- Email: [email protected]
Corticotropin and cortisol responses to corticotropin-releasing factor in the chronically hypoxemic ovine fetus
Corticotropin and cortisol responses to corticotropin-releasing factor in the chronically hypoxemic ovine fetus DeWana R. Kerr, PhD: Maria I. Castro, PhD:' b Nancy K. Valego, PhD: Nayel M. Rawashdeh, MD, PhD, and James C. Rose, PhD"' b Winston-Salem, North Carolina OBJECTIVE: The purpose of this study was to determine if mild hypoxemia (- 25% below normal) of at least 5 days' duration alters corticotropin and cortisol responses to corticotropin-releasing factor. STUDY DESIGN: We studied 14 (hypoxemic, n = 5; normoxemic, n = 9) fetuses of 135 ± 1 (mean ± SEM) days' gestational age. Fetuses were placed in the experimental group if arterial P02 was :5 16 mm Hg for 5 days. In normoxemic animals arterial P02 was ~ 17 mm Hg. Plasma hormone responses were compared by analysis of variance. RESULTS: Resting corticotropin levels were not different (hypoxemic 26 ± 5 pg/ml, normoxemic 29 ± 12 pg/ml), and corticotropin-releasing factor (530 ± 30 ng/kg) increased (p = 0.01) corticotropin levels similarly in both groups. Basal plasma cortisol levels (hypoxemic 20 ± 10 ng/ml, normoxemic, 30 ± 7 ng/ml) were not significantly different. Both groups had similarly increased (p < 0.01) plasma cortisol levels after corticotropin-releasing factor administration. CONCLUSION: Mild hypoxemia lasting 5 days does not significantly alter corticotropin and cortisol responses to corticotropin-releasing factor in the late-gestation ovine fetus. (AM J OBSTET GYNECOL 1992;167:1686-90.)
Key words: Hypoxemia, fetus, sheep, corticotropin, cortisol
The stress of a reduction in oxygen supply leading to hypoxemia is a potentially serious danger faced by the fetus during gestation and labor. J Most fetal studies of hypoxemia have involved reducing fetal arterial P0 2 30% to 45% below normal. We refer to such conditions as a certain percent hypoxemia (e.g., 30% to 45% hypoxemia). Induction of such levels of hypoxemia and their maintenance for up to 7 hours increase plasma corticotropin concentrations in ovine fetuses between gestational ages of 96 and 145 days (term - 145 days),"·6 Corticosteroid responses to such hypoxemia are undetectable or low at the younger ages and greater later in gestation. 2 . 6 It has also been shown that adrenal secretion rates of corticosteroids are increased by profound hypoxemia. 7 The previously mentioned studies involve the acute induction of fetal hypoxemia late in gestation. The effects of long-term naturally occurring From the Departments of PhysiologylPharmacology" and ObstetricslGynecologyb and the Perinatal Research Laboratories, Bowman Gray School of Medicine, Wake Forest University. Supported by National Institutes of Health grant HD 11210. Presented in part at the Thirty-Seventh Annual Meeting of the Society for Gynecologic Investigation, St. Louis, Missouri, March 21-24, 1990. Reprint requests: James c. Rose, PhD, Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1083. 616/41932
1686
fetal hypoxemia on circulating corticotropin and cortisol levels and their responses to corticotropin-releasing factor (CRF) are unknown. In this report we describe results from a group of fetuses that manifested mild hypoxemia ( < 30% hypoxemia) in the absence of hypercapnia, acidosis, or elevations in hematocrit. We studied these animals to determine if mild hypoxemia, possibly naturally occurring, of ;;:: 5 days' duration altered resting plasma corticotropin and cortisol levels and the responses to synthetic ovine CRF.
