Plasma Adrenocorticotropin and Cortisol Concentrations during Acute ...

0013-7227/01/$03.00/0 Endocrinology Copyright © 2001 by The Endocrine Society

Vol. 142, No. 2 Printed in U.S.A.

Plasma Adrenocorticotropin and Cortisol Concentrations during Acute Hypoxemia after a Reversible Period of Adverse Intrauterine Conditions in the Ovine Fetus During Late Gestation* D. S. GARDNER, A. J. W. FLETCHER, A. L. FOWDEN,

AND

D. A. GIUSSANI

The Physiological Laboratory, University of Cambridge, Cambridge, United Kingdom CB2 3EG ABSTRACT The present study determined the pituitary-adrenal responses to acute hypoxemia after a period of reversible adverse intrauterine conditions produced by partial compression of the umbilical cord for 3 days in the sheep fetus during late gestation. At 118 ⫾ 2 days gestation (term is ⬃145 days), 12 sheep fetuses were instrumented under halothane anesthesia with an occluder cuff around the umbilical cord, amniotic and vascular catheters, and a transit-time flow probe around an umbilical artery. In 6 of the fetuses at 125 days, umbilical blood flow was reduced by about 30% from baseline for 3 days (UCC), after which the occluder was deflated. The remaining 6 fetuses acted as sham-operated controls in which the occluder was not inflated. All fetuses were then subsequently subjected to 2 periods of acute hypoxemia, elicited by reducing the maternal inspired fraction of oxygen (FiO2) at 2 ⫾ 1 and 5 ⫾ 2 days after the end of cord compression or sham compression. In addition, 4 fetuses from each group were subjected to an ACTH challenge 1–2 days after the final episode of acute hypoxemia. Maternal and fetal arterial blood samples were taken at appropriate intervals during cord compression, acute hypoxemia, and ACTH challenge for analyses of blood gases, pH, and plasma ACTH and cortisol concentrations. Partial compression of the umbilical cord produced reversible mild fetal asphyxia, a transient

increase in fetal plasma ACTH, and a progressive increase in fetal plasma cortisol. At 5 ⫾ 2 days after the end of compression, despite similar blood gas status between the groups, basal plasma cortisol, but not ACTH, concentrations were significantly greater in compressed fetuses relative to sham controls. However, this dissociation did not affect a similar increment in fetal plasma ACTH and cortisol concentrations during acute hypoxemia or in the fetal plasma cortisol response to the ACTH challenge in either group. An increase in adrenocortical mass occurred in fetuses preexposed to partial compression of the umbilical cord relative to sham controls. The data suggest that fetal exposure to a reversible period of adverse intrauterine conditions produced by partial compression of the umbilical cord does not affect the magnitude of the fetal hypothalamic-pituitary-adrenal axis response to subsequent acute hypoxemia, but it leads to resetting of basal hypothalamic-pituitary-adrenal axis function in the fetus. The mechanism for this resetting may include an increase in adrenocortical steroidogenic synthetic capacity, but it is not due to a change in adrenocortical sensitivity to ACTH. Inappropriate fetal glucocorticoid exposure after reversible periods of adverse intrauterine conditions has important implications for fetal and postnatal development. (Endocrinology 142: 589 –598, 2001)

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trauterine complications, such as those produced by partial compression of the umbilical cord, may increase the susceptibility of the fetus to perinatal complications and, potentially, neurodevelopmental handicap (12). Furthermore, episodes of acute hypoxemia are common during the actual processes of labor and delivery (13) and are therefore just as likely to occur after as during the period of adverse intrauterine conditions in complicated pregnancies. The aims of the present study were therefore to determine the fetal plasma ACTH and cortisol responses to acute hypoxemia after exposure of the fetus to a reversible period of adverse intrauterine conditions produced by controlled, partial compression of the umbilical cord in sheep during late gestation. In addition, to address possible mechanisms mediating any alteration in these endocrine responses, control and compressed fetuses were subjected to an ACTH challenge, and the adrenal glands from both groups of fetuses were harvested for histological analysis.

HE FETAL ENDOCRINE responses to stress, whether acute (i.e. minutes to hours) induced by hemorrhage (1, 2), hypotension (3), or hypoxemia (4, 5), or chronic (days to weeks) induced by removal of the majority of endometrial caruncles [the sites of formation of the ovine placentomes (carunclectomy)] (6), feto-placental embolization (7), or reduced uterine blood flow (8), have been clearly documented, and all are characterized by an increase in fetal plasma ACTH and cortisol concentrations. Although a few studies have investigated the effects of preexisting adverse intrauterine conditions on the fetal hypothalamic-pituitary-adrenal (HPA) axis response to a superimposed acute challenge (acute-on-chronic) (9 –11), no study has investigated the fetal HPA axis response to acute stress after the reestablishment of basal status following a period of adverse intrauterine conditions (acute-after-chronic). This is important, because it has been proposed that mild, undiagnosed, antecedent inReceived August 18, 2000. Address all correspondence and requests for reprints to: Dr. Dino A. Giussani, The Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, United Kingdom CB2 3EG. E-mail: [email protected]. * This work was supported by the British Heart Foundation.

Materials and Methods Surgical preparation Twelve Welsh Mountain ewes carrying singleton pregnancies of known gestational age were used in the study. All procedures were

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FETAL HPA AXIS ACTIVITY AFTER REVERSIBLE INTRAUTERINE STRESS

performed under the United Kingdom Animals (Scientific Procedures) Act, 1986. All food, but not water, was withdrawn from the animals for 24 h before surgery. Surgery was performed under aseptic conditions at 118 ⫾ 2 days gestation (dGA; term is ⬃145 dGA). Anesthesia was induced with sodium thiopentone (20 mg/kg, iv; Intraval Sodium, Rhone Me´rieux, Dublin, Ireland) and was maintained with 1–2% halothane in O2/N2O (50:50). In brief, after midline abdominal and uterine incisions, the fetal head was exteriorized for insertion of carotid artery and jugular vein catheters (id, 0.86 mm; od, 1.52 mm; Critchly Electrical Products, Auburn, NSW, Australia) with the tips of the catheters extended to the ascending aorta and superior vena cava, respectively. The catheters were plugged with sterile brass pins, and the uterine incision was closed in layers. The fetal hindlimbs were subsequently exteriorized through a second uterine incision for insertion of femoral artery (id, 0.86 mm; od, 1.52 mm) and femoral vein (id, 0.56 mm; od, 0.96 mm) catheters, which were extended into the descending aorta and inferior vena cava, respectively. A further catheter was anchored onto the fetal hindlimb in the amniotic cavity for recording of the reference pressure. A transit-time flow transducer (Transonics, Inc., Ithaca, NY) was placed around an umbilical artery (4RS) within the fetal abdominal cavity as previously described (14). In addition, an inflatable occluder cuff (OC20HD, In Vivo Metrics, CA) was positioned around the proximal end of the umbilical cord and anchored to the fetal abdominal wall so as to avoid contact with the cord when not inflated (14). The second uterine incision was closed in layers. A Teflon catheter was placed in the maternal femoral artery and extended to the descending aorta. Antibiotics were administered to the fetus through the femoral vein (300 mg ampicillin; Penbritin, SmithKline Beecham Animal Health, Surrey, UK) and amniotic catheters (300 mg ampicillin). All catheters were filled with heparinized saline (80 IU heparin/ml in 0.9% NaCl), plugged with brass pins. Then, together with

