol, 6.6 min. Ionization was in positive mode, and Q2 had 1.2 mTorr of argon for all steroids. The mass transitions followed, for internal standard and steroid, respectively, were as follows: cortisol-d2, 365.3–121.2 at 28 V, and cortisol, 363.3–122.2 at 28 V; corticosterone-d8, 355.3–125.2 at 24 V, and cortisone, 361.1–163.0 at 28 V; DHEA, 271.2–197.0 at 18 V, and progesterone, 315.1–109.0 at 26 V; 11-deoxycortisol-d2, 349.2–109.1 at 26 V, and 11-deoxycortisol, 347.2–109.1 at 26 V. Mean inter- and intraassay coefficient of variation values were as follows: cortisol, 11.2 and 7.1%; cortisone, 20.4 and 10.3%; DHEA, 28.5 and 19.6%; progesterone, 11.7 and 7.6%; and 11-deoxycortisol, 16.8 and 10.7%.
Statistics
Statistical analyses were performed using JMP 5.1 (SAS Institute Inc., Cary, NC).
Maternal data were compared using a two-way ANOVA with nutritional group, single/twin-bearing, and their interaction as independent variables.
For the fetal CRH/AVP and metyrapone challenges, area under the curve (AUC) was calculated from baseline, taken as the average of the two baseline samples, and the data were log-transformed where required to approximate normality. Fetal data were compared using a two-way ANOVA with twinning, nutritional group, and the interaction between the factors as independent variables and with sheep number nested within nutritional group and single/twins to allow for the nonindependence of twins. The Tukey-Kramer method was used to correct for multiple comparisons. A similar two-way repeated-measures ANOVA was used to compare longitudinal data from the fasting (121–124 d) and refeeding (124–125 d) periods.
Twins were divided into heavy or light based on postmortem weight. Heavy vs. light comparisons were made by two-way ANOVA with heavy/light, nutritional group, and their interaction as independent variables.
Sex differences were investigated within singleton and twin pregnancies using a two-way ANOVA with sex, nutritional group, and their interaction as independent variables. In twin pregnancies, the effect of the sex mix of the pair, designated as mixed, male or female, was evaluated using ANOVA in male and female twin fetuses separately.
Data are presented as mean ± SEM. Geometric mean and SEM are used where appropriate.
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Results |
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The average weight loss due to undernutrition was 15.1 ± 0.4% in singleton-bearing ewes and 17.3 ± 0.5% in twin-bearing ewes (Table 1). There were no significant weight differences between nutritional groups or single- and twin-bearing ewes by d 110.
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). Plasma progesterone levels increased over the duration of the experiment but remained higher in twin-bearing ewes (Table 2). Maternal plasma ACTH, cortisol, and cortisone levels did not change significantly between the start and end of the experiment, but both the cortisol to cortisone and cortisol to ACTH ratios declined (Table 2).
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Maternal plasma ACTH levels did not change with fasting or refeeding, but cortisol and cortisone levels increased with fasting and then decreased with refeeding (Fig. 2). Twinning and nutritional group did not affect these changes. Plasma progesterone levels also increased with fasting and decreased with refeeding in singleton-bearing ewes but continued to increase in twin-bearing ewes. Progesterone levels in twin-bearing ewes remained elevated at d 131 compared with d 121 (16.6 ± 1.7 vs. 14.5 ± 1.2 ng/ml, P = 0.02), whereas they had returned to baseline in singleton-bearing ewes (9.83 ± 0.74 vs. 10.07 ± 0.54 ng/ml, P = 0.45).
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). However, there were no differences in plasma ACTH or steroid levels between nutritional groups in twins. Fetal plasma ACTH and DHEA levels did not change between 114–121 and 128–131 d, but cortisol, cortisone, the cortisol to ACTH ratio, and the cortisol to cortisone ratio all increased. There was no difference in cortisol levels between twin and singleton fetuses at 128–131 d, but cortisone levels remained higher in control singletons than in PCUN singletons and all twins.
There was no effect of fetal sex or sex mix of a twin pair on baseline values and no differences between heavy and light twins in a pair.