Material and methods All surgical and experimental protocols were approved by the Animal Care and Use Committee of the Bowman Gray School of Medicine. We studied 14 chronically cannulated ovine fetuses at 135 ± 1 (mean ± SEM) days of gestation. Term in the sheep is approximately 145 days. Fetuses were placed in an experimental group (hypoxemic, n = 5) if the arterial P0 2 was ::;; 16 mm Hg for ;;:: 5 days. The arterial P0 2 levels were also lower at the time of surgery during maternal ventilation with 100% oxygen, which suggests that the condition probably existed before our receiving the animals in the laboratory. The control group (normoxemic, n = 9) consisted of fetuses with an arterial P0 2 of ;;:: 17 mm Hg. In an attempt to avoid the
Volume 167 Number 6
complicating effects of acidosis and hypercapnia on plasma corticotropin and cortisol levels, only fetuses with normal arterial pH and Pc0 2 values were included in this report. Surgery. Ewes with known single insemination dates in which pregnancy was confirmed at 50 days of gestation by ultrasonography were brought to the vivarium to acclimate them to the environment approximately 5 days before surgery. Food was withheld for 48 hours and water for 24 hours before surgery. Gentamicin (80 mg) (Elkins-Sinn, Cherry Hill, N.J.) was administered intravenously to the ewe before surgery. Anesthesia was induced with intravenous ketamine hydrochloride (Butler Veterinary Supply, Kernersville, N.C.) and pentobarbital sodium (Barber Veterinary Supply, Richmond, Va.) and maintained with 0.75% to 1.00% halothane delivered in 100% oxygen. Strict aseptic precautions were observed. Mter anesthesia, an intravneous infusion of Ringer's solution was started and continued throughout the course of surgery. The uterus was exposed through a midline abdominal incision, delivered, and covered in warm saline solution-moistened towels. The fetal hind limbs were located and exposed through a small uterine incision. Polyvinyl catheters were inserted into the right and left fetal tibial arteries and right saphenous vein and advanced to the descending aorta and inferior vena cava, respectively. A 0.5 ml sample of arterial blood was taken for determination of pH, Pco 2 , P0 2 , and hematocrit to assess animal well-being. Catheters were filled with 1000 D/ml heparin sodium. The exterioized portion of the fetus was then returned to the uterus, after which a polyvinyl catheter was placed in the amniotic cavity. Mter the injection of 80 mg of gentamicin into the amniotic fluid, the uterus was closed with a double row of sutures. Mter completion of the abdominal surgery, all fetal catheters were led to the left flank of the ewe and exteriorized. Polyvinyl chloride catheters were placed into the left maternal femoral artery and vein and advanced to the abdominal aorta and inferior vena cava, respectively. These catheters were filled with heparin, plugged, and exited through a flank incision. Daily the fetal and maternal vascular catheters were drained of heparin, flushed with sterile saline solution, filled with heparin, and plugged to maintain patency. Postoperatively the ewes were housed in metabolic carts in air-conditioned, light-cycled rooms. Experiment. Mter a 5-day recovery period from surgery, the animals were studied with the ewe in a portable metabolic cart. Food and water were withdrawn 1 to 2 hours before the experiment. Each experiment was preceded by 80 mg of gentamicin administered intravenously to the ewe. All experiments were initiated between 11 AM and 1 PM hours. Care was taken to conduct the experiments in a quiet environment. An aliquot of lyophilized ovine CRF (BaChem, Phil-
Plasma corticotropin and cortisol in chronic fetal hypoxemia
1687
adelphia) was diluted with a solution of 0.1 % bovine serum albumin in normal saline solution immediately before each experiment. Fetal weight was estimated on the basis of gestational age and later determined by back calculation from birth or postmortem weight, by assuming a 2% weight gain per day.s Control arterial blood samples were taken. A I-minute intravenous injection of ovine CRF (530 ± 30 ng/kg) in 2 ml of 0.1 % bovine serum albumin in normal saline solution was administered and followed by additional arterial sampling from the ewe and fetus at 30, 60, 120, 240, and 360 minutes after the injection. Arterial blood samples were taken at all sample times from the ewe and fetus for cortisol, blood gas, and hematocrit measurements. Fetal blood samples were also taken for corticotropin at these times. The total volume removed at each sample time was 2.0 m!. Hormone assays. Plasma immunoreactive corticotropin and cortisol concentrations were measured by radioimmunoassays, the characteristics of which have been described in detail elsewhere. g • 10 Blood gas determination. All blood gases and pH were measured at 39% C with a Radiometer ABL30 (Radiometer, Copenhagen) instrument. Statistical procedures. Weight, basal plasma hormone levels, blood gases, and hematocrit were compared with independent group t tests. Two-way analysis of variance for repeated measures was used to assess differences between hypoxemic and normoxemic groups in hormone changes across the length of the experiment. A significance level of p < 0.05 was used. Results
Weight, age, ovine CRF dose, and basal values of blood gases, pH, and hematocrit are shown in Table I. Weight was not different between the two groups. Resting pH, Pc0 2 , and hematocrit were not significantly different and did not change across the experiment. All were within normal limits. Arterial P0 2 levels during surgery (5 days before the experiment), 1 and 2 days before the experiment (hypoxemic group only), and at the beginning of the experiment are shown in Table II. Arterial P0 2 levels were lower in the hypoxemic group during catheterization in the presence of maternal ventilation. Values on days 1 and 2 before the study were not different from those on the day of the experiment in the hypoxemic group. At the beginning of the experiment arterial P0 2 levels remained significantly lower in the hypoxemic group than in the normoxemic group (p < 0.02). During the experiment arterial P0 2 levels did not change in either group. Fig. 1 shows fetal plasma corticotropin responses to the ovine CRF injection. Resting plasma corticotropin levels were not different. The increase in plasma corticotropin levels after ovine CRF administration was sig-
1688 Kerr et al.
December 1992 Am J Obstet Gyneco1
900
•
700
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600
"'tlO
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500
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400
'-"
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o
o
800
•
Hypoxemic Normoxemic
300 200 100
0 -100 -80
80
0
120
240
180
300
420
380
Time ( min) Fig. 1. Fetal plasma corticotropin responses to ovine CRF bolus injection. Values are presented as means ± SEM. Where there are no bars, SEM lies within symbol. Basal values before each ovine CRF injection were not different. Plasma corticotropin levels increased similarly from baseline in both groups after ovine CRF (p = 0.030).
Table II. Arterial P0 2 values
Table I. Weight, age, ovine CRF dose, basal arterial pH, Pc0 2 , and hematocrit Normoxemic Weight (kg) Age (days of gestation) Ovine CRF dose (ng/kg) pH Pe0 2 (mm Hg) Hematocrit (%) No.
