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1Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge, and 2Imperial College School of Medicine Endocrine Unit, Hammersmith Hospital, London, United Kingdom
Submitted 17 May 2006 ; accepted in final form 16 July 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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145 days). Chemoreflex function and plasma concentrations of vasoconstrictor hormones were measured. Reductions in fetal arterial PO2 during hypoxemia were similar at all ages. At 125–130 days, hypoxemia elicited transient bradycardia, femoral vasoconstriction, and increases in plasma concentrations of catecholamines, neuropeptide Y (NPY), AVP, ACTH, and cortisol. Close to full term, in association with the prepartum increase in fetal basal cortisol, there was a developmental increase in the magnitude and persistence of fetal bradycardia and in the magnitude of the femoral constrictor response to hypoxemia. The mechanisms mediating these changes close to full term included increases in the gain of chemoreflex function and in the magnitudes of the fetal NPY and AVP responses to hypoxemia. Data combined irrespective of gestational age revealed significant correlations between fetal basal cortisol and fetal bradycardia, femoral resistance, chemoreflex function, and plasma AVP concentrations. The data show that the fetal cardiovascular defense to hypoxemia changes in pattern and magnitude just before full term because of alterations in the gain of the neural and endocrine mechanisms mediating them, in parallel with the prepartum increase in fetal basal cortisol.
fetus; prepartum cortisol surge; glucocorticoid
130 days (full term 145 days) (3, 43), and increases most dramatically within the last 5 days before delivery, between 140 and 145 days. The prepartum cortisol surge is known to be an important factor in the developmental maturation of fetal tissues and physiological systems in preparation for the transition to neonatal life (23). For example, the prepartum maturation of basal cardiovascular function in the fetus is influenced by cortisol. The developmental increase in ovine fetal mean arterial blood pressure (FBP) and fall in fetal heart rate (FHR) are prevented by fetal adrenalectomy and restored to normal by cortisol replacement (63). Similarly, the prepartum cortisol surge has been associated with developmental changes in the basal activity of many fetal endocrine vasoconstrictor mechanisms, such as the noradrenergic and vasopressinergic systems (1, 8, 24, 37, 51, 56, 59–61). Although the developmental changes in basal cardiovascular function in the fetus have been well documented, less is known about the maturation of the fetal cardiovascular defense to hypoxemic stress (see below), and there is no information about the period just before full term.
Acute hypoxemia is a physiological stressor that may challenge the fetus during late gestation and, in particular, during labor and delivery (32). The ovine fetal cardiovascular defense to acute hypoxemia during late gestation has been a vibrant field of research for 
30 years but has been most extensively studied at 120–130 days, before the prepartum cortisol surge (4, 7–9, 19, 24–28, 33, 34, 37, 48, 51, 56, 58, 65). Within this gestational age range, episodes of acute hypoxemia elicit integrated fetal cardiovascular and endocrine responses. The cardiovascular responses include transient bradycardia at the onset of the hypoxemic challenge, a progressive increase in FBP, and redistribution of the fetal combined ventricular output in favor of the adrenal, myocardial, and cerebral circulations at the expense of reduced perfusion to peripheral vascular beds, such as those of the fetal carcass (7, 9, 48). The initial fetal bradycardia and increase in peripheral vascular resistance involve activation of the parasympathetic and sympathetic nervous systems, respectively, which is initiated by a carotid chemoreflex (4, 28). The outflow in sympathetic nervous activity results in increases in peripheral vascular tone, including femoral vasoconstriction, which is mediated by 
-adrenergic efferent pathways (28, 53). As the period of hypoxemia progresses, the reflex-initiated cardiovascular responses are modified. Release of epinephrine (E) into the fetal circulation opposes vagal tone and returns FHR to basal levels (10). The fetal peripheral vasoconstriction is maintained by increased release of vasoactive agents into the fetal circulation, such as catecholamines, neuropeptide Y (NPY), and AVP (1, 8, 20, 27, 50, 51, 55, 56, 59).