FIG. 1. Change in umbilical blood flow during umbilical cord compression or sham compression. Values are the mean ⫾ SEM of cardiovascular data (30 min average) for a baseline period (1 day before umbilical cord compression), for the 3 days during umbilical cord compression or sham compression (bar), and for 1 day of recovery after umbilical cord compression or sham compression. A, Control fetuses (n ⫽ 6); B, UCC fetuses (n ⫽ 6). *, P ⬍ 0.05, baseline vs. compression or recovery. ˙ Q umb, Umbilical blood flow.

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the flow probes and occluder leads, the catheters were exteriorized through an incision in the maternal flank and housed in a pouch sutured to the maternal skin.

Postoperative care Animals were housed in individual pens with access to hay and water ad libitum. Concentrates were fed twice daily (100 g; Sheep Nuts no. 6, H&C Beart Ltd., Kings Lynn, UK). All ewes received antibiotics (0.20 – 0.25 mg/kg, im; Depocillin, Mycofarm, Cambridge, UK) and analgesia (10 –20 mg/kg, orally, phenylbutazone; Equipalozone Paste, Arnolds Veterinary Products Ltd., Shropshire, UK) immediately after surgery and daily for 3 days. The patency of fetal vascular catheters was maintained by a slow continuous infusion of heparinized saline (25 IU heparin/ml at 0.1 ml/h in 0.9% NaCl) containing antibiotic (1 mg/ml benzylpenicillin; Crystapen, Schering-Plough Corp., Welwyn Garden City, UK).

Experimental procedure At least 5 days after surgery, at 124 ⫾ 0.5 dGA, baseline mean unilateral umbilical blood flow was determined over a 24-h period in all fetuses. The animals were then divided randomly into two experimental groups. In six fetuses the occluder cuff was inflated to reduce umbilical blood flow by about 30% from the predetermined baseline for 3 days (Fig. 1). Compression of the umbilical cord was achieved either by manual inflation of the occluder cuff with saline (n ⫽ 2) or by an automated servo-controlled system that inflated or deflated the occluder cuff according to the umbilical blood flow reading (n ⫽ 4) (15). After 3 days of compression the occluder cuff was deflated, allowing return of umbilical blood flow to baseline. In the remaining six fetuses the oc-

FETAL HPA AXIS ACTIVITY AFTER REVERSIBLE INTRAUTERINE STRESS cluder cuff remained deflated throughout the duration of the experimental procedure. These animals were designated sham-operated control animals. After umbilical cord compression or sham compression, all fetuses were subjected to two separate episodes of acute hypoxemia, induced by reducing maternal inspired fraction of oxygen (FiO2) at 130 ⫾ 1 dGA (2 ⫾ 1 days after umbilical cord compression or sham compression; hypoxemia I) and again at 134 ⫾ 1 dGA (5 ⫾ 2 days; hypoxemia II). The rationale for the first episode of acute hypoxemia was to determine whether fetal preexposure to adverse intrauterine conditions produced by partial umbilical cord compression had influenced the plasma ACTH and cortisol responses to subsequent acute stress. The rationale for the second episode of acute hypoxemia was to determine whether any measured effect was transient, persistent, or progressive. The protocol for acute fetal hypoxemia involved a 3-h experiment consisting of 1 h of normoxia, 1 h of hypoxemia, and 1 h of recovery, as previously described in detail (16). Briefly, a large, transparent, polythene bag was placed over the ewe’s head, into which air was passed at a rate of approximately 40 liter/min for the first 1 h. After this control period, fetal hypoxemia was induced by changing the concentrations of gases breathed by the ewe to 9% O2 in N2 with 2–3% CO2. This mixture was designed to reduce fetal carotid PaO2 to approximately 12 mmHg, while maintaining arterial isocapnia. After the 1-h period of hypoxemia the ewe was returned to breathing air for the 1-h recovery period. Between 1–2 days after the second hypoxemia protocol, a 2.5-␮g bolus dose of synthetic ACTH (Synacthen, Ciba Pharmaceuticals, London, UK) was injected iv in four control and four UCC fetuses to determine adrenocortical steroidogenic sensitivity to ACTH. The dose of Synacthen used was based on previous studies from this laboratory (17).

Measurements and biochemical analyses Daily maternal descending aortic and fetal carotid blood samples (0.4 ml) were drawn into sterile syringes and analyzed for arterial blood gases, percent saturation of O2 in hemoglobin (% Sat Hb), hemoglobin concentration, and acid/base status using an ABL5 blood gas analyzer and OSM2 hemoximeter (Radiometer, Copenhagen, Denmark). Measurements in maternal and fetal blood were corrected to 38 and 39.5 C, respectively. In addition, maternal and fetal arterial blood samples (4 ml) were taken simultaneously before umbilical cord compression at ⫺1 day and ⫺1 h; during umbilical cord compression at ⫹1 h, ⫹8 h, ⫹1 day, ⫹2 days, and ⫹3 days; and subsequently at 1 day after deflation of the occluder cuff for measurement of blood gases, acid/base status and hormone concentrations. During the hypoxemia protocol, paired maternal and fetal arterial blood samples (4 ml) were collected at 15 and 45 min of normoxia, after 15 and 45 min of hypoxemia, and after 45 min of recovery for measurement of blood gases, acid/base status, and hormones. Fetal arterial blood samples (2 ml) were also taken during the ACTH challenge at ⫺15 and ⫺5 min and subsequently at 5, 15, and 30 min after injection. This blood-sampling regimen did not significantly affect fetal hemoglobin concentration over the period of study in either group. All blood samples for hormone analysis were collected into K⫹/EDTA-treated tubes, kept on ice, and centrifuged at 4000 rpm for 4 min at 4 C. Plasma samples were stored at ⫺70 C until analyses.