2.7 135 536 7.341 45.9 32.9
± 0.2 ± 0.5 ± 65
± 0.007
± 1.7
± 1.7 9
Hypoxemic
2.4 133 598 7.356 49.2 36.3
0.2 0.8 39 0.002 ± 0.9 ± 2.3 5 ± ± ± ±
Days before study
Normoxemic (N (mm Hg)
5* 2
27.5 ± 1.7
o
19.7 ± 1.1
1
=
9)
Hypoxemic (N = 5) (mm Hg)
22.3 15.3 14.7 14.5
± LOt ± 0.3
± 0.6
± 0.8
*At surgery during ventilation of ewe with 100% oxygen. tNormoxemic versus hypoxemic (p < 0.05).
Values are mean ± SE.
nificant (F = 4.823, P < 0.003) and similar in the two groups. Basal plasma cortisol concentrations were not significantly different. Plasma cortisol increased in both groups (F = 9.190,p < 0.0001) similarly subsequent to ovine CRF (Fig. 2). Comment
These results suggest that naturally occurring, mild (- 25%) hypoxemia of at least 5 days' duration does not significantly alter plasma corticotropin and cortisol basal levels or responses to moderate doses of ovine CRF in the late-gestation ovine fetus. This is consistent with a recent report· in which induction of approximately 30% fetal hypoxemia and its maintenance for up to 28 days did not change fetal plasma cortisol levels. II The discrepancy between our results and those of
others, which show increases in plasma corticotropin in response to artificially induced hypoxemia, may be explained in part by the degree of hypoxemia achieved or by the acute onset of the stimulus. 12. 13 When corticotropin responses are observed, the hypoxemia is acutely induced and the fetal arterial P0 2 is lowered by ~ 30%.2-4. 6 It is possible the difference in the effect of various levels of hypoxemia on corticotropin release is related to a threshold of hypoxemia-induced increases in peripheral oxygen chemoreceptor firing rates. Blanco et al. 14 have demonstrated chemoreceptor discharge during basal conditions and increases in firing rate as arterial P0 2 decreases in anesthesized fetal lambs of 90 to 143 days' gestation. The threshold for increased firing appears to be in the vicinity of 30% hypoxemia. Likewise, the threshold for acute-onset, hypoxemia-induced corticotropin release has been estimated to be between 12% and 28% hypoxemia l5 in late gestation, and modest cortisol responses have been
Plasma corticotropin and cortisol in chronic fetal hypoxemia
Volume 167 l'\umber 6
1689
70
-.
60
"0
50
....:l ~
Z
'--"
....:l
40
•
0
rn ..... E--t
o
30
• Normoxemic 0 Hypoxemic
~
0
U
20
10
0 -60
0
60
120
180
240
300
360
420
Time ( min) Fig. 2. Fetal plasma cortisol responses to ovine CRF bolus injection. Resting levels were not different. Plasma cortisol increased similarly in both groups subsequent to ovine CRF administration (P < 0.0001).
observed with a 22% reduction in PO Z • 16 The difference between our findings and those of other studies may also be explained by decreases in arterial pH occurring in some of these studies, which could have served as an additional stimulus to corticotropin release. 2.7 It appears therefore that naturally occurring chronic 25% hypoxemia in the absence of acidosis is not an adequate stimulus for prolonged elevations in plasma corticotropin and cortisol. The possibility remains, although it is unlikely, that 25% hypoxemia initially stimulated plasma corticotropin or cortisol increases, which, with adaptation, returned to normal. 17 Additional findings that reinforce the results of the current study are the observations, in fetal lambs of 125 to 140 days' gestation, that artificial induction of shortterm 20% hypoxemia does not result in increased circulating levels of the catecholamines norepinephrine and epinephrine, whereas more severe hypoxemia does. 1M. 19 It has also been shown plasma arginine vasopressin levels do not increase in response to approximately 20% hypoxemia but do increase at 30% hypoxemia. 15 . 20·22 In addition to CRF, the catecholamines and arginine vasopressin are secreted in response to a variety of stresses and have been implicated as corticotropin-releasing factors. 2". 24 The current study reveals no hypoxemia-induced alteration in plasma corticotropin and cortisol responses to a moderate ovine CRF injection. Consistent with this observation are the findings of Hashimoto et al. 25 who demonstrated that in adult rats, plasma corti-
cotropin and corticosterone responses to exogenous CRF are not altered by the stress of chronic immobilization. In addition to effects on pituitary-adrenal function, hypoxemia has been associated with diminished fetal weight. 26 . 27 The current study suggests chronic, naturally occurring 25% hypoxemia in fetal lambs has no effect on weight. Consistent with this finding is the recent report that artifically induced 30% hypoxemia of 7 to 28 days' duration does not alter fetal weight in late gestation. II However, when lamb fetuses are maintained at 30% hypoxemia from 30 to 135 days of gestation 26 or at very low levels of arterial oxygen tension during the last month of gestation,27 growth retardation is observed. In both ovine and human fetuses certain levels of acute hypoxemia also lead to increases in hematocrit, which help to maintain fetal oxygenation. 2M Although there is a tendency in the current study toward increased hematocrit levels with chronic 25% hypoxemia, significant elevations are not detected. This is unlike artificially induced 30% hypoxemia maintained for > 28 days in late gestation, which raises hematocrit. II These findings, in combination with our results, suggest the hypoxemia threshold for a significant hematocrit increase (presumably through hematopoietin stimulation) lies between 25% and 30% hypoxemia. We thank S. Block and J Eisenach for consultation and K. Barnes, H. Booze, B. Brooks, and B. Tucker for technical support. The corticotropin used for iodina-