Maternal fast and refeed.
The 72-h fast halved fetal plasma glucose levels, from 0.79 ± 0.02 to 0.43 ± 0.01 mmol/liter in twins and from 0.97 ± 0.04 to 0.53 ± 0.02 mmol/liter in singletons.
Fetal plasma ACTH levels increased with fasting and decreased quickly in response to maternal refeeding (both P < 0.01), with no additional effect of twinning or PCUN (Fig. 3A). Fetal plasma cortisol (Fig. 3B; P < 0.01) and cortisone (Fig. 3C; P < 0.01) levels increased in response to the fasting, with levels increasing more in twins than singletons (P < 0.05), and decreased quickly in response to refeeding (both P < 0.01). Plasma DHEA levels (Fig. 3D) decreased in response to fasting (P < 0.01) but less in twins than singletons (P < 0.01). On refeeding, DHEA levels increased in singletons (P < 0.01) but continued to decrease in twins (P < 0.01).
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Nutritional group did not affect the changes in fetal plasma ACTH, cortisol, or cortisone levels in response to fasting and refeeding. However, plasma DHEA levels decreased further in control than PCUN fetuses during the fasting period (Fig. 3D).
There was no effect of sex or sex mix of a twin pair on fetal responses to maternal fasting and refeeding.
CRH/AVP challenge.
There were no differences in maternal plasma ACTH and cortisol levels at baseline. Fetal plasma ACTH levels rose rapidly after iv CRH and AVP in all animals. Twins had a greater ACTH (Fig. 4A) and cortisol response (Fig. 4B) than singletons.
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). There was also a decreased subsequent cortisol response (Fig. 4B). Twin fetuses had a smaller cortisol AUC to ACTH AUC ratio than singletons (68.7 ± 7.8 vs. 87.1 ± 16.4, P < 0.05), but PCUN fetuses were not different from controls (77.1 ± 13.6 vs. 73.6 ± 8.4, P = 0.82).
There was no effect of fetal sex or sex mix of a twin pair on responses to the CRH and AVP and no differences between heavy and light twins within a pair.
Metyrapone challenge.
There were no differences in maternal ACTH or cortisol levels at baseline. The metyrapone challenge resulted in decreased fetal plasma cortisol levels at 30 min in all groups, with no difference between groups in the cortisol trough (P = 0.26) (twin control 3.23 ± 0.45 to 0.97 ± 0.15, twin PCUN 4.06 ± 1.22 to 1.69 ± 0.73, singleton control 2.91 ± 0.51 to 0.90 ± 0.23, singleton PCUN 3.18 ± 0.68 to 1.77 ± 0.57 ng/ml, P < 0.01 for time effect). Twinning resulted in a greater ACTH response to this fall than singletons (Fig. 5A), and a subsequent greater 11-deoxycortisol response (Fig. 5B) but did not affect the ratio of 11-deoxycortisol AUC to ACTH AUC (118 ± 22 vs. 200 ± 92, P = 0.14).
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), but there was a greater 11-deoxycortisol response (Fig. 5B), although the ratio of 11-deoxycortisol AUC to ACTH AUC was similar (166 ± 48 vs. 123 ± 34, P = 0.44). There was no effect of fetal sex or sex mix of a twin pair on responses to the metyrapone challenge and no differences between heavy and light twins within a pair.
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Discussion |
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Maternal HPA axis
The maternal HPA axis is usually up-regulated by pregnancy, with elevated cortisol levels due to decreased pituitary sensitivity to negative feedback by cortisol (25). This may be due, at least in part, to the anti-mineralocorticoid actions of progesterone (26, 27), leading to decreased negative feedback by cortisol. Our finding of an increase in maternal cortisol concentrations in response to fasting, without any significant change in circulating ACTH concentrations, is consistent with decreased cortisol negative feedback at the hypothalamus and pituitary.