Hormone analyses All hormone measurements were performed within 2 months of sample collection. Plasma ACTH and cortisol concentrations were determined by RIA validated for use in ovine plasma. ACTH. Maternal and fetal plasma ACTH concentrations were measured using a commercially available double antibody 125I RIA kit (INCSTAR Corp., Wokingham, UK). The lower limit of detection for the assay was between 10 –25 pg/ml. The intraassay coefficients of variation for two plasma pools (37 and 150 pg/ml) were 3.6% and 4.1%, respectively. The interassay coefficient of variation was 8.4%. The cross-reactivities for the assay were less than 0.01% for ␣MSH, ␤-endorphin, ␤-lipotropin, leucine enkephalin, methionine enkephalin, bombesin, calcitonin, PTH, FSH, arginine vasopressin, oxytocin, and substance P. Cortisol. Maternal and fetal plasma cortisol concentrations were measured by RIA validated for use in ovine plasma, as described previously (18). The lower limit of detection for the assay was 1.0 –1.5 ng/ml. The

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intra- and interassay coefficients of variations were 5.3% and 7.8%, respectively. The cross-reactivities of the antiserum at 50% binding with other cortisol-related compounds were 0.5% cortisone, 2.3% corticosterone, 0.3% progesterone, and 4.6% deoxycortisol.

Microscopy At least 1 day after the ACTH challenge, between 136 –137 dGA, all ewes were injected iv with a terminal anesthetic (20 mg/kg sodium pentobarbitone), and the fetal body and adrenal weights were determined. In addition, an adrenal gland was fixed in 10% formaldehyde and, within 2 days, embedded in paraffin wax for sectioning (UCC fetuses, n ⫽ 5; sham control fetuses, n ⫽ 3). Adrenal glands from an additional two age-matched control fetuses without hormone measurements were also fixed, embedded, and included in the analyses for adrenal morphology. Each adrenal gland was cut in half along its longitudinal axis, and a total of 6 ⫻ 7-␮m sections from the midline were taken with a microtome, mounted on individual slides, and stained with hemalum. Total adrenal, adrenocortical, and adrenomedullary widths were determined with a calibrated eye-piece graticule under low power (⫻4) light microscopy. At least six measurements were taken from each of the adrenal sections.

Data collection and analyses Analog signals for calibrated umbilical blood flow were recorded continuously in control and UCC fetuses for 1 day before umbilical cord compression, during the 3 days of compression, and for 1 day after deflation of the occluder cuff using a data acquisition system. The signal was digitized, displayed, and subsequently stored at 8-sec intervals on disk by custom software (NI-DAQ, National Instruments, Austin, TX) running on a personal computer. Files were subsequently analyzed using Microsoft Corp. Excel spreadsheets. Unilateral umbilical blood flow was measured on a T201 or T206 flow box (Transonic).

Statistical analyses Values for all variables are expressed as the mean ⫾ sem unless otherwise stated. All measured variables were first analyzed for normality of distribution. All data obtained were parametric and were analyzed using two-way ANOVA with repeated measures (Sigma-Stat, SPSS, Inc., Chicago, IL) followed by an appropriate post-hoc test. A comparison between the slopes and intercepts of regression curves was conducted according to Armitage and Berry (19). For all comparisons, statistical significance was accepted when P ⬍ 0.05.

Results Effect of umbilical cord compression on umbilical blood flow, arterial blood gas and acid/base status, and plasma ACTH and cortisol concentrations

Umbilical blood flow. Baseline unilateral umbilical blood flow, calculated at ⫺1 ⫾ 0.5 day before umbilical cord compression (124 ⫾ 0.5 dGA), was similar in sham control and UCC fetuses (171 ⫾ 18 vs. 170 ⫾ 21 ml/min, respectively). Unilateral umbilical blood flow in sham control fetuses remained unaltered from baseline throughout the experimental protocol. In contrast, inflation of the occluder cuff in UCC fetuses at 125 ⫾ 0.5 dGA produced a reduction in unilateral umbilical blood flow by the desired level of approximately 30% from baseline and maintained this reduction throughout the 3 days of umbilical cord compression (Fig. 1). After umbilical cord compression, unilateral umbilical blood flow returned to a level significantly greater than that measured during baseline (Fig. 1). Arterial blood gas and metabolic status.

Fetal: Baseline arterial blood gas and acid/base status were similar in sham control and UCC fetuses (Table 1). Arterial

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TABLE 1. Fetal arterial blood gas and acid/base status during umbilical cord compression Baseline

pHa Control UCC Pa, CO2 (mmHg) Control UCC Pa, O2 (mmHg) Control UCC SatHb (%) Control UCC Hb (g/dl) Control UCC Base excess (mEq/liter) Control UCC