1690 Kerr et al.
tion was kindly provided by the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases. REFERENCES 1. Parer ]T, Livingston EG. What is fetal distress? AM ] OBSTET GYNECOL 1990;162:1421-7. 2. Alexander DP, Forsling ML, Martin M], et al. The effect of maternal hypoxia on fetal pituitary hormone release in the sheep. Bioi Neonate 1972;21:219-28. 3. Boddy K, Jones CT, Mantell C, Ratcliffe ]G, Robinson ]S. Changes in plasma ACTH and corticosteroid of the maternal and fetal sheep during hypoxia. Endocrinology 1974;94:588-91. 4. Jones CT, Ritchie ]WK. Endocrine and metabolic changes associated with periods of spontaneous hypoxia in fetal sheep. Bioi Neonate 1976;29:286-93. 5. Jones CT, Boddy K, Robinson ]S, Ratcliffe ]G. Developmental changes in the responses of the adrenal glands of foetal sheep to endogenous adrenocorticotropin, as indicated by hormone responses to hypoxemia. ] Endocrinol 1977;72:279-92. 6. Challis ]RG, Richardson BS, Rurak D, W10dek ME, Patrick ]E. Plasma adrenocroticotropin hormone and adrenal blood flow during sustained hypoxemia in fetal sheep. AM ] OBSTET GYNECOL 1986;155:1332-6. 7. Jackson BT, Morrison SH, Cohn HE, Piasecki GJ. Adrenal secretion of glucocorticoids during hypoxemia in fetal sheep. Endocrinology 1989;125:2751-7. 8. ]obert DM. A study of pre-natal growth and development in the sheep.] Agr Sci 1965;47:390-428. 9. Rose ]C, MacDonald AA, Heyman MA, Rudolph PM. Developmental aspects of the pituitary-adrenal responses to hemorrhagic stress in lamb fetuses in utero.] Clin Invest 1978;61 :424-8. 10. Rose ]C, Meis P], Morris M. Ontogeny of endocrine responses to hypotension in lamb fetuses. Am ] Physiol 1981; 140:E656-61. 11. Kitanaka T, Alonso ]G, Gilbert RD, Sui BL, Clemons GK, Longo LD. Fetal responses to long-term hypoxemia in sheep. Am] PhysioI1989;256:RI348-54. 12. Cook DM, Kendall], Greer MA, Kramer RM. The effect of acute or chronic ether stress on plasma ACTH concentration in the rat. Endocrinology 1973;92: 10 19-24. 13. Raff H, Shinsake ], Keil LC, Dallman MF. Vasopressin, ACTH, and blood pressure during hypoxia induced at different times. Am] Physiol 1983;245:E489-93. 14. Blanco CE, Dawes GS, Hanson MA, McCooke HB. The response to hypoxia of arterial chemoreceptors in fetal
December 1992 Am J Obstet Gyneco1
sheep and newborn lambs. ] Physiol (Lond) 1984;351 :2537. 15. Ahagi K, Challis ]RG. Threshold of hormonal and biophysical responses to acute hypoxemia in fetal sheep at different gestational ages Can] Physiol Pharmacol 1990; 68:549-55. 16. Towell ME, Figueroa], Manhowitz S, Elias B, Nathanielsz P. The effect of mild hypoxemia maintained for twentyfour hours on maternal and fetal glucose, lactate, cortisol, and arginine vasopressin in pregnant sheep at 122 and 139 days gestation. AM ] OBSTET GYNECOL 1987;157: 1550-7. 17. Rivier C, Vale W. Diminished responsiveness of the hypothalamic-pituitary-adrenal axis of the rat during exposure to prolonged stress: a pituitary-mediated mechanism. Endocrinology 1987;121:1320-8. 18. Cohn WR, Piasecki G], Jackson BT. Plasma catecholamines during hypoxemia in fetal lambs. Am] Physiol 1982;243:R520-5. 19. Jones CT, Robinson RO. Plasma catecholamines in foetal and adult sheep.] Physiol 1975;248:15-33. 20. Nakamura T, Ayres NA, Gomez RA, Robillard ]E. Renal responses to hypoxemia during renin-angiotensin system inhibition in fetal lambs. Am] Physiol 1985;249:RI16-24. 21. Rurak DW. Plasma vasopressin levels during hypoxemia and the cardiovascular effects of exogenous vasopressin in fetal and adult sheep.] Physiol (Lond) 1978;277:341-57. 22. Challis ]RG, Brooks AN. Maturation and activation of hypothalamic-pituitary-adrenal function in fetal sheep. Endocr Rev 1989;10:182-203. 23. Rivier C, Vale W. Roles of corticotropin releasing factor, catecholamines and vasopressin in modulating stress-induced ACTH release in the adult rat. Nature 1983;305: 325-7. 24. Axelrod], Reisine TD. Stress hormones: their interaction and regulation. Science 1984;224:452-9. 25. Hashimoto K, Suemaru S, Takao T, Sugawara M, Makino S, Ota Z. Corticotropin-releasing hormone and pituitaryadrenocortical responses in chronically stressed rats. Regul Pept 1988;23:117-26. 26. Jacobs R, Falconer], Robinson]S, Webster MED. Effect of hypoxia on the initiation of secondary wool follicles in the fetus. Aust] Bioi Sci 1986;39:79-88. 27. Jacobs R, Owens ]A, Falconer j, Webster MED, Robinson ]S. Changes to metabolite concentration in fetal sheep subjected to prolonged hypobaric hypoxia.] Dev Physiol 1988; 10: 113-21. 28. Linderkamp O. Placental transfusion: determinants and effects. Clin Perinatol 1982;9:559-92.