Although the cardiovascular responses to acute hypoxemia or asphyxia are known to change between mid and late gestation (7, 30, 33, 34, 64, 65), no study has investigated developmental changes in cardiovascular responses to hypoxemia after 130 days, when fetal basal cortisol concentrations rise. This study, therefore, tested the hypothesis that the fetal cardiovascular defense to hypoxemia matures close to full term, in association with the prepartum surge in fetal basal cortisol. The hypothesis was tested by characterizing the ovine fetal cardiovascular responses to acute hypoxemia and addressing the neural and endocrine mechanisms mediating them in association with changes in fetal basal cortisol within three narrow gestational age ranges: 1) between 125 and 130 days, immediately before the elevation in endogenous basal cortisol concentration, 2) between 135 and 140 days, during which fetal basal cortisol concentration is known to rise, and 3) at >140 days, coinciding with the main fetal plasma cortisol surge immediately before full term.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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All surgical and experimental procedures were performed under the United Kingdom Animals (Scientific Procedures) Act 1986 and were approved by the Ethical Review Committee of the University of Cambridge. Separate groups of pregnant sheep were studied at three gestational ages before and during the prepartum increase in fetal plasma cortisol (3, 43). Thirteen Welsh Mountain sheep fetuses (7 males and 6 females) were surgically prepared at 117 days (SD 2) and studied between 125 and 130 days of gestation (full term 145 days). Another 12 fetuses were instrumented at 130 days (SD 1). Half of these fetuses were studied between 135 and 140 days (n = 6, 3 males and 3 females), and the other half were studied separately at >140 days (n = 6, 3 males and 3 females) before the onset of labor, as assessed by amniotic fluid pressure fluctuations (47). Each study group comprised separate individuals, and no individual was studied twice. Briefly, sheep fetuses were surgically prepared for long-term recording under general anesthesia, as described previously in detail (20). Food, but not water, was withheld from the ewes for 24 h before surgery. After induction with thiopentone sodium (20 mg/kg iv; Intraval Sodium, Rhone Mérieux, Dublin, Ireland), general anesthesia (1.5–2.0% halothane in 50:50 O2-N2O) was maintained by positive-pressure ventilation. A lower abdominal midline incision was made, and the gravid uterus was exposed. Fetal instrumentation was achieved in two stages. The first uterine incision exposed the fetal head, and PVC catheters (0.58 or 0.86 mm ID and 0.96 or 1.52 mm OD, respectively; Critchly Electrical Products, NSW, Australia) were inserted into a fetal carotid artery and jugular vein. The second uterine incision exposed the fetal hindlimbs, and catheters were inserted into the femoral artery and femoral vein. An ultrasonic flow transducer (model 2R or 3S, Transonic Systems, Ithaca, NY) was positioned around the contralateral femoral artery. Another catheter was anchored to the hindlimb for measurement of amniotic cavity pressure. The uterine incisions were closed in layers, and the catheters were filled with heparinized saline (80 IU heparin/ml in 0.9% NaCl) and sealed with brass pins. A Teflon catheter was also inserted into the maternal femoral artery and advanced to the caudal aorta. All catheters and flow probe leads were exteriorized via a small incision in the maternal flank.
Ewes were housed in individual pens, had free access to water and hay, were fed concentrates twice daily (100 g; Sheep Nuts no. 6, H and C Beart, Kings Lynn, UK), and generally resumed normal feeding patterns within 24 h of surgery. The ewes received 2 days of postoperative analgesia (3 g phenylbutazone daily by mouth; Equipalozone Paste E-pp, Arnolds Veterinary Products, Shropshire, UK) if required. Antibiotics were administered daily to the ewe (procaine penicillin, 0.20–0.25 mg/kg im; Depocillin, Mycofarm, Cambridge, UK), to the fetus (150 mg/kg iv ampicillin;Penbritin, SmithKline Beecham Animal Health, Surrey, UK), and into the amniotic cavity (300 mg ampicillin). Daily maternal caudal aortic and fetal ascending aortic blood samples (0.4 ml) were taken for analysis of blood gas and acid-base status. After recovery from surgery, the patency of the vascular catheters was maintained by a slow infusion of heparinized saline at 0.1 ml/h.
Pressure transducers (COBE, Argon, TX) were attached to the fetal femoral artery and amniotic cavity catheters. Pressure data were recorded digitally by a custom-programmed computerized data acquisition system (Cornell University, Ithaca, NY) via an electronic analog-to-digital converter (NIDAQ, National Instruments, Austin, TX). Femoral blood flow (FBF) was monitored via a monitor (model T201/T206, Transonics, Ithaca, NY). FHR readings were triggered from FBP or FBF pulse.
Acute Hypoxemia Protocol
After 5 days of postoperative recovery, fetal hypoxemia was induced by reduction of maternal fraction of inspired O2, a well-established method for inducing hypoxemia in the sheep fetus that has been used widely for 30 yr (27). In contrast to other methods of inducing fetal hypoxemia (e.g., umbilical cord compression, placental embolization, uterine artery ligation, and hypobaria), maternal inhalational hypoxia induces relatively pure isocapnic fetal hypoxemia and, therefore, provides a unifactorial stimulus for physiological study. The challenge comprised a 3-h protocol: 1 h of normoxia, 1 h of hypoxia, and 1 h of recovery (28). At the start of the 1st h, a large transparent respiratory hood was placed over the ewe's head, and air was passed through the hood at a rate of 40 l/min. After 1 h of normoxia, the gas mixture breathed by the ewe was switched to 9% O2 in N2 (18 l/min air and 22 l/min N2) with small amounts of CO2 (1–2 l/min) added to maintain fetal arterial PCO2 (PaCO2) constant, and the fetus was subjected to 1 h of hypoxemia. This gas mixture reduced fetal arterial PO2 (PaO2) to within 12–15 mmHg. After 1 h of hypoxemia, the hood was removed, and the ewe breathed room air for the recovery period.