Umbilical cord compression

⫺1 day

⫺1 h

⫹1 h

⫹8 h

⫹1 day

⫹2 day

⫹3 day

Recovery ⫹4 day

7.35 ⫾ 0.01 7.34 ⫾ 0.01

7.35 ⫾ 0.01 7.33 ⫾ 0.01

7.34 ⫾ 0.01 7.30 ⫾ 0.01a

7.34 ⫾ 0.01 7.31 ⫾ 0.01a,b

7.34 ⫾ 0.01 7.30 ⫾ 0.01a,b

7.34 ⫾ 0.01 7.32 ⫾ 0.01a

7.33 ⫾ 0.01 7.32 ⫾ 0.01a

7.33 ⫾ 0.01 7.36 ⫾ 0.01

50.0 ⫾ 1.7 51.3 ⫾ 1.3

51.2 ⫾ 1.6 49.8 ⫾ 1.8

51.6 ⫾ 2.4 53.0 ⫾ 2.2a

53.0 ⫾ 2.3 53.5 ⫾ 1.7a

54.2 ⫾ 1.0 54.6 ⫾ 1.2a

53.3 ⫾ 1.7 55.3 ⫾ 1.4a

53.3 ⫾ 1.5 55.8 ⫾ 1.3a

53.5 ⫾ 0.9 49.8 ⫾ 2.0

22.6 ⫾ 1.5 22.6 ⫾ 1.5

22.0 ⫾ 1.9 22.6 ⫾ 2.0

22.8 ⫾ 1.9 21.1 ⫾ 1.7

22.0 ⫾ 2.2 20.3 ⫾ 0.9a

20.6 ⫾ 1.2 19.5 ⫾ 1.4a

22.1 ⫾ 1.8 20.1 ⫾ 1.0a

22.6 ⫾ 1.8 19.3 ⫾ 1.4a

21.5 ⫾ 1.0 22.3 ⫾ 1.1

67.1 ⫾ 2.7 65.3 ⫾ 3.0

66.9 ⫾ 4.0 66.5 ⫾ 5.2

69.9 ⫾ 4.0 58.8 ⫾ 5.2a

65.6 ⫾ 4.0 55.5 ⫾ 3.1a

62.4 ⫾ 1.3 52.9 ⫾ 2.7a,b

65.8 ⫾ 3.2 55.9 ⫾ 3.7a

65.5 ⫾ 3.7 56.1 ⫾ 4.3a

64.1 ⫾ 3.2 62.0 ⫾ 4.2

8.8 ⫾ 0.3 8.9 ⫾ 0.4

8.8 ⫾ 0.3 8.6 ⫾ 0.4

8.7 ⫾ 0.1 8.9 ⫾ 0.5

8.7 ⫾ 0.2 9.1 ⫾ 0.5

8.6 ⫾ 0.3 9.2 ⫾ 0.5

9.0 ⫾ 0.3 8.9 ⫾ 0.5

8.7 ⫾ 0.3 8.7 ⫾ 0.4

8.8 ⫾ 0.3 8.6 ⫾ 0.6

1.5 ⫾ 1.1 1.3 ⫾ 0.7

1.4 ⫾ 0.6 ⫺0.3 ⫾ 1.4

1.2 ⫾ 0.6 ⫺1.2 ⫾ 1.4

1.4 ⫾ 1.1 ⫺0.6 ⫾ 1.1

2.2 ⫾ 0.3 ⫺0.3 ⫾ 1.1

1.8 ⫾ 0.9 1.0 ⫾ 0.8

0.6 ⫾ 1.0 1.3 ⫾ 0.8

1.0 ⫾ 0.5 1.8 ⫾ 1.3

Fetal carotid arterial blood gas and acid/base status during umbilical cord compression or sham compression. Fetal blood samples were collected for measurement of blood gases during baseline at ⫺1 day and ⫺1 h and subsequently at ⫹1 h, ⫹8 h, ⫹1 day, ⫹2 days, and ⫹3 days during umbilical cord compression or sham compression and at ⫹1 and ⫹3 days of recovery after umbilical cord compression or sham compression. Values are the mean ⫾ SEM for control (n ⫽ 6) and UCC (n ⫽ 6) fetuses. Fetal blood gas values were corrected to 39.5 C. pHa, Arterial pH; PaCO2, arterial CO2 partial pressure; PaO2, arterial O2 partial pressure, SatHb, percent oxygen saturation of hemoglobin; UCC, umbilical cord compression. a P ⬍ 0.05, baseline vs. umbilical cord compression/recovery. b P ⬍ 0.05, control vs. UCC fetuses.

blood gas status remained unchanged from baseline throughout the experimental protocol in sham control fetuses. In UCC fetuses, umbilical cord compression, to reduce umbilical blood flow by approximately 30% of baseline, caused falls in pHa, PaO2, and %SatHb and an immediate rise in PaCO2. These changes were maintained until the end of the 3-day compression period (Table 1). After umbilical cord compression, arterial blood gas values and acid/base status returned to baseline conditions by 1 day recovery in UCC fetuses (Table 1). Maternal: Maternal arterial blood gas and acid-base status were similar in control and UCC ewes during the baseline period (control: pHa, 7.49 ⫾ 0.01; PaCO2, 34.3 ⫾ 1.1 mmHg; PaO2, 103 ⫾ 3 mmHg; %SatHb, 93.9 ⫾ 1.7%; UCC: pHa, 7.48 ⫾ 0.01; PaCO2, 33.4 ⫾ 0.7 mmHg; PaO2, 98.1 ⫾ 4.1 mmHg; %SatHb, 94.1 ⫾ 1.3%). All values for arterial blood gas and acid/base status remained unaltered from baseline throughout the duration of the experimental protocol in control and UCC ewes. Plasma ACTH and cortisol.

Fetal: Before umbilical cord compression, baseline ACTH and cortisol concentrations were similar in control and UCC fetuses (Fig. 2). In control fetuses, both ACTH and cortisol concentrations remained unaltered from baseline for the duration of the sham compression period. In contrast, umbilical cord compression elicited an immediate increase in plasma ACTH concentration; however, the effect was transient, with values returning toward baseline concentrations 1 day after compression (Fig. 2). In contrast, plasma cortisol concentra-

tions increased progressively during the period of compression, with values remaining significantly higher than baseline 3 days after the onset of umbilical cord compression. Maternal: Baseline ACTH and cortisol concentrations in control and UCC ewes were 39.2 ⫾ 8.3 vs. 48.9 ⫾ 5.6 pg/ml for ACTH (P ⫽ NS) and 10.3 ⫾ 1.5 vs. 14.0 ⫾ 2.2 ng/ml for cortisol (P ⫽ NS), respectively. Daily concentrations of maternal ACTH and cortisol were unaltered from baseline during the period of umbilical cord compression or sham compression. Effect of acute hypoxemia after the period of reversible adverse intrauterine conditions on arterial blood gas and acid/base status and ACTH and cortisol concentrations Arterial blood gas and acid/base status during acute hypoxemia.

Fetal: Baseline arterial blood gas and acid/base status were similar in sham control and UCC fetuses during both acute hypoxemia protocols (Table 2). A similar reduction in PaO2, %SatHb, pHa and acid-base excess (ABE) occurred in sham control and UCC fetuses during both hypoxemia I and II (Table 2). These changes generally occurred without any alteration in PaCO2 from baseline in either group of fetuses; however, a mild hypercapnia developed in control fetuses early during the first hypoxemic challenge (Table 2). Arterial blood gas status returned to baseline levels during recovery; however, both groups of fetuses remained mildly acidic by the end of each of the hypoxemic protocols (Table 2). Maternal: Basal arterial blood gas and acid/base status


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FIG. 2. Values are the mean ⫾ SEM for control (E; n ⫽ 6) and UCC (F; n ⫽ 6) fetuses. Fetal blood samples were collected for measurement of ACTH and cortisol during the baseline period at ⫺1 day and ⫺1 h; at ⫹1 h, ⫹8 h, ⫹1 day, ⫹2 days, and ⫹3 days during umbilical cord compression or sham compression (bar); and at ⫹1 day of recovery after umbilical cord compression or sham compression. a, P ⬍ 0.05, baseline vs. umbilical cord compression; b, P ⬍ 0.05, control vs. UCC fetuses.

were similar in sham control and UCC ewes during both hypoxemia I and II (Table 3). Similar falls in maternal PaO2 and %SatHb and increases in hemoglobin concentration occurred during each of the hypoxemia protocols in both groups of ewes. These changes occurred without any alteration in PaCO2 from baseline. After each of the hypoxemia protocols, arterial blood gas status returned to baseline levels by the end of recovery period in both groups of ewes (Table 3). Plasma ACTH and cortisol during acute hypoxemia.