Key words: Hypoxemia, fetus, sheep, corticotropin, cortisol
The stress of a reduction in oxygen supply leading to hypoxemia is a potentially serious danger faced by the fetus during gestation and labor. J Most fetal studies of hypoxemia have involved reducing fetal arterial P0 2 30% to 45% below normal. We refer to such conditions as a certain percent hypoxemia (e.g., 30% to 45% hypoxemia). Induction of such levels of hypoxemia and their maintenance for up to 7 hours increase plasma corticotropin concentrations in ovine fetuses between gestational ages of 96 and 145 days (term - 145 days),"·6 Corticosteroid responses to such hypoxemia are undetectable or low at the younger ages and greater later in gestation. 2 . 6 It has also been shown that adrenal secretion rates of corticosteroids are increased by profound hypoxemia. 7 The previously mentioned studies involve the acute induction of fetal hypoxemia late in gestation. The effects of long-term naturally occurring From the Departments of PhysiologylPharmacology" and ObstetricslGynecologyb and the Perinatal Research Laboratories, Bowman Gray School of Medicine, Wake Forest University. Supported by National Institutes of Health grant HD 11210. Presented in part at the Thirty-Seventh Annual Meeting of the Society for Gynecologic Investigation, St. Louis, Missouri, March 21-24, 1990. Reprint requests: James c. Rose, PhD, Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1083. 616/41932
1686
fetal hypoxemia on circulating corticotropin and cortisol levels and their responses to corticotropin-releasing factor (CRF) are unknown. In this report we describe results from a group of fetuses that manifested mild hypoxemia ( < 30% hypoxemia) in the absence of hypercapnia, acidosis, or elevations in hematocrit. We studied these animals to determine if mild hypoxemia, possibly naturally occurring, of ;;:: 5 days' duration altered resting plasma corticotropin and cortisol levels and the responses to synthetic ovine CRF.
Material and methods All surgical and experimental protocols were approved by the Animal Care and Use Committee of the Bowman Gray School of Medicine. We studied 14 chronically cannulated ovine fetuses at 135 ± 1 (mean ± SEM) days of gestation. Term in the sheep is approximately 145 days. Fetuses were placed in an experimental group (hypoxemic, n = 5) if the arterial P0 2 was ::;; 16 mm Hg for ;;:: 5 days. The arterial P0 2 levels were also lower at the time of surgery during maternal ventilation with 100% oxygen, which suggests that the condition probably existed before our receiving the animals in the laboratory. The control group (normoxemic, n = 9) consisted of fetuses with an arterial P0 2 of ;;:: 17 mm Hg. In an attempt to avoid the
Volume 167 Number 6
complicating effects of acidosis and hypercapnia on plasma corticotropin and cortisol levels, only fetuses with normal arterial pH and Pc0 2 values were included in this report. Surgery. Ewes with known single insemination dates in which pregnancy was confirmed at 50 days of gestation by ultrasonography were brought to the vivarium to acclimate them to the environment approximately 5 days before surgery. Food was withheld for 48 hours and water for 24 hours before surgery. Gentamicin (80 mg) (Elkins-Sinn, Cherry Hill, N.J.) was administered intravenously to the ewe before surgery. Anesthesia was induced with intravenous ketamine hydrochloride (Butler Veterinary Supply, Kernersville, N.C.) and pentobarbital sodium (Barber Veterinary Supply, Richmond, Va.) and maintained with 0.75% to 1.00% halothane delivered in 100% oxygen. Strict aseptic precautions were observed. Mter anesthesia, an intravneous infusion of Ringer's solution was started and continued throughout the course of surgery. The uterus was exposed through a midline abdominal incision, delivered, and covered in warm saline solution-moistened towels. The fetal hind limbs were located and exposed through a small uterine incision. Polyvinyl catheters were inserted into the right and left fetal tibial arteries and right saphenous vein and advanced to the descending aorta and inferior vena cava, respectively. A 0.5 ml sample of arterial blood was taken for determination of pH, Pco 2 , P0 2 , and hematocrit to assess animal well-being. Catheters were filled with 1000 D/ml heparin sodium. The exterioized portion of the fetus was then returned to the uterus, after which a polyvinyl catheter was placed in the amniotic cavity. Mter the injection of 80 mg of gentamicin into the amniotic fluid, the uterus was closed with a double row of sutures. Mter completion of the abdominal surgery, all fetal catheters were led to the left flank of the ewe and exteriorized. Polyvinyl chloride catheters were placed into the left maternal femoral artery and vein and advanced to