Calibrated mean fetal caudal aortic arterial and amniotic cavity pressures, FHR, and mean FBF were recorded continually at 1-s intervals throughout the protocol with a computerized data acquisition system. Maternal and fetal arterial blood samples (5 ml) were taken every 30 min, starting from 15 min of normoxia, for measurement of blood gas and acid-base status and plasma hormone concentrations. An additional fetal ascending aortic sample was taken 5 min after the onset of hypoxemia to confirm a rapid and appropriate fall in fetal PaO2.
Measurements and Calculations
Fetal arterial pH (pHa), PaO2, and PaCO2 and calculated base excess were obtained using a blood gas analyzer (model ABL 5, Radiometer, Copenhagen, Denmark); measurements were corrected to 39.5°C for fetal blood and 38°C for maternal blood. Blood Hb concentration ([Hb]) and percent saturation of Hb with O2 were determined using a hemoximeter (model OSM2, Radiometer) calibrated for ovine fetal blood. Hematocrit was obtained using a microhematocrit centrifuge (Hawksley).
Amniotic cavity pressure was used as the zero-pressure reference level. The rate-pressure product, an index of myocardial work (18, 40), was calculated as FBP x FHR (mmHg·beats·min–1). Fetalfemoral vascular resistance (FVR) was calculated using Ohm's principle by dividing FBP, corrected for amniotic pressure, by mean FBF (22).
Maternal and Fetal Plasma Hormone Concentrations
Maternal and fetal plasma concentrations of norepinephrine (NE), E, NPY, AVP, ACTH, and cortisol were measured in appropriately treated plasma stored at –80°C until assay within 4 mo from collection. The renin-angiotensin system was not investigated in the present study to limit the total amount of blood that needed to be sampled from the fetuses during the experimental protocol.
Catecholamine assays. Maternal and fetal plasma NE and E concentrations were measured by high-pressure liquid chromatography with electrochemical detection (22). The samples were prepared by absorption of 250 µl of plasma onto acid-washed alumina, and 20-µl aliquots of the 100-µl perchloric acid elutes were injected onto the column. Dihydroxybenzylamine was added as the internal standard to each plasma sample before absorption. Recovery ranged from 63% to 97%, and all catecholamine values were corrected for their respective recovery. The interassay coefficients of variation for NE and E were 6.2% and 7.3%, respectively, and the minimum detectable concentration was 10 pg/ml.
NPY assay. Maternal and fetal plasma NPY concentrations were measured by radioimmunoassay, as described previously (20). All samples were assayed in duplicate simultaneously. The assay used rabbit antiserum and 125I-labeled porcine peptide. Antiserum was produced in-house, and the animals were humanely killed at the end of antiserum production. Free and bound fractions were separated with dextran-coated charcoal. The assay, which was validated for use in ovine plasma using stripped ovine plasma, could detect <1 pmol/l (95% confidence interval). The interassay coefficient of variation was 6.8%. There was no detectable cross-reactivity of the anti-NPY antiserum with peptide YY.
AVP assay. Maternal and fetal plasma AVP concentrations were measured using a commercially available double-antibody radioimmunoassay kit (Nichols Institute Diagnostics, Saffron Walden, Essex, UK) after separation from plasma proteins by methanol extraction and chromatography (22). The lower detection limit of the assay was 1.3 pg/ml. The intra-assay coefficients of variation for four control plasma pools (mean concentrations 3.2, 9.9, 12.2, and 28.9 pg/ml) were 10.0%, 6.7%, 3.7%, and 4.6%, respectively. The interassay coefficient of variation was 6.9% for a mean value of 10.8 pg/ml. The anti-AVP antiserum (Nichols Institute Diagnostics) showed cross-reactivities of <0.1% with lysine vasopressin, oxytocin, and vasotocin.
ACTH assay.
Maternal and fetal plasma ACTH concentrations were measured using a commercially available double antibody 125I RIA kit (Incstar, Wokingham, UK). The lower limit of detection of the assay was 10–25 pg/ml. The interassay coefficient of variation was 8.4%. The intra-assay coefficient of variations for two plasma pools (37 and 150 pg/ml) were 3.6 and 4.1%, respectively. The assay had negligible (<0.01%) cross-reactivities with -MSH, 
-endorphin, -lipotropin, leucine enkephalin, methionine enkephalin, bombesin, calcitonin, parathyroid hormone, follicle-stimulating hormone, arginine vasopressin, oxytocin, and substance P.
Cortisol assay. Plasma cortisol concentrations were measured as described previously (20). The lower limit of detection of the assay was 1.0–1.5 ng/ml. The intra- and interassay coefficients of variation were 5.3% and 13.0%, respectively. The cross-reactivities of the antiserum at 50% binding with other cortisol-related compounds were 0.5% for cortisone, 2.3% for corticosterone, 0.3% for progesterone, and 4.6% for deoxycortisol.