Fetal: Changes in fetal plasma ACTH and cortisol concentrations during each of the acute hypoxemia protocols are shown in Fig. 3. Basal plasma ACTH and cortisol concentrations were similar in sham control and UCC fetuses during hypoxemia I. However, in the hypoxemia II protocol, basal plasma cortisol, but not ACTH, concentrations were elevated in UCC fetuses relative to those in sham controls. Despite the elevated basal plasma cortisol values in the UCC fetuses produced by hypoxemia II, significant increases in

fetal plasma ACTH and cortisol occurred in both groups of fetuses during each acute hypoxemia experiment. During recovery after hypoxemia I and II, plasma ACTH and cortisol remained elevated relative to baseline in both groups of fetuses. However, plasma cortisol, but not ACTH, concentrations remained elevated in UCC relative to those in sham control fetuses during recovery after hypoxemia II. Correlation of paired plasma ACTH and cortisol concentrations showed that both the slope and intercept of the relationship were similar for control and UCC fetuses during hypoxemia I (Fig. 4). However, a significant change in the intercept, but not the slope, of the relationship between plasma ACTH and cortisol occurred in UCC fetuses during hypoxemia II (Fig. 4). Maternal: Basal plasma ACTH and cortisol were similar in sham control and UCC ewes during hypoxemia I (ACTH, 35.4 ⫾ 12.9 vs. 64.6 ⫾ 7.3 pg/ml; cortisol, 28.5 ⫾ 5.0 vs. 30.0 ⫾ 5.6 ng/ml, respectively) and hypoxemia II (ACTH, 38.4 ⫾ 9.3 vs. 57.3 ⫾ 11.0 pg/ml; cortisol, 32.0 ⫾ 8.4 vs. 25.5 ⫾ 6.0 ng/ml, respectively). Maternal plasma ACTH and cortisol concentrations remained unaltered from baseline during both hy-

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TABLE 2. Fetal arterial blood gas and acid/base status during acute hypoxemia at 2 and 5 days after UCC Hypoxemia I Baseline

pHa Control UCC PaCO2 (mmHg) Control UCC PaO2 (mmHg) Control UCC SatHb (%) Control UCC Hemoglobin (g/dl) Control UCC Base excess (mEq/liter) Control UCC