The increased maternal cortisol concentrations in response to acute fasting differs from the suppression of the HPA axis reported by ourselves (22) and others (28) in response to a prolonged fast in gestation. This simply reflects the difference between an acute and a chronic stressor; indeed, in our study of prolonged undernutrition, we demonstrated an acute up-regulation of the maternal HPA axis (22), followed by suppression as fasting continued.
The acute rise in circulating progesterone concentrations with late-gestation fasting seen in this study and the sustained elevation in twin-bearing ewes in late gestation are novel observations. Differences in progesterone levels may be due to altered production or clearance. Placental production of progesterone is related to placental mass and is therefore higher in twin-bearing ewes (29). Metabolic clearance of progesterone is via the liver and has been shown to be inversely related to feed intake in nonpregnant sheep (30), probably due to altered hepatic portal blood flow (31). Thus a decrease in clearance may have contributed to the rise in progesterone levels that we observed with maternal fasting. Similarly, decreased clearance may contribute to the continued rise in progesterone levels that we observed in twin-bearing ewes after refeeding, because we have previously shown that feed intake is lower in twin-bearing than singleton-bearing ewes in late gestation (14).
An alternative explanation for the rise in progesterone with fasting could be reduced uterine blood flow. Although we were not able to measure uterine blood flow in this experiment, a previous study reported a 25% reduction in uterine blood flow with a 5-d fast in pregnant ewes (32). If placental progesterone production and uterine uptake were unchanged, then, according to the Fick principle, circulating concentrations of progesterone would increase.
Fetal HPA axis
The prepartum cortisol surge in fetal sheep begins around 128 d gestation in singleton pregnancies and 135 d in twin pregnancies (9). The lower glucocorticoid levels and the lower ratio of cortisol to ACTH in twin fetuses compared with singletons at 114 –121 d in this study are therefore unlikely to relate to differences in timing of the cortisol surge. Our results are in contrast to a previous study (9) that found lower ACTH levels in twin fetuses at 115–122 d but no differences in cortisol levels, although in that study only one fetus of a twin pair was catheterized, making it difficult to exclude bias from sampling the twin that would not initiate labor and would therefore be likely to have less advanced adrenal maturation even before the prepartum cortisol surge. However, our findings of low cortisol but similar ACTH levels in twin fetuses, with no evidence of blunting of responses higher up the HPA axis, are similar to those reported at 133 d (8).
Although blunting of adrenal steroidogenesis is the most likely explanation for the lower circulating cortisol concentrations in twin fetuses, the increased cortisol response to maternal fasting in twin fetuses at 121–124 d demonstrates that the axis is fully functional and able to respond to a hypoglycemic stress. Including baseline glucose concentration in an analysis of the cortisol response to fasting does not alter the results, suggesting that the magnitude of the hypoglycemic stress does not account for the similar response. The decreased ratio of cortisol to ACTH suggests that the adrenals of twin fetuses are less sensitive to ACTH stimulation, as has been observed previously (8), which may be due to altered levels of the ACTH receptor, steroidogenic acute regulatory protein, or adrenocortical steroidogenic enzyme activity. Although twin fetal adrenals at 53–56 d gestation have been shown to have decreased mRNA levels of P450c17, the rate-limiting enzyme in cortisol biosynthesis, and also of IGF-I, IGFII, and the IGF receptors (33), which are thought to be important in stimulating growth of the fetal adrenal gland (34), we are not aware of any published data comparing the levels of these factors in late-gestation singleton and twin fetuses.
An alternative explanation for the differences in circulating cortisol concentrations is either altered production at extraadrenal sites or altered transplacental transfer from the mother. Bioactive cortisol can be produced in the liver from cortisone by the 11β-hydroxysteroid dehydrogenase (11βHSD) type 1 enzyme. Although the 11βHSD isozymes are thought to play an important role in controlling local cortisol levels (35), the influence on systemic levels is not known. Hepatic size is reduced in twins (14), and metabolic activity of the liver may also be altered, but whether hepatic 11βHSD-1 activity is different in twin and singleton fetuses is not known. The liver also produces cortisol-binding globulin (CBG), levels of which influence total cortisol levels. CBG is thought to play an important role in negative feedback of the fetal HPA axis (36) and also binds progesterone (37). Although it is not known whether CBG concentrations are different in twins and singletons, the higher progesterone concentrations in twins may result in competition for CBG binding between progesterone and cortisol, thereby resulting in lower circulating cortisol concentrations in twin fetuses.