Data and Statistical Analyses
Values are means ± SE unless otherwise indicated. Statistical significance for comparisons between blood gases, acid-base status, cardiovascular variables, and hormone concentrations was assessed using two-way repeated-measures ANOVA and Tukey's post hoc test, to which the data were well suited. For fetal cardiovascular variables, absolute values and absolute changes from mean normoxic baseline during the acute hypoxemia protocol were first analyzed by the summary measures method (44). This well-documented process focuses on the number of comparisons, simplifies the data analysis, and helps prevent statistical invalidity when multiple measurements are made on the same individuals (25, 26, 44). Functional chemoreflex analysis was performed to assess the effects of advancing gestation on fetal cardiac and vasomotor chemoreflex responses. For this analysis, linear regression lines were plotted for the cardiovascular chemoreflex response of each fetus, and the individual values for slopes were compared between groups using one-way ANOVA (22). Statistical significance of correlations was assessed using the Pearson product-moment correlation coefficient test. For all tests, significance was accepted when P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Fetal basal arterial plasma cortisol concentrations at 125–130, 135–140, and >140 days gestation were 13.5 ± 1.2, 55.2 ± 8.7, and 97.6 ± 17.6 ng/ml, respectively, confirming that they were within the normal range expected for each gestational age group and that there was a surge in fetal plasma cortisol immediately before full term (3, 43).
Blood Gases and Acid-Base Status
Values for maternal caudal aortic blood gases and pHa were similar between gestational age groups under baseline conditions (Table 1). Reductions in maternal caudal aortic PaO2 were similar in all gestational age groups during the hypoxemic challenge. In all groups, maternal caudal aortic pHa and PaCO2 remained unchanged from baseline throughout the protocol (Table 1).
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Absolute values and the statistical analyses for the absolute changes from individual normoxic baselines for fetal cardiovascular variables during the protocol are shown in Figs. 2 and 3, respectively. Basal FHR and FBF declined toward full term, with significantly lower values during normoxia in fetuses at >140 days (149 ± 7 beats/min and 24.0 ± 4.1 ml/min) than at 125–130 days (165 ± 5 beats/min and 35.9 ± 2.9 ml/min, P < 0.05; Fig. 2). Calculation of the rate-pressure product during basal conditions revealed lower values in fetuses at >140 than at 125–130 and 135–140 days: 6.8 ± 0.1 vs. 7.5 ± 0.3 and 7.6 ± 0.1 mmHg·beats·min–1 x 10–3 (P < 0.05). Fetal cardiovascular responses to acute hypoxemia at 125–130 days were similar to those reported previously at equivalent gestational ages (4, 7, 9, 24–28, 33, 34, 37, 48, 51, 56, 58, 65). These responses included transient bradycardia, a progressive increase in arterial blood pressure, a sustained fall in FBF, and a concomitant sustained increase in calculated FVR during hypoxemia (Figs. 2 and 3). The nadir of the absolute heart rate values, occurring within 15 min from the onset of hypoxemia, was greater in fetuses exposed to acute hypoxemia at >140 days than at 125–130 days: 100 ± 6 vs. 120 ± 5 beats/min (P < 0.05). Toward full term, there was a developmental shift in the persistence of bradycardia throughout acute hypoxemia (Figs. 2 and 3). Although absolute basal arterial blood pressure was similar between gestational age groups, the increment in blood pressure into the recovery period was greater in fetuses at >140 than at 125–130 days gestation (Fig. 3). Within 15 min after the onset of hypoxemia, the rate-pressure product decreased to similar values at 125–130 days (4.9 ± 0.4), 135–140 days (5.9 ± 0.9), and >140 days (5.2 ± 0.7 mmHg·beats·min–1 x 10–3). After these initial 15 min, the rate-pressure product became elevated above baseline in fetuses at 125–130 days (7.5 ± 0.3 vs. 8.3 ± 0.4 mmHg·beats·min–1 x 10–3, P < 0.05), returned toward baseline at 135–140 days (7.6 ± 0.1 vs. 7.6 ± 0.6 mmHg·beats·min–1 x 10–3), and remained below baseline at >140 days (6.8 ± 0.1 vs. 6.4 ± 0.6 mmHg·beats·min–1 x 10–3, P < 0.05) during the remaining 45 min of the hypoxemic challenge. Despite the developmental reductions in basal FBF toward full term, further reductions during acute hypoxemia were similar in magnitude to that of each of the gestational age groups studied (Figs. 2 and 3). These developmental changes in arterial blood pressure and FBF responses to acute hypoxemia resulted in an increase in the magnitude of the calculated FVR response to acute hypoxemia toward full term (Figs. 2 and 3).