Hypoxemia II

Hypoxemia

Recovery

Baseline

7.29 ⫾ 0.01a 7.27 ⫾ 0.01a

7.30 ⫾ 0.01a 7.30 ⫾ 0.02a

53.3 ⫾ 1.8a 51.0 ⫾ 1.5

52.2 ⫾ 2.1 51.6 ⫾ 1.8

22.1 ⫾ 0.7 20.2 ⫾ 1.4

12.2 ⫾ 0.7a 12.0 ⫾ 0.6a

61.9 ⫾ 2.6 54.8 ⫾ 6.5

H15

H45

7.33 ⫾ 0.01 7.33 ⫾ 0.01

7.31 ⫾ 0.01a 7.32 ⫾ 0.01a

51.0 ⫾ 1.8 51.5 ⫾ 1.3

Hypoxemia

Recovery

H15

H45

7.34 ⫾ 0.01 7.33 ⫾ 0.01

7.32 ⫾ 0.01a 7.31 ⫾ 0.01a

7.29 ⫾ 0.01a 7.28 ⫾ 0.01a

7.30 ⫾ 0.01a 7.31 ⫾ 0.01a

50.3 ⫾ 2.2 49.8 ⫾ 1.7

54.2 ⫾ 0.8 53.7 ⫾ 1.7

54.2 ⫾ 1.0 52.8 ⫾ 2.4

54.4 ⫾ 1.0 51.4 ⫾ 2.7

53.6 ⫾ 1.0 49.2 ⫾ 2.4

12.6 ⫾ 0.2a 11.6 ⫾ 0.5a

20.0 ⫾ 1.0 20.6 ⫾ 1.5

20.2 ⫾ 1.0 20.9 ⫾ 1.2

11.4 ⫾ 0.5a 11.4 ⫾ 0.2a

11.6 ⫾ 0.2a 12.4 ⫾ 0.5a

19.2 ⫾ 1.7 21.4 ⫾ 1.9

30.5 ⫾ 4.1a 30.1 ⫾ 4.3a

30.7 ⫾ 3.1a 27.5 ⫾ 4.5a

56.5 ⫾ 3.7 50.6 ⫾ 7.2a

57.5 ⫾ 3.0 59.4 ⫾ 4.9

29.4 ⫾ 3.9a 29.4 ⫾ 3.2a

28.9 ⫾ 3.2a 31.2 ⫾ 4.2a

51.5 ⫾ 4.6a 56.5 ⫾ 5.1

8.3 ⫾ 0.3 8.0 ⫾ 0.3

7.1 ⫾ 1.9 8.3 ⫾ 0.3

9.0 ⫾ 0.5 8.4 ⫾ 0.5

8.2 ⫾ 0.1 7.3 ⫾ 0.2a

9.2 ⫾ 0.5 7.6 ⫾ 0.1

9.9 ⫾ 0.5a 8.2 ⫾ 0.3

9.6 ⫾ 0.5 7.8 ⫾ 0.2

8.7 ⫾ 0.5a 7.5 ⫾ 0.3

0.3 ⫾ 0.6 0.8 ⫾ 0.8

⫺0.8 ⫾ 0.7 ⫺1.0 ⫾ 0.7a

⫺2.8 ⫾ 1.1a ⫺3.5 ⫾ 1.2a

⫺1.8 ⫾ 1.0 ⫺2.5 ⫾ 1.1a

2.3 ⫾ 0.7 1.1 ⫾ 0.4

0.8 ⫾ 1.0a ⫺0.4 ⫾ 0.5a

⫺1.6 ⫾ 1.0a ⫺3.2 ⫾ 0.6a

⫺0.6 ⫾ 0.8a ⫺2.0 ⫾ 0.6a

Fetal carotid arterial blood gas and acid/base status during acute hypoxemia at 2 ⫾ 1 days (mean ⫾ SD, 130 ⫾ 1.3 dGA; hypoxemia I) and at 5 ⫾ 2 days (134 ⫾ 1.2 dGA; hypoxemia II) after umbilical cord compression or sham compression. Data represent combined values at 15 and 45 min of normoxia (baseline), at 15 (H15) and 45 min (H45) of hypoxemia and at 45 min (R45) of recovery. Values are the mean ⫾ SEM for control (n ⫽ 6) and UCC (n ⫽ 6) fetuses. a P ⬍ 0.05, baseline vs. hypoxemia or recovery. TABLE 3. Maternal arterial blood gas and acid/base status during hypoxemia at 2 and 5 days after UCC Hypoxemia I Baseline

pHa Control UCC PaCO2 (mmHg) Control UCC PaO2 (mmHg) Control UCC SatHb (%) Control UCC Hemoglobin (g/dl) Control UCC Base excess (mEq/liter) Control UCC

Hypoxemia II

Hypoxemia

Recovery

H15

H45

7.48 ⫾ 0.01 7.50 ⫾ 0.01

7.49 ⫾ 0.01 7.49 ⫾ 0.01

7.49 ⫾ 0.02 7.48 ⫾ 0.01

7.49 ⫾ 0.01 7.47 ⫾ 0.02

35.7 ⫾ 0.9 32.6 ⫾ 0.5

36.5 ⫾ 1.0 34.0 ⫾ 1.5

37.6 ⫾ 1.6 34.0 ⫾ 1.6

102.4 ⫾ 3.5 98.1 ⫾ 2.4

42.4 ⫾ 2.5a 42.1 ⫾ 3.6a

93.8 ⫾ 2.6 94.6 ⫾ 1.5

Baseline

Hypoxemia

Recovery

H15

H45

7.48 ⫾ 0.01 7.50 ⫾ 0.01

7.47 ⫾ 0.02 7.51 ⫾ 0.01

7.48 ⫾ 0.02 7.50 ⫾ 0.01

7.48 ⫾ 0.01 7.49 ⫾ 0.02

35.5 ⫾ 0.6 33.2 ⫾ 1.4

36.2 ⫾ 0.8 34.9 ⫾ 1.7

37.8 ⫾ 1.3 33.4 ⫾ 0.4

37.6 ⫾ 0.6 34.1 ⫾ 0.7

35.4 ⫾ 1.3 33.4 ⫾ 0.2

41.0 ⫾ 2.1a 39.1 ⫾ 2.9a

99.3 ⫾ 2.5 97.8 ⫾ 7.2

101.0 ⫾ 0.8 98.3 ⫾ 2.9

44.2 ⫾ 2.0a 41.5 ⫾ 1.9a

40.4 ⫾ 1.9a 39.6 ⫾ 0.6a

104.6 ⫾ 2.6 96.3 ⫾ 3.4

64.0 ⫾ 5.1a 68.4 ⫾ 3.0a

61.6 ⫾ 3.6a 63.6 ⫾ 4.6a

94.3 ⫾ 2.4 94.4 ⫾ 1.1

93.3 ⫾ 2.5 93.9 ⫾ 0.9

63.4 ⫾ 6.0a 66.5 ⫾ 2.9a

59.9 ⫾ 4.9a 65.3 ⫾ 0.9a

93.2 ⫾ 2.9 91.9 ⫾ 0.7

7.8 ⫾ 0.5 8.3 ⫾ 0.2

8.7 ⫾ 0.5a 9.1 ⫾ 0.4a

8.9 ⫾ 0.7a 9.2 ⫾ 0.4a

8.0 ⫾ 0.6 8.1 ⫾ 0.3

8.2 ⫾ 0.6 8.4 ⫾ 0.1

9.0 ⫾ 0.7a 9.4 ⫾ 0.3a

9.1 ⫾ 0.6a 9.3 ⫾ 0.3a

7.8 ⫾ 0.6a 8.3 ⫾ 0.2

4.0 ⫾ 1.2 3.2 ⫾ 0.8

5.3 ⫾ 1.2a 3.5 ⫾ 0.9

5.6 ⫾ 1.1a 2.8 ⫾ 1.1

4.1 ⫾ 0.9 2.0 ⫾ 0.8a

4.0 ⫾ 1.0 4.5 ⫾ 0.2

4.2 ⫾ 1.2 5.0 ⫾ 0.7

4.8 ⫾ 1.4 4.3 ⫾ 0.7

3.6 ⫾ 1.5 3.5 ⫾ 0.2

Maternal arterial blood gas and acid/base status during acute hypoxemia at 2 ⫾ 1 days (mean ⫾ SD, 130 ⫾ 1.3 dGA; hypoxemia I) and at 5 ⫾ 2 days (134 ⫾ 1.2 dGA; hypoxemia II) after umbilical cord compression or sham compression. Data represent combined values at 15 and 45 min of normoxia (baseline), at 15 (H15) and 45 min (H45) of hypoxemia, and at 45 min (R45) of recovery. Values are the mean ⫾ SEM for control (n ⫽ 6) and UCC (n ⫽ 6) ewes. Maternal blood gas values were corrected to 38 C. a P ⬍ 0.05, baseline vs. hypoxemia or recovery.

poxemia I and II. However, during recovery from each hypoxemic episode, plasma cortisol, but not ACTH, concentrations fell below baseline in both groups of ewes (hypox-

emia I, 11.3 ⫾ 2.4 and 12.7 ⫾ 4.2 ng/ml; hypoxemia II, 11.2 ⫾ 1.9 and 10.0 ⫾ 2.2 ng/ml for plasma cortisol concentration in sham control and UCC ewes, respectively).


595

FIG. 3. Fetal plasma ACTH and cortisol concentrations during acute hypoxemia at 2 ⫾ 1 days (mean ⫾ SD, 130 ⫾ 1.3 dGA; hypoxemia I) and at 5 ⫾ 2 days (134 ⫾ 1.2 dGA; hypoxemia II) after umbilical cord compression or sham compression. Data represent combined values at 15 and 45 min of normoxia (baseline), at 15 (H15) and 45 min (H45) of hypoxemia, and at 45 min (R45) of recovery. Values are the mean ⫾ SEM for control (䡺; n ⫽ 6) and UCC (f; n ⫽ 6) fetuses. a, P ⬍ 0.05, baseline vs. hypoxemia or recovery; b, control vs. UCC fetuses.