the abdominal aorta and inferior vena cava, respectively. These catheters were filled with heparin, plugged, and exited through a flank incision. Daily the fetal and maternal vascular catheters were drained of heparin, flushed with sterile saline solution, filled with heparin, and plugged to maintain patency. Postoperatively the ewes were housed in metabolic carts in air-conditioned, light-cycled rooms. Experiment. Mter a 5-day recovery period from surgery, the animals were studied with the ewe in a portable metabolic cart. Food and water were withdrawn 1 to 2 hours before the experiment. Each experiment was preceded by 80 mg of gentamicin administered intravenously to the ewe. All experiments were initiated between 11 AM and 1 PM hours. Care was taken to conduct the experiments in a quiet environment. An aliquot of lyophilized ovine CRF (BaChem, Phil-
Plasma corticotropin and cortisol in chronic fetal hypoxemia
1687
adelphia) was diluted with a solution of 0.1 % bovine serum albumin in normal saline solution immediately before each experiment. Fetal weight was estimated on the basis of gestational age and later determined by back calculation from birth or postmortem weight, by assuming a 2% weight gain per day.s Control arterial blood samples were taken. A I-minute intravenous injection of ovine CRF (530 ± 30 ng/kg) in 2 ml of 0.1 % bovine serum albumin in normal saline solution was administered and followed by additional arterial sampling from the ewe and fetus at 30, 60, 120, 240, and 360 minutes after the injection. Arterial blood samples were taken at all sample times from the ewe and fetus for cortisol, blood gas, and hematocrit measurements. Fetal blood samples were also taken for corticotropin at these times. The total volume removed at each sample time was 2.0 m!. Hormone assays. Plasma immunoreactive corticotropin and cortisol concentrations were measured by radioimmunoassays, the characteristics of which have been described in detail elsewhere. g • 10 Blood gas determination. All blood gases and pH were measured at 39% C with a Radiometer ABL30 (Radiometer, Copenhagen) instrument. Statistical procedures. Weight, basal plasma hormone levels, blood gases, and hematocrit were compared with independent group t tests. Two-way analysis of variance for repeated measures was used to assess differences between hypoxemic and normoxemic groups in hormone changes across the length of the experiment. A significance level of p < 0.05 was used. Results
Weight, age, ovine CRF dose, and basal values of blood gases, pH, and hematocrit are shown in Table I. Weight was not different between the two groups. Resting pH, Pc0 2 , and hematocrit were not significantly different and did not change across the experiment. All were within normal limits. Arterial P0 2 levels during surgery (5 days before the experiment), 1 and 2 days before the experiment (hypoxemic group only), and at the beginning of the experiment are shown in Table II. Arterial P0 2 levels were lower in the hypoxemic group during catheterization in the presence of maternal ventilation. Values on days 1 and 2 before the study were not different from those on the day of the experiment in the hypoxemic group. At the beginning of the experiment arterial P0 2 levels remained significantly lower in the hypoxemic group than in the normoxemic group (p < 0.02). During the experiment arterial P0 2 levels did not change in either group. Fig. 1 shows fetal plasma corticotropin responses to the ovine CRF injection. Resting plasma corticotropin levels were not different. The increase in plasma corticotropin levels after ovine CRF administration was sig-
1688 Kerr et al.
December 1992 Am J Obstet Gyneco1
900
•
700
..--. ......
600
"'tlO
S
500
0..
400
'-"
..c: ...., CJ -<
o
o
800
•
Hypoxemic Normoxemic
300 200 100
0 -100 -80
80
0
120
240
180
300
420
380
Time ( min) Fig. 1. Fetal plasma corticotropin responses to ovine CRF bolus injection. Values are presented as means ± SEM. Where there are no bars, SEM lies within symbol. Basal values before each ovine CRF injection were not different. Plasma corticotropin levels increased similarly from baseline in both groups after ovine CRF (p = 0.030).
Table II. Arterial P0 2 values
Table I. Weight, age, ovine CRF dose, basal arterial pH, Pc0 2 , and hematocrit Normoxemic Weight (kg) Age (days of gestation) Ovine CRF dose (ng/kg) pH Pe0 2 (mm Hg) Hematocrit (%) No.
2.7 135 536 7.341 45.9 32.9
± 0.2 ± 0.5 ± 65
± 0.007
± 1.7
± 1.7 9
Hypoxemic
2.4 133 598 7.356 49.2 36.3
0.2 0.8 39 0.002 ± 0.9 ± 2.3 5 ± ± ± ±
Days before study
Normoxemic (N (mm Hg)
5* 2
27.5 ± 1.7
o
19.7 ± 1.1
1
=
9)
Hypoxemic (N = 5) (mm Hg)
22.3 15.3 14.7 14.5
± LOt ± 0.3
± 0.6
± 0.8
*At surgery during ventilation of ewe with 100% oxygen. tNormoxemic versus hypoxemic (p < 0.05).