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Functional Chemoreflex Curves
The change from normoxic baseline in fetal PaO2 and the changes in FHR and FVR during the first 15 min of hypoxemia, a period governed by neural mechanisms (27), are shown as scatter plots. Analysis of slopes on individual regression lines for change in FVR vs. change in PaO2 [0.13 ± 0.03, 0.31 ± 0.14, and 0.69 ± 0.18 mmHg·(ml·min–1)–1·mmHg–1 at 125–130, 135–140, and >140 days, respectively, P < 0.05] and change in FHR vs. change in PaO2 (2.36 ± 0.40, 2.44 ± 1.07, and 4.07 ± 0.60 beats·min–1·mmHg–1 at 125–130, 135–140, and >140 days, respectively, P < 0.05) indicated significantly steeper slopes for fetuses exposed to hypoxemia at >140 days than at 125–130 and 135–140 days (Fig. 4).
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Fetal arterial plasma concentrations of NE, E, NPY, and AVP significantly increased hypoxemia in all groups (Fig. 5). Although the magnitudes of the fetal plasma NE responses were similar at all gestational ages, the increments during acute hypoxemia toward full term in plasma NPY (4.15 ± 1.42, 6.27 ± 2.73, and 37.94 ± 18.42 pmol/l increment from normoxia to 45 min of hypoxemia at 125–130, 135–140, and >140 days, respectively, P < 0.05) and AVP (130 ± 34, 238 ± 60, and 355 ± 8 pg/ml increment from normoxia to 45 min of hypoxemia at 125–130, 135–140, and >140 days, respectively, P < 0.05) were enhanced (Fig. 5). The plasma E response at 45 min of hypoxemia tended to be greater in fetuses exposed to hypoxemia at >140 days than earlier in gestation, but this difference fell outside statistical significance (Fig. 5). When data were combined irrespective of gestational age, significant positive correlations were found between the changes from baseline in FVR and plasma NE, E, NPY, and AVP concentrations during acute hypoxemia: 
for NE, 
for E, 
for NPY, and 
for AVP (n = 25, P < 0.01).
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ACTH and Cortisol Responses to Acute Hypoxemia
Although maternal basal plasma cortisol concentrations were elevated at 135–140 compared with 125–130 days gestation, there were no significant changes in maternal plasma ACTH or cortisol concentrations during acute hypoxemia in any of the gestational age groups (Fig. 6). Fetal basal plasma ACTH concentrations and the increases in ACTH concentrations during acute hypoxemia were similar between the gestational age groups (Fig. 6). In contrast, despite elevations in basal fetal plasma cortisol concentrations with advancing gestational age, the magnitude of the increment in fetal plasma cortisol concentrations during acute hypoxemia was greater in fetuses at >140 than at 125–130 days: 80 ± 17 vs. 18 ± 3 ng/ml (P < 0.05; Fig. 6).
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Consistent with a shift toward full term in the persistence of the fetal bradycardia during acute hypoxemia, there were significant negative correlations between fetal basal cortisol concentration and mean FHR between 15 and 60 min of hypoxemia: 
, n = 25, P < 0.01; Fig. 7A). Furthermore, there was a significant positive correlation between fetal basal cortisol concentration and the change in FVR during hypoxemia: 
, n = 25, P < 0.01; Fig. 7B). A significant relation was also obtained for basal fetal plasma cortisol and the femoral chemoreflex response, assessed by the slope of the relation between the change in fetal FVR and the change in fetal PaO2 for all individual fetuses: 
, n = 25, P < 0.01; Fig. 7C). In contrast, there was no relation for basal fetal plasma cortisol and the slope between the change in FHR and the change in fetal PaO2 for all individual fetuses: r = 0.17 (P > 0.05).
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, n = 18, P < 0.01; Fig. 7D). |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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FHR Response to Hypoxemia
Previous research showed a transition in the pattern of the fetal cardiac response to acute hypoxemia between 80 and 120 days of ovine gestation, coinciding with the establishment of functional chemoreflexes and myocardial chronotropic control mechanisms (7, 33, 34, 64, 65). In the ovine fetus at 84–102 days gestation, episodes of acute hypoxemia elicit no change or increases in FHR (7, 33, 34). As gestation advances toward 120 days, the pattern of the FHR response changes to transient bradycardia, which corresponds to a shift toward vagal dominance in the autonomic control of heart rate during acute hypoxemia (65). The influence of cholinergic mechanisms on FHR is less before 120 days than later in gestation, and cholinergic and humoral adrenergic chronotropic drives are relatively well balanced during acute hypoxemia, thereby preventing fetal bradycardia (65). In fetuses at >120 days gestation, bradycardia at the onset of hypoxemia is mediated by increased vagal drive (7, 28), overriding humoral adrenergic stimulation of the myocardium (65). The results of the present study extend these findings by demonstrating further developmental changes in patterns and magnitudes of the mammalian FHR response to acute hypoxemia at even later gestational ages that, importantly, coincide with the increase in fetal plasma cortisol concentrations that occurs close to full term (3, 43). These changes may indicate a continuation of the trend toward increasing vagal dominance over humoral sympathetic drive to the heart (65). One mechanism that accounts for the gestational age-dependent increase in negative chronotropic drive during acute hypoxemia is enhancement of fetal carotid chemoreflex gain. In the present study, we assessed fetal cardiac chemoreflex function by plotting absolute changes in FHR against PaO2 during the first 15 min of hypoxemia, when neuronal reflexes are known to dominate in regulating the fetal cardiovascular responses to acute hypoxemia (28). The results of the present study indicate an increase in the fetal cardiac chemoreflex functional sensitivity to acute hypoxemia at >140 days compared with 125–130 days in vivo. These results are consistent with previous findings, but in acutely exteriorized, anesthetized fetal sheep, where shifts in setting and sensitivity of carotid chemoreceptor afferent activity from the fetal toward the adult ranges were observed as full term approached (6).