Plasma cortisol during the ACTH challenge. Before the ACTH challenge, plasma ACTH concentrations were similar in control and UCC fetuses (54.2 ⫾ 8.4 vs. 50.7 ⫾ 8.7 pg/ml). Concentrations of plasma cortisol were 39% greater in UCC fetuses relative to controls (35.5 ⫾ 10.0 vs. 49.5 ⫾ 13.0 ng/ml), although this difference fell outside statistical significance. A similar increment in plasma cortisol occurred after the ACTH challenge in control and UCC fetuses (Fig. 5). Fetal body and adrenal weights and adrenal morphology

Fetal body weights were similar between sham control and UCC fetuses (Table 4). However, the adrenal glands were larger in UCC than in sham control fetuses when expressed in absolute terms or relative to body weight (P ⬍ 0.05). The width of the adrenal cortex was greater, and the width of the adrenal medulla narrower, in UCC fetuses relative to sham controls (P ⬍ 0.05). Consequently, when expressed as a proportion of total adrenal width, the percentage of the adrenal occupied by the adrenal cortex was significantly greater, and that occupied by the adrenal medulla was significantly lower, in UCC fetuses relative to sham controls (P ⬍ 0.05).

Discussion

Investigation of how the fetal HPA axis responsiveness may be altered during adverse intrauterine conditions in complicated pregnancies has concentrated solely on stimulating the axis with an acute stressor superimposed on preexisting chronic stress. The few studies that have investigated fetal HPA axis function during acute-on-chronic stress suggest that the fetal adrenal is sensitized to ACTH. For example, after carunclectomy, the increase in fetal plasma cortisol during superimposed acute hypoxemia was greater in small than in normal-sized carunclectomized fetuses in sheep (9). In addition, after chronic embolization of the sheep placenta, the fetal plasma cortisol response to superimposed acute hypoxemia was similar in control and embolized fetuses despite lower plasma ACTH and adrenal blood flow in embolized fetuses relative to controls (11). To date, no study has addressed the fetal HPA axis response to an acute stress after the period of adverse intrauterine conditions has resolved. The present study reports that the magnitude of the fetal plasma ACTH and cortisol responses to 1 h of acute hypoxemia after a 3-day period of adverse intrauterine conditions

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FIG. 5. Fetal plasma cortisol concentrations after a 2.5-␮g bolus dose of exogenous ACTH (Synacthen) between 6 –7 days after umbilical cord compression/sham compression. Values are the mean ⫾ SEM for control (䡺; n ⫽ 4) and UCC (f; n ⫽ 4) fetuses expressed as the change from mean baseline (combined values at ⫺15 and ⫺5 min) and at 5, 15, and 30 min after the ACTH bolus (time zero). ⴱ, P ⬍ 0.05 compared with baseline. TABLE 4. Adrenal weight and morphology

FIG. 4. Linear regression for fetal plasma ACTH and cortisol in control (E, dashed line) and UCC (F, solid line) fetuses. Data points are for all paired ACTH and cortisol samples in individual fetuses obtained during either the first episode of acute hypoxemia at 2 ⫾ 1 days (mean ⫾ SD, 130 ⫾ 1.3 dGA; hypoxemia I) or during the second episode of acute hypoxemia at 5 ⫾ 2 days (134 ⫾ 1.2 dGA; hypoxemia II) after umbilical cord compression or sham compression. There was a significant change in the intercept (P ⬍ 0.05), but not the slope, of the relationship between plasma ACTH and cortisol in UCC fetuses during hypoxemia II.

produced by partial compression of the umbilical cord is not altered relative to that in sham control fetuses. However, after adverse intrauterine conditions, the basal plasma cortisol, but not ACTH, concentration becomes elevated. This suggests that in contrast to the effects of acute-on-chronic stress on the fetal HPA axis, fetal adrenocortical sensitivity is unaltered, but a resetting of the HPA axis occurs during acute-after-chronic stress. Factors affecting the set-point and sensitivity of the axis are, therefore, differentially modified by the timing, duration, and degree of fetal exposure to adverse intrauterine conditions. A sustained elevation in basal fetal plasma cortisol despite unaltered ACTH levels is a feature of many experimental models that produces prolonged adverse intrauterine conditions. For example, in sheep, sustained fetal hypercortisolemia with only transient elevations in fetal plasma ACTH concentration occur after carunclectomy (20), feto-placental embolization (7, 21), and reduced utero-placental blood flow (22). The mechanism(s) mediating sustained elevations in fetal plasma cortisol in the absence of significant elevations in fetal plasma ACTH during or after adverse intrauterine

Fetal wt (kg) Adrenal wt (mg) Adrenal wt:fetal wt (mg/kg) Total width of adrenal (mm) Adrenocortical width (mm) Adrenomedullary width (mm) Cortex:total adrenal width ratio (%) Medulla:total adrenal width ratio (%)

Control

UCC

P

2.96 ⫾ 0.22 323 ⫾ 9 113 ⫾ 10 3.70 ⫾ 0.34 0.71 ⫾ 0.05 2.15 ⫾ 0.23 19 ⫾ 1

2.64 ⫾ 0.21 519 ⫾ 58 202 ⫾ 29 3.69 ⫾ 0.15 1.01 ⫾ 0.09 1.47 ⫾ 0.12 27 ⫾ 1

NS 0.02 0.04 NS 0.02 0.02 0.01

58 ⫾ 2

40 ⫾ 4

0.01

Adrenal weight and morphology in control and UCC fetuses. All values are the mean ⫾ SEM. Body weight and adrenal weight were determined in control (n ⫽ 5) and UCC (n ⫽ 5) fetuses postmortem (between 136 –137 dGA).