Differences in circulating maternal levels of glucocorticoid between twin- and singleton-bearing ewes are not responsible for the different fetal levels seen in our study, although differences in the placental 11βHSD-2 barrier could influence the amount of maternal cortisol that crosses to the fetus. At 53–56 d gestation, there is greater expression of 11βHSD-2 mRNA in the placenta of twin fetuses than singletons, although there is no difference in protein expression, and enzyme activity levels were not measured (33). We are not aware of any data on placental 11βHSD-2 activity in singletons and twins in late gestation.
This is the first study to assess higher centers of the HPA axis in both fetuses of a twin pair. The assessment took place at 127–128 d gestation, a time when the prepartum cortisol surge may just be starting (9, 38) and when CRH has its greatest effect on pituitary function (39). We found that central HPA axis sensitivity to stimulation is greater in twins than in singletons, whether assessed by the corticotrophic response to CRH/AVP or by the negative feedback response to a fall in circulating cortisol concentrations. It is not clear whether this represents a permanent increase in pituitary responsiveness to stimulation in twins or a delay in the normal prepartum decrease in sensitivity of pituitary ACTH production to hypothalamic CRH stimulation that accompanies the cortisol surge (4, 38), which would be consistent with a longer gestation in twin-bearing ewes (10).
Basal ACTH secretion is regulated differently from stimulated ACTH secretion (40), with basal secretion probably still under cortisol negative feedback control, whereas stimulated secretion is not. The greater ACTH response in twin fetuses to direct stimulation by CRH/AVP may be due in part to changes in receptor density, the relative proportions of the corticotroph subpopulations in the pituitary that do, or do not, express the CRH receptor (41), or ACTH production from proopiomelanocortin (POMC). Farrand et al. (41) demonstrated that placental restriction in sheep, induced by removal of the majority of placental attachment sites before pregnancy, increased the proportion of corticotrophs expressing POMC, ACTH, and the CRH type 1 receptor, but it is not known whether twinning also has an effect on these cells.
The greater ACTH response after decreased cortisol concentrations in the metyrapone challenge in twin fetuses could result from several factors. The similar decline in cortisol, which signifies successful inhibition of 11β-hydroxylase by metyrapone, in all groups suggests that differences in cortisol response to metyrapone are not responsible. It also implies that the fetal adrenal is supplying similar proportions of circulating cortisol in the different groups. The difference, therefore, may lie in altered cortisol negative feedback sensitivity. The normal feedback relationship between ACTH and cortisol is known to be suspended in late gestation because both increase simultaneously (42). Negative feedback takes place at the hippocampus, hypothalamus, and pituitary through the glucocorticoid receptor (GR) and mineralocorticoid receptors (43, 44), the occupancy of which is thought to be protected by local activity of the 11βHSD-2 isozyme. Decreased pituitary expression of GR in late gestation may be a mechanism for the altered feedback (44). GR levels in the higher centers of the HPA axis are known to be affected by antenatal dexamethasone (45) and prenatal undernutrition (46) in guinea pigs and prenatal stress in rats (47), but the effect of twinning is not known. Another possible mechanism for the diminished feedback in late gestation would be changes in 11βHSD activity in the higher centers of the HPA axis. 11βHSD-2 expression in the brain is much more limited than 11βHSD-1, at least in the rat (48, 49), although dehydrogenase activity of 11βHSD-1 may be significant (50). 11βHSD-1-deficient mice demonstrate exaggerated responses to stress, suggesting diminished glucocorticoid feedback (51). The greater ACTH response to metyrapone in twins, therefore, may be due to increased sensitivity of the negative feedback system, which in turn may relate to delayed parturition or permanent alteration of the axis.