In the present study, enhancement of functional chemoreflex activity may have resulted from maturation at several levels of the chemoreflex pathway, including increases in the gain of carotid body chemoreceptor transduction, changes in the gain of central signal integration in the brain stem, and enhancement of cardiac vagal efferent activity. Ovine fetal carotid chemoreflex afferent activity is known to increase in response to reductions in PaO2 and increases in PaCO2 (6, 13, 14). It is unclear whether acidemia per se acts as a fetal carotid chemostimulant and the extent to which it may summate or potentiate with other carotid chemoreceptor stimuli (14). However, such summation between pHa and PaO2 has been demonstrated in adult cats (13), and, if a similar situation exists in fetal sheep, the gestational age-dependent increase in the degree of acidemia achieved during acute hypoxemia toward full term may increase the level of stimulation experienced by the carotid chemoreceptors, despite equivalent reductions in fetal carotid PaO2 in each of the gestational age groups.
Recent work in our laboratory using a Langendorff preparation showed greater sensitivity to muscarinic agonists but less sensitivity to -adrenergic agonists in isolated hearts from full-term than from younger fetal sheep (21). Thus reciprocal changes in cardiac sensitivity to vagal and humoral sympathetic stimulation provide a mechanism contributing to persistence of bradycardia during acute hypoxemia in the ovine fetus at full term. Alterations in cardiac sensitivity to opposing autonomic influences may involve glucocorticoid-mediated changes, inasmuch as exposure to glucocorticoids has been shown to increase rat myocardial muscarinic acetylcholine receptor affinity in vivo (36) and in vitro (52), and glucocorticoids are known to modify cellular sensitivity to -adrenergic agonists (11). Furthermore, treatment of immature fetal sheep with dexamethasone, a synthetic glucocorticoid, shifts the pattern of the FHR response to acute hypoxemia from transient to persistent bradycardia (22). Finally, in the present study, the inverse correlation between fetal basal cortisol concentrations and the mean FHR between 15 and 60 min of hypoxemia is consistent with a glucocorticoid-mediated developmental shift toward full term in persistence in the FHR response to acute hypoxemia. In our previous study using the Langendorff technique (21), although advancing gestational age augmented the magnitude of the myocardial responses to carbachol and attenuated responsiveness to isoprenaline, treatment of preterm fetal sheep with cortisol had no significant effect on these responses. The reasons for the disparity between these findings are unclear but may include failure of the cortisol infusion regimen to fully induce changes during late gestation with the endogenous prepartum increase in cortisol (23). This may result from insufficient duration of fetal cortisol exposure or failure to reach a threshold level necessary to induce changes in the myocardium. Alternatively, gestation-dependent effects in the fetal heart may be cortisol independent or may require concomitant activity in additional endocrine systems, such as the thyroid axis (5, 49). The insignificant relation between fetal basal cortisol and the slope of the relation between changes in PaO2 and changes in heart rate for all individual fetuses supports the hypothesis that agents in addition to cortisol contribute to developmental changes in the chemoreflex-induced bradycardic response to hypoxemia in the full-term ovine fetus.
During normoxia and under degrees of hypoxemia where aerobic metabolism is maintained, myocardial O2 consumption correlates with the rate-pressure product of cardiac work in neonatal sheep (19). During acute hypoxemia, fetal bradycardia is important in preventing increases in the rate-pressure product and, hence, cardiac work (19), whereas increases in myocardial blood flow maintain myocardial O2 delivery (19, 48, 54, 62), allowing maintenance of aerobic metabolism. The persistence of the fetal bradycardia response to acute hypoxemia toward full term may therefore contribute to reducing cardiac work and maintaining aerobic myocardial metabolism, provided reductions in blood O2 content are appropriately offset by increases in myocardial blood flow and the duration of diastolic coronary perfusion (19).