Values are mean ± SE.
nificant (F = 4.823, P < 0.003) and similar in the two groups. Basal plasma cortisol concentrations were not significantly different. Plasma cortisol increased in both groups (F = 9.190,p < 0.0001) similarly subsequent to ovine CRF (Fig. 2). Comment
These results suggest that naturally occurring, mild (- 25%) hypoxemia of at least 5 days' duration does not significantly alter plasma corticotropin and cortisol basal levels or responses to moderate doses of ovine CRF in the late-gestation ovine fetus. This is consistent with a recent report· in which induction of approximately 30% fetal hypoxemia and its maintenance for up to 28 days did not change fetal plasma cortisol levels. II The discrepancy between our results and those of
others, which show increases in plasma corticotropin in response to artificially induced hypoxemia, may be explained in part by the degree of hypoxemia achieved or by the acute onset of the stimulus. 12. 13 When corticotropin responses are observed, the hypoxemia is acutely induced and the fetal arterial P0 2 is lowered by ~ 30%.2-4. 6 It is possible the difference in the effect of various levels of hypoxemia on corticotropin release is related to a threshold of hypoxemia-induced increases in peripheral oxygen chemoreceptor firing rates. Blanco et al. 14 have demonstrated chemoreceptor discharge during basal conditions and increases in firing rate as arterial P0 2 decreases in anesthesized fetal lambs of 90 to 143 days' gestation. The threshold for increased firing appears to be in the vicinity of 30% hypoxemia. Likewise, the threshold for acute-onset, hypoxemia-induced corticotropin release has been estimated to be between 12% and 28% hypoxemia l5 in late gestation, and modest cortisol responses have been
Plasma corticotropin and cortisol in chronic fetal hypoxemia
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Time ( min) Fig. 2. Fetal plasma cortisol responses to ovine CRF bolus injection. Resting levels were not different. Plasma cortisol increased similarly in both groups subsequent to ovine CRF administration (P < 0.0001).
observed with a 22% reduction in PO Z • 16 The difference between our findings and those of other studies may also be explained by decreases in arterial pH occurring in some of these studies, which could have served as an additional stimulus to corticotropin release. 2.7 It appears therefore that naturally occurring chronic 25% hypoxemia in the absence of acidosis is not an adequate stimulus for prolonged elevations in plasma corticotropin and cortisol. The possibility remains, although it is unlikely, that 25% hypoxemia initially stimulated plasma corticotropin or cortisol increases, which, with adaptation, returned to normal. 17 Additional findings that reinforce the results of the current study are the observations, in fetal lambs of 125 to 140 days' gestation, that artificial induction of shortterm 20% hypoxemia does not result in increased circulating levels of the catecholamines norepinephrine and epinephrine, whereas more severe hypoxemia does. 1M. 19 It has also been shown plasma arginine vasopressin levels do not increase in response to approximately 20% hypoxemia but do increase at 30% hypoxemia. 15 . 20·22 In addition to CRF, the catecholamines and arginine vasopressin are secreted in response to a variety of stresses and have been implicated as corticotropin-releasing factors. 2". 24 The current study reveals no hypoxemia-induced alteration in plasma corticotropin and cortisol responses to a moderate ovine CRF injection. Consistent with this observation are the findings of Hashimoto et al. 25 who demonstrated that in adult rats, plasma corti-
cotropin and corticosterone responses to exogenous CRF are not altered by the stress of chronic immobilization. In addition to effects on pituitary-adrenal function, hypoxemia has been associated with diminished fetal weight. 26 . 27 The current study suggests chronic, naturally occurring 25% hypoxemia in fetal lambs has no effect on weight. Consistent with this finding is the recent report that artifically induced 30% hypoxemia of 7 to 28 days' duration does not alter fetal weight in late gestation. II However, when lamb fetuses are maintained at 30% hypoxemia from 30 to 135 days of gestation 26 or at very low levels of arterial oxygen tension during the last month of gestation,27 growth retardation is observed. In both ovine and human fetuses certain levels of acute hypoxemia also lead to increases in hematocrit, which help to maintain fetal oxygenation. 2M Although there is a tendency in the current study toward increased hematocrit levels with chronic 25% hypoxemia, significant elevations are not detected. This is unlike artificially induced 30% hypoxemia maintained for > 28 days in late gestation, which raises hematocrit. II These findings, in combination with our results, suggest the hypoxemia threshold for a significant hematocrit increase (presumably through hematopoietin stimulation) lies between 25% and 30% hypoxemia. We thank S. Block and J Eisenach for consultation and K. Barnes, H. Booze, B. Brooks, and B. Tucker for technical support. The corticotropin used for iodina-