However, the fact that we have previously demonstrated an increased pituitary response to CRH/AVP in postpubertal twin sheep compared with singletons, and that HPA axis activity was related to birth weight within twin pairs, suggests that the changes in HPA axis function in twin fetuses are not due simply to a delay in the prepartum cortisol surge (13). Indeed, increased sensitivity of the HPA axis to insulin-induced hypoglycemia, similar to our findings in twins, is reported to be associated with advanced rather than delayed maturation in sheep (52) and horses (53). Furthermore, we have also demonstrated changes in the glucose-insulin axis in twin fetal sheep consistent with advanced pancreatic maturation (14).
We therefore suggest that our data are consistent with the hypothesis that twinning may have long-term effects on postnatal HPA axis physiology, particularly at the level of the pituitary. However we cannot exclude the possibility that the observed changes are reflective of delayed prepartum cortisol surge as well as more permanent alterations in HPA axis function.
In contrast to the effects of twinning, fetuses of PCUN ewes had pituitary resistance to CRH/AVP stimulation, but the increased 11-deoxycortisol response to decreased cortisol negative feedback was a similar response to that of twins. The 11-deoxycortisol response to metyrapone may be due to greater pituitary ACTH response to loss of negative cortisol feedback, as has been previously demonstrated (5) and/or greater adrenal response to ACTH. We have previously demonstrated increased basal cortisol concentrations at 127–128 d gestation in singleton fetuses following a similar PCUN regimen, no change in cortisol response to an ACTH challenge, an increased ACTH and 11-deoxycortisol response to metyrapone (5), an earlier prepartum cortisol rise, and preterm birth (6). The pituitary resistance to direct stimulation could be due to decreased CRH receptor levels in the pituitary, as occurs as parturition approaches (4), altered corticotroph populations, or altered POMC processing, because this also changes approaching term (54). Whether these changes relate to ontogeny, permanent changes in HPA axis function, or both is not clear from this experiment, although increased basal cortisol levels and increased ACTH response to CRH stimulation have been found in lambs whose mothers were undernourished from mating until 30 d gestation (55).
Conclusions
This study demonstrates that twinning and PCUN have profound but very different effects on fetal HPA axis function in late gestation. Further research into the mechanisms underlying these differences may provide insights into the different effects of these periconceptional events on gestation length. In sheep, gestation length is decreased by PCUN but increased by twinning. However, in human pregnancy, there is increasing evidence that poor nutrition around the time of conception (56) and twinning (57) both decrease gestation length. Therefore, by understanding the different effects of these periconceptional events on fetal HPA axis function, insights may be gained into the onset of parturition in human preterm birth.
The changes in fetal HPA axis function that we describe may reflect altered maturation of the axis before birth but may also indicate altered long-term function of the HPA axis, particularly at the level of the pituitary. It is also possible that both are manifestations of the same underlying alterations in the HPA axis. Antenatal glucocorticoids, for example, affect both gestation length and postnatal HPA axis function in guinea pigs (45).
Despite evidence of long-term alterations in physiology as a result of twinning and lower birth weights, evidence linking twinning to increased rates of cardiovascular disease or its risk factors in later life in human studies remains conflicting (20). The substantially different fetal development of twins compared with both control and undernourished singletons, as we have shown in the glucose-insulin and the HPA axis, may be responsible for some of this conflict. These findings emphasize that the underlying causes of altered fetal development are more important in the relationship with adult health and disease than the gross effects on fetal growth.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online November 29, 2007
Abbreviations: AUC, Area under the curve; AVP, arginine vasopressin; CBG, cortisol-binding globulin; DHEA, dehydroepiandrosterone; GR, glucocorticoid receptor; HPA, hypothalamic-pituitary-adrenal; 11βHSD, 11β-hydroxysteroid dehydrogenase; PCUN, periconceptional undernutrition; POMC, proopiomelanocortin.
Received September 20, 2007.
Accepted for publication November 19, 2007.
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, Singleton control (n = 11); 
, singleton PCUN (n = 12); 
, twin control (n = 11); 
, twin PCUN (n = 8). 
, P < 0.05 for twinning x time interaction.