Fetal Peripheral Vasoconstrictor Response to Hypoxemia
Although episodes of acute hypoxemia elicit increases in adrenal, myocardial, and cerebral blood flows at all gestational ages examined to date, the pattern of the peripheral blood flow response varies at the different gestational ages. For example, in fetuses before 100 days, acute hypoxemia elicits a reduction in umbilical blood flow but fails to decrease blood flow to the gastrointestinal, renal, musculoskeletal, and cutaneous circulations (33, 34). This contrasts with the well-characterized changes in combined ventricular output distribution in response to hypoxemia after 120 days, where, depending on the severity of the hypoxemic challenge, blood flow to the fetal carcass, spleen, gastrointestinal tract, pancreas, and kidneys is reduced (9, 28, 48). Although the magnitude of ovine fetal defense responses to asphyxia produced by arrest of uterine blood flow is known to change between 0.6 and 0.9 gestation (37) and Dawes and colleagues showed in 1968 (12) that the decrement in FBF in response to asphyxia resulting from compression of the umbilical cord for 1.5 min increased with advancing gestational age in the acutely exteriorized, anesthetized fetal sheep, there were no data on the ovine in vivo unanesthetized fetal vascular responses to acute hypoxemia just before full term in relation to the fetal cortisol surge. Experiments in chickens have provided most of the information on how the magnitude of the embryonic responses to acute hypoxemia changes in the period immediately before hatching (46). Mulder and colleagues (46) used fluorescent microspheres injected into the chorioallantoic veins of chick embryos to assess fractional distribution of the combined ventricular output before and during 5 min of acute hypoxemia. With advancing incubation time (from 10 to 19 days; normal incubation time 21 days), the magnitude of the redistribution of the combined ventricular output increased, favoring the brain and myocardium at the expense of perfusion to the intestines, liver, and carcass. The enhancement of femoral vasoconstriction in response to acute hypoxemia toward full term reported in the present study extends these findings for the first time to the mammalian fetus.
Multiple mechanisms may account for the developmental reduction in basal FBF and enhancement toward full term in the magnitude of the FVR response to acute hypoxemia. Such mechanisms include structural and functional changes at the level of the peripheral vasculature, enhancements in neural and endocrine inputs to the peripheral vasculature, and changes in vascular sensitivity to neural, endocrine, and paracrine constrictor and dilator influences. The present study confirms the well-known finding that circulating levels of AVP are greater in the fetal than maternal circulation, even during basal conditions, supporting a comparatively stronger endocrine constrictor input to the fetal peripheral vasculature (1, 59). The increase in the slopes of the vasomotor chemoreflex function curves in the fetuses at full term relative to that earlier in gestation supports an increase in gain of carotid chemoreflex function as full term approaches. In addition, when data were combined irrespective of gestational age, positive correlations were also found between increments in fetal plasma NE, E, NPY, and AVP concentrations and the peak increment in fetal FVR during acute hypoxemia. This is consistent with a role for these agents in contributing to the hypoxemia-induced fetal femoral vasoconstriction. Accordingly, Raff and colleagues (50) reported a greater plasma AVP response to hypoxemia in older (133 days) than in younger (122 days) ovine fetuses. The developmental increase in magnitude of the fetal plasma AVP response to acute hypoxemia in past and present studies may partly result from developmental increases in hypothalamic AVP levels with advancing gestational age (60). In addition, in unanesthetized adult rats, injection of NPY into the supraoptic nucleus drives secretion of AVP by hypothalamic vasopressinergic neurons (67). Therefore, if the enhanced fetal plasma NPY response to acute hypoxemia reflects a corresponding increase in hypothalamic NPY activity, then the enhancement in the plasma AVP response toward full term may, at least in part, be driven by an upregulation of the bioavailability of NPY.
NPY is a neuropeptide transmitter that is colocalized and coreleased with NE from sympathetic nerve terminals (17) and has potent vasoconstrictor activity, acting directly via Y1 receptors or indirectly by potentiating the effects of noradrenergic neurotransmission (31). The increase in fetal plasma NPY concentration during acute hypoxemia may not only contribute to promoting peripheral vasoconstriction by acting as an endocrine agent, but it may also be a useful marker of fetal sympathetic nervous system activity (42). In the present study, despite a gestational age-dependent increase in the magnitude of the fetal plasma NPY response to acute hypoxemia, there was no corresponding enhancement in the magnitude of the noradrenergic response. This contrasts with the findings in chicken embryos in which the noradrenergic response to acute hypoxemia increases with advancing incubation time (45). However, in the ovine fetus during the last third of gestation, the majority (90%) of the plasma NE response and the entire plasma E response to acute hypoxemia originate from the adrenal medulla, rather than as overspill from sympathetic nerve terminals (39). Taken together, these data indicate that there may be enhancement in the levels of sympathetic efferent nerve activity during episodes of acute hypoxemia with advancing gestational age in the ovine fetus. The pattern of sympathetic nerve discharge governs relative amounts of NPY and NE released from sympathetic nerve terminals, with NPY being favored at higher stimulation frequencies (42). Therefore, the increase in the magnitude of the fetal plasma NPY response to acute hypoxemia with advancing gestational age may indicate enhancement of sympathetic efferent discharge frequency and/or a shift in the dominance of signal transmission from noradrenergic to peptidergic mechanisms.