conditions remains unclear, but may involve increased adrenocortical steroidogenic capacity or sensitivity to ACTH, altered trans-placental cortisol passage, decreased negative feedback at the level of the adrenal, an increase in the activity or concentration of ACTH-independent steroidogenic factor, or changes in the ratio of bioactive/immunoreactive ACTH. To address the first possibility, cord-compressed and control fetuses were subjected to an ACTH challenge, and at the end of all experiments, their adrenals were harvested and prepared for histological analysis. Cord compression induced a marked increase in adrenal weight, which was predominantly due to an increase in adrenocortical mass, suggesting a greater potential for increased adrenocortical steroidogenic capacity. Maintained adrenal growth against a declining rate of growth of the fetal body during chronic adverse intrauterine conditions may be achieved through a specific redistribution of the fetal circulation favoring cerebral, myocardial, and adrenal perfusion (23). Larger adrenal glands may reflect an increase in cell number (hyperplasia) or cell size (hypertrophy); however, only adrenal hypertrophy was reported in response to reductions in utero-placental blood flow for 48 h in fetal sheep at a similar gestational age (24), suggesting that increased cell size, rather than num-


ber, may explain the increase in adrenocortical mass in UCC fetuses. However, although the results of the ACTH challenge show that the adrenocortical response in compressed fetuses is not sensitized to ACTH under basal conditions, they do not exclude the possibility that the adrenal cortex in cord-compressed relative to sham control fetuses may be sensitized to ACTH under stimulated conditions. Neural innervation of the adrenal gland by the splanchnic nerve comprises one of the components mediating adrenocortical sensitivity to ACTH. Stimulation of the splanchnic innervation to the adrenal gland potentiates adrenocortical steroidogenesis in response to an exogenous infusion of ACTH (25). In addition, studies in fetal and adult animals have provided clear evidence that tonic activity in the splanchnic nerve maintains the sensitivity of the adrenal cortex to ACTH. In calves (25), lambs (26), and fetal sheep (27), section of the splanchnic nerve reduces the sensitivity of ACTHinduced cortisol output from the adrenal cortex. Therefore, it is possible that fetal exposure to umbilical cord compression may increase sympathetic outflow, thereby increasing adrenocortical sensitivity to circulating ACTH. However, as only the intercept, not the slope, of the relationship between ACTH and cortisol was altered by cord compression, the data suggest that a change in the setting, but not the sensitivity, of the adrenal cortex to ACTH mediates sustained elevations in fetal plasma cortisol after preexposure to umbilical cord compression. Although the extent to which chronic elevations of fetal plasma cortisol concentration reflect greater materno-fetal trans-placental passage cannot be determined in the present study, the potential for this mechanism to maintain higher circulating fetal plasma cortisol concentrations is negligible, because maternal plasma cortisol concentrations remained unaltered from baseline during cord compression and the majority of circulating cortisol in fetuses greater than 120 dGA is of fetal origin (28). Indeed, in sheep, such a scenario is actively prevented by the placental enzyme complex 11␤hydroxysteroid dehydrogenase, which metabolizes bioactive cortisol to its inactive, stable metabolite cortisone (29). It is possible that enhanced fetal cortisol output after preexposure to umbilical cord compression may be due to a reduction in adrenocortical sensitivity to negative feedback by cortisol. Potentially, the level of cortisol-induced negative feedback to the adrenal is highest during late gestation in fetal sheep, as the adrenocortical density of glucocorticoid receptor (GR) is highest at this time (30). A down-regulation of fetal adrenocortical GR can be promoted in late gestation under certain circumstances, for example, after 2-day exposure to dexamethasone (31). However, dexamethasone is a potent synthetic glucocorticoid, and whether a down-regulation of adrenocortical GR occurs after exposure to high concentrations of endogenous glucocorticoid remains to be determined. Increased action of an ACTH-independent steroidogenic factor during cord compression could in part account for potentiation of cortisol output from the adrenal cortex. Possible candidates include several neuropeptides, such as vasoactive intestinal peptide, CRH, and the eicosanoid PGE2, as all have been shown to promote steroidogenesis in the absence of changes in circulating ACTH (32–34). Of these fac-

597

tors, PGE2 is the most likely candidate in fetal sheep, because fetal plasma PGE2 concentrations increase in parallel with plasma cortisol when plasma ACTH is depressed during chronic adverse intrauterine conditions, such as those produced by embolization (11, 35, 36) and reduced uteroplacental blood flow (35). Alternatively the possibility remains that hypothalamic processing of ACTH is altered by umbilical cord compression. ACTH is cosecreted from the fetal sheep pituitary with higher mol wt precursor forms of ACTH, such as pro-ACTH and POMC, which may also exhibit a degree of ACTH-like biological activity (37). The processing of POMC is gestational age dependent (38) and can be affected by fetal stress (39). Therefore, it is possible that fetal exposure to umbilical cord compression may have led to a change in the ratio of bioactive/immunoreactive ACTH in our study, leading to greater adrenocortical stimulation of cortisol output despite lower measured concentrations of ACTH. Finally, in sheep, an ontogenic increase in fetal plasma cortisol occurs toward term that induces maturation of fetal organ systems in preparation for postnatal life and initiates labor and delivery (40). In the present study the concentrations of fetal plasma cortisol measured 7 days after umbilical cord compression (⬃135 dGA) are commensurate with the concentrations of fetal plasma cortisol at approximately 2–3 days before term (⬃141–142 dGA) in control sheep fetuses of the same breed as those used in the present study (40). This may suggest a forward shift in the mechanisms that account for the normal prepartum increment in plasma cortisol in cord-compressed fetuses. However, in the present study this was insufficient to initiate labor, as determined by intrauterine pressure fluctuations, by the end of the experimental protocol. In conclusion, the data reported in this study show that partial compression of the umbilical cord for 3 days in late gestation fetal sheep elevates basal fetal plasma cortisol, but not ACTH, concentrations and does not affect the magnitude of the pituitary-adrenal response to a subsequent period of acute hypoxemia. Additional analysis of the data suggests that the mechanism mediating maintained elevations in fetal plasma cortisol, in the absence of increased ACTH concentrations, is a change in the set-point of the HPA axis and/or increased adrenal steroidogenic capacity, rather than an increase in adrenocortical sensitivity to ACTH. These findings imply that acute stress after, rather than superimposed during, a period of adverse intrauterine conditions has a differential effect on the HPA axis. Sustained elevations of circulating cortisol in the fetus have important implications not only for fetal development (41), but also for adult health, as inappropriate glucocorticoid overexposure in fetal life has been associated with hypertension and insulin resistance in adult life (42, 43). Acknowledgments The authors acknowledge Mr. Paul Hughes for his help during surgery, Mrs. Sue Nicholls and Miss Victoria Johnson for their routine care of the animals used in this study, Mr. Malcolm Bloomfield and Mrs. Kate Clarke for performing the RIAs, and Mrs. Anita Shelley and Mrs. Mel Quy for help in preparation of adrenals for histology.

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