1690 Kerr et al.
tion was kindly provided by the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases. REFERENCES 1. Parer ]T, Livingston EG. What is fetal distress? AM ] OBSTET GYNECOL 1990;162:1421-7. 2. Alexander DP, Forsling ML, Martin M], et al. The effect of maternal hypoxia on fetal pituitary hormone release in the sheep. Bioi Neonate 1972;21:219-28. 3. Boddy K, Jones CT, Mantell C, Ratcliffe ]G, Robinson ]S. Changes in plasma ACTH and corticosteroid of the maternal and fetal sheep during hypoxia. Endocrinology 1974;94:588-91. 4. Jones CT, Ritchie ]WK. Endocrine and metabolic changes associated with periods of spontaneous hypoxia in fetal sheep. Bioi Neonate 1976;29:286-93. 5. Jones CT, Boddy K, Robinson ]S, Ratcliffe ]G. Developmental changes in the responses of the adrenal glands of foetal sheep to endogenous adrenocorticotropin, as indicated by hormone responses to hypoxemia. ] Endocrinol 1977;72:279-92. 6. Challis ]RG, Richardson BS, Rurak D, W10dek ME, Patrick ]E. Plasma adrenocroticotropin hormone and adrenal blood flow during sustained hypoxemia in fetal sheep. AM ] OBSTET GYNECOL 1986;155:1332-6. 7. Jackson BT, Morrison SH, Cohn HE, Piasecki GJ. Adrenal secretion of glucocorticoids during hypoxemia in fetal sheep. Endocrinology 1989;125:2751-7. 8. ]obert DM. A study of pre-natal growth and development in the sheep.] Agr Sci 1965;47:390-428. 9. Rose ]C, MacDonald AA, Heyman MA, Rudolph PM. Developmental aspects of the pituitary-adrenal responses to hemorrhagic stress in lamb fetuses in utero.] Clin Invest 1978;61 :424-8. 10. Rose ]C, Meis P], Morris M. Ontogeny of endocrine responses to hypotension in lamb fetuses. Am ] Physiol 1981; 140:E656-61. 11. Kitanaka T, Alonso ]G, Gilbert RD, Sui BL, Clemons GK, Longo LD. Fetal responses to long-term hypoxemia in sheep. Am] PhysioI1989;256:RI348-54. 12. Cook DM, Kendall], Greer MA, Kramer RM. The effect of acute or chronic ether stress on plasma ACTH concentration in the rat. Endocrinology 1973;92: 10 19-24. 13. Raff H, Shinsake ], Keil LC, Dallman MF. Vasopressin, ACTH, and blood pressure during hypoxia induced at different times. Am] Physiol 1983;245:E489-93. 14. Blanco CE, Dawes GS, Hanson MA, McCooke HB. The response to hypoxia of arterial chemoreceptors in fetal
December 1992 Am J Obstet Gyneco1
sheep and newborn lambs. ] Physiol (Lond) 1984;351 :2537. 15. Ahagi K, Challis ]RG. Threshold of hormonal and biophysical responses to acute hypoxemia in fetal sheep at different gestational ages Can] Physiol Pharmacol 1990; 68:549-55. 16. Towell ME, Figueroa], Manhowitz S, Elias B, Nathanielsz P. The effect of mild hypoxemia maintained for twentyfour hours on maternal and fetal glucose, lactate, cortisol, and arginine vasopressin in pregnant sheep at 122 and 139 days gestation. AM ] OBSTET GYNECOL 1987;157: 1550-7. 17. Rivier C, Vale W. Diminished responsiveness of the hypothalamic-pituitary-adrenal axis of the rat during exposure to prolonged stress: a pituitary-mediated mechanism. Endocrinology 1987;121:1320-8. 18. Cohn WR, Piasecki G], Jackson BT. Plasma catecholamines during hypoxemia in fetal lambs. Am] Physiol 1982;243:R520-5. 19. Jones CT, Robinson RO. Plasma catecholamines in foetal and adult sheep.] Physiol 1975;248:15-33. 20. Nakamura T, Ayres NA, Gomez RA, Robillard ]E. Renal responses to hypoxemia during renin-angiotensin system inhibition in fetal lambs. Am] Physiol 1985;249:RI16-24. 21. Rurak DW. Plasma vasopressin levels during hypoxemia and the cardiovascular effects of exogenous vasopressin in fetal and adult sheep.] Physiol (Lond) 1978;277:341-57. 22. Challis ]RG, Brooks AN. Maturation and activation of hypothalamic-pituitary-adrenal function in fetal sheep. Endocr Rev 1989;10:182-203. 23. Rivier C, Vale W. Roles of corticotropin releasing factor, catecholamines and vasopressin in modulating stress-induced ACTH release in the adult rat. Nature 1983;305: 325-7. 24. Axelrod], Reisine TD. Stress hormones: their interaction and regulation. Science 1984;224:452-9. 25. Hashimoto K, Suemaru S, Takao T, Sugawara M, Makino S, Ota Z. Corticotropin-releasing hormone and pituitaryadrenocortical responses in chronically stressed rats. Regul Pept 1988;23:117-26. 26. Jacobs R, Falconer], Robinson]S, Webster MED. Effect of hypoxia on the initiation of secondary wool follicles in the fetus. Aust] Bioi Sci 1986;39:79-88. 27. Jacobs R, Owens ]A, Falconer j, Webster MED, Robinson ]S. Changes to metabolite concentration in fetal sheep subjected to prolonged hypobaric hypoxia.] Dev Physiol 1988; 10: 113-21. 28. Linderkamp O. Placental transfusion: determinants and effects. Clin Perinatol 1982;9:559-92.