In the present study, when data were combined irrespective of gestational age, a positive correlation was found between the magnitude of the FVR achieved during the hypoxemic episode and the corresponding fetal basal cortisol concentration. The significant relation between fetal basal cortisol and the slope of the relation between changes in PO2 and changes in FVR for all individual fetuses suggests a glucocorticoid-dependent effect on in vivo vasomotor chemoreflex function toward full term. In addition, it is well established that cortisol and other glucocorticoids modify vascular sensitivity to vasoactive agents (2, 66). For example, it has been shown that 24 h of cortisol infusion into the ovine fetus enhances the pressor response to exogenous angiotensin II administration (61). Studies in our laboratory have shown that treatment of immature fetal sheep with dexamethasone enhances the magnitude of the femoral vasoconstrictor response to acute hypoxemia to values obtained in fetuses at full term (22). Studies using in vitro wire myography have demonstrated developmental changes in the sensitivity and responsiveness of isolated ovine fetal resistance arteries to vasoconstrictor and vasodilator agents (15, 16). Although vascular sensitivity to NE declined in the ovine fetal middle cerebral and femoral arteries, the magnitude of the maximum femoral arterial contractile response elicited by NE increased with advancing gestational age (15). Application of depolarizing K+ solutions also indicated a gestational age-dependent increase in the contractile capacity of the middle cerebral and femoral arteries that was associated with enhanced vascular sensitivity to endothelin-1 (16). Furthermore, although femoral arteries responded to acetylcholine in all age groups, the magnitude of the relaxation declined with advancing gestational age (16). Similar studies in the chick embryo have demonstrated increases in carotid and femoral -adrenergic sensitivity with advancing incubation time (41). Taken together, past and present data suggest that developmental increases in femoral arterial constrictor capacity, vascular responsiveness to constrictor adrenergic and peptidergic agonists, and ontogenic decreases in responsiveness to cholinergic dilator stimulation may contribute to the enhanced ovine fetal FVR response to acute hypoxemia toward full term.
Fetal ACTH and Cortisol Responses to Hypoxemia
The results of this study demonstrate an ontogenic increase in the increment in ovine fetal plasma cortisol concentration in response to episodes of acute hypoxemia close to full term. Despite the gestational age-dependent increases in basal plasma cortisol concentration and the lack of differences in the fetal plasma ACTH response to acute hypoxemia between the gestational age groups, the increment in fetal plasma cortisol concentration during acute hypoxemia was greater at >140 than at 125–130 days gestation. These findings extend those of previous investigators who have reported ontogenic increases in the increments in fetal plasma cortisol concentration during acute hypoxemia (35, 38) by emphasizing that developmental changes in adrenocortical responses to acute hypoxemia occur not only in late gestation, but also immediately before full term.
In conclusion, the data show developmental changes in the pattern of the ovine fetal cardiovascular defense to acute hypoxemia during the last 15–20 days of gestation, right up to full term, in association with the prepartum fetal cortisol surge. Prepartum maturation of the fetal cardiovascular defense mechanisms may be an important protection against intrapartum hypoxia. Studies in which basal plasma cortisol concentrations in the preterm and full-term ovine fetus are manipulated by exogenous infusion and adrenalectomy are needed to address the role of fetal endogenous glucocorticoid in the maturation of the fetal cardiovascular defense response to hypoxic stress just before full term.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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-receptor responsiveness and function during late gestation. Pediatr Res 31: 468–473, 1992.[Web of Science][Medline]-adrenergic blockade on blood flow distribution during hypoxaemia in fetal sheep. J Dev Physiol 6: 349–358, 1984.[Web of Science][Medline]-adrenergic receptors by steroid hormones. Annu Rev Physiol 46: 119–130, 1984.[CrossRef][Web of Science][Medline]-adrenergic receptors, adenylate cyclase and ornithine decarboxylase in rat heart and kidney. J Dev Physiol 16: 251–261, 1991.[Medline]-adrenergic blockade during hypoxia in fetal sheep. Am J Obstet Gynecol 142: 410–415, 1982.[Web of Science][Medline]This article has been cited by other articles:
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) in fetal PaO2 and fetal FVR or FHR during the first 15 min of acute hypoxemia protocol is plotted at 125–130 days gestation (dGA; n = 13), 135–140 dGA (n = 6), and >140 dGA (n = 6). Values are individual data points for each fetus at 15 and 45 min of normoxia and 5 and 15 min of hypoxemia. Lines represent best fit for each group. r, Regression coefficient. *P < 0.05, >140 dGA vs. 125–130 dGA (by analysis of slopes and Student's t-test for unpaired